The 28 + 28 day design is an effective sampling time for analyzing mutant frequencies in rapidly proliferating tissues of MutaMouse animals.

The Organisation for Economic Co-Operation and Development Test Guideline 488 (TG 488) uses transgenic rodent models to generate in vivo mutagenesis data for regulatory submission. The recommended design in TG 488, 28 consecutive daily exposures with tissue sampling three days later (28 + 3d), is optimized for rapidly proliferating tissues such as bone marrow (BM). A sampling time of 28 days (28 + 28d) is considered more appropriate for slowly proliferating tissues (e.g., liver) and male germ cells. We evaluated the impact of the sampling time on mutant frequencies (MF) in the BM of MutaMouse males exposed for 28 days to benzo[a]pyrene (BaP), procarbazine (PRC), isopropyl methanesulfonate (iPMS), or triethylenemelamine (TEM) in dose-response studies. BM samples were collected + 3d, + 28d, + 42d or + 70d post exposure and MF quantified using the lacZ assay. All chemicals significantly increased MF with maximum fold increases at 28 + 3d of 162.9, 6.6, 4.7 and 2.8 for BaP, PRC, iPMS and TEM, respectively. MF were relatively stable over the time period investigated, although they were significantly increased only at 28 + 3d and 28 + 28d for TEM. Benchmark dose (BMD) modelling generated overlapping BMD confidence intervals among the four sampling times for each chemical. These results demonstrate that the sampling time does not affect the detection of mutations for strong mutagens. However, for mutagens that produce small increases in MF, sampling times greater than 28 days may produce false-negative results. Thus, the 28 + 28d protocol represents a unifying protocol for simultaneously assessing mutations in rapidly and slowly proliferating somatic tissues and male germ cells.

Analysis of Biomarkers of DNA Damage and Mutagenicity in Mice Exposed to Acrylonitrile.

Acrylonitrile (ACN), which is a widely used industrial chemical, induces cancers in the mouse via unresolved mechanisms. For this report, complementary and previously described methods were used to assess in vivo genotoxicity and/or mutagenicity of ACN in several mouse models, including (i) female mice devoid of cytochrome P450 2E1 (CYP2E1), which yields the epoxide intermediate cyanoethylene oxide (CEO), (ii) male lacZ transgenic mice, and (iii) female (wild-type) B6C3F1 mice. Exposures of wild-type mice and CYP2E1-null mice to ACN at 0, 2.5 (wild-type mice only), 10, 20, or 60 (CYP2E1-null mice only) mg/kg body weight by gavage for 6 weeks (5 days/week) produced no elevations in the frequencies of micronucleated erythrocytes, but induced significant dose-dependent increases in DNA damage, detected by the alkaline (pH >13) Comet assay, in one target tissue (forestomach) and one nontarget tissue (liver) of wild-type mice only. ACN exposures by gavage also caused significant dose-related elevations in the frequencies of mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) reporter gene of T-lymphocytes from spleens of wild-type mice; however, Hprt mutant frequencies were significantly increased in CYP2E1-null mice only at a high dose of ACN (60 mg/kg) that is lethal to wild-type mice. Similarly, drinking water exposures of lacZ transgenic mice to 0, 100, 500, or 750 ppm ACN for 4 weeks caused significant dose-dependent elevations in Hprt mutant frequencies in splenic T-cells; however, these ACN exposures did not increase the frequency of lacZ transgene mutations above spontaneous background levels in several tissues from the same animals. Together, the Comet assay and Hprt mutant frequency data from these studies indicate that oxidative metabolism of ACN by CYP2E1 to CEO is central to the induction of the majority of DNA damage and mutations in ACN-exposed mice, but ACN itself also may contribute to the carcinogenic modes of action via mechanisms involving direct and/or indirect DNA reactivity.

Analysis of DNA Adducts and Mutagenic Potency and Specificity in Rats Exposed to Acrylonitrile.

