Commentary AReturn to theOrigin of the EMGS:Rejuvenating theQuest for HumanGermCellMutagens andDetermining theRisk to Future Generations FrancescoMarchetti ,*George R.Douglas, andCarole L.Yauk Environmental Health Science Research Bureau, Health Canada, Ottawa, Ontario, Canada 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 muta- gens exist. We argue that it is time to reevaluate the available data and conclude that we already have evidence for the existence of environmental expo- sures 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 environ- mental 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 Key words:mutation; germ cell; transgene; test guideline; heritable; next generation sequencing ROOTSOFEMGS INGERMCELLMUTAGENESIS Fifty years ago, the Environmental Mutagen Society (now Environmental Mutagenesis and Genomics Society, EMGS) was founded with the goal of identifying and elim- inating agents from the environment that cause mutations in germ cells to protect the health of human populations (DeMarini 2020). Soon after, a committee was established to formalize and publicize the fundamental role and mis- sion of the EMGS (Committee 17 1975). Most of the con- ferences and publications from this period emphasized the health hazards of germ cell mutations (e.g., Workshop on “The Evaluation of Chemical Mutagenicity Data in Rela- tion to Population Risk, Research Triangle Park, North Carolina, April 26–28, 1973”; Environmental Health Per- spectives, Vol. 6, December 1973). Despite the main emphasis on germ cell-mediated genetic effects, there was one paper at the workshop (Ames 1973) that profoundly changed the direction and intent of mutagenicity testing from a germ cell orientation to the prediction of carcinoge- nicity. As a result of the ground-breaking invention of the Ames assay and the demonstrated high degree of correla- tion between mutagenesis and carcinogenesis, the vast majority of genetic toxicology test development and appli- cation became centered on the relationship between muta- tion and cancer. Notwithstanding the increased emphasis on cancer, germ cell mutation has remained an endpoint of regulatory con- cern. Numerous reports have been published and regulatory practices promulgated related to the application and use of germ cell mutagenicity data (see review by Singer and Yauk 2010). Indeed, there are examples of regulatory assessment of germ cell data at a frequency similar to that for the regulatory assessment of cancer data (Yauk et al. 2015a). Furthermore, there is renewed interest in the Reproduced with the permission of the Minister of Health Canada Grant sponsor: Health Canada. *Correspondence to: Francesco Marchetti, Environmental Health Science and Research Bureau, Health Canada, 50 Colombine Driveway, Ottawa, ON K1A 0K9, Canada. E-mail: francesco.marchetti@canada.ca Received 29 July 2019; Revised 21 August 2019; Accepted 28 August 2019 DOI: 10.1002/em.22327 Published online 31 August 2019 in Wiley Online Library (wileyonlinelibrary.com). Environmental andMolecularMutagenesis 61:42^54 (2020) © 2019HerMajesty theQueen in Right of Canada https://orcid.org/0000-0002-9435-4867 mailto:francesco.marchetti@canada.ca http://crossmark.crossref.org/dialog/?doi=10.1002%2Fem.22327&domain=pdf&date_stamp=2019-10-10 regulatory assessment of germ cell hazard and risk because of the wide international implementation of the Globally Harmonized System (GHS) of classification and labelling of chemicals (United Nations 2017), which considers germ cell mutagenicity for hazard classification. This commentary provides a brief summary of the issues surrounding the use of germ cell mutagenicity data in regu- latory hazard and risk assessment, the application of new facilitating technologies, and the prospects for a germ cell genotoxicity renaissance. IMPACTOFGERMCELLMUTATIONS The role of mutations in the carcinogenic process is well established and thus, mutagenicity testing has been routinely conducted in order to identify agents that are capable of inducing mutations and may initiate cancer (Heflich et al. 2020). In this context, it should be noted that mutation is used a proxy for the ability of a compound to induce cancer and not as a direct determinant of cancer development. Also, although somatic mutations are associ- ated with a broad spectrum of human diseases (Erickson 2014; Goschalk et al., 2020), there is still limited evidence that a somatic mutation induced by an environmental expo- sure may result in an adverse health effect other than cancer. The impact of mutations in germ cells is dramatically different. A single mutation in a germ cell will produce an individual where every cell in the organism contains that mutation. Thus, the chance of that mutation impacting the health of the affected individual is much larger and it is not restricted to cancer. Indeed, the Human Gene Mutation Database (Stenson et al. 2017) currently catalogues over 260,000 different adverse mutational events affecting approximately 10,000 genes (accessed June 2019). Although point mutations represent the largest group of mutations, genomic changes affecting a few to a few thou- sand base pairs, translocations and complex chromosomal aberrations are also observed. The database is a critical resource to gather insights into the landscape of mutations that are linked to human diseases, and while it provides no information on the causes of the mutations, it would be hard to imagine that not a single one of those mutations was induced by an exogenous factor. Although germ cell mutations are relatively rare at the individual level, they can have a profound impact when considered at the population level. It was recently estimated that de novo mutations contribute to developmental disor- ders in as many as 400,000 births per year (Deciphering Developmental Disorders Study 2017). At the population level, even a relatively small increase in mutation rate cau- sed by an environmental