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equently, the Barrett's and Esophageal Adenocarcinoma Consortium (BEACON) identified risk loci for BE and esophageal adenocarcinoma near CRTC1 and BARX1, and within 100 kb of FOXP1. We aimed to identify further SNPs that increased BE risk and to validate previously reported associations. METHODS: We performed a genome-wide association study (GWAS) to identify variants associated with BE and further analyzed promising variants identified by BEACON by genotyping 10,158 patients with BE and 21,062 controls. RESULTS: We identified 2 SNPs not previously associated with BE: rs3072 (2p24.1; odds ratio [OR] = 1.14; 95% CI: 1.09-1.18; P = 1.8 x 10(-11)) and rs2701108 (12q24.21; OR = 0.90; 95% CI: 0.86-0.93; P = 7.5 x 10(-9)). The closest protein-coding genes were respectively GDF7 (rs3072), which encodes a ligand in the bone morphogenetic protein pathway, and TBX5 (rs2701108), which encodes a transcription factor that regulates esophageal and cardiac development. Our data also supported in BE cases 3 risk SNPs identified by BEACON (rs2687201, rs11789015, and rs10423674). Meta-analysis of all data identified another SNP associated with BE and esophageal adenocarcinoma: rs3784262, within ALDH1A2 (OR = 0.90; 95% CI: 0.87-0.93; P = 3.72 x 10(-9)). CONCLUSIONS: We identified 2 loci associated with risk of BE and provided data to support a further locus. The genes we found to be associated with risk for BE encode transcription factors involved in thoracic, diaphragmatic, and esophageal development or proteins involved in the inflammatory response.

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otection effects. We used a beta-cyclodextrin (beta-CD) drug modification method in reduce of the necessary dose of the SHSST. As the results, the FAS-FADD expressions leaded apoptosis in CCl4 intraperitoneal (IP) injection induced acute liver injury in rats. Silymarin, baicalein, SHSST, and SHSST beta-CD complex (SHSSTc) pretreatments protected liver through the decreasing of the expressions of FAS-FADD and downstream caspase-3 and caspase-8. Particularly, SHSSTc (30 mg/kg day) treatment enhanced cell survival pathway activation through the PI3K, Akt and Bad phosphorylation. Compared with SHSST as well as silymarin and baicalein, SHSSTc provided a magnificent liver protection effect, especially in survival pathway activation/TUNEL-apoptotic cell reduction/serum cholesterol level suppression. All these data suggested that beta-CD complex modified the SHSST and promoted the bioavailability and liver protection effects. (c) 2014 Wiley Periodicals, Inc. Environ Toxicol, 2014.

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% of the MWF aerosol particles had diameters in the range of 0.42-4.6 microm, with geometric mean diameter of 1.56 microm. The body weight and pulmonary function parameters were measured every week during the 8 weeks of exposure, whereas bronchoalveolar lavage (BAL) fluid was prepared to measure the inflammatory markers and cytokines after the 8 weeks of exposure. There were no changes in body weight and respiratory function (tidal volume and respiratory frequency) during the 8 weeks of exposure to the MWF containing endotoxins, yet lung weight increased significantly (P < 0.05) in the rats exposed to the MWF both with and without endotoxins. The number of polymorphonuclear (PMN) cells in the BAL fluid increased significantly (P < 0.05) in the rats exposed to MWF with endotoxins, and the levels of cytokines such as IL-4, INF-gamma, IL-1beta, and TNF-alpha also were significantly increased (P < 0.05) compared to the control. The NOx production activity of the BAL cells increased significantly (P < 0.05) in the rats exposed to the MWF with and without endotoxins. Increases in lung weight, number of PMN cells, and levels of extracellular cytokines and NOx were all more significant in the rats exposed to the MWF with endotoxins rather than in those exposed to MWF without endotoxins. In spleen cell cultures, T-cell proliferation activity was decreased, yet cytokine levels (INF-gamma, IL-1beta, IL-4, and TNF-alpha) remained unchanged after repeated exposure to MWF with and without endotoxins. Although the levels of total IgG(1), IgG(2a), and IgE antibodies in the serum were not changed, the levels of endotoxin-specific antibodies, including IgG(2a) and IgE, were increased significantly (P < 0.05) in the rats exposed to endotoxins, but there was not a significant increase in endotoxin-specific IgG(1). When taken together, the results indicate that lung inflammatory responses can be induced without changing pulmonary function after repeated exposure to MWFs contaminated with endotoxins. In addition, endotoxin-specific IgG(2a) and IgE may be effective biomarkers for workers exposed to MWFs contaminated with endotoxins in the workplace.

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re have not been clarified. In the present study, we investigated the effect of repeated painting of VOCs onto mouse skin. Various concentrations of toluene, xylene, and formaldehyde (FA) were applied once a week for 5 weeks. While FA solution (2-10%) induced remarkable ear swelling and caused evident infiltration of inflammatory cells, high concentrations of toluene and xylene (50 or 100%) evoked mild ear swelling and marginal inflammatory cell invasion. In addition, FA exposure markedly increased the expression of interleukin-4 (IL-4), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and transient receptor potential vanilloid-1 (TRPV-1) mRNAs in the ears and IL-4 and NT-3 mRNAs in the cervical lymph nodes. Furthermore, capsazepine, a TRPV-1 antagonist, significantly suppressed ear swelling caused by repeated painting of 5% FA. These findings demonstrate that FA has more potent irritancy against skin than toluene or xylene and suggest that the Th2 response, neurotrophins and TRPV-1 play important roles in FA-induced skin inflammation.

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mal blood glucose levels as nondiabetic controls. Among T2DM patients, 1116 have MVCDs, 266 have a history of T2DM of >10 years but never developed MVCDs. Eighty-two single-nucleotide polymorphisms (SNPs) in 54 candidate genes were genotyped. Discrete association studies were performed by the PLINK program for T2DM and MVCDs. Significant associations were found among candidate gene SNPs and T2DM, including rs1526167 of the TOX gene (allele A, P = 2.85 × 10(-9), OR = 1.44). The SNP rs10811661 of the CDKN2A/B gene was also associated with T2DM (allele T, P = 4.09 × 10(-7), OR = 1.36). When we used control patients with >10 years of T2DM history without MVCD, we found that the G allele of SNP rs1526167 of the TOX gene was associated with MVCD (nominal P = 4.33 × 10(-4)). In our study, significant associations were found between TOX and CDKN2A/B gene SNPs and T2DM. The TOX polymorphism might account for the higher risk of T2DM and the lower risk of MVCDs in the Han Chinese population.

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ship between TGF-β and GSN in aging induced cardiac remodeling is still unknown. We evaluated the expressions of TGF-β and GSN in HFD fed 22 months old aging SD rats, followed by the administration of either probucol or alcalase potato protein hydrolysate (APPH). Western blotting and Masson trichrome staining showed that APPH (45 and 75 mg/kg/day) and probucol (500 mg/kg/day) treatments significantly reduced the aging and HFD-induced hypertrophy and fibrosis. Echocardiograph showed that the performance of the hearts was improved in APPH, and probucol treated HFD aging rats. Serum from all rats was collected and H9c2 cells were cultured with collected serums separately. The GSN dependent hypertrophy was inhibited with an exogenous TGF-β in H9c2 cells cultured in HFD+ APPH treated serum. Thus, we propose that along with its role in cardiac fibrosis, TGF-β also acts as an upstream activator of GSN dependent hypertrophy. Hence, TGF-β in serum could be a promising therapeutic target for cardiac remodeling in aging and/or obese subjects.

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leviate OXA-induced peripheral neurotoxicity by selectively inhibiting these uptake transporters in vitro and in vivo. Our results revealed that organic cation transporter 2 (OCT2), organic cation/carnitine transporter 1 (OCTN1) and organic cation/carnitine transporter 2 (OCTN2) were involved in the uptake of OXA in dorsal root ganglion (DRG) neurons and mitochondria, respectively. L-THP (1-100 μM) reduced OXA (40 μM) induced cytotoxicity in MDCK-hOCT2 (Madin-Darby canine kidney, MDCK), MDCK-hOCTN1, MDCK-hOCTN2, and rat primary DRG cells, and decreased the accumulation of OXA in above cells and rat DRG mitochondria, but did not affect its efflux from MDCK-hMRP2 cells. Furthermore, Co-administration of L-THP (5-20 mg/kg for mice, 10-40 mg/kg for rats; twice a week, iv or ig) attenuated OXA (8 mg/kg for mice, 4 mg/kg for rats; twice a week, iv) induced peripheral neurotoxicity and reduced the platinum concentration in the DRG. Whereas, L-THP (1-100 μM for cells; 10-20 mg/kg for mice) did not impair the antitumour efficacy of OXA (40 μM for cells; 8 mg/kg for mice) in HT29 tumour-bearing nude mice nor in tumour cells (HT29 and SW620 cells). In conclusion, OCT2, OCTN1 and OCTN2 contribute to OXA uptake in the DRG and mitochondria. L-THP attenuates OXA-induced peripheral neurotoxicity via inhibiting OXA uptake but without impairing the antitumour efficacy of OXA. L-THP is a potential candidate drug to attenuate OXA-induced peripheral neurotoxicity.

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ethane dimethane sulfone (EDS) to eliminate all Leydig cells in the testis and then were divided into 4 groups of 0 (control), 10, 100, and 1000 mg/kg/day DCHP. Rats were gavaged either vehicle (corn oil, control) or DCHP from post-EDS day 7 to day 21 and 28. On post-EDS day 21 and day 28, rats were euthanized and serum testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH) levels were measured, and Leydig cell number, cell size, gene, and protein expression were evaluated. During the course of exposure, DCHP did not cause the general toxicity to rats. On post-EDS day 21, DCHP significantly increased serum testosterone level at 10 and 100 mg/kg and increased Leydig cell number at 10 mg/kg via stimulating their mitosis. On post-EDS day 28, DCHP lowered serum testosterone levels and Leydig cell number at 1000 mg/kg. DCHP dose-dependently down-regulated the expression of many Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, and Insl3) and their proteins, especially at 1000 mg/kg. DCHP also lowered the pAKT1/AKT1 and pERK1/2/ERK1/2 ratios. In conclusion, DCHP at low doses (10 and 100 mg/kg) increased Leydig cell number during the initial regeneration and inhibited Leydig cell regeneration during the course of its exposure.

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s previous report. In this work, 90-day administration of RPS induced liver injury. (1) H-NMR- and GC/MS-based metabonomic analyses in conjunction with histopathological examinations, blood biochemistry and hepatic phase I and II enzymes assays were performed to evaluate the toxic mechanisms of RPS induced in rats. As a result, oral administration of RPS possessed certain liver toxicity in SD rats. (1) H-NMR and GC/MS data indicated that RPS inhibited the oxidation of fatty acids, glycolysis, and TCA cycle pathway, and disturbed glycine, serine, and threonine metabolism. Low expression of TG, T-CHO, and LDL-C and high levels of ALT and AST indicated that chronic exposure to RPS caused hepatocyte damage, synthesis dysfunction, and transportation failure of lipoproteins. In addition, RPS downregulated the mRNA levels of CYP1A2, CYP2E1, and UGTs. In conclusion, we used metabonomics approach to study the toxicity of RPS for the first time. This research demonstrated that metabonomics method was a promising tool to study and diagnose TCM-induced toxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 99-108, 2017.

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onse induced by chronic lead exposure and the possible mechanisms are still unknown. The aim of this study was to explore the change of the sympathoexcitatory response induced by chronic lead exposure via the P2X4 receptor in the stellate ganglion (SG). Rats were given lead acetate through drinking water freely at doses of 0 g/L (control group), 0.5 g/L (low lead group), and 2 g/L (high lead group) for 1 year. Our results demonstrated that lead exposure caused autonomic nervous dysfunction, including blood pressure and heart rate increased and heart rate variability (HRV) decreased. Western blotting results indicated that after lead exposure, the protein expression levels in the SG of P2X4 receptor, IL-1β and Cx43 were up-regulated, the phosphorylation of p38 mitogen-activated protein kinase (MAPK) was activated. Real-time PCR results showed that the mRNA expression of P2X4 receptor in the SG was higher in lead exposure group than that in the control group. Double-labeled immunofluorescence results showed that P2X4 receptor was co-expressed with glutamine synthetase (GS), the marker of satellite glial cells (SGCs). These changes were positively correlated with the dose of lead exposure. The up-regulated expression of P2X4 receptor in SGCs of the SG maybe enhance the sympathoexcitatory response induced by chronic lead exposure.

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public health concern. The modes of action of lead neurotoxic effects include disturbance of neurotransmitter storage and release, damage of mitochondria, as well as induction of apoptosis in neurons, cerebrovascular endothelial cells, astroglia and oligodendroglia. Our studies here, from a novel point of view, demonstrates that lead specifically caused induction of COX-2, a well known inflammatory mediator in neurons and glia cells. Furthermore, we revealed that COX-2 was induced by lead in a transcription-dependent manner, which relayed on transcription factor NFAT, rather than AP-1 and NFκB, in glial cells. Considering the important functions of COX-2 in mediation of inflammation reaction and oxidative stress, our studies here provide a mechanistic insight into the understanding of lead-associated inflammatory neurotoxicity effect via activation of pro-inflammatory NFAT3/COX-2 axis.

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350, or 700mg/m3 of 1,2-DCE, via inhalation, 6h/day for 28days. Increased liver/body weight ratios, as well as serum AST and serum ALT activity were observed in the 350 and 700mg/m3 1,2-DCE exposure group mice, compared with the control group mice. In addition, decreased body weights were observed in mice exposed to 700mg/m3 1,2-DCE, compared with control mice. Exposure to 350 and 700mg/m3 1,2-DCE also led to significant accumulation of hepatic glycogen, free fatty acids (FFA) and triglycerides, elevation of blood triglyceride and FFA levels, and decreases in blood glucose levels. Results from microarray analysis indicated that the decreases in glucose-6-phosphatase catalytic subunit (G6PC) and liver glycogen phosphorylase (PYGL) expression, mediated by the activation of AKT serine/threonine kinase 1 (Akt1), might be responsible for the hepatic glycogen accumulation and steatosis. Further in vitro study demonstrated that 2-chloroacetic acid (1,2-DCE metabolite), rather than 1,2-DCE, up-regulated Akt1 phosphorylation and suppressed G6PC and PYGL expression, resulting in hepatocellular glycogen accumulation. These results suggest that hepatic glucose and lipid homeostasis are impaired by 1,2-DCE exposure via down-regulation of PYGL and G6PC expression, which may be primarily mediated by the 2-chloroacetic acid-activated Akt1 pathway.

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nhibit fetal adrenal corticosterone production, but the underlying mechanism is unknown. This study investigated the effects of prenatal caffeine ingestion on corticosterone and its associated synthesized enzymes (steroidogenic acute regulatory protein, StAR; 3beta-hydroxysteroid dehydrogenase, 3beta-HSD; cytochrome P450 cholesterol side chain cleavage, P450scc; P450c21; and P450c11) in the fetal adrenal in rats and further explored the underlying mechanism by analyzing the epigenetic modification and expression of the key transcription factor steroidogenic factor-1 (SF-1). The pregnant rats were intragastrically treated with 120 mg/kg.d caffeine from gestational day 11-20. The results showed that the IUGR rate was 51.2% after caffeine treatment. The contents of corticosterone and the mRNA levels of StAR, P450scc, P450c21, and P450c11 were decreased significantly in the fetal adrenal. Furthermore, caffeine reduced both the protein and the mRNA expression of SF-1 in the fetal adrenal. The epigenetic analysis showed that caffeine treatment can significantly enhance the mRNA expression of DNA methyltransferase (Dnmt) 1, Dnmt3a, histone deacetylases (Hdac) 1, and Hdac2. The detection of DNA methylation by bisulfite-sequencing PCR uncovered a notably increased total methylation rate in the SF-1 promoter. The ChIP assay showed decreased acetylation levels of H3K9 and H3K14 in the SF-1 promoter. In conclusion, prenatal caffeine ingestion is able to induce aberrant DNA methylation and histone acetylation of the SF-1 promoter in the rat fetal adrenal. These effects may contribute to the inhibition of the expression of SF-1 and its associated steroidogenic enzymes and the production of corticosterone during fetal development.

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yle='font-weight:700;'>toxicity. Acetaminophen was co-administered orally to rats for 3 days following 28 days of arsenic pre-exposure (Phase-I) and thereafter, acetaminophen was withdrawn, but arsenic exposure was continued for another 28 days (Phase-II). Arsenic increased lipid peroxidation and reactive oxygen species (ROS) generation, depleted glutathione (GSH), and decreased superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glutathione reductase (GR) activities. Acetaminophen caused exacerbation of arsenic-mediated lipid peroxidation and ROS generation and further enhancement of serum alanine aminotransferase and aspartate aminotransferase activities. In Phase-I, acetaminophen caused further GSH depletion and reduction in SOD, catalase, GPx and GR activities, but in Phase-II, only GPx and GR activities were more affected. Arsenic did not alter basal and inducible nitric oxide synthase (iNOS)-mediated NO production, but decreased constitutive NOS (cNOS)-mediated NO release. Arsenic reduced expression of endothelial NOS (eNOS) and iNOS genes. Acetaminophen up-regulated eNOS and iNOS expression and NO production in Phase-I, but reversed these effects in Phase-II. Results reveal that acetaminophen increased the risk of arsenic-mediated hepatic oxidative damage. Withdrawal of acetaminophen administration also increased susceptibility of liver to hepatotoxicity. Both ROS and NO appeared to mediate lipid peroxidation in Phase-I, whereas only ROS appeared responsible for peroxidative damage in Phase-II.

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(LPS) and therapeutic doses of diclofenac (Dcl), an analgesic associated with drug idiosyncrasy in patients, induced severe hepatotoxicity in rats. Here, we used an integrated discovery to targeted LC-MS proteomics approach to identify mechanistically relevant liver and plasma proteins modulated by LPS/Dcl treatment, potentially applicable as early markers for IDRs. Based on pre-screening results and their role in liver toxicity, 47 liver and 15 plasma proteins were selected for targeted LC-MS analysis. LPS alone significantly changed the levels of 19 and 3 of these proteins, respectively. T-kininogen-1, previously suggested as a marker of drug-induced liver injury, was markedly elevated in plasma after repeated Dcl treatment in the absence of hepatotoxicity, possibly indicating clinically silent stress. Dcl both alone and in combination with LPS, caused up-regulation of the ATP synthase subunits (ATP5J, ATPA, and ATPB), suggesting that Dcl may sensitize cells against additional stress factors, such as LPS through generation of mitochondrial stress. Additionally, depletion of plasma fibrinogen was observed in the co-treatment group, consistent with an increased hepatic fibrin deposition and suspected contribution of the hemostatic system to IDRs. In contrast, several proteins previously suggested as liver biomarkers, such as clusterin, did not correlate with liver injury in this model. Taken together, these analyses revealed proteomic changes in a rat model of LPS/Dcl co-administration that could offer mechanistic insight and may serve as biomarkers or safety alert for a drug's potential to cause IDRs.

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acological properties and thus can be used as promising candidate against various ailments. In the current study, we aimed at demonstrating the protective effect of zingerone against experimental colon carcinogenesis and elucidating its possible mechanism by studying inflammatory and Nrf-2 signaling cascade. Four groups of animals (I-IV) were made with six animals each. Group I (control) was given normal saline orally. Group II was given 1,2-dimethylhydrazine (DMH) at the dose rate of 20 mg/kg body weight. Group III and IV were treated with DMH at the dose rate of 20 mg/kg body weight and also received oral treatment of zingerone at a dose rate of 50 and 100 mg/kg body weight, respectively, for first 5 weeks and animals were euthanized after 16 weeks. Our results reveal that DMH treated rats exhibited elevated ROS and MDA levels, increased activity of cytochrome P450 2E1 and serum marker enzyme carcinoembreyonic antigen (CEA), increased no of aberrant crypts of foci (ACF), and elevated expression of inflammatory and proliferative proteins. Nrf-2 was downregulated by DMH treatment. Treatment with zingerone to DMH treated rats, resulted in alterations in the activity of the cytochrome P450 2E1 and CEA. In addition, immunostaining of NF-kB-p65, COX-2, iNOS, and PCNA, Ki-67 was suppressed by zingerone. Furthermore, zingerone administration also attenuated the level of IL-6 and TNF-α and it also helps in preserving mucous layer. Thus, zingerone could be considered as a good chemopreventive agent in experimental model of colon carcinogenesis. Further studies are required to study other pathways involved in colon carcinogenesis and their modulation buy zingerone.

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h BBOP (0, 10, 100, 250, and 500 mg/kg body weight) for 21 days. BBOP did not affect the weight of body and adrenal glands. BBOP significantly reduced serum corticosterone levels at 250 and 500 mg/kg, and lowered aldosterone level at 500 mg/kg without affecting adrenocorticotropic hormone. BBOP did not alter the thickness of the adrenal cortex. BBOP significantly down-regulated the expression of steroidogenesis-related genes (Scarb1, Star, Cyp11a1, Cyp21, Cyp11b1, Cyp11b2, Nr5a1, Nr4a1, and Nr4a2) and proteins, and antioxidant enzymes (Sod1, Sod2, Gpx1, and Cat) and their proteins, while up-regulating the expression of Mc2r and Agtr1a at various doses. BBOP reduced the phosphorylation of AKT1, AKT2, and ERK1/2, as well as the levels of SIRT1 and PGC1α without affecting the phosphorylation of AMPK. BBOP significantly induced the production of reactive oxygen species and apoptosis rate in H295R cells at 100 μM and higher after 24 h of treatment. In conclusion, male rats exposed to BBOP in puberty have significant reduction of steroid biosynthesis with a potential mechanism that is involved in the decrease in the phosphorylation of AKT1, AKT2, ERK1/2, as well as SIRT1 and PGC1α and increase in ROS.

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ansgenic mice (iDTR) in which Cre-mediated excision of a STOP cassette renders cells sensitive to DT. We tested the iDTR strain by crossing to the T cell- and B cell-specific CD4-Cre and CD19-Cre strains, respectively, and observed efficient ablation of T and B cells after exposure to DT. In MOGi-Cre/iDTR double transgenic mice expressing Cre recombinase in oligodendrocytes, we observed myelin loss after intraperitoneal DT injections. Thus, DT crosses the blood-brain barrier and promotes cell ablation in the central nervous system. Notably, we show that the developing DT-specific antibody response is weak and not neutralizing, and thus does not impede the efficacy of DT. Our results validate the use of iDTR mice as a tool for cell ablation in vivo.

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6 predominantly colorectal cancer patients treated by FPs with early development of high (grade 3-4) hematological and/or gastrointestinal toxicity. Six previously described [85T>C (C29R), 496A>G (M166V), 775A>G (K259E), 1601G>A (S534N), 1627A>G (I543V), IVS14+1G>A, 2194G>A (V732I)] and two novel [187A>G (K63E) and 1050 G>A (R357H)] non-synonymous DPYD variants were found in 56/76 (73.7%) high-toxicity patients. Subsequently, these alterations were analyzed in 48 patients with excellent long-term tolerance of FPs and in 243 controls and were detected in 37/48 (77.1%) and 166/243 (68.3%) cases, respectively. Analysis of these alterations as risk factors for development of toxicity in pooled FPs-treated population demonstrated that C29R negatively correlated with overall gastrointestinal toxicity (OR = 0.48; 95%CI 0.23-1.0) and M166V in women protected against overall hematological toxicity and neutropenia (both OR = 0.26; 95%CI 0.07-0.89), whereas IVS14+1G>A (found in five high-toxicity patients only) increased risk of mucositis in overall population (OR = 7.0; 95%CI 1.1-44.53), and thrombocytopenia in women (OR = 10.8; 95%CI 1.24-93.98). Moreover, we identified a strong association of V732I with leucopenia (OR = 8.17; 95%CI 2.44 - 27.31) and neutropenia (OR=2.78; 95% CI 1.03-7.51). Our data enabled characterization of "high risk" haplotypes (carriers of IVS14+1G>A or V732 lacking M166V) representing small (22% female and 11% male patients), population in high risk of serious hematological toxicity development, and in patients with "lower risk" that unlikely develop serious hematological toxicity [carriers of M166V without IVS14+1G>A and V732I in females (32% women), and non-carriers of C29R, M166V, IVS14+1G>A, and V732I in males (46% men)]. Our results indicate that genotyping of several DPYD variants may lead to stratification of patients with respect to the risk of serious hematological toxicity development during FPs treatment.

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, which leads to significant anorectal malformations (ARMs) in male rat offspring. The incidence of ARMs was 39.5% in male offspring and all abnormal pups were complicated with secondary megacolon. General images, histological analysis and anatomy examination confirmed the malformation. The development abnormalities such as decreased bodyweight (BW) and anogenital distance (AGD), shortened body lengths (with tail removed), as well as increased abdominal circumference were observed at different developmental stages of ARMs in male rat. The developmental abnormalities in both solid organs (brain, heart, liver, spleen, lung and kidney) and reproductive organs (testes and epididymis) of abnormal pubs on PND35 were also investigated. In addition, the serum testosterone (T) level of ARMs in male rats on PND1 was significantly lower than that of controls with accompanying reduced expression of androgen receptor (AR), sonic hedgehog (Shh) and bone morphogenetic protein 4 (Bmp4) mRNA from tissues of the terminal rectum. These results conclusively demonstrate for the first time that in utero exposure to DBP leads to an increased likelihood for the development of ARMs and subsequent complicating megacolon in male rat offspring.

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its receptor, EGFR, play roles in cancer metastasis. CTX III, a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity; however, the effect of CTX III on the EMT of cancer cells remains elusive. CTX III treatment resulted in morphological changes from elongated and spindle shape to rounded and epithelial-like shape, induced upregulation of E-cadherin and concurrent downregulation of N-cadherin and Vimentin protein levels, corresponding to observed decreases in cell migration and invasion. CTX III treatment also decreased the expression of Snail and Twist in EGF-induced MDA-MB-231 cells. Concurrently, CTX III efficiently inhibited the EGFR phosphorylation and downstream activation of phosphatidylinositol 3-kinase (PI3K)/Akt and ERK1/2. The EGFR specific inhibitor AG1478 significantly suppressed ERK1/2 and Akt phosphorylation, cell migration and invasion, as well as the expressional changes associated with EMT markers in EGF-induced MDA-MB-231 cells. CTX III inhibitory effect on EGF-evoked invasion of MDA-MB-231 cells is mediated through suppressing EGF/EGFR activation and EMT process.

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of this topic. Eighty-seven records provided serum or blood measurements of one or more solutes in patients with CKD. These records described 32 previously known uremic toxins and 56 newly reported solutes. The articles most frequently reported concentrations of β2-microglobulin, indoxyl sulfate, homocysteine, uric acid, and parathyroid hormone. We found most solutes (59%) in only one report. Compared with previous results, more recent articles reported higher uremic concentrations of many solutes, including carboxymethyllysine, cystatin C, and parathyroid hormone. However, five solutes had uremic concentrations less than 10% of the originally reported values. Furthermore, the uremic concentrations of four solutes did not exceed their respective normal concentrations, although they had been previously described as uremic retention solutes. In summary, this review extends the classification of uremic retention solutes and their normal and uremic concentrations, and it should aid the design of experiments to study the biologic effects of these solutes in CKD.

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n. The aim of this study was to determine the effects of chronic PFDoA exposure (110 days) on testosterone biosynthesis and the expression of genes related to steroidogenesis in male rats. In this study, we examined the serum levels of sex hormones, growth hormone, and insulin in male rats. Testicular morphology and the expression of key genes and proteins in testosterone biosynthesis were also analyzed. Markedly decreased serum testosterone levels were recorded after 110 days of PFDoA exposure at 0.2 and 0.5mg PFDoA/kg/d, and cast-off cells were observed in some seminiferous tubules in testes exposed to 0.5mg PFDoA/kg/d. PFDoA exposure resulted in significantly decreased protein levels of steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme (P450scc), along with significantly reduced mRNA levels of insulin-like growth factor I (IGF-I), insulin-like growth factor I receptor (IGF-IR), and interleukin 1alpha (IL-1alpha) in rat testes at 0.2 and 0.5mg/kg/d. In addition, PFDoA exposure also affected the expression of some genes in the hypothalamo-neurohypophyseal system. However, PFDoA did not affect the expression of 5alpha-reductase, 3alpha-hydroxysteroid dehydrogenase, or aromatase in testis and liver. These findings demonstrate that chronic PFDoA exposure disrupts testicular steroidogenesis and expression of related genes in male rats. Multiple factors may be involved in the inhibition of testosterone by PFDoA.

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OX for 6 weeks from 2 weeks after DEN treatment. All animals were subjected to two-thirds partial hepatectomy 1 week after OX treatment. The numbers and areas of glutathione S-transferase placental form (GST-P)-positive foci were significantly increased in the livers of rats treated with OX, with concomitantly increased cell proliferation, compared with those in the livers of the DEN alone group. Quantitative real-time RT-PCR analysis revealed that OX induced not only mRNA expression of phase I enzymes Cyp1a1, Cyp1a2, but also Nrf2-regulated phase II enzymes such as Gpx2, Nqo1, Yc2, Akr7a3 and Gstm1, presumably due to an adaptive response against OX-induced oxidative stress. Reactive oxygen species production increased in microsomes isolated from the livers of OX-treated rats. Furthermore, OX enhanced oxidative DNA damage (as assessed by 8-hydroxydeoxyguanosine; 8-OHdG) and lipid peroxidation (as assessed by thiobarbituric acid-reactive substances; TBARS). These results suggest that administration of OX at a high dose and for a long term enhances oxidative stress responses, which may contribute to its tumor-promoting potential in rats.

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e renal Ang II and endothelin-1 (ET-1) receptor expression and the action of telmisartan, an ARB, on DNR-induced nephrotoxicity. Sprague-Dawley rats were treated with a cumulative dose of 9mg/kg DNR (i.v.). Telmisartan was administered orally every day for 6 weeks. DNR rats showed nephrotoxicity as evidenced by worsening renal function, which was evaluated by measuring protein in urine, levels of urea and creatinine in serum, lipid profiles, malondialdehyde level, and the glutathione peroxidase activity in kidney tissue. These changes were reversed by treatment with telmisartan, which resulted in significant improvement in renal function. Furthermore, telmisartan increased nephrin protein expression, and down-regulated renal expression of Ang II and its receptor Ang II type I. Renal protein expressions of ET-1 and its receptor ET-receptor type A were increased in DNR rats, and treatment with telmisartan attenuated these increased expressions. Telmisartan mediated a further increase in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma). In addition, the expressions of cyclooxygenase-2 and cellular adhesion molecules were increased in DNR rats, which were attenuated by telmisartan. In conclusion, telmisartan has a protective effect on DNR-induced nephrotoxicity through Ang II and ET-1, with the alteration of their receptor expressions, which is associated with its anti-inflammatory and anti-oxidant effects at least in part through PPAR-gamma agonistic actions.

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style='font-weight:700;'>toxic carcinogens, but DNA damage secondary to the oxidative stress resulting from reactive oxygen species generation is suspected in rodents given these chemicals. To examine whether WY or PBO have DNA-damaging potential in livers of rats subjected to repeated oral administration for 14 days, the in vivo liver comet assay was performed in partially hepatectomized rats, and the expression of some DNA-repair genes was examined. Then, to examine whether they have genotoxic potential, the in vivo liver initiation assay was performed in rats. In the comet assay, positive results were obtained at 3 h after the last treatment of WY, and some DNA-repair genes such as Apex1, Mlh1, Xrcc5, and Gadd45 were up-regulated in the liver. In the liver initiation assay, negative results were obtained for both WY and PBO. The results of the present study suggest that WY, but not PBO, causes some DNA damage in livers of rats, but such DNA damage was repaired by the increased activity of some DNA repair genes and may not lead to a DNA mutation.

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d to allow a rapid screening to classify NPs in terms of toxicity. We adapted the cytochrome c assay to take into account NP interference, to improve its sensitivity and to develop a high-throughput method. The intrinsic oxidative ability of a panel of NPs (carbon black, Mn2O3, Cu, Ag, BaSO4, CeO2, TiO2 and ZnO) was measured with this enhanced test and compared to other acellular redox assays. To assess whether their oxidative potential correlates with cellular responses, we studied the effect of insoluble NPs on the human bronchial epithelial cell line NCI-H292 by measuring the cytotoxicity (WST-1 assay), pro-inflammatory response (IL-8 cytokine production and expression) and antioxidant defense induction (SOD2 and HO-1 expression). The adapted cytochrome c assay had a greatly increased sensitivity allowing the ranking of NPs in terms of their oxidative potential by using the developed high-throughput technique. Besides, a high oxidative potential revealed to be predictive for toxic effects as Mn2O3 NPs induced a strong oxidation of cytochrome c and a dose-dependent cytotoxicity, pro-inflammatory response and antioxidant enzyme expression. BaSO4, which presented no intrinsic oxidative potential, had no cellular effects. Nevertheless, CeO2 and TiO2 NPs demonstrated no acellular oxidant-generating capacity but induced moderate cellular responses. In conclusion, the novel cytochrome c oxidation assay could be used for high-throughput screening of the intrinsic oxidative potential of NPs. However, acellular redox assays may not be sufficient to discriminate among low-toxicity NPs, and additional tests are thus needed.

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l group (n = 6) were subjected to physiologic saline by intragastric (i.g.) route. Group Ethanol (n = 6) was given 1 ml of 80% ethanol (v/v) in distilled water through i.g. route. Group Vitamin E (Vit E), (n = 6) was given vitamin E (100 mg kg(-1) day(-1)) by intra peritonealy. Group Vitamin E + Ethanol (n = 6) was given vitamin E 2 h before the administration of ethanol. At the end of 4 weeks, blood samples were taken and plasma malondialdehyde (MDA), protein carbonyls (PCs), aspartate aminotransferase (AST), tumor necrosis factor-alpha (TNF-alpha) and pMMP-9 levels were measured. Chronic ethanol administration increased the AST, MDA, PCs, TNF-alpha and pMMP-9 levels when compared to those in control group (p < 0.05, p < 0.01, p < 0.01, p < 0.05, p < 0.05, respectively). Vitamin E treatment was found to decrease lipid peroxidation and protein oxidation (p < 0.01, p < 0.01, respectively). Also TNF-alpha and pMMP-9 levels returned to normal by vitamin E treatment. Within all subjects, there was positive correlation between pMMP-9 levels and MDA, PCs levels (p = 0.045, r = 0.454; p = 0.004, r = 0.574, respectively). We conclude that since antioxidant supplementation decreases the alcohol-induced pMMP-9 levels, oxidative stress could be one of the mediators of the generation of MMP-9.

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r morphology. The placebo caused an increase in the incidence of intimal hyperplasia compared to saline controls in female rats; octreotide increased the incidence and severity of intimal hyperplasia in males and females. Intimal hyperplasia consisted of increased numbers of cells located between the endothelial cell layer and the internal elastic lamina. Severity was based on the degree of compromise of the vascular lumen (regardless of vessel size and number), with severely affected vessels having no visible lumen. Intimal hyperplasia in rats treated with octreotide was considered to be an unexpected and adverse finding, given that this compound and other somatostatin analogs have been investigated as reducers of intimal proliferation or restenosis after angioplasty in humans and that no such lesion has been reported in the literature for this class of compound to date. The induction of intimal hyperplasia by the placebo is also a notable finding; this may be because of the low pH of the formulation.

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lly) daily for 12 weeks and (2) 20% ethanol (4 g kg(-1), orally) daily for 12 weeks. The mean arterial blood pressure was recorded every week. The animals were anesthetized with pentobarbital after 12 weeks; thoracic aorta were isolated and analyzed for aortic reactivity response, inflammatory mediators, oxidant/antioxidant enzyme protein expression and endothelial nitric oxide-generating system. The results show that the mean BP was significantly elevated 12 weeks after ethanol ingestion. The increased BP was related to increased aortic inflammation (tumor necrosis factor [TNF]-alpha; nitric oxide synthase [iNOS], COX-2 and MCP-1 protein expression) and elevated angiotensin II levels in alcohol-treated group compared to control. Aortic Nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity, membrane and cytosolic subunits p22(pho) (x) and p47(pho) (x) expression and Mn-SOD activity and protein expression significantly increased, whereas nitric oxide (NO), endothelial NO synthase (eNOS), vascular endothelial growth factor (VEGF)-A and CuZn-SOD activity and protein expression significantly decreased in alcohol-treated group compared to control. The acetylcholine-mediated vasorelaxation response was depressed in the aorta of ethanol-treated rats compared to control. In conclusion, chronic ethanol-induced elevation in BP is related to increased aortic inflammation, elevated angiotensin II levels, induction of NADPH oxidase causing endothelial injury, depletion of CuZn-SOD, down-regulation of endothelial NO generating system and impaired vascular relaxation in rats.