Acrylonitrile (ACN), which is a widely used industrial chemical, induces cancers in multiple organs/tissues of rats by unresolved mechanisms. For this report, evidence for ACN-induced direct/indirect DNA damage and mutagenesis was investigated by assessing the ability of ACN, or its reactive metabolite, 2-cyanoethylene oxide (CEO), to bind to DNA in vitro, to form select DNA adducts [N7-(2'-oxoethyl)guanine, N2,3-ethenoguanine, 1,N6-ethenodeoxyadenosine, and 3,N4-ethenodeoxycytidine] in vitro and/or in vivo, and to perturb the frequency and spectra of mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene in rats exposed to ACN in drinking water. Adducts and frequencies and spectra of Hprt mutations were analyzed using published methods. Treatment of DNA from human TK6 lymphoblastoid cells with [2,3-14C]-CEO produced dose-dependent binding of 14C-CEO equivalents, and treatment of DNA from control rat brain/liver with CEO induced dose-related formation of N7-(2'-oxoethyl)guanine. No etheno-DNA adducts were detected in target tissues (brain and forestomach) or nontarget tissues (liver and spleen) in rats exposed to 0, 3, 10, 33, 100, or 300 ppm ACN for up to 105 days or to 0 or 500 ppm ACN for ∼15 months; whereas N7-(2'-oxoethyl)guanine was consistently measured at nonsignificant concentrations near the assay detection limit only in liver of animals exposed to 300 or 500 ppm ACN for ≥2 weeks. Significant dose-related increases in Hprt mutant frequencies occurred in T-lymphocytes from spleens of rats exposed to 33-500 ppm ACN for 4 weeks. Comparisons of "mutagenic potency estimates" for control rats versus rats exposed to 500 ppm ACN for 4 weeks to analogous data from rats/mice treated at a similar age with N-ethyl-N-nitrosourea or 1,3-butadiene suggest that ACN has relatively limited mutagenic effects in rats. Considerable overlap between the sites and types of mutations in ACN-exposed rats and butadiene-exposed rats/mice, but not controls, provides evidence that the carcinogenicity of these epoxide-forming chemicals involves corresponding mutagenic mechanisms.

Quantitative relationships between lacZ mutant frequency and DNA adduct frequency in Muta™Mouse tissues and cultured cells exposed to 3-nitrobenzanthrone.

The frequency of stable DNA adducts in a target tissue can be used to assess biologically effective dose; however, the utility of the metric in a risk assessment context depends on the likelihood that the DNA damage will be manifested as mutation. Previously, we employed the Muta™Mouse system to examine the induction of lacZ mutants and DNA adducts following exposure to the well-studied mutagenic carcinogen 3-nitrobenzanthrone (3-NBA). In this follow-up work, we examined the empirical relationships between total adduct frequency and mutant frequency (MF) in tissues and cultured cells following acute 3-NBA exposure. The results show a significant induction of DNA damage and lacZ mutants in liver, colon and bone marrow, as well as FE1 pulmonary epithelial cells. In contrast, lung and small intestine samples had low, but significantly elevated adduct levels, with no significant increases in lacZ MF. Additional analyses showed a significant relationship between the mutagenic efficiency of total adducts, measured as the slope of the relationships between MF and total adduct frequency, and tissue-specific mitotic index (MI). The lack of mutation response in lung, in contrast to the high in vitro MF in FE-1 lung cells, is likely related to the 100-fold difference in MI. The lack of small intestine mutagenic response may be related to limited metabolic capacity, differences in DNA repair, and /or chemically induced apoptosis that has been observed for other potent mutagens. The results indicate that interpretation of adduct frequency values in a risk assessment context can be improved by considering the MI of the target tissue; however, more generalised interpretation is hampered by tissue-specific variations in metabolic capacity and damage processing. The work provides a proof of principle regarding the use of the Muta™Mouse system to critically examine the health risks associated with tissue-specific adduct loads.