exposure can have astounding consequences. For example, an analysis that considered only the spectrum of mutations that are expected to be induced by tobacco smoke and the roughly 700 genes that have been associated so far with intellectual disability showed that even a modest 25% increase in tobacco- induced sperm mutations would lead to over 500,000 affected children per generation across the global popula- tion (Beal et al. 2017). The economic consequences of these tobacco-induced de novo mutations for the health care system can be easily estimated in the dozens of billion US dollars. The above example provides just a glimpse of the socie- tal and economic impacts of environmentally induced de novo mutations and of the importance of identifying and eliminating exposure to environmental agents that can induce these effects. THE ENIGMAOF THE EXISTENCEOFHUMANGERMCELL MUTAGENS Despite many decades of research, regulatory decisions based on germ cell mutagenic effects are stymied by the lack of even one established, and widely acknowledged, human germ cell mutagen. As noted by DeMarini (2012), this enigma may stem from the lack of an organization that is specifically tasked with assessing the available data to deter- mine whether the weight of evidence is sufficient to conclude that an agent is a human germ cell mutagen (e.g., similarly to what the International Agency for Research on Cancer (IARC) does for carcinogens). We wholeheartedly endorse this assessment and argue that we already have sufficient data to say that human germ cell mutagens exist and that the cur- rent notion to the contrary is not grounded in facts. Below, we briefly discuss the evidence that substantiates the exis- tence of human germ cell mutagens. Over the past 80 years, a large body of literature has accumulated on the existence of many germ cell mutagens in animals. These studies demonstrate that exposure of lab- oratory rodents to genotoxic agents spanning different modes of action not only results in the induction of genetic damage in the germ cells themselves, but also in the trans- mission of de novo genetic changes to the offspring. Several reviews have been published on agents that cause genetic damage in rodent germ cells resulting in the induc- tion of chromosomal structural aberrations (Marchetti and Wyrobek 2005), aneuploidy (Pacchierotti et al. 2019), gene mutations (Marchetti et al. 2018b), dominant lethal mutations (Green et al. 1985) and inherited mutations (Russell 2004). The information contained in these reviews is sufficient to identify 84 agents as rodent germ cell muta- gens (Table I), which is likely a large underestimate of the true number of agents that are known to be genotoxic in rodent germ cells. Thirty-one of the mammalian germ cell mutagens have been shown to significantly increase inherited genetic changes in the offspring of exposed males (Table I). Thus, it is unquestionable that many envi- ronmental agents when tested under controlled laboratory Environmental and Molecular Mutagenesis. DOI 10.1002/em 43Quest for Human Germ Cell Mutagens 10982280, 2020, 1, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1002/em .22327 by H ealth C anada, W iley O nline L ibrary on [15/03/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense conditions damage the genetic material of rodent germ cells and result in the transmission of genetic defects to the offspring. Is it realistic then to think that none of these agents possess these characteristics when humans are exposed? We believe that the answer to this question is an unequivocal “no” and that there are many factors that are TABLE I. Mammalian Germ Cell Mutagensa Agent Germ cellsb Embryoc Offspringd Agent Germ cellsb Embryoc Offspringd Acrylamide M, R M M 7,12-Dimethylbenz[a]anthracene M M Ionizing radiation H, M M He, M Busulfan M M Tobacco smoke H, M M Hf, M Carbendazim DH, SH M Benzo(a)pyrene M M M Chemotherapy cocktails H, M M Bleomycin M M M Colchicine CH, DH, M M Chlorambucil M M M Di-(2-ethylhexyl)-phthalate M M Cyclophosphamide M M M Diepoxybutane M M Ethyl methanesulfonate M M M Ethanol H, M M Ethylene oxide M M, R M Hycantone methanesulfonate M R Etoposide CH, H, M M M Nocodazole M M Glycidamide M M M Taxol M M Isopropyl methanesulfonate M M M Vinblastine sulfate CH, M M Melphalan M M M 6-Mercaptopurine M Methyl methanesulfonate M M M Captan M Mitomycin C M M M Chloramphenicol M N-ethyl-N-nitrosourea M M M Colcemid M N-methyl-N-nitrosourea M M M Di-2-ethyl-hexyladipate M Procarbazine M M M Dihydroergotoxin methanesulfonate M Triethylenemelamine M M M Dimethylmyrelan M Trophosphamide M M M Ergotamine tartrate M 1-(2-Chroethyl) 3-cycloehxyl-1- nitrosourea R M Forfestrol tetrasodium M 1,3-Butadiene M M Fotrin M Chlormethine M M Hexamethylphosphoramide M Dacarbazine M M Methyl mercury chloride SH, M Diethyl sulfate M M Methysergide hydrogen maleate M Ifosfamide M M Mitomen M n-Propyl methanesulfonate M M N-methyl-N0-nitro-N- nitrosoguanidine M ThioTEPA M M Rubratoxin B M Trimethyl phosphate M M Saccarin M N-propyl-N-nitrosourea M M Triflupromazine M Trichlorfon M Hg Tris(2-methylazidiniyl)phosphine oxide M 1,2-Dibromo-3-chloropropane M M 4-Nitroquinoline-1-oxide M Acrylonitrile H Griseofulvin M Air pollution M, HG Hydroquinone M Amsacrine M Hydroxyurea M Benomyl M Merbarone M Benzene H Nitrilotriacetic acid M Chloral hydrate M Orthovanadate M Diethylstilbestrol M Podophyllotoxin CH Econazole M Teniposide M Epirubicin hydrochloride M Thiabendazole M Fenvalerate H Vincristine sulfate CH aAgents reported as having positive results in the following papers: Green et al. (1985); Russell (2004); Marchetti and Wyrobek (2005); Marchetti et al. (2018a); Marchetti et al. (2018b); Pacchierotti et al. (2019). For each agent, the species used in the studies is reported: CH (Chinese hamster); DH (Djungarian Hamster); H (Humans); HG (Herring gull); M (Mouse); R (Rat); SH (Syrian Hamster). bIncludes results obtained with the cytogenetic