Genotoxicity assessment in HepaRG™ cells as a new approach methodology follow up to a positive response in the human TK6 cell micronucleus assay: Naphthalene case study.

We are evaluating the use of metabolically competent HepaRG™ cells combined with CometChip® for DNA damage and the micronucleus (MN) assay as a New Approach Methodology (NAM) alternative to animals for follow up genotoxicity assessment to in vitro positive genotoxic response. Naphthalene is genotoxic in human TK6 cells inducing a nonlinear dose-response for the induction of micronuclei in the presence of rat liver S9. of naphthalene. In HepaRG™ cells, naphthalene genotoxicity was assessed using either 6 (CometChip™) or 12 concentrations of naphthalene (MN assay) with the top dose used for assessment of genotoxicity for the Comet and MN assay was 1.25 and 1.74 mM respectively, corresponding to approximately 45% cell survival. In contrast to human TK6 cell with S9, naphthalene was not genotoxic in either the HepaRG™ MN assay or the Comet assay using CometChip®. The lack of genotoxicity in both the MN and comet assays in HepaRG™ cells is likely due to Phase II enzymes removing phenols preventing further bioactivation to quinones and efficient detoxication of naphthalene quinones or epoxides by glutathione conjugation. In contrast to CYP450 mediated metabolism, these Phase II enzymes are inactive in rat liver S9 due to lack of appropriate cofactors causing a positive genotoxic response. Rat liver S9-derived BMD10 over-predicts naphthalene genotoxicity when compared to the negative genotoxic response observed in HepaRG™ cells. Metabolically competent hepatocyte models like HepaRG™ cells should be considered as human-relevant NAMs for use genotoxicity assessments to reduce reliance on rodents.

Early microRNA responses in rodent liver mediated by furan exposure establish dose thresholds for later adverse outcomes.

To reduce reliance on long-term in vivo studies, short-term data linking early molecular-based measurements to later adverse health effects is needed. Although transcriptional-based benchmark dose (BMDT) modeling has been used to estimate potencies and stratify chemicals based on potential to induce later-life effects, dose-responsive epigenetic alterations have not been routinely considered. Here, we evaluated the utility of microRNA (miRNA) profiling in mouse liver and blood, as well as in mouse primary hepatocytes in vitro, to indicate mechanisms of liver perturbation due to short-term exposure of the known rodent liver hepatotoxicant and carcinogen, furan. Benchmark dose modeling of miRNA measurements (BMDmiR) were compared to the referent transcriptional (BMDT) and apical (BMDA) estimates. These analyses indicate a robust dose response for 34 miRNAs to furan and involvement of p53-linked pathways in furan-mediated hepatotoxicity, supporting mRNA and apical measurements. Liver-sourced miRNAs were also altered in the blood and primary hepatocytes. Overall, these results indicate mechanistic involvement of miRNA in furan carcinogenicity and provide evidence of their potential utility as accessible biomarkers of exposure and disease.

Error-corrected duplex sequencing enables direct detection and quantification of mutations in human TK6 cells with strong inter-laboratory consistency.

Error-corrected duplex sequencing (DS) enables direct quantification of low-frequency mutations and offers tremendous potential for chemical mutagenicity assessment. We investigated the utility of DS to quantify induced mutation frequency (MF) and spectrum in human lymphoblastoid TK6 cells exposed to a prototypical DNA alkylating agent, N-ethyl-N-nitrosourea (ENU). Furthermore, we explored appropriate experimental parameters for this application, and assessed inter-laboratory reproducibility. In two independent experiments in two laboratories, TK6 cells were exposed to ENU (25-200 µM) and DNA was sequenced 48, 72, and 96 h post-exposure. A DS mutagenicity panel targeting twenty 2.4-kb regions distributed across the genome was used to sample diverse, genome-representative sequence contexts. A significant increase in MF that was unaffected by time was observed in both laboratories. Concentration-response in the MF from the two laboratories was strongly positively correlated (r = 0.97). C:G>T:A, T:A>C:G, T:A>A:T, and T:A>G:C mutations increased in consistent, concentration-dependent manners in both laboratories, with high proportions of C:G>T:A at all time points. The consistent results across the three time points suggest that 48 h may be sufficient for mutation analysis post-exposure. The target sites responded similarly between the two laboratories and revealed a higher average MF in intergenic regions. These results, demonstrating remarkable reproducibility across time and laboratory for both MF and spectrum, support the high value of DS for characterizing chemical mutagenicity in both research and regulatory evaluation.

