Application of the transcriptomic biomarker, TGx-DDI, in genotoxicity screening
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE107162 | GEO | 2017/11/21
SECONDARY ACCESSION(S): PRJNA419190
REPOSITORIES: GEO
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Project description:Application of the transcriptomic biomarker, TGx-DDI, in genotoxicity screening
| PRJNA419190 | ENA
Project description:Gene expression biomarkers are now available for application in the identification of genotoxic hazards. The TGx-DDI transcriptomic biomarker can accurately distinguish DDI from non-DDI exposures based on changes in the expression of 64 biomarker genes. The bioamarker was originally derived from DNA microarray gene expression profiles of TK6 human lymphoblastoid cells post 4-hour exposure to 28 reference DDI and non-DDI agents [Li et al. 2015]. To broaden the applicability of TGx-DDI, the biomarker was tested using quantitative RT-PCR (qPCR), which is accessible to most molecular biology laboratories. To assess the classification capability of the biomarker using qPCR, a custom 96-well TaqMan qPCR array (TGx-DDI qPCR array) was constructed using the 64 biomarker genes. TK6 cells were exposed to each of the 28 reference agents and their vehicle controls for 4 hours and the expression level of the TGx-DDI genes were profiled using the TaqMan arrays. This study provides reference qPCR expression profiles of the TGx-DDI biomarker for DDI chemical classification using qPCR.
2018-12-21 | GSE121532 | GEO
Project description:The TGx-DDI is a transcriptomic biomarker used to predict the DNA damage-inducing (DDI) capability of chemicals by providing mechanistic information to aid in the interpretation of positive genotoxicity data. The biomarker was developed within the Health and Environmental Sciences Institute (HESI) and measures transcriptional changes in a set of primarily p53-responsive genes. We investigated three antibiotic drugs, nitrofurantoin (NIT), novobiocin sodium salt (NOV) and metronidazole (MTZ), with a long history of clinical use in human and/or veterinary applications. While all three drugs are considered safe, there is some evidence of chromosome damage with NIT and NOV. We conducted high-throughput CometChip and TGx-DDI assays to evaluate the drugs in a parallel test strategy to explore their toxicity using these newer mechanism-based tests. An extended concentration-response analysis was conducted in human TK6 cells (from 0 to 1 mM). The alkaline CometChip assay was used to measure DNA damage, while TGx-DDI was applied using TempO-Seq and NanoString technologies (in two different laboratories) to confirm reproducibility of the results. The CometChip was negative for all three drugs except at overtly cytotoxic concentrations, which were eliminated from the gene expression analysis. NIT and MTZ were classified as non-DDI by TGx-DDI at all concentrations tested. NOV classified as DDI at the highest concentrations tested. NOV is bacterial DNA-gyrase inhibitor that also acts as a topoisomerase II inhibitor at high concentrations. The TGx-DDI classification results were identical across laboratories/technologies. This case study demonstrates the utility of TGx-DDI to de-risk NIT and NOV, and the reproducibility of the biomarker.
2022-08-02 | GSE196369 | GEO
Project description:The TGx-DDI is a transcriptomic biomarker used to predict the DNA damage-inducing (DDI) capability of chemicals by providing mechanistic information to aid in the interpretation of positive genotoxicity data. The biomarker was developed within the Health and Environmental Sciences Institute (HESI) and measures transcriptional changes in a set of primarily p53-responsive genes. We investigated three antibiotic drugs, nitrofurantoin (NIT), novobiocin sodium salt (NOV) and metronidazole (MTZ), with a long history of clinical use in human and/or veterinary applications. While all three drugs are considered safe, there is some evidence of chromosome damage with NIT and NOV. We conducted high-throughput CometChip and TGx-DDI assays to evaluate the drugs in a parallel test strategy to explore their toxicity using these newer mechanism-based tests. An extended concentration-response analysis was conducted in human TK6 cells (from 0 to 1 mM). The alkaline CometChip assay was used to measure DNA damage, while TGx-DDI was applied using TempO-Seq and NanoString technologies (in two different laboratories) to confirm reproducibility of the results. The CometChip was negative for all three drugs except at overtly cytotoxic concentrations, which were eliminated from the gene expression analysis. NIT and MTZ were classified as non-DDI by TGx-DDI at all concentrations tested. NOV classified as DDI at the highest concentrations tested. NOV is bacterial DNA-gyrase inhibitor that also acts as a topoisomerase II inhibitor at high concentrations. The TGx-DDI classification results were identical across laboratories/technologies. This case study demonstrates the utility of TGx-DDI to de-risk NIT and NOV, and the reproducibility of the biomarker.
2022-08-02 | GSE196372 | GEO
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.