Severity of effect considerations regarding the use of mutation as a toxicological endpoint for risk assessment: A report from the 8th International Workshop on Genotoxicity Testing (IWGT).

Exposure levels without appreciable human health risk may be determined by dividing a point of departure on a dose-response curve (e.g., benchmark dose) by a composite adjustment factor (AF). An "effect severity" AF (ESAF) is employed in some regulatory contexts. An ESAF of 10 may be incorporated in the derivation of a health-based guidance value (HBGV) when a "severe" toxicological endpoint, such as teratogenicity, irreversible reproductive effects, neurotoxicity, or cancer was observed in the reference study. Although mutation data have been used historically for hazard identification, this endpoint is suitable for quantitative dose-response modeling and risk assessment. As part of the 8th International Workshops on Genotoxicity Testing, a sub-group of the Quantitative Analysis Work Group (WG) explored how the concept of effect severity could be applied to mutation. To approach this question, the WG reviewed the prevailing regulatory guidance on how an ESAF is incorporated into risk assessments, evaluated current knowledge of associations between germline or somatic mutation and severe disease risk, and mined available data on the fraction of human germline mutations expected to cause severe disease. Based on this review and given that mutations are irreversible and some cause severe human disease, in regulatory settings where an ESAF is used, a majority of the WG recommends applying an ESAF value between 2 and 10 when deriving a HBGV from mutation data. This recommendation may need to be revisited in the future if direct measurement of disease-causing mutations by error-corrected next generation sequencing clarifies selection of ESAF values.

No evidence for transgenerational genomic instability in the F1 or F2 descendants of Muta™Mouse males exposed to N-ethyl-N-nitrosourea.

Exposure of male mice to genotoxic agents can increase mutation frequencies in their unexposed descendants. This phenomenon, known as transgenerational genomic instability (TGI), can persist for several generations. However, little is known about the underlying mechanisms. Chemically-induced TGI has been demonstrated in non-coding unstable tandem repeat DNA regions, but it is unclear whether it extends to other genetic endpoints. We investigated whether exposure of Muta™Mouse males to a single dose of 75mg/kg N-ethyl-N-nitrosourea (ENU) increased the spontaneous frequency of gene mutations or chromosome damage in their offspring. Treated males were mated with untreated females 3 days, 6 weeks or 10 weeks post-exposure to produce the F1 generation. Offspring were thus conceived from germ cells exposed to ENU as mature spermatozoa, dividing spermatogonia, or spermatogonial stem cells, respectively. F2 mice were generated by mating F1 descendants with untreated partners. Mutations in the lacZ transgene were quantified in bone marrow and micronucleus frequencies were evaluated in red blood cells by flow-cytometry for all F0 and their descendants. LacZ mutant frequencies were also determined in sperm for all exposed males and their male descendants. In F0 males, lacZ mutant frequencies were significantly increased in bone marrow at least 10-fold at all three time points investigated. In sperm, lacZ mutant frequency was significantly increased 7-11-fold after exposure of dividing and stem cell spermatogonia, but not in replication-deficient haploid sperm. Micronucleus frequencies assessed two days after ENU treatment were increased 5-fold in F0 males, but returned to control levels after 10 weeks. Despite the strong mutagenic response in F0 males, pre- and post-meiotic ENU exposure did not significantly increase lacZ mutant or micronucleus frequencies in F1 or F2 offspring. These findings suggest that TGI may not extend to all genetic endpoints and that further investigation of this phenomenon and its health relevance will require multiple measures of genomic damage.

Harnessing genomics to identify environmental determinants of heritable disease.