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amine initiation. Two-third partial hepatectomy was performed 1 week after the FF treatment. Histopathologically, the number of hepatocellular altered foci significantly increased in the FF-treated groups with a concomitant increase in the number of hepatocytes positive for anti-Ki-67 antibody, but the number and area of glutathione S-transferase placental form (GST-P)-positive foci decreased in these groups, as compared to those in the controls. Microarray analysis or quantitative real-time reverse transcription-polymerase chine reaction demonstrated the significant up-regulations of Aco and Cyp4a1 (genes related to lipid metabolism); Gpx2, Yc2, Cat, Cyp2b15, and Ugt1a6 (metabolic oxidative stress-related genes); Apex1, Mgmt, Xrcc5, Nbn, and Gadd45a (DNA repair-related genes); and Ccnd1 (cell cycle-related genes) in the FF-treated groups, and the significant down-regulations of Cyp1a2, Gsta2, Gstm2, and Gstm3 (phase I or II metabolism-related genes); Mlh1 and Top1 (DNA repair-related genes); and Cdkn1a, Cdkn1b, Chek2, and Gadd45b (cell cycle/apoptosis-related genes) in these rats. FF-treatment increased the activity of enzymes such as carnitine acetyltransferase, carnitine palmitoyltransferase, fatty acyl-CoA oxidizing system, and catalase in the liver, but not superoxide dismutase in the liver. In addition, 8-OHdG level in liver DNA, lipofuscin deposition in hepatocytes, and in vitro reactive oxygen species production in microsomes significantly increased due to FF treatment. These results suggest that oxidative stress is involved in the development of FF-induced hepatocellular preneoplastic foci in rats.

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ts containing 0, 0.015, 0.03, 0.06, 0.125, 0.25, or 0.5% PBO after an i.p. injection of N-diethylnitrosamine (DEN) to initiate hepatocarcinogenesis. Histopathologically, Glutathione S-transferase placental form (GST-P)-positive foci were significantly increased in a dose-dependent manner in rats given 0.25% PBO or higher. The formation of microsomal ROS in the liver was significantly increased in 0.25 and 0.5% PBO. Real-time RT-PCR showed that the expression of the CYP1A1, UDPGTr-2, and Mrp3 genes was significantly upregulated in rats given 0.03% PBO or higher. These results suggest that 0.25% is the threshold dose of PBO that induces ROS-mediated hepatocarcinogenesis in rats, although the CYP1A1 gene that is related to ROS generation and the UDPGTr-2 and Mrp3 genes that are involved in protection against ROS were induced in the livers of rats even at a PBO dose of 0.03%.

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CNS and has been shown to induce behavioral, biochemical and immunological effects. The purpose of this study was to investigate the effects of AMPH on pulmonary cellular influx, vascular permeability and airway reactivity. AMPH effects on adhesion molecule expression, IL-10 and IL-4 release and mast cell degranulation were also studied. Male Wistar rats were sensitized with ovalbumin (OVA) plus alum via subcutaneous injection. One week later, the rats received another injection of OVA-alum (booster). Two weeks after this booster, the rats were subjected to AMPH treatment 12hours prior to the OVA airway challenge. In rats treated with AMPH, the OVA challenge reduced cell recruitment into the lung, the vascular permeability and the cellular expression of ICAM-1 and Mac-1. Additionally, elevated levels of IL-10 and IL-4 were found in samples of lung explants from allergic rats. AMPH treatment, in comparison, increased IL-10 levels but reduced those of IL-4 in the lung explants. Moreover, the tracheal responsiveness to methacholine (MCh), as well as to an in vitro OVA challenge, was reduced by AMPH treatment, and levels of PCA titers were not modified by the drug. Our findings suggest that single AMPH treatment down-regulates several parameters of lung inflammation, such as cellular migration, vascular permeability and tracheal responsiveness. These results also indicate that AMPH actions on allergic lung inflammation include endothelium-leukocyte interaction mechanisms, cytokine release and mast cell degranulation.

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te the role of IL-10 in liver injury by a pretreatment with anti-IL-10 neutralizing antibody (IL-10Ab). Rats were given goat IgG or IL-10Ab before being treated with CHX (CHX group or IL-10Ab/CHX group). In the CHX group, the CHX treatment markedly induced hepatic mRNA and serum protein levels of IL-10. The up-regulation of IL-10 was significantly suppressed in the IL-10Ab/CHX group. Blocking IL-10 in the IL-10Ab/CHX group led to greater increases in hepatic mRNA and serum levels of proinflammatory cytokines, such as TNF-alpha and IL-6. The IL-10Ab/CHX group developed more severe hepatocellular apoptosis, neutrophil transmigration, and necrotic change of hepatocytes compared with the CHX group. The caspase activities and mRNA levels of Cc120, LOX-1, and E-selectin in the livers were significantly higher in the IL-10Ab/CHX group than the CHX group. These results demonstrate that IL-10 plays an important role in counteracting the effect of proinflammatory cytokines, such as a TNF signaling cascade, and in attenuating the CHX-induced liver injury.

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fferences were observed. To study the regulation of CYP2S1 mRNA expression by AhR agonists, rats were treated with 3-methylcholanthrene (3-MC; 25mg/kg/dayx3 days) or with a single intraperitoneal injection of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at various dosages (0, 1, 5, 10, 50, 100mug/kg). CYP2S1 mRNA levels were increased in lung, stomach, jejunum and ileum following treatment with 3-MC and in lung, liver and kidney tissues following treatment with TCDD. Induction of CYP2S1 mRNA was greater with TCDD than 3-MC treatment and was more pronounced in lung than other tissues. Antiserum raised against a peptide corresponding to the C-terminus of CYP2S1 was used to measure relative CYP2S1 protein expression by immunoblot analysis. An immunoreactive CYP2S1 protein band with an approximate molecular weight of 50kDa was detected in microsomes of rat lung, stomach and kidney, but not other tissues. Unlike CYP2S1 mRNA, CYP2S1 protein levels were not increased after treatment with 3-MC or the highest dosage of TCDD, indicating that CYP2S1 protein expression was less sensitive than mRNA expression to AhR-mediated regulation. Our study is the first to characterize CYP2S1 mRNA and protein expression in rats, and from the results obtained, we conclude that AhR is involved in the transcriptional regulation of CYP2S1 in rats.

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ugh determination of certain biochemical markers including total urea, creatinine, and kidney marker enzyme, that is, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In addition histopathological examination of intoxicated and treated kidney with both proteins was performed. The present results show a significant increase in serum total urea and creatinine, while significant decrease in GAPDH. Improvement in all biochemical parameters studied was demonstrated, which was documented by the amelioration signs in rats kidney architecture. Thus, both plant protein extracts can counteract the nephrotoxic process, minimize damage to the kidney, delay disease progression, and reduce its complications.

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n time, and enhanced anticoagulation factor activity, such as antithrombin III and protein C (P<0.05). These results suggested that the anticoagulant action of DAT-rich garlic oil was due to inhibition and/or inactivation of thrombin. In addition, DAT-rich garlic oil benefits blood anticoagulation factors, which might further prevent the development of thrombus formation. However, the intake of garlic oil at high dose significantly increased plasma fibrinogen concentration (P<0.05), and affected the levels of several hematological parameters such as erythrocyte count, hemoglobin and platelets (P<0.05). The adverse effect of high doses of garlic oil might further influence the hemostatic balance. Therefore, the concentration of DAT-rich garlic oil should be carefully considered in its application. Supplementation of garlic oil at 5mg/kg bodyweight has anticoagulation effect in this animal study.

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intravenous injection of lead nitrate. Western blotting and immunohistochemistry revealed that FTL was increased in hepatocytes around the central veins and strongly expressed in nonparenchymal cells. Some FTL-positive nonparenchymal cells were identified as Kupffer cells that were positive for CD68. FTL-positive Kupffer cells occupied about 60% of CD68-positive cells in the periportal and perivenous areas. The relationships between FTL expression and apoptosis induction or the engulfment of apoptotic cells were examined. TUNEL-positive cells were increased in the treatment group, and enhanced expression of milk fat globule EGF-like 8 was demonstrated in some Kupffer cells and hepatocytes, indicating enhanced apoptosis induction and phagocytosis of apoptotic cells. FTL-positive Kupffer cells were not detected without lead nitrate treatment or in rat livers treated with clofibrate, which induces hepatocyte proliferation but not apoptosis. These results suggest that FTL expression in Kupffer cells after lead treatment is dependent on phagocytosis of apoptotic cells.

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for next three days. A third group of 8 rats was given three injections of normal saline and served as control. All the animals were sacrificed on eighth day and assessed for hematological changes, heme synthesizing pathway enzymes, hepatic drug metabolizing status and sulfhydryl levels in blood and liver. Lead administration resulted in decreased hemoglobin, increased reticulocytosis, depression of delta aminolevulinic acid dehydratase (ALAD) and uroporphyrinogen I synthetase (UPS) activity in blood and liver. In vitro metabolism of drugs aminopyrine, aniline and p-nitroanisole by liver homogenate and in vivo metabolism of pentabarbitone was also reduced in lead exposed rate. Zinc treated rats showed improved hematological profile and activated ALAD and UPS activity, recovery of N-demethylation of aminopyrine and O-demethylation of p-nitroanisole and partial restoration of free thiol levels in blood and liver thereby indicating that zinc could confer protection against lead toxicity.

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igh-fat diet for 4 weeks. After 4 weeks of feeding, the hemodynamic parameters in the rats fed with HFD were significantly increased as compared with control rats. Rats fed with HFD had elevated levels of serum lipids, insulin, leptin, glucose and apolipoprotein B. Lipid peroxides and caspase-3 levels were increased while serum apolipoprotein A1 and antioxidant enzymes levels in heart tissues were decreased in HFD-induced obesity in rats as compared to normal healthy control rats fed on standard rat pellet diet. This model of diet-induced obesity will be a useful tool for studying the mechanisms by which dietary fat induces the obesity in humans.

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ntratracheally instilled with DDAC to the lung and the bronchoalveolar lavage (BAL) fluid and lung tissues were collected to assess dose- and time-related pulmonary injury. Exposure to 1500mug/kg of DDAC caused severe morbidity with pulmonary congestive oedema. When the BAL fluid from survivors was examined on day 3 after treatment, exposure to 150mug/kg of DDAC caused weakly induced inflammation, and exposure to 15mug/kg did not cause any visible effects. Next, we observed pulmonary changes that occurred up to day 20 after 150mug/kg of DDAC exposure. Pulmonary inflammation peaked on day 7 and was confirmed by expression of interleukin-6, monocyte chemotactic protein-1, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and regulated upon activation, normal T-cell expressed and secreted in the BAL fluid; these changes were accompanied by altered gene expression of their chemokine (C-C motif) receptor (Ccr) 1, Ccr2, Ccr3, and Ccr5. Cytotoxicity evoked by DDAC was related to the inflammatory changes and was confirmed by an in vitro study using isolated mouse lung fibroblasts. The inflammatory phase was accompanied or followed by pulmonary remodeling, i.e., fibrosis, which was evident in the mRNA expression of type I procollagen. These results suggest that administering DDAC by intratracheal instillation causes pulmonary injury in mice, and occupational exposure to DDAC might be a potential hazard to human health.

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ry gland. Tumors were induced with various chemical carcinogens including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 7-12-dimethylbenz[a]anthracene (DMBA), N-nitrosomethylurea (NMU), and 4-aminobiphenyl. The global gene expression profiles in carcinomas and adenomas were distinguishable by hierarchical clustering and multi-dimensional scaling analyses. Permutation analysis revealed 110 clones statistically differentially expressed between benign and malignant tumors (p < 0.0005). Carcinomas showed relatively high expression of several genes associated with mammary epithelial cell growth and proliferation (e.g., cyclin D1, PDGFalpha) and relatively low expression of differentiation marker genes (e.g., beta -casein, whey acidic protein, transferrin). Other categories of genes showing differential expression between carcinomas and adenomas were associated with protein homeostasis, cytoskeleton, extracellular matrix, and cell metabolism (fatty acid metabolism, oxidative phosphorylation, and glycolysis). Major gene families implicated in malignancy by over-expression in carcinomas included the annexins (annexin A1 and A4) and Stat family of transcription factors (Stat3 and Stat5a). The elevated expression of the prolactin receptor in carcinomas concomitant with several components of the mitogenic prolactin signaling pathway implicated prolactin/prolactin receptor/Stat5a/cyclin D1 in rat mammary gland malignancy.

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dominant-negative mutant of p85 subunit of PI3K failed to inhibit CT from inducing COX-2 expression. CT did not activate PI3K/AKT signaling pathway whereas LY29 and WM decreased the activity of PI3K. LY303511 (LY30), a structural analogue and a negative control for PI3K inhibitory activity of LY29, also suppressed COX-2 induction. These data suggest PI3K-independent mechanisms in regulating CT-induced COX-2 expression. LY29 and LY30 do not inhibit glucocorticoid receptor transactivity. Both compounds have been reported to inhibit Casein Kinase 2 activity and modulate potassium and calcium levels independent of PI3K, while LY29 has been reported to inhibit mammalian Target of Rapamycin (mTOR), and DNA-dependent Protein Kinase (DNA-PK). Inhibitor of Casein Kinase 2 (CK2), mTOR or DNA-PK failed to prevent CT from inducing COX-2 expression. Tetraethylammonium (TEA), a potassium channel blocker, and nimodipine, a calcium channel blocker, both attenuated CT from inducing COX-2 gene expression. CT was found to increase intracellular Ca(2+) concentration, which can be inhibited by LY29, TEA or nimodipine. These data suggest a possible role of calcium instead of PI3K in CT-induced COX-2 expression in cardiomyocytes.

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and HHM model rats produced by implantation of PTHrP-expressing tumor (LC-6) cells were prepared. HHM rats fractured its incisor, and histopathologically, restrict population of odontoblasts showed findings classified as "shortening of high columnar odontoblasts" and "dentin niche." The incisors were immunostained against PTHrP and PTHR1. In normal rats, PTHrP and PTHR1 colocalized in ameloblasts, cementoblasts, and odontoblastic cells from mesenchymal cells to columnar odontoblasts. In high columnar odontoblasts, PTHrP solely expressed. In the HHM animals, although the expression patterns were identical to those of the normal rats in normal area, the shortened high columnar odontoblasts maintained PTHR1 expression and dentin niche comprising odontoblastic cells expressed both proteins. In the HHM model, the protein expression patterns changed in the odontoblastic cells with histological anomalies, and thus direct relations between the anomalies and PTHrP/PTHR1 axis are suggested.

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outer membrane protein (OMP) from faecal flora of healthy and rheumatic humans. Anti-cyclic citrullinated peptide (anti-CCP2), C-reactive protein (CRP) and rheumatoid factor (RF) values increased (p < 0.05) in both arthritic groups as compared to normal values. The rheumatic markers anti-CCP2, CRP and RF values decreased significantly in E- and GCM-treated groups compared to arthritic none-treated acute or chronic groups. The results of RF values of GCM-treated groups in acute and chronic models decreased exhibiting no statistical difference compared with the normal value. Histological examinations of the hind paw sections revealed moderate inflammation, oedema and mild proliferation of synovial cells in acute arthritic rats and more damage to cartilage and bone with severe inflammation in chronic ones. Echinacea acute treated group showed edema with proliferated synovial membrane and partial damage in cartilage and bone. While in the E-chronic treated group, rough edge with destructed cartilage and bone existed. However, the acute GCM group revealed mild cartilage damage. But the chronic GCM group showed mild synovial cells proliferation and revealed no inflammation with mild cartilage damage edge. Results demonstrated the OMP arthropathic property and through promising light on arthritis treatment using E- or GCM, with the advantage of GMC results over that of E-. The composite GCM is needed for further studies over the dose and duration to assess its preventive effects against the bacterial OMP arthrogenicity.

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denosine triphosphate (ATP)-binding cassette G2 transporter efflux activity results in a marked increase in systemic exposure to various statins. Importantly, the effects of these genetic polymorphisms differ depending on the specific statin that is used. This provides a rational basis for the individualization of lipid-lowering therapy.

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4 experienced the treadmill running of 15 to 22 m/min for 25 to 64 minutes, 5 times a week for 8 weeks. Groups 1 to 4 received lead acetate (20 mg/kg), the sham group received curcumin solvent (ethyl oleat), and the curcumin and training + curcumin groups received curcumin solution (30 mg/kg) intraperitoneally. Lead administration resulted in significant increases in high-sensitivity C-reactive protein (hs-CRP), creatine kinase-MB (CK-MB), malondialdehyde (MDA), and low-density lipoprotein (LDL), and significantly decreased glutathione peroxidase (GPx), Total Antioxidant Capacity (TAC), and high-density lipoprotein (HDL) levels. Treadmill running and\or curcumin supplementation resulted in a significant decrease in hs-CRP, CK-MB, MDA, and LDL levels and significantly increased GPx, TAC, and HDL levels. These results suggest a lifestyle-induced cardioprotective potential in ameliorating lead-induced cardiotoxicity.

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ss of lens clarity with time. alpha-Crystallin, a major lens structural protein and molecular chaperone, undergoes various post-translational modifications upon aging which may contribute to decreased chaperone function and contribute to loss of lens clarity. This study evaluated the impact of 5 weeks of oral Pb(2+) exposure (peripheral Pb(2+) level approximately 30 microg/dL) on the alphaA-crystallin protein profile of the lens from Fisher 344 rats. Decreases in relative protein spot intensity of more acidic forms of alphaA- and betaA(4)-crystallin and of truncated forms of alphaA-crystallin were noted. This data indicates that changes in post-translational processing of crystallins do occur in vivo following short courses of clinically relevant Pb(2+)-exposure. In addition, organ culture of lenses from 4.5-month-old rats in 5 microM Pb(2+) resulted in opacities, demonstrating that lead is toxic to the lens and can induce a loss of lens clarity.

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odules and carcinomas relative to surrounding tissue and tissues obtained from control animals. Myc expression was similar in hyperplastic nodules and carcinomas induced by DCA, but was significantly higher in TCA-induced carcinomas than in hyperplastic nodules and carcinomas produced by DCA. In carcinomas from animals whose TCA treatment was suspended at 37 weeks, c-myc expression remained high relative to control and surrounding liver tissue at 52 weeks. In contrast, the expression of c-H-ras was consistently elevated in carcinomas from both treatments relative to hyperplastic nodules and non-tumor tissue. Within carcinomas from both treatments, focal areas could be located which expressed even higher levels of c-myc. This heterogeneity was not observed in carcinomas hybridized to c-H-ras-probes. These data suggest that elevated expression of c-H-ras and c-myc might play an important role in the development of hepatic tumors in B6C3F1 mice. Elevated expression of c-H-ras was closely associated with malignancy. Increased c-myc expression does not seem necessary for progression to the malignant state. On the other hand, the increased expression of c-myc appears related to the earlier progression of TCA-induced tumors to the malignant state.

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unclear. To investigate the changes in gene expression at the onset of PQ injury, we used the differential display-polymerase chain reaction (PCR) method. Rats were treated intraperitoneally with 20 mg/kg PQ, and after 3 h the lungs were immediately excised. Samples of mRNA from normal and treated rats were used to prepare radiolabeled cDNAs, which were electrophoresed. Then the transcription levels were compared. We isolated 26 fragments of cDNA that were potentially affected by PQ, and determined their nucleotide sequences. Six clones of interest were selected and analyzed further. The reverse transcript-PCR based on their sequence information confirmed the differential expression for five clones: four clones were up-regulated and one was down-regulated. We were particularly interested in two genes that had homology with the known gene: TATA box-binding protein-associated factor, RNA polymerase II, B, 150 kDa (TAFIIB), and a candidate gene for lipodystrophy, Lpin2. Both genes were significantly up-regulated within 3 h of PQ intake and the stimulation continued during our 24-h observation period. In addition, up-regulation of Lpin2 was observed in the lungs, but not in the liver and kidneys. In situ hybridization using lung sections showed that the expression of both genes was strongly visualized in Clara cells and in alveolar macrophages. These findings suggest a stimulation of transcription levels and changes in lipid metabolism in Clara cells and in macrophages in the lungs, which result in their playing a crucial role at the onset of PQ-driven pulmonary injury.

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ivation of PPARalpha. Fenofibrate and clofibrate reduced the expression of 10 and 12 proteins, respectively with the expression of ACSL1, SLC25A4, A1BG, HADHA, ATP2A2, BDH1, ETFDH, HADHB, and CPT2 being reduced in common with both of fibrate-treated groups. The approach adopted in this study provides an efficient method for monitoring global changes in protein expression.

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etermine if the endogenously expressed NQO1 in cardiovascular cells could scavenge O2(.-). We observed that NQO1 was highly expressed in cardiovascular cells, including rat aortic smooth muscle A10 and cardiac H9c2 cells, as well as normal human aortic smooth muscle and endothelial cells. NQO1, but not SOD in the cardiovascular cells was highly inducible by 3H-1,2-dithiole-3-thione (D3T). Cytosols from H9c2 and human aortic smooth muscle cells (HASMCs) were isolated to determine the O2(.-) scavenging ability of the endogenously expressed NQO1 by using pyrogallol autooxidation assay. We showed that cytosols from the above cells inhibited pyrogallol autooxidation in an NADPH or NADH-dependent manner. The NADH/NADPH-dependent inhibition of pyrogallol autooxidation by the cytosols was completely abolished by the NQO1-specific inhibitor, ES936, suggesting that the endogenously expressed NQO1 could scavenge O2(.-). In the presence of NADH/NADPH, cytosols from D3T-treated cells showed increased ability to scavenge O2(.-) as compared to cytosols from untreated cells. This increased ability to scavenge O2(.-) was also completely reversed by ES936. 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide spin-trapping experiments using potassium superoxide as a O2(.-) generator further confirmed the ability of NQO1 from HASMCs to scavenge O2(.-). The spin-trapping experiments also showed that induction of NQO1 by D3T in HASMCs augmented the O2(.-) scavenging ability. Taken together, these results demonstrate that the highly expressed and inducible endogenous NQO1 in cardiovascular cells may act as a potential O2(.-) scavenger.

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asma but also locally in tissues from precursors and substrates either locally expressed or imported from the circulation. In most areas of the brain, only locally generated angiotensins can exert effects on their receptors owing to the blood-brain barrier. Other tissue renin-angiotensin-aldosterone systems are found in cardiovascular organs such as kidney, heart, and vessels and play important roles in the function of these organs and in the deleterious actions of hypertension and diabetes on these tissues. Novel components with mostly opposite actions to the classical renin-angiotensin-aldosterone systems have been described and need functional characterization to evaluate their suitability as novel drug targets.

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(AVP), and proopiomelanocortin (POMC) expression in rat brain. To observe time-dependent changes in these neuropeptides, male Sprague-Dawley rats were given TCDD (50 microg/kg) and terminated 1, 2, 4, or 7 days later. In addition, to observe dose-dependent changes in feeding and neuropeptides, rats were also given a range of TCDD doses (12.5, 25, or 50 microg/kg) and terminated 14 days later. TCDD suppressed food and water intake over 14 days in a dose-dependent manner. TCDD treatment also increased CRF and POMC mRNA levels in the hypothalamic paraventricular nucleus (PVN) and arcuate nucleus, respectively, in a dose- and time-dependent manner. These increases were related to decreased food intake following TCDD administration. TCDD treatment increased AVP and CRF mRNA levels in the PVN, and these increases were related to decreased water intake. Interestingly, the increases in CRF, AVP and POMC expression were observed 7 to 14 days after TCDD administration. These results suggest that a single administration of TCDD induced long-lasting increases in CRF, AVP, and POMC mRNA levels in the hypothalamus and that these changes are related to reduced food and water intake 7 to 14 days after TCDD administration.

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ls following O(3) exposure may shed some light on this subject. To investigate the role of TNF-alpha in the O(3)-induced pulmonary insult, we intraperitoneally injected rats with either rabbit preimmune serum or rabbit antirat TNF-alpha 1 h prior to O(3) exposure. Approximately 12 h after the end of O(3) exposure the animals were sacrificed, the lungs lavaged, and tissue samples collected for expression of cytokine genes relevant to inflammation. The bronchoalveolar lavage fluid (BALF) was analyzed for albumin as a marker of pulmonary epithelial permeability changes and for fibronectin for its role in lung injury and repair. The lavage cells were collected, counted, and identified to quantitate the inflammatory response. Ozone exposure resulted in a significant increase in BALF albumin and fibronectin as compared to air-exposed controls and a significant increase in BALF polymorphonuclear leukocytes (PMNs). Antibody treatment produced a significant decrease in BALF albumin and PMNs as compared to O(3)-exposed rats given preimmune serum. Antibody treatment did not affect the BALF fibronectin concentration or the total cell count in the BAL. Tissue analysis for gene arrays revealed an activation of IL-1alpha, IL-6, and IL-10 in animals exposed to O(3). The gene expression was downregulated in animals treated with anti-TNF-alpha antibody prior to O(3) exposure. The results suggest a central role for TNF-alpha in the mechanistic pathways critical to lung inflammation. The significance of TNF-alpha in the inflammation and epithelial injury produced by ozone exposure reflects its overall contribution through modulation of other cytokines.

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se the expression of 1 gene (Atp1b3) and decrease the expression of 1 gene (Tap1) in the cerebrum, increase the expression of 1 gene (Dnaja2) in the cerebellum, increase the expression of 2 genes (Actb and Timm23) and decrease the expression of 1 gene (Spink3) in the liver, increase the expression of 2 genes (RT1-Bb and Mgat5) and decrease the expression of 6 genes (Tnfaip8, Rara, Slc2a4, Wdr12, Pias4 and Timm13) in the kidney.

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, in situ hybridization, and electron microscopy. ECIs were strongly positive for surfactant protein D (SP-D) and weakly positive for Clara cell specific protein (CCSP). Clara cell cytoplasm was positive for CCSP mRNA regardless of ECIs, but not within ECIs. Corticosteroid treatment and ECI presence did not affect the immunohistochemistry and in situ hybridization staining intensities. Electron microscopy revealed large intracytoplasmic granules with an irregular limiting membrane. The ECI number was microscopically quantified in rats from three-, six-, and twenty-four-month studies. The mean ECI counts in treated rats increased from three- to fifty-four-fold with a positive dose-related trend, when compared with vehicle controls. Although the mechanism is unclear, SP-D and to a lesser extent CCSP accumulate in the ECIs. As human bronchial epithelium does not appear to contain structures analogous to the ECI, it is suggested that the observation of an increased number of ECIs in the treated rats is not likely to be relevant for human clinical risk assessment.

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ed in MNU-initiated rats with or without subsequent AA administration were investigated. Frequencies of mutations in codon 12 from GGA to GAA were significantly (p < 0.05) higher in rats with AA administration (82%, 23 out of 28 tumors) as compared to those without AA (50%, 9 out of 18 tumors), but the latency and volume of H-ras mutation-harboring tumors were similar to those of the mutation-lacking tumors. No mutations in codons 13 or 61 were detected in either treatment groups. The results thus indicate that H-ras gene mutations in codon 12 play a pivotal role in initiation of carcinogenesis and it appears possible that AA administration may selectively co-stimulate and/or maintain initiated cells via other genomic or non-genomic events in MNU-treated rats.

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examined the effects of inorganic arsenic and inorganic mercury, which induce apoptosis in proximal tubular cells, on cellular protein level of p53 and gene expression of Ube2d family. Inorganic arsenic induced apoptosis with p53 accumulation, and suppressed Ube2d1, Ube2d2 and Ube2d4 expression, but not Ube2d3. On the other hand, although apoptosis was induced in response to inorganic mercury in proximal tubular cells, protein level of p53 was not elevated by inorganic mercury. These results suggest that inorganic arsenic, but not inorganic mercury, may induce p53-dependent apoptotic pathways through downregulation of gene expression of Ube2d family in proximal tubular cells.

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ol. Appl. Pharmacol. 114, 97-107, 108-117, 118-126), and searched for the most sensitive and reliable among several indices of TCDD toxicity. Pregnant Holtzman rats were given a single oral dose of 0, 12.5, 50, 200, or 800 ng TCDD/kg body weight on gestational day (GD) 15, and male offspring were sacrificed on postnatal day (PND) 49 or 120. GC-MS analysis of the abdominal fat tissue and testis clearly showed increased amounts of TCDD in these offspring. However, there was no TCDD effect on body weight of offspring. There were no changes on testicular or epididymal weights by TCDD administration, even at the 800-ng/kg dose in rats sacrificed on either PND 49 or 120. In addition, TCDD administration resulted in no changes in daily sperm production or sperm reserve at any of the doses used. However, the weight of the urogenital complex, including the ventral prostate, was significantly reduced at doses of 200 and 800 ng TCDD/kg in rats sacrificed on PND 120. Moreover, the anogenital distance (AGD) of male rats sacrificed on PND 120 showed a significant decrease in the groups receiving doses greater than 50 ng TCDD/kg. TCDD administration resulted in no apparent dose-dependent changes in levels of either serum testosterone or luteinizing hormone. Interestingly, reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that, in the ventral prostates of the PND 49 group, TCDD administration resulted in both a dose-dependent increase in 5alpha-reductase type 2 (5alphaR-II) mRNA level and a dose-dependent decrease in androgen receptor (AR) mRNA level. These results suggest that low-dose TCDD administration had a greater effect on the development of the external genital organs and ventral prostate than on development of the testis and other internal genital organs. Moreover, it is highly suggested that the decrease in the size of the ventral prostate by maternal TCDD exposure might be due to decreased responsiveness of the prostate to androgen due to an insufficient expression level of androgen receptor during puberty.

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ema. We have found that both ovCNP-39 and the endogenous C-type natriuretic peptide, CNP-22, produce oedema in the rat paw and release histamine from rat peritoneal mast cells. Two synthetic peptides, ovCNP-39(1-17) and ovCNP-39(18-39), corresponding to the N- and C-termini, respectively, are equipotent histamine releasers, suggesting that ovCNP-39 and other natriuretic peptides do not act through conventional natriuretic peptide receptors on mast cells.

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(-3) M). As three people had been poisoned following ingestion of H. r. we wanted to investigate the effects of PA in rats and in cell cultures. Rats given PA via probang (100-800 mg/ kg) within 24 h developed strongly reduced locomotor activity, depressed visual placing response and impaired wire manoeuvre. Laboratory investigation of blood revealed hepatorenal failure, metabolic acidosis as well as hypokalaemia and hypocalcaemia. All symptoms closely paralleled the effects seen in the poisoned people. Proliferation of cultured cells (including rat brain neurons and glia, fibroblasts, tumour cells) was depressed at 10(-4)-10(-3) M PA. We conclude that the intoxication of people poisoned with H. r. is due to the high content of the DHO-DH inhibitor PA.

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lation between sPLA(2)-IIA activity and the severity of phosgene-induced acute lung injury. Adult male rats were randomly exposed to either normal room air (control group) or a concentration of 400 ppm phosgene (phosgene-exposed group) for there are 5 phosgene-exposed groups altogether. For the time points of 1, 3, 6, 12 and 24 h post-exposure, one phosgene-exposed group was sacrificed at each time point. The severity of acute lung injury was assessed by Pa(O2)/F(IO2) ratio, wet-to-dry lung-weight ratio, and bronchoalveolar lavage (BAL) fluid protein concentration. sPLA(2)-IIA activity in BAL fluid markedly increased between 1 h and 12 h after phosgene exposure, and reached its highest level at 6 h. Moreover, the trend of this elevation correlated well with the severity of lung injury. These results indicate that sPLA(2)-IIA probably participates in phosgene-induced acute lung injury.

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its mechanism of action is unclear, arsenic is known to exert at least some of its toxic effects through interaction with sulfhydryl groups, and the non-protein sulfhydryl glutathione (GSH) appears to play an important role in detoxication of arsenic. Similarly, metallothionein (MT), a metal-binding protein with high sulfhydryl content, often functions in defense against metal-induced or oxidative cellular injury. Therefore, we examined the relationship among GSH, MT gene expression, and arsenic-induced toxicity or c-myc expression in cultured rat myoblast (L6) cells. In initial toxicity studies, arsenic was used in both the trivalent (arsenite) and pentavalent (arsenate) forms. The role of GSH was studied by pretreating cells with L-buthionine sulfoximine (BSO), which induces a marked depletion of GSH. In vitro exposure of L6 cells to BSO (1 to 25 microM) resulted in dose-dependent decreases in GSH. GSH depletion sensitized cells to both arsenite and arsenate. Zinc pretreatment, at levels which highly activated MT expression, had no effect on arsenite-induced cytotoxicity. Arsenite (1 microM) alone modestly increased c-myc expression from 1 to 4 h after treatment (maximum of 2.0-fold over control). After GSH depletion cells responded to arsenite exposure with much larger increases in c-myc transcription (3.2-fold over control). Zinc pretreatment had no reductive effect on arsenite-induced c-myc expression despite markedly activating the MT gene. Thus, it appears that the cellular levels of GSH, but not MT gene expression, play an important role in resistance to arsenic toxicity and aberrant gene activation. Moreover, depletion of GSH enhances arsenic-induced proto-oncogene activation, which might contribute to subsequent transformation.

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these mechanisms in experimental rat HT induced by the anti-thyroid drug methimazole (MMI-HT rat), we carried out gene expression analysis (sonic hedgehog (Shh), reelin, and Bax) using quantitative real-time PCR. Histological examination revealed cerebellar abnormalities, including reductions in the thickness of the molecular layer and delayed disappearance of the external granular layer (EGL), as well as excess bulges or sublobules in the internal granular layer (IGL). At Postnatal Day (P) 6, Shh expression in MMI-HT rat was comparable to that in controls, thus suggesting that Shh expression was sufficient to form the lobes in the initial phase. However, Shh expression decreased in the later phases, as compared with age-matched controls. This demonstrated that stronger and sustained signaling is necessary for partitioning of the cardinal lobes into lobes and sublobes. Although reelin expression was not clearly different from that in controls, Bax expression decreased at P 15. The attrition of Bax at P 15 as well as Shh in the later phase may be related to irregularities in the IGL and the relatively large numbers of internal granular cells. Taken together, these results suggest that Shh expression is related to the morphological cerebellar changes in experimental hypothyroidism and that sustained signaling by Shh may play a key role in normal development, particularly lobulation, in the cerebellum.

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y in pregnancy when fetal immunity is least well developed. Genetic analysis could provide unique insight into events in utero that are otherwise difficult to determine. We tested the hypothesis that propensity for T. gondii to cause eye disease is associated with genes previously implicated in congenital or juvenile onset ocular disease. Using mother-child pairs from Europe (EMSCOT) and child/parent trios from North America (NCCCTS), we demonstrated that ocular and brain disease in congenital toxoplasmosis associate with polymorphisms in ABCA4 encoding ATP-binding cassette transporter, subfamily A, member 4 previously associated with juvenile onset retinal dystrophies including Stargardt's disease. Polymorphisms at COL2A1 encoding type II collagen, previously associated with Stickler syndrome, associated only with ocular disease in congenital toxoplasmosis. Experimental studies showed that both ABCA4 and COL2A1 show isoform-specific epigenetic modifications consistent with imprinting, which provided an explanation for the patterns of inheritance observed. These genetic and epigenetic risk factors provide unique insight into molecular pathways in the pathogenesis of disease.