Evaluation of the herbicide glyphosate, (aminomethyl)phosphonic acid, and glyphosate-based formulations for genotoxic activity using in vitro assays.

Glyphosate, the most heavily used herbicide world-wide, is applied to plants in complex formulations that promote absorption. The National Toxicology Program reported in 1992 that glyphosate, administered to rats and mice at doses up to 50,000 ppm in feed for 13 weeks, showed little evidence of toxicity, and no induction of micronuclei was observed in the mice in this study. Subsequently, mechanistic studies of glyphosate and glyphosate-based formulations (GBFs) that have focused on DNA damage and oxidative stress suggest that glyphosate may have genotoxic potential. However, few of these studies directly compared glyphosate to GBFs, or effects among GBFs. To address these data gaps, we tested glyphosate, glyphosate isopropylamine (IPA), and (aminomethyl)phosphonic acid (AMPA, a microbial metabolite of glyphosate), 9 high-use agricultural GBFs, 4 residential-use GBFs, and additional herbicides (metolachlor, mesotrione, and diquat dibromide) present in some of the GBFs in bacterial mutagenicity tests, and in human TK6 cells using a micronucleus assay and a multiplexed DNA damage assay. Our results showed no genotoxicity or notable cytotoxicity for glyphosate or AMPA at concentrations up to 10 mM, while all GBFs and herbicides other than glyphosate were cytotoxic, and some showed genotoxic activity. An in vitro to in vivo extrapolation of results for glyphosate suggests that it is of low toxicological concern for humans. In conclusion, these results demonstrate a lack of genotoxicity for glyphosate, consistent with observations in the NTP in vivo study, and suggest that toxicity associated with GBFs may be related to other components of these formulations.

Using the HepaCometChip Assay for Broad-Spectrum DNA Damage Analysis.

Exposure to DNA damaging agents can lead to mutations that cause cancer. The liver is particularly vulnerable because it contains high levels of Cytochrome P450 enzymes that can convert xenobiotics into DNA reactive metabolites that form potentially carcinogenic bulky DNA adducts. As such, current requirements for preclinical testing include in vivo testing for DNA damage in the liver, which often requires many animals. Given that efforts are underway in many countries to reduce or eliminate the use of animals in research, there is a critical need for fast and robust in vitro tests to discern whether xenobiotics or potential pharmaceutical agents can damage the hepatocyte genome. One possible approach is to leverage the alkaline comet assay, which is used to assess genotoxicity based on the ability of damaged DNA to become free to migrate toward the anode during electrophoresis. The comet assay, however, has several limitations. The assay is (i) slow and (ii) vulnerable to experimental noise, (iii) it is difficult to detect bulky DNA adducts since they do not directly affect DNA migration, and (iv) cell types typically used do not have robust metabolic capacity. To address some of these concerns, we have developed the "HepaCometChip" (a.k.a. the HepaRG CometChip), wherein metabolically competent cells are incorporated into a higher throughput CometChip platform. Repair trapping is used to increase sensitivity for bulky lesions: undetectable bulky lesions are converted into repair intermediates (specifically, single-strand breaks) that can be detected with the assay. Here, we describe a protocol for performing the HepaCometChip assay that includes handling and dosing of HepaRG cells and performing the CometChip assay. With its higher throughput, ability to capture metabolic activation, and sensitivity to bulky lesions, the HepaCometChip offers a potential alternative to the use of animals for genotoxicity testing. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: HepaRG cell culturing and dosing Basic Protocol 2: CometChip assay.

The common indoor air pollutant α-pinene is metabolised to a genotoxic metabolite α-pinene oxide.

α-Pinene caused a concentration-responsive increase in bladder hyperplasia and decrease in sperm counts in rodents following inhalation exposure. Additionally, it formed a prospective reactive metabolite, α-pinene oxide.To provide human relevant context for data generated in animal models and explore potential mechanism, we undertook studies to investigate the metabolism of α-pinene to α-pinene oxide and mutagenicity of α-pinene and α-pinene oxide.α-Pinene oxide was formed in rat and human microsomes and hepatocytes with some species differences. Based on area under the concentration versus time curves, the formation of α-pinene oxide was up to 4-fold higher in rats than in humans.While rat microsomes cleared α-pinene oxide faster than human microsomes, the clearance of α-pinene oxide in hepatocytes was similar between species.α-Pinene was not mutagenic with or without induced rat liver S9 in Salmonella typhimurium or Escherichia coli when tested up to 10 000 µg/plate while α-pinene oxide was mutagenic at ≥25 µg/plate.α-Pinene was metabolised to α-pinene oxide under the conditions of the bacterial mutation assay although the concentration was approximately 3-fold lower than the lowest α-pinene oxide concentration that was positive in the assay, potentially explaining the lack of mutagenicity observed with α-pinene.

