Detection of genotoxic and non-genotoxic renal carcinogens in vitro in NRK-52E cells using a transcriptomics approach. (2012).

There is a need to develop quick, cheap, sensitive and specific methods to detect the carcinogenic potential of chemicals. Currently there is no in vitro model system for reliable detection of non-genotoxic carcinogens (NGTX) and current tests for detection of genotoxic carcinogens (GTX) can have low specificity. A transcriptomics approach holds promise and a few studies have utilised this technique. However, the majority of these studies have examined liver carcinogens with little work on renal carcinogens which may act via renal-specific NGTX mechanisms. In this study the normal rat renal cell line (NRK-52E) was exposed to sub-toxic concentrations of selected rat renal carcinogens and non-carcinogens (NC) for 6 h, 24 h and 72 h. Renal carcinogens were classified based on their presumed mode of action into GTX and NGTX classes. A whole-genome transcriptomics approach was used to determined genes and pathways as potential signatures for GTX, NGTX and those common to both carcinogenic events in vitro. For some of the GTX compounds an S9 drug metabolising system was included to aid pro-carcinogen activation. Only three genes were commonly deregulated after carcinogen (GTX + NGTX) exposure, one Mdm2 with a detection rate of 67%, and p21 and Cd55 with a detection rate of 56%. However, examination of enriched pathways showed that 3 out of 4 NGTX carcinogens and 4 out of 5 GTX carcinogens were related to known pathways involved in carcinogenesis giving a detection rate of 78%. In contrast, none of the NC chemicals induced any of the above genes or well-established carcinogenic pathways. Additionally, five genes (Lingo1, Hmox1, Ssu72, Lyrm and Usp9x) were commonly altered with 3 out of 4 NGTX carcinogens but not with NC or GTX carcinogens. However, there was no clear separation of GTX and NGTX carcinogens using pathway analysis with several pathways being common to both classes. The findings presented here indicate that the NRK-52E cell line has the potential to detect carcinogenic chemicals, although a much larger number of chemicals need to be used to confirm these findings

Transcriptomic alterations induced by Monuron in rat and human renal proximal tubule cells in vitro and comparison to rat renal-cortex in vivo

Monuron (1,1-dimethyl-3-(4-chlorophenyl)urea) is a non-selective phenylurea herbicide, widely used in developing countries although concerns have been raised about its toxicity and carcinogenicity. Monuron was evaluated by the National Toxicology Program in 1988 and shown to be a male rat-specific renal carcinogen. We report that oral administration of Monuron to male rats for 3 days, leads to a larger number of genes being differentially expressed in the renal-cortex than in the liver. Further, we observed up-regulation of cell cycle genes and genes involved in cell proliferation in the renal-cortex while in the liver xenobiotic metabolising enzymes were up-regulated. We also identified one commonly down-regulated gene in both organs – fragile histidine triad gene (Fhit), a putative tumour suppressor gene; however the down-regulation was only significant at the protein level in the liver. In addition, we conducted in vitro whole-genome transcriptomics studies with human and rat renal cortical cells. Rat cells exposed to Monuron showed down-regulation of sterol biosynthesis, spliceosome and cell cycle genes and up-regulation of genes involved in amino acid metabolism and transport. No genes were found to be differentially expressed in human cells exposed to Monuron. Overall, the findings from the in vitro studies showed very little overlap with the whole animal findings

Effect of Chemical Mutagens and Carcinogens on Gene Expression Profiles in Human TK6 Cells

Characterization of toxicogenomic signatures of carcinogen exposure holds significant promise for mechanistic and predictive toxicology. In vitro transcriptomic studies allow the comparison of the response to chemicals with diverse mode of actions under controlled experimental conditions. We conducted an in vitro study in TK6 cells to characterize gene expression signatures of exposure to 15 genotoxic carcinogens frequently used in European industries. We also examined the dose-responsive changes in gene expression, and perturbation of biochemical pathways in response to these carcinogens. TK6 cells were exposed at 3 dose levels for 24 h with and without S9 human metabolic mix. Since S9 had an impact on gene expression (885 genes), we analyzed the gene expression data from cells cultures incubated with S9 and without S9 independently. The ribosome pathway was affected by all chemical-dose combinations. However in general, no similar gene expression was observed among carcinogens. Further, pathways, i.e. cell cycle, DNA repair mechanisms, RNA degradation, that were common within sets of chemical-dose combination were suggested by clustergram. Linear trends in dose–response of gene expression were observed for Trichloroethylene, Benz[a]anthracene, Epichlorohydrin, Benzene, and Hydroquinone. The significantly altered genes were involved in the regulation of (anti-) apoptosis, maintenance of cell survival, tumor necrosis factor-related pathways and immune response, in agreement with several other studies. Similarly in S9+ cultures, Benz[a]pyrene, Styrene and Trichloroethylene each modified over 1000 genes at high concentrations. Our findings expand our understanding of the transcriptomic response to genotoxic carcinogens, revealing the alteration of diverse sets of genes and pathways involved in cellular homeostasis and cell cycle control

An untargeted multi-technique metabolomics approach to studying intracellular metabolites of HepG2 cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

<p>Abstract</p> <p>Background</p> <p><it>In vitro </it>cell systems together with omics methods represent promising alternatives to conventional animal models for toxicity testing. Transcriptomic and proteomic approaches have been widely applied <it>in vitro </it>but relatively few studies have used metabolomics. Therefore, the goal of the present study was to develop an untargeted methodology for performing reproducible metabolomics on <it>in vitro </it>systems. The human liver cell line HepG2, and the well-known hepatotoxic and non-genotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were used as the <it>in vitro </it>model system and model toxicant, respectively.</p> <p>Results</p> <p>The study focused on the analysis of intracellular metabolites using NMR, LC-MS and GC-MS, with emphasis on the reproducibility and repeatability of the data. State of the art pre-processing and alignment tools and multivariate statistics were used to detect significantly altered levels of metabolites after exposing HepG2 cells to TCDD. Several metabolites identified using databases, literature and LC-nanomate-Orbitrap analysis were affected by the treatment. The observed changes in metabolite levels are discussed in relation to the reported effects of TCDD.</p> <p>Conclusions</p> <p>Untargeted profiling of the polar and apolar metabolites of <it>in vitro </it>cultured HepG2 cells is a valid approach to studying the effects of TCDD on the cell metabolome. The approach described in this research demonstrates that highly reproducible experiments and correct normalization of the datasets are essential for obtaining reliable results. The effects of TCDD on HepG2 cells reported herein are in agreement with previous studies and serve to validate the procedures used in the present work.</p