Next-generation sequencing technologies can now be used to directly measure heritable de novo DNA sequence mutations in humans. However, these techniques have not been used to examine environmental factors that induce such mutations and their associated diseases. To address this issue, a working group on environmentally induced germline mutation analysis (ENIGMA) met in October 2011 to propose the necessary foundational studies, which include sequencing of parent-offspring trios from highly exposed human populations, and controlled dose-response experiments in animals. These studies will establish background levels of variability in germline mutation rates and identify environmental agents that influence these rates and heritable disease. Guidance for the types of exposures to examine come from rodent studies that have identified agents such as cancer chemotherapeutic drugs, ionizing radiation, cigarette smoke, and air pollution as germ-cell mutagens. Research is urgently needed to establish the health consequences of parental exposures on subsequent generations.

Assessing the quality and making appropriate use of historical negative control data: A report of the International Workshop on Genotoxicity Testing (IWGT).

Historical negative control data (HCD) have played an increasingly important role in interpreting the results of genotoxicity tests. In particular, Organisation for Economic Co-operation and Development (OECD) genetic toxicology test guidelines recommend comparing responses produced by exposure to test substances with the distribution of HCD as one of three criteria for evaluating and interpreting study results (referred to herein as "Criterion C"). Because of the potential for inconsistency in how HCD are acquired, maintained, described, and used to interpret genotoxicity testing results, a workgroup of the International Workshops for Genotoxicity Testing was convened to provide recommendations on this crucial topic. The workgroup used example data sets from four in vivo tests, the Pig-a gene mutation assay, the erythrocyte-based micronucleus test, the transgenic rodent gene mutation assay, and the in vivo alkaline comet assay to illustrate how the quality of HCD can be evaluated. In addition, recommendations are offered on appropriate methods for evaluating HCD distributions. Recommendations of the workgroup are: When concurrent negative control data fulfill study acceptability criteria, they represent the most important comparator for judging whether a particular test substance induced a genotoxic effect. HCD can provide useful context for interpreting study results, but this requires supporting evidence that (i) HCD were generated appropriately, and (ii) their quality has been assessed and deemed sufficiently high for this purpose. HCD should be visualized before any study comparisons take place; graph(s) that show the degree to which HCD are stable over time are particularly useful. Qualitative and semi-quantitative assessments of HCD should also be supplemented with quantitative evaluations. Key factors in the assessment of HCD include: (i) the stability of HCD over time, and (ii) the degree to which inter-study variation explains the total variability observed. When animal-to-animal variation is the predominant source of variability, the relationship between responses in the study and an HCD-derived interval or upper bounds value (i.e., OECD Criterion C) can be used with a strong degree of confidence in contextualizing a particular study's results. When inter-study variation is the major source of variability, comparisons between study data and the HCD bounds are less useful, and consequentially, less emphasis should be placed on using HCD to contextualize a particular study's results. The workgroup findings add additional support for the use of HCD for data interpretation; but relative to most current OECD test guidelines, we recommend a more flexible application that takes into consideration HCD quality. The workgroup considered only commonly used in vivo tests, but it anticipates that the same principles will apply to other genotoxicity tests, including many in vitro tests.

Error-corrected next generation sequencing - Promises and challenges for genotoxicity and cancer risk assessment.

Error-corrected Next Generation Sequencing (ecNGS) is rapidly emerging as a valuable, highly sensitive and accurate method for detecting and characterizing mutations in any cell type, tissue or organism from which DNA can be isolated. Recent mutagenicity and carcinogenicity studies have used ecNGS to quantify drug-/chemical-induced mutations and mutational spectra associated with cancer risk. ecNGS has potential applications in genotoxicity assessment as a new readout for traditional models, for mutagenesis studies in 3D organotypic cultures, and for detecting off-target effects of gene editing tools. Additionally, early data suggest that ecNGS can measure clonal expansion of mutations as a mechanism-agnostic early marker of carcinogenic potential and can evaluate mutational load directly in human biomonitoring studies. In this review, we discuss promising applications, challenges, limitations, and key data initiatives needed to enable regulatory testing and adoption of ecNGS - including for advancing safety assessment, augmenting weight-of-evidence for mutagenicity and carcinogenicity mechanisms, identifying early biomarkers of cancer risk, and managing human health risk from chemical exposures.

