Miniaturized flow cytometry‐based in vitro primary human lymphocyte micronucleus assay—validation study

Most in vitro mammalian genotoxicity assays show a low specificity (high rate of irrelevant positive results), and therefore, lead to an increase in follow‐up in vivo genotoxicity testing. One of the sources of the high rate of in vitro irrelevant positive results that find no confirmation in in vivo studies may be the characteristics of the test system used. It has been shown that cells that are p53 deficient or carry an alteration in DNA repair genes may be more prone to produce high rate of false/irrelevant positive results. Primary human lymphocytes (HuLy) are considered to show a higher specificity in predicting the in vivo genotoxic potential of a tested compound. We recently developed a flow cytometry‐based primary human T‐lymphocyte micronucleus test (MNT) and showed that the technology is promising and reliable in detecting genotoxic compounds. The purpose of the present work was to develop and validate a miniaturized format of the assay. For validation purposes of the flow cytometry HuLy MNT a wide selection of compounds with different mechanisms of genotoxicity was used. The evaluation covered 30 compounds: 19 commercially available genotoxicants and nongenotoxicants and 11 early pharmaceutical development compounds. Being faster and less tedious than the microscopic analysis, the miniaturized flow cytometry‐based methodology showed very promising results. Conveniently, cell division is verified within the same sample as the MN frequency. Moreover analysis of hypodiploid events may provide an indication for a mode of action, i.e. clastogenic versus aneugenic mechanism, for further follow‐up testing. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.     

In vivo pig‐a and micronucleus study of the prototypical aneugen vinblastine sulfate

The Pig‐a assay is being used in regulatory studies to evaluate the potential of agents to induce somatic cell gene mutations and an OECD test guideline is under development. A working group involved with establishing the guideline recently noted that representative aneugenic agents had not been evaluated, and to help fill this data gap Pig‐a mutant phenotype and micronucleated reticulocyte frequencies were measured in an integrated study design to assess the mutagenic and cytogenetic damage responses to vinblastine sulfate exposure. Male Sprague Dawley rats were treated for twenty‐eight consecutive days with vinblastine dose levels from 0.0156 to 0.125 mg/kg/day. Micronucleated reticulocyte frequencies in peripheral blood were determined at Days 4 and 29, and mutant cell frequencies were determined at Days ?4, 15, 29, and 46. Vinblastine affected reticulocyte frequencies, with reductions noted during the treatment phase and increases observed following cessation of treatment. Micronucleated reticulocyte frequencies were significantly elevated at Day 4 in the high dose group. Although a statistically significant increase in mutant reticulocyte frequencies were found for one dose group at a single time point (Day 46), it was not deemed biologically relevant because there was no analogous finding in mutant RBCs, it occurred at the lowest dose tested, and only 1 rat exceeded an upper bound tolerance interval established with historical negative control rats. Therefore, whereas micronucleus induction reflects vinblastine's well‐established aneugenic effect on hematopoietic cells, the lack of a Pig‐a response indicates that this tubulin‐binding agent does not cause appreciable mutagenicity in this same cell type. Environ. Mol. Mutagen. 59:30–37, 2018. © 2017 Wiley Periodicals, Inc.     

In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability

The in vitro micronucleus test has received considerable attention in recent years for its use in drug safety assessment and toxicological research. The less tedious nature of the assay relative to chromosome aberration analyses is a driving force, and explains why many chemical and drug safety programs have adopted the endpoint. Development of a high-throughput micronucleus scoring system would further enhance the utility of the assay for lead optimization and other early drug development work. Although several variations of a flow cytometric (FCM) method for scoring cell-culture-derived micronuclei (MN) have been described in the literature, they have been unable to distinguish true MN from apoptotic and necrotic chromatin (Nüsse M and Marx K 1997: Mutat Res 392: 109-115). Here, we report advances to this methodology whereby a sequential staining procedure is used to differentially label these types of sub-2n particles. With the use of ethidium monoazide (EMA), the chromatin of dead and dying cells is labeled. After a photoactivation step that covalently binds EMA to chromatin, cytoplasmic membranes are digested and resulting lysates are incubated with RNase plus a pan-nucleic acid dye (SYTOX Green). This process provides a suspension of free nuclei and sub-2n particles that are labeled with either SYTOX or SYTOX and EMA. Preliminary studies with heat-shocked L5178Y mouse cells demonstrated that EMA stains necrotic and mid- through late-stage apoptotic cells. Importantly, the sequential labeling procedure provided reliable micronucleus enumeration, even when cultures contained high percentages of dead cells. Subsequently, experiments with the following diverse genotoxicants were performed: hydroxyurea, methyl methanesulfonate, benzo[a]pyrene, etoposide, cyclophosphamide, and vinblastine. Additionally, the nongenotoxicants sucrose, tributyltin methoxide, and dexamethasone were tested up to 5 mg/ml, or to cytotoxic concentrations. FCM data were found to correspond closely with microscopy-based measurements. Collectively, these data suggest that this sequential EMA/SYTOX staining procedure provides reliable, high-throughput enumeration of in vitro MN.     