or FISH analyses of either sperm or oocytes. cIncludes results obtained with the dominant lethal assay. dIncludes results obtained in rodents with the specific locus test, heritable translocation assay in F1 males or tandem repeats and studies with human families. eDubrova et al. (1996, 2002, 2006). fSecretan et al. (2009). gCzeizel et al. (1993). Environmental and Molecular Mutagenesis. DOI 10.1002/em 44 Marchetti et al. 10982280, 2020, 1, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1002/em .22327 by H ealth C anada, W iley O nline L ibrary on [15/03/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense responsible for the apparent lack of human germ cell mutagens. Undoubtedly, there are many experimental variables that make the chance of identifying rodent germ cell mutagens more likely than doing the same in humans. First, studies in laboratory animals are generally conducted at dose levels that seldom occur in human populations. These studies use primarily acute or subacute regimens rather than the chronic exposure that is more common in humans. Acute exposures use much higher doses than those that can be applied under chronic conditions and are more likely to result in a measurable effect because the amount of DNA lesions induced at one time can overwhelm the DNA repair defense mechanisms (O’Brien et al. 2015; Yauk et al. 2015b). Second, these experiments generally use animals with a homogenous genetic background that is not repre- sentative of the genetic diversity present in the human pop- ulation. A genetically homogenous population reduces the variability in the response and increases the statistical power of detecting an effect. Third, exposure parameters and experimental conditions can be very carefully con- trolled in the laboratory to significantly reduce confounding factors that may affect the response. Indeed, major chal- lenges of conducting studies on the effects of environmen- tal chemicals in human populations are the precise characterization of the exposure and the identification of a true control population. Fourth, technological limitations have contributed to the challenges of identifying human germ cell mutagens as the most common methods used for investigating induced mutations in rodent germ cells (e.g., the specific locus test [SLT]; Russell et al. 1998) are not applicable to humans. Thus, both technological and methodological difficulties associated with human studies may be in part responsible for the current lack of consensus on the existence of human germ cell mutagens. Despite these caveats, rodent germ cell mutagens are pri- mary candidates for human germ cell mutagens and, indeed, there are several examples of agents that induce effects in both rodent and human gametes. In his commen- tary, DeMarini (2012) identified ionizing radiation, chemo- therapeutic agents, tobacco smoking and air pollution as potential human germ cell mutagens. The latter two expo- sures constitute complex mixtures that contain dozens of potential candidate germ cell mutagens, the most notable being genotoxic polycyclic aromatic hydrocarbons (PAHs) like benzo[a]pyrene (BaP). As briefly summarized below, the available human data for these agents do indeed support their categorization as human germ cell mutagens. Since the pioneering work of Russell et al. with the SLT (reviewed in Russell et al. 1998), ionizing radiation has been one of the best characterized and most studied rodent germ cell mutagen. In the early 90s, Dubrova et al. (1996) reported increased numbers of mutations in tandem repeat DNA sequences (minisatellites) in the offspring of men who had been exposed to ionizing radiation during the Chernobyl accident. These results were highly controversial at the time because of criticisms on the selection of concur- rent controls. However, follow up work with better mat- ched control populations and longitudinal study designs confirmed the initial findings (Dubrova 2002; Dubrova et al. 2002, 2006) and were further supported by studies with laboratory rodents demonstrating that ionizing radia- tion induced the same types of mutations under controlled laboratory conditions (Dubrova et al. 1993, 1998). The findings of Dubrova et al. were also criticized because of the type of mutations that were measured. It was thought that alterations in minisatellite sequences had no health impact and, thus, were of little significance for human health. Since then, we have a much better understanding of how duplications and deletions in noncoding genomic regions (including tandem repeats) can impact human health (Mirkin 2007; Mori et al. 2013; Lupski 2015). In light of today’s knowledge and understanding that point mutations and genomic alterations in coding genes repre- sent only a portion of the possible genetic changes with health-related implications, we contend that a re-evaluation of the original results of Dubrova et al. leads to the conclu- sion that these findings can be considered as evidence supporting the induction of heritable de novo mutations in humans by ionizing radiation. Chemotherapy cocktails generally contain chemicals that are known to be genotoxic and have been shown to induce both chromosomal abnormalities (reviewed in Wyrobek et al. 2005) and tandem repeat mutations (Vilarino-Guell et al. 2003; Glen et al. 2008; Glen and Dubrova 2012) in rodent sperm. Studies in cancer patients undergoing chemo- therapy have also shown significant increases in the fre- quency of sperm carrying either aneuploidy or structural chromosomal abnormalities (Robbins et al. 1997; Frias et al. 2003). Although in the majority of cases these increases were temporary with the frequencies of sperm with chromosomal abnormalities returning to baseline levels with increasing time from the end of chemotherapy (Martin et al. 1997; Robbins et al. 1997; Frias et al. 2003), a few studies reported significantly higher levels of abnor- mal sperm many years after chemotherapy (De Mas