James T. MacGregor

The induction of micronuclei as a measure of genotoxicity

Abstract There are many possible micronucleus assays involving different test organisms and tissues. Because micronuclei arise from chromosomal fragments or chromosomes that are not incorporated into daughter nuclei at the time of cell division, the assay detects both clastogens and agents that affect the spindle apparatus. We know of no case in which micronuclei and chromosomal breakage (or loss) have been shown to occur independently of one another in any dividing cell population. This relationship is so close that false-positives and false-negatives (insofar as the detection of tissue-specific chromosome damage is concerned) should be determined primarily by the statistics of sampling. The production of micronuclei in various experimental organisms has been reviewed. Although there are several promising experimental approaches such as the use of meiotic plant cells or human cells in culture, only one form of the assay, the in vivo mammalian bone-marrow polychromatic erythrocyte (PCE) assay, has been sufficiently developed to be considered a standard assay. More than 150 chemicals have been tested in this assay, with varying degrees of rigor. The data from the literature have been summarized and evaluated in light of the work Groups recommendations for an adequate test. The standards for an adequate test are an important part of the recommendations. These standards, although based on the most recent information available to us, are subject to change because this assay is still evolving. The most important recommendations in this report are: (1) at least 500 PCE should be examined from each of 8 animals to detect an increase of about 4‰ (per thousand) PCE when the background is less than 4 per 1000, (2) sampling should be extended to at least 72 h after the initial treatment, with sampling intervals no greater than 24 h, and (3) the highest possible doses should be used. The success rate of the assay to detect chemicals designated by the Environmental Protection Agency (EPA) as carcinogens is difficult to estimate for several reasons. First, few chemicals designated as noncarcinogens were studied, although in routine testing noncarcinogens are expected to be much more common than carcinogens. Hence, the rate of false-positives (insofar as the detection of cancer is concerned), which ought to be one of the strongest features of the assays, could not be estimated. Second, few chemicals have been tested as rigorously as this report recommends. Hence, the rate of false-negatives is almost certainly overestimated. (It is, nevertheless, obvious that false-negatives are to be expected for any tissue-specific in vivo assay like the micronucleus assay. For example diethylnitrosamine, which produces chromosomal aberrations, micronuclei, and cancer in the liver, is not detected in the bone-marrow micronucleus assay.) Third, many carcinogens are species-specific and this fact has not been taken into account. Considering these caveats, the uncorrected detection rate of the chemicals designated as carcinogens by EPA is about 50%. We believe that this would have been significantly higher had all tests been performed according to the test criteria. Further improvements in the assay are to be expected and these may lead to improvements in its success rate. Recent developments are discussed.

In vivo rodent erythrocyte micronucleus assay

The following summary represents a consensus of the working group except where noted. The items discussed are listed in the order in which they appear in the OECD guideline (474) for easy reference. Introduction, purpose, scope, relevance, application and limits of test. The analysis of immature erythrocytes in either bone marrow or peripheral blood is equally acceptable for those species in which the spleen does not remove micronucleated erythrocytes. In the mouse, mature erythrocytes are also an acceptable cell population for micronucleus analysis when the exposure duration exceeds 4 weeks. Test substances. Organic solvents such as DMSO are not recommended. Freshly prepared solutions or suspensions should be used unless stability data demonstrate the acceptability of storage. Vegetable oils are acceptable as solvents or vehicles. Suspension of the test chemicals is acceptable for p.o. or i.p. administration but not for i.v. injection. The use of any unusual solvent should be justified. Selection of species. Any commonly used laboratory rodent species is acceptable. There is no strain preference. Number and sex. The size of experiment (i.e., number of cells per animal, number of animals per group) should be finalized based on statistical considerations. Although a consensus was not achieved, operationally it was agreed that 2000 cells per animal and four animals per group was a minimum requirement. In general, the available database suggests that the use of one gender is adequate for screening. However, if there is evidence indicating a significant difference in the toxicity between male and female, then both sexes should be used. Treatment schedule. No unique treatment schedule can be recommended. Results from extended dose regimens are acceptable as long as positive. For negative studies, toxicity should be demonstrated or the limit dose should be used, and dosing continued until sampling. Dose levels. At least three dose levels separated by a factor between 2 and square root of 10 should be used. The highest dose tested should be the maximum tolerated dose based on mortality, bone marrow cell toxicity, or clinical symptoms of toxicity. The limit dose is 2 g/kg/day for treatment periods of 14 days or less and 1 g/kg/day for treatment periods greater than 14 days. A single dose level (the limit dose) is acceptable if there is no evidence of toxicity. Controls. Concurrent solvent (vehicle) controls should be included at all sampling times. A pretreatment sample, however, may also be acceptable only in the short treatment period peripheral blood studies. A concurrent positive control group should be included for each experiment.(ABSTRACT TRUNCATED AT 400 WORDS)

