Methods for quantifying DNA damage, as well as repair of that damage, in a high-throughput format are lacking. Single cell gel electrophoresis (SCGE; comet assay) is a widely-used method due to its ...technical simplicity and sensitivity, but the standard comet assay has limitations in reproducibility and throughput. We have advanced the SCGE assay by creating a 96-well hardware platform coupled with dedicated data processing software (CometChip Platform). Based on the original cometchip approach, the CometChip Platform increases capacity ~200 times over the traditional slide-based SCGE protocol, with excellent reproducibility. We tested this platform in several applications, demonstrating a broad range of potential uses including the routine identification of DNA damaging agents, using a 74-compound library provided by the National Toxicology Program. Additionally, we demonstrated how this tool can be used to evaluate human populations by analysis of peripheral blood mononuclear cells to characterize susceptibility to genotoxic exposures, with implications for epidemiological studies. In summary, we demonstrated a high level of reproducibility and quantitative capacity for the CometChip Platform, making it suitable for high-throughput screening to identify and characterize genotoxic agents in large compound libraries, as well as for human epidemiological studies of genetic diversity relating to DNA damage and repair.
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.
Black cohosh (BC; Actaea racemosa L.), a top‐selling botanical dietary supplement, is marketed to women primarily to ameliorate a variety of gynecological symptoms. Due to widespread usage, limited ...safety information, and sporadic reports of hepatotoxicity, the Division of the National Toxicology Program (DNTP) initially evaluated BC extract in female rats and mice. Following administration of up to 1000 mg/kg/day BC extract by gavage for 90 days, dose‐related increases in micronucleated peripheral blood erythrocytes were observed, along with a nonregenerative macrocytic anemia resembling megaloblastic anemia in humans. Because both micronuclei and megaloblastic anemia may signal disruption of folate metabolism, and inadequate folate levels in early pregnancy can adversely affect neurodevelopment, the DNTP conducted a pilot cross‐sectional study comparing erythrocyte micronucleus frequencies, folate and B12 levels, and a variety of hematological and clinical chemistry parameters between women who used BC and BC‐naïve women. Twenty‐three women were enrolled in the BC‐exposed group and 28 in the BC‐naïve group. Use of any brand of BC‐only supplement for at least 3 months was required for inclusion in the BC‐exposed group. Supplements were analyzed for chemical composition to allow cross‐product comparisons. All participants were healthy, with no known exposures (e.g., x‐rays, certain medications) that could influence study endpoints. Findings revealed no increased micronucleus frequencies and no hematological abnormalities in women who used BC supplements. Although reassuring, a larger, prospective study with fewer confounders (e.g., BC product diversity and duration of use) providing greater power to detect subtle effects would increase confidence in these findings.
Response to Letter to the Editor Smith‐Roe, Stephanie L.; Shockley, Keith R.; Bucher, John R. ...
Environmental and molecular mutagenesis,
February 2020, 2020-Feb, 2020-02-00, 20200201, Letnik:
61, Številka:
2
Journal Article
Abstract
The in vitro MultiFlow DNA Damage assay multiplexes p53, γH2AX, phospho-histone H3, and polyploidization biomarkers into 1 flow cytometric analysis (Bryce, S. M., Bernacki, D. T., Bemis, J. ...C., and Dertinger, S. D. (2016). Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach. Environ. Mol. Mutagen. 57, 171–189). The work reported herein evaluated the generalizability of the method, as well as several data analytics strategies, to a range of chemical classes not studied previously. TK6 cells were exposed to each of 103 diverse chemicals, 86 of which were supplied by the National Toxicology Program (NTP) and selected based upon responses in genetic damage assays conducted under the Tox21 program. Exposures occurred for 24 h over a range of concentrations, and cell aliquots were removed at 4 and 24 h for analysis. Multiplexed response data were evaluated using 3 machine learning models designed to predict genotoxic mode of action based on data from a training set of 85 previously studied chemicals. Of 54 chemicals with sufficient information to make an a priori call on genotoxic potential, the prediction models’ accuracies were 79.6% (random forest), 88.9% (logistic regression), and 90.7% (artificial neural network). A majority vote ensemble of the 3 models provided 92.6% accuracy. Forty-nine NTP chemicals were not adequately tested (maximum concentration did not approach assay’s cytotoxicity limit) and/or had insufficient conventional genotoxicity data to allow their genotoxic potential to be defined. For these chemicals MultiFlow data will be useful in future research and hypothesis testing. Collectively, the results suggest the MultiFlow assay and associated data analysis strategies are broadly generalizable, demonstrating high predictability when applied to new chemicals and classes of compounds.
Currently, approximately 80,000 chemicals are used in commerce. Most have little-to-no toxicity information. The U.S. Toxicology in the 21st Century (Tox21) program has conducted a battery of in ...vitro assays using a quantitative high-throughput screening (qHTS) platform to gain toxicity information on environmental chemicals. Due to technical challenges, standard methods for providing xenobiotic metabolism could not be applied to qHTS assays. To address this limitation, we screened the Tox21 10,000-compound (10K) library, with concentrations ranging from 2.8 nM to 92 µM, using a p53 beta-lactamase reporter gene assay (p53-bla) alone or with rat liver microsomes (RLM) or human liver microsomes (HLM) supplemented with NADPH, to identify compounds that induce p53 signaling after biotransformation. Two hundred and seventy-eight compounds were identified as active under any of these three conditions. Of these 278 compounds, 73 gave more potent responses in the p53-bla assay with RLM, and 2 were more potent in the p53-bla assay with HLM compared with the responses they generated in the p53-bla assay without microsomes. To confirm the role of metabolism in the differential responses, we re-tested these 75 compounds in the absence of NADPH or with heat-attenuated microsomes. Forty-four compounds treated with RLM, but none with HLM, became less potent under these conditions, confirming the role of RLM in metabolic activation. Further evidence of biotransformation was obtained by measuring the half-life of the parent compounds in the presence of microsomes. Together, the data support the use of RLM in qHTS for identifying chemicals requiring biotransformation to induce biological responses.
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