Flow cytometric 96‐well microplate‐based in vitro micronucleus assay with human TK6 cells: Protocol optimization and transferability assessment

An automated approach for scoring in vitro micronuclei (MN) has been described in which flow cytometric analysis is combined with compound exposure, processing, and sampling in a single 96‐well plate (Bryce SM et al. [2010]: Mutat Res 703:191‐199). The current report describes protocol optimization and an interlaboratory assessment of the assay's transferability and reproducibility. In a training phase, the methodology was refined and collaborating laboratories were qualified by repeatedly testing three compounds. Second, a set of 32 chemicals comprised of reference genotoxicants and presumed non‐genotoxicants was tested at each of four sites. TK6 cells were exposed to 10 closely spaced compound concentrations for 1.5‐ to 2‐cell population doublings, and were then stained and lysed for flow cytometric analysis. MN frequencies were determined by evaluating ≥5,000 cells per replicate well, and several indices of cytotoxicity were acquired. The prevalence of positive results varied according to the MN‐fold increase used to signify a genotoxic result, as well as the endpoint used to define a cytotoxicity limit. By varying these parameters, assay sensitivity and specificity values ranged from 82 to 98%, and 86 to 97%, respectively. In a third phase, one laboratory tested a further six genotoxicants and five non‐genotoxic apoptosis inducers. In these experiments assay specificity was markedly improved when top concentration selection was based on two cytotoxicity endpoints—relative survival and quantification of ethidium monoazide‐positive events. Collectively, the results indicate that the miniaturized assay is transferable across laboratories. The 96‐well format consumes considerably less compound than conventional in vitro MN test methods, and the high information content provided by flow cytometry helps guard against irrelevant positive results arising from overt toxicity. Environ. Mol. Mutagen. 54:180–194, 2013. © 2013 Wiley Periodicals, Inc.     

Interpreting in vitro micronucleus positive results: Simple biomarker matrix discriminates clastogens,aneugens, and misleading positive agents

The specificity of in vitro mammalian cell genotoxicity assays is low, as they yield a high incidence of positive results that are not observed in animal genotoxicity and carcinogenicity tests, that is, “misleading” or “irrelevant” positives. We set out to develop a rapid and effective follow‐up testing strategy that would predict whether apparent in vitro micronucleus‐inducing effects are due to a clastogenic, aneugenic, or secondary irrelevant mode(s) of action. Priority was given to biomarkers that could be multiplexed onto flow cytometric acquisition of micronucleus frequencies, or that could be accomplished in parallel using a homogeneous‐type assay. A training set of 30 chemicals comprised of clastogens, aneugens, and misleading positive chemicals was studied. These experiments were conducted with human TK6 cells over a range of closely spaced concentrations in a continuous exposure design. In addition to micronucleus frequency, the following endpoints were investigated, most often at time of harvest: cleaved Parp‐positive chromatin, cleaved caspase 3‐positive chromatin, ethidium monoazide bromide‐positive chromatin, polyploid nuclei, phospho‐histone H3‐positive (metaphase) cells, tetramethylrhodamine ethyl ester‐negative cells, cellular ATP levels, cell cycle perturbation, and shift in γ‐H2AX fluorescence relative to solvent control. Logistic regression was used to identify endpoints that effectively predict chemicals' a priori classification. Cross validation using a leave‐one‐out approach indicated that a promising base model includes γ‐H2AX shift and change in phospho‐histone H3‐positive events (25/30 correct calls). Improvements were realized when one or two additional endpoints were included (26—30/30 correct calls). These models were further evaluated with a test set of 10 chemicals, and also by evaluating 3 chemicals at a collaborating laboratory. The resulting data support the hypothesis that a matrix of high throughput‐compatible biomarkers can effectively delineate two important modes of genotoxic action as well as identify cytotoxicity that can lead to irrelevant positive results. Environ. Mol. Mutagen. 55:542–555, 2014. © 2014 Wiley Periodicals, Inc.     