Toxicology and Genetic Toxicology in the New Era of “Toxicogenomics”: Impact of “-omics” Technologies

The unprecedented advances in molecular biology during the last two decades have resulted in a dramatic increase in knowledge about gene structure and function, an immense database of genetic sequence information, and an impressive set of efficient new technologies for monitoring genetic sequences, genetic variation, and global functional gene expression. These advances have led to a new sub-discipline of toxicology: “toxicogenomics”. We define toxicogenomics as “the study of the relationship between the structure and activity of the genome (the cellular complement of genes) and the adverse biological effects of exogenous agents.” This broad definition encompasses most of the variations in the current usage of this term, and in its broadest sense includes studies of the cellular products controlled by the genome (messenger RNAs, proteins, metabolites, etc.). The new “global” methods of measuring families of cellular molecules, such as RNA, proteins, and intermediary metabolites have been termed “-omic” technologies, based on their ability to characterize all, or most, members of a family of molecules in a single analysis. With these new tools, we can now obtain complete assessments of the functional activity of biochemical pathways, and of the structural genetic (sequence) differences among individuals and species, that were previously unattainable. These powerful new methods of high-throughput and multi-endpoint analysis, include gene expression arrays that will soon permit the simultaneous measurement of the expression of all human genes on a single “chip”. Likewise, there are powerful new methods for protein analysis (proteomics: the study of the complement of proteins in the cell) and for analysis of cellular small molecules (metabonomics: the study of the cellular This article has been reproduced from Mutation Research, Vol 499, 2002, pp 13–25, Aardema & MacGregor, by the permission of Elsevier Science, Ltd. metabolites formed and degraded under genetic control). This will likely be extended in the near future to other important classes of biomolecules such as lipids, carbohydrates, etc. These assays provide a general capability for global assessment of many classes of cellular molecules, providing new approaches to assessing functional cellular alterations. These new methods have already facilitated significant advances in our understanding of the molecular responses to cell and tissue damage, and of perturbations in functional cellular systems.

Trivalent Chromium: Assessing the Genotoxic Risk of an Essential Trace Element and Widely Used Human and Animal Nutritional Supplement

Trivalent chromium [Cr(III)] is recognized as an essential nutrient, and is widely used as a nutritional supplement for humans and animals. Recent reports of the induction of genetic damage in cultured cells exposed to Cr(III) compounds in vitro have heightened the concern that Cr(III) compounds may exert genotoxic effects under certain conditions, raising the question of the relative benefit versus risk of dietary and feed supplementation practices. We have reviewed the literature since 1990 on genotoxic effects of Cr(III) compounds to determine whether recent findings provide a sufficient weight of evidence to modify the conclusions about the safety of this dietary supplement reached in the several comprehensive reviews conducted during the period 1990–2004. The extensive literature on genotoxic effects of Cr(III) compounds includes many instances of conflicting information, with both negative and positive findings often reported in similar test systems. Outcomes of in vitro tests conducted with Cr(III) in cultured cells are quite variable regardless of the chemical form of the chromium compound tested. The in vitro data show that Cr(III) has the potential to react with DNA and to cause DNA damage in cell culture systems, but under normal circumstances, restricted access of Cr(III) to cells in vivo limits or prevents genotoxicity in biological systems. The available in vivo evidence suggests that genotoxic effects are very unlikely to occur in humans or animals exposed to nutritional or to moderate recommended supplemental levels of Cr(III). However, excessive intake of Cr(III) supplements does not appear to be warranted at this time. Thus, like other nutrients that have exhibited genotoxic effects in vitro under high exposure conditions, nutritional benefits appear to outweigh the theoretical risk of genotoxic effects in vivo at normal or modestly elevated physiological intake levels.