Impact of sampling time on the detection of mutations in rapidly proliferating tissues using transgenic rodent gene mutation models: A review.

The OECD Test Guideline 488 (TG 488) for the Transgenic Rodent Gene Mutation Assay has undergone several revisions to update the recommended design for studying mutations in somatic tissues and male germ cells. The recently revised TG recommends a single sampling time of 28 days following 28 days of exposure (i.e., 28 + 28 days) for all tissues, irrespective of proliferation rates. An alternative design (i.e., 28 + 3 days) is appropriate when germ cell data is not required, nor considered. While the 28 + 28 days design is clearly preferable for slowly proliferating somatic tissues and germ cells, there is still uncertainty about the impact of extending the sampling time to 28 days for rapidly somatic tissues. Here, we searched the available literature for evidence supporting the applicability and utility of the 28 + 28 days design for rapidly proliferating tissues. A total of 79 tests were identified. When directly comparing results from both designs in the same study, there was no evidence that the 28 + 28 days regimen resulted in a qualitatively different outcome from the 28 + 3 days design. Studies with a diverse range of agents that employed only a 28 + 28 days protocol provide further evidence that this design is appropriate for rapidly proliferating tissues. Benchmark dose analyses demonstrate high quantitative concordance between the 28 + 3 and 28 + 28 days designs for rapidly proliferating tissues. Accordingly, our review confirms that the 28 + 28 days design is appropriate to assess mutagenicity in both slowly and rapidly proliferating somatic tissues, and germ cells, and provides further support for the recommended design in the recently adopted TG 488.

Suitable top concentration for tests with mammalian cells: mouse lymphoma assay workgroup.

The Mouse Lymphoma Expert Workgroup of the International Workshop for Genotoxicity Tests (IWGT) met in Basel, Switzerland in August of 2009. The Workgroup (WG) was tasked with discussing the appropriate top concentration for non-pharmaceuticals that would be required for the conduct of the mouse lymphoma assay (MLA) when sufficient cytotoxicity [to between 10 and 20% relative total growth (RTG)] has not been attained. The WG approached this task by (1) enumerating the various regulatory decisions/use for MLA data, (2) discussing the appropriate assays to which MLA data and assay performance should be compared and (3) discussing all the proposals put forth concerning the top concentration for non-pharmaceuticals. In addition, one of the members presented a summary of a re-evaluation of the National Toxicology Program MLA data using the IWGT harmonized guidance that was underway as a separate (non IWGT) activity, being conducted by two members of the Expert WG. The WG was asked to vote on each of the various proposals for top concentration for when cytotoxicity is not concentration limiting. While there was general agreement that the top concentration for non-pharmaceuticals should be re-evaluated and likely lowered from the current recommended levels, there was no agreement on a specific new recommendation.

Assessment of subclinical, toxicant-induced hepatic gene expression profiles after low-dose, short-term exposures in mice.