et al. 2001; Tempest et al. 2008). These results suggest a perma- nent effect of the chemotherapeutic cocktails on human stem cell spermatogonia. Importantly, these effects mirror those observed in the sperm of laboratory animals and demonstrate that when exposure levels are comparable, similar effects are seen in both rodent and human germ cells. Thus, for chemotherapeutic agents there is clear evi- dence that results obtained in rodent germ cells are predic- tive of similar effects in human germ cells. Tobacco smoke is one of the most widespread environ- mental exposures in human populations. The detrimental effects of tobacco smoking have been known for many decades, but in recent years there has been a renewed appreciation for its effects in the germ cells of smokers and Environmental and Molecular Mutagenesis. DOI 10.1002/em 45Quest for Human Germ Cell Mutagens 10982280, 2020, 1, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1002/em .22327 by H ealth C anada, W iley O nline L ibrary on [15/03/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense their potential consequences for the offspring. A recent review on the effects of tobacco smoking identified increases in DNA strand breaks, aneuploidy and mutations as some of the genetic changes that are present in the sperm of tobacco smokers (Beal et al. 2017). Also, studies in both rodents (Yauk et al. 2007; Marchetti et al. 2011) and humans (Linschooten et al. 2013) have consistently reported increases in tobacco-induced tandem repeat muta- tions. Perhaps surprisingly, there are yet no human data showing an increase in de novo mutations in the offspring of smokers, although whole genome analysis of cohorts of families with smoking fathers was identified as a priority research area (Yauk et al. 2013). Nevertheless, support for the hereditary effects of tobacco smoke can be found in the IARC’s assessment that preconception paternal smoking is causally linked to an increased risk of childhood leukemia in the offspring (Secretan et al. 2009). Among the four potential human germ cell mutagens proposed by DeMarini, air pollution is the one with the more limited data in humans. However, multiple studies in rodents (Somers et al. 2002; Yauk et al. 2008) and wildlife species (Yauk and Quinn 1996; Yauk et al. 2000) have consistently shown the impact of air pollution on the germ cell genome, including the demonstration that the muta- genic effects of air pollution in germ cells reside with par- ticulate matter (Somers et al. 2004). Importantly, all these studies of environmentally exposed animals resulted in her- itable tandem repeat mutations induced by ambient levels of air pollutants. Even in the absence of direct evidence in humans, the available animal data would be considered, when evaluated within an IARC style assessment, to be sufficient to conclude that air pollution is likely to be a human germ cell mutagen. PAHs are exemplary mutagens in tobacco smoke and air pollution. A large body of literature demonstrates that BaP is highly mutagenic to adult rodent germ cells. Studies clearly show that BaP causes both tandem repeat and gene mutations in rodent spermatogonial stem cells and differen- tiating spermatogonia (Olsen et al. 2010; Verhofstad et al. 2011; Xu et al. 2014; O’Brien et al. 2016a, b; Rowan- Carroll et al. 2017). Moreover, a recent study applying modern genomic technologies provides clear evidence that BaP induced de novo mutations (both single nucleotides and large duplications) in the offspring of exposed male mice (Beal et al. 2019). These data also lend weight to the relevance of tandem repeat mutations as biomarkers of potential mutagenicity in other locations in the genome. In parallel with this strong evidence of germ cell mutagenicity in rodents, PAH DNA adducts are detected in human sperm samples of individuals exposed to air pollution (Jeng et al. 2015; Oliveri Conti et al. 2017). Sperm of human smokers exhibit high levels of BaP DNA adducts as well as increased burdens of bulky adducts and oxidative lesions in general (reviewed in Beal et al. 2017). Therefore, the presence of BaP and other bulky PAH adducts in human sperm, and strong evidence supporting that BaP induces mutations in rodent sperm that are transmitted to their off- spring, strongly imply that BaP and PAHs are likely to cause germ cell mutagenicity in humans. Evidence for the induction of genetic damage in human germ cells also comes from many studies that have reported significant increases in genetic abnormali- ties in the sperm of workers occupationally exposed to benzene (Xing et al. 2010; Marchetti et al. 2012), acrylo- nitrile (Xu et al. 2003) and fenvalerate (Xia et al. 2004) among others. When the available human germ cell data are evaluated as a whole, the unavoidable conclusion is that we already have sufficient data to say that human germ cell mutagens exist. Admittedly, what is missing is the demonstration that some of these effects observed in human germ cells are transmitted to the next generation. One possible such an example is the reported case of a cluster of Down syndrome cases in a human population linked to environmental exposure to trichlorfon (Czeizel et al. 1993). However, this finding is outweighed by the current failure of epidemiological studies in children of atomic bomb (Schull 2003) and cancer (Mulvihill 2012) survivors to demonstrate an increase in inherited diseases in the offspring. Although these human epidemiological studies have shortcomings (discussed in detail in Schull 2003; DeMarini 2012; Mulvihill 2012), data demonstrat- ing the induction of de novo mutations in the offspring of exposed parents will be necessary to change this notion that there are no human germ cell mutagens. As discussed later, we believe that, as a scientific commu- nity, we are at the cusp of applying the latest genomic approaches to demonstrate transmission of environmen- tally induced de novo mutations to human offspring