Project description:In vitro gene expression signatures to predict toxicological responses can provide mechanistic context for human health risk assessment purposes. We previously developed the TGx-28.65 genomic biomarker from a database of gene expression profiles in human TK6 cells exposed to 28 well-known compounds, and it comprises 65 genes that can classify chemicals as DNA damaging or non-DNA damaging. In this study, we applied the TGx-28.65 genomic biomarker in parallel with the in vitro micronucleus (MN) assay to determine if two chemicals of regulatory interest at Health Canada, disperse orange (DO: the orange azo dye 3-[[4-[(4-Nitrophenyl)azo]phenyl]benzylamino]propanenitrile) and 1,2,4-benzenetriol (BT: a metabolite of benzene) are genotoxic or non-genotoxic. Both chemicals caused dose-dependent declines in relative survival (RS) and increases in apoptosis. A strong significant increase in micronucleus induction was observed for all concentrations of BT; the top two concentrations of DO also caused a statistically significant increase in MN, but these increases were less than 2-fold above controls. TGx-28.65 analysis classified BT as genotoxic at all three concentrations and DO as genotoxic at the mid and high concentrations. Thus, although DO only induces a small increase in MN, this response is sufficient to induce a cellular DNA damage response that is likely relevant for risk assessment. Benchmark dose modeling revealed that BT is much more potent than DO; the BT benchmark dose for MN induction was similar to that of benzo[a]pyrene, (BaP: a genotoxic carcinogen), which was used as a positive control. The results strongly suggest that follow-up work is required to assess whether DO and BT are also genotoxic in vivo. This is particularly important for DO, which may require metabolic activation by bacterial gut flora to fully induce its genotoxic potential. Our previously published data and this proof of concept study suggest that the TGx-28.65 genomic biomarker has the potential to add significant value to existing approaches used to assess genotoxicity.

Project description:Two-year rodent bioassays play a central role in evaluating both the carcinogenic potential of a chemical and generating quantitative information on the dose-response behavior for chemical risk assessments. The bioassays involved are expensive and time-consuming, requiring nearly lifetime exposures (two years) in mice and rats and costing $2 to $4 million per chemical. Since there are approximately 80,000 chemicals registered for commercial use in the United States and 2,000 more are added each year, applying animal bioassays to all chemicals of concern is clearly impossible. To efficiently and economically identify carcinogens prior to widespread use and human exposure, alternatives to the two-year rodent bioassay must be developed. In this study, animals were exposed for 13 weeks to 10 chemicals that were positive for liver tumors in the two-year rodent bioassay, 14 chemicals that were negative for liver tumors, and two chemicals that produced an equivocal response. Matched vehicle control groups were run concurrently with each chemical treatment. Gene expression analysis was performed on the livers of the animals to assess the potential for identifying gene expression biomarkers and signaling pathways that can predict tumor formation in a two-year bioassay following a 13 week exposure. Five-week-old female B6C3F1 mice were exposed for 13 weeks to 26 chemicals. The chemical and dose information are provided with the individual sample annotations. With each chemical treatment, a matched vehicle control group was run concurrently with the exposure. A subset of five chemicals, methylene chloride, naphthalene, 1,2,3-trichloropropane, propylene glycol mono-t-butyl ether, and 1,4-dichlorobenzene, were performed in a five-point dose response with matched control groups. The concentrations for these exposures overlapped those in the original cancer bioassay. Gavage exposures were administered 5 days per week and feed exposures were provided 7 days per week. For inhalation exposures, mice were exposed 6 hr per day, 5 days per week. After 13 weeks, animals were euthanized and livers were collected. The right, caudate and median liver lobes were minced together and used for microarray analysis. Microarray analysis was performed on the livers of three to five mice per treatment group.