Gene expression profiling that examines critical, toxicologically-relevant gene and signal-response pathways promises to improve risk assessment and safety evaluation of low-dose chemical exposures. As an approach to achieving this goal, mechanistic interpretations based upon gene expression changes that are determinants of adverse toxicological outcomes were applied to the analysis of low-dose gene expression profiles. RNA for expression profiling was obtained from mice given short-term gavage exposures to diminishing doses of four toxicants: 3,3',4,4',5-pentachlorobiphenyl (PCB126), phenobarbital (PB), isoproterenol (IPR), and lead acetate (PbAc). Lowest doses were below the no-observable effects levels established using standard clinical toxicology parameters. Hepatic gene expression profiles were analyzed using a custom, focused oligonucleotide DNA microarray, the HC ToxArray™, containing toxin-responsive and toxicologically-determinant genes. Expression data were compared to changes in conventional clinical chemistry parameters and drug metabolism activities. PCB126 and PB demonstrated a dose-dependent correlation between minimal changes in biochemical markers, hepatic metabolism and induction of gene expression profiles. PbAc exposure gave a small adaptive profile at the highest dose. IPR- and PCB126-induced changes were detected at doses below those required to alter the traditional biochemical endpoints and included genes with causal roles in hepatic toxicity, insulin resistance, atherosclerosis, angiogenesis and hypertension. Likely adverse phenotypic consequences resulting from expression changes lead to assignments of "Lowest Observed Adverse Transcriptional Expression Levels" (LOATEL) for each agent. These results support the suggestion that altered expression profiles of genes contributing to toxicologically-relevant pathways provide useful tools for reducing uncertainty in establishing no-effect levels and for designing longer-term toxicity studies.

Germ-line mutations, DNA damage, and global hypermethylation in mice exposed to particulate air pollution in an urban/industrial location.

Particulate air pollution is widespread, yet we have little understanding of the long-term health implications associated with exposure. We investigated DNA damage, mutation, and methylation in gametes of male mice exposed to particulate air pollution in an industrial/urban environment. C57BL/CBA mice were exposed in situ to ambient air near two integrated steel mills and a major highway, alongside control mice breathing high-efficiency air particulate (HEPA) filtered ambient air. PCR analysis of an expanded simple tandem repeat (ESTR) locus revealed a 1.6-fold increase in sperm mutation frequency in mice exposed to ambient air for 10 wks, followed by a 6-wk break, compared with HEPA-filtered air, indicating that mutations were induced in spermatogonial stem cells. DNA collected after 3 or 10 wks of exposure did not exhibit increased mutation frequency. Bulky DNA adducts were below the detection threshold in testes samples, suggesting that DNA reactive chemicals do not reach the germ line and cause ESTR mutation. In contrast, DNA strand breaks were elevated at 3 and 10 wks, possibly resulting from oxidative stress arising from exposure to particles and associated airborne pollutants. Sperm DNA was hypermethylated in mice breathing ambient relative to HEPA-filtered air and this change persisted following removal from the environmental exposure. Increased germ-line DNA mutation frequencies may cause population-level changes in genetic composition and disease. Changes in methylation can have widespread repercussions for chromatin structure, gene expression and genome stability. Potential health effects warrant extensive further investigation.

Characterisation of Muta™Mouse λgt10-lacZ transgene: evidence for in vivo rearrangements.

The multicopy λgt10-lacZ transgene shuttle vector of Muta™Mouse serves as an important tool for genotoxicity studies. Here, we describe a model for λgt10-lacZ transgene molecular structure, based on characterisation of transgenes recovered from animals of our intramural breeding colony. Unique nucleotide sequences of the 47 513 bp monomer are reported with GenBank® assigned accession numbers. Besides defining ancestral mutations of the λgt10 used to construct the transgene and the Muta™Mouse precursor (strain 40.6), we validated the sequence integrity of key λ genes needed for the Escherichia coli host-based mutation reporting assay. Using three polymerase chain reaction (PCR)-based chromosome scanning and cloning strategies, we found five distinct in vivo transgene rearrangements, which were common to both sexes, and involved copy fusions generating ∼10 defective copies per haplotype. The transgene haplotype was estimated by Southern hybridisation and real-time-polymerase chain reaction, which yielded 29.0 ± 4.0 copies based on spleen DNA of Muta™Mouse, and a reconstructed CD2F(1) genome with variable λgt10-lacZ copies. Similar analysis of commercially prepared spleen DNA from Big Blue® mouse yielded a haplotype of 23.5 ± 3.1 copies. The latter DNA is used in calibrating a commercial in vitro packaging kit for E.coli host-based mutation assays of both transgenic systems. The model for λgt10-lacZ transgene organisation, and the PCR-based methods for assessing copy number, integrity and rearrangements, potentially extends the use of Muta™Mouse construct for direct, genomic-type assays that detect the effects of clastogens and aneugens, without depending on an E.coli host, for reporting effects.