and conclusively demonstrate the existence of human germ cell mutagens. AREHUMANGERMCELLS FUNDAMENTALLYDIFFERENT FROMRODENTGERMCELLS? The results obtained in rodents may not be predictive of similar effects in humans if there are fundamental differ- ences between human and rodent spermatogenesis or if human germ cells have a more efficient DNA damage response than rodent germ cells. However, there is no solid evidence for either of these two possibilities. An extensive description of the process of spermatogenesis in mamma- lian species, including DNA repair competency, is outside of the scope of this commentary and the reader is directed to a few comprehensive reviews on the topic (Olsen et al. 2005; Amann 2008; Hermo et al. 2010). Here, we aim to succinctly argue that there are no physiological differences in spermatogenic processes that can be invoked to cause differences in the response of humans and rodents to envi- ronmental germ cell mutagens. Environmental and Molecular Mutagenesis. DOI 10.1002/em 46 Marchetti et al. 10982280, 2020, 1, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1002/em .22327 by H ealth C anada, W iley O nline L ibrary on [15/03/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense The process of spermatogenesis has been studied in detail in humans and in many laboratory animals. Sper- matogenesis is remarkably similar among mammalian spe- cies with the major difference being the time necessary to produce mature sperm from stem cell spermatogonia, which is species specific (Adler 1996). Furthermore, many of the genes that are known to be essential for spermato- genesis are conserved across species (Bonilla and Xu 2008; White-Cooper and Bausek 2010) and defects in a particular gene can produce the same phenotype in multiple species (Vangompel and Xu 2011). Also conserved across species is the fact that the DNA repair capacity is maximum in spermatogonia and gradually disappears during the postmeiotic phase of spermatogenesis (Olsen et al. 2005). Data in rodents clearly show that this period is most vul- nerable to the induction of genetic lesions that are retained in the fertilizing sperm and that result in the formation of de novo genetic defects in the developing embryo (Marchetti and Wyrobek 2005). The apparent lack of human germ cell mutagens cannot be ascribed to a higher resistance of human germ cells to the detrimental impact of genotoxic agents. As already dis- cussed, exposure to comparable doses of ionizing radiation or chemotherapeutic agents induce similar effects in both rodent and human sperm. Furthermore, it is broadly accepted that aging is a human germ cell mutagen. An increase in the risk of having a child with a genetic syn- drome as function of paternal age has been known for decades (Crow 2000). Studies with next generation sequencing (NGS) have unequivocally shown the presence of a paternal age effect for the induction of de novo muta- tions that are transmitted to the offspring (Kong et al. 2012; Francioli et al. 2015; Rahbari et al. 2016). More recent studies have also demonstrated an increase in de novo mutations, albeit smaller than the paternal effect, as function of maternal age (Maretty et al. 2017; Gao et al. 2019). In these genomic studies, the stronger paternal age effect is attributed to the continuous replication of stem cell spermatogonia throughout the male’s reproductive life and to the possibility of introducing an error during DNA repli- cation. Interestingly, the potential contribution of an envi- ronmental exposure in the paternal age effect is seldom considered. For example, a study that analyzed the paternal age effect in three human families with multiple children showed that the slope of the paternal age effect varied greatly among the fathers (Rahbari et al. 2016). While dif- ferent baseline efficiencies in DNA repair activities could contribute to this variability, so could differential exposure of the fathers to an environmental mutagen. Finally, analy- sis of the types of de novo mutations transmitted to human offspring show clustering of mutations (Francioli et al. 2015) as observed in the offspring of mutagen-exposed ani- mals (Adewoye et al. 2015; Beal et al. 2019) and signatures of mutational processes that point to DNA damage as the source of de novo mutations (Gao et al. 2019). It is difficult to imagine a process by which the DNA repair machinery of human germ cells is able to distinguish between lesions that are induced by endogenous processes vs. those induced by exogenous agents and are able to completely repair the latter but not the former. In summary, there is little biological plausibility to sup- port the notion that the response of human germ cells to an environmental insult is fundamentally different from that occurring in rodent germ cells, which would negate the possibility that established rodent germ cell mutagens would also be human germ cell mutagens. TECHNICAL LIMITATIONSOFAVAILABLEGERMCELL MUTATIONTESTS The search for human germ cell mutagens has been ham- pered by the lack of efficient test methods to detect the impact of environmental agents on the germline. Even in laboratory rodents, the study of germ cell mutagenesis has suffered from the limitations of available tests, such as the need for an exceedingly large number of animals. A prime example is the SLT developed in the late 1950s at Oak Ridge National Laboratory. For many decades, it was the only available test to detect de novo mutations in the offspring of exposed mice. The SLT was instrumental in establishing occupational exposure limits for radiation workers (Neel and Lewis 1990) and made fundamental contributions to the elucidation of the varying sensitivity of the different phases of spermatogenesis to environmental exposure (Russell 2004). However, a typical experiment required the analysis of several hundred thousand animals, something that would not be considered remotely feasible today, both for