A flow cytometry based in vitro micronucleus assay in TK6 cells—Validation using early stage pharmaceutical development compounds

The micronucleus test (MNT) is a well established test for detecting clastogenic and aneugenic compounds. Despite the assay's advantages, the MNT may produce false positive and false negative results in some conditions. This fact may be related to the underestimation of apoptosis or necrosis, the p53 status of the cell system or the cytotoxicity assay, and the top dose selection. The purpose of our studies was to contribute to the validation efforts of the flow cytometry based MNT. To identify the most reliable cytotoxicity assay for the top dose selection five parameters for relative survival were tested: relative cell count, relative population doubling, trypan blue supravital staining, relative ratio of scored nuclei to latex beads, and ethidium monoazide staining. For all compounds the least sensitive method was the relative cell count and the most reliable was the nuclei/beads ratio. The comparative evaluation of micronuclei induction in TK6 cells, analyzed with microscopy and flow cytometry, was performed with reference compounds and internal Novartis early development compounds with positive, weak positive, equivocal, and negative genotoxic effects. Our data document a good correlation between the MNT results obtained by flow cytometry and by microscopy. The results confirm that the method may be applied for routine testing in the pharmaceutical industry for the tested group of compounds, including compounds which require metabolic activation. However, further validation and miniaturization may be required. Environ. Mol. Mutagen., 2011. © 2010 Wiley‐Liss,Inc.     

Diethylnitrosamine genotoxicity evaluated in sprague dawley rats using Pig‐a mutation and reticulocyte micronucleus assays

Diethylnitrosamine (DEN) is a genotoxic carcinogen, but in vivo DNA‐damaging activities are not usually evident in hematopoietic cells because the short‐lived active metabolite is formed mainly in the liver. DEN therefore represented an interesting case for evaluating the performance characteristics of blood‐based endpoints of genotoxicity that have been automated using flow cytometric analysis—frequency of micronucleated reticulocytes and Pig‐a mutant phenotype reticulocytes (RET^CD59?) and erythrocytes (RBC^CD59?). Male Sprague Dawley rats were treated for 28 consecutive days with DEN at levels up to 12.5 mg/kg/day. Serial blood samples were collected and micronucleus frequencies were determined on Days 4 and 29, while RET^CD59? and RBC^CD59? frequencies were determined on Days 15, 29, and 42. The Pig‐a analyses were conducted with an enrichment step based on immunomagnetic column separation to increase the statistical power of the assay. Modest but significant reductions to reticulocyte frequencies demonstrated that bone marrow was exposed to reactive intermediates. Even so, DEN did not affect micronucleus frequencies at any dose level tested. However, RET^CD59? frequencies were significantly elevated in the high dose group on Day 29, and RBC^CD59? were increased at this same dose level on Days 29 and 42. These results demonstrate that the Pig‐a assay is sufficiently sensitive to evaluate chemicals for genotoxic potential, even in the case of a promutagen that has traditionally required direct assessment(s) of liver tissue for detection of DNA‐damage. Environ. Mol. Mutagen. 55:400–406, 2014. © 2014 Wiley Periodicals, Inc.     