Serum Troponins as Biomarkers of Drug-Induced Cardiac Toxicity

Member of the Expert Working Group and Chair of the Expert Working Group and Corresponding Author, Professor, Department of Biochemistry & Molecular Biology, University of Minnesota School of Medicine, Duluth, MN FDA Liaison and FDA Center for Drug Evaluation and Research, Rockville, MD 20852 Center for Drug Evaluation and Research, FDA, Laurel, MD 20708 Principal Scientist, Oxford GlycoSciences, Montgomery Village, MD 20886-1265 FDA National Center for Toxicological Research, Rockville, MD 20857 Drug Safety Evaluation, Global Research and Development, Ann Arbor Laboratories, Pfizer Inc., Ann Arbor, MI 48105 Laboratory of Molecular Carcinogenesis, National Institutes of Environmental Health Sciences, Research Triangle Park, NC 27709 Director, General Toxicology, Drug Safety and Metabolism, Schering-Plough Research Institute, Lafayette, NJ 07848, and Manager, Clinical Pathology Laboratory, Preclinical Safety Sciences, GlaxoSmithKline, Hertfordshire, SG12, ODP, United Kingdom

Quantitative approaches for assessing dose–response relationships in genetic toxicology studies

Genetic toxicology studies are required for the safety assessment of chemicals. Data from these studies have historically been interpreted in a qualitative, dichotomous “yes” or “no” manner without analysis of dose–response relationships. This article is based upon the work of an international multi‐sector group that examined how quantitative dose–response relationships for in vitro and in vivo genetic toxicology data might be used to improve human risk assessment. The group examined three quantitative approaches for analyzing dose–response curves and deriving point‐of‐departure (POD) metrics (i.e., the no‐observed‐genotoxic‐effect‐level (NOGEL), the threshold effect level (Td), and the benchmark dose (BMD)), using data for the induction of micronuclei and gene mutations by methyl methanesulfonate or ethyl methanesulfonate in vitro and in vivo. These results suggest that the POD descriptors obtained using the different approaches are within the same order of magnitude, with more variability observed for the in vivo assays. The different approaches were found to be complementary as each has advantages and limitations. The results further indicate that the lower confidence limit of a benchmark response rate of 10% (BMDL10) could be considered a satisfactory POD when analyzing genotoxicity data using the BMD approach. The models described permit the identification of POD values that could be combined with mode of action analysis to determine whether exposure(s) below a particular level constitutes a significant human risk. Subsequent analyses will expand the number of substances and endpoints investigated, and continue to evaluate the utility of quantitative approaches for analysis of genetic toxicity dose–response data. Environ. Mol. Mutagen., 2013.

Integration of mutation and chromosomal damage endpoints into 28-day repeat dose toxicology studies.

Two endpoints of genetic toxicity, mutation at the X-linked Pig-a gene and chromosomal damage in the form of micronucleated reticulocytes (MN-RETs), were evaluated in blood samples obtained from 28-day repeat-dosing studies typical of those employed in toxicity evaluations. Male Wistar Han rats were treated at 24-h intervals on days 1 through 28 with one of five prototypical genotoxicants: N-ethyl-N-nitrosourea, 7,12-dimethyl-12-benz[a]anthracene, 4-nitroquinoline-1-oxide (4NQO), benzo(a)pyrene, and N-methyl-N-nitrosourea. Flow cytometric scoring of CD59-negative erythrocytes (indicative of glycosylphosphatidylinositol anchor deficiency and hence Pig-a mutation) was performed using blood specimens obtained on days -1, 15, 29, and 56. Blood specimens collected on days 4 and 29 were evaluated for MN-RET frequency using flow cytometry-based MicroFlow Kits. With the exception of 4NQO, each chemical induced significant increases in the frequency of MN-RETs on days 4 and 29. All five agents increased the frequency of mutant phenotype (CD59 negative) reticulocytes (RETs) and erythrocytes. Mutation responses in RETs occurred earlier than in erythrocytes and tended to peak, or nearly peak, at day 29. In contrast, the mutant phenotype erythrocyte responses were modest on day 29 and required additional time to reach their maximal value. The observed kinetics were expected based on the known turnover of RETs and erythrocytes. The data show that RETs can serve as an appropriate indicator cell population for 28-day studies. Collectively, these data suggest that blood-based genotoxicity endpoints can be effectively incorporated into routine toxicology studies, a strategy that would reduce animal usage while providing valuable genetic toxicity information within the context of other toxicological endpoints.

Micronucleated erythrocyte frequency in peripheral blood of B6C3F1 mice from short‐term, prechronic, and chronic studies of the NTP carcinogenesis bioassay program