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and sex steroids including CYP2C11 and CYP2C12. We hypothesized that PP exposure would also lead to similar changes in CYP2C7, a retinoic acid and testosterone hydroxylase. CYP2C7 gene expression was dramatically down-regulated in the livers of rats treated for 13 weeks by WY-14,643 (WY; 500 ppm) or gemfibrozil (GEM; 8000 ppm). In the same tissues, exposure to WY and GEM and to a lesser extent di-n-butyl phthalate (20,000 ppm) led to decreases in CYP2C7 protein levels in both male and female rats. An examination of the time and dose dependence of CYP2C7 protein changes after PP exposure revealed that CYP2C7 was more sensitive to compound exposure compared to other CYP2C family members. Protein expression was decreased after 1, 5 and 13 weeks of PP treatment. CYP2C7 protein expression was completely abolished at 5 ppm WY, the lowest dose tested. GEM and DBP exhibited dose-dependent decreases in CYP2C7 protein expression, becoming significant at 1000 ppm or 5000 ppm and above, respectively. These results show that PP exposure leads to changes in CYP2C7 mRNA and protein levels. Thus, in addition to known effects on steroid metabolism, exposure to PP may alter retinoic acid metabolism.

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C5B as well as several other secretory products. Mucous differentiation and expression of MUC2, MUC5AC, MUC5B and MUC7, but not MUCi, MUC4, and MUC8 mucin genes, were shown to be retinoic acid- (RA) or retinol-dependent. We found that RA control of mucin genes was mediated by the retinoid acid receptors RAR alpha and, to a lesser extent, by RAR gamma. Our studies also showed that other important bioregulators such as thyroid hormone (T3) and epidermal growth factor (EGF) modulate basal expression of mucin genes, interacting with RA in a concentration-dependent manner. T3, which binds to thyroid receptors (TRs) belonging to the same superfamily of steroid hormone nuclear receptors as the RARs, inhibits mucin gene expression, particularly MUC5AC. One possible mechanism of this T3 effect is downregulation of RAR proteins, which are critical for mucin gene expression. However, we also found that T3 inhibits MUC5AC transcription.EGF, which had previously been shown to stimulate mucin expression and mucin secretion in cultured rat tracheal epithelial (RTE) cells, inhibited mucin secretion in human bronchial epithelial cell cultures. This effect was EGF concentration- and time-dependent and was progressively abolished by increasing the RA concentration. Subsequent studies suggested that the inhibitory effects of high concentrations of EGF may result from selective reduction of MUC5AC expression. These studies thus point to potentially important species differences in the mechanisms regulating mucous production, and they also confirm previous findings indicating differential regulation of MUC5AC and MUC5B gene expression.

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lly curated from the literature are integrated to generate expanded networks and predict many novel associations between different data types. CTD now contains over 15 million toxicogenomic relationships. To navigate this sea of data, we added several new features, including DiseaseComps (which finds comparable diseases that share toxicogenomic profiles), statistical scoring for inferred gene-disease and pathway-chemical relationships, filtering options for several tools to refine user analysis and our new Gene Set Enricher (which provides biological annotations that are enriched for gene sets). To improve data visualization, we added a Cytoscape Web view to our ChemComps feature, included color-coded interactions and created a 'slim list' for our MEDIC disease vocabulary (allowing diseases to be grouped for meta-analysis, visualization and better data management). CTD continues to promote interoperability with external databases by providing content and cross-links to their sites. Together, this wealth of expanded chemical-gene-disease data, combined with novel ways to analyze and view content, continues to help users generate testable hypotheses about the molecular mechanisms of environmental diseases.

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wo existing classes of drug used to treat type 2 diabetes, i.e., the biguanides and thiazolidinediones. However, in both cases these activate AMPK indirectly, and an interesting question concerns whether a drug that directly activated AMPK would retain the therapeutic benefits of the existing drugs while eliminating unwanted side effects. AMPK activators also now have potential as anticancer drugs.

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DNA microarray. Changes in gene expression that were considered to reflect damage to the lung, a change in the balance of electrolytes and fluid, and alveolar remodeling were observed. The products of these genes were: CSF-1 receptor, which is a receptor of inflammatory cytokines that activates monocyte/macrophages; TGF-beta type II receptor, which is a receptor of TGF-betas involved in wound healing and fibrosis; a subunit of Na+/K(+)-ATPase, an amiloride-sensitive cation channel, and a subunit of the potassium channel, all of which regulate the alveolar fluid balance and play a role in clearing lung edema; the adenosine A2a receptor, which has a protective function in the lung and interacts with dopamine D1 and D2 receptors to regulate the function of amiloride-sensitive cation channels; cofilin, which is involved in the depolymerization and cleavage of actin filaments; LIM motif-containing protein kinase 1, which negatively regulates the activity of cofilin; SHPS-1, which regulates the integrin-mediated reorganization of the cytoskeleton; and sodium channel beta 2, which is involved in cell adhesion and migration. These results indicate that PQ-induced pulmonary fibrosis does not merely terminate as cicatrices three months after the discontinuation of PQ treatment, but that dynamic functional change continues in the lung.

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inger protein 415 isoform 5 (ZNF415-5). Reverse-transcription (RT)-polymerase chain reaction (PCR) analysis revealed that the ZfLp mRNA is expressed in the breast, lung, stomach, small intestine colon and ovary, but not in the liver. When expressed in Xenopus laevis oocytes, ZfLp mediated the high affinity transport of [(3)H]paclitaxel (taxol) in a sodium-independent manner (K(m) = 336.7 +/- 190.0 nM). The uptake of [(3)H]paclitaxel (taxol) by ZfLp was trans-stimulated by glutarate and glutathione (GSH). A cis-inhibition experiment revealed that ZfLp-mediated transport of [(3)H]paclitaxel (taxol) is inhibited by several organic solutes specifically clotrimazole. Using several clotrimazole derivatives, we found that N-benzylimidazole would be a minimum unit for producing the inhibition of ZfLp-mediated drug uptake. Our results may provide insights into the novel role of soluble protein, such as ZNF, in the human body. Our results, therefore, would be expected to facilitate research on the novel role of ZNFs and on the discovery of novel drugs for targeting ZNF-related proteins such as ZfLp.

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a single intraperitoneal injection of N-diethylnitrosamine for initiation treatment. Two weeks after the initiation, rats were given no tumor-promoting agents (DEN alone), I3C (2,500 or 5,000 ppm in diet), PB (60 or 120 ppm in drinking water), or 2,500 ppm I3C + 60 ppm PB for 6 weeks. One week after the I3C/PB treatments, all animals underwent a two-thirds partial hepatectomy. The number and area of liver cell foci positive for glutathione S-transferase placental form (GST-P(+) foci) were not significantly fluctuated in the PB+I3C group in the isoadditive statistical model. On the contrary, the mRNA levels of Cyp2b1/2 and Nqo1 were suppressed and enhanced, respectively, in the PB+I3C group in the isoadditive model, but there was no enhancement in the microsomal reactive oxygen species (ROS) production, thiobarbituric acid-reactive substance levels, and Ki-67(+) cell ratio in this group. The results suggest that the co-administration of I3C and PB causes no modifying effects in liver tumor promotion in rats.

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melatonin treated (Mel), vitamin E plus vitamin C plus CE treated (Vit+CE), and melatonin plus CE treated (Mel+CE). The rats in the CE, Vit+CE and Mel+CE groups were administered orally with CE in two equal doses of 41 mg/kg body weight (0.25 LD50). Melatonin and vitamins E and C were administrated intramuscularly at the doses of 10, 150 and 200 mg/kg, respectively. The levels of thiobarbituric acid reactive substance (TBARS) and antioxidant potential (AOP), and the activities of glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) were studied in the homogenates of kidney tissue. There were no significant differences in the activities of SOD and CAT between the experimental groups. The level of TBARS increased significantly (P<0.05) while AOP decreased significantly (P<0.05) in the CE group compared with the C group. GSH-Px activity was significantly (P<0.05) lower in the CE group and higher in the melatonin group than the control group. Histopathological changes were found in the kidney tissue of rats treated with CE. These were infiltration in mononuclear cells at perivascular and peritubular areas, hydropic degenerations in tubule epithelium and glomerular sclerosis. The severity of the lesions was reduced by administration of vitamins and melatonin. These results suggest that CE increases lipid peroxidation and decreases AOP by increasing oxidative stress, and that high doses of melatonin and a combination of vitamin E plus vitamin C considerably reduce the toxic effect of CE on kidney tissue of rats.

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the AhR regulates the appearance and numbers of ovarian follicles. Ovaries were harvested from AhR-deficient (AhRKO) and wild-type mice on gestational day 18 (GD 18) and postnatal days (PND) 2-3, 8, 32-35, and 53. Complete serial sections of ovaries were evaluated histologically for the presence of germ cells and follicles. On GD 18, there was no difference in the number of germ cells per ovary between AhRKO and wild-type fetuses. However, by PND 2-3, AhRKO mice had significantly more fully formed primordial follicles (AhRKO = 38,440 +/- 3632 versus wild-type = 21,120 +/- 2688) and fewer single germ cells than wild-type mice (AhRKO = 12,696 +/- 1192 vs. wild-type = 18,160 +/- 720). On PND 8 and 32-35, there was no difference in the number of follicles between AhRKO and wild-type mice but by PND 53, AhRKO mice had significantly fewer antral follicles than wild-type (AhRKO = 3416 +/- 480 vs. wild-type = 6776 +/- 1024). Taken together, these results suggest that the AhR may play a role in the formation of primordial follicles and the regulation of antral follicle numbers.

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aptic plasticity by binding to the multifunctional ApoE receptors apolipoprotein E receptor 2 (Apoer2) and very low density lipoprotein receptor (Vldlr). We have previously shown that the presence of ApoE4 renders neurons unresponsive to Reelin by impairing the recycling of the receptors, thereby decreasing its protective effects against amyloid beta (Abeta) oligomer-induced synaptic toxicity in vitro. We showed that when Reelin was knocked out in adult mice, these mice behaved normally without overt learning or memory deficits. However, they were strikingly sensitive to amyloid-induced synaptic suppression and had profound memory and learning disabilities with very low amounts of amyloid deposition. Our findings highlight the physiological importance of Reelin in protecting the brain against Abeta-induced synaptic dysfunction and memory impairment.

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enzyme kinetic constants, the IC(50) of warfarin for VKOR was determined in human, murine, porcine, and rat hepatic microsomes. Interspecies differences were observed when comparing the K(m app) (range, 2.41-6.46 microM), V(max) (range, 19.5-85.7 nmol/mg/min), and Cl(int app) (range, 8.2-18.4 ml/mg/min) values. Comparison of the IC(50) values of warfarin, across the four species tested, revealed a significant species difference between murine microsomes (0.17 microM) and rat microsomes (0.07 microM). Overall, this study indicates that there are interspecies differences regarding the in vitro reduction of vitamin K(1) 2,3-epoxide by the warfarin-sensitive enzyme vitamin K(1) 2,3-epoxide reductase. Significant differences between the IC(50) values of murine and rat microsomes suggest differences in the susceptibility of these species to warfarin.

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es using enzyme activities (CYP1A2 (ethoxyresorufin), 2B (benzoxyresorufin for rat and bupropion for human) and CYP3A (testosterone)) and Taqmantrade mark Low Density Array (TLDA) analysis. There was a good correlation between the induction of CYP1A2, CYP2B6 and CYP3A4 enzyme activities and mRNA expression in human hepatocytes. In contrast, BROD activities and mRNA expression in rat hepatocytes correlated poorly. However, bupropion hydroxylation correlated well with Cyp2b1 expression in rat hepatocytes. TLDA analysis of a panel of mRNAs encoding for CYPs, phase 2 enzymes, nuclear receptors and transporters revealed that the main genes induced by the 8 compounds tested were the CYPs. AhR ligands also induced UDP-glucuronosyltransferases and glutathione S-transferases in rat and human hepatocytes. The transporters, MDR1, MDR3 and OATPA were the only transporter genes significantly up-regulated in human hepatocytes. In rat hepatocytes Bsep, Mdr2, Mrp2, Mrp3 and Oatp2 were up-regulated. We could then show a good in vivo:in vitro correlation in the induction response of isolated rat hepatocytes and ex-vivo hepatic microsomes for the drug development candidate, EMD392949. In conclusion, application of TLDA methodology to investigate the potential of compounds to induce enzymes in rat and human hepatocytes increases the throughput and information gained from one assay, without reducing the predictive capacity.

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of interleukin-1alpha (IL-1alpha), tumor necrosis factor (TNF-alpha) and monocyte chemoattractant protein-1 (MCP-1) in the skin and blood of hairless rats. Dermal exposures were carried out by occlusive application of chemicals (230 microl for 1 h, using Hill Top Chambers) for 1 h. The expression of IL-1alpha, TNF-alpha and MCP-1 was measured by enzyme immunoassay (EIA), and the regulatory proteins NFkappaB and IkappaBalpha were measured by Western blot analysis. The skin irritation and TEWL data indicate that the irritation was in the following decreasing order: nonane > dodecane > tetradecane. Likewise, nonane significantly increased the expression of IL-1alpha, TNF-alpha and MCP-1 in skin and blood as compared to control at different time points. Dodecane and tetradecane did not show any increase in the expression of IL-1alpha and MCP-1 as compared to control (P > 0.05), but the expression of TNF-alpha by dodecane and tetradecane was significantly higher than control at all time points. The release of cytokines by nonane exposure was further supported by activation of NFkappaB p65 and corresponding degradation of IkappaBalpha in the skin. In conclusion, this study demonstrates that the biophysical parameters (TEWL and erythema scores) were correlated to the biomarker expressions after dermal exposures with nonane but not with dodecane and tetradecane. Dodecane produced only mild irritation in response to experimental conditions of the present study and further did not show significant differences in IL-1alpha and MCP-1 levels in skin and blood. However, TNF-alpha was well expressed in response to all the chemicals. Tetradecane did not show any visible signs of skin irritation and also did not produce any significant difference in IL-1alpha and MCP-1 release profiles as compared with control. The expression of TNF-alpha in skin due to tetradecane support the fact that visually indistinguishable skin irritation reactions can induce significant changes in the biological marker profile.

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to investigate the combined nephrotoxic effects of TCDD and PCBs on rats and to reveal the potential interactions between TCDD and PCBs. Male Sprague-Dawley rats were intragastrically administered TCDD (10 mug/kg), PCBs (Aroclor 1254, 10 mg/kg), or the combination (10 mug/kg TCDD + 10 mg/kg Aroclor 1254). After 12 consecutive days of exposure, all treatments induced nephrotoxicity, as evidenced by significant increases in the levels of serum creatinine and blood urea nitrogen, changes of kidney histopathology, and significant renal oxidative stress. Most of these effects were more remarkable in the combined-exposure group. Furthermore, all treatments induced renal cytochrome P450 1A1 (CYP1A1) protein expression, and the induction was more conspicuous in the combined-exposure group. These findings suggested that the nephrotoxicity induced by TCDD and PCBs in the present study might be attributable to the high expression of CYP1A1. In addition, the result of the two-way analysis of variance revealed that the combined effects of TCDD and PCBs were complicated, being additive, synergistic, or antagonistic depending on the selection of toxicity end points under the present experimental condition. This study demonstrates that combined exposure to TCDD and PCBs induced significant nephrotoxicity in rats, and there were complicated interactions between the two pollutants on the nephrotoxicity.

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0 ppm) in their drinking water for 7 months. The expression of nAChRs at mRNA and protein levels, neurotoxicity and oxidative stress were analyzed in the study. The results indicated that there were no significant changes at mRNA level of the nAChR alpha3, alpha7, beta2 subunits in PC12 cells, and alpha4, alpha7, beta2 subunits in rat brains between the groups with fluorosis and controls. A significant decline in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, and increased levels of protein oxidation and lipid peroxidation were observe in PC12 cells treated with high doses of fluoride or rat brains with chronic fluorosis. The decreases of nAChR alpha3 and alpha7 subunit proteins in PC12 cells resulted from fluoride toxicity were mostly prevented by a pretreatment with antioxidant. The results suggest that the deficit of nAChRs induced by fluoride toxicity occurs at the level of post-transcription of the receptor gene, in which a mechanism might be involved in the damage by oxidative stress.

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e), chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate), and a fungicide, thiram (tetramethylthiuram disulphide) individually or in combination caused significant inhibition of the growth of R. japonicum No. 16. The effects of insecticide-fungicide seed treatments on the nitrogenase activity of soybean plants in nitrogen-fixing capacity, weights of leaves, stems, and nodules were determined. Thiram, singly or in combination with lindane and/or chlorpyrifos, significantly delayed growth of the plants and affected the activity of nitrogenase in the fixation of nitrogen 3 weeks after treatments. No drastic effect of any of the pesticide treatments on soybean plant growth was observed after 8 weeks.

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The data indicates significant alterations in the parameters related to haeme synthesis pathway like inhibition of blood delta-aminolevulinic acid dehydratase, delta-aminolevulinic acid synthetase, oxidative stress like depletion of glutathione (GSH) and increase in oxidized glutathione (GSSG) and thiobarbituric acid reactive substances. These changes were accompanied by depletion in GSH:GSSG ratio, whole brain biogenic amine levels and a dose-dependent increase in fluoride concentration. Interestingly and most significantly, these changes were more pronounced at lower concentrations of fluoride compared with higher fluoride dose. Biochemical changes were supported by the histological observations, which also revealed that at high concentrations of fluoride, toxic effects and damages to organs were more pronounced. These changes support our earlier findings regarding the role of decreased ionic mobility of fluoride ion at higher concentrations, leading to less pronounced toxicity.

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o produce a proinflammatory response. This mechanism results in the activation of NF-kappa B and subsequent inflammatory gene induction. In order to determine the effect of combustion source particle matter (PM), rich in zinc and nickel but with negligible endotoxin, on a possible activation of TLR4-mediated cell signaling and inflammation, we intratracheally (IT) instilled 3.3 mg/kg of PM into 12-w-old healthy male Wistar Kyoto (WKY) and susceptible spontaneously hypertensive (SH) rats. Inflammation, inflammatory-mediator gene expression, bronchoalveolar lavage fluid (BALF) protein and LDH, TLR4 and CD14 protein, and NF-kappa B activation in the lung were determined after 24 h. Dose-response data (0.0, 0.83, 3.33, and 8.3 mg/kg PM) for BALF LDH were obtained as a marker of lung cell injury in SH rats. BALF neutrophils, but not macrophages, were significantly increased in the PM-exposed WKY and SH rats. SH rats showed a greater PMN increase than WKY rats. Similarly, BALF protein and LDH levels were also increased following PM exposure but to a significantly greater extent in SH rats. Plasma fibrinogen was increased only in SH rats exposed to PM. The increased inflammation seen in PM-exposed SH rats was accompanied by a significant increase in TLR4 protein in the lung tissue, which was primarily localized in alveolar macrophages and epithelial cells. CD14 was also increased by PM exposure in both SH and WKY rats but was significantly greater in the SH rats. These increases were associated with greater translocation of NF-kappa B in the lungs of SH rather than WKY rats. This was accompanied by increased macrophage inhibitory protein (MIP)-2 mRNA expression at 24 h of exposure. These data suggest that the increased inflammation in the lungs of PM-exposed SH rats compared to WKY rats is accompanied by an increase in TLR4-mediated cell signaling. Thus, one of the mechanisms for greater susceptibility of SH rats to PM exposure may involve an increased activation of the TLR4 signaling pathway.

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been linked to asthma exacerbation in many cities with vehicular traffic congestion. We tested the hypothesis that DEP-induced enhancement of the hallmark features of allergic airway disease in a murine model is dependent on the function of the pan neurotrophin receptor p75. Ovalbumin (OVA)-sensitized C57B1/6J mice were intranasally instilled with an antibody against the p75 receptor or saline alone 1 h before OVA challenge. The mice were then exposed nose-only to the PM2.5 fraction of SRM2975 DEP or air alone for 5 h beginning 1 h after OVA challenge. Two days later, air-exposed OVA-allergic mice developed a small but insignificant increase in methacholine-induced airflow obstruction relative to air-exposed, vehicle-sensitized mice. DEP-exposed OVA-allergic mice had a significantly greater degree of airway obstruction than all other groups. Instillation of anti-p75 significantly attenuated the DEP-induced increase in airway obstruction in OVA-allergic mice to levels similar to non-sensitized mice. The DEP-induced exacerbation of allergic airway responses may, in part, be mediated by neurotrophins.

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receptor (NR) subunit mRNAs and proteins in rat striatum when rats were in manganism. The rats were divided randomly into four groups of six males and six females each: control group (group 1) and 8, 40, and 200 micromol/kg Mn-treated groups (groups 2-4). The control group rats were subcutaneously (s.c.) injected with normal saline. Manganese-treated rats were s.c. injected with respectively 8, 40, and 200 micromol/kg of MnCl(2) . 6H(2)O in normal saline. The administration of MnCl(2) . 6H(2)O for 4 weeks significantly increased Mn concentration in the striatum. With the increase in administered MnCl(2) dosage, Glu concentration and cell apoptosis rate increased significantly. The relative intensity of NR2A mRNA decreased significantly in 8 micromol/kg Mn-treated rats. However, relative intensities of NR1 and NR2B mRNAs decreased significantly in 40 micromol/kg Mn-treated rats. Similarly, the relative intensity of NR2A protein showed a significant decrease in 40 micromol/kg Mn-treated rats whereas those of NR1 and NR2B decreased significantly in 200 micromol/kg Mn-treated rats. Therefore, the expression of NR2A mRNA and protein were much more sensitive to Mn than that of NR1 and NR2B. In conclusion, the results suggested that Mn induced nerve cell damage by increasing extracellular Glu level and altered expression of NR subunit mRNAs and proteins in rat striatum.

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romide to address the type of cell death induced by nano-Se in HCC-bearing rats. To predict the oxidative stress and DNA damage, the generation of 8-hydroxy-2-deoxyguanosine (8-OHdG) and 2-deoxyguanosine (2-dG) was examined. Moreover, the expression of some HCC-related genes was investigated such as aldo-keto reductase 1B10 (Akr1b10), ING3 and Foxp1 genes. As well as the histopathological study of liver tissue sections was performed. The results obtained from this study revealed that (HCC+Nano Se) group shows the highest number of damaged cancerous cells. Furthermore, the necrotic/apoptotic rate was significantly higher in (nano-Se+HCC), (HCC+Doxo) and (HCC+Doxo+nano-se) compared to that in the untreated HCC group. Treatment of HCC group with nano-se decreased the ratio of 8-OHdG/2-dG generation significantly with respect to the untreated HCC group. The opposite was observed regarding the gene expression of AKr1b10 and ING3. The treatment of HCC group with nano-se elicited significant increase in the expression of Akr1b10 and ING3 genes compared with untreated HCC group. On the other hand, the expression of Foxp1 gene was significantly decreased in HCC group treated with nano-se in comparison with the untreated HCC group. The histopathological study provided a supportive evidence for the molecular genetics study. Our data shed light on the molecular mechanisms of nano-se in attenuating HCC in the experimental model.

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tigated using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach. Genotyping was performed on DNA isolated from white blood cells from 134 patients with prostate cancer and 184 healthy control subjects. Both the prostate cancer patients and the controls demonstrated similar frequencies of the variant allele SULT1A1*2 (35.1% vs 39.1%). Homozygosity for the variant allele was slightly less frequent in cancer patients than controls (12.7% vs 17.4%). Our study does not support the hypothesis that the phenol sulphotransferase variant allele SULT1A1*2 with a G/A transition at nucleotide 638 is a risk modifier for prostate cancer in the Caucasian population.

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air-fed control groups. The reduced aldolase activity following PenCB-treatment was due to the marked suppression of the expression of aldolase B shown by immunoblot analysis after SDS-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. The suppression of rat liver aldolase B could be a key biochemical lesion caused by PenCB.

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ith phenobarbital, a potent CYP inducer, increased the predominance of expression of these three mRNAs in WI rats (by 26-, 4-, and 2-fold, respectively) along with the predominance of increased microsomal total P450 contents and smooth-surface endoplasmic reticulum in the centrilobular hepatocytes. CYP1A enzymatic activity was also higher in WI rats than in SD rats. No strain differences were observed in phenobarbital induction of CYP2B1/2, CYP2C6, or CYP3A1. CYP3A2 mRNA was more strongly induced by dexamethasone, a typical inducer of CYP3A, together with CYP3A1 mRNA, in WI rats than in SD rats (by 2-fold), whereas the CYP1A1 and CYP1A2 mRNA expression induced by beta-naphtoflavone, a typical inducer of CYP1A, did not differ between the two strains. Furthermore, WI rats exhibited predominantly arylhydrocarbon receptor, pregnane X receptor, and constitutive androstane receptor mRNAs, responsible for CYP1A or CYP3A induction, with phenobarbital or dexamethasone induction. In conclusion, significant, predominant expression of hepatic CYP1A and CYP3A mRNAs in WI rats was observed, possibly related to nuclear receptor-mediated induction. Considering the pharmacokinetic and toxicological importance of CYP1A and CYP3A, different outcomes might arise depending on the rat strains used in preclinical studies of drugs metabolized typically or mainly by both isoforms.

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e RT-PCR, which revealed that genes related to steroidogenesis including cytochrome P450 17A1 (CYP17A1), steroidogenic acute regulatory protein (StAR) and CYP11A1 were significantly increased. Besides the testes were histopathologicaly examined, which revealed testicular destruction and degeneration represented by a layer of columnar epithelium, oedematous changes surrounding the seminiferous tubules besides vacuolated spermatogonial cells and more elongated Leydig cells. These data suggest that profenofos considered as one of the male reproductive toxicants. Furthermore, we propose that the above three steroidogenic-related genes and the gene of acrosomal reaction as potential biomarkers of testicular toxicity.

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verdose. The pathophysiology of renal toxicity in acetaminophen poisoning has been attributed to cytochrome P-450 mixed function oxidase isoenzymes present in the kidney, although other mechanisms have been elucidated, including the role of prostaglandin synthetase and N-deacetylase enzymes. Paradoxically, glutathione is considered an important element in the detoxification of acetaminophen and its metabolites; however, its conjugates have been implicated in the formation of nephrotoxic compounds. Acetaminophen-induced renal failure becomes evident after hepatotoxicity in most cases, but can be differentiated from the hepatorenal syndrome, which may complicate fulminant hepatic failure. The role of N-acetylcysteine therapy in the setting of acetaminophen-induced renal failure is unclear. This review will focus on the pathophysiology, clinical features, and management of renal insufficiency in the setting of acute acetaminophen toxicity. CASE: A 47-year-old female was found lethargic at home and brought by ambulance to an emergency department. History from family members suggested an inadvertent acetaminophen overdose, and she had last been seen a few hours earlier. She reportedly ingested 18 tablets of 500 mg acetaminophen (APAP) over the previous two days because she had run out of her prescription pain medication. Her past medical history was significant for fibromyalgia, arthritis, and a prior gastric bypass procedure. She had no history of alcohol abuse or renal insufficiency. She was lethargic. Vital signs: BP 128/96 mmHg, pulse 112/min, respirations 32/min; pulse oximetry 98% on 2L nasal cannula oxygen. Laboratory studies: BUN 9 mg/dL, creatinine 0.9 mg/dl, acetaminophen 12 mcg/mL, AST 5409 u/L and ALT 1085 u/L. A urinalysis was negative for blood with trace protein and ketones. A urine drug screen was positive for marijuana and opioid metabolites. At the initial hospital, she was treated with N-acetylcysteine (NAC) orally. Subsequently, she developed fulminant hepatic failure with elevated transaminases, hypoglycemia, and coagulopathy (Tables 1A and 1B). She was transferred to our facility two days after initial presentation for liver transplant evaluation. At that time, her APAP level was 2.0 mg/L. Oral NAC therapy was continued after transfer. The patient's liver function subsequently improved and she ultimately did not require transplantation. She did develop acute renal failure during the course of her hospitalization, with a creatinine of 2.3 mg/dL on transfer, which increased to 8.1 mg/dL nine days later (approximately 11-13 days post-ingestion). Medical toxicology was consulted by the intensive care unit team to address whether this was acetaminophen-induced renal failure and if there was a role for NAC in this setting.

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1-1467) and are formed primarily through O-sulfonation of alpha-hydroxytamoxifen (alpha-OHTAM). To explore the genotoxicic mechanisms of TAM, TAM was incubated with one of multiple human cytochrome P450 enzymes, i.e., P450 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, 3A7, 4A11, 4F2, 4F3A, or 4F3B, in a NADPH regenerating system, and the metabolites were identified using HPLC/UV analysis with authentic standards. Among the 18 human P450 enzymes, P450 3A4 generated a significant amount of alpha-OHTAM. When some rat P450 enzymes were examined, P450 3A2 also catalyzed alpha-hydroxylation of TAM. Similarly, human P450 3A4 and rat P450 3A1 and 3A2 converted toremifene (TOR, a chlorinated TAM analogue) to alpha-hydroxytoremifene (alpha-OHTOR). The formation of alpha-OHTAM and alpha-OHTOR by these P450 enzymes was confirmed by tandem mass spectroscopy. Only the P450 3A subfamily enzymes are able to alpha-hydroxylate TAM and TOR. Although the formation of alpha-OHTOR by these enzymes was much higher than that of alpha-OHTAM, TOR is known to be much less genotoxic than TAM. The results support our proposed mechanism that the lower genotoxicity of TOR is due to limited O-sulfonation of alpha-OHTOR by hydroxysteroid sulfotransferases, resulting in the poor formation of DNA adducts (Shibutani, S., et al. (2001) Cancer Res. 61, 3925-3931).

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tudy, to assess effects of maternal exposure to ethinylestradiol (EE) on brain sexual differentiation of offspring, region-specific mRNA expression of two estrogen-responsive genes, gamma-aminobutyric acid transporter type 1 (GAT-1) and anti-apoptotic bcl-xL was measured in the medial preoptic area (MPOA), including sexually dimorphic nucleus (SDN), at the late stage of brain sexual differentiation in rats. Pregnant Sprague-Dawley animals were fed diets containing EE at concentrations of 0, 0.02, 0.1, and 0.5 ppm from day 15 of pregnancy to day 9 after delivery. In another group, neonates were directly injected with estradiol benzoate (EB: 10 microg/pup, sc) on postnatal day (PND) 2. The MPOA on PND 9 was microdissected from methacarn-fixed paraffin-embedded brain sections to measure mRNA levels by competitive RT-PCR, followed by plate hybridization. EE-exposure decreased GAT-1 expression dose-dependently from 0.02 ppm in females and at 0.5 ppm in males, while EB-treatment caused reduction only in females. EE-exposure did not alter Bcl-xL levels. At week 11, EE-exposed females exhibited a similar spectrum of histopathological changes in endocrine-linked organs as with EB, evident from 0.1 ppm, while in males EE-exposure did not cause histopathological alteration despite clear change with EB-treatment. Measurement of SDN-POA dimensions at week 11 revealed volume reduction in males exposed to 0.5 ppm EE or EB. The results suggest that GAT-1 expression in the developing MPOA is a sensitive measure for the level of disruption of brain sexual differentiation due to maternal dietary exposure to estrogens, despite definite reproductive abnormalities may not be detectable in males with this exposure protocol.

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inone was administered (0.1 mg/kg/day; i.p.) following GEN or saline injections. Blood and urine samples were collected to measure the renal function tests. Kidneys were excised for histological evaluation and for the measurement of malondialdehyde (MDA), glutathione (GSH), myeloperoxidase (MPO) activity, and chemiluminescence (CL). Halofuginone treatment to animals with GEN-induced renal injury caused a significant decrease in serum blood urea nitrogen level and reduced the elevated MDA, GSH content, and MPO activity. It was also effective in reversing the elevated CL values of rats with GEN-induced nephrotoxicity and preserving renal morphology, as examined microscopically. In conclusion, halofuginone was beneficial in GEN-induced acute nephrotoxicity. The mechanism could be attributed, at least in part, to decreased tissue leukocyte infiltration and reactive metabolite production.

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on liver gluconeogenesis using an isolated perfused rat liver system. Gluconeogenesis from 2.5 mM lactate was inhibited by 1 mM MCA and was completely abolished after 2 h of perfusion. Levels of citric acid cycle intermediates such as citrate, isocitrate, and 2-oxoglutarate (2-OG) were significantly reduced by MCA. The finding that the levels of citrate and 2-OG were similarly reduced (to 31 and 36% of control, respectively) indicates that aconitase was not inhibited by MCA. On the contrary, gluconeogenesis from glycerol, which can be converted to glucose without glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was not inhibited by MCA. GAPDH was inactivated by MCA in vitro, but enolase, phosphoglycerate mutase, and phosphoglycerate kinase were not inactivated at the same or higher concentrations of MCA. Furthermore, GAPDH activity in the MCA-perfused liver decreased to 33-42% of control and that in the liver of rats exposed to MCA was reduced to 19% of control. We concluded that MCA inactivates GAPDH, and this is the cause of the inhibition of liver gluconeogenesis.

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uctive toxicants on protein expression profiles in the rat testes and to identify potential biomarker candidates. Four well-known reproductive toxicants, ethylene glycol monomethyl ether (EGME), cyclophosphamide (CP), sulfasalazine (SASP) and 2,5-hexanedione (2,5-HD), were administered to male rats in a single dose, and protein expression profiles were investigated after 24 hr by two-dimensional gel electrophoresis (2DE). Histopathological examination of the testes and serum concentration analysis were also performed. From the results of the comparison of 2D-gels among different doses of a compound and among compounds, 52, 20, 24 and 111 spots were nominated as differentially expressed spots with EGME, CP, SASP and 2,5-HD treatments, respectively. Several spermatogenesis-involved proteins were identified, including glutathione S-transferase (GST), testis-specific heat shock protein 70-2 (HSP70-2), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and phosphatidylethanolamine-binding protein (PEBP). Some of them were altered by more than one compound. In summary, remarkable histopathological findings were observed only in the EGME high-dose group, and most of the protein changes were detected before histopathological changes occurred. Therefore, the proteins identified in this study could potentially serve as biomarkers to evaluate male reproductive toxicity at an early stage of drug discovery and development.

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We have previously reported high levels of black carbon in AM from biomass smoke-exposed children and adults. We therefore aimed to use a mouse model to test the hypothesis that high levels of carbon loading of AM in vivo increases susceptibility to pneumococcal pneumonia. METHODS: Female outbred mice were treated with either intranasal phosphate buffered saline (PBS) or ultrafine carbon black (UF-CB in PBS; 500 mug on day 1 and day 4), and then infected with S. pneumoniae strain D39 on day 5. Survival was assessed over 72 h. The effect of UF-CB on AM carbon loading, airway inflammation, and a urinary marker of pulmonary oxidative stress was assessed in uninfected animals. RESULTS: Instillation of UF-CB in mice resulted a pattern of AM carbon loading similar to that of biomass-smoke exposed humans. In uninfected animals, UF-CB treated animals had increased urinary 8-oxodG (P = 0.055), and an increased airway neutrophil differential count (P < 0.01). All PBS-treated mice died within 72 h after infection with S. pneumoniae, whereas morbidity and mortality after infection was reduced in UF-CB treated animals (median survival 48 h vs. 30 h, P < 0.001). At 24 hr post-infection, UF-CB treated mice had lower lung and the blood S. pneumoniae colony forming unit counts, and lower airway levels of keratinocyte-derived chemokine/growth-related oncogene (KC/GRO), and interferon gamma. CONCLUSION: Acute high level loading of AM with ultrafine carbon black particles per se does not increase the susceptibility of mice to pneumococcal infection in vivo.

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e-mutagenic 7,8-dihydro-8-oxoguanine (8-oxoG) from DNA. We demonstrate that a single Cd aerosol exposure of adult male Lewis rats causes time- and dose-dependent down-regulation in the pulmonary levels of rOGG1 mRNA and OGG1 protein, quantified by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assays and western analyses, respectively. Immunohistochemical studies confirmed that Cd inhalation reduces the relative amount of OGG1 in lungs of exposed animals without altering its over-all distribution within the lung, which appears to be more prominent within the alveolar epithelium. In agreement with our in vivo studies, we show that OGG1 expression is also attenuated in alveolar epithelial cell cultures exposed to CdCl(2) either acutely or by repeated passaging in Cd-containing medium. The effects caused by Cd were observed in cells that show no loss in viability, as assessed by colony forming ability, the MTT assay, and propidium iodide membrane permeability studies. Nuclear extracts prepared from Cd-treated cells also exhibit a reduction in the ability to nick a synthetic oligonucleotide containing 8-oxoG. We conclude from these studies that Cd causes suppression of OGG1 in the lung and that this mechanism may, in part, play a role in the Cd carcinogenic process.