Safety evaluation of carbon tetrafluoride as an inert hyperbaric breathing gas in Sprague-Dawley rats.

Carbon tetrafluoride (CF4) is an inert gas with higher molecular weight and lower water solubility than commonly used hyperbaric breathing gases. These inert gas properties decrease time required to decompress and avoid decompression sickness after deep dives. To assess CF4 toxicity, Sprague-Dawley rats were exposed to 8 atm absolute (ATA) air (10 males, 10 females) or 8 ATA 79% CF4/21% O2 (25 males, 25 females). Exposures were 30 min daily for 5 days. Rat behavior was normal throughout the testing period. There were no gross or microscopic pathology abnormalities following repeat dose exposure. Male body weight trends were similar between groups. Female body weight trends were 0.5 ± 0.8% day-1 for hyperbaric air exposure and - 0.2 ± 0.8% day-1 for hyperbaric CF4 exposure (P = 0.01) but remained within literature cited norms. Organ weights and hematologic indices remained within or near literature normal ranges. Clinical chemistry panels showed no signs of toxicity in renal or hepatic biomarkers. Polychromatic erythrocyte micronucleus frequency showed no chromosomal damage. Comet assay showed no DNA damage in lung tissue. Females exposed to CF4 had 2.5 times greater percent tail DNA in liver tissue than controls (P = 0.009). However this result remained within the normal range of local negative controls. A bacterial reverse mutation assay with exposure to 1 ATA 79% CF4/21% O2 for 72 h was nonmutagenic in four strains of Salmonella typhimurium and one strain of Escherichia coli. Overall, there was no evidence that CF4 caused organ toxicity or genetic toxicity.

A Modern Genotoxicity Testing Paradigm: Integration of the High-Throughput CometChip® and the TGx-DDI Transcriptomic Biomarker in Human HepaRG™ Cell Cultures.

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.

Flow cytometric micronucleus assay and TGx-DDI transcriptomic biomarker analysis of ten genotoxic and non-genotoxic chemicals in human HepaRG™ cells.

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.

Sensitive CometChip assay for screening potentially carcinogenic DNA adducts by trapping DNA repair intermediates.

Genotoxicity testing is critical for predicting adverse effects of pharmaceutical, industrial, and environmental chemicals. The alkaline comet assay is an established method for detecting DNA strand breaks, however, the assay does not detect potentially carcinogenic bulky adducts that can arise when metabolic enzymes convert pro-carcinogens into a highly DNA reactive products. To overcome this, we use DNA synthesis inhibitors (hydroxyurea and 1-ß-d-arabinofuranosyl cytosine) to trap single strand breaks that are formed during nucleotide excision repair, which primarily removes bulky lesions. In this way, comet-undetectable bulky lesions are converted into comet-detectable single strand breaks. Moreover, we use HepaRG™ cells to recapitulate in vivo metabolic capacity, and leverage the CometChip platform (a higher throughput more sensitive comet assay) to create the 'HepaCometChip', enabling the detection of bulky genotoxic lesions that are missed by current genotoxicity screens. The HepaCometChip thus provides a broadly effective approach for detection of bulky DNA adducts.

Integrated in silico and in vitro genotoxicity assessment of thirteen data-poor substances.

The Canadian Domestic Substances List (DSL) contains chemicals that have not been tested for genotoxicity as their use pre-dates regulatory requirements. In the present study, (quantitative) structure-activity relationships ((Q)SAR) model predictions and in vitro tests were conducted for genotoxicity assessment of 13 data-poor chemicals from the DSL (i.e. CAS numbers 19286-75-0, 13676-91-0, 2478-20-8, 6408-20-8, 74499-36-8, 26694-69-9, 29036-02-0, 120-24-1, 84696-48-9, 4051-63-2, 5718-26-3, 632-51-9, and 600-14-6). First, chemicals were screened by (Q)SAR models in Leadscope® and OASIS TIMES; two chemicals were excluded from (Q)SAR as they are complex mixtures. Six were flagged by (Q)SAR as potentially mutagenic and were subsequently confirmed as mutagens using the Ames assay. Of nine chemicals with clastogenic (Q)SAR flags, eight induced micronuclei in TK6 cells. Benchmark dose analysis was used to evaluate the potency of the chemicals. Four chemicals were bacterial mutagens with similar potencies. Three chemicals were more potent in micronuclei induction than the prototype alkylating agent methyl methanesulfonate and three were equipotent to the mutagenic carcinogen benzo[a]pyrene in the presence of rat liver S9. Overall, 11 of the 13 DSL chemicals demonstrated at least one type of genotoxicity in vitro. This study demonstrates the application of genotoxic potency analysis for prioritizing further investigations.