Application of the adverse outcome pathway framework to genotoxic modes of action

In May 2017, the Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee hosted a workshop to discuss whether mode of action (MOA) investigation is enhanced through the application of the adverse outcome pathway (AOP) framework. As AOPs are a relatively new approach in genetic toxicology, this report describes how AOPs could be harnessed to advance MOA analysis of genotoxicity pathways using five example case studies. Each of these genetic toxicology AOPs proposed for further development includes the relevant molecular initiating events, key events, and adverse outcomes (AOs), identification and/or further development of the appropriate assays to link an agent to these events, and discussion regarding the biological plausibility of the proposed AOP. A key difference between these proposed genetic toxicology AOPs versus traditional AOPs is that the AO is a genetic toxicology endpoint of potential significance in risk characterization, in contrast to an adverse state of an organism or a population. The first two detailed case studies describe provisional AOPs for aurora kinase inhibition and tubulin binding, leading to the common AO of aneuploidy. The remaining three case studies highlight provisional AOPs that lead to chromosome breakage or mutation via indirect DNA interaction (inhibition of topoisomerase II, production of cellular reactive oxygen species, and inhibition of DNA synthesis). These case studies serve as starting points for genotoxicity AOPs that could ultimately be published and utilized by the broader toxicology community and illustrate the practical considerations and evidence required to formalize such AOPs so that they may be applied to genetic toxicity evaluation schemes. Environ. Mol. Mutagen. 61:114–134, 2020. © 2019 Wiley Periodicals, Inc.     

In vivo flow cytometric Pig‐a and micronucleus assays: Highly sensitive discrimination of the carcinogen/noncarcinogen pair benzo(a)pyrene and pyrene using acute and repeated‐dose designs

Combining multiple genetic toxicology endpoints into a single in vivo study, and/or integrating one or more genotoxicity assays into general toxicology studies, is attractive because it reduces animal use and enables comprehensive comparative analysis using toxicity, metabolism, and pharmacokinetic information from the same animal. This laboratory has developed flow cytometric scoring techniques for monitoring two blood‐based genotoxicity endpoints—micronucleated reticulocyte frequency and gene mutation at the Pig‐a locus—thereby making combination and integration studies practical. The ability to effectively monitor these endpoints in short‐term and repeated dosing schedules was investigated with the carcinogen/noncarcinogen pair benzo(a)pyrene (BP) and pyrene (Pyr). Male Sprague‐Dawley rats were treated via oral gavage for 3 or 28 consecutive days with several dose levels of Pyr, including maximum tolerated doses. BP exposure was administered by the same route but at one dose level, 250 or 125 mg/kg/day for 3‐day and 28‐day studies, respectively. Serial blood samples were collected up to Day 45, and were analyzed for Pig‐a mutation with a dual labeling method (SYTO 13 in combination with anti‐CD59‐PE) that facilitated mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. A mutant cell enrichment step based on immunomagnetic column separation was used to increase the statistical power of the assay. BP induced robust mutant reticulocyte responses by Day 15, and elevated frequencies persisted until study termination. Mutant erythrocyte responses lagged mutant reticulocyte responses, with peak incidences observed on Day 30 of the 3‐day study (43‐fold increase) and on Day 42 of the 28‐day study (171‐fold increase). No mutagenic effects were apparent for Pyr. Blood samples collected on Day 4, and Day 29 for the 28‐day study, were evaluated for micronucleated reticulocyte frequency. Significant increases in micronucleus frequencies were observed with BP, whereas Pyr had no effect. These results demonstrate that Pig‐a and micronucleus endpoints discriminate between these structurally related carcinogenic and noncarcinogenic agents. Furthermore, the high sensitivity demonstrated with the enrichment protocol indicates that the Pig‐a endpoint is suitable for both repeated‐dose and acute studies, allowing integration of mutagenic and clastogenic endpoints into on‐going toxicology studies, and use as a short‐term assay that provides efficient screening and mechanistic information in vivo. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.     

Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach

Several endpoints associated with cellular responses to DNA damage as well as overt cytotoxicity were multiplexed into a miniaturized, “add and read” type flow cytometric assay. Reagents included a detergent to liberate nuclei, RNase and propidium iodide to serve as a pan‐DNA dye, fluorescent antibodies against γH2AX, phospho‐histone H3, and p53, and fluorescent microspheres for absolute nuclei counts. The assay was applied to TK6 cells and 67 diverse reference chemicals that served as a training set. Exposure was for 24 hrs in 96‐well plates, and unless precipitation or foreknowledge about cytotoxicity suggested otherwise, the highest concentration was 1 mM. At 4‐ and 24‐hrs aliquots were removed and added to microtiter plates containing the reagent mix. Following a brief incubation period robotic sampling facilitated walk‐away data acquisition. Univariate analyses identified biomarkers and time points that were valuable for classifying agents into one of three groups: clastogenic, aneugenic, or non‐genotoxic. These mode of action predictions were optimized with a forward‐stepping process that considered Wald test p‐values, receiver operator characteristic curves, and pseudo R² values, among others. A particularly high performing multinomial logistic regression model was comprised of four factors: 4 hr γH2AX and phospho‐histone H3 values, and 24 hr p53 and polyploidy values. For the training set chemicals, the four‐factor model resulted in 94% concordance with our a priori classifications. Cross validation occurred via a leave‐one‐out approach, and in this case 91% concordance was observed. A test set of 17 chemicals that were not used to construct the model were evaluated, some of which utilized a short‐term treatment in the presence of a metabolic activation system, and in 16 cases mode of action was correctly predicted. These initial results are encouraging as they suggest a machine learning strategy can be used to rapidly and reliably predict new chemicals' genotoxic mode of action based on data from an efficient and highly scalable multiplexed assay. Environ. Mol. Mutagen. 57:171–189, 2016. © 2016 Wiley Periodicals, Inc.     

Pig‐a gene mutation and micronucleated reticulocyte induction in rats exposed to tumorigenic doses of the leukemogenic agents chlorambucil,thiotepa, melphalan,and 1,3‐propane sultone

To evaluate whether blood‐based genotoxicity endpoints can provide temporal and dose‐response data within the low‐dose carcinogenic range that could contribute to carcinogenic mode of action (MoA) assessments, we evaluated the sensitivity of flow cytometry‐based micronucleus and Pig‐a gene mutation assays at and below tumorigenic dose rate 50 (TD50) levels. The incidence of micronucleated reticulocytes (MN‐RET) was used to evaluate chromosomal damage, and the frequency of CD59‐negative reticulocytes (RET^CD59?) and erythrocytes (RBC^CD59?) served as phenotypic reporters of mutation at the X‐linked Pig‐a gene. Several leukemogenic agents with a presumed genotoxic MoA were studied. Specifically, male Sprague Dawley rats were treated via oral gavage for 28 days with chlorambucil, thiotepa, melphalan, and 1,3‐propane sultone at doses corresponding to 0.33x, 1x, and 3x TD50, as well as at the maximum tolerated dose. Frequencies of MN‐RET were determined at Days 4 and 29, and RET^CD59? and RBC^CD59? data were collected pretreatment as well as Days 15/16, 29, and 56/57. Dose‐related increases were observed for each endpoint, and time to maximal effect was consistently: MN‐RET < RET^CD59? < RBC^CD59?. For each of the chemicals studied, the genotoxic events occurred long before tumors or preneoplastic lesions would be expected. Furthermore, in the case of Pig‐a gene mutation, the responses were observed at or below the TD50 dose for three out of the four chemicals studied. These data illustrate the potential for quantitative blood‐based analyses to provide dose‐response and temporality information that relates genetic damage to cancer induction. Environ. Mol. Mutagen. 55:299–308, 2014. © 2014 Wiley Periodicals, Inc.     

Mouse Pig‐a and micronucleus assays respond to N‐ethyl‐N‐nitrosourea,benzo[a]pyrene,and ethyl carbamate,but not pyrene or methyl carbamate