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for the detoxification of catecholestrogens. In the present study, we examined what role COMT gene polymorphisms may play in the development of breast cancer in a case-control study of 130 sporadic unrelated premenopausal Turkish breast cancer patients with 233 unrelated healthy controls. The frequency of COMT-L allele was more significantly represented in the breast cancer cases (48.08%) than in the controls (38.20%). The genotype frequencies of COMT-HH, HL and LL were 25.4, 53.1 and 21.5% in the breast cancer subjects and 26.6, 62.7 and 10.7% in the controls respectively. In conclusion, the COMT-L allele and COMT-LL genotype are genetic risk factors for sporadic breast cancer in premenopausal Turkish women.

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problem of public safety for the populations living on these territories that are exposed to low levels of 137Cs during a long period through food. Then it is necessary to study potential effect of this chronic contamination. To mimic this situation, the authors investigate the potential biological effects of chronic exposure to 137Cs at a postaccidental dose (150 Bq/rat/day) on hepatic metabolism of cholesterol in rat. Plasma lipid level, gene expression and activity were analyzed. It was observed that in 137Cs-exposed rats, gene expression of low-density lipoprotein receptor (LDLr), apolipoprotein B (apoB), and liver X receptor alpha (LXRalpha) are increased (95%, p < .05; 34%, p < .05; 20%, p < 0.05, respectively), whereas transporter adenosine triphosphate-binding cassette transporter G5 (ABCG5) is decreased (42%, p < .05). In addition, cytochrome P450 27A1 (CYP27A1) activity is increased (34%, p < .05) in contaminated rat liver. In conclusion, the results suggest that 137Cs contamination at low-level induces molecular modifications of the liver cholesterol metabolism without leading to a dysregulation of its homeostasis. These results suggest that chronic long term exposure at low-level of 137Cs may evolve to lipid disorder.

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e and female F344 rats were compared. Ethoxyphenoxazone deethylation was induced much more by HCB than by OCS in both of these strains of mice (although neither OCS nor HCB greatly induced deethylation in the DBA/2 strain). In rats ethoxyphenoxazone deethylase was induced 26-fold by HCB but only four-fold by OCS, whereas dealkylation of pentoxyphenoxazone (an indicator of phenobarbital-like induction) increased 43- and 36-fold, respectively. Both chemicals were poor inducers of dealkylation of pentoxyphenoxazone in mice. When fed HCB continuously but not when given OCS, C57BL/6 and C57BL/10 mice (both after pretreatment with iron) and F344 rats developed porphyria with a depression of hepatic uroporphyrinogen decarboxylase activity. The results illustrate that in these species OCS and HCB cannot be considered as equally efficient agents for inducing ethoxyphenoxazone deethylation or causing porphyria. If these effects are mediated through binding to the aromatic hydrocarbon responsiveness (Ah) receptor, HCB would appear to have a much greater affinity than OCS despite the face that neither chemical possesses a structure currently considered to be necessary for efficient binding.

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. Another set of experiments were carried out using cell lines, derived from the monkey kidney (COS-7), and from human tumors of the lung (A549) and liver (HepG2). Cells were cultured for 4 days in the permanent presence of 100, 200, or 400 microM NiCl2. In control rats, stress proteins pattern was found to be tissue specific: two protein bands of 96 and 94 kDa were immunodetected with the anti-GRP94 antibody in kidney and liver extracts, whereas only the 96 kDa band was present in ovary extracts. HSP73 was present in kidney, liver, and ovary whereas HSP72 was only found in kidney. In kidney of nickel-treated animals, HSP73 and the 96 kDa proteins were overexpressed whereas HSP72 was strongly down regulated. No such effect was observed in liver or ovary. Similarly, in nickel-treated cell lines, HSP72 was downregulated and GRP94 (96 kDa protein) was overexpressed. HSP73 expression appeared moderately increased in A549 cells but decreased in COS-7 cells. Because long-term caloric restriction was reported to reduce free radical generation in cells, the effect of 1 month food restriction (50%) was tested in rats as a possible way to lower oxidative damages induced by Ni. No significant effect on HSP expression was observed.

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re reduced (P < 0.05), whereas the alkaline phosphatase level was augmented (P < 0.05) in lead fed rats compared with controls. Kinetic studies with sucrase revealed a low Vmax (0.224 in control and 0.160 units mg(-1) protein in lead exposed) with no change in Km (12.6-13.5 mM). Western blot analysis for alkaline phosphatase yielded intense staining of enzyme protein in lead fed rats compared with controls, however, the intensity of the antigen signal was reversed for sucrase under these conditions. These findings suggest that ingestion of lead may interfere with the crypt cell differentiation process thus affecting enzyme functions in the rat intestine.

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bone, kidney, and liver) after acute or chronic contamination and show that it produces chemical or radiological toxicity or both. The literature is essentially devoid of information about uranium-related cellular and molecular effects on metabolic functions such as xenobiotic detoxification. The present study thus evaluated rats chronically exposed to depleted uranium in their drinking water (1mg/(ratday)) for 9 months. Our specific aim was to evaluate the hepatic and extrahepatic mRNA expression of CYP3A1/A2, CYP2B1, and CYP1A1 as well as of the nuclear receptors PXR, CAR, and RXR in these rats. CYP3A1 mRNA expression was significantly higher in the brain (200%), liver (300%), and kidneys (900%) of exposed rats compared with control rats, while CYP3A2 mRNA levels were higher in the lungs (300%) and liver (200%), and CYP2B1 mRNA expression in the kidneys (300%). Expression of CYP1A1 mRNA did not change significantly during this study. PXR mRNA levels increased in the brain (200%), liver (150%), and kidneys (200%). Uranium caused CAR mRNA expression in the lungs to double. Expression of RXR mRNA did not change significantly in the course of this study, nor did the hepatic activity of CYP2C, CYP3A, CYP2A, or CYP2B. Uranium probably affects the expression of drug-metabolizing CYP enzymes through the PXR and CAR nuclear receptors. These results suggest that the stimulating effect of uranium on these enzymes might lead to hepatic or extrahepatic toxicity (or both) during drug treatment and then affect the entire organism.

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tal day 1 (PND 1). Dams and pups were continuously exposed to lead through the drinking water of the dam until PND 20. Postnatal exposure in the pups resulted in altered mRNA levels of the following apoptotic and neurotrophic factors: caspase 2 and 3, bax, bcl-x, brain-derived neurotrophic factor (BDNF). Ribonuclease protection assays were conducted to measure the factors simultaneously at the following postnatal time points: 9, 12, 15, 20, 25, days. Our results suggest a brain region- and time-specific response following lead acetate exposure. The region most vulnerable to alterations occurs in the hippocampus with alterations beginning at PND 12, in which caspase 3, bcl-x, BDNF increase with lead exposure. Significant treatment effects were not observed for both the cortex and cerebellum.

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gents are compromised by number of limitations due to their lipophobic nature, particularly in case of chronic poisoning. Combination therapy is a new approach to ensure enhanced removal of metal from the body, reduced doses of potentially toxic chelators, and no redistribution of metal from one organ to another, following chronic metal exposure. The present study attempts to investigate dose-related effects of two thiol chelators, DMSA and one of its new analogues, monoisoamyl dimercaptosuccinic acid (MiADMSA), when administered in combination with the aim of achieving normalization of altered biochemical parameters suggestive of oxidative stress and depletion of inorganic arsenic following chronic arsenic exposure. Twenty-five adult male Wistar rats were given 25 ppm arsenic for 10 weeks followed by chelation therapy with the above chelating agents at a dose of 0.3 mmol/kg (orally) when administered individually or 0.15 mmol/kg and 0.3 mmol/kg (once daily for 5 consecutive days), respectively, when administered in combination. Arsenic exposure led to the inhibition of blood delta-aminolevulinic acid dehydratase (ALAD) activity and depletion of glutathione (GSH) level. These changes were accompanied by significant depletion of hemoglobin, RBC and Hct as well as blood superoxide dismutase (SOD) acitivity. There was an increase in hepatic and renal levels of thiobarbituric acid-reactive substances, while GSH:GSSG ratio decreased significantly, accompanied by a significant increase in metallothionein (MT) in hepatocytes. DNA damage based on denaturing polyacrylamide gel electrophoresis revealed significant loss in the integrity of DNA extracted from the liver of arsenic-exposed rats compared to that of normal animals. These changes were accompanied by a significant elevation in blood and soft-tissue arsenic concentration. Co-administration of DMSA and MiADMSA at lower dose (0.15 mmol/kg) was most effective not only in reducing arsenic-induced oxidative stress but also in depleting arsenic from blood and soft tissues compared to other treatments. This combination was also able to repair DNA damage caused following arsenic exposure. We thus recommend combined administration of DMSA and MiADMSA for achieving optimum effects of chelation therapy.

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sed by toluene exposure, and if so, if effects are age-dependent. OS parameters were selected to measure the production of reactive oxygen species (NADPH Quinone oxidoreductase 1 (NQO1), NADH Ubiquinone reductase (UBIQ-RD)), antioxidant homeostasis (total antioxidant substances (TAS), superoxide dismutase (SOD), gamma-glutamylcysteine synthetase (gamma-GCS), glutathione transferase (GST), glutathione peroxidase (GPX), glutathione reductase (GRD)), and oxidative damage (total aconitase and protein carbonyls). In this study, Brown Norway rats (4, 12, and 24months) were dosed orally with toluene (0, 0.65 or 1g/kg) in corn oil. Four hours later, frontal cortex, cerebellum, striatum, and hippocampus were dissected, quick frozen on dry ice, and stored at -80 degrees C until analysis. Some parameters of OS were found to increase with age in select brain regions. Toluene exposure also resulted in increased OS in select brain regions. For example, an increase in NQO1 activity was seen in frontal cortex and cerebellum of 4 and 12month old rats following toluene exposure, but only in the hippocampus of 24month old rats. Similarly, age and toluene effects on glutathione enzymes were varied and brain-region specific. Markers of oxidative damage reflected changes in oxidative stress. Total aconitase activity was increased by toluene in frontal cortex and cerebellum at 12 and 24months, respectively. Protein carbonyls in both brain regions and in all age groups were increased by toluene, but step-down analyses indicated toluene effects were statistically significant only in 12month old rats. These results indicate changes in OS parameters with age and toluene exposure resulted in oxidative damage in frontal cortex and cerebellum of 12month old rats. Although increases in oxidative damage are associated with increases in horizontal motor activity in older rats, further research is warranted to determine if these changes in OS parameters are related to neurobehavioral and neurophysiological effects of toluene in animal models of aging.

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ally, in the incisors of the rats, the enamel organ was disorganized with the remaining enamel matrix residing within the enamel space. The expanding cysts derived from the enamel organ were formed in the periosteal connective tissue on the labial side. At the bottom of the tooth germs, the precursor cells of the epithelial root sheath were arranged regularly and the enamel organs were preserved to the same degree as those of normal rats. In the molar teeth of the affected rats an enamel matrix remained on the neck and crown of the erupted teeth; however, no abnormality was observed at the tooth root. Although an animal model of AI has been developed from mutants of the SHR-SP rat strain, the present cases represent another potential model of the disease because of the differences in the way the enamel matured and the odontogenic cyst formation in the incisors.

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pesticides and other xenobiotics, but its acinar distribution is unknown. In this study we have analysed the activity of N-acetyltransferase in cell lysates obtained from the periportal or perivenous region by digitonin treatment during in situ liver perfusion. Livers from control animals were compared with rats chronically exposed either to ethanol by liquid diet or to lipopolysaccharide (endotoxin) by intravenous administration. The activity of N-acetyltransferase in the perivenous region was slightly (+ 20%) higher than in the periportal region. Although chronic ethanol exposure did not change total activity, the acinar distribution was reversed to a higher activity in the periportal region. In contrast, chronic endotoxin significantly increased N-acetyltransferase activity, but did not affect the acinar distribution. This increase was counteracted by simultaneous ethanol treatment. N-Acetyltransferase activity in perivenous lysates was significantly reduced after the co-administration of ethanol and endotoxin compared to that after endotoxin alone. Thus, the perivenous zonation of liver N-acetyltransferase is moderate compared to other transferases or P450 isozymes, and the cellular capacity for N-acetylation in the perivenous region, where xenobiotic activation to reactive intermediates dominates, may be insufficient.

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ment of transporters is still very scarce. This work aimed to contribute to the characterization of membrane transport mechanisms, namely, extraneuronal monoamine transporter (EMT), the multidrug resistant protein 1 (MRP1), and P-glycoprotein (P-gp) in freshly isolated cardiomyocytes from adult rats. These transporters may be accountable for uptake and/or efflux of adrenaline and an adrenaline oxidation product, 5-(glutathion-S-yl)adrenaline, in cardiomyocyte suspensions. Our results showed that 5-(glutathion-S-yl)adrenaline efflux was mediated by MRP1. Additionally, we demonstrated that the adduct formation occurs within the cardiomyocytes, since EMT inhibition reduced the intracellular adduct levels. The classical uptake2 transport in rat myocardial cells was inhibited by the typical EMT inhibitor, corticosterone, and surprisingly was also inhibited by low concentrations of another drug, a well-known P-gp inhibitor, GF120918. The P-gp activity was absent in the cells since P-gp-mediated efflux of quinidine was not blocked by GF120918. In conclusion, this work showed that freshly isolated cardiomyocytes from adult rats constitute a good model for the study of catecholamines and catecholamines metabolites membrane transport. The cardiomyocytes maintain EMT and MRP1 fully active, and these transporters contribute to the formation and efflux of 5-(glutathion-S-yl)adrenaline. In the present experimental conditions, P-gp activity is absent in the isolated cardiomyocytes.

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ontaining 1457 and 10,155 ppm butylated hydroxytoluene (BHT). Treatment with TB and BHT resulted in dose-dependent increases in relative liver weight. TB was found to be a mixed inducer of cytochrome P450 (CYP) forms in the CYP1A and CYP2B subfamilies. The administration of high doses of TB resulted in the induction of 7-ethoxyresorufin O-deethylase and 7-pentoxyresorufin O-depentylase activities, CYP1A1, CYP1A2, CYP2B1 and CYP2B1/2 mRNA levels and CYP1A2 and CYP2B1/2 apoprotein levels. In contrast, BHT was a CYP2B form inducer, increasing 7-pentoxyresorufin O-depentylase activity, CYP2B1 and CYP2B1/2 mRNA levels and CYP2B1/2 apoprotein levels. Both TB and BHT induced GSH S-transferase activities towards a range of substrates. In addition, TB and BHT markedly induced GSTP1 mRNA levels, but had only a small effect on GSTT1 mRNA levels. In summary, these results demonstrate that TB induces both phase I and II xenobiotic metabolising enzymes in rat liver.

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ydrocarbon receptor (AhR) mediates the toxic and estrogenic effects of a wide variety of environmental contaminants and industrial pollutants. Both the ERR1 and the AhR are known to modulate the ER's signalling pathways in multiple ways. Organic chemicals with a certain structural relationship to steroid hormones often induce a tissue- or cell-specific variety of responses distinct from estrogenic responses and this may involve ERR1 and AhR. The UV-screens benzophenone-2 and benzophenone-3 (BP2, BP3), structurally related to known steroid receptor ligands, are used in cosmetics and plastics to improve product stability and durability. Both BP2 and BP3 were shown to exert uterotrophic effects and BP2 was shown to bind to the estrogen receptors. Whether such effects are also exerted in other organs is unknown. Therefore, an approach to a multi-organic risk assessment for these substances was made by measuring the gene-expression of the four mentioned receptors in the pituitary, the uterus and the thyroid after a five-day treatment in comparison to estradiol. Though BP2 seems to exert an estrogen-like effect while BP3 does not, there are regulatory effects on receptor expression for both substances that indicate a kind of endocrine disruption that is not assessed by "classical" estrogenic markers.

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style='font-weight:700;'>toxicity, and carcinogenicity. The objective of the present study is to evaluate whether or not perinatal maternal exposure to BPA at 0.05, 0.5, 5, 50, and 200 mg/kg/d affects N-methyl-D-aspartate (NMDA) receptor (NMDAR) subunits NR1, NR2A, 2B, estrogen receptor beta (ERbeta), and aromatase cytochrome P450 (P450arom) protein expressions of hippocampus in male rat offspring during postnatal development. Western-blotting analyses showed that perinatal exposure to BPA significantly affected the expression of NMDAR subunits. At the lower doses of 0.05 to 50 mg/kg/d, BPA concentration dependently inhibited the expression of NMDAR subunits. However, at the higher dose (200 mg/kg/d), the effects of BPA on these subunits were different, with a stronger inhibition of NR1 expression and a slighter inhibition of NR2A, 2B expression when compared with those at the lower dosage of BPA. In addition, perinatal exposure to BPA inhibited the expression of ERbeta protein, but increased P450arom protein expression in a concentration-dependent manner, especially during the early postnatal period (the first 1-3 postnatal weeks). No significant influence of BPA on P450arom was observed at postnatal week 8. These data suggest that environmental BPA exposure may affect the development of the brain, enhancing the local biosynthesis of estrogen in the brain, inhibiting ERbeta and NMDAR expressions.

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Trap screen. We find that TOX3 interacts with both cAMP response element (CRE)-binding protein (CREB) and CREB-binding protein (CBP), and knockdown of the endogenous TOX3 by RNAi leads to significant reduction of calcium-induced c-fos expression and complete inhibition of calcium activation of the c-fos promoter. The effects of TOX3 on calcium-dependent transcription require the CRE elements. These observations identify TOX3 as an important regulator of calcium-dependent transcription and suggest that TOX3 exerts its effect on CRE-mediated transcription via its association with the CREB-CBP complex.

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that uranium could affect the reproductive system. To distinguish chemical versus radiological effects of uranium on the metabolism of the steroids in the testis, rats were contaminated via their drinking water with depleted or enriched uranium. Animals were exposed to radionuclides for 9 months at a dose of 40 mg/L (560 Bq/L for depleted uranium, 1680 Bq/L for enriched uranium). Whereas depleted uranium did not seem to significantly affect the production of testicular steroid hormones in rats, enriched uranium significantly increased the level of circulating testosterone by 2.5-fold. Enriched uranium contamination led to significant increases in the mRNA levels of StAR (Steroidogenic Acute Regulatory protein; 3-fold, p = .001), cyp11a1 (cytochrome P45011a1; 2.2-fold, p < .001), cyp17a1 (cytochrome P45017a1; 2.5-fold, p = .014), cyp19a1 (cytochrome P45019a1; 2.3-fold, p = .021), and 5alpha -R1 (5alpha reductase type 1; 2.0-fold, p = .02), whereas depleted uranium contamination induces no changes in the expression of these genes. Moreover, expression levels of the nuclear receptors LXR (Liver X Receptor) and SF-1 (Steroidogenic Factor 1), as well as the transcription factor GATA-4, were modified following enriched uranium contamination. Altogether, these results show for the first time a differential effect among depleted or enriched uranium contamination on testicular steroidogenesis. It appears that the deleterious effects of uranium are mainly due to the radiological activity of the compound.

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mation. The hypothesized link between reactive oxygen species and the effects of FA was also studied. To do so, male Wistar rats were exposed to FA inhalation (1%, 90min daily) for 3 days, and subsequently sensitized with ovalbumin (OVA)-alum by subcutaneous route. One week later the rats received another OVA-alum injection by the same route (booster). Two weeks later the rats were challenged with aerosolized OVA. The OVA challenge of rats upon FA exposure induced an elevated release of LTB(4), TXB(2), IL-1beta, IL-6 and VEGF in lung cells, increased phagocytosis and lung vascular permeability, whereas the cell recruitment into lung was reduced. FA inhalation induced the oxidative burst and the nitration of proteins in the lung. Vitamins C, E and apocynin reduced the levels of LTB(4) in BAL-cultured cells of the FA and FA/OVA groups, but increased the cell influx into the lung of the FA/OVA rats. In OVA-challenged rats, the exposure to FA was associated to a reduced lung endothelial cells expression of ICAM-1 (intercellular cell adhesion molecule 1). In conclusion, our findings suggest that FA down regulate the cellular migration into the lungs after an allergic challenge and increase the ability of resident lung cells likely macrophages to generate inflammatory mediators, explaining the increased lung vascular permeability. Our data are indicative that the actions of FA involve mechanisms related to endothelium-leukocyte interactions and oxidative stress, as far as the deleterious effects of this air pollutant on airways are concerned.

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-treated or -untreated rats for 3 or 14 days. In the BLM 0.2 microg group, slight alveolar mononuclear cell infiltration was observed, although PF was not noted. In the BLM 20-microg and 2,000-microg groups, diffuse alveolar damage along with neutrophil infiltration and subsequent PF were observed. In the saline + G-CSF group and BLM 0.2 microg + G-CSF group, a marked increase in the number of alkaline phosphatase (ALP)-positive neutrophils was noted in the alveolar capillaries, although there was neither neutrophil infiltration in alveoli nor exacerbation of lung injury. In the BLM 20 microg + G-CSF and BLM 2,000 microg + G-CSF groups, an exacerbation of lung injury along with an increase in the number of ALP-positive neutrophils in the alveoli was observed. These results indicate that the administration of G-CSF to rats with slight lung injury bearing no PF does not exacerbate the lung injury. The exacerbating effects of G-CSF seem to be associated not only with the marked infiltration of activated neutrophils but also with the severity of underlying lung injury.

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e) was administered as positive control. Experiments in vitro were also performed to elucidate the possible mechanism of action. The aminolevulinic acid dehydratase was almost completely inhibited in vitro from 0.5mM concentration, and the IC(50) was stated at 0.265 mM, 20 times higher than lead (IC(50) stated at 0.013 mM). Reduced glutathione, partially but significantly, reactivated in vitro the enzyme treated with cis-platin (0.5 and 5.0mM), whereas zinc showed a positive, significant effect with the higher dose (5.0mM) only. On the contrary, inhibition caused by lead (0.005 mM) was partially, but significantly restored by reduced glutathione, and, almost completely, by zinc. The experiments in vivo show that cis-platin causes a dose- and time-dependent inhibition of ALAD activity with 5.0 and 10.0mg/kg dose, until 66 and 33% of the control activity 96 h after treatment, respectively. The results show that erythrocyte ALAD is sensitive to cis-platin and suggest that the mechanism of enzyme inhibition is a direct interaction with sulfhydryl groups, whereas zinc site appears involved with the higher doses only. This mechanism appears different from lead that prevalently inhibits ALAD removing zinc from the enzyme, other than interacting with sulfhydryl groups.

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enzymes are expressed in probably all life forms. In humans, polymorphism in GST genes has been associated with susceptibility to various diseases though some recent data indicate that these genotypes modify disease phenotype. Thus, GST genotypes alone and in combination have been linked with clinical outcome.

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the possible involvement of Fe(2)O(3) in benzo[a]pyrene (BaP)/Fe(2)O(3) mixtures on the induction of CYP1A1 enzymes in the lung. Male Sprague-Dawley rats were instilled intratracheally with haematite ((56)Fe(2)O(3) or (54)Fe(2)O(3), 3 mg), BaP (3 mg) or BaP (3 mg) coated onto haematite ((56)Fe(2)O(3) or (54)Fe(2)O(3)) particles (3 mg). Firstly, mRNA expressions of cyp1a1 were studied. Secondly, protein concentrations and catalytic activities (7-ethoxyresorufin O-deethylase: EROD) of CYP1A1 were determined. Thirdly, (54)Fe from BaP/(54)Fe(2)O(3) mixtures in microsomal proteins was studied using time-of- flight laser microprobe mass spectrometry (ToF-LMMS). Statistically significant increases in mRNA expressions, protein concentrations and catalytic activities of CYP1A1 were observed in animals exposed to BaP, to BaP coated onto (56)Fe(2)O(3) particles or to BaP coated onto (54)Fe(2)O(3) particles versus controls. Both of the BaP/Fe(2)O(3) mixtures induced higher CYP1A1 protein levels and EROD activities than BaP alone. Iron oxide particles per se did not affect mRNA levels of cyp1a1 but only enhanced BaP-mediated increases of CYP1A1 protein levels and activity. The ToF-LMMS spectrum pro fi les showed that the (54)Fe/(56)Fe ratio in the microsomes of BaP coated onto (54)Fe(2)O(3) particle-instilled animals was 1.3 instead of the theoretical ratio (i.e. 0.063) observed in BaP coated onto (56)Fe(2)O(3) particle-instilled animals. Taken together, these novel data support the hypothesis that the Fe(2)O(3)-induced increases of the metabolic activation of BaP might rely on the property of Fe(2)O(3) particles to enhance the BaP-induced translation rate of the cyp1a1 gene into functional haemoproteins.

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etSeO were synthesized and characterized by 1H-NMR and ESI/MS. These reference compounds were then used to develop a sensitive HPLC assay to monitor SeMet oxidation to MetSeO. The formation of MetSeO in rat liver microsomes was time-, protein concentration-, SeMet concentration-, and NADPH-dependent. The microsomal activity exhibited a SeMet Km value (mean +/- S.D.; n = 4) of 0.91 +/- 0.29 mM and a Vmax value of 44 +/- 8.0 nmol MetSeO/mg protein/min. The inclusion of 1-benzylimidazole, superoxide dismutase, or deferoxamine caused no inhibition of the rat liver microsomal activity. Because these results suggested the involvement of FMOs in the oxidation of SeMet in rat liver microsomes, the formation of MetSeO was also examined using cDNA-expressed human and purified rat FMOs. The results showed that both rat and human FMO1 and FMO3 but not FMO5 can catalyze the reaction. The SeMet kinetic constants were obtained with purified rat liver FMO3 (Km = 0.11 mM, Vmax = 280 nmol/mg protein/min) and rat liver FMO1 (Km = 7.8 mM, Vmax = 1200 nmol/mg protein/min). Because SeMet has anti-cancer, chemopreventive, and toxic properties, the kinetic results suggest that FMO3 is likely to play a role in the biological activities of SeMet at low exposure conditions.

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olyacrylamide electrophoresis, mitochondrial protein bands appeared, especially in the range of molecular weight 60 kDa and higher, whereas protein bands disappeared in the 20-40 kDa range on the 4th day after paraquat exposure. The protein profile was normalized on the 7th day and no remarkable changes were seen thereafter up to the 56th day. The changes seen during the observation period were thought to reflect the course of paraquat-induced dysfunction and subsequent repair. The malondialdehyde concentration in mitochondria decreased until the 7th day but subsequently increased and recovered to normal levels by the 56th day. The relative density of HSP60 increased until the 7th day but subsequently decreased and recovered to normal levels by the 56th day. These two parameters therefore showed symmetrical changes. The change in the malondialdehyde concentration was thought to reflect the course of activation of the antioxidation function in mitochondria and the progression of repair. The change in the relative density of HSP60 was thought to have increased to repair the proteins affected by the paraquat radical and to have normalized with the progression of healing. These results suggest that HSP60 may play an important role in preventing the progression of pulmonary fibrosis induced by paraquat.

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ishable from rat liver cytosolic sEH. Cytosol from rat liver or inflammatory cells and recombinant rat sEH were incubated with trans-diphenylpropene oxide (tDPPO), a selective substrate for sEH. The tDPPO hydration activity we observed in inflammatory cell cytosol was lower than that from liver. The Km for tDPPO hydration observed in rat inflammatory cell cytosol was the same as the Km for rat liver cytosol (10 microM). Recombinant rat sEH and cytosol from rat liver or inflammatory cells were incubated with the sEH inhibitors, chalcone oxide, 4-fluorochalcone oxide, and 4-phenylchalcone oxide. The IC50 values were 40, 8, and 0.4 microM, respectively, in all samples. Furthermore, sEH activity could be completely immunoprecipitated out of the samples, and the amount of antibody required to do so was apparently identical, regardless of the source of enzyme. SDS-polyacrylamide gel electrophoresis followed by Western blot analysis revealed a single sEH band with a molecular weight of 62 kDa. Isoelectric focusing followed by Western blot analysis revealed multiple bands containing tDPPO-hydrating activity. Although the inflammatory cell bands had the same pattern as those from liver cytosol, the recombinant sEH showed a different banding pattern. These multiple bands were not an artifact of the IEF gel selected. Furthermore, in a 2-dimensional IEF gel, the bands re-migrated to the same position. The presence of sEH in inflammatory cells suggests that this enzyme may have an important endogenous function.

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< 0.05) were augmented in purified brush borders, whereas sucrase, trehlase (P < 0.01) and glutamyl transpeptidase (P < 0.05) were reduced in nickel fed animals compared with controls. Kinetic and heat inactivation studies with brush border sucrase and alkaline phosphatase confirmed these findings. Western blot analysis of alkaline phosphatase showed a strong signal for the enzyme protein but a reduced level of sucrase antigen in nickel fed rat intestine compared with the controls. These findings suggest that the expression of various brush border enzymes along the crypt-villus axis is modulated in rat intestine exposed to nickel, which may disrupt the digestive functions of the intestinal tissue.

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n of statins. We studied the effects of fluvastatin (Lescol), a member of the statin family, on hepatic protein regulation. Male F344 rats treated with 0.8 mg/kg per day fluvastatin or 24 mg/kg per day fluvastatin for 7 days showed treatment-related changes in 58 liver proteins (P<0.005). Major effects were evident in the cholesterol biosynthesis pathway including the induction of enzymes upstream and downstream of the target enzyme HMG CoA reductase. Treatment also triggered alterations in key enzymes of carbohydrate metabolism and was associated with changes in a heterogeneous set of cellular stress proteins involved in cytoskeletal structure, calcium homeostasis and protease activity. The latter set of protein alterations indicates that hepatotoxicity is associated with high-dose treatment. Based on the results it is suggested that HMG-CoA synthase and isopentenyl-diphosphate delta-isomerase may be explored as alternative drug targets and that the induction levels of these enzymes may serve as a measure of potency of individual statin drugs. It is proposed that efficacy and cellular stress markers discovered in this study may be used in a high throughput screen (HTS) assay format to compare efficiently and accurately the therapeutic windows of different members of the statin family.

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ne-targeted oxidation of protein. In addition, using ebselen as an electrophilic probe, we characterized the cysteine residues required for posttranslational modification into an electrophile sensor protein in the phase 2 detoxification response. Ebselen showed a potent antioxidant effect against the spontaneous and 4-hydroxy-2-nonenal-stimulated production of intracellular reactive oxygen species in rat liver epithelial RL34 cells. Meanwhile, upon in vitro incubation with a redox-active sulfhydryl protein (thioredoxin), ebselen showed a strong electrophilic potential of mediating the formation of selenenylsulfide and intra- and intermolecular disulfide linkages within the protein. By taking advantage of this antioxidant and electrophilic property of ebselen, we characterized posttranslational modification of Kelch-like ECH-associated protein 1 (Keap1), an electrophile sensor protein, which represses the ability of the transcription factor NF-E2-related factor 2 (Nrf2) upon induction of the phase 2 detoxification response. Ebselen potently induced the gene expression of a series of phase 2 enzymes in rat liver epithelial RL34 cells, which was associated with the formation of a high molecular weight complex of Keap1. Furthermore, a cysteine residue in Keap1, C151, was found to be uniquely required not only for the formation of the complex but also for the induction of the phase 2 response by ebselen. Thus, this unique antioxidant and electrophilic property of ebselen giving rise to the cysteine-targeted oxidation enabled us to evaluate the role of sensor cysteines in redox regulation of protein function under electrophile stress.

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llantoic placenta over the course of pregnancy. Three methods have been employed: real-time RT-PCR, western blotting and immunohistochemistry. The expression of mdr1a and mdr1b genes was demonstrated as early as on the 11th gestation day (gd) and increased with advancing gestation. Western blotting analysis revealed the presence of P-gp in the rat placenta starting from gd 13 onwards. P-gp was localized in the developing labyrinth zone of the placenta on gd 13; from gd 15 up to the term P-gp was seen as a dot like continuous line in the syncytiotrophoblast layers. Our data confirm the presence of P-gp in the rat chorioallantoic placenta starting soon after its development, which may signify the involvement of P-gp in transplacental pharmacokinetics during the whole period of placental maturing.

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ion (1.85 mM), the order was rat (4.21 +/- 0.72) > mouse (2.72 +/- 0.11) > human (1.91 +/- 0.84). Kinetic analysis indicated the presence of at least two forms of styrene-metabolizing cytochrome P450s with different Km values in human liver microsomes. Styrene was also metabolized in human lung microsomes: the rate of styrene glycol formation was higher in the lung microsomes from smokers than in those from current nonsmokers. The P450 forms responsible for transforming styrene to styrene glycol were determined by analyzing cDNA-expressed individual P450 forms produced in cultured hepatoma G2 cells by recombinant vaccinia viruses. Of the 12 human P450 forms studied, CYP2B6 and CYP2E1 existing in human liver and/or lungs and CYP2F1 in human lungs were the most active in the forming of styrene glycol, followed by CYP1A2 and CYP2C8. Human CYP3A3, CYP3A4, CYP3A5, and CYP4B1 also catalyzed the metabolism but were much less active. CYP2A6, CYP2C9, and CYP2D6 had only a little detectable activity. CYP1A2, CYP2B6, CYP2C8, CYP2E1, and CYP3A4/3A3 were expressed in human liver microsomes, and CYP2C8 was expressed in human lung microsomes, although the expression of CYP2F1 and CYP4B1 could not be investigated. These data indicate that several human hepatic and/or pulmonary P450 forms are capable of metabolizing styrene, albeit at different rates.

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is not clear whether the MPP(+)-induced overproduction of ROS and RNS occurs in the intraneuronal and/or extracellular compartment. Early steps in the neurotoxic mechanism evoked by MPP(+) include a profound dopaminergic energy impairment, which mediates a massive release of dopamine (DA), glutathione (GSH), and cysteine (CySH). In the event that MPP(+) mediates extracellular generation of ROS (such as superoxide and/or hydroxyl radicals) and/or peroxynitrite, released DA, GSH, and CySH should be oxidized forming thioethers of DA and disulfides. Using microdialysis experiments in which MPP(+) was perfused into the striatum of awake rats, the present study was unable to detect the presence of such biomarkers of extracellular ROS and/or RNS generation. However, MPP(+) induced a transient, concentration-dependent rise of extracellular l-3,4-dihydroxyphenylalanine (l-DOPA), identified on the basis of dialysate analysis using several HPLC methods and its conversion to DA by purified l-DOPA decarboxylase (DDC). Methamphetamine (30 mg/kg, i.p.) similarly caused a significant but transient rise of l-DOPA in the rat striatum. Antioxidants such as salicylate and mannitol had no effect on the MPP(+)-mediated elevation of extracellular l-DOPA, suggesting that it is not formed by nonenzymatic hydroxylation of l-tyrosine by ROS or RNS. Rather, in vivo, but not in vitro, MPP(+) caused rapid inhibition of DDC, which appears to result in intraneuronal accumulation and subsequent release of l-DOPA. Because l-DOPA can mediate l-glutamate release, as well as be an excitotoxin, the possibility is raised that l-DOPA may play a role in the dopaminergic neurotoxicity of MPP(+).

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d DBP in corn oil orally at 0, 250, 500, 1000, and 2000 mg/kg daily for 30 days. Serum T and GC were measured by radioimmunoassay and enzyme-linked immunosorbent assay, respectively. The responses, including glucocorticoid receptor (GR), type I 11beta-hydroxysteroid dehydrogenase (11beta-HSD1), and steroidogenesis acute regulatory protein (StAR) in the testes tissues, were determined by Western blotting and reverse transcriptase PCR. DBP exposure resulted in testicular toxicity, such as seminiferous tubule degeneration and a decrease in the number of spermatogenic cells. T was decreased and GC was increased in a DBP concentration-dependent manner in the exposure group. The expression of GR and 11beta-HSD1 was significantly increased, with an associated decrease in expression of StAR. Neither the expression of the GR nor 11beta-HSD1 and StAR were statistically significantly different in the postexposure group compared with the control. However, the weight and morphology of the testes did not recover in the postexposure group. These data suggest that DBP inhibits testosterone production through a GC-mediated pathway in prepubertal male rats, and after exposure to DBP ceases, testosterone biosynthesis returns.

