Drug-induced Liver Fibrosis: Testing Nevirapine in a Viral-like Liver Setting Using Histopathology, MALDI IMS, and Gene Expression.

Nevirapine (NVP) is associated with hepatotoxicity in 1-5% of patients. In rodent studies, NVP has been shown to cause hepatic enzyme induction, centrilobular hypertrophy, and skin rash in various rat strains but not liver toxicity. In an effort to understand whether NVP is metabolized differently in a transiently inflamed liver and whether a heightened immune response alters NVP-induced hepatic responses, female brown Norway rats were dosed with either vehicle or NVP alone (75 mg/kg/day for 15 days) or galactosamine alone (single intraperitoneal [ip] injection on day 7 to mimic viral hepatitis) or a combination of NVP (75/100/150 mg/kg/day for 15 days) and galactosamine (single 750 mg/kg ip on day 7). Livers were collected at necropsy for histopathology, matrix-assisted laser desorption/ionization imaging mass spectrometry and gene expression. Eight days after galactosamine, hepatic fibrosis was noted in rats dosed with the combination of NVP and galactosamine. No fibrosis occurred with NVP alone or galactosamine alone. Gene expression data suggested a viral-like response initiated by galactosamine via RNA sensors leading to apoptosis, toll-like receptor, and dendritic cell responses. These were exacerbated by NVP-induced growth factor, retinol, apoptosis, and periostin effects. This finding supports clinical reports warning against exacerbation of fibrosis by NVP in patients with hepatitis C.

An initial characterization of N-terminal-proatrial natriuretic peptide in serum of Sprague Dawley rats.

In the clinical setting, natriuretic peptides (NPs) have proven to be reliable noninvasive markers for diagnostic, prognostic, and therapeutic monitoring of heart failure. Given their proven utility in humans, NPs are potential candidates for translational biomarkers during drug development to detect drug-induced hemodynamic stress resulting in cardiac hypertrophy in preclinical species. We evaluated the intra- and interassay precision and the stability of serum N-terminal-proatrial natriuretic peptide (NT-proANP) using a commercially available enzyme-linked immunoassay (EIA). We then measured NT-proANP concentrations in 532 serum samples from 337 male Crl:CD(SD) rats with or without pressure-induced cardiac hypertrophy. Additionally, we established a reference range using samples from control animals across multiple studies. The data demonstrate that the NT-proANP EIA is a robust and reproducible assay for the measurement of NT-proANP. The noninvasive translational utility, minimal sample volume requirement, and the lack of existing hypertrophic biomarkers in the male rat make NT-proANP an excellent candidate for further interrogation as a biomarker of cardiac hypertrophy in preclinical toxicology investigations.

Transformation of SV40-immortalized human uroepithelial cells by 3-methylcholanthrene increases IFN- and Large T Antigen-induced transcripts.

BACKGROUND: Simian Virus 40 (SV40) immortalization followed by treatment of cells with 3-methylcholanthrene (3-MC) has been used to elicit tumors in athymic mice. 3-MC carcinogenesis has been thoroughly studied, however gene-level interactions between 3-MC and SV40 that could have produced the observed tumors have not been explored. The commercially-available human uroepithelial cell lines were either SV40-immortalized (HUC) or SV40-immortalized and then 3-MC-transformed (HUC-TC). RESULTS: To characterize the SV40 - 3MC interaction, we compared human gene expression in these cell lines using a human cancer array and confirmed selected changes by RT-PCR. Many viral Large T Antigen (Tag) expression-related changes occurred in HUC-TC, and it is concluded that SV40 and 3-MC may act synergistically to transform cells. Changes noted in IFP 9-27, 2'-5' OAS, IF 56, MxA and MxAB were typical of those that occur in response to viral exposure and are part of the innate immune response. Because interferon is crucial to innate immune host defenses and many gene changes were interferon-related, we explored cellular growth responses to exogenous IFN-gamma and found that treatment impeded growth in tumor, but not immortalized HUC on days 4 - 7. Cellular metabolism however, was inhibited in both cell types. We conclude that IFN-gamma metabolic responses were functional in both cell lines, but IFN-gamma anti-proliferative responses functioned only in tumor cells. CONCLUSIONS: Synergism of SV40 with 3-MC or other environmental carcinogens may be of concern as SV40 is now endemic in 2-5.9% of the U.S. population. In addition, SV40-immortalization is a generally-accepted method used in many research materials, but the possibility of off-target effects in studies carried out using these cells has not been considered. We hope that our work will stimulate further study of this important phenomenon.

