Time Dependent Gene Expression Changes in the Liver of Mice Treated with Benzene

Benzene is used as a general purpose solvent. Benzene metabolism starts from phenol and ends with p-benzoquinone and o-benzoquinone. Liver injury inducted by benzene still remains a toxicologic problem. Tumor related genes and immune responsive genes have been studied in patients suffering from benzene exposure. However, gene expression profiles and pathways related to its hepatotoxicity are not known. This study reports the results obtained in the liver of BALB/C mice (SLC, Inc., Japan) administered 0.05 ml/100 g body weight of 2% benzene for six days. Serum, ALT, AST and ALP were determined using automated analyzer (Fuji., Japan). Histopathological observations were made to support gene expression data. c-DNA microarray analyses were performed using Affymetrix Gene-chip system. After six days of benzene exposure, twenty five genes were down regulated whereas nineteen genes were up-regulated. These gene expression changes were found to be related to pathways of biotransformation, detoxification, apoptosis, oxidative stress and cell cycle. It has been shown for the first time that genes corresponding to circadian rhythms are affected by benzene. Results suggest that gene expression profile might serve as potential biomarkers of hepatotoxicity during benzene exposure

Reference Electrode at Molten Salt: A Comparative Analysis of Electroceramic Membranes

A reference electrode is important for controlling electrochemical reactions. Evaluating properties such as the reduction potential of the elements is necessary to optimize the electrochemical processes in pyroprocessing, especially in a multicomponent environment. In molten chloride systems, which are widely used in pyroprocessing, a reference electrode is made by enclosing the silver wire and molten salt solution containing silver chloride into the membranes. However, owing to the high temperature of the molten salt, the choice of the membrane for the reference electrode is limited. In this study, three types of electroceramic, mullite, Pyrex, and quartz, were compared as reference electrode membranes. They are widely used in molten salt electrochemical processes. The potential measurements between the two reference electrode systems showed that the mullite membrane has potential deviations of approximately 50 mV or less at temperatures higher than 650??C, Pyrex at temperatures lower than 500??C, and quartz at temperatures higher than 800??C. Cyclic voltammograms with different membranes showed a significant potential shift when different membranes were utilized. This research demonstrated the uncertainties of potential measurement by a single membrane and the potential shift that occurs because of the use of different membranes

miRNA regulation of cytotoxic effects in mouse Sertoli cells exposed to nonylphenol

Background: It is known that some environmental chemicals affect the human endocrine system. The harmful effects of endocrine disrupting chemical (EDC) nonylphenol (NP) have been studied since the 1980s. It is known that NP adversely affects physiological functions by mimicking the natural hormone 17 beta-estradiol. In the present study, we analyzed the expression of miRNAs and their target genes in mouse Sertoli TM4 cells to better understand the regulatory roles of miRNAs on Sertoli cells after NP exposure. Methods: Mouse TM4 Sertoli cells were treated with NP for 3 or 24 h, and global gene and miRNA expression were analyzed using Agilent mouse whole genome and mouse miRNA v13 arrays. Results: We identified genes that were > 2-fold differentially expressed in NP-treated cells and control cells (P < 0.05) and analyzed their functions through Gene Ontology analysis. We also identified miRNAs that were differentially expressed in NP-treated and control cells. Of the 186 miRNAs the expression of which differed between NP-treated and control cells, 59 and 147 miRNAs exhibited 1.3-fold increased or decreased expression at 3 and 24 h, respectively. Network analysis of deregulated miRNAs suggested that Ppara may regulate the expression of certain miRNAs, including miR-378, miR-125a-3p miR-20a, miR-203, and miR-101a, after exposure to NP. Additionally, comprehensive analysis of predicted target genes for miRNAs showed that the expression of genes with roles in cell proliferation, the cell cycle, and cell death were regulated by miRNA in NP-treated TM4 cells. Levels of expression of the miRNAs miR-135a* and miR-199a-5p were validated by qRT-PCR. Finally, miR-135a* target gene analysis suggests that the generation of reactive oxygen species (ROS) following exposure to NP exposure may be mediated by miR-135a* through regulation of the Wnt/beta-catenin signaling pathway. Conclusions: Collectively, these data help to determine NP's actions on mouse TM4 Sertoli cells and increase our understanding of the molecular mechanisms underlying the adverse effects of xenoestrogens on the reproductive system.This work was supported an Eco-Technopia 21 project grant from the Ministry of Environment (Development of Decision Method of Chromosomal Abnormality in Reproductive System by Toxic Substances at the Korea Institute of Toxicology)

Gene expression profiling in the lung tissue of cynomolgus monkeys in response to repeated exposure to welding fumes

Many in the welding industry suffer from bronchitis, lung function changes, metal fume fever, and diseases related to respiratory damage. These phenomena are associated with welding fumes; however, the mechanism behind these findings remains to be elucidated. In this study, the lungs of cynomolgus monkeys were exposed to MMA-SS welding fumes for 229 days and allowed to recover for 153 days. After the exposure and recovery period, gene expression profiles were investigated using the Affymetrix GeneChip® Human U133 plus 2.0. In total, it was confirmed that 1,116 genes were up-or down-regulated (over 2-fold changes, P < 0.01) for the T1 (31.4 ± 2.8 mg/m3) and T2 (62.5 ± 2.7 mg/m3) dose groups. Differentially expressed genes in the exposure and recovery groups were analyzed, based on hierarchical clustering, and were imported into Ingenuity Pathways Analysis to analyze the biological and toxicological functions. Functional analysis identified genes involved in immunological disease in both groups. Additionally, differentially expressed genes in common between monkeys and rats following welding fume exposure were compared using microarray data, and the gene expression of selected genes was verified by real-time PCR. Genes such as CHI3L1, RARRES1, and CTSB were up-regulated and genes such as CYP26B1, ID4, and NRGN were down-regulated in both monkeys and rats following welding fume exposure. This is the first comprehensive gene expression profiling conducted for welding fume exposure in monkeys, and these expressed genes are expected to be useful in helping to understand transcriptional changes in monkey lungs after welding fume exposure