This laboratory previously described a method for scoring the incidence of peripheral blood Pig‐a mutant phenotype rat erythrocytes using immunomagnetic separation in conjunction with flow cytometric analysis (In Vivo MutaFlow®). The current work extends the method to mouse blood, using the frequency of CD24‐negative reticulocytes (RET^CD24−) and erythrocytes (RBC^CD24−) as phenotypic reporters of Pig‐a gene mutation. Following assay optimization, reconstruction experiments demonstrated the ability of the methodology to return expected values. Subsequently, the responsiveness of the assay to the genotoxic carcinogens N‐ethyl‐N‐nitrosourea, benzo[a]pyrene, and ethyl carbamate was studied in male CD‐1 mice exposed for 3 days to several dose levels via oral gavage. Blood samples were collected on Day 4 for micronucleated reticulocyte analyses, and on Days 15 and 30 for determination of RET^CD24− and RBC^CD24− frequencies. The same design was used to study pyrene, with benzo[a]pyrene as a concurrent positive control, and methyl carbamate, with ethyl carbamate as a concurrent positive control. The three genotoxicants produced marked dose‐related increases in the frequencies of Pig‐a mutant phenotype cells and micronucleated reticulocytes. Ethyl carbamate exposure resulted in moderately higher micronucleated reticulocyte frequencies relative to N‐ethyl‐N‐nitrosourea or benzo[a]pyrene (mean ± SEM = 3.0 ± 0.36, 2.3 ± 0.17, and 2.3 ± 0.49%, respectively, vs. an aggregate vehicle control frequency of 0.18 ± 0.01%). However, it was considerably less effective at inducing Pig‐a mutant cells (e.g., Day 15 mean no. RET^CD24− per 1 million reticulocytes = 7.6 ± 3, 150 ± 9, and 152 ± 43 × 10⁻⁶, respectively, vs. an aggregate vehicle control frequency of 0.6 ± 0.13 × 10⁻⁶). Pyrene and methyl carbamate, tested to maximum tolerated dose or limit dose levels, had no effect on mutant cell or micronucleated reticulocyte frequencies. Collectively, these results demonstrate the utility of the cross‐species Pig‐a and micronucleated reticulocyte assays, and add further support to the value of studying both endpoints in order to cover two distinct genotoxic modes of action. Environ. Mol. Mutagen. 57:28–40, 2016. © 2015 Wiley Periodicals, Inc.     

Assessment of 5‐fluorouracil and 4‐nitroquinoline‐1‐oxide in vivo genotoxicity with Pig‐a mutation and micronucleus endpoints

Genotoxicity assessments were conducted on male Sprague Dawley rats treated with 5‐fluorouracil (5‐FU) and 4‐nitroquinoline‐1–oxide (4NQO) as part of an international validation trial of the Pig‐a mutant phenotype assay. Rats were orally exposed to 0, 11.5, 23, or 46 mg/kg/day 5‐FU for three consecutive days (Days 1–3); blood was sampled on Days ?1, 4, 15, 29, and 45. Pig‐a mutant phenotype reticulocyte (RET^CD59?) and mutant phenotype erythrocyte (RBC^CD59?) frequencies were determined on Days ?1, 15, 29, and 45, and percent micronucleated reticulocytes (%MN‐RET) were measured on Day 4. Rats were treated with 4NQO for 28 consecutive days by oral gavage, at doses of 1.5, 3, or 6 mg/kg/day. RBC^CD59? and RET^CD59? frequencies were determined on Days ?1, 15, and 29, and MN‐RET were quantified on Day 29. Whereas 5‐FU was found to increase %MN‐RET, no significant increases were observed for RBC^CD59? or RET^CD59? at any of the time points studied. The high dose of 4NQO (6 mg/kg/day) was observed to markedly increase RBC^CD59? and RET^CD59? frequencies, and this same dose level caused a weak but significantly elevated increase in MN‐RET (approximately twofold). Collectively, the results provide additional support for the combination of Pig‐a mutation and MN‐RET into acute and 28‐day repeat‐dose studies. Environ. Mol. Mutagen. 55:735–740, 2014. © 2014 Wiley Periodicals, Inc.     

Flow cytometric analysis of Pig‐a gene mutation and chromosomal damage induced by procarbazine hydrochloride in CD‐1 mice

Procarbazine is a genotoxic carcinogen whose DNA‐damaging activities are not reliably detected in vitro. We evaluated the in vivo genotoxic effects of procarbazine on hematopoietic cells of male CD‐1 mice using a multi‐endpoint study design that scored micronucleated reticulocyte (MN‐RET) frequency and gene mutation at the Pig‐a locus. CD‐1 mice were treated for 3 days with procarbazine, up to 150 mg/kg/day. Blood samples collected on Day 3 exhibited robust induction of MN‐RETs, with the high dose group exhibiting a mean 29‐fold increase. Blood collected 15 and 30 days after treatment began was analyzed for Pig‐a mutation with a dual labeling method that facilitated mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. Procarbazine significantly increased mutant reticulocyte frequencies by Day 15. Mutant erythrocyte responses were also apparent, with a peak incidence observed for the high dose group on Day 30. These results demonstrate that the complex metabolism and resulting genotoxicity of procarbazine is best evaluated in intact animal models, and show that the flow cytometric methods employed offer a means to efficiently monitor both in vivo chromosomal damage and mutation. Environ. Mol. Mutagen. 54:294–298, 2013. © 2013 Wiley Periodicals, Inc.     