5-hydroxytryptamine (5HT)-induced valvulopathy: compositional valvular alterations are associated with 5HT2B receptor and 5HT transporter transcript changes in Sprague-Dawley rats.

Several drugs have been linked to valvulopathy in humans, including therapeutic agents for obesity, Parkinson's disease and migraine. There is increasing evidence that the 5-hydroxytryptamine 2B receptor (5HT2BR) activation and/or increased circulating 5HT (5-hydroxytryptamine) may play a significant role in the pathogenesis of drug-induced valvulopathy. In the present study, we investigated whether 7-day 5HT subcutaneous injections led to structural and compositional abnormalities in conjunction with transcriptomic modulation of 5HT2BR and 5HT transporter (5HTT) genes in the aortic and mitral valves of Sprague-Dawley (SD) rats. Subcutaneous injections of 5HT for 7 days resulted in thickening and compositional alteration of aortic and mitral valves in SD rats. More specifically, valve-leaflets from 5HT-treated rats had greater valve thickness, a higher amount of glycosaminoglycans (GAGs) and a lower amount of collagen. The compositional alteration was associated with up-regulation and down-regulation of 5HT2BR and 5HTT genes, respectively. The present study strongly suggests that the activation of 5HT2BR and inhibition of 5HTT played a significant role in the pathogenesis of 5HT-induced valvulopathy in SD rats. Thus, these findings further highlight the necessity and/or utilization of animal models to screen potential valvular effects of serotonergic compounds.

5-Hydroxytryptamine (5HT) receptors in the heart valves of cynomolgus monkeys and Sprague-Dawley rats.

5-Hydroxytryptamine-2B receptor (5HT2BR) stimulation is known to cause fibroblast mitogenesis, and the mitogenic effect has been proposed to trigger valvular heart disease in humans. In this study, we used real-time polymerase chain reaction (TaqMan) to quantify transcript levels of 5HT2B, 5HT2C, and 5HT1B receptors and immunohistochemistry (IHC) to detect the tissue localization of these receptors in the normal heart valves of cynomolgus (CM) monkeys and Sprague-Dawley (S-D) rats. In both species, positive immunostaining was noted for 5HT1B and 5HT2B receptors in mitral, tricuspid, aortic, and pulmonary valves, and the cell types showing positive staining were interstitial cells and endothelial cells lining the valve leaflet. In CM monkeys, 5HT2CR was expressed only in the endothelial cells lining the leaflet, whereas S-D valves were negative for this receptor. IHC results were correlated with 5HT2B and 5HT1B receptor transcripts for all four valves. However, 5HT2C receptor transcripts were lower than 5HT2B or 5HT1B in all CM monkey valves, whereas 5HT2C transcripts were below the level of detection in any of the S-D rat valves. Our data showed the expression of 5HT2B, 5HT1B, and 5HT2C receptors in the normal heart valves of CM monkeys and S-D rats, and IHC and TaqMan techniques may be used to study the potential mechanism of compounds with 5HT2BR agonist activity.

Clofibrate-induced gene expression changes in rat liver: a cross-laboratory analysis using membrane cDNA arrays.

Microarrays have the potential to significantly impact our ability to identify toxic hazards by the identification of mechanistically relevant markers of toxicity. To be useful for risk assessment, however, microarray data must be challenged to determine reliability and interlaboratory reproducibility. As part of a series of studies conducted by the International Life Sciences Institute Health and Environmental Science Institute Technical Committee on the Application of Genomics to Mechanism-Based Risk Assessment, the biological response in rats to the hepatotoxin clofibrate was investigated. Animals were treated with high (250 mg/kg/day) or low (25 mg/kg/day) doses for 1, 3, or 7 days in two laboratories. Clinical chemistry parameters were measured, livers removed for histopathological assessment, and gene expression analysis was conducted using cDNA arrays. Expression changes in genes involved in fatty acid metabolism (e.g., acyl-CoA oxidase), cell proliferation (e.g., topoisomerase II-Alpha), and fatty acid oxidation (e.g., cytochrome P450 4A1), consistent with the mechanism of clofibrate hepatotoxicity, were detected. Observed differences in gene expression levels correlated with the level of biological response induced in the two in vivo studies. Generally, there was a high level of concordance between the gene expression profiles generated from pooled and individual RNA samples. Quantitative real-time polymerase chain reaction was used to confirm modulations for a number of peroxisome proliferator marker genes. Though the results indicate some variability in the quantitative nature of the microarray data, this appears due largely to differences in experimental and data analysis procedures used within each laboratory. In summary, this study demonstrates the potential for gene expression profiling to identify toxic hazards by the identification of mechanistically relevant markers of toxicity.