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f the antioxidant response are induced in rodent models of nonalcoholic fatty liver disease. To simulate simple fatty liver and NASH, respectively, male Sprague-Dawley rats were fed a high-fat (HF) or a methionine and choline-deficient (MCD) diet for 8 weeks. Key marker genes of the antioxidant response that are known to undergo upregulation via activation of Nuclear Factor Erythroid 2-Related Factor 2 were measured using the branched DNA signal amplification assay. Messenger RNA levels of the antioxidant response, including NAD(P)H:quinone oxidoreductase-1 (Nqo1), Glutamate cysteine ligase catalytic (Gclc), and Heme oxygenase-1 (Ho-1), were significantly induced in MCD rat liver but not in HF rat liver. Furthermore, Nqo1 protein expression and activity underwent significant upregulation in MCD rat liver but not in HF rat liver. These data strongly indicate that the pathology induced by the MCD dietary model of NASH results in upregulation of the antioxidant response in rats.

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g(-1) b.w.). A quantitative immunoblotting procedure was applied to determine the levels of GR, Hsp90 and two nucleocytoplasmic Hsp70 isoforms (constitutive Hsp73 and inducible Hsp72) in the renal cytosol, as well as the amounts of these proteins within GR heterocomplexes immunoprecipitated by anti-GR antibody. Mercury was found to stimulate GR association with all the examined Hsps. The most prominent effect of the metal was stimulation of Hsp72 interaction with GR. On the other hand, the metal administration led to an increase of Hsp90 level in the cytosol, while the cytosolic levels of Hsp70 isoforms remained unaltered. These findings suggest that association of Hsps, at least Hsp70, with the GR might be ascribed to changes in the affinity of their interaction rather than to changes in the Hsp availability in the cytosol. Therefore, GR heterocomplex assembly seems to be a controlled process enabling chaperoning and functioning of the GR to be in concert with physiological demands.

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igase (GLCL) catalytic subunit (GLCLC) to transfect Chinese hamster ovary (CHO) cells. CHO cell lines (CHOhi) with stably enhanced GLCL activities (14.61+/-0.82 mU/mg protein) and greater GSH contents (45.7+/-1.37 nmol/mg protein) than observed in wild-type CHO K1 cells (0.26+/-0.01 mU/mg protein and 20.7+/-1.15 nmol/mg protein, respectively) were developed and were confirmed to have integrated the GLCLC cDNA into their genomic DNA and to exhibit increased GLCLC mRNA levels, by Southern and northern analyses, respectively. Similarly treated and selected CHO cell lines that showed no increases in GLCL activities (CHOun) were studied as controls for the effects of GLCLC transgene expression. CHOhi cells showed significantly greater resistance to oxidant stress caused by exposure to tert-butyl hydroperoxide (tBuOOH) than did CHO or CHOun cells. Twenty-four hours after exposure to 400 or 800 microM tBuOOH, wild-type CHO cells had released more cellular lactate dehydrogenase (67.3+/-14.5% and 94.4+/-2%) than had CHOhi cells (5.11+/-0.5% and 46.0+/-5.4%, n=4, P<0.05). The present data demonstrate improved resistance to oxidant injury of CHO cells stably transfected with the GLCLC cDNA. Although additional enhancements in GLCL activities are possible by transfection with cDNAs for both catalytic and regulatory GLCL subunits, our results demonstrate that the increases in GLCL activities that can be attained by transfection of the GLCLC cDNA alone can enhance cellular antioxidant defense function.

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tudy period, the kidneys were examined histopathologically. Concentrations up to and including 50 ppm did not induce any adverse effect. At 250 ppm, growth of male and female animals was markedly retarded. Significantly increased activities of the cytosolic phosphohexose isomerase were excreted by males and females receiving 250 ppm at all timepoints from week 13. The values of the mitochondrial glutamate dehydrogenase were mostly elevated from week 1 to 57, however, due to a wide scatter range, were only occasionally significantly different from control values. The brush border enzymes (gamma-glutamyl transferase, alkaline phosphatase and leucine arylamidase) were not changed in a relevant manner in female rats, while in 250 ppm males the excreted activity of ALP and LAP from week 1 to week 18, and that of GGT during the entire study period were significantly lower than the control values. Excretion of the lysosomal enzymes aryl sulfatase A, beta-galactosidase, and beta-N-acetyl-D-glucosaminidase was at no time influenced in a noteworthy manner. Histopathology after 72 weeks revealed chronic but also acute degenerative changes in the kidneys of 250 ppm males and females. A comparison of published data on persons having undergone high cadmium exposure with the results presented here shows remarkable differences.

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esticular changes and began to explore the possible mechanisms involved. Morphological examination revealed the presence of many abnormally large, multi-nucleated germ cells by 24 h posttreatment with DEHP and with its metabolite, mono-ethylhexyl phthalate (MEHP), but not with another metabolite, 2-ethylhexanol (2-EH; all at 1.28 mmol/kg) or with vehicle alone. These cells persisted through 48 h posttreatment, the longest time point examined in our study. We also assessed the rate of Sertoli cell proliferation in pups at intervals after dosage with either chemical or vehicle by administering bromodeoxy uridine (BrdU) 3 h before euthanasia. By 24 h after treatment with DEHP or MEHP, but not 2-EH or vehicle, the number of BrdU-labeled Sertoli cells was obviously diminished in testicular sections. Quantitation of DEHP-treated pups and controls indicates that a dose-response relationship exists between chemical treatment and labeling index (LI) of Sertoli cells, with a LI at the highest DEHP dose tested that was only 20% of that in controls. In addition, when we examined the time course of the effect of an intermediate dose of DEHP, we found that there the LI of Sertoli cells rebounds by 48 h after dosage, when we found the rate of proliferation in treated pups to be significantly higher than in controls. We also explored the potential mechanism involved in the response to DEHP and found serum levels of FSH to be unaffected by the chemical. In addition, study of cell cycle-related proteins including p27kip1 and cyclins D1, D2, and D3 with Western and Northern analysis indicated that cyclin D2 mRNA is specifically down-regulated by DEHP in a dose-dependent manner, and this decrease is manifest as a small, transient but reproducible reduction in the amount of cyclin D2 protein detectable in samples from treated pups compared to controls. Our findings characterize the changes in neonatal Sertoli cells and gonocytes that follow in vivo to low levels of DEHP and its metabolite, MEHP, as well as providing new information on the underlying mechanism and highlighting the extreme sensitivity of the neonatal testis to injury by this toxicant.

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the NQO1 gene, which encodes a NAD(P)H quinone dehydrogenase and the Calumenin gene between bromadiolone-resistant and anticoagulant-susceptible rats upon saline and bromadiolone administration. Additionally, we established the effect of bromadiolone on the gene expression in the resistant and susceptible phenotype. Bromadiolone had no effect on VKORC1 and NQO1 expression in resistant rats, but induced significantly Calumenin expression in the susceptible rats. Calumenin expression was similar between the resistant and the susceptible rats upon saline administration but twofold lower in resistant rats after bromadiolone treatment. These results indicate that Danish bromadiolone resistance does not involve an overexpression of calumenin. Independent of the treatment, we observed a low VKORC1 expression in resistant rats, which in conjugation with the Y139C polymorphism most likely explains the low VKOR activity and the enhanced need for vitamin K observed in Danish resistant rats. Furthermore the bromadiolone resistance was found to be associated with a low expression of the NQO1 gene.

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x of inflammatory cells into the renal interstitium is mediated by adhesion molecules. In this study, the development of fibrosis in the UUO model of the rat was examined and its relation to the time course of LFA-1 and ICAM-1 expression was assessed by immunohistochemistry. An increase in interstitial connective tissue was detected on day 10 after UUO, with a maximum on day 35. After unilateral ureteral obstruction, LFA-1 was prominently expressed in interstitial infiltrates, and to a lesser degree in glomerular areas. An initial increase in LFA-1-positive cells was noted already on day 10, with a maximum on day 20 and a decline on day 25. During the time course of 35 days after UUO, we observed an increase in ICAM-1 expression in the vascular endothelium, in tubular epithelium and in interstitial areas. This study shows that LFA-1 expression and ICAM-1 expression are concordant and that this process is associated with increasing interstitial fibrosis. ICAM-1 interstitial tissue may facilitate the homing and persistence of an interstitial infiltrate by ICAM-1/LFA-1 interactions, thereby preceding the development of renal interstitial fibrosis.

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tively. After a 3-day total food deprivation delta-aminolevulinic acid dehydratase activity remained unchanged in red blood cells, but an increase by 52% was found in the liver. The erythrocyte activity of porphobilinogen deaminase was reduced by 42% and its hepatic activity--by 16%.

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he effect of Co, WC, and WC-Co particles on the release by lung phagocytes of interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), fibronectin, and cystatin-c. The responses were compared with those induced by two other lung toxicants, i.e., crystalline silica (DQ12) and arsenic trioxide (As2O3). IL-1 and TNF-alpha activities produced in the presence and absence of LPS stimulation were measured with the aid of bioassays while fibronectin and cystatin-c were determined by latex immunoassays. In vitro, maximal noncytotoxic doses of As2O3, Co, WC, or WC-Co did not significantly affect the production of these mediators by rat alveolar macrophages. In contrast, DQ12 enhanced the production of TNF-alpha (with and without LPS stimulation) and IL-1 (after LPS stimulation) and decreased cystatin-c release (in the absence of LPS). Following a single intratracheal instillation of the different test preparations in the rat, the response of the lung phagocytes obtained by bronchoalveolar lavage (BAL) 24 hr later was examined. We were unable to detect any consistent effect of Co (0.06 mg/100 g body wt), WC (1 mg/100 g body wt), or WC-Co treatment (1 mg/100 g body wt) on the production of the above mediators. In contrast, after LPS stimulation, As2O3 (0.5 mg/100 g body wt) and DQ12 (1 mg/100 g body wt) stimulated the production of TNF-alpha and IL-1. In the absence of LPS, As2O3 stimulated fibronectin and cystatin-c production and DQ12 stimulated cystatin-c release. Since the dose of WC-Co used in vivo (1 mg/100 g body wt) caused pronounced lung inflammation (increased LDH, protein, and albumin levels in BAL fluid), we conclude that the acute lung toxicity of WC-Co particles is not mediated through enhanced production of the examined mediators by lung phagocytes.

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been studied in more detail. A significant fraction of genes in most organisms and tissues is targets of these small ncRNAs. Because these tiny RNAs are turning out to be important regulators of gene and genome expression, their aberrant expression profiles are expected to be associated with cellular dysfunction and disease. In fact, an ever-increasing number of studies have implicated miRNAs and siRNAs in human health and disease ranging from metabolic disorders to diseases of various organ systems as well as various forms of cancer. Nevertheless, despite the flurry of research on these small ncRNAs, many aspects of their biology still remain to be understood. The following discussion focuses on some aspects of the biogenesis and function of small ncRNAs with major emphasis on miRNAs since these are the most widespread endogenous small ncRNAs that have been called "micromanagers" of gene expression. Their emerging significance in toxicology is also discussed.

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unknown. Because hypophysectomy leads to a depletion of multiple pituitary hormones besides GH, a study was designed to investigate the possible involvement of GH in EB-mediated hepatic P450 expression using GH-deficient dwarf rats as a more specific animal model. In these rats, pituitary GH was selectively reduced to about 10% of normal levels and other pituitary trophic hormones including thyroid-stimulating hormone, adrenocorticotropic hormone, luteinizing hormone, follicle-stimulating hormone, and prolactin are largely unchanged. Male control and HsdOla:DWARF-dw-4 (Harlan, UK) rats were subjected to a single ip injection of EB (10 mmol/kg). CYP2E1- and CYP2B-dependent activities, protein, and RNA levels were measured 10 and 24 h afterward. The results indicated that dwarf rats without EB exposure expressed higher CYP2E1. Although EB treatment induced CYP2E1 activity, protein, and mRNA both in controls and dwarf rats, the magnitude of the response to EB exposure was greater 10 h after the treatment in dwarf rats. Hypophysectomy also increased CYP2E1 protein induction by EB compared to intact rats. This effect was reversed by GH supplementation to hypophysectomized rats. Overall, responses of CYP2B to EB exposure in dwarf rats did not display basic differences from controls. In conclusion, the results demonstrate that (1) the suppression of CYP2B induction found in the multi-hormone-deficient HX rats is not found in the more specific GH-deficient rat model, confirming that GH does not have a major influence on CYP2B expression and (2) both hypophysectomized and GH-deficient rats show an altered inducibility of CYP2E1 after EB treatment.

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searched for protein abundance changes induced by Mn exposure. The electrophoretic separation resolved over 300 distinct spots as visualized by colloidal Coomassie blue (CCB), of which three spots were induced and three spots were inhibited after Mn exposure in all the five brain mitochondria preparations. Analysis by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) indicated that these spots are calcium-transporting ATPase type 2C (ATP-dependent Ca(2+) pump PMR1); 60-kDa heat shock protein; Mitochondrial transmembrane GTPase FZO1B; ATP-binding cassette, sub-family b; Long-chain-fatty-acid-CoA ligase; ATP Synthase Beta Chain; and Succinate dehydrogenase flavoprotein subunit. The changes of the mitochondrial ATP synthase beta-subunit and Succinate dehydrogenase flavoprotein subunit indicate an effected level of mitochondrial ATP content and/or ATP-producing capacity. This result provides suggestion that respiratory chain complexes were implicated in the mitochondrial dysfunction induced by Mn intoxicity. And the changes of 60-kDa heat shock protein and ATP-dependent Ca(2+) pump PMR1 expression indicate that the Ca homeostasis and stress effect were involved in Mn intoxicity.

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mediated by GST in different species as well as the isoform specificity and reaction products of the GST pathway. Conjugation activity of CHBrCl(2) with GSH in mouse liver cytosol was time- and protein-dependent, was not inhibited by the GST alpha, mu and pi inhibitor S-hexyl-GSH, and correlated with GST T1-1 activity toward the substrate 1,2-epoxy-3-(4'-nitrophenoxy)propane. Conjugation activities in hepatic cytosols of different species toward CHBrCl(2) followed the order mouse > rat > human. As compared with CH(2)Cl(2), the catalytic efficiency (k(cat)/K(m)) of conjugation of CHBrCl(2) with GSH by pure recombinant rat GST T1-1 was approximately 3-6-fold less. Taken together, this suggests that GST T1-1 is the primary catalyst for conjugation of CHBrCl(2) with GSH and that flux through this pathway is less than for CH(2)Cl(2). The initial GSCHCl(2) conjugate formed was unstable and degraded to several metabolites, including GSCH(2)OH, S-formyl-GSH, and HCOOH. Addition of NAD(+) to cytosol did not alter the rate of conjugation of CHBrCl(2) with GSH; however, it did increase the amount of [(14)C]HCOOH produced ( approximately 10-fold). A similar result was seen in a reaction containing pure rat GST T1-1 and GSH-dependent formaldehyde dehydrogenase, indicating that GSCH(2)OH was formed as a precursor to S-formyl-GSH. The half-life of synthetic S-formyl-GSH in pH 7.4 buffer was approximately 1 h at ambient temperature and decreased to approximately 7 min in pH 9.0 buffer, and it does not react with deoxyguanosine. In conclusion, GST T1-1 conjugation of CHBrCl(2) has been definitively demonstrated and the kinetics of conjugation of CHBrCl(2) with GSH characterized in mouse, rat, and human hepatic cytosols. The significance of this GST pathway is that reactive GSH conjugates are produced resulting in possible formation of DNA adducts. Comparisons with CH(2)Cl(2) suggest that the reactive intermediates specific to GSH conjugation of CHBrCl(2) are more mutagenic/genotoxic than those derived from CH(2)Cl(2).

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0). Rats in infection group were infected with 5 x 10(4) tachyzoites by intraperitoneal injection, while those in control group received same volume of PBS. On the 3rd, 6th, 9th and 12th day post infection, ten rats from infection group and two from control group were sacrificed, the thymus glands were removed. The thymus tissue sections were stained with hematoxylin and eosin (HE) for observation on histopathological changes. Single thymus cell suspensions were prepared. Cell cycle analysis was performed by flow cytometry, and proliferation index was calculated. Thymus frozen sections were stained with Hoechst 33258, and morphologic changes in apoptotic nuclei were observed under fluorescence microscope. Expression of Bcl-2 and Bax proteins were determined by using immunohistochemistry.
RESULTS: Microscopic examination showed that pathological changes occurred in thymus grand on the 3rd day after infection. The space between connective tissue capsules was widened, cells in cortex and medulla cells were sparse, and more phagocytes and extravasated blood were found in thymus. On the 6th day post infection the thymus damage was aggravated, and no significant improvement was seen on day 12. On the 3rd, 6th, 9th and 12th day after infection, thymocyte proliferation index was (11.15 +/- 0.99)%, (6.17 +/- 1.02)%, (5.45 +/- 0.96)% and (6.63 +/- 1.52)%, respectively, and each of them was significantly lower than that of the control [(13.81 +/- 1.18)%] (P < 0.01). On the 3rd day after infection, the number of apoptotic cells increased, significantly increased on day 6, and there was no much difference in the number of apoptotic cells between day 6 and day 12. The immunohistochemistry results showed that on the 3rd, 6th, 9th and 12th day post-infection, the gray scale value of Bax positive cells was 88.21 +/- 4.74, 64.69 +/- 6.82, 83.62 +/- 5.79, and 101.09 +/- 6.72, respectively, and each of them was significantly lower than that of the control (128.69 +/- 8.95) (P < 0.01), while there was no significant change in the Bcl-2 protein level (P > 0.05).
CONCLUSION: T. gondii causes severe pathological damage in host thymus tissue with a decrease in the proliferation index, an increase in the number of apoptotic cells, and high expression of Bax protein.

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nase, and kynurenine aminotransferases) in their tissues were determined. The results showed that exposure to carbon disulfide caused a significant increase in the activities of kynureninase and kynurenine-2-oxoglutarate aminotransferase in the kidneys, but only a slight increase in their activities in the liver. The activities of L-tryptophan 2,3-dioxygenase and kynurenine 3-hydroxylase also tended to increase, but the increases were not statistically significant. These results suggests that the kynurenine pathway of tryptophan metabolism in the kidneys of rats exposed to carbon disulfide is activated and that the increased activities of kynurenine-2-oxoglutarate aminotransferase in the kidneys may cause the increased excretion of tryptophan metabolites after tryptophan loading as shown in an earlier study (A. Okayama, L. Fun, A. Yamatodani, Y. Ogawa, H. Wada, and S. Goto, 1987, Arch. Toxicol. 60, 460-463.

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lymerase chain reaction (RT-PCR) in male and female rats. Significant increases in the gene expression level of SREBP-2, a transcription factor for the HMGR gene, occurred at 6-12 h in male and at 24-36 h in female rats after LN-treatment. The gene expression level of HMGR, a rate-limiting enzyme for cholesterol biosynthesis, significantly increased at 3-48 h in male rats and 12-48 h in female rats. Subsequently, significant increases in the amount of hepatic total cholesterol in male and female rats were also observed at 3-48 h and 24-48 h, respectively. The present findings demonstrate that increases in gene expressions of hepatic SREBP-2 and HMGR and the amount of hepatic total cholesterol by LN occur earlier in male rats than in the females, and that increases in the gene expression level of HMGR and the amount of hepatic total cholesterol occur prior to the increase in the gene expression level of SREBP-2 in either sex of rats. Copyright (c) 2006 John Wiley & Sons, Ltd.

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ntly increased the cumulative food intake over 1-, 2- and 4-hr periods. Ad lib feeding decreased significantly two days after pertussis toxin (PT) administration, but recovered to nearly normal levels on the fourth day. PT had no immediate effect on NPY-induced feeding; however, four days after PT was injected NPY (2 micrograms) did not increase the food intake compared to control. In vitro investigations showed that isoproterenol-stimulated adenylate cyclase activity in the hypothalamus of control rats was inhibited by NPY. In PT-treated rats, however, no inhibition of cAMP production was observed. These results suggest that cAMP may mediate NPY-induced feeding and that a PT-sensitive G protein may be involved in this signal transduction.

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-weight:700;'>toxicity is not fully understood. In the present study, the role of the sodium hydrogen exchanger (NHE) in MTX-induced increases in intracellular Ca(2+) and subsequent cell death were investigated in cultured rat cortical neurons. Intracellular Ca(2+) concentrations ([Ca(2+)](i)) were measured fluorimetrically using FURA-2 as the fluorescence indicator. Cell death was measured with the alamarBlue cell viability assay and the vital dye ethidium bromide (EB) uptake assay. Results showed that MTX increased, in a concentration dependent manner, both [Ca(2+)](i) and cell death in cortical neurons. Decreasing the pH of the treatment medium from 7.5 to 6.0 diminished MTX-induced cell death. The protection offered by lowering extracellular pH was not due to MTX degradation, because it was still effective even if the cells were treated with MTX in normal pH and then switched to a lower pH. Pretreatment of cells with the specific NHE inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), prevented MTX-induced increases in [Ca(2+)](i), as well as cell death in a concentration dependent manner. Furthermore, knockdown of NHE1 by SiRNA transfection suppressed MTX-induced cell death in human embryonic kidney (HEK) cells. Together, these results suggest that NHE1 plays a major role in MTX-induced neurotoxicity.

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ly eliciting tumor promoter effects in others. This study examines the disposition of I3C in the pregnant rat model, specifically to determine whether I3C can traverse the maternal placenta, and what effects, if any, are elicited in the neonate. We now report that dietary I3C treatment of pregnant female rats results in appearance of I3C acid condensation products in both maternal and neonatal livers. Livers from I3C-fed maternal rats showed CYP1A1 protein induction; however, no CYP1B1 protein was detected. No CYP1A1 or CYP1B1 protein was detected in the livers of pregnant controls or their offspring. We also report a sex-specific induction of CYP1A1 and CYP1B1 protein in livers from newborns born to I3C-fed dams. CYP1A1 protein was significantly induced in male neonatal liver, but not in females. Conversely, hepatic CYP1B1 protein was induced to high levels in female neonates, with no CYP1B1 protein detected in male littermates. Our results demonstrate that dietary I3C acid condensation products can cross the maternal placenta and differentially induce neonatal hepatic CYP1A1 and CYP1B1 in a sex-specific manner. The results highlight the potential of I3C to effect changes in the overall metabolic profile of xenobiotics to which the fetus is exposed transplacentally and indicate the possible involvement of sex-specific modulators in Ah receptor-mediated responses in this model.

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xposure to certain chemicals, such as 3-methylcholanthrene, benzo[a]pyrene, beta-naphthoflavone, and oltipraz elevates UGT1A6 mRNA in liver and to a lesser extent gastrointestinal tract and kidney, but not in other tissues. The mechanisms underlying this complex pattern of expression have been elusive. We have identified a new type of UGT1A6 mRNA (class 2) that differs in its 5' untranslated sequence. The class 2 transcript is the more abundant type expressed in liver, gastrointestinal tract, and kidney. Transcription of the class 2 mRNA is initiated 107 bases 5' of the UGT1A6 coding exon. The promoter region flanking the transcription start site contains an HNF1-like binding site identical to that in the human UGT1A6 gene. Both class 1 and class 2 mRNAs were elevated in liver by 3-methylcholanthrene, benzo[a]pyrene, beta-naphthoflavone, and oltipraz, with preferential elevation of class 1 occurring after 3-methylcholanthrene and benzo[a]pyrene treatment. These data suggest that transcription from a second promoter contributes to tissue- and inducer-specific expression of rat UGT1A6.

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akes up a significant percentage of these rare adverse effects, with fatalities occurring mainly in children receiving polypharmacy. Previous studies have highlighted the different pharmacological effects of acute valproate exposure, a combination of which are likely to underpin its observed broad-spectrum anticonvulsant efficacy. However, limited studies have been undertaken to investigate the subacute effects of this compound and how genomic effects may underlie the observed hepatotoxic effects. Investigation into the mild hepatoxicity observed in rats exposed to high doses of VPA may provide important information on the human situation. Male Sprague-Dawley rats were dosed with 500 mg/kg/day sodium valproate: after necropsy, mRNA was subjected to suppression PCR subtractive hybridization, identifying 8 up-regulated and 14 down-regulated mRNA species. The down-regulation of several mRNA species coding for enzymes involved in cellular energetics (e.g., succinate dehydrogenase, aldolase B) was of particular interest, as mitochondrial dysfunction is a key feature of valproate hepatotoxicity. In vitro studies were then undertaken to examine the dose and time dependence of these changes and also their effect on the overall energy levels within the cell. We demonstrate that, both in vivo and in vitro, valproate exposure in rats results in a significant decrease in pathways involved in cellular energy homeostasis. These changes may provide insight into the rare human hepatoxicity associated with this compound.

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t exposure of rats to 3-methylcholanthrene (MC) causes sustained induction of hepatic cytochrome P4501A expression for up to 45 days. Here, we tested the hypothesis that MC elicits persistent induction of other genes that are regulated by the Ah receptor (AHR). Female Sprague-Dawley rats were treated with MC (100 micromol/kg) ip once daily for 4 days, and gene expression patterns were investigated using total liver RNA isolated from animals at 1, 15, and 28 days after MC withdrawal. Gene expression was studied by cDNA microarray analyses using 4608 unique clones from liver-derived expressed sequence tag (EST) libraries fortified with clones of known liver genes representing approximately 4000 genes. Several phase I (P4501A1, -1A2) and phase II [e.g., glutathione-S-transferase (GST)-M1, UDP-glucuronosyl transferases (UGT)] genes were persistently induced (3-10-fold) by MC for 15-28 days. The persistent induction of P4501A1 gene expression was confirmed by real time reverse transcriptase polymerase chain reaction (RT-PCR) experiments. MC also elicited a 5-fold persistent augmentation of acute phase genes such as orosomucoid 1 and alpha-1-acid glycoprotein (AGP), and this was accompanied by sustained liver damage and inflammation in the MC-exposed rats. In conclusion, our results strongly suggest that sustained induction of P4501A1 by MC is accompanied by persistent expression of other genes belonging to the Ah gene battery, as well as certain other genes involved in toxic responses. Elucidating the mechanisms of persistent induction of P4501A1 and other genes by MC might lead to a better understanding of the mechanisms of toxicity mediated by PAHs.

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ferase IV increased 3- to 8-fold in >95% O2 treated rat lungs. However, hyperoxic exposure did not change the mRNA and protein levels of aryl sulfotransferase IV in lungs as revealed by Western blot and RT-PCR. This suggests that oxidative regulation occurs at the level of protein modification. The increase of nonprotein soluble thiol and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios in treated lung cytosols correlated well with the aryl sulfotransferase IV activity increase. In vitro, rat liver cytosol 2-naphthol sulfation activity was activated by GSH and inactivated by GSSG. Our results suggest that Cys residue chemical modification is responsible for the in vivo and in vitro oxidative regulation. The molecular modeling structure of aryl sulfotransferase IV supports this conclusion. Our gel filtration chromatography results demonstrated that neither GSH nor GSSG treatment changed the existing aryl sulfotransferase IV dimer status in cytosol, suggesting that oxidative regulation of aryl sulfotransferase IV is not caused by dimer-monomer status change.

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m creatinine (Scr) and the number of TUNEL-positive cells in the outer stripe of the outer medulla in rats, which reached peak levels at 5 days after CDDP. The expressions of cyclin-dependent kinase inhibitors (p21 and p27), cyclin B1, cyclin D1, PCNA, GADD 45, and GADD 153 were significantly increased in the outer medulla, reaching peak levels at 3 days after CDDP. Increments of p27 and PCNA were observed in the same nuclei. Sodium arsenite (SA), a heavy metal, attenuated tubular damage and increased Scr- and TUNEL-positive cells at 5 days after CDDP. SA augmented CDDP-induced increment of p27 but suppressed the increased expression of cyclin B1 and cyclin D1 at 3 days after CDDP. SA-induced attenuation of nephrotoxicity was associated with enhanced expression of proliferating cell nuclear antigen (PCNA) and growth-arrest and DNA damage (GADD) 153 in damaged tubular cells. Our findings indicated that (1) proteins related to cell cycle regulation and DNA repair are induced in CDDP nephrotoxicity, (2) the SA-induced attenuation of CDDP nephrotoxicity is associated with increased expression of p27 and decreased expression of cyclin B1 and cyclin D1, they all induce cell cycle arrest at G1/S and G2/M, and (3) enhanced expression of DNA repair-related proteins is also associated with attenuation of CDDP-nephrotoxicity.

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Performing the counts microscopically on representative hematoxylin and eosin (H&E)-stained sections of ovary is tedious and error-prone. The ability to mark oocyte nuclei distinctly with an antibody significantly increases speed and accuracy of counting. We have identified a rabbit polyclonal antibody directed against a synthetic 14-amino acid sequence from human cytochrome P-450 1B1 (CYP1B1) that unequivocally marks rodent oocyte nuclei, in addition to nuclei of some ovarian granulosa and theca cells. Follicles of all degrees of maturity are easily distinguished from ovarian background; ability to detect and identify primordial follicles is particularly enhanced. High-contrast and high-resolution labeling was achieved with routine immunohistochemical procedures using an avidin-biotin-peroxidase method on rat and mouse tissues fixed in 10% neutral buffered formalin.

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nal protein kinases CaMKI, II, and IV play pivotal roles. Our review focuses on this class of CaM kinases to illustrate the structural and biochemical basis for Ca(2+)/CaM interaction with and regulation of its target enzymes. Gene transcription has been chosen as the functional endpoint to illustrate the recent advances in Ca(2+)/CaM-mediated signal transduction mechanisms.

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cated that mononuclear phagocytes from hamsters, in contrast to those from rats, lack the iNOS pathway. Therefore, the purpose of this study was to investigate whether rats and hamsters differ in lung iNOS expression in vivo upon exposure to asbestos fibers and whether differences in iNOS induction are associated with differences in the acute pulmonary inflammatory reaction. Body weight, alveolar-arterial oxygen difference, differential cell count in bronchoalveolar lavage fluid, total protein leakage, lung myeloperoxidase activity and lipidperoxidation, wet/dry ratio, iNOS mRNA and protein expression, and nitrotyrosine staining of lung tissue were determined 1 and 7 days after intratracheal instillation of asbestos fibers in CD rats and Syrian golden hamsters. Exposure of rats to asbestos fibers resulted in enhanced pulmonary iNOS expression and nitrotyrosine staining together with an acute inflammation that was characterized by an influx of neutrophils, enhanced myeloperoxidase activity and lipid peroxidation, damage of the alveolar-capillary membrane, edema formation, and impairment of gas exchange. In comparison, instillation of asbestos fibers in hamsters resulted in a significantly milder inflammatory reaction of the lung with no induction of iNOS in pulmonary cells. The data obtained provide important information to understand the underlying mechanisms of species differences in the pulmonary response upon exposure to asbestos fibers.

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earlier study, exposure of rats to 4 mg Hg0 vapor/m3, 2 h per day for 10 days, did not produce pathological alterations in the lung but increased metallothionein and glutathione S-transferase in the kidney. This study was undertaken to examine pulmonary gene expression associated with Hg0 vapor inhalation. Total RNA was extracted from lung tissues of rats, previously exposed to air or Hg0 vapor, and subjected to microarray analysis. Hg0 vapor exposure increased the expression of genes encoding inflammatory responses, such as chemokines, tumor necrosis factor-alpha (TNFalpha), TNF-receptor-1, interleukin-2 (IL-2), IL-7, prostaglandin E2 receptor, and heat-shock proteins. As adaptive responses, glutathione S-transferases (GST-pi, mGST1), metallothionein, and thioredoxin peroxidase were all increased in response to Hg exposure. Some transporters, such as multidrug resistance-associated protein (MRP), P-glycoprotein, and zinc transporter ZnT1, were also increased in an attempt to reduce pulmonary Hg load. The expression of transcription factor c-jun/AP-1 and PI3-kinases was suppressed, while the expression of protein kinase-C was increased. Expression of epidermal fatty acid-binding protein was also enhanced. Real-time RT-PCR and Western blot analyses confirmed the microarray results. In summary, genomic analysis revealed an array of gene alterations in response to Hg0 vapor exposure, which could be important for the development of pulmonary adaptation to Hg during Hg0 vapor inhalation.

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span>icant, on S100B protein content in cerebrospinal fluid (CSF) of adult rats. In addition, the performance of animals in an open field (number of squares crossing and rearings) was also analyzed in order to obtain a possible link between alteration in S100B protein content in CSF and parameters related to neurological injury. MeHg treatment increased serum mercury and S100B protein levels in the CSF. A decrease in the numbers of crossings and rearings was observed in MeHg-treated animals when compared to control group, which suggests a possible neurological injury. The present data show, for the first time, increased S100B levels in CSF after exposure to a neurotoxic metal. Authors discuss the possibility of astrocytic involvement in MeHg-induced neurotoxicity.

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otrimeric enzyme that specifically binds to double-strand breaks and phosphorylates its target proteins. Because both DNA breaks and apoptosis are known to occur following irradiation, whole-body irradiation was administered to find out the temporal pattern and dose-response of PARP expression and the activity pattern of DNA-PK. To assess the temporal response, male Wistar rats were subjected to a radiation dose of 3Gy and killed at various time intervals (1-24 hours). Both the PARP activity and expression were enhanced 4 hours after irradiation. Fragmented PARP was not observed until 24 hours after irradiation. The differential expression at DNA-PK various doses (0.1-5.0 Gy) was examined. The maximum expression of PARP was noted at 1 Gy, whereas the activation of DNA-PK was maximally observed at 3 Gy. We did not observe any increase in the expression of PARP until the dose of 3Gy was reached, which contradicted the findings in previous in vitro reports of PARP activation at high radiation doses. DNA-PK, however, showed a dose-dependent increase. Our results indicate that although both the PARP and the DNA-PK are nuclear enzymes with similar roles, the activation of these enzymes is dependent on the dose, and any extrapolation of data from in vitro observations can lead to misinterpretation.

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ations exposed to [14C]2,5-HD. Purified M and H proteins from 2,5-HD-treated NFs were subjected to cyanogen bromide (CNBr) cleavage, and the resultant peptides were separated by Tris-tricine SDS-PAGE and electroblotted to PVDF membranes. Peptides were identified by direct sequencing of stained bands and the relative radiolabeling of each peptide was determined by comparing band intensities in fluorographed blots. For NF-M, the highest label was found in CNBr 10, a peptide corresponding to residues 678-846 at the extreme carboxyl terminus. This region of the protein includes three highly conserved lysine-containing sequences believed to be critical to its function. For NF-H, the greatest binding was localized in CNBr 7 + 8, representing an incomplete cleavage product of residues 390-810. This peptide contains essentially all of the phosphorylation sites in the carboxyl terminus of NF-H, a domain believed to control NF interactions in the axon. Only minor radiolabeling was observed in other M or H peptides. Extensive dephosphorylation of NFs prior to 2,5-HD exposure had no effect on relative adduct levels in each protein. These results provide additional support for limited and specific binding of 2,5-HD to neurofilaments and indicate that the phosphorylation state of the protein may not substantially influence this binding.

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ound to be minimal over the course of nine weeks. In the second part, the reproducibility of cardiovascular response to three successive administration of sotalol, an antihypertensive agent, was studied. In the last part, cardiovascular parameters determined by telemetry were compared to those obtained by direct arterial catheterization and showed a good linear correlation between those two methods.

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y, the effects of PFDoA exposure on testes ultrastructure, testosterone levels, and steroidogenic gene expression were investigated. Male rats were orally dosed for 14 days with 1, 5, or 10 mg PFDoA/kg/day or with vehicle. Absolute testis weight was diminished at the highest dose while relative testes weight was markedly increased at doses of 5 and 10 mg/kg/day. Total serum cholesterol levels were significantly increased at the highest dose. While luteinizing hormone was significantly decreased at the highest dose, testosterone was markedly decreased at doses of 5 and 10 mg PFDoA/kg/day. Serum levels of follicle-stimulating hormone were not significantly affected by PFDoA, and estradiol levels were markedly decreased only at 5 mg/kg/day. Leydig cells, Sertoli cells, and spermatogenic cells from rats that received 5 or 10 mg PFDoA/kg/day, exhibited apoptotic features including dense irregular nuclei, condensed chromatin, ill-defined nuclear membranes, and abnormal mitochondria. PFDoA exposure resulted in significant declines in mRNA expression of several genes involved in cholesterol transport and steroid biosynthesis at doses of 5 and 10 mg PFDoA/kg/day, while the gene expression of luteinizing hormone receptor and aromatase was not significantly changed. Our results demonstrate that PFDoA affects the reproduction function of male rats via alterations in steroidogenesis genes, testosterone levels, and testes ultrastructure.