A Return to the Origin of the EMGS: Rejuvenating the Quest for Human Germ Cell Mutagens and Determining the Risk to Future Generations.

Fifty years ago, the Environmental Mutagen Society (now Environmental Mutagenesis and Genomics Society) was founded with a laser-focus on germ cell mutagenesis and the protection of "our most vital assets"-the sperm and egg genomes. Yet, five decades on, despite the fact that many agents have been demonstrated to induce inherited changes in the offspring of exposed laboratory rodents, there is no consensus on whether human germ cell mutagens exist. We argue that it is time to reevaluate the available data and conclude that we already have evidence for the existence of environmental exposures that impact human germ cells. What is missing are definite data to demonstrate a significant increase in de novo mutations in the offspring of exposed parents. We believe that with over two decades of research advancing knowledge and technologies in genomics, we are at the cusp of generating data to conclusively show that environmental exposures cause heritable de novo changes in the human offspring. We call on the research community to harness our technologies, synergize our efforts, and return to our Founders' original focus. The next 50 years must involve collaborative work between clinicians, epidemiologists, genetic toxicologists, genomics experts and bioinformaticians to precisely define how environmental exposures impact germ cell genomes. It is time for the research and regulatory communities to prepare to interpret the coming outpouring of data and develop a framework for managing, communicating and mitigating the risk of exposure to human germ cell mutagens. Environ. Mol. Mutagen. 61:42-54, 2020. © 2019 Her Majesty the Queen in Right of Canada.

In utero Exposure to Genotoxicants Leading to Genetic Mosaicism: An Overlooked Window of Susceptibility in Genetic Toxicology Testing?

In utero development represents a sensitive window for the induction of mutations. These mutations may subsequently expand clonally to populate entire organs or anatomical structures. Although not all adverse mutations will affect tissue structure or function, there is growing evidence that clonally expanded genetic mosaics contribute to various monogenic and complex diseases, including cancer. We posit that genetic mosaicism is an underestimated potential health problem that is not fully addressed in the current regulatory genotoxicity testing paradigm. Genotoxicity testing focuses exclusively on adult exposures and thus may not capture the complexity of genetic mosaicisms that contribute to human disease. Numerous studies have shown that conversion of genetic damage into mutations during early developmental exposures can result in much higher mutation burdens than equivalent exposures in adults in certain tissues. Therefore, we assert that analysis of genetic effects caused by in utero exposures should be considered in the current regulatory testing paradigm, which is possible by harmonization with current reproductive/developmental toxicology testing strategies. This is particularly important given the recent proposed paradigm change from simple hazard identification to quantitative mutagenicity assessment. Recent developments in sequencing technologies offer practical tools to detect mutations in any tissue or species. In addition to mutation frequency and spectrum, these technologies offer the opportunity to characterize the extent of genetic mosaicism following exposure to mutagens. Such integration of new methods with existing toxicology guideline studies offers the genetic toxicology community a way to modernize their testing paradigm and to improve risk assessment for vulnerable populations. Environ. Mol. Mutagen. 61:55-65, 2020. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

Mutation as a Toxicological Endpoint for Regulatory Decision-Making.

Mutations induced in somatic cells and germ cells are responsible for a variety of human diseases, and mutation per se has been considered an adverse health concern since the early part of the 20th Century. Although in vitro and in vivo somatic cell mutation data are most commonly used by regulatory agencies for hazard identification, that is, determining whether or not a substance is a potential mutagen and carcinogen, quantitative mutagenicity dose-response data are being used increasingly for risk assessments. Efforts are currently underway to both improve the measurement of mutations and to refine the computational methods used for evaluating mutation data. We recommend continuing the development of these approaches with the objective of establishing consensus regarding the value of including the quantitative analysis of mutation per se as a required endpoint for comprehensive assessments of toxicological risk. Environ. Mol. Mutagen. 61:34-41, 2020. © 2019 Wiley Periodicals, Inc.