Effects of minimally toxic levels of carbonyl cyanide P-(trifluoromethoxy) phenylhydrazone (FCCP), elucidated through differential gene expression with biochemical and morphological correlations.

Uncouplers of oxidative phosphorylation have relevance to bioenergetics and obesity. The mechanisms of action of chemical uncouplers of oxidative phosphorylation on biological systems were evaluated using differential gene expression. The transcriptional response in human rhabdomyosarcoma cell line (RD), was elucidated following treatment with carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), a classical uncoupling agent. Changes in mitochondrial membrane potential were used as the biological dosimeter. There was an increase in membrane depolarization with increasing concentrations of FCCP. The concentration at 75% uncoupling (20 microM) was chosen to study gene expression changes, using cDNA-based large-scale differential gene expression (LSDGE) platforms. At the above concentration, subtle light microscopic and clear gene expression changes were observed at 1, 2, and 10 h. Statistically significant transcriptional changes were largely associated with protein synthesis, cell cycle regulation, cytoskeletal proteins, energy metabolism, apoptosis, and inflammatory mediators. Bromodeoxyuridine (BrdU) and propidium iodide (PI) assays revealed cell cycle arrest to occur in the G1 and S phases. There was a significant initial decrease in the intracellular adenosine triphosphate (ATP) concentrations. The following seven genes were selected as potential molecular markers for chemical uncouplers: seryl-tRNA synthetase (Ser-tRS), glutamine-hydrolyzing asparagine synthetase (Glut-HAS), mitochondrial bifunctional methylenetetrahydrofolate dehydrogenase (Mit BMD), mitochondrial heat shock 10-kDa protein (Mit HSP 10), proliferating cyclic nuclear antigen (PCNA), cytoplasmic beta-actin (Act B), and growth arrest and DNA damage-inducible protein 153 (GADD153). Transcriptional changes of all seven genes were later confirmed with reverse transcription-polymerase chain reaction (RT-PCR). These results suggest that gene expression changes may provide a sensitive indicator of uncoupling in response to chemical exposure.

Etomoxir-induced oxidative stress in HepG2 cells detected by differential gene expression is confirmed biochemically.

Although they are known to be effective antidiabetic agents, little is published about the toxic effects of carnitine palmitoyltransferase-1 (CPT-1) inhibitors, such as etomoxir (ET). These compounds inhibit mitochondrial fatty acid beta-oxidation by irreversibly binding to CPT-1 and preventing entry of long chain fatty acids into the mitochondrial matrix. Treatment of HepG2 cells with 1 mM etomoxir for 6 h caused significant modulations in the expression of several redox-related and cell cycle mRNAs as measured by microarray analysis. Upregulated mRNAs included heme oxygenase 1 (HO1), 8-oxoguanine DNA glycosylase 1 (OGG1), glutathione reductase (GSR), cyclin-dependent kinase inhibitor 1A (CDKN1 [p21(waf1)]) and Mn+ superoxide dismutase precursor (SOD2); while cytochrome P450 1A1 (CYP1A1) and heat shock 70kD protein 1 (HSPA1A) were downregulated. Real time quantitative PCR (RT-PCR) confirmed the significant changes in 4 of 4 mRNAs assayed (CYP1A1, HO1, GSR, CDKN1), and identified 3 additional mRNA changes; 2 redox-related genes, gamma-glutamate-cysteine ligase modifier subunit (GCLM) and thioredoxin reductase (TXNRD1) and 1 DNA replication gene, topoisomerase IIalpha (TOP2A). Temporal changes in selected mRNA levels were examined by RT-PCR over 11 time points from 15 min to 24 h postdosing. CYP1A1 exhibited a 38-fold decrease by 4 h, which rebounded to a 39-fold increase by 20 h. GCLM and TXNRD1 exhibited 13- and 9-fold increases, respectively at 24 h. Etomoxir-induced oxidative stress and impaired mitochondrial energy metabolism were confirmed by a significant decrease in reduced glutathione (GSH), reduced/oxidized glutathione ratio (GSH/GSSG), mitochondrial membrane potential (MMP), and ATP levels, and by concurrent increase in oxidized glutathione (GSSG) and superoxide generation. This is the first report of oxidative stress caused by etomoxir.