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scher-344 rats were exposed to PTFE fumes generated by temperatures ranging from 450 to 460 degrees C for 15 min at an exposure concentration of 5 x 10(5) particles/cm3, equivalent to approximately 50 micrograms/m3. Responses were examined 4 hr post-treatment when these rats demonstrated 60-85% neutrophils (PMNs) in their lung lavage. Increases in abundance for messages encoding the antioxidants manganese superoxide dismutase and metallothionein (MT) increased 15- and 40-fold, respectively. For messages encoding the pro- and anti-inflammatory cytokines: inducible nitric oxide synthase, interleukin 1 alpha, 1 beta, and 6 (IL-1 alpha, IL-1 beta, and IL-6), macrophage inflammatory protein-2, and tumor necrosis factor-alpha (TNF alpha) increases of 5-, 5-, 10-, 40-, 40-, and 15-fold were present. Vascular endothelial growth factor, which may play a role in the integrity of the endothelial barrier, was decreased to 20% of controls. In situ sections were hybridized with 33P cRNA probes encoding IL-6, MT, surfactant protein C, and TNF alpha. Increased mRNA abundance for MT and IL-6 was expressed around all airways and interstitial regions with MT and IL-6 demonstrating similar spatial distribution. Large numbers of activated PMNs expressed IL-6, MT, and TNF alpha. Additionally, pulmonary macrophages and epithelial cells were actively involved. These observations support the notion that PTFE fumes containing ultrafine particles initiate a severe inflammatory response at low inhaled particle mass concentrations, which is suggestive of an oxidative injury. Furthermore, PMNs may actively regulate the inflammatory process through cytokine and antioxidant expression.

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acute silicosis. In animal models, histiocytic granulomas develop within days after high-dose intratracheal (IT) silica instillation. However, following chronic inhalation of occupationally relevant doses of silica, discrete granulomas resembling human silicosis arise months after the final exposure without significant lung inflammation/apoptosis. To identify molecular events associated with chronic silicosis, lung RNA samples from controls or subchronic silica-exposed rats were analyzed by Affymetrix at 28 wk after silica exposures. Results suggested a significant upregulation of 144 genes and downregulation of 7 genes. The upregulated genes included complement cascade, chemokines/chemokine receptors, G-protein signaling components, metalloproteases, and genes associated with oxidative stress. To examine the kinetics of gene expression relevant to silicosis, quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), Luminex-bead assays, Western blotting, and/or zymography were performed on lung tissues from 4 d, 28 wk, and intermediate times after subchronic silica exposure and compared with 14-d acute silicosis samples. Results indicated that genes regulating fibrosis (secreted phosphoprotein-1, Ccl2, and Ccl7), redox enzymes (superoxide dismutase-2 and arginase-1), and the enzymatic activities of matrix metalloproteinases 2 and 9 were upregulated in acute and chronic silicosis models. However, proinflammatory cytokines were strongly upregulated only in acute silicosis. Thus, inflammatory cytokines are associated with acute but not chronic silicosis. Data suggest that genes regulating fibrosis, oxidative stress, and metalloproteases may contribute to both acute and chronic silicosis.

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susceptible than the 7-day-old larvae. Resistance to copper increased with age. Low concentrations of 5 and 20 micrograms/liter copper were more inhibitory to 1-day and 7-day old larvae, respectively, than were higher copper levels. The responses of 1-day-old larvae to copper levels from 0 to 20 micrograms/liter and 7-day-old to copper levels from 0 to 100 micrograms/liter copper were complex. Increasing salinity 7.6 to 30.5% reduced both copper toxicity and protozoa/bacteria attacks.

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cell death in rat cerebellar granule cells in vitro using fura-2 single-cell microfluorimetry and viability assays, respectively. The nonspecific muscarinic receptor antagonist atropine significantly delayed the onset of MeHg-induced Ca2+ elevations and reduced the amount of Ca2+ released into the cytosol. Depletion of the smooth endoplasmic reticulum (SER) Ca2+ pool with thapsigargin or down-regulation of muscarinic receptors and inositol-1,3,4-triphosphate (IP3) receptors with bethanechol (BCh) caused similar reductions in the amplitude of the MeHg-induced Ca2+ increase, suggesting that MeHg interacts with muscarinic receptors to cause Ca2+ release from the SER through activation of the IP3 receptors. To determine whether this Ca2+ release plays a role in MeHg-induced cell death, cells were exposed to MeHg in the presence of specific muscarinic receptor inhibitors. Acute exposure to increasing concentrations of MeHg (0.2-1.0 microM) caused a corresponding increase in cell death at 24.5 h post-exposure. Prior down-regulation of muscarinic and IP3receptors with BCh protected against cell death. Protection was ablated by atropine and the M3 receptor antagonist 4-diphenylacetoxyl-N-methylpiperidine methiodide (DAMP), but not by the neuronal nicotinic receptor antagonist dihydro-beta-erythroidine hydrobromide (DHE). Thus activation of M3 muscarinic receptors with subsequent generation of IP3 evidently contributes to elevated [Ca2+]i and subsequent cytotoxicity of cerebellar granule cells by MeHg.

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aldehyde toxicity, early development of aldehyde dehydrogenases (ALDHs) in lung has been poorly studied. The expression of ALDH in liver and lung across ages (postnatal day 1, 8, 22, and 60) was investigated in Wistar-Han rats. In adult, the majority of hepatic ALDH activity was found in mitochondria, while cytosolic ALDH activity was the highest contributor in lung. Total aldehyde oxidation capability in liver increases with age, but stays constant in lung. These overall developmental profiles of ALDH expression in a tissue appear to be determined by the different composition of ALDH isoforms within the tissue and their independent temporal and tissue-specific development. ALDH2 showed the most notable tissue-specific development. Hepatic ALDH2 was increased with age, while the pulmonary form did not. ALDH1 was at its maximum value at postnatal day 1 (PND1) and decreased thereafter both in liver and lung. ALDH3 increased with age in liver and lung, although ALDH3A1 was only detectible in lung. Collectively, the present study indicates that, in the case of aldehyde exposure, the in vivo responses would be tissue and age dependent.

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c)-lesioned rats using the microdialysis technique. We also investigated the effect of acute COX inhibition on the damaged SNc neurons. Our results indicate a significant increase in dopaminergic neurotransmission and a decrease in glutamatergic neurotransmission (P < 0.05) only after selective COX-2 inhibition in the striatum of normal and hemiparkinsonian rats. Nonetheless, neither COX-1 nor COX-2 inhibitors showed any improvement in the damaged SNc neurons.

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c injury. In the present studies, we investigated the molecular mechanism for the neuro-protective effect of Huwentoxin-I (HWTX-I), a spider toxin selectively blocking N-type voltage-dependent Ca((2+)) channel, on rat models with global cerebral ischemia-reperfusion injury. Our studies demonstrated that HWTX-I could maintain the morphological stability of pyramidal cells in this model. Furthermore, HWTX-I could decrease the concentration of malon-dialdehyde, but increase the activity of superoxide dismutase and glutathione peroxidase. It also reduced the expression level of related factors of Fas and tumor necrosis factor death receptor apoptosis pathways in the hippocampus. In summary, HWTX-I has an obvious neuroprotective effect, which may act through its inhibition on a certain apoptosis pathway.

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onstrated that CdO inhalation caused time- and dose-dependent increases in the steady-state levels of gamma-GCS-HS mRNA that were highly correlated with lung Cd burden. Observed increases in gamma-GCS-HS gene expression were maximal 2 h following a single aerosol exposure to Cd and appeared to be triggered by an oxidant stress, characterized by a decline in the reduced to oxidized glutathione ratio. Immunoblotting of proteins in lung extracts from treated and untreated animals produced a single protein band corresponding to a molecular weight of 73 kDa. Elevated levels of gamma-GCS-HS mRNA and gamma-GCS-HS protein in lungs of Cd-exposed animals were also accompanied by higher gamma-GCS enzymatic activity and elevations in glutathione (GSH). Immunohistochemical and in situ hybridization studies were used to identify compartments in the lung where Cd-induced expression of gamma-GCS-HS was localized. The most prominent staining for gamma-GCS-HS protein and gamma-GCS-HS mRNA was observed in the alveolar epithelium of Cd-exposed animals. Quantitative image analysis confirmed a good agreement between relative levels of protein and mRNA transcripts for gamma-GCS-HS. These observations suggest that resistance to Cd toxicity in the lung may reflect the ability of specific lung cells to upregulate gamma-GCS expression and increase de novo GSH synthesis as an adaptive response.

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nge with Penicillium chrysogenum extract (PCE) induces dose-dependent allergic asthma-like responses in mice. The aim of this study was to investigate whether neurotrophins are involved in the PCE-induced allergic airway response in mice. Mice were exposed to 10, 20, 50, or 70 microg PCE by involuntary aspiration 4 times over 1 mo. Bronchial alveolar lavage fluid (BALF) was collected immediately before and after the final exposure. The levels of NGF, NT-3, and NT-4 were determined by enzyme-linked immunosorbent assay (ELISA). The lungs were fixed and processed for immunohistochemical examination of NGF production. PCE-exposed mice had dose-dependent increases in NGF, NT-3, and NT-4 in both BALF and sera. Exposures to PCE produced elevation in positive immunohistochemical staining for NGF in the airway epithelium and smooth muscle cells, in addition to infiltrated cells such as mononuclear cells, eosinophils, and macrophages. Taken together, this is the first study to link fungal allergic asthma in an experimental model with enhanced production of neurotrophins in the airways, and suggests that neurotrophins may play a role in the etiology of mold-induced asthma in humans.

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were investigated in cultured rat embryonic midbrain cells. Twelve-day rat embryo midbrain cells were cultured for 48 h. OTA (0.5 or 1 microg/ml) and/or 1.5-deoxy PGJ2 (0.5 microM) were then added for 48 h. Cell number and neurite outgrowth were determined to assess the neurotoxicity of OTA. AP-1 and NF-kappaB activation was determined by gel mobility shift assay after 3 h of exposure to OTA and/or 15-deoxy PGI2. OTA caused concentration-dependent reductions in neurite outgrowth and cell number, and induced AP-1 and NF-kappaB activation. Cotreatment with 15-deoxy PGJ2 (0..5 microM) blocked OTA-induced decrease in neurite outgrowth and cell number and inhibited AP-1 and NF-kappaB activation. 15-Deoxy PGJ2 (0.5 microM) caused the expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in the cells. Results show that 1.5-deoxy PGJ2 blocked OTA-induced neurotoxicity by inhibiting AP-1 and NF-kappaB activation in cultured rat embryonic midbrain cells.

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often counteracts PKC-dependent effects, can prevent and reverse these alterations. The pro-oxidant compounds tert-butylhydroperoxide (tBOOH, 100 microM) and 2,3-dimethoxy-1,4-naphthoquinone (30 microM), reduced by -41% and -29%, respectively, the percentage of couplets accumulating the fluorescent bile salt analog, cholyl-lysylfluorescein in their canalicular vacuoles (p < 0.01). tBOOH-induced bile salt secretory failure was accompanied by internalization of the canalicular bile salt export pump (Bsep), and disarrangement of cytoskeletal F-actin. All these deleterious effects were fully prevented by the intracellular Ca2+ chelator BAPTA/AM (20 microM), the pan-specific PKC inhibitors H7 (100 microM) and staurosporine (1 microM), the inhibitor of Ca2+-dependent PKCs, Go6976 (2 microM), and the PKA activator dibutyryl-cAMP (500 microM). H7, Go6976, and dibutyryl-cAMP not only prevented but also fully reversed the decrease in the cholyl-lysyl-fluorescein accumulation. In conclusion, these results suggest that low levels of oxidative stress impair bile salt secretion by internalizing Bsep through a Ca2+-dependent, PKC-mediated mechanism, and that inhibition of PKC, or activation of PKA, prevents and reverses these effects. Alterations in actin organization may be a causal factor.

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cidate whether nephropathy could be caused by materials deposited in the kidney. Furthermore, analysis of the renal deposits in rats was also performed. Consequently, interstitial nephritis comprising interstitial inflammatory cell infiltration, dilatation, basophilia and epithelial necrosis of renal tubules and collecting ducts, deposits in renal tubules and collecting ducts, and so forth was seen in six of the eight rats and in all eight rats in the 1 and 3 mg/kg FYX-051 alone groups, respectively, with the intensity in the 3 mg/kg group being moderate to severe. In the simultaneous treatment with citrate group, however, no alterations were observed in the kidney, except for minimal interstitial nephritis in one instance in the 3 mg/kg FYX-051 + citrate group along with an increased urinary pH, leading to an increase in xanthine solubility. Analysis of intrarenal deposits showed that the entity would be composed of xanthine crystals. The present study, therefore, showed that nephropathy in rats occurring after the administration of FYX-051 was a secondary change caused by xanthine crystals being deposited in the kidney, and no other causes could be implicated in this kidney lesion.

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in PP signaling. The ribosomal protein L11 (rpL11), a component of the large 60S subunit, was identified as a PPARalpha-associated protein. Since rpL11 is a regulator of p53 and the cell cycle, the association between this protein and PPARalpha was examined in detail. PPARalpha-rpL11 interaction was confirmed using yeast and mammalian two-hybrid systems as well as in vitro pull-down assays. The association with rpL11 occurs within the D-domain (hinge-region) of PPARalpha. Unlike PPARalpha, the two closely related isoforms PPARbeta and gamma do not interact with rpL11. Cotransfection of mammalian cells with rpL11 resulted in ligand-dependent inhibition of transcriptional activity of PPARalpha. Ribosomal protein L11-mediated inhibition of gene expression is associated with decreased binding to the PPAR-response element (PPRE) DNA sequence. Release of rpL11 from the ribosome by serum deprivation or low-dose actinomycin D did not dramatically affect PPRE-driven luciferase activity when PPARalpha was overexpressed by cotransfection. However, when endogenous levels of PPARalpha are examined and rpL11 concentration is manipulated by expression by small interference RNA, the ability of peroxisome proliferator to induce PPRE-driven reporter activity and target gene mRNA is affected. These studies show that rpL11 inhibits PPARalpha activity and adds further evidence that ribosomal proteins play roles in the control of transcriptional regulation.

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PERK mediates many of the unfolded protein response (UPR)-induced events, including degradation of the type 1 interferon (IFN) receptor IFNAR1 in vitro. Here we report that whole-body or pancreas-specific Perk ablation in mice leads to an increase in IFNAR1 protein levels and signaling in pancreatic tissues. Concurrent IFNAR1 deletion attenuated the loss of PERK-deficient exocrine and endocrine pancreatic tissues and prevented the development of diabetes. Experiments using pancreas-specific Perk knockouts, bone marrow transplantation, and cultured pancreatic islets demonstrated that stabilization of IFNAR1 and the ensuing increased IFN signaling in pancreatic tissues represents a major driver of injury triggered by Perk loss. Neutralization of IFNAR1 prevented pancreatic toxicity of PERK inhibitor, indicating that blocking the IFN pathway can mitigate human genetic disorders associated with PERK deficiency and help the clinical use of PERK inhibitors.

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ood gene expression profiles were determined after latency periods (0-16 weeks). Silica exposure resulted in pulmonary toxicity as evidenced by histological and biochemical changes in the lungs. The number of significantly differentially expressed genes in the blood, identified by microarray analysis, correlated with the severity of silica-induced pulmonary toxicity. Functional analysis of the differentially expressed genes identified activation of inflammatory response as the major biological signal. Induction of pulmonary inflammation, as suggested by the blood gene expression data, was supported by significant increases in the number of macrophages and infiltrating neutrophils as well as the activity of pro-inflammatory chemokines observed in the lungs of the silica-exposed rats. A gene expression signature developed using the blood gene expression data predicted the exposure of rats to lower, minimally toxic and nontoxic concentrations of silica. Taken together, our findings suggest the potential application of peripheral blood gene expression profiling as a minimally invasive surrogate approach to detect pulmonary toxicity induced by silica in the rat. However, further research is required to determine the potential application of our findings specifically to monitor human exposure to silica and the resulting pulmonary effects.

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c acid (MMAs(V)), dimethylarsinic acid (DMAs(V)), and trimethylarsine oxide (TMAs(V)O) in mammals. We examined the effects of chronic exposure to methylated arsenicals on arsenic tolerance by using rat normal liver TRL 1215 cells. TRL 1215 cells were exposed for 20 weeks to MMAs(V), DMAs(V), or TMAs(V)O at levels that produced submicromolar cellular concentrations of arsenic. On chronic exposure to these methylated arsenicals, the cells acquired tolerance to acute arsenic cytolethality. Cellular arsenic uptake was reduced in these cells compared to passage-matched control cells. The long-term arsenic exposure increased glutathione S-transferase (GST) activity and cellular glutathione (GSH) levels. Glutathione S-transferase, multidrug resistance-associated proteins (Mrps; efflux transporters encoded by Mrp genes), and P-glycoprotein [P-gp; efflux transporter encoded by multidrug resistance gene (MDR)] had also increased in these cells at the transcript and protein levels. The depletion of cellular GSH and the inhibition of Mrps and P-gp functions increased cellular arsenic uptake and reduced arsenic tolerance in these cells. These results indicate that chronic exposure to methylated arsenicals induces a generalized arsenic tolerance that is caused by increased arsenic excretion. Because accumulation of methylated arsenicals may occur in patients with chronic arsenic poisoning and arsenic-treated APL patients, this study may provide important information regarding chronic arsenic poisoning and the latent risk of developing multidrug resistance in APL therapy using inorganic arsenite.

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oli cell injury. In this study, we show that germ cells from B6.SMNC3H-Fas(gld,gld) (gld) mice that express a dysfunctional form of FasL still undergo significant apoptosis, albeit at a lower incidence than seen in B6 mice, following mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell injury. In addition, we show the presence of Fas, TRAIL-R1 (Death Receptor-4, DR4), and TRAIL-R2 (DR5) in the testis of C57BL/6 (B6) and gld mice (4 weeks old), and Sprague-Dawley rats (5 weeks old) and their responsiveness after MEHP treatment. More importantly, Western blot analysis of cellular fractions showed an increase in death receptor localization on the membrane fractions taken from Sprague-Dawley rats. Immunohistochemical analysis indicated localization of Fas and DR5 primarily to the spermatocyte subpopulation of germ cells. Examination of downstream receptor-mediated signals (i.e., cleavage of procaspase-8 and NFkappaB activation) revealed an early increase in NFkappaB-DNA binding and an increase in procaspase-8 processing in mutant gld mice. In summary, germ cell-associated death receptors, as well as downstream signaling elements, appear to be responsive to MEHP-induced Sertoli cell injury. Whether this is directly responsible for the increases in germ cell apoptosis after MEHP exposure is yet to be determined. The observed robust and early increase in Fas in wild-type testis and diminished rates of germ cell apoptosis in mutant testis (gld and lpr(cg)) reiterates the importance of the Fas signaling pathway.

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ncentrations in rat plasma were examined after intravenous injection of NP (100 microg/kg) at different time intervals. In addition, rat Leydig cells were challenged with different concentrations of NP (4.25-127.5 microM) to evaluate its influences on testosterone steroidogenesis. Administration of NP showed a decrease of hCG-induced plasma testosterone. Moreover, in vitro experiments revealed that NP (127.5 microM) alone stimulated testosterone release through increase of both protein levels and activities of the StAR and P450(SCC). In contrast, NP inhibited hCG-induced testosterone release in rat Leydig cells. The inhibitory effect was also observed after incubation of the Leydig cells in the presence of different precursors. These results suggested that NP had differential effects on testosterone synthesis.

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ated the effect of TDI in a murine model of TDI-induced asthma. Mice were skin-sensitized with TDI on 2 consecutive days and challenged intranasally 8 days later. Lung expression and activity of CYP were assessed 24 h after the challenge. A significant increase in Cyp1a1 protein expression was detected by western blotting in lung from mice sensitized and challenged with TDI, whereas no modification of expression of other CYP, namely Cyp2b, Cyp2e1 and Cyp3a was observed. Increase in Cyp1a1 protein was associated with an increase in Cyp1a1 mRNA, as assessed by polymerase chain reaction after reverse transcription of total lung RNA. However, a decreased Cyp1a1 activity, as measured by O-deethylation of ethoxyresorufin was observed in lung from TDI-sensitized and challenged mice, suggesting that TDI may inhibit Cyp1a1 function. In agreement with this hypothesis, in vitro experiments conducted on liver microsomes overexpressing Cyp1a1 after treatment of mice with 3-methylcholanthrene showed that TDI markedly inhibited in a concentration-dependent manner Cyp1a1 activity. In conclusion, expression of Cyp1a1, known to exhibit rather negative functions in the lung, is increased in mice sensitized and challenged with TDI. However, this effect is associated with a decreased enzyme activity, which might limit the toxicological consequences of increased Cyp1a1 expression.

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f melittin were investigated on the tyrosine phosphorylation of platelet-derived growth factor (PDGF) beta receptor (Rbeta) and its downstream intracellular signal transduction. When combined with PDGF-Rbeta inhibitor, melittin exhibited a synergic inhibitory effect on PDGF-BB-induced rat aortic VSMC proliferation. In addition, melittin inhibited PDGF-Rbeta phosphorylation in a concentration-dependent manner. Accordingly, the downstream signal transduction of PDGF-Rbeta, such as ERK1/2, Akt, and PLCgamma1 phosphorylation, was also inhibited by melittin in the same manner. These findings suggest that, in addition to suppressing NF-kappaB activation, the antiproliferative effect of melittin in VSMC may be mediated, at least in part, by the inhibition of PDGF-Rbeta tyrosine phosphorylation and its downstream intracellular signal transduction.

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lagen and elastin in the ECM, in conjunction with upregulation of other Cu-binding proteins including Cu,Zn-superoxide dismutase (SOD1), copper chaperone for SOD1 (CCS1), metallothionein (MT), and Menkes P-type ATPase (ATP7A), a Cu transporter in the membrane of the Golgi apparatus, as well as gamma-glutamylcysteine synthetase (gamma-GCS), an enzyme for glutathione biosynthesis. Reduction and loss of cytoplasmic distribution of LO in CdR cells were accompanied by its dislocation with the Menkes P-type ATPase and the endoplasmic reticulum marker. CdR cells displayed a defect in LO catalytic activity but an enhancement in Cu,Zn-SOD catalytic activity consistent with the protein expression levels of these enzymes. Although long-term Cd exposure of cells enhanced the Menkes P-type ATPase protein expression, actually, it reduced Cu-dependent catalytic activity of this enzyme in parallel with the deficiency of LO. The low level of 64Cu bound to the LO fraction and the high level of 64Cu bound to the MT fraction provide direct evidence for limitation of Cu bioavailability for LO existing in the CdR cells. These results suggest that downregulation of LO is linked with upregulation of other Cu-binding proteins and with alteration in Cu homeostasis in the CdR phenotype.

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sm disturbances and the development of experimental acute hepatic porphyria, as well as the role of reactive oxygen species (ROS) through assays on hepatic key gluconeogenic and glycogenolytic enzymes; phosphoenolpyruvate carboxykinase (PEPCK) and glycogen phosphorylase (GP), respectively. Female Wistar rats were treated with three different doses of the porphyrinogenic drug 2-allyl-2-isopropylacetamide (AIA) and with a single dose of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Thus, rats were divided into the following groups: group L (100 mg AIA + 50 mg DDC/kg body wt.); group M (250 mg AIA + 50 mg DDC/kg body wt.) and group H (500 mg AIA + 50 mg DDC/kg body wt.). The control group (group C) only received vehicles (saline solution and corn oil). Acute hepatic porphyria markers ALA-synthase (ALA-S) and ferrochelatase, heme precursors ALA and porphobilinogen (PBG), and oxidative stress markers superoxide dismutase (SOD) and catalase (CAT) were also measured in hepatic tissue. On the other hand, hepatic cytosolic protein carbonyl content, lipid peroxidation and urinary chemiluminescence were determined as in vivo oxidative damage markers. All these parameters were studied in relation to the different doses of AIA/DDC. Results showed that enzymes were affected in a drug-dose-dependent way. PEPCK activity decreased about 30% in group H with respect to groups C and L, whereas GP activity decreased 53 and 38% in group H when compared to groups C and L, respectively. On the other hand, cytosolic protein carbonyl content increased three-fold in group H with respect to group C. A marked increase in urinary chemiluminescence and a definite increase in lipid peroxidation were also detected. The activity of liver antioxidant enzyme SOD showed an induction of about 235% in group H when compared to group C, whereas CAT activity diminished due to heme depletion caused by both drugs. Based on these results, we can speculate that the alterations observed in glucose metabolism enzymes could be partly related to the damage caused by ROS on their enzymatic protein structures, suggesting that they could be also linked to the beneficial role of glucose administration in acute hepatic porphyria cases.

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ly induces apoptosis in olfactory sensory neurons of the nose. The PC-12 rat pheochromocytoma cell model was used to elucidate potential mechanisms of SG-induced neuronal cell death. Agarose gel electrophoresis revealed that exposure to SG at 10 ng/ml or higher for 48-h induced DNA fragmentation characteristic of apoptosis in PC-12 cells. SG-induced apoptosis was confirmed by microscopic morphology, hypodiploid fluorescence and annexin V-fluorescein isothiocyanate (FITC) uptake. Messenger RNA expression of the proapoptotic genes p53, double-stranded RNA-activated protein kinase (PKR), BAX, and caspase-activated DNAse was significantly elevated from 6 to 48 h after SG treatment. SG also induced apoptosis and proapoptotic gene expression in neural growth factor-differentiated PC-12 cells. Although SG-induced caspase-3 activation, caspase inhibition did not impair apoptosis. Moreover, SG induced nuclear translocation of apoptosis-inducing factor (AIF), a known contributor to caspase-independent neuronal cell death. SG-induced apoptosis was not affected by inhibitors of oxidative stress or mitogen-activated protein kinases but was suppressed by the PKR inhibitor C16 and by PKR siRNA transfection. PKR inhibition also blocked SG-induced apoptotic gene expression and AIF translocation but not caspase-3 activation. Taken together, SG-induced apoptosis in PC-12 neuronal cells is mediated by PKR via a caspase-independent pathway possibly involving AIF translocation.

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bjective of this study was to identify alterations in gene expression within the testis and epididymis associated with abnormal Wolffian duct development and to correlate changes in gene expression with the gross morphology of the affected epididymides. Pregnant Sprague-Dawley rats were administered either corn oil vehicle or linuron (50 mg/kg/day) by gavage from GD 12 to 21 (n = 3-6 controls, n = 5-10 linuron-treated dams per time point). Changes in gene expression were evaluated in testes on GD 21 and in epididymides on GD 21 and postnatal day (PND) 7, using cDNA microarrays and confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) analyses. RNA was isolated from intact epididymides with reduced or no ductal coiling from the linuron groups, and epididymides with noncontiguous ducts were excluded. In the fetal testis, exposure to linuron did not result in reduced mRNA expression of the AR or that of several steroidogenic enzymes, supporting the hypothesis that linuron does not reduce fetal testosterone production. Linuron induced a significant decrease in AR mRNA expression in GD 21 epididymides. Significant changes in mRNA expression in GD 21 and PND 7 epididymides were also identified in the epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and Notch signaling pathways. These pathways are involved in tissue morphogenesis. Changes in the expression of AR and IGF-1 receptors were detected by immunostaining in malformed epididymides from linuron-exposed rats. Linuron induced changes in epididymal gene expression suggestive of altered paracrine interactions between the mesenchyme and epithelial cells during development. The EGF, Notch, IGF-1, BMP4, and FGF signaling pathways may be involved in normal testosterone-mediated development of the Wolffian duct.

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crease of the buoyant density of several lysosomal enzymes, indicating lysosomal lipid overload. Biochemical analysis confirmed an increased cellular content of major phospholipids and sphingomyelin, but not of cholesterol. Cocaine, a membrane-permeant weak base, is concentrated by acidotropic sequestration, because its accumulation was abrogated by the proton ionophore, monensin and the vacuolar ATPase inhibitor, bafilomycin A1. At its estimated lysosomal concentration, cocaine almost completely inhibited phospholipase A1 activity on liposomes. Cell incubation with cocaine, but not with its inactive metabolite, benzoylecgonine, rapidly inactivated acid sphingomyelinase, as reflected by a 10-fold decrease in Vmax with identical Km. Acid sphingomyelinase inactivation was fully prevented by the thiol proteinases inhibitors, leupeptin and E64, indicating that cocaine induces selective sphingomyelinase proteolysis. Upon cocaine removal, acid sphingomyelinase activity was rapidly restored, pointing to its fast turnover. In contrast, the cellular content of several other lysosomal hydrolases was increased up to 2-fold. Together, these data show that acidotropic accumulation of cocaine in lysosomes rapidly inhibits acid phospholipase A1 and inactivates acid sphingomyelinase, which can explain induction of a mixed lysosomal lipidosis.

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re to air pollutants. The lungs are the primary source of circulating ET-1, but the direct effects of individual air pollutants and their interaction in modulating the pulmonary endothelin system are unknown. Fischer-344 rats were exposed to particles (0, 5, 50 mg/m3 EHC-93), ozone (0, 0.4, 0.8 ppm), or combinations of particles and ozone for 4 h. Changes in gene expression were measured using real-time reverse transcription polymerase chain reaction immediately after exposure and following 24 h recovery in clean air. Both pollutants individually increased preproET-1, endothelin converting enzyme-1, and endothelial nitric oxide synthase mRNA levels in the lungs shortly after exposure, consistent with the concomitant increase in plasma of the 21 amino acid ET-1[1-21] peptide measured by HPLC-fluorescence. PreproET-1 mRNA remained elevated 24 h after exposure to particles but not after ozone, in line with previously documented changes of the peptide in plasma. Both pollutants transiently increased endothelin-B receptor mRNA expression, while ozone decreased endothelin-A receptor mRNA levels. Coexposure to particles plus ozone increased lung preproET-1 mRNA but not plasma ET-1[1-21], suggesting alternative processing or degradation of endothelins. This coincided with an increase in the lungs of matrix metalloproteinase-2 (MMP-2), an enzyme that cleaves bigET-1 to ET-1[1-32]. Taken together, our data indicate that ozone and particulate matter independently regulate the expression of lung endothelin system genes, but show complex toxicological interaction with respect to plasma ET-1.

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mg/kg or flutamide (1, 12.5, or 25 mg/kg/d) as a positive control. The male offspring were sacrificed at 31 d of age. DBP and flutamide dose-dependently significantly increased the incidence of hypospadias and cryptorchidism in F1 male offspring. The weights of testes and accessory sex organs (epididymides, seminal vesicles, ventral prostate, levator ani plus bulbocavernosus muscles (LABC), and Cowper's glands) were significantly reduced in DBP-treated animals. Furthermore, cauda agenesis of epididymides and ventral prostate atrophy were observed in high-dose 700-mg/kg DBP males. Anogenital distance (AGD) and levels of dihydrotestosterone (DHT) and testosterone were significantly decreased in the DBP (700 mg/kg/d)-treated groups. In particular, the expression of androgen receptor (AR) and 5alpha-reductase type 2 in the proximal penis was markedly depressed following administration of DBP (700 mg/kg/d) or flutamide (25 mg/kg/d). The expression of sonic hedgehog (Shh) in the urethral epithelium of the proximal penis was significantly less in the DBP (700 mg/kg/d)- or flutamide (25 mg/kg/d)-treated groups. In addition, DBP dose-dependently significantly increased the expression of estrogen receptor (ER alpha) in the undescended testis. Data demonstrated that in utero exposure to DBP produced several abnormal responses in male reproductive organs, and these effects may be due to disruption of the stage-specific expression of genes related to androgen-dependent organs development.

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ts and the underlying mechanism. Rats were given 5mg/(kg day) methylmercury chloride (MMC) orally on postnatal day (PND) 7, PND14, PND28, and PND60 for consecutive 7 days. A control group was treated with 0.9% sodium chloride solution 5 ml/(kg day) instead. On PND69, spatial learning and memory was evaluated by Morris water maze test. Behavior deficits were found in MMC-treated rats of PND7 and PND14 groups (p<0.01). N-methyl-D-aspartate (NMDA) receptor 2 subunits mRNA expressions were evaluated 3 days after the last administration. In hippocampus, the mRNA expression of NR2A and NR2B decreased, but the NR2C expression increased in PND14 group following MMC-treatment (p<0.01). In cerebral cortex, mRNA expression of NR2A decreased, with NR2C expression elevating in PND14 group following MMC-treatment (p<0.05). These observations suggest that the postnatal exposure to MeHg during PND7-20 could cause neurobehavioral deficits which extend to adulthood. Furthermore, the abnormal expression of NMDAR 2 subunits might associate with the impairment.

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eceptor (AhR) and subsequent effects on gene transcription. TCDD causes cancer in multiple tissues in different animal species and is classified as a class 1 human carcinogen. In initiation-promotion studies TCDD was shown to be a potent liver tumor promotor. Among other theories it has been hypothesized that TCDD acts as a tumor promotor by preventing initiated cells from undergoing apoptosis. We examined the effects of TCDD on ultraviolet C (UV-C) light-induced apoptosis in primary rat hepatocytes and Huh-7 human hepatoma cells. TCDD inhibits UV-C light-induced apoptosis in both cell types. This effect is seen with chromatin condensation and fragmentation and appears to be mediated by the AhR in rat hepatocytes. Apoptosis induced by UV-C light in these cells is caspase-dependent and is accompanied by alterations in apoptosis-related gene expression such as up-regulation of proapoptotic bcl-2 family genes like bak and bax, and a marked down regulation of the expression of the antiapoptotic bcl-2. TCDD treatment of irradiated hepatocytes induces the expression of some apoptosis-related genes (birc3, dad1, pycard, tnf). Upstream apoptotic events, namely caspase activation and caspase substrate cleavage are not inhibited by TCDD treatment. We hypothesize that TCDD inhibits late-stage apoptotic events that lead to internucleosomal DNA fragmentation, maintaining chromosomal integrity probably in order to sustain metabolic capacity and hepatic elimination of substrates despite of an initiation of apoptosis.

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perfluorooctane sulfonate (PFOS) at doses of 0.1, 0.6 and 2.0 mg/kg/d and 0.05% Tween 80 as control by gavage from gestation days 2-21. The dams were allowed to give nature delivery and then heart tissues from weaned (postnatal day 21) offspring rats were analyzed for mitochondrial injury through ultrastructure observation by electron microscope, global gene expression profile by microarray, as well as related mRNA and proteins expression levels by quantitative PCR and western blot. Ultrastructural analysis revealed significant vacuolization and inner membrane injury occurred at the mitochondria of heart tissues from 2.0 mg/kg/d dosage group. Meanwhile, the global gene expression profile showed significant difference in level of some mRNA expression associated with mitochondrial function at 2.0 mg/kg/d dosage group, compared to the control. Furthermore, dose-response trends for the expression of selected genes were analyzed by quantitative PCR and western blot analysis. The selected genes were mainly focused on those encoding for proteins involved in energy production, control of ion levels, and maintenance of heart function. The down-regulation of mitochondrial ATP synthetase (ATP5E, ATP5I and ATP5O) implicated a decrease in energy supply. This was accompanied by down-regulation of gene transcripts involved in energy consumption such as ion transporting ATPase (ATP1A3 and ATP2B2) and inner membrane protein synthesis (SLC25A3, SLC25A4, SLC25A10, SLC25A29). The up-regulation of gene transcripts encoding for uncoupling proteins (UCP1 and UCP3), epidermal growth factor receptor (EGFR) and connective tissue growth factor (CTGF), was probably a protective process to maintain heart function. The results indicate PFOS prenatal exposure can induce cardiac mitochondrial injury and gene transcript change, which may be a significant mechanism of the developmental toxicity of PFOS to rat.