Project description:Introduction: Higher-throughput mode-of-action-based assays provide a valuable approach to expedite chemical evaluation for human health risk assessment. In this study, we combined the high-throughput alkaline CometChip® assay with the TGx-DDI transcriptomic biomarker (DDI = DNA damage-inducing) using high-throughput TempO-Seq® as an integrated genotoxicity testing approach. Objective: To determine if the high-throughput CometChip® assay and TGx-DDI biomarker analysis can be combined using human HepaRG™ cells to provide an efficient next-generation genotoxicity screening approach to identify DNA damage-inducing (DDI) chemicals. Methods: Metabolically competent human HepaRG™ cell cultures were exposed to increasing concentrations of 12 chemicals (9 DDI and 3 non-DDI) using a repeat exposure design (0 hr, 24 hr, 48 hr) in 96-well plates to measure and classify chemicals based on their ability to damage DNA. After a 4 hr recovery period, exposed cells were used to perform the Trevigen CometChip assay to assess DNA damage or used for TempO-Seq® S1500+ Targeted Transcriptome Sequencing for TGx-DDI classification purposes. Bioinformatics and statistical tools were used to classify chemicals as DDI or non-DDI using three separate analyses. Benchmark concentration analysis was also conducted for both assays for the purposes of potency ranking. Results: In combination, the CometChip® assay and the TGx-DDI were 100% accurate in identifying chemicals that induce DNA damage. Quantitative benchmark concentration (BMC) modeling was applied to evaluate chemical potencies for both assays. The BMCs for the CometChip® assay and the TGx-DDI for all 12 chemicals were highly concordant (within four-fold) and resulted in identical potency rankings. Conclusions: These results suggest that these two assays can be integrated for efficient, high-throughput identification of DNA damage in HepaRG™ cells. This study provides evidence for the complementarity of the high-throughput CometChip® assay with TGx-DDI biomarker analysis. Integration of these two tests provides an effective and high-throughput approach to identify DDI agents. This work is a first step in accomplishing a more integrated genotoxicity testing strategy to derive mechanistic information to better inform human health risk assessment in a high-throughput manner.

Project description:Background: Modern testing paradigms seek to apply human-relevant cell culture models and integrate data from multiple test systems to accurately inform potential hazards and modes of action for chemical toxicology. In genetic toxicology, the use of metabolically competent human hepatocyte cell culture models provides clear advantages over other more commonly used cell lines that require the use of external metabolic activation systems, such as rat liver S9. HepaRG™ cells are metabolically competent cells that express Phase I and II metabolic enzymes and differentiate into mature hepatocyte-like cells, making them ideal for toxicity testing. We assessed the performance of the flow cytometry in vitro micronucleus (MN) test and the TGx-DDI transcriptomic biomarker to detect DNA damage-inducing (DDI) chemicals in human HepaRG™ cells after a 3-day repeat exposure. The biomarker, developed for use in human TK6 cells, is a panel of 64 genes that accurately classifies chemicals as DDI or non-DDI. Herein, the TGx-DDI biomarker was analyzed by Ion AmpliSeq whole transcriptome sequencing to assess its classification accuracy using this more modern gene expression technology as a secondary objective. Methods: HepaRG™ cells were exposed to increasing concentrations of 10 test chemicals (six genotoxic chemicals, including one aneugen, and four non-genotoxic chemicals). Cytotoxicity and genotoxicity were measured using the In Vitro MicroFlow® kit, which was run in parallel with the TGx-DDI biomarker. Results: A concentration-related decrease in relative survival and a concomitant increase in MN frequency were observed for genotoxic chemicals in HepaRG™ cells. All five DDI and five non-DDI agents were correctly classified (as genotoxic/non-genotoxic and DDI/non-DDI) by pairing the test methods. The aneugenic agent (colchicine) yielded the expected positive result in the MN test and negative (non-DDI) result by TGx-DDI. Conclusions: This next generation genotoxicity testing strategy is aligned with the paradigm shift occurring in the field of genetic toxicology. It provides mechanistic insight in a human-relevant cell-model, paired with measurement of a conventional endpoint, to inform the potential for adverse health effects. This work provides support for combining these assays in an integrated test strategy for accurate, higher throughput genetic toxicology testing in this metabolically competent human progenitor cell line.

Dataset Information

Expression Data for HT-1080 cells exposed to ETP, QUE and MMS

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