The genotoxicity of mainstream and sidestream marijuana and tobacco smoke condensates.

While the prevalence of tobacco use has decreased in Canada over the past decade, that of marijuana use has increased, particularly among youth. However, the risks of adverse health effects from marijuana smoke exposure, specifically as compared to tobacco, are currently not well understood. The objectives of this study were to evaluate the relative ability of matched marijuana and tobacco condensates to induce (geno)toxic responses in three in vitro test systems. This study provides comparative data for matched sidestream and mainstream condensates, as well as condensates prepared under both a standard and an extreme smoking regime designed to mimic marijuana smoking habits. The results indicate that tobacco and marijuana smoke differ substantially in terms of their cytotoxicity, Salmonella mutagenicity, and ability to induce chromosomal damage (i.e., micronucleus formation). Specifically, the marijuana condensates were all found to be more cytotoxic and more mutagenic in the presence of S9 than the matched tobacco condensates. In contrast, the tobacco condensates appeared to induce cytogenetic damage in a concentration-dependent manner, whereas the matched marijuana condensates did not. In addition, when corrected for total particulate matter yield, little difference was observed in the mutagenic activity of samples smoked under the extreme vs the standard regime for both tobacco and marijuana condensates.

Mutagenicity testing for chemical risk assessment: update of the WHO/IPCS Harmonized Scheme.

Since the publication of the International Programme on Chemical Safety (IPCS) Harmonized Scheme for Mutagenicity Testing, there have been a number of publications addressing test strategies for mutagenicity. Safety assessments of substances with regard to genotoxicity are generally based on a combination of tests to assess effects on three major end points of genetic damage associated with human disease: gene mutation, clastogenicity and aneuploidy. It is now clear from the results of international collaborative studies and the large databases that are currently available for the assays evaluated that no single assay can detect all genotoxic substances. The World Health Organization therefore decided to update the IPCS Harmonized Scheme for Mutagenicity Testing as part of the IPCS project on the Harmonization of Approaches to the Assessment of Risk from Exposure to Chemicals. The approach presented in this paper focuses on the identification of mutagens and genotoxic carcinogens. Selection of appropriate in vitro and in vivo tests as well as a strategy for germ cell testing are described.

Toxicogenomic analysis of mainstream tobacco smoke-exposed mice reveals repression of plasminogen activator inhibitor-1 gene in heart.

Tobacco smoking is associated with cardiovascular pathology. However, the molecular mechanisms of tobacco smoke exposure that lead to initiation or exacerbation of cardiovascular disease are unclear. In this study, the effects of mainstream tobacco smoke (MTS) on global transcription in the heart were investigated. Male C57B1/CBA mice were exposed to MTS from 2 cigarettes daily, 5 days/wk for 6 or 12 wk. Mice were sacrificed immediately, or 6 wk following the last cigarette. High-density DNA microarrays were used to characterize global gene expression changes in whole heart. Fifteen genes were significantly differentially expressed following exposure to MTS. Among these genes, cytochrome P-450 1A1 (Cyp1A1) was upregulated by 12-fold, and Serpine-1 (plasminogen activator inhibitor-1, PAI-1) was downregulated by 1.7-fold. Concomitant increase in Cyp1A1 protein levels and decrease in total and active PAI-1 protein was observed in tissue extracts by Western blot assay and enzyme-linked immunosorbent assay (ELISA), respectively. Observed changes were transient and were partially reversed during break periods. Thus, gene expression profiling of heart tissue revealed a novel cardiovascular mechanism operating in response to MTS. Our results suggest a potential role for PAI-1 in MTS-induced cardiovascular pathology.