2020-02-18 | GSE136009 | GEO
Project description:The conventional battery for genotoxicity testing is not well suited to assessing the large number of chemicals needing evaluation. Traditional in vitro tests lack throughput, provide little mechanistic information, and have poor specificity in predicting in vivo genotoxicity. New Approach Methodologies (NAMs) aim to accelerate the pace of hazard assessment and reduce reliance on in vivo tests that are time-consuming and resource-intensive. As such, high-throughput transcriptomic and flow cytometry-based assays have been developed for modernized in vitro genotoxicity assessment. This includes: the TGx-DDI transcriptomic biomarker (i.e., 64-gene expression signature to identify DNA damage-inducing (DDI) substances), the MicroFlow® assay (i.e., a flow cytometry-based micronucleus (MN) test), and the MultiFlow® assay (i.e., a multiplexed flow cytometry-based reporter assay that yields mode-of-action (MoA) information). The objective of this study was to investigate the utility of the TGx-DDI transcriptomic biomarker, multiplexed with the MicroFlow® and MultiFlow® assays, as an integrated NAM-based testing strategy for screening data-poor compounds prioritized by Health Canada’s New Substances Assessment and Control Bureau. Human lymphoblastoid TK6 cells were exposed to 3 control and 10 data-poor substances, using a 6-point concentration range. Gene expression profiling was conducted using the targeted TempO-SeqTM assay, and the TGx-DDI classifier was applied to the dataset. Classifications were compared with those based on the MicroFlow® and MultiFlow® assays. Benchmark Concentration (BMC) modeling was used for potency ranking. The results of the integrated hazard calls indicate that five of the data-poor compounds were genotoxic in vitro, causing DNA damage via a clastogenic MoA, and one via a pan-genotoxic MoA. Two compounds were likely irrelevant positives in the MN test; two are considered possibly genotoxic causing DNA damage via an ambiguous MoA. BMC modeling revealed nearly identical potency rankings for each assay. This ranking was maintained when all endpoint BMCs were converted into a single score using the Toxicological Prioritization (ToxPi) approach. Overall, this study contributes to the establishment of a modernized approach for effective genotoxicity assessment and chemical prioritization for further regulatory scrutiny. We conclude that the integration of TGx-DDI, MicroFlow®, and MultiFlow® endpoints is an effective NAM-based strategy for genotoxicity assessment of data-poor compounds.
2022-11-15 | GSE213454 | GEO
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.
2017-10-16 | GSE95188 | GEO
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.
2017-10-16 | GSE95187 | GEO
Project description:Transcriptomic signatures, or biomarkers, of toxicity can facilitate rapid mechanistic analysis of chemicals using high-content transcriptomic data and identification of potential hazards to human health. We developed an 81-gene transcriptomic biomarker, named TGx-HDACi, to detect histone deacetylase inhibitors (HDACi) in TK6 human lymphoblastoid cells after 4 hours of chemical exposure. This work leveraged an established transcriptomic biomarker of DNA damage, TGx-DDI, which was developed by Heng Hong Li et al. (2015); TGx-DDI contains 63 genes and can distinguish between DNA damage-inducing (DDI) and non-DDI agents after 4 hours of exposure in TK6 cells. TGx-HDACi was derived from Templated Oligo-Sequencing (TempO-Seq) whole transcriptome gene expression profiles of 20 reference compounds, consisting of 10 HDACi and 10 non-HDACi compounds. Fourteen of the TGx-HDACi reference compounds are also part of the 28-compound reference set for TGx-DDI. The nearest shrunken centroid (NSC) method was applied to the gene expression profiles and 81 genes were identified that accurately identified HDACi compounds in the NSC probability analysis. The classification performance of TGx-HDACi was validated using an additional set of 11 test compounds (4 HDACi and 7 non-HDACi). Thus far, TGx-HDACi has demonstrated 100% accuracy. The availability of TGx-HDACi increases the diversity of tools that can facilitate mode of action analysis of toxicants using gene expression profiling.
2021-04-14 | GSE164478 | GEO
Project description: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 DNA damage-sensing CometChip® assay with the TGx-DDI transcriptomic biomarker (DDI = DNA damage-inducing) using high-throughput TempO-Seq®, as an integrated genotoxicity testing approach. We used metabolically competent differentiated human HepaRG™ cell cultures to enable the identification of chemicals that require bioactivation to cause genotoxicity. We studied 12 chemicals (nine DDI, three non-DDI) in increasing concentrations to measure and classify chemicals based on their ability to damage DNA. The CometChip® classified 10/12 test chemicals correctly, missing a positive DDI call for aflatoxin B1 and propyl gallate. The poor detection of aflatoxin B1 adducts is consistent with the insensitivity of the standard alkaline comet assay to bulky lesions (a shortcoming that can be overcome by trapping repair intermediates). The TGx-DDI biomarker accurately classified 10/12 agents. TGx-DDI correctly identified aflatoxin B1 as DDI, demonstrating efficacy for combined used of these complementary methodologies. Zidovudine, a known DDI chemical, was misclassified as it inhibits transcription, which prevents measurable changes in gene expression. Eugenol, a non-DDI chemical known to render misleading positive results at high concentrations, was classified as DDI at the highest concentration tested. When combined, the CometChip® assay and the TGx-DDI biomarker 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 biomarker were highly concordant (within 4-fold) and resulted in identical potency rankings. These results demonstrate that these two assays can be integrated for efficient identification and potency ranking of DNA damaging agents in HepaRG™ cell cultures.
| S-EPMC8416458 | biostudies-literature