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ad effects. The present study was designed to test the hypothesis that arsenite alters signaling involved in focal adhesion structure and function in cultured myoblasts. H9C2 cells were exposed to 1, 2.5, 5, or 10 microM sodium arsenite for 48 h. MTT metabolism and staining by neutral red, trypan blue, and propidium iodide showed that sodium arsenite treatments of 5 microM or less were not overtly cytotoxic. At these doses, sodium arsenite did not affect the amount of polymerized actin in cells, rate of protein synthesis, or amounts of vinculin, talin, paxillin, and focal adhesion kinase (FAK) in cells. However, sodium arsenite-treated cells contained fewer focal adhesions with an altered distribution pattern. Sodium arsenite exposure caused a dose-dependent reduction in cell migration and cell attachment rates. The average area of substrate covered by a cell was also reduced, although the average volume of cells was not significantly affected. Sodium arsenite exposure resulted in reduced tyrosine phosphorylation of FAK, its substrate paxillin and the FAK auto- phosphorylation site, Tyr397. Our results indicate that sodium arsenite can alter focal adhesion structure and function, thus affecting cell attachment and migration and possibly other aspects of focal adhesion function such as integrin signaling. These diverse consequences may be mediated by a relatively specific inhibition of FAK tyrosine phosphorylation, modifying scaffolding proteins.

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n fully developed tissue of rats. We designed four groups (control, MCT, RNAi + MCT, and solvent + MCT) of experiments in series 1 and seven groups (control, MCT, MCT + CP-96345-3.4, MCT + CP-96345-10, MCT + CP-96344-10, MCT + SR-48968, and MCT + SR-48965) of experiments in series 2. Rats in the control groups received saline injection. MCT-treated rats received a single MCT injection (60 mg/kg sc). One day prior to MCT, bilateral nodose ganglia were microinjected with ds PPT RNA in rats of the RNAi + MCT group or with solvent in the solvent + MCT group. Beginning from 1 day post-MCT, MCT-treated rats received a daily injection of the NK(1) receptor antagonist, CP-96345 (3.4 or 10 mg/kg ip) or its inactive enantiomer CP-96344 (10 mg/kg ip). The NK(2) receptor antagonist SR-48968 (3 mg/kg ip) or its inactive enantiomer SR-48965 (3 mg/kg ip) was injected to MCT-treated rats every other day starting 1 day post-MCT. Functional study was carried out 2 weeks (series 1) or 3 weeks (series 2) after MCT. MCT induced right ventricular hypertrophy, as well as increases in pulmonary arterial pressure, PPT mRNA (nodose ganglia and lung tissue), and lung tissue substance P level. All of the above MCT-induced alterations were attenuated by either RNAi or NK receptor antagonists. We conclude that tachykinins play an important role in MCT-induced pulmonary hypertension.

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pment is synaptogenesis, which is essential for the progression of neuronal differentiation and the establishment of neuronal network. We therefore aim to test the hypothesis that toluene may interfere with synaptogenesis by applying toluene to cultured hippocampal neurons dissected from embryonic rat brains. In the presence of toluene, hippocampal neurons displayed a significant loss of the immunostaining of synapsin and densin-180 punctas. Notably, a dramatic reduction was also discerned for the colocalization of the two synaptic markers. Moreover, Western blotting analyses revealed that toluene exposure resulted in considerable down-regulation of the expression of synapse-specific proteins. None of the preceding observations can be attributed to toluene-induced cell death effects, since toluene treatments failed to affect the viability of hippocampal neurons. Overall, our data are consistent with the idea that toluene may alter the expression and localization of essential synaptic proteins, thereby leading to a disruption of synapse formation and maintenance.

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PROP and GABA. Ace and Meth, but not IL-1, inhibited AChE activity, while all three compounds; (1) increased ACh and CRF mRNA levels in and CRF release from; (2) activated the CRE promoter region of CRF-gene in: and (3) increased cFos binding to the AP-1 region of the CRF-gene in the hypothalamus. Dex suppressed the effects of IL-1, possibly by inducing the nGRE regulatory sites of the CRF-gene. Dex, however, did not modulate the effects of Ace and Meth on the hypothalamus, which may be attributed to the failure of Dex to modulate the CRF-gene's nGRE regulatory sites. Atrop caused 80-90% inhibition of the effects of IL-1, but caused only 50-65% inhibition of the effects of Ace or Meth on CRF mRNA levels in and CRF release from the hypothalamus. PTL did not affect, while PROP slightly attenuated the effects of IL-1 and the insecticides on the hypothalamus. GABA attenuated the effects of the insecticides but not the effects of IL-1 on the hypothalamus. This suggests that the IL-1-induced augmentation of CRF synthesis in and release from the hypothalamus is mediated through a cholinergic pathway, while the insecticide-induced augmentation of CRF synthesis in and release from the hypothalamus is mediated through the cholinergic and GABAergic pathways. The insecticides, but not IL-1, disrupt feedback regulation of CRF synthesis in and release from the hypothalamus.

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ion of PPAR-regulated genes involved in testicular cells apoptosis. Sprague-Dawley male rats were treated orally with 250, 500, or 750 mg/kg/d DEHP for 28 d, while control rats were given corn oil. The levels of cell cycle regulators (pRb, cyclins, CDKs, and p21) and apoptosis-related proteins were analyzed by Western blot analysis. The role of PPAR-gamma (PPAR-gamma), class B scavenger receptor type 1 (SR-B1), and ERK1/2 was further studied to examine the signaling pathway for DEHP-induced apoptosis. Results showed that the levels of pRB, cyclin D, CDK2, cyclin E, and CDK4 were significantly lower in rats given 500 and 750 mg/kg/d DEHP, while levels of p21 were significantly higher in rat testes. Dose-dependent increases in PPAR-gamma and RXRalpha proteins were observed in testes after DEHP exposure, while there was a significant decrease in RXRgamma protein levels. In addition to PPAR-gamma, DEHP also significantly increased SR-B1 mRNA and phosphorylated ERK1/2 protein levels. Furthermore, DEHP treatment induced pro-caspase-3 and cleavage of its substrate protein, poly(ADP-ribose) polymerase (PARP), in a dose-dependent manner. Data suggest that DEHP exposure may induce the expression of apoptosis-related genes in testes through induction of PPAR-gamma and activation of the ERK1/2 pathway.

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lding block of cardiac gap junctions and hemichannels, plays an important role in cardioprotection. Recent reports confirmed the presence of Cx43 in the mitochondria as well. In this study, the role of mitochondrial Cx43 was evaluated 3 or 6 h after Doxorubicin administration to the rat heart cell line H9c2. Pharmacological inhibition of Hsp90 demonstrated that the mitochondrial Cx43 conferred cardioprotection by reducing cytosolic and mitochondrial reactive oxygen species production, mitochondrial calcium overload and mitochondrial membrane depolarization and cytochrome c release. In conclusion, our study demonstrates that Cx43 plays an important role in the protection of cardiac cells from Doxorubicin-induced toxicity.

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or 50 mg Cd/l for 6, 12 and 24 weeks. Concentrations of cytochrome P450 and cytochrome b(5) as well as activities of NADPH-cytochrome P450 reductase and NADH-cytochrome b(5) reductase were determined in the kidney microsomal fraction. Protein content of CYP1A1, CYP2E1 and CYP3A1 cytochrome P450 isoforms was evaluated as well. In the rats exposed to 5 mg Cd/l, the concentration of cytochrome P450 decreased (by 41%) after 24 weeks of the experiment. The activity of NADPH-cytochrome P450 reductase decreased (by 24%) after 6 and 12 weeks, whereas after 24 weeks it remained unchanged, compared with the control group. Moreover, a decrease in the concentration of cytochrome b(5) (by 25, 15 and 26% at 6, 12 and 24 weeks, respectively) and the activity of its NADH reductase (by 26 and 31% at 6 and 24 weeks, respectively) was noted in these animals. At the exposure to 50 mg Cd/l, the concentrations of cytochrome P450 and cytochrome b(5) and the activities of their corresponding reductases were decreased at each time-point. Western blot analysis revealed that all isoforms of cytochrome P450 studied were affected by Cd and the effect was dependent on the level and the duration of exposure. The results of this study indicate that chronic exposure to Cd in a dose- and time-dependent manner affects the kidney cytochrome P450-dependent monooxygenase system by decreasing the concentrations of cytochrome P450 and cytochrome b(5) and inhibiting the activities of their corresponding reductases. The effect of Cd on the cytochrome P450 content is associated with its ability to stimulate or inhibit of various P450 isoforms. A very important finding of this study is that Cd affects the kidney cytochrome P450-dependent monooxygenase system at relatively low exposure and low kidney Cd accumulation (2.40+/-0.15 microg/g). As the experimental model used reflects human exposure to Cd, we conclude that Cd can affect the kidney cytochrome P450-dependent monooxygenase system in environmentally exposed humans. Previously we have reported disorders in the system in the liver of rats at the same levels of exposure as in this study. Thus, we hypothesize that the metabolism and detoxification of many substances, including xenobiotics, may be seriously affected in Cd-exposed subjects.

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tic expression associated with the affected physiological processes to identify potential targets for future pharmacological/toxicological intervention. Male rats (n = 24) were implanted with temperature transmitters, which measure core temperature every 5 min. After an 8-d recovery, the rats were fed an endophyte-free diet for 5 d. During the following 5-d treatment period, rats were fed either an endophyte-free or an endophyte-infected (91.5 microg of ergovaline.kg of BW(-1).d(-1)) diet. At the end of treatment, rats were euthanized and a sample of the liver was obtained. Feed conversion efficiency was calculated for both treatment groups. Serum prolactin concentrations were measured using ELISA. Liver tissue RNA was reverse transcribed and hybridized to an oligonucleotide microarray chip. Microarray data were analyzed using a 2-step ANOVA model and validated by quantitative real-time PCR. Significant reductions in mean core temperature, feed intake, feed conversion efficiency, BW, liver weight per unit of BW, and serum prolactin concentrations were observed in endophyte-infected rats. There was downregulation (P < 0.05) of various genes associated with energy metabolism, growth and development, and antioxidant protection, as well as an upregulation of genes associated with gluconeogenesis, detoxification, and biotransformation. This study demonstrated that even short-term exposure of rats to tall fescue endophytic toxins under thermoneutral conditions can result in physiological responses associated with altered gene expression within the liver.

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nical studies. Whereas these vascular lesions in rats have been well characterized histologically, little is known about their pathogenesis and in turn, sensitive and specific biomarkers for preclinical and clinical monitoring do not exist. In order to investigate the early molecular mechanisms underlying vascular injury, time-course studies were performed by treating rats for 2-24 h with high doses of the PDE4 inhibitor CI-1044. Transcriptomics analyses in mesenteric tissue were performed using oligonucleotide microarray and real-time RT-PCR technologies and compared to histopathological observations. In addition, protein measurements were performed in serum samples to identify soluble biomarkers of vascular injury. Our results indicate that molecular alterations preceded the histological observations of inflammatory and necrotic lesions in mesenteric arteries. Some gene expression changes suggest that the development of the lesions could follow a primary modulation of the vascular tone in response to the pharmacological effect of the compound. Activation of genes coding for pro- and antioxidant enzymes, cytokines, adhesion molecules, and tissue inhibitor of metalloproteinase 1 (TIMP-1) indicates that biomechanical stimuli may contribute to vascular oxidant stress, inflammation, and tissue remodeling. TIMP-1 appeared to be an early and sensitive predictive biomarker of the inflammatory and the tissue remodeling components of PDE4 inhibitor-induced vascular injury.

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aimed to investigate the in vivo role of PPARalpha in elevating STATs as well as oxidative/nitrosative stress in a model of lipopolysaccharide (LPS)-induced acute hepatic inflammatory injury. Using age-matched Ppara-null and wild-type (WT) mice, we demonstrate that the deletion of PPARalpha aggravates LPS-mediated liver injury through activating STAT1 and NF-kappaB-p65 accompanied by increased levels of pro-inflammatory cytokines. Furthermore, the activities of key anti-oxidant enzymes and mitochondrial complexes were significantly decreased while lipid peroxidation and protein nitration were elevated in LPS-exposed Ppara-null mice compared to WT. These results indicate that PPARalpha is important in preventing LPS-induced acute liver damage by regulating STAT1 inflammatory signaling pathways and oxidative/nitrosative stress.

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2.5, 25, and 250muM), the effects of which on cell proliferation and differentiation were investigated. In undifferentiated PC12 cells, MEHP inhibited cell proliferation in a dose-dependent manner. After 24h of MEHP treatment, there was a dose-dependent G2/M cell cycle arrest as well as a sharp drop of DNA synthesis. During the process of NGF-induced differentiation of the cell line, 4 days of MEHP exposure (2.5-250muM) increased membrane and cytoskeletal protein contents, enhanced NGF-induced neurite outgrowth, up-regulated the choline acetyl transferase (ChAT) mRNA and down-regulated tyrosine hydroxylase (TH) mRNA levels, which suggested the promoted differentiation towards the acetylcholine (ACh) phenotype at the expense of the dopamine (DA) phenotype. Take together, our results indicate that MEHP has a potential to disturb neurodevelopment by suppressing cell proliferation and promote cell differentiation in neurocytes.

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100 mg/kg) were evaluated in male and female Sprague-Dawley rats using Affymetrix Rat Genome U34A gene arrays. In addition to changes in the hepatic expression of well-characterized drug-metabolizing enzymes, an increase in Mdm2 mRNA was observed with both compounds after single or repeat dosing (5 days). However, gene array analyses did not reveal changes in other p53-dependent genes, suggesting that induction of Mdm2 occurred in a p53-independent manner. Real-time polymerase chain reaction confirmed the microarray results, as PB increased Mdm2 mRNA approximately twofold after single or repeat doses in male and female rats. PCN treatment increased Mdm2 mRNA levels up to 5- and 12-fold in male and female rats, respectively, after 5 days of dosing. Hepatic Mdm2 protein levels were increased, and immunohistochemical evaluation of rat liver demonstrated nuclear localization of Mdm2, suggesting an interaction with p53. Consequently, p53 protein levels were also decreased by approximately 35 and 50% after 5 days of PB and PCN treatment, respectively. In direct contrast to rats, PB and PCN (100 mg/kg) did not induce Mdm2 mRNA in mouse liver after 5 days of dosing. Finally, although Mdm2 in mice and humans is reported to migrate electrophoretically as two proteins with molecular weights of 76 and 90 kDa, rat Mdm2 protein was detected primarily as a 120-kDa species. Follow-up experiments indicated that rat hepatic Mdm2 was subject to posttranslational modification with small ubiquitin-modifying (SUMO) proteins. Although the molecular mechanisms controlling Mdm2 induction by PB and PCN in rats have not yet been determined, these results suggest that early effects on cell cycle regulation, response to DNA damage or cell transformation may contribute to liver tumor development.

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s were also analyzed. Male rats had significantly higher liver microsomal cytochrome P-450 content and NADPH-cytochrome P-450 reductase activities than females at 14 to 26 wk. Microsomal cytochrome P-450 content was decreased in senescence in both genders compared with postpubertal and adulthood stages. The activity of 6beta-testosterone hydroxylase in male rats, which was significantly higher than those in females at all ages, decreased after 52 wk. After 26 wk, the levels of CYP3A1 protein markely declined in both genders, which resulted in a large gender difference (male greater than female). The protein levels and mRNA of CYP3A2 were constitutively expressed in 2- to 52-wk-old male rats, but they decreased after 76 wk, and decreased in females after 6 wk. The expression of CYP3A1 or CYP3A2 in males are generally higher than in females. The metabolites of territrems MA1, MAX, MA2, MB2, MB4, and MC were measured by high-performance chromatography (HPLC). Formation of MA1, MAX, and MA2 decreased after 52 wk in males, and MAX and MA2 were not formed after 6 wk in females. The amount of MB2 formed in females was less than in males, but the amount of MC (TRC metabolites) formed in females was higher than in males. The gender differences in metabolism of TRA were related to the protein and mRNA expression of CYP3A2. The protein levels and mRNA expression of CYP3A2 and efficiency of territrems metabolism were decreased after 76 wk. The results suggested that the effects of age and gender on territrem metabolism are due to differences in CYP3A1 and CYP3A2 expression in the liver microsomes.

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lucidate the mechanism underlying the stress response in the duodenum--one of the tissues that is most sensitive to external stress. DNA microarray analysis revealed that the immobilization for 2 weeks caused great changes in gene expression in the rat duodenum: 165 genes exhibited more than a two-fold change in expression level (103 up-regulated; 62 down-regulated). In addition, functional classification of these genes showed that immobilization preferentially stimulated the expression of genes related to lipid metabolism, including genes encoding mitochondrial HMG-CoA synthase, a key enzyme in ketogenesis; solute carrier 27A2, a fatty acid transporter; and dienoyl CoA reductase, a key enzyme in beta-oxidation. To elucidate the factors mediating these immobilization-induced changes, we treated rats and small intestinal IEC-6 cells with dexamethasone and hydrogen peroxide. In both rats and IEC-6 cells, treatment with dexamethasone induced changes in gene expression that mimicked the immobilization-mediated increase in expression of the mitochondrial HMG-CoA synthase and dienoyl CoA reductase transcripts, suggesting that stress-induced synthesis of glucocorticoid hormones mediates, at least in part, the stress response in the duodenum. These results suggest that immobilization may alter lipid metabolism in the small intestine by modifying the expression of specific genes through which the small intestine may seek to protect itself from stress-induced damage.

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ion permeation through this pathway. In the present study, immunohistochemical analysis revealed a striking overlap of the expression pattern of LTCC and ZnT-1 in cardiac tissue and brain. Using siRNA to silence ZnT-1 expression, we then assessed the role of ZnT-1 in regulating cation permeation through the L-type Ca(2+) channels in cells that are vulnerable to heavy metal permeation. Transfection of cortical neurons with ZnT-1 siRNA resulted in about 70% reduction of ZnT-1 expression and increased Ca(2+) influx via LTCC by approximately fourfold. Moreover, ZnT-1 siRNA transfected neurons showed approximately 30% increase in synaptic release, monitored using the FM1-43 dye. An increased cation influx rate, through the LTCC, was also recorded for Zn(2+) and Cd(2+) in cells treated with the ZnT-1 siRNA. Furthermore, Cd(2+)-induced neuronal death increased by approximately twofold after transfection with ZnT-1 siRNA. In addition, ZnT-1 siRNA transfection of the ovarian granulosa cell line, POGRS1, resulted in a twofold increase in Cd(2+) influx rate via the LTCC. Finally, a robust nimodipine-sensitive Cd(2+) influx was observed using a low extracellular Cd(2+) concentration (5 muM) in neurons and testicular slice cultures, attesting to the relevance of the LTCC pathway to heavy metal toxicity. Taken together, our results indicate that endogenously-expressed ZnT-1, by modulating LTCC, has a dual role: regulating calcium influx, and attenuating Cd(2+) and Zn(2+) permeation and toxicity in neurons and other cell types.

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may underlie the pathogenesis of aluminium induced neurodegeneration. Thus, the present study was undertaken to reveal the effects of chronic aluminium exposure (10mg/kg b.wt, intragastrically for 12 weeks) on the oxidative damage to mitochondrial proteins in male albino Wistar rats. Chronic aluminium exposure resulted in decrease in the activity of mitochondrial superoxide dismutase (MnSOD) and aconitase in different regions of rat brain suggesting increased oxidative stress. This decrease in MnSOD activity in turn might be responsible for the increased protein oxidation as observed in our study. All these processes taken together may cause increased oxidative damage to mitochondrial proteins in general. By taking the advantage of recent immunochemical probe for oxidatively modified proteins, we identified MnSOD to be susceptible to oxidative damage in aluminium treated animals. The quantitative RT-PCR analysis for Lon protease, a protease involved in the removal of oxidatively modified proteins from mitochondria, showed decreased mRNA expression suggesting increased oxidative damage and decreased removal of mitochondrial proteins. The identification of specific proteins as targets of oxidative damage may provide new therapeutic measures to reverse the effects of aluminium induced neurodegeneration.

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nt with downregulation of lysyl oxidase (LO), a copper-dependent enzyme for crosslinking collagen and elastin in the extracellular matrix (ECM). Cd downregulation of LO in treated cells was closely accompanied by suppression of synthesis of collagen, a major structure component of the lung ECM. Using rats intratracheally instilled with cadmium chloride (30 microg, once a week) as an animal model, we further demonstrated that although 2-week Cd instillation induced a non-significant change in the lung LO activity and collagen synthesis, 4- and 6-week Cd instillation resulted in a steady decrease in the lung LO and collagen expression. The lung MT and total GSH levels were both upregulated upon the long-term Cd exposure. Emphysematous lesions were generated in lungs of 6-week Cd-dosed rats. Increases of cellular thiols by transfection of cells with MT-II expression vectors or treatment of cells with GSH monoethyl ester, a GSH delivery system, markedly inhibited LO mRNA levels and catalytic activities in the cell model. Thus, Cd upregulation of cellular thiols may be a critical cellular event facilitating downregulation of LO, a potential mechanism for Cd-induced emphysema.

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re has focused on the respiratory system, showing the importance of inhalation as the primary route for TiO2 NP exposure in the workplace. TiO2 NPs may translocate to systemic organs from the lung and gastrointestinal tract (GIT) although the rate of translocation appears low. There have also been studies focusing on other potential routes of human exposure. Oral exposure mainly occurs through food products containing TiO2 NP-additives. Most dermal exposure studies, whether in vivo or in vitro, report that TiO2 NPs do not penetrate the stratum corneum (SC). In the field of nanomedicine, intravenous injection can deliver TiO2 nanoparticulate carriers directly into the human body. Upon intravenous exposure, TiO2 NPs can induce pathological lesions of the liver, spleen, kidneys, and brain. We have also shown here that most of these effects may be due to the use of very high doses of TiO2 NPs. There is also an enormous lack of epidemiological data regarding TiO2 NPs in spite of its increased production and use. However, long-term inhalation studies in rats have reported lung tumors. This review summarizes the current knowledge on the toxicology of TiO2 NPs and points out areas where further information is needed.

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10 mg/kg), or their combination by intraperitoneal injection 30 min prior to ANP induction. At 1, 3, 6, and 12 hours after ANP induction, lung NO production, and inducible NO synthase (iNOS) expression were measured. Lung histopathological changes, bronchoalveolar lavage (BAL) protein concentration, proinflammatory mediators tumor necrotic factor alpha (TNF-alpha), and lung tissue myeloperoxidase (MPO) activity were examined. Results showed that NO production and iNOS mRNA expression in alveolar macrophages (AMs) were significantly increased along with significant increases in lung histological abnormalities and BAL proteins in the ANP group, all of which were further enhanced by pretreatment with L-Arg and attenuated by pretreatment with L-NAME, respectively. These markers were slightly attenuated by pretreatment with combination of L-Arg + L-NAME, suggesting that NO is required for initiating the acute lung damage in ANP rats, and also that L-Arg-enhanced lung injury is mediated by its NO generation rather than its direct effect. MPO activity and TNF-alpha expression in lung were upregulated in the ANP rats and further enhanced by pretreatment with L-Arg and attenuated by pretreatment with L-NAME, respectively. These results suggest that overproduction of NO mediated by iNOS in the lung is required for the acute lung inflammation and damage secondary to ANP.

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aled that there were 14, 41, 110, 386, and 916 genes significantly changed in the 0.125, 0.25, 0.5, 1.0, and 2.0 g/kg treatment groups, respectively. There were 16 drug metabolizing genes altered in all three high-dose treatment groups, among which seven genes belong to cytochrome P450 isozymes. While gene expression of Cyp1a1, 1a2, 2c6, 3a1, and 3a3 increased; Cyp 2c23 and 2c40 decreased, all in a dose-dependent manner. Real-time PCR analyses of several genes verified these results. Our results indicate that kava extract can significantly modulate drug metabolizing enzymes, particularly the CYP isozymes, which could cause herb-drug interactions and may potentially lead to hepatotoxicity.

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ible mediation of cytochrome P450 (CYP) in producing reactive metabolites. In this paper, clozapine toxicity was, respectively, examined in two cultures of rat hepatocytes. Gel entrapment culture of hepatocytes with higher expression on CYP activities showed higher sensitivity to clozapine treatment than hepatocyte monolayer, indicating the possible involvement of CYP in hepatotoxicity of clozapine. Moreover, in each culture, CYP inhibitors were used to confirm the possible mediation of CYP enzymes. Pretreatment of hepatocytes with CYP 3A inhibitor (ketoconazole), CYP 2E1 inhibitor (diethyldithiocarbamate, DDC) and non-specific inhibitor (cimetidine) significantly reduced the toxicity of clozapine. But the pretreatment with CYP 1A2 inhibitor (fluvoxamine) had no such protective effect indicative of non-function of CYP 1A2 in clozapine toxicity. In addition, glycyrrhizic acid (GA), a scavenger of reactive oxygen species (ROS), also inhibited the adverse response to clozapine, suggesting the positive involvement of oxidant pressure. Thus, it could be concluded that clozapine-induced toxicity was mediated by CYP, particularly CYP 3A and CYP 2E1, and oxidant pressure.

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icroM) resulted in a significantly decreased activity of adenylyl cyclase enzyme which was followed by a decreased synthesis of cAMP. This decreased cAMP level resulted in the disruption of entire steroidogenic cascade causing a depressed synthesis of testosterone. However, TCS-induced decrease in the production of testosterone returned to normalcy when cells were treated with forskolin (an adenylyl cyclase activator). Transcription followed by translational of four prominent steroidogenic enzyme/proteins, cytochrome P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and steroidogenic acute regulatory (StAR) protein, also decreased in a dose-dependent manner in TCS-treated Leydig cells as determined by RT-PCR, enzyme assay and Western blot. These results suggested that the disruption of the activity of adenylyl cyclase enzyme by TCS in turn leads to the disruption of intermediate steroidogenic cascade causing a depressed testosterone production. The study further confirmed the anti-androgenic activity of TCS in Leydig cells with highest effective concentration at 1 microM.

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ubated with 20, 40, and 80 mg/l sodium fluoride for 24 h in vitro. The results showed that the cell survival rate in the 80 mg/l fluoride-treated group was significantly lower than that of the control group. Forty and 80 mg/l of fluoride induced significantly increased lactate dehydrogenase release, intracellular reactive oxygen species, and the percentage of apoptosis. Compared with control group, the malondialdehyde levels were significantly elevated while glutathione levels and glutathione peroxidase activities were decreased in all fluoride-treated groups, accompanied by the markedly reduced superoxide dismutase activity in 80 mg/l fluoride-treated group. With respect to NCAM mRNA expression levels, a significant dose-dependent decrease was observed in 40 and 80 mg/l fluoride-treated groups against the control group. In addition, as compared to the control group, the protein expression levels of NCAM-180 in 40 and 80 mg/l fluoride-treated groups, NCAM-140 in all fluoride-treated groups, and NCAM-120 in the 80 mg/l fluoride-treated group were significantly decreased. Our study herein suggested that fluoride could cause oxidative stress, apoptosis, and decreased mRNA and protein expression levels of NCAM in rat hippocampal neurons, contributing to the neurotoxicity induced by fluoride.

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kappaB and procaspase-1 activation. In this study, intratracheal instillation of silica in rats was found to produce transient upregulation of mRNA levels of the PAAD family of proteins, PYPAF7 (PYRIN containing Apaf1-like protein; Apaf stands for apoptosis activating factor) and MEFV (for Mediterranean fever), in bronchoalveolar lavage (BAL) cells. The levels were markedly elevated at 4 h, returning to basal levels by 24 h. In contrast, intratracheal instillation of LPS produced a sustained upregulation of the two genes in BAL cells. In vitro exposure of BAL cells to silica or lipopolysaccharide (LPS) produced no changes in the expression of these genes, indicating that silica or LPS exposure in vivo induces some factors that are responsible for the upregulation of PYPAF7 and MEFV. The mRNA levels of these two genes in peripheral blood monocytes and PMN following LPS exposure did not change, indicating that AM and peripheral blood cells show similar response to LPS exposure in vitro. This study provides the basis for a physiological model to study the effects of these two genes in modulating the inflammatory response after particle exposure.

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r NF-E2-related factor 2 (Nrf2) regulates the expression of a battery of antioxidative enzymes and Nrf2 signaling can be activated by small-molecule drugs that disrupt Keap1-mediated repression of Nrf2 signaling. Therefore, genetic and pharmacologic approaches were used to activate Nrf2 signaling to assess protection against inflammatory liver injury. Profound increases in indicators of cell death were observed in both Nrf2 wild-type (Nrf2-WT) mice and Nrf2-disrupted (Nrf2-KO) mice 24 h following intravenous injection of concanavalin A (12.5 mg/kg, ConA), a model for T cell-mediated acute inflammatory liver injury. However, hepatocyte-specific conditional Keap1 null (Alb-Cre:Keap1(flox/-), cKeap1-KO) mice with constitutively enhanced expression of Nrf2-regulated antioxidative genes as well as Nrf2-WT mice but not Nrf2-KO mice pretreated with three daily doses of a triterpenoid that potently activates Nrf2 (30 mumol/kg, cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl-imidazolide [CDDO-Im]) were highly resistant to ConA-mediated inflammatory liver injury. CDDO-Im pretreatment of both Nrf2-WT and Nrf2-KO mice resulted in equivalent suppression of serum proinflammatory soluble proteins suggesting that the hepatoprotection afforded by CDDO-Im pretreatment of Nrf2-WT mice but not Nrf2-KO mice was not due to suppression of systemic proinflammatory signaling, but instead was due to activation of Nrf2 signaling in the liver. Enhanced hepatic expression of Nrf2-regulated antioxidative genes inhibited inflammation-mediated oxidative stress, thereby preventing hepatocyte necrosis. Attenuation of hepatocyte death in cKeap1-KO mice and CDDO-Im pretreated Nrf2-WT mice resulted in decreased late-phase proinflammatory gene expression in the liver thereby diminishing the sustained influx of inflammatory cells initially stimulated by the ConA challenge. Taken together, these results clearly illustrate that targeted cytoprotection of hepatocytes through Nrf2 signaling during inflammation prevents the amplification of inflammatory responses in the liver.

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s and xenobiotics. This classic detoxification process, which led to the discovery nearly 50 years ago of the cosubstrate UDP-glucuronic acid (19), is now known to be carried out by 15 human UGTs. Characterization of the individual gene products using cDNA expression experiments has led to the identification of over 350 individual compounds that serve as substrates for this superfamily of proteins. This data, coupled with the introduction of sophisticated RNA detection techniques designed to elucidate patterns of gene expression of the UGT superfamily in human liver and extrahepatic tissues of the gastrointestinal tract, has aided in understanding the contribution of glucuronidation toward epithelial first-pass metabolism. In addition, characterization of the UGT1A locus and genetic studies directed at understanding the role of bilirubin glucuronidation and the biochemical basis of the clinical symptoms found in unconjugated hyperbilirubinemia have uncovered the structural gene polymorphisms associated with Crigler-Najjar's and Gilbert's syndrome. The role of the UGTs in metabolism and different disease states in humans is the topic of this review.

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nding potential of reactive intermediates generated by GSTT1-1-mediated metabolism of CHBrCl(2). First, rodent hepatic cytosol incubations containing [(14)C]CHBrCl(2), supplemented glutathione (GSH), and calf thymus DNA resulted in approximately 3-fold (rat liver cytosol) and 7-fold (mouse liver cytosol) greater amounts of total radioactivity (RAD) associated with the purified DNA as compared to a control (absence of rodent cytosol) following liquid scintillation counting (LSC) of isolated DNA. The relative increase in DNA labeling is consistent with the conjugation activity of these rodent cytosols toward CHBrCl(2). Second, exposure of GSTT1-1-expressing S. typhimurium to [(14)C]CHBrCl(2) resulted in a concentration-dependent increase of bacterial DNA-associated total radioactivity. Characterization of DNA-associated radioactivity could not be assigned to a specific deoxynucleoside adduct(s) following enzymatic hydrolysis of DNA and subsequent HPLC analysis. A possible explanation for this observation was formation of a 'transient' adduct that was unstable in the DNA isolation and hydrolysis procedures employed. To circumvent problems of adduct instability, reactions of [(14)C]CHBrCl(2) with GSH catalyzed by recombinant rat GSTT1-1 were performed in the presence of calf thymus DNA or, alternatively, the model nucleophile deoxyguanosine. Hydroxyapatite chromatography of [(14)C]-labeled DNA or HPLC chromatography of [(14)C]-labeled deoxyguanosine derivatives demonstrated the covalent binding of [(14)C]CHBrCl(2)-derived metabolites to DNA and deoxyguanosine in low yield (approximately 0.02% of [(14)C]CHBrCl(2) biotransformed by GSTT1-1 resulted in DNA adducts). Cytochrome P450 (CYP)- and GST-catalyzed biotransformation of CHBrCl(2) in rat tissues (kidney and large intestine) that develop tumors following chronic CHBrCl(2) exposure were compared with rat liver (a nontarget tissue). Rat liver had a significant capacity to detoxify CHBrCl(2) (to carbon dioxide) compared with kidney and large intestine as a result of CYP-catalyzed oxidation, liver was approximately 16-fold more efficient than kidney and large intestine when intrinsic clearance values (V(max)/K(m)) were compared. In contrast, the efficiency of GST-mediated GSH conjugation of CHBrCl(2) in kidney and large intestine was only slightly lower than liver (approximately 2- to 4-fold lower), thus, the relative amounts of reactive intermediates that are produced with the capacity to covalently modify DNA may be enhanced in these extrahepatic tissues. The significance of these findings is that conjugation of CHBrCl(2) with GSH can result in the covalent modification of DNA and that cancer target tissues in rats have a much reduced detoxification capacity, but only a modest decrease in bioactivation capacity, as compared to the liver (a nontarget tissue in rats).

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activation of the NLRP3 inflammasome, a pathway also implicated in AMD. Additionally, iron-induced degeneration of the retinal pigmented epithelium (RPE) is suppressed in mice lacking inflammasome components caspase-1/11 or Nlrp3 or by inhibition of caspase-1. Iron overload increases abundance of RNAs transcribed from short interspersed nuclear elements (SINEs): Alu RNAs and the rodent equivalent B1 and B2 RNAs, which are inflammasome agonists. Targeting Alu or B2 RNA prevents iron-induced inflammasome activation and RPE degeneration. Iron-induced SINE RNA accumulation is due to suppression of DICER1 via sequestration of the co-factor poly(C)-binding protein 2 (PCBP2). These findings reveal an unexpected mechanism of iron toxicity, with implications for AMD and neurodegenerative diseases associated with excess iron.

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d dose-dependent hepatic estrogenicity of o,p'-DDT was investigated using complementary DNA microarrays in immature ovariectomized Sprague-Dawley rats with complementary histopathology and tissue-level analysis. Animals were gavaged with 300 mg/kg o,p'-DDT either once or once daily for 3 consecutive days. Liver samples were examined 2, 4, 8, 12, 18, or 24 h after a single dose or following three daily doses. For dose-response studies, a single dose of 3, 10, 30, 100, or 300 mg/kg body weight o,p'-DTT was administered for 3 consecutive days. Genes associated with drug metabolism (Cyp2b2 and Cyp3a2), the nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR), cell proliferation (Ccnd1, Ccnb1, Ccnb2, and Stmn1), and oxidative stress (Gclm and Hmox1) were significantly induced. Cyp2b2 exhibited dose-dependent regulation and was significantly induced across all time points, while cell proliferation- and oxidative stress-related genes exhibited transient induction. The induction of Cyp2b2 and Cyp3a2 mRNA levels suggest PXR/CAR activation, consistent with expression of genes associated with oxidative stress. Few genes known to be estrogen receptor (ER) regulated were differentially expressed when compared to the hepatic gene expression profile elicited by ethynyl estradiol in immature ovariectomized C57BL/6 mice using the same study design and analysis methods. These data indicate that o,p'-DDT elicits PXR/CAR-, not ER-, mediated gene expression in the rat liver. Based on the species-specific differences in CAR regulation, the extrapolation of rodent DDT hepatocarcinogenicity to humans warrants further investigation.