Genotoxicity evaluation of the naturally-derived food colorant, gardenia blue, and its precursor, genipin.

Gardenia blue is widely used in Eastern Asia as a natural food colorant. To evaluate the genotoxic potential of gardenia blue, as well as genipin, the natural starting material from which it is produced, a GLP-compliant test battery was conducted according to OECD guidelines. No evidence of mutagenicity of gardenia blue was detected in a 5-strain bacterial reverse mutation assay, with or without metabolic activation; an equivocal response for genipin occurred in S. typhimurium TA97a without metabolic activation. In in vitro micronucleus and chromosome aberration assays, genipin tested positive under some test conditions; however, gardenia blue tested negative in both assays. In combined micronucleus/comet assays conducted in male and female B6C3F1 mice, exposure to genipin at doses reaching maximal toxicity (74 and 222 mg/kg bw/day for males and females, respectively) or gardenia blue tested up to the limit dose (2000 mg/kg bw/day) did not induce micronuclei in peripheral blood or DNA damage in several examined tissues. Modified ("reverse") comet assays showed no evidence of DNA crosslinking potential of either genipin, known to form crosslinks with other macromolecules, or gardenia blue. Our results indicate that consumption of gardenia blue in food products does not pose a significant genotoxic concern for humans.

Black cohosh extracts and powders induce micronuclei, a biomarker of genetic damage, in human cells.

Black cohosh extract (BCE) is a widely used dietary supplement marketed to women to alleviate symptoms of gynecological ailments, yet its toxicity has not been well characterized. The National Toxicology Program (NTP) previously reported significant increases in micronucleated erythrocytes in peripheral blood of female Wistar Han rats and B6C3F1/N mice administered 15-1,000 mg BCE/kg/day by gavage for 90 days. These animals also developed a dose-dependent nonregenerative macrocytic anemia characterized by clinical changes consistent with megaloblastic anemia. Both micronuclei (MN) and megaloblastic anemia can arise from disruption of the folate metabolism pathway. The NTP used in vitro approaches to investigate whether the NTP's test lot of BCE, BCEs from various suppliers, and root powders from BC and other cohosh species, were genotoxic in general, and to gain insight into the mechanism of action of BCE genotoxicity. Samples were tested in human TK6 lymphoblastoid cells using the In Vitro MicroFlow® MN assay. The NTP BCE and a BC extract reference material (XRM) were tested in the MultiFlow® DNA Damage assay, which assesses biomarkers of DNA damage, cell division, and cytotoxicity. The NTP BCE and several additional BCEs were tested in bacterial mutagenicity assays. All samples induced MN when cells were grown in physiological levels of folic acid. The NTP BCE and BC XRM produced activity patterns consistent with an aneugenic mode of action. The NTP BCE and five additional BCEs were negative in bacterial mutagenicity tests. These findings show that black cohosh preparations induce chromosomal damage and may pose a safety concern. Environ. Mol. Mutagen. 59:416-426, 2018. © 2018 Published 2018. This article is a US Government work and is in the public domain in the USA.

Comprehensive evaluation of the flavonol anti-oxidants, alpha-glycosyl isoquercitrin and isoquercitrin, for genotoxic potential.

Quercetin and its glycosides possess potential benefits to human health. Several flavonols are available to consumers as dietary supplements, promoted as anti-oxidants; however, incorporation of natural quercetin glycosides into food and beverage products has been limited by poor miscibility in water. Enzymatic conjugation of multiple glucose moieties to isoquercitrin to produce alpha-glycosyl isoquercitrin (AGIQ) enhances solubility and bioavailability. AGIQ is used in Japan as a food additive and has been granted generally recognized as safe (GRAS) status. However, although substantial genotoxicity data exist for quercetin, there is very little available data for AGIQ and isoquercitrin. To support expanded global marketing of food products containing AGIQ, comprehensive testing of genotoxic potential of AGIQ and isoquercitrin was conducted according to current regulatory test guidelines. Both chemicals tested positive in bacterial reverse mutation assays, and exposure to isoquercitrin resulted in chromosomal aberrations in CHO-WBL cells. All other in vitro mammalian micronucleus and chromosomal aberration assays, micronucleus and comet assays in male and female B6C3F1 mice and Sprague Dawley rats, and Muta™ Mouse mutation assays evaluating multiple potential target tissues, were negative for both chemicals. These results supplement existing toxicity data to further support the safe use of AGIQ in food and beverage products.