financial and ethical considerations. Other available tests, although not requiring as many animals as the SLT, did not provide information on the heritability of mutations in live animals (i.e., the dominant lethal test) or assayed only on a specific type of genetic damage (i.e., reciprocal translocation test; results with both tests reviewed in Marchetti and Wyrobek 2005). Over the years, a variety of tests have been developed to assess different types of genetic damage in germ cells and applied to the question of environmentally induced germ cell mutagenesis. Corollary assays from some of the current rodent germ cell tests are available for use with human sperm and have allowed a direct comparison of the response to environmental insult across species. An in- depth review of the strengths and limitations of these tests is provided in the 2013 IWGT report on germ cell testing (Yauk et al. 2015a). Here, we specifically highlight two approaches that represented important breakthroughs in the field of germ cell mutagenesis. As discussed, the analysis of tandem repeat mutations in sperm has been instrumental in demonstrating consistency of effects across species and contributed to the overall Environmental and Molecular Mutagenesis. DOI 10.1002/em 47Quest for Human Germ Cell Mutagens 10982280, 2020, 1, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1002/em .22327 by H ealth C anada, W iley O nline L ibrary on [15/03/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense weight of evidence that ionizing radiation, chemotherapeu- tic drugs, tobacco smoking and air pollution are human germ cell mutagens. The analysis of tandem repeat muta- tions provided, for the first time, an opportunity to study heritable mutagenesis in humans using a reasonable and manageable population size. Furthermore, the assay can be conducted in any species, including wild animals, and is equally applicable to the analysis of sperm of exposed fathers and their children enabling a direct demonstration of the heritability of these mutations. Unfortunately, uncer- tainties over the impact of these types of mutations for human health and a technically demanding protocol has hampered the broad acceptance of tandem repeat assays among the regulatory and research communities. However, extensive efforts are ongoing to develop advanced genomic approaches (e.g., long-read sequencing; Chaisson et al. 2019; Levy-Sakin et al. 2019) for rapidly and efficiently characterizing structural variation in the genome and it is envisioned that these methods will become important tools for analyzing environmentally induced structural variation in the germ cell genome (Salk and Kennedy 2020). A breakthrough for germ cell mutagenicity for regulatory testing also occurred at the end of the 20th century when transgenic rodent (TGR) models were developed (reviewed in Lambert et al. 2005). These models have bacterial reporter genes integrated into their genome that can be recovered from any tissue and used in an in vitro assay to measure mutations that were induced in vivo. The use of TGR models for mutagenicity testing was later codified in an Organisation for Economic Co-Operation and Develop- ment Test Guideline (OECD 2013). TGR models provided the opportunity to conduct in vivo mutagenicity testing using a reasonable number of animals, in line with other well-established genotoxicity tests for somatic cells, and to directly compare the response in germ cells with that in somatic tissues. This is particularly important because germ cell mutagenicity is a health hazard criterion in the GHS classification and labelling of chemicals (United Nations 2017) and has resulted in an increased demand for testing in this area. The use of TGR models for germ cell muta- genesis was recently summarized together with recommen- dations on proper experimental design for integration with somatic tissue mutagenicity testing (Marchetti et al. 2018a, b). A major limitation of existing germ cell methods is the inability to account for postfertilization events. There is a wealth of data showing that the manifestation of damage induced in postmeiotic germ cells into a fixed genetic change, whether point mutation or chromosomal aberra- tion, is not completed until after the sperm fertilizes the egg (Marchetti and Wyrobek 2005). There is also an example of a meiotic DNA lesion that does not manifest itself as a permanent genetic abnormality until after fertili- zation (Marchetti et al. 2015). These data point to the importance of the perifertilization period in determining the amount and types of lesions that originate de novo genetic changes, and in particular, of the role that an effi- cient DNA repair system in the fertilized egg can play in fundamentally mediating how much of the sperm DNA lesions are correctly repaired (Marchetti et al. 2007; Derijck et al. 2008). We believe that recent developments in genomic methodologies (described later) are well posi- tioned to fill this key research gap and also address another current limitation of germ cell tests; i.e., the lack of efficient and practical methods to analyze the induction of mutations in female germ cells that has resulted in a paucity of experimental data on female germ cell mutagenicity. RISKASSESSMENTOFGERMCELLMUTATIONSOVERTHE NEXT50 YEARSOF THE EMGS During the first 50 years of the EMGS, the mutagenic activity of chemicals has been evaluated primarily in a yes/no fashion. However, a paradigm shift is ongoing in genetic toxicology to move away from a purely qualitative analysis of genotoxic responses (Dearfield et al. 2017). The proposed path forward implements refined dose–response modeling approaches to establish point of departure (PoD) metrics for potency comparisons, with the long-term vision of using mutation as a PoD in human health risk assess- ment (Johnson et al. 2015; Wills et al. 2016). The chal- lenges of this paradigm shift and the necessary improvement in methods to measure