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sus C57Bl/6J wild-type (WT) mice to silica were compared. Male mice (8-10 wk old, mean body weight 24.0 g) were anesthetized and exposed, by aspiration, to silica (40 mg/kg) or saline. At 24 h and 42 d postexposure, lungs were lavaged with saline. The first bronchoalveolar lavage (BAL) fluid supernatant was analyzed for lactate dehydrogenase (LDH) activity, levels of albumin, tumor necrosis factor-alpha (TNF-alpha), and macrophage inflammatory protein-2 (MIP-2), as well as total antioxidant capacity (TAC). The cellular fraction of the total BAL was used to determine alveolar macrophage (AM) and polymorphonuclear leukocyte (PMN) counts, and zymosanstimulated AM chemiluminescence (AM-CL). In separate mice, lung histopathological changes were evaluated 42 d postexposure. Acute (24-h) silica exposure decreased AMs, increased PMNs, increased LDH activity and levels of albumin, TNF-alpha, and MIP-2 in BAL fluid, and enhanced AM-CL in both iNOS KO and WT mice. However, iNOS KO mice exhibited less AM activation (defined as increased AM-CL and decreased AM yield) than WT. Furthermore, TAC following acute silica decreased in WT but was maintained in iNOS KO mice. Pulmonary reactions to subchronic (42 d) silica exposure were similar to acute. However, histopathological and BAL fluid indices of lung damage and inflammation, AM activation, and lung hydroxyproline levels were significantly less in iNOS KO compared to WT mice. These results suggest that iNOS-derived NO contributes to the pathogenesis of silica-induced lung disease in this mouse model.

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ic dose levels in drinking water resulted in a progressive coproporphyrinuria, reaching highest rates of coproporphyrin excretion 5 weeks after initiation of exposure. The development of coproporphyrinuria was accompanied by substantial metal accumulation in the kidney and a significant decrease in renal, but not hepatic, coproporphyrinogen oxidase activity. During prolonged exposure to either metal, the rates of coproporphyrin excretion and metal accumulation by the kidney continued to increase for 2 to 3 weeks following maximal inhibition of renal coproporphyrinogen oxidase. Acute treatment studies and studies in vitro support the conclusion that the kidney is the principal source of excess urinary coproporphyrin during metal exposure. These observations demonstrate that metal-induced coproporphyrinuria is predominantly of renal etiology and that impairment of renal coproporphyrinogen oxidase is a principal cause of this effect.

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diated NS, we examined myelin basic protein in CO poisoned XOR-depleted rats and performed radial maze studies to evaluate the alteration of cognitive function. Carbon monoxide poisoned XOR-depleted rats did not exhibit myelin basic protein alterations or impaired cognitive function, both found in CO poisoned control rats. These results indicate that XOR is essential to the pathological cascade of CO-mediated NS.

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enetic polymorphisms in NAT1 and/or NAT2 modify breast cancer risk in women exposed to these carcinogens. p-Aminobenzoic acid (selective for rat NAT2) and sulfamethazine (SMZ; selective for rat NAT1) N-acetyltransferase catalytic activities were both expressed in primary cultures of rat mammary epithelial cells. PABA, 2-aminofluorene, and 4-aminobiphenyl N-acetyltransferase and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine and N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline O-acetyltransferase activities were two- to threefold higher in mammary epithelial cell cultures from rapid than slow acetylator rats. In contrast, SMZ (a rat NAT1-selective substrate) N-acetyltransferase activity did not differ between rapid and slow acetylators. Rat mammary cells cultured in the medium supplemented 24 h with 10muM ABP showed downregulation in the N-and O-acetylation of all substrates tested except for the NAT1-selective substrate SMZ. This downregulation was comparable in rapid and slow NAT2 acetylators. These studies clearly show NAT2 acetylator genotype-dependent N- and O-acetylation of aromatic and heterocyclic amine carcinogens in rat mammary epithelial cell cultures to be subject to downregulation by the arylamine carcinogen ABP.

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uch kind of effects of B(a)P on lungs in relation with soluble epoxide hydrolase (sEH) and thioredoxin reductase (TrxR) activities. Glycyrrhizic acid (GA), an active principle of Glycyrrhiza glabra (Licorice), is known to modulate various molecular processes. In the present study, we investigated the protective effects of GA against B(a)P induced debilities in lungs of Wistar rats. Intratracheal instillation of B(a)P significantly suppressed NF-kappaB translocation, sEH, TrxR and catalase activities in lung tissue. A marked induction of H(2)O(2) levels along with caspases activation (caspases-2, -3, -6, -8, and -9) in lung tissue after B(a)P exposure was observed. Lung injury was assessed by measuring lactate dehydrogenase (LDH), alkaline phosphatase (ALP), total cell count, total protein, neutrophil elastase activity in bronchoalveolar lavage fluid (BALF). Reduction in phospholipid content further potentiated these parameters. GA oral administration (50 and 100mg/kg b.wt.) significantly showed protection of lung epithelium by suppression of caspases activities in lung tissue and reduction of total protein, total cells, elastase activity, LDH and ALP activities along with fortification of phospholipids in BALF. Histological observations also confirm the findings in above mentioned parameters. Results indicate a strong correlation between amelioration of sEH and TrxR activities, and NF-kappaB activation. The present investigation gives an insight into probable mechanisms of lung injuries induced by short term exposures of B(a)P and prevention by glycyrrhizic acid.

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doses of 0.1, 0.6 and 2.0 mg/kg/d and 0.05% Tween 80 as control by gavage from gestation days 2 to 21. The dams were allowed to give birth and liver samples from weaned (postnatal day 21) offspring rats were analyzed for PFOS content, relative liver weight, global DNA methylation, methylation of LINE-1 regulatory region, tumor suppressor gene glutathione S-transferase pi (GSTP) and p16 promoter methylation level, as well as related genes expression level. In PFOS-exposed weaned rats, compared to the control, global DNA methylation and methylation of LINE-1 regulatory region decreased significantly only in the 2.0 mg/kg/d group. Up to 30% of critical CpG sites (+79, 81 and 84) in GSTP promoter region were methylated in the livers of PFOS-treated rats, while p16 promoter methylation was not affected. In addition, the up-regulated expression of GSTP was observed and this increase was associated with its main pathway of transcription regulation: Keap1-Nrf2/MafK. Thus, early-induced changes in critical cytosines within the GSTP gene promoter region may be a biomarker of hepatic PFOS burden, though their direct role in PFOS-induced hepatotoxicity, including its potential carcinogenic action, needs further research.

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e identified. Amyloid beta (Abeta) plaques develop in the brains of LOAD patients and are considered to be a pathological hallmark of this disease. Recently 12 new Abeta toxicity modifier genes (ADSSL1, PICALM, SH3KBP1, XRN1, SNX8, PPP2R5C, FBXL2, MAP2K4, SYNJ1, RABGEF1, POMT2, and XPO1) have been identified that potentially play a role in LOAD risk. In this study, we have examined the association of 222 SNPs in these 12 candidate genes with LOAD risk in 1291 LOAD cases and 958 cognitively normal controls. Single site and haplotype analyses were performed using PLINK. Following adjustment for APOE genotype, age, sex, and principal components, we found single nucleotide polymorphisms (SNPs) in PPP2R5C, PICALM, SH3KBP1, XRN1, and SNX8 that showed significant association with risk of LOAD. The top SNP was located in intron 3 of PPP2R5C (P=0.009017), followed by an intron 19 SNP in PICALM (P=0.0102). Haplotype analysis revealed significant associations in ADSSL1, PICALM, PPP2R5C, SNX8, and SH3KBP1 genes. Our data indicate that genetic variation in these new candidate genes affects the risk of LOAD. Further investigation of these genes, including additional replication in other case-control samples and functional studies to elucidate the pathways by which they affect Abeta, are necessary to determine the degree of involvement these genes have for LOAD risk.

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y of inhaled PD is unknown, these studies were conducted to characterize the toxicity of inhaled PD across a range of concentrations in rodents. Male and female Wistar-Han rats and B6C3F1 mice were exposed to 0, 50, 100, or 200 ppm PD 6 h/d, 5 d/wk for up to 2 wk. Bronchoalveolar lavage fluid (BALF) was collected after 1, 3, 5, and 10 exposures, and histopathology was evaluated after 12 exposures. MCP-1, MCP-3, CRP, FGF-9, fibrinogen, and OSM were increased 2- to 9-fold in BALF of rats exposed for 5 and 10 days to 200 ppm. In mice, only fibrinogen was increased after 5 exposures to 200 ppm. The epithelium lining the respiratory tract was the site of toxicity in all mice and rats exposed to 200 ppm. Significantly, PD also caused both intraluminal and intramural fibrotic airway lesions in rats. The histopathological and biological changes observed in rats raise concerns that PD inhalation may cause BO in exposed humans.

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osures of rats would significantly increase lung tumor incidence. Male and female F344 rats (n = 81 to 178/gender) were exposed whole-body 6 h/day, 5 days/week for up to 30 months to smoke from 1R3 research cigarettes diluted to 100 (LS) or 250 (HS) mg total particulate matter/m(3), or sham-exposed to clean air (C). Gross respiratory tract lesions and standard lung and nasal sections were evaluated by light microscopy. A slight reduction of survival suggested that the HS level was at the maximum tolerated dose as commonly defined. Cigarette smoke exposure significantly increased the incidences of non-neoplastic and neoplastic proliferative lung lesions in females, while nonsignificant increases were observed in males. The combined incidence of bronchioloalveolar adenomas and carcinomas in females were: HS = 14%; LS = 6%; and C = 0%. These incidences represented minima because only standard lung sections and gross lesions were evaluated. Mutations in codon 12 of the K-ras gene occurred in 4 of 23 (17%) tumors. Three mutations were G to A transitions and one was a G to T transversion. The incidence of neoplasia of the nasal cavity was significantly increased at the HS, but not the LS level in both males and females (HS = 6%, LS = 0.3%, C = 0.4% for combined genders). These results demonstrate that chronic whole-body exposure of rats to cigarette smoke can induce lung cancer.

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erum enzymatic activity of AST, ALT, ALP, ACP and LDH. In addition, treatment with ascorbic acid prevented elevated production of LPO, PC and NO and restored the depletion of reduced SOD and CAT activities. Interestingly, ascorbic acid markedly upregulated lymphocytes relative mRNA expression of lymphocytes SOD2 gene corresponding to GAPDH, house keeping candidate gene in arsenic-treated rat, which might provide anti-oxidative activity in the blood.

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s were treated with corn oil vehicle under the same conditions. Animals were killed at 24h after the last treatment. Another group of rats treated with DEHP or corn oil vehicle (control group) for 2 days were held for 30 days without treatment to observe recovery. Testes were analyzed for histopathology, TUNEL staining, immunofluorescence (IF) and Western blot analyses. Animals exposed to DEHP for 2 days or 7 days showed severe alterations of seminiferous tubules characterized by germ cell sloughing. Animals from the longer term recovery group treated with DEHP showed foci of delayed spermatogenesis. A linear and continuous pattern of N-cadherin was observed in the basal compartment of the seminiferous tubules. A similar pattern but with higher IF intensity was observed for N-cadherin in rats treated with DEHP for 2 days or 7 days, compared to control animals. The alpha-, beta- and p120-catenins were detected in the basal compartment of seminiferous tubules in similar localization and IF pattern for DEHP and control groups. A significant increase in testicular N-cadherin and alpha-catenin levels was detected by Western blot analysis in DEHP-exposed versus control rats. No variations in N-cadherin or catenin expression were detected in the recovery groups. These findings demonstrate that DEHP induces an up-regulation of N-cadherin and alpha-catenin expression and may perturb cell-cell adhesion phenomena in the seminiferous tubule.

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n the yolk sacs of rat embryos on gestational days 10 and 11. Commencing at conception and throughout pregnancy, half of the streptozotocin-diabetic and half of the control rats received daily FA injections. Maternal diabetes impaired vascular morphology and decreased CuZnSOD and GPX-1 gene expression in yolk sacs. Maternal diabetes also increased the levels of CuZnSOD protein, increased the Bax/Bcl-2 protein ratio and decreased Vegf-A protein distribution. FA treatment normalized vascular morphology, decreased mRNA levels of all three SOD isoforms and increased Vegf-A mRNA levels, rectified CuZnSOD protein distribution and Bax/Bcl-2 ratio. A teratogenic diabetic environment produces a state of vasculopathy, oxidative stress, and mild apoptosis in the yolk sac. FA administration normalizes vascular morphology, diminishes apoptotic rate, and increases Vegf-A gene expression and protein distribution in the yolk sac of diabetic rats.

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al lipopolysaccharide (LPS), results in hepatocellular injury. We tested the hypothesis that hepatic gene expression changes could be distinguished among vehicle-, LPS-, RAN- and LPS/RAN-treated rats before the onset of significant liver injury in the LPS/RAN-treated rats (i.e., 3 h post-treatment). Rats were treated with LPS (44 x 10(6) EU/kg, i.v.) or its vehicle, then two hours later with RAN (30 mg/kg, i.v.) or its vehicle. They were killed 3 h after RAN treatment, and liver samples were taken for evaluation of liver injury and RNA isolation. Hepatic parenchymal cell injury, as estimated by increases in serum alanine aminotransferase (ALT) activity, was not significant at this time. Hierarchal clustering of gene expression data from Affymetrix U34A rat genome array grouped animals according to treatment. Relative to treatment with vehicle alone, treatment with RAN and/or LPS altered hepatic expression of numerous genes, including ones encoding products involved in inflammation, hypoxia, and cell death. Some were enhanced synergistically by LPS/RAN cotreatment. Real-time PCR confirmed robust changes in expression of B-cell translocation gene 2, early growth response-1, and plasminogen-activator inhibitor-1 (PAI-1) in cotreated rats. The increase in PAI-1 mRNA was reflected in an increase in serum PAI-1 protein concentration in LPS/RAN-treated rats. Consistent with the antifibrinolytic activity of PAI-1, significant fibrin deposition occurred only in livers of LPS/RAN-treated rats. The results suggest the possibility that expression of PAI-1 promotes fibrin deposition in liver sinusoids of LPS/RAN-treated rats and are consistent with the development of local ischemia and consequent tissue hypoxia.

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has become a major social problem, especially in developing urban areas. Further formaldehyde is classified as a genotoxicant in the respiratory tract of rats and humans. To better understand the molecular mechanisms involved in formaldehyde intoxication, we sought differentially regulated genes by formaldehyde exposure to Hs 680.Tr human trachea cells, using polymerase chain reaction (PCR)-based suppression subtractive hybridization. We identified 27 different formaldehyde-inducible genes, including those coding for the major histocompatibility complex, class IA, calcyclin, glutathione S-transferase pi, mouse double minute 2 (MDM2), platelet-derived growth factor receptor alpha, and which are known to be associated with cell proliferation and differentiation, immunity and inflammation, and detoxification. Induction of these genes by formaldehyde treatment was confirmed by reverse transcription PCR and western blot analysis. Further, the expression of calcyclin, glutathione S-transferase pi, PDGFRA and MDM2 were significantly induced in the tracheal epithelium of Sprague Dawley rats after formaldehyde inhalation. Our results suggest that the elevated levels of these genes may be associated with the formaldehyde-induced toxicity, and that they deserve evaluation as potential biomarkers for formaldehyde intoxication.

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tion in rodents are less clear. We therefore wished to evaluate, in a comparative fashion, a prototype immunoassay for serum CRP, a commercial assay for serum Hp, and an automated assay for Fbgn, using a model of acute inflammation in the rat. Additionally, pro-inflammatory cytokines and serum protein fractions were also measured. The model of inflammation used was the intraperitoneal injection of Freund's complete adjuvant (FCA). In a concluding experiment, findings with Hp in the FCA rat model were validated in a toxicologically relevant study involving the induction of acute hepatic inflammation using the model hepatotoxicant carbon tetrachloride (CCl(4)). Female Wistar Han rats were treated with a single injection of FCA in a dose-response study (1.25-10.0 ml/kg, sampling at 36 h) and two time-course studies (over 40 h and 21 days). In a final experiment, rats were dosed with CCl(4) at 0.8 ml/kg and sampled over a 17-day period. In FCA and CCl(4) experiments, serum/plasma was prepared and tissues taken at autopsy for histological assessment (CCl(4) study only). In the dose-response study, serum CRP, Hp and plasma Fbgn were increased at all FCA dose levels at 36 h post-dosing. Serum alpha(2) and beta(1) globulin fractions were also increased, while albumin levels were decreased. In the 40-h time-course study, CRP levels peaked at 25-40 h post-dosing, to approximately 120% of control (as 100%). Hp levels increased to a maximum at 25 and 40 h post-dosing with values greater than 400% of control, and alpha(2) and beta(1) globulin fractions peaked at 30 and 40 h post-dosing to 221 and 187% of control, respectively. Increased serum interleukin-6 (IL-6) and interleukin-1beta (IL-1beta) levels peaked at 20 h (11-fold) and 25 h (19-fold), respectively. In a 21-day time-course study, no increased CRP levels were measured despite elevated levels of Hp, which peaked at 36 h (approximately 7-fold above control), and remained elevated up to 21 days. IL-6 and IL-1beta levels peaked at 12 h (19-fold) and 24 h (28-fold), respectively. Liver histopathology of animals treated with CCl(4) showed centrilobular hepatocellular degeneration and necrosis (most significant at 36 h) with an inflammatory response (most significant at 48 h). Resolution of the lesion was complete by 4 days post-dosing. Serum alanine aminotransferase, aspartate aminotransferase and glutamate dehydrogenase levels peaked at 36 h post-dosing. Hp levels increased maximally at 48 h (426% of control). We conclude that serum CRP is a poor marker of acute inflammation in the rat in comparison with serum Hp and plasma Fbgn. Between Hp and Fbgn, serum Hp is shown to be the most sensitive and useful marker of acute inflammation.

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er with that of the expression of metallothionein (MT) isoforms in a mouse model [superoxide dismutase1 transgenic (SOD1 Tg) mouse] of ALS. The expression of MT-I and MT-II (MT-I/II) isoforms were measured together with Western blotting, copper level, and lipid peroxides amounts increased in an age-dependent manner in the spinal cord, the region responsible for motor paralysis. A significant increase was already seen as early as 8-week-old SOD1 Tg mice, at which time the mice had not yet exhibited motor paralysis, and showed a further increase at 16 weeks of age, when paralysis was evident. Inversely, the spinal zinc level had significantly decreased at both 8 and 16 weeks of age. The third isoform, the MT-III level, remained at the same level as an 8-week-old wild-type mouse, finally increasing to a significant level at 16 weeks of age. It has been believed that a mutant SOD1 protein, encoded by a mutant SOD1, gains a novel cytotoxic function while maintaining its original enzymatic activity, and causes motor neuron death (gain-of-toxic function). Copper-mediated oxidative stress seems to be a probable underlying pathogenesis of gain-of-toxic function. Taking the above current concepts and the classic functions of MT into account, MTs could have a disease modifying property: the MT-I/II isoform for attenuating the gain-of-toxic function at the early stage of the disease, and the MT-III isoform at an advanced stage.

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on of the pathogenesis has not been clearly established. To understand the mechanism of PQ in pulmonary toxicity, we developed an animal model of PQ-induced lung injury by intranasal instillation of PQ solution using C57Black/6J mice. Twenty microliters of PQ solution (0.01, 0.01, and 0.04 mg/mouse) was applied through the nares, and the same amount of vehicle was applied in control mice. The pathological progression of lung pathology in our mouse model was very similar to that of patients suffering from PQ poisoning. The lungs of some animals exposed to PQ showed acute fulmination, resulting in death from 5 days post-exposure, but others showed a more protracted injury, resulting in typical pulmonary fibrosis at 3 weeks. Using this PQ-poisoned mouse model, we examined the gene expression at the initial destructive phase (within 5 days) that fibrosis has not completely developed. We prepared RNAs after 6h, 24h, and 5 days and examined the changes of the expression levels for 45 selected genes. The genes showing >2-fold increase at 6h or a time-dependent decrease during this experimental period may be the early markers for the destructive phase. These genes are Mt1, Mt2, Hmox1, Gcl, GR, IL-6, IL-13, Txn1, Fas, FasL, Lpin2, Mmp1a, Mmp12, Sfp-B, Sfp-D, CAT, EC-SOD, GST, and Pltp. On the other hand, the genes involved in the development of fibrosis, such as procollagen, Fn1, Eln, SMA, and Mmp9, Timp1 were significantly increased on day 5, not at 6h nor at 24h, after PQ treatment (the late marker). The genes showing a significant increase (Mmp3 and Mmp8) or decrease (VEGFA) at 24h and 5 days and not at 6h may be also the late markers. These changes in gene expression, which are equalled to functional activities of proteins, will be the targets for future studies focused on the development on PQ-induced pulmonary damage.

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ost of its toxic effects through the aryl hydrocarbon receptor (AhR). OBJECTIVES: The aims of the present study were to quantitatively evaluate changes in bone geometry, mineral density and biomechanical properties following long-term exposure to TCDD, and to further investigate the role of AhR in TCDD-induced bone alterations. To this end, tissue material used in the study was derived from TCDD-exposed Long-Evans (L-E) and Han/Wistar (H/W) rats, which differ markedly in sensitivity to TCDD-induced toxicity due to a strain difference in AhR structure. METHODS: Ten weeks old female L-E and H/W rats were administered TCDD s.c. once per week for 20 weeks, at doses corresponding to calculated daily doses of 0, 1, 10, 100 and 1000ngTCDD/kgbw (H/W only). Femur, tibia and vertebra from the L-E and H/W rats were analyzed by peripheral quantitative computed tomography (pQCT) and biomechanical testing at multiple sites. Dose-response modeling was performed to establish benchmark doses for the analyzed bone parameters, and to quantify strain sensitivity differences for those parameters, which were affected by TCDD exposure in both rat strains. RESULTS: Bone geometry and bone biomechanical parameters were affected by TCDD exposure, while bone mineral density parameters were less affected. The trabecular area at proximal tibia and the endocortical circumference at tibial diaphysis were the parameters that showed the highest maximal responses. Significant strain differences in response to TCDD treatment were observed, with the L-E rat being the most sensitive strain. For the parameters that were affected in both strains, the differences in sensitivity were quantified, showing the most pronounced (about 49-fold) strain difference for cross-sectional area of proximal tibia. CONCLUSION: The study provides novel information about TCDD-induced bone alterations at doses, which are of relevance from a health risk assessment point of view. In addition, the obtained results provide further support for a distinct role of the AhR in TCDD-induced bone alterations, and suggest that the benchmark dose modeling approach is appropriate for quantitative evaluation of bone toxicity parameters.

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ction of free radicals. Lycopene belongs to the group of natural carotenoids, which are found in many fruits, vegetables and other green plants. Lycopene, the most potent antioxidant protects against oxidative damage. The present study was conducted to elucidate the protective role of lycopene against Aroclor 1254-induced changes in Leydig cellular steroidogenic acute regulatory (StAR) protein, cytochrome P450 side chain cleavage (P450 scc) enzyme expression and 3beta-hydroxy steroid dehydrogenase (3beta-HSD) activity. The rats were divided into four groups. Each group consists of six animals. Group I rats were administered with corn oil intraperitoneally (i.p.) for 30 days. Group II rats were treated with Aroclor 1254 (i.p.) 2mgkg(-1)body weight (bwt)day(-1) for 30 days. Group III rats were treated with Aroclor 1254 (i.p.) 2mgkg(-1)bwtday(-1) along with simultaneous supplementation of lycopene 4mgkg(-1)bwtday(-1) (gavage) for 30 days. Group IV rats administered with lycopene alone at the dose of 4mgkg(-1)bwt day(-1) (gavage) for 30 days. After 24h of the last treatment, animals were decapitated, blood was collected and serum testosterone level was estimated by radioimmunoassay (RIA). Testes were removed and Leydig cells were isolated in aseptic condition. StAR protein, cytochrome P450 scc enzyme expression were studied by Western blot analysis and 3beta-HSD activity was estimated spectrophotometrically. Aroclor 1254 treatment significantly reduced the serum testosterone level. Simultaneous supplementation of lycopene maintained the serum testosterone to near normal. Aroclor 1254 exposure decreased Leydig cellular StAR protein, cytochrome P450 scc enzyme expression and activity of 3beta-HSD. However, simultaneous supplementation of lycopene improved Leydig cellular StAR protein, cytochrome P450 scc expression and activity of 3beta-HSD. These results suggested that lycopene have ameliorative role against Aroclor 1254 induced Leydig cell dysfunction.

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(PbAc) or sodium acetate (NaAc; control), beginning 30 days prior to breeding and continuing until their pups were weaned. For the first part of this study, animals from both groups were killed when 31 days old, at 0800 h, for assessment of basal ovarian StAR gene expression. Results indicated Pb decreased (p < 0.01) both StAR transcripts. In the second part of the study, pregnant mare serum gonadotropin (PMSG) was administered to half of the Pb-treated and control animals at 0800 h. These animals, and animals from both groups that did not receive PMSG, were killed and ovaries and blood collected at 1600 h to assess ovarian StAR protein and E2 responsiveness to gonadotropin stimulation. Pb decreased (p < 0.0001) basal StAR protein expression and lowered (p < 0.001) E2 levels in animals that did not receive PMSG. PMSG induced (p < 0.0001) StAR protein in both the Pb-treated and control animals, an action associated with increased (p < 0.001) serum levels of E2. These results are the first to show that Pb alters basal StAR synthesis, but does not alter gonadotropin-stimulated StAR synthesis, hence, suggesting the primary action of Pb to suppress E2 is through its known action to suppress the serum levels of luteinizing hormone and not due to decreased responsiveness of StAR synthesizing machinery.

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ungs of Sprague-Dawley rats 24 h following exposure to varying doses of silica by intratracheal instillation. Arginase expression was evaluated by activity assay, quantification of arginase I and arginase II mRNA levels using real-time polymerase chain reaction (PCR), and immunohistochemistry. Analyses of cells and fluid obtained by bronchoalveolar lavage (BAL) showed that markers of pulmonary inflammation, tissue damage, activation of alveolar macrophages (AM) and NO production were significantly increased by all silica doses. Arginase activity was increased also in AMs isolated from BAL fluid of silica-treated rats. Silica produced two- and three-fold increases in arginase activity of whole lung at doses of 1 and 5 mg/100 g body weight, respectively. Levels of arginase I mRNA, but not of arginase II mRNA, were similarly elevated. In control lungs, arginase I immunoreactivity was observed only in AMs sparsely dispersed throughout the lung; no inducible nitric oxide synthase (iNOS) immunoreactivity was detected. In silica-treated lungs, arginase I and iNOS were co-expressed in most AMs that were abundantly clustered at inflammatory foci. The rapid induction of arginase I expression in inflammatory lung cells, similar to induction of arginase in other inflammatory lung diseases, implicates elevated arginase activity as a factor in the development of lung damage following exposure to silica.

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chemopreventive efficiency of diclofenac, which is a preferentially selective COX-2 inhibitor in lung cancer. Female Wistar rats were divided into 4 groups. Group 1 served as control and received saline intratracheally, once. In group 2 lung cancer was induced by a single intratracheal instillation of dimethybenz(a)anthracene (DMBA) (20 mg/kg body weight). Group 3 was given the intervention of diclofenac (8 mg/kg body weight) daily by oral gavage, in addition to DMBA. Group 4 received diclofenac alone. After 18 weeks of treatment, animals were sacrificed and various studies done. COX-2 expression as seen by western immunoblot and immunohistochemistry (IHC) was increased in the DMBA group, while diclofenac intervention was able to bring down the levels of the enzyme. Apoptosis studies by DNA fragmentation, TUNEL and fluorescent dyes reveal the lowered number of apoptotic cells in group 2. The levels were restored by diclofenac treatment in group 3. There was also a significant reduction in tumor incidence in DMBA+Diclofenac treated animals. All these results indicate that diclofenac acts as an effective chemopreventive agent that mediates its effects by the induction of apoptosis in cancer tissue and suppression of COX-2 enzyme.

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was found to be maximum at the fourth week of treatment, and the sedimentation pattern of DNA was found to return to near normal size by the seventh week of NDEA treatment. Simultaneously, there were perturbations in the nuclear enzymes involved in DNA replication and repair. Activities of DNA polymerase beta, DNA ligase, and topoisomerase were found to increase in as early as the first week of NDEA treatment and reached the maximum at the fourth week, and thereafter declined to normal level by the eighth week of treatment. Concomitantly, the activities of DNA polymerase alpha, DNA primase, and RNA polymerase which were unaltered in the initial period of carcinogen treatment recorded a marked increase after sixth week of NDEA treatment. Results suggest that administration of NDEA inflicts DNA damage, which is manifested as increase in DNA repair enzymes in the initial period and activated DNA replicative enzymes at a later period, indicating the active proliferation of transformed cells.

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nt roles in progesterone synthesis. The Wistar rats were exposed to cadmium in vivo (at 2.5, 5, 7.5mg/kg, as a single s.c. dose). We showed that the serum P(4) and granule cells P(4) of rats were significantly lower than control group. Ovaries granule cells were incubated in Dulbecco-modified Eagle medium +15% fetal bovine serum with 0, 10, 20, or 40 microM CdCl(2) in vitro, progesterone levels were declined in a dose-dependent manner. Our data showed that the expression of StAR and P450scc in vivo or in vitro were inhibited when treated with CdCl(2) (p<0.05). Coculture with 8-bromo-cAMP enhanced progesterone secretion in untreated cultures and reversed the decline in progesterone secretion induced by CdCl(2) treatment; the expression of StAR mRNA and P450scc mRNA in 8-Br-cAMP+40 microM CdCl(2) were significantly higher than 40 microM CdCl(2), and were lower than control group. We concluded that StAR, which delivers cholesterol to the inner mitochondrial membrane, is one site at which Cd interferes with progesterone production in cultured rats ovarian granule cells; P450scc, which conveys cholesterol to pregnenolone, is anther site. The mechanisms were mainly controlled by the cAMP-dependent pathway.

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' of development. Male rats were injected with 2.4 microg per pup per day of BPA from postnatal days (PND) 1-5 and controls received vehicle. During development, the testicular expression pattern of SRs (AR, ERbeta and ERalpha), coactivators (SRC-1, SRC-2 and SRC-3) and corepressors (NCoR and SMRT) in BPA-exposed rats were compared. A significant decrease in the expression of SRs was seen in the BPA group. SRC-1 showed a significant decrease, whereas SRC-2 and SRC-3 showed a significant increase in the protein expression whereas corepressor expression remained unaltered in the BPA-exposed groups. Such impairments in the expression pattern can be a putative mechanism of adversities on fertility as a result of BPA exposure. Copyright (c) 2013 John Wiley & Sons, Ltd.

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from a diffuse perinuclear and cytoplasmic distribution to a pattern decorating F-actin filaments. Over this time filaments were decreased in number in many cells, and membrane ruffling was initiated. Western blotting and 125I-F-actin gel overlays demonstrated an increase in actin-binding gelsolin activity in the cytoskeletal fraction of cell extracts following Cd2+ treatment. In in vitro polymerization assays, gelsolin acted as a nucleating factor and increased the rate of polymerization. Cytosolic extracts also increased the polymerization rate. Addition of Cd2+ together with gelsolin further increased the rate of polymerization. Gelsolin enhanced depolymerization of purified actin, and Cd2+ partially suppressed this effect. However, cytoskeletal extracts from Cd2+-treated cells also markedly increased depolymerization, suggesting further that Cd2+ may activate cellular component(s) such as gelsolin for actin binding. We conclude that a major effect of Cd2+ on the mesangial cell cytoskeleton is manifest through activating the association of gelsolin with actin, with gelsolin's severing properties predominating under conditions found in Cd2+-treated cells.

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traffic density. A decrease in lead levels was also noted in vegetation as distance from highway increased.

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='font-weight:700;'>toxicity, we studied the effects of NHE-1 inhibitors on nerve and dorsal root ganglion (DRG) neurons isolated from the adult rat. Compound action potentials (CAPs) were recorded from electrically stimulated sections of isolated sciatic nerve/DRG/root preparations. Whole-cell patch-clamp technique was used to record fast and slow voltage-dependent Na+ currents from dissociated DRG neurons (29-41 microm). Exposures to 1 and 10 microM of a selective NHE-1 inhibitor reduced the amplitude of the CAP recorded from the dorsal root by 33% and 58%, respectively (p < 0.05). The compound had no effect on CAPs recorded from the ventral root. Perfusion of dissociated DRG neurons with NHE-1 inhibitors at 10 and 100 microM shifted voltage-dependent inactivation curves of fast Na+ current by as much as 11 mV (p < 0.001) in the hyperpolarizing direction. No shift was observed in slow Na+ currents. No statistically significant drug effects were observed on voltage-dependent activation or recovery from inactivation of either fast or slow Na+ currents. These results suggest that NHE-1 inhibitors may reduce peripheral neuronal excitability by shifting fast Na+ channels into the inactivated state under physiological conditions. Such effects may underlie peripheral neuropathies reported in rats and dogs with NHE-1 inhibitors.

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ported that sinapic acid has antioxidant efficacy as metal chelators due to the orientation of functional groups. However, it has not yet been examined in experimental animals. In light of this fact, the purpose of this study was to characterize the protective role of sinapic acid against arsenic induced toxicity in rats. Rats were orally treated with arsenic alone (5mg/kg body weight (bw)/day) plus sinapic acid at different doses (10, 20 and 40mg/kg bw/day) for 30days. Hepatotoxicity was measured by the increased activities of serum hepatospecific enzymes namely aspartate transaminase, alanine transaminase, alkaline phosphatase, gamma glutamyl transferase, lactate dehydrogenase and total bilirubin along with increased elevation of lipid peroxidative markers, thiobarbituric acid reactive substances, lipid hydroperoxides, protein carbonyl content and conjugated dienes. The toxic effect of arsenic was also indicated by significantly decreased activities of enzymatic antioxidants like superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, glutathione reductase and glucose-6-phosphate dehydrogenase along with non-enzymatic antioxidant like reduced glutathione. Administration of sinapic acid exhibited significant reversal of arsenic induced toxicity in hepatic tissue. The effect at a dose of 40mg/kgbw/day was more pronounced than the other two doses (10 and 20mg/kgbw/day). All these changes were supported by reduction of arsenic concentration and histopathological observations of the liver. These results suggest that sinapic acid has a protective effect over arsenic induced toxicity in rat.

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mutated into a cocaine hydrolase (CocH) can maintain enzyme levels that destroy cocaine within seconds after appearance in the blood stream, allowing little to reach the brain. Rapid enzyme action causes a sharp rise in plasma levels of two cocaine metabolites, benzoic acid (BA) and ecgonine methyl ester (EME), a smooth muscle relaxant that is mildly hypotensive and, at best, only weakly rewarding. The present study, utilizing Balb/c mice, tested reward effects and cardiovascular effects of administering EME and BA together at molar levels equivalent to those generated by a given dose of cocaine. Reward was evaluated by conditioned place preference. In this paradigm, cocaine (20 mg/kg) induced a robust positive response but the equivalent combined dose of EME + BA failed to induce either place preference or aversion. Likewise, mice that had undergone gene transfer with mouse CocH (mCocH) showed no place preference or aversion after repeated treatments with a near-lethal 80 mg/kg cocaine dose. Furthermore, a single administration of that same high cocaine dose failed to affect blood pressure as measured using the noninvasive tail-cuff method. These observations confirm that the drug metabolites generated after CocH gene transfer therapy are safe even after a dose of cocaine that would ordinarily be lethal.

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for 3 h or 12 h by HPLC. Flow cytometry (FCM) and RT-PCR technology were used to detect the levels of tyrosine hydroxylase (TH) mRNA and protein in PC12 cells treated with different doses of deltamethrin for 3 h or 12 h. Deltamethrin decreased the levels of dopamine, TH mRNA and TH protein in PC12 cells, and the increased dopamine levels induced by l-Dopa at 20-50 microM were also decreased by deltamethrin for 3 h or 12 h. These results indicate that deltamethrin decreases dopamine content by the inhibition of TH mRNA and protein level in PC12 cells and maybe also have inhibitory effect on aromatic l-amino acid decarboxylase.