Integration of the TGx-28.65 genomic biomarker with the flow cytometry micronucleus test to assess the genotoxicity of disperse orange and 1,2,4-benzenetriol in human TK6 cells.

In vitro gene expression signatures to predict toxicological responses can provide mechanistic context for regulatory testing. We previously developed the TGx-28.65 genomic biomarker from a database of gene expression profiles derived from human TK6 cells exposed to 28 well-known compounds. The biomarker 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 and increases in apoptosis. A strong significant increase in MN 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 <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 caused a small increase in MN, this response was sufficient to induce a cellular DNA damage response. Benchmark dose modeling confirmed that BT is much more potent than DO. 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.

Application of the TGx-28.65 transcriptomic biomarker to classify genotoxic and non-genotoxic chemicals in human TK6 cells in the presence of rat liver S9.

In vitro transcriptional signatures that predict toxicities can facilitate chemical screening. We previously developed a transcriptomic biomarker (known as TGx-28.65) for classifying agents as genotoxic (DNA damaging) and non-genotoxic in human lymphoblastoid TK6 cells. Because TK6 cells do not express cytochrome P450s, we confirmed accurate classification by the biomarker in cells co-exposed to 1% 5,6 benzoflavone/phenobarbital-induced rat liver S9 for metabolic activation. However, chemicals may require different types of S9 for activation. Here we investigated the response of TK6 cells to higher percentages of Aroclor-, benzoflavone/phenobarbital-, or ethanol-induced rat liver S9 to expand TGx-28.65 biomarker applicability. Transcriptional profiles were derived 3 to 4 hr following a 4 hr co-exposure of TK6 cells to test chemicals and S9. Preliminary studies established that 10% Aroclor- and 5% ethanol-induced S9 alone did not induce the TGx-28.65 biomarker genes. Seven genotoxic and two non-genotoxic chemicals (and concurrent solvent and positive controls) were then tested with one of the S9s (selected based on cell survival and micronucleus induction). Relative survival and micronucleus frequency was assessed by flow cytometry in cells 20 hr post-exposure. Genotoxic/non-genotoxic chemicals were accurately classified using the different S9s. One technical replicate of cells co-treated with dexamethasone and 10% Aroclor-induced S9 was falsely classified as genotoxic, suggesting caution in using high S9 concentrations. Even low concentrations of genotoxic chemicals (those not causing cytotoxicity) were correctly classified, demonstrating that TGx-28.65 is a sensitive biomarker of genotoxicity. A meta-analysis of datasets from 13 chemicals supports that different S9s can be used in TK6 cells, without impairing classification using the TGx-28.65 biomarker.

Genetic and rat toxicity studies of cyclodextrin glucanotransferase.

INTRODUCTION: Microbiologically derived cyclodextrin glucanotransferase (CGTase) is used commercially as a processing agent in manufacture of food, pharmaceuticals, and cosmetics. Its toxic potential was evaluated in anticipation of use in the production of alpha-glycosyl isoquercitrin, a water-soluble form of quercetin. METHODS: Following OECD guidelines, CGTase, produced by Bacillus pseudalcaliphilus DK-1139, was evaluated in a genotoxicity battery consisting of a bacterial reverse mutation assay, an in vitro micronucleus (MN) assay and MN and comet assays using B6C3F1 male and female mice. These same genotoxicity assays were also conducted for sodium sulfate, a contaminant of CGTase preparation. In a 90-day Sprague Dawley rat toxicity study, CGTase was administered by gavage in water at daily doses of 0, 250, 500, and 1000 mg/kg/day. RESULTS: CGTase did not induce mutations with or without metabolic activation in the bacterial reverse mutation assay. Formation of micronuclei was not induced in either in vitro or in vivo MN assays with or without metabolic activation. No induction of DNA damage was detected in male or female mouse liver, stomach, or duodenum in the comet assay. Sodium sulfate also tested negative in these same genotoxicity assays. In the 90-day repeated dose rat study there were no treatment-related adverse clinical or pathological findings. CONCLUSION: The genotoxicity assays and repeated dose toxicity study support the safe use of CGTase in production of alpha-glycosyl isoquercitrin.