Accumulation of diacylglycerol induced by CCl_4-derived radicals in rat liver membrane and its inhibition with radical trapping reagent : FT-IR spectroscopic and HPLC chromatographic observations

We have investigated the accumulation of diacylglycerol (DAG) induced by carbon tetrachloride (CCl_4)-derived radicals in the liver of female Sprague-Dawley (SD) rats after intraperitoneally injecting CCl_4. DAG is an intracellular activator of protein kinase C (PKC) which regulates cell proliferation and differentiation. The electron spin resonance (ESR) study gave the signal of the PBN-CCl_3 adduct in the liver of the rats which were pretreated with PBN, confirming that CCl_4 was metabolized into CCl_3 radicals with cytochrome P450 enzyme and indicating that PBN could trap them. The blood biochemical assay supported the trapping of the CCl_3 radicals ; the pretreatment of rats with PBN inhibited the increase in the GOT and GPT values upon exposure to CCl_4. The Fourier transform-infrared (FT-IR) study indicated in comparison with the model compounds that the CCl_4-injected rats accumulated DAG in addition to phosphatidylcholine, phosphatidylethanolamine and triglyceride (TG) in the lipid membrane fraction of the liver homogenate. DAG was found to be ca. 10-15 % of the membrane phospholipids by weight. However, DAG was not found in the lipid of the liver microsomes, suggesting that it is formed only in the cell membrane of liver. Also, neither DAG nor TG was found in the lipid membrane of the rats that were pretreated with PBN followed by an injection of CCl_4. The formation of DAG was confirmed by an HPLC study. The activation of PKC was observed in liver homogenate in the rats that were injected with CCl_4. On the basis of the above findings, it was concluded that the CCl_4-derived radicals stimulate PKC through the accumulation of DAG in the liver membrane of the rats. Furthermore, it was shown that PBN has a protective and therapeutic effect against CCl_4-induced damage

Application of FT-IR and ESR spectroscopic techniques to the study of CCl_4-induced peroxidation in rat liver microsomes

FT-IR and ESR were used for on the investigation of the CCl_4-induced peroxidation of rat liver microsomes in combination with biochemical methods. Lipid peroxidation was assayed by TBA reagent in the presence of CCl_4 and NADPH. The CCl_3 radical was detected by ESR spectroscopy with a spin trapping reagent of PBN. The FT-IR spectroscopy revealed that absorption band of -C-H in -C=C-H decreased in intensity at 3012 cm^, but the absorption bands of the phosphate head and choline in the phospholipids did not significantly change between 1300 and 900 cm^. These findings were interpreted to be due to the removal of H from -C=C-H by radicals as the first step of lipid peroxidation, and to the absence of dephosphorylation of phospholipids in the microsomal membrane. This is the first IR spectroscopic evidence indicating the nature of damage to a microsomal membrane caused by CCl_4 treatment. The spectroscopies used here demonstrated that they are useful tools to observe the damage to microsomal membranes

Diclofenac Disrupts the Circadian Clock and through Complex Cross-Talks Aggravates Immune-Mediated Liver Injury—A Repeated Dose Study in Minipigs for 28 Days

Diclofenac effectively reduces pain and inflammation; however, its use is associated with hepato- and nephrotoxicity. To delineate mechanisms of injury, we investigated a clinically relevant (3 mg/kg) and high-dose (15 mg/kg) in minipigs for 4 weeks. Initially, serum biochemistries and blood-smears indicated an inflammatory response but returned to normal after 4 weeks of treatment. Notwithstanding, histopathology revealed drug-induced hepatitis, marked glycogen depletion, necrosis and steatosis. Strikingly, the genomic study revealed diclofenac to desynchronize the liver clock with manifest inductions of its components CLOCK, NPAS2 and BMAL1. The > 4-fold induced CRY1 expression underscored an activated core-loop, and the dose dependent > 60% reduction in PER2mRNA repressed the negative feedback loop; however, it exacerbated hepatotoxicity. Bioinformatics enabled the construction of gene-regulatory networks, and we linked the disruption of the liver-clock to impaired glycogenesis, lipid metabolism and the control of immune responses, as shown by the 3-, 6- and 8-fold induced expression of pro-inflammatory CXCL2, lysozyme and ß-defensin. Additionally, diclofenac treatment caused adrenocortical hypertrophy and thymic atrophy, and we evidenced induced glucocorticoid receptor (GR) activity by immunohistochemistry. Given that REV-ERB connects the circadian clock with hepatic GR, its > 80% repression alleviated immune responses as manifested by repressed expressions of CXCL9(90%), CCL8(60%) and RSAD2(70%). Together, we propose a circuitry, whereby diclofenac desynchronizes the liver clock in the control of the hepatic metabolism and immune response