Convenient,multi‐well plate‐based DNA damage response analysis using DT40 mutants is applicable to a high‐throughput genotoxicity assay with characterization of modes of action

Chemists continually synthesize myriad new chemicals (～2,000/year), some of which make their way into the environment or otherwise pose possible threats to humans who potentially become exposed to the compounds. Regulators must determine whether these, along with the glut (～80,000) of existing, chemicals are toxic and at what exposure levels. An important component of this determination is to ascertain the mode of action (MOA) of each compound as it relates to the pathway the compound uses to induce genotoxicity. Several assays have traditionally been used to reveal these effects to the genome: the Ames test, tests with yeast and mammalian cell lines, and animal studies. Previously, we described a new multi‐well plate‐based method which makes use of the DT40 isogenic cell line and its dozens of available mutants knocked out in DNA repair and cell cycle pathways and we now provide a detailed protocol of the further improvement of the assay. Although the DT40 line has existed for some time and has been used in numerous studies of DNA repair pathways, little use has been made of this valuable resource for toxicological investigations. Our method introduces the 2,3‐bis (2‐methoxy‐4‐nitro‐5‐sulfophenyl)?5‐[(phenylamino) carbonyl]?2H‐tetrazolium hydroxide dye scheme determination of cell survival in a manner that greatly increases throughput and reduces cost while maintaining reasonable sensitivity. Although this new genotoxicity assay requires validation with many more mutagens before becoming an established, regulatory decision‐making analysis tool, we believe that this method will be very advantageous if eventually added to the repertoire of those investigating MOAs of potentially genotoxic substances. Environ. Mol. Mutagen. 52:153–160, 2011. © 2010 Wiley‐Liss, Inc.     

Etoposide (VP-16) is a potent inducer of micronuclei in male rat meiosis: Spermatid micronucleus test and DNA flow cytometry after etoposide treatment

The genotoxic and cytotoxic effects of etoposide (VP-16), a topoisomerase II inhibitor, on male rat spermatogenic cells were studied by analysing induction of micronuclei during meiosis. Micronuclei (MN) were scored in early spermatids offer different time intervals corresponding to exposure of different stages of meiotic prophase. Etoposide had a strong effect on diplotene-diakinesis I cells harvested 1 day after exposure, and a significant effect also on late pachytene cells harvested 3 days after exposure. The effect at 18 days corresponding to exposure of preleptotene stage of meiosis (S-phase) was weaker but also statistically significant. Adriamycin was used as a positive control in this study. The results indicate a different mechanism of action of etoposide compared with adriamycin and other chemicals studied previously with the spermatid micronucleus test. DMA flow cytometry was carried out to assess cytotoxic damage at the same time intervals (1, 3, and 18 days after treatment) at stages I and VII of the seminiferous epithelial cycle allowing a study of cytotoxicity to different spermatogenic cell stages. Damage of differentiating sper-matogonia was observed by a decrease in the cell numbers of the 2C peak 1 and 3 days after treatment and by a reduction of the number of 4C cells (primary spermatocytes) 18 d after etoposide treatment. Adriamycin also killed differentiating spermatogonia. Since the cell population which showed a high induction of MN by etoposide was not reduced in number, the genotoxic effect is remarkable. We conclude that etoposide is a potent inducer of genotoxicity and patients treated with this agent during cancer chemotherapy are at a risk of genetic damage. © 1994 Wiley-Liss, Inc.     