mutagenicity, and how to refine risk assessment approaches to encourage uptake by regulatory bodies, are discussed in Heflich et al. (2020). This paradigm shift has important implications for the risk assessment of germ cell mutations and the need to conduct germ cell mutagenesis testing. A general assump- tion in risk assessment is that evaluations based on somatic cell mutation assays protect the germline by default. Historical and experimental data support that somatic cell mutation assays predict putative mutagenic effects in the germline. Indeed, there is currently no evi- dence for a unique germ cell mutagen, although caution should be exercised in drawing broad conclusions because chemicals that are negative in somatic cells are rarely tested in germ cells (Yauk et al. 2015a). This new empha- sis on dose–response modeling requires that reliance on somatic mutagenicity tests to predict effects in germ cells now extends also to establishing PoDs that protect the germline as well. However, we already know that this is incorrect. Several examples exist demonstrating germ cell effects at doses that do not elicit adverse effects in somatic cells; thus, PoD based on somatic endpoints may underes- timate the risk to the germline. For example, Witt et al. (2003) reported strong dominant lethal effects in mice exposed to N-hydroxyacrylamide while no significant Environmental and Molecular Mutagenesis. DOI 10.1002/em 48 Marchetti et al. 10982280, 2020, 1, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1002/em .22327 by H ealth C anada, W iley O nline L ibrary on [15/03/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense effects could be detected using the bone marrow micronu- cleus (MN) analysis, one of the most commonly con- ducted tests for regulatory purpose. Similarly, Marchetti et al. (2011) showed induction of tandem repeat mutations in the sperm of mice exposed to either mainstream or side- stream tobacco smoke at doses that did not increase MN frequencies in bone marrow. Finally, a review of the gen- otoxicity of acrylamide showed that the response in germ cells is stronger than the response in somatic cells (Dearfield et al. 1995). It could be said that the above examples suffer from the shortcoming of comparing different endpoints in germ cells vs. somatic cells and, thus, the observed differential responses are not a true indication of an increased sensitiv- ity of germ cells to those agents, but simply reflect method- ological differences in the sensitivity to detect an effect. However, even when the same endpoint is analyzed, there are still four cases of chemicals that failed to induce MN in the mouse bone marrow, while significantly increasing their frequency in spermatids (Cliet et al. 1993). While recognizing the potential impact of induced germline mutations on human populations, national and international efforts have concentrated on the identifica- tion of germ cell mutagen hazards, eliminating them, or otherwise reducing their potential exposure to human populations (Committee 17 1975; IPCS 1985; World Health Organization 1986). Efforts also extended to identifying epidemiologic associations between human male exposures and mutation-based diseases in offspring (Narod et al. 1988) to identify potential human germline mutagens. In recognition of such potential hazards, national and international regulatory authorities have established guidelines and regulations that include the goal of identifying and controlling human exposure to germline mutagens (Cimino 2006). Paramount among such regulatory schemes is the inter- national adoption of the GHS of Classification and Label- ling of Chemicals (United Nations 2017) by most industrialized nations. The GHS includes human germ cell mutation as a toxicological endpoint equal to other end- points such as cancer and reproductive effects, thus, indi- cating its universal international acceptance. While such regulatory schemes do establish a basis for germline hazard identification, they do not provide a clear path to the actual estimation of the human risk in terms of induced mutation rate in offspring associated with such hazards. Various germ cell risk estimation methods have relied primarily on the use of animal studies and some form of quantitative extrapolation to a related class of human genetically based disease (reviewed extensively in Yauk et al. 2015a). There are limitations to the currently available approaches for quantitative assessment of germ cell risk from chemical exposures. For example: (1) animal studies do not directly measure or quantitate effects in humans, while current methods do not routinely assess environmentally caused de novo mutations in specific human target genes, or groups/categories of genes; (2) there are no accepted, or validated, allometric and exposure fac- tors for extrapolation of animal germline mutation data to individual humans, or human population groups, and there is insufficient information on the means to extrapolate mutation data in animal genes to analogous human genes and (3) there is no accepted de minimis risk level for the estimation of unacceptable exposure levels for germline mutation effects to offspring. Despite such historically based shortcomings, we are now at a turning point facilitated by the accelerated devel- opment of new genomic technologies that will finally enable us to make significant advances in the application of genetic risk assessment in both the academic and regulatory contexts. THE FUTURE:NEXTGENERATIONSEQUENCING Studies on the heritable effects of environmental agents in humans have historically relied on measuring phenotypic endpoints rather than detecting the underlying genetic cause (DeMarini 2012). As not all de novo mutations have a discernable phenotypic consequence, these approaches were limited to measuring only dominant effects in early life with a resulting loss of statistical power. In addition, such approaches cannot quantify