The mouse peripheral blood micronucleus (MN) test was performed on samples collected from 20 short‐term, 67 subchronic, and 5 chronic toxicity and carcinogenicity studies conducted by the National Toxicology Program (NTP). Data are presented for studies not previously published. Aspects of protocol that distinguish this test from conventional short‐term bone marrow MN tests are duration of exposure, and absence of repeat tests and concurrent positive controls. Furthermore, in contrast to short‐term bone marrow MN tests where scoring is limited to polychromatic erythrocytes (PCE), longer term studies using peripheral blood may evaluate MN in both, or either, the normochromatic (NCE) or PCE populations. The incidence of MN‐PCE provides an index of damage induced within 72 hr of sampling, whereas the incidence of MN in the NCE population at steady state provides an index of average damage during the 30‐day period preceding sampling. The mouse peripheral blood MN test has been proposed as a useful adjunct to rodent toxicity tests and has been effectively incorporated as a routine part of overall toxicity testing by the NTP. Data derived from peripheral blood MN analyses of dosed animals provide a useful indication of the in vivo potential for induced genetic damage and supply an important piece of evidence to be considered in the overall assessment of toxicity and health risk of a particular chemical. Although results indicate that the test has low sensitivity for prediction of carcinogenicity, a convincingly positive result in this assay appears to be highly predictive of rodent carcinogenicity. Environ. Mol. Mutagen. 36:163–194, 2000

Derivation of point of departure (PoD) estimates in genetic toxicology studies and their potential applications in risk assessment

Genetic toxicology data have traditionally been employed for qualitative, rather than quantitative evaluations of hazard. As a continuation of our earlier report that analyzed ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS) dose–response data (Gollapudi et al., 2013), here we present analyses of 1‐ethyl‐1‐nitrosourea (ENU) and 1‐methyl‐1‐nitrosourea (MNU) dose–response data and additional approaches for the determination of genetic toxicity point‐of‐departure (PoD) metrics. We previously described methods to determine the no‐observed‐genotoxic‐effect‐level (NOGEL), the breakpoint‐dose (BPD; previously named Td), and the benchmark dose (BMD10) for genetic toxicity endpoints. In this study we employed those methods, along with a new approach, to determine the non‐linear slope‐transition‐dose (STD), and alternative methods to determine the BPD and BMD, for the analyses of nine ENU and 22 MNU datasets across a range of in vitro and in vivo endpoints. The NOGEL, BMDL10 and BMDL1SD PoD metrics could be readily calculated for most gene mutation and chromosomal damage studies; however, BPDs and STDs could not always be derived due to data limitations and constraints of the underlying statistical methods. The BMDL10 values were often lower than the other PoDs, and the distribution of BMDL10 values produced the lowest median PoD. Our observations indicate that, among the methods investigated in this study, the BMD approach is the preferred PoD for quantitatively describing genetic toxicology data. Once genetic toxicology PoDs are calculated via this approach, they can be used to derive reference doses and margin of exposure values that may be useful for evaluating human risk and regulatory decision making. Environ. Mol. Mutagen. 55:609–623, 2014.

Efficient monitoring of in vivo pig-a gene mutation and chromosomal damage: summary of 7 published studies and results from 11 new reference compounds.

The ability to effectively monitor gene mutation and micronucleated reticulocyte (MN-RET) frequency in short-term and repeated dosing schedules was investigated using the recently developed flow cytometric Pig-a mutation assay and flow cytometric micronucleus analysis. Eight reference genotoxicants and three presumed nongenotoxic compounds were studied: chlorambucil, melphalan, thiotepa, cyclophosphamide, azathioprine, 2-acetylaminofluorene, hydroxyurea, methyl methanesulfonate, o-anthranilic acid, sulfisoxazole, and sodium chloride. These experiments extend previously published results with seven other chemicals. Male Sprague Dawley rats were treated via gavage for 3 or 28 consecutive days with several dose levels of each chemical up to the maximum tolerated dose. Blood samples were collected at several time points up to day 45 and were analyzed for Pig-a mutation with a dual-labeling method that facilitates mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. An immunomagnetic separation technique was used to increase the efficiency of scoring mutant cells. Blood samples collected on day 4, and day 29 for the 28-day study, were evaluated for MN-RET frequency. The three nongenotoxicants did not induce Pig-a or MN-RET responses. All genotoxicants except hydroxyurea increased the frequency of Pig-a mutant reticulocytes and erythrocytes. Significant increases in MN-RET frequency were observed for each of the genotoxicants at both time points. Whereas the highest Pig-a responses tended to occur in the 28-day studies, when total dose was greatest, the highest induction of MN-RET was observed in the 3-day studies, when dose per day was greatest. There was no clear relationship between the maximal Pig-a response of a given chemical and its corresponding maximal MN-RET response, despite the fact that both endpoints were determined in the same cell lineage. Taken with other previously published results, these data demonstrate the value of integrating Pig-a and micronucleus endpoints into in vivo toxicology studies, thereby providing information about mutagenesis and chromosomal damage in the same animals from which toxicity, toxicokinetics, and metabolism data are obtained.