A predictive toxicogenomics signature to classify genotoxic versus non-genotoxic chemicals in human TK6 cells.

Genotoxicity testing is a critical component of chemical assessment. The use of integrated approaches in genetic toxicology, including the incorporation of gene expression data to determine the DNA damage response pathways involved in response, is becoming more common. In companion papers previously published in Environmental and Molecular Mutagenesis, Li et al. (2015) [6] developed a dose optimization protocol that was based on evaluating expression changes in several well-characterized stress-response genes using quantitative real-time PCR in human lymphoblastoid TK6 cells in culture. This optimization approach was applied to the analysis of TK6 cells exposed to one of 14 genotoxic or 14 non-genotoxic agents, with sampling 4 h post-exposure. Microarray-based transcriptomic analyses were then used to develop a classifier for genotoxicity using the nearest shrunken centroids method. A panel of 65 genes was identified that could accurately classify toxicants as genotoxic or non-genotoxic. In Buick et al. (2015) [1], the utility of the biomarker for chemicals that require metabolic activation was evaluated. In this study, TK6 cells were exposed to increasing doses of four chemicals (two genotoxic that require metabolic activation and two non-genotoxic chemicals) in the presence of rat liver S9 to demonstrate that S9 does not impair the ability to classify genotoxicity using this genomic biomarker in TK6cells.

Genotoxicity evaluation of the flavonoid, myricitrin, and its aglycone, myricetin.

Myricitrin, a flavonoid extracted from the fruit, leaves, and bark of Chinese bayberry (Myrica rubra SIEBOLD), is currently used as a flavor modifier in snack foods, dairy products, and beverages in Japan. Myricitrin is converted to myricetin by intestinal microflora; myricetin also occurs ubiquitously in plants and is consumed in fruits, vegetables, and beverages. The genotoxic potential of myricitrin and myricetin was evaluated in anticipation of worldwide marketing of food products containing myricitrin. In a bacterial reverse mutation assay, myricetin tested positive for frameshift mutations under metabolic activation conditions whereas myricitrin tested negative for mutagenic potential. Both myricitrin and myricetin induced micronuclei formation in human TK6 lymphoblastoid cells under conditions lacking metabolic activation; however, the negative response observed in the presence of metabolic activation suggests that rat liver S9 homogenate may detoxify reactive metabolites of these chemicals in mammalian cells. In 3-day combined micronucleus/Comet assays using male and female B6C3F1 mice, no induction of micronuclei was observed in peripheral blood, or conclusive evidence of damage detected in the liver, glandular stomach, or duodenum following exposure to myricitrin or myricetin. Our studies did not reveal evidence of genotoxic potential of myricitrin in vivo, supporting its safe use in food and beverages.

Integration of metabolic activation with a predictive toxicogenomics signature to classify genotoxic versus nongenotoxic chemicals in human TK6 cells.

The use of integrated approaches in genetic toxicology, including the incorporation of gene expression data to determine the molecular pathways involved in the response, is becoming more common. In a companion article, a genomic biomarker was developed in human TK6 cells to classify chemicals as genotoxic or nongenotoxic. Because TK6 cells are not metabolically competent, we set out to broaden the utility of the biomarker for use with chemicals requiring metabolic activation. Specifically, chemical exposures were conducted in the presence of rat liver S9. The ability of the biomarker to classify genotoxic (benzo[a]pyrene, BaP; aflatoxin B1, AFB1) and nongenotoxic (dexamethasone, DEX; phenobarbital, PB) agents correctly was evaluated. Cells were exposed to increasing chemical concentrations for 4 hr and collected 0 hr, 4 hr, and 20 hr postexposure. Relative survival, apoptosis, and micronucleus frequency were measured at 24 hr. Transcriptome profiles were measured with Agilent microarrays. Statistical modeling and bioinformatics tools were applied to classify each chemical using the genomic biomarker. BaP and AFB1 were correctly classified as genotoxic at the mid- and high concentrations at all three time points, whereas DEX was correctly classified as nongenotoxic at all concentrations and time points. The high concentration of PB was misclassified at 24 hr, suggesting that cytotoxicity at later time points may cause misclassification. The data suggest that the use of S9 does not impair the ability of the biomarker to classify genotoxicity in TK6 cells. Finally, we demonstrate that the biomarker is also able to accurately classify genotoxicity using a publicly available dataset derived from human HepaRG cells.