Analysis of genotoxicity and the carcinogenic mode of action for ortho-phenylphenol

Ortho-phenylphenol (OPP) and its sodium salt (SOPP) are commercial products that have wide human exposure and have been shown in several studies to be rodent carcinogens. Genetic toxicology data were assessed in an attempt to understand the carcinogenic mode of action of OPP and SOPP. More than 130 studies were evaluated to determine if OPP, SOPP, or any of their enzymatic or nonenzymatic breakdown products react directly with DNA to induce mutation, changes in chromosome structure or number, DNA repair, or nonspecific DNA damage including strand breakage or covalent binding. The genotoxicity databases for OPP and SOPP are not only large but heterogeneous, requiring weight-of-evidence methods to arrive at a conclusion regarding their genotoxic properties and potential. Evidence derived from the available studies leads to the conclusion that study results showing OPP/SOPP directly interacting with DNA are equivocal. Clastogenicity was the most consistent type of genetic toxicity produced by OPP/SOPP (and their break-down products) and was consistently associated with other intracellular preneoplastic toxicity produced at super-threshold concentrations. The weight of evidence from the combined database supports the hypothesis that OPP/SOPP-induced DNA damage is a threshold-dependent response associated with target tissue toxicity, most likely induced by their breakdown products phenylhydroquinone and phenylbenzoquinone. It is possible that this threshold-dependent clastogenicity could contribute to the carcinogenic mode of action for OPP or SOPP.     

Evaluation of the Pig‐a,micronucleus, and comet assay endpoints in a 28‐day study with ethyl methanesulfonate

Ethyl methanesulfonate (EMS) was evaluated as part of the validation effort for the rat Pig‐a mutation assay and compared with other well‐established in vivo genotoxicity endpoints. Male Sprague‐Dawley (SD) rats were given a daily dose of 0, 6.25, 12.5, 25, 50, or 100 mg/kg/day EMS for 28 days, and evaluated for a variety of genotoxicity endpoints in peripheral blood, liver, and colon. Blood was sampled pre‐dose (Day 1) and at various time points up to Day 105. Pig‐a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBC^CD59? and RET^CD59? frequencies. The first statistically significant increases in mutant frequencies were seen in RETs on Day 15 and in RBCs on Day 29 with the maximum RET^CD59? on Day 29 and of RBC^CD59? on Day 55. The lowest dose producing a statistically significant increase of RET^CD59? was 12.5 mg/kg on Day 55 and 25 mg/kg for RBC^CD59? on Day 55. EMS also induced significant increases in % micronucleated RETs (MN‐RETs) in peripheral blood on Days 3, 15, and 28. No statistically significant increases in micronuclei were seen in liver or colon. Results from the in vivo Comet assay on Day 29 showed generally weak increases in DNA damage in all tissues evaluated with little evidence for accumulation of damage seen over time. The results with EMS indicate that the assessment of RBC^CD59? and/or RET^CD59? in the Pig‐a assay could be a useful and sensitive endpoint for a repeat dose protocol and complements other genotoxicity endpoints. Environ. Mol. Mutagen. 55:492–499, 2014. © 2014 Wiley Periodicals, Inc.     

Evaluating genotoxicity data to identify a mode of action and its application in estimating cancer risk at low doses: A case study involving carbon tetrachloride

In the new USEPA cancer risk assessment guidelines, mode of action (MoA) information, combined with a determination of whether or not a chemical is mutagenic, plays an important role in determining whether a linear or nonlinear approach should be used to estimate cancer risks at low doses. In this article, carbon tetrachloride (CT) is used as an example to illustrate how mixed genotoxicity data can be evaluated and used to identify a likely MoA. CT is essentially negative in inducing gene mutations in Salmonella, but is consistently positive in inducing recombination and aneuploidy in fungi. Negative or equivocal results were seen in most in vitro and in vivo studies in mammals, including mutation studies in transgenic mice. However, DNA adducts, primarily those derived from oxidation- and lipid-peroxidation-derived products as well as DNA double-strand breaks and micronucleated cells, have been seen repeatedly in the liver of CT-treated animals. On the basis of the weight of evidence, CT should not be considered a directly mutagenic agent. Mutagenic as well as other genotoxic effects, as they occur, will most likely be generated through indirect mechanisms resulting from oxidative and lipid peroxidative damage and/or damage occurring during necrosis or apoptosis. As key events in this process are expected to occur in a nonlinear fashion, the expected relationship between CT dose and carcinogenic response in the liver is likely to be nonlinear with a steep dose response. This conclusion is consistent with rodent cancer bioassay results in which steep nonlinear dose responses have been seen.