the impact of recessive mutations on health until subsequent generations, or muta- tions that contribute to multigenic disorders. The advent of NGS approaches, together with great gains in bioinformat- ics pipelines to handle, analyze and validate sequenced data, are allowing the analysis of mutations over the entire genome and are promising to revolutionize the way germ cell mutagenicity is investigated. NGS complements other genomic approaches such as array comparative genomic hybridization to capture a variety of genomic changes that can impact human health (Carvalho and Lupski 2016). NGS approaches can also provide critical mechanistic information on the types of mutations that are induced and increase the confidence that these de novo mutations are indeed the consequence of an exogenous exposure. Impor- tantly, these approaches are feasible with a relatively small number of samples and are equally applicable to laboratory animals as well as human families (Webster et al. 2018). In fact, experiments conducted in laboratory animals can inform studies in human families on the proper experimen- tal design, power of analysis and confounding factors. There are already several examples of the use of these genomic approaches for germ cell mutagenesis studies in rodents. Adewoye et al. (2015) reported significant induc- tion of copy number variants (CNVs) in the offspring of irradiated animals. Interestingly, both irradiated spermato- gonia and sperm caused similar levels of CNVs in off- spring. In the same study, although there was evidence for Environmental and Molecular Mutagenesis. DOI 10.1002/em 49Quest for Human Germ Cell Mutagens 10982280, 2020, 1, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1002/em .22327 by H ealth C anada, W iley O nline L ibrary on [15/03/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense clustering of mutations after irradiation of male germ cells, the authors could not detect an overall significant increase in de novo single nucleotide variants (SNVs). However, a recent study (Beal et al. 2019) reported significant increases in CNV gains and SNVs in the offspring of male mice exposed to BaP with evidence of clustering of mutations. The study also demonstrated that there was a significant increase in constitutional SNVs (i.e., present at fertilization) when spermatogonia were exposed to BaP, while there was a significant increase in embryonic SNVs (i.e., originating after fertilization) when postmeiotic germ cells were exposed. Importantly, the types of mutations observed in the offspring matched the expected mutational signature of BaP and were consistent with the pattern of mutations observed in the sperm of exposed males, conclusively dem- onstrating that the increase in de novo mutation was linked to the paternal exposure to BaP. Finally, there is also evi- dence that exome sequencing may be sufficient to detect significant increases in de novo mutations (Masumura et al. 2016a, b). These studies found that N-ethyl-N-nitrosourea (ENU) significantly increased de novo mutations in the exomes of offspring from exposed mice and that the inherited mutations exhibited a spectrum characteristic of ENU-induced mutations. Together, these rodent studies: (1) provide foundational examples for the application of genomic technologies for investigating the induction of heritable mutations following exposure to environmental mutagens; (2) demonstrate that different mutagenic agents can differentially affect various types of genetic changes; (3) provide support for the herita- bility of mutations observed in sperm; and (4) confirm the important role of postfertilization events in determining the genetic load that is transmitted to the offspring. Application of NGS to human families to study the underlying causes of de novo mutations has focused mostly on measuring the increase in de novo mutations as function of paternal or maternal age. These studies are nevertheless important because they provide information on the baseline levels of de novo mutations and how they change as a function of age. Clearly, paternal age is a critical con- founding factor that must be controlled for in any study assessing the impact of environmental exposure in the human population. Indeed, a recent statistical power analy- sis showed that controlling for the paternal age effect and modeling family-to-family variability significantly reduced the number of families needed to detect an increase in de novo mutations (Webster et al. 2018). The analysis also demonstrated that inclusion of families with multiple chil- dren confers additional sensitivity to detect an effect. For example, sequencing of just 6 four-child families per study group provides an 80% power to detect a 30% increase in de novo mutations (Webster et al. 2018). These data show the feasibility of designing studies to evaluate the effects of environmental exposures in human populations that do not require large cohorts and significant financial investment. It is easy to envision that these types of studies will become common over the next few years and will provide funda- mental data on the susceptibility of human germ cells to environmental agents. One such an example is a recent study that demonstrated a significant correlation between levels of paternal exposure to dioxin and incidence of de novo SNVs in their offspring (Ton et al. 2018). THENEXT50 YEARS: ACALLTOACTION Fifty years ago, the founding members of the EMGS would not have predicted that half a century would pass and we would still be waiting for the demonstration of the exis- tence of human germ cell mutagens. Yet, it is a testament of the rightfulness and importance of the original mission of the Society that the pursuit of that goal has survived. During these 50 years, incredible progress has been made in our understanding of the mechanisms of germ cell mutagenesis and in the tools that we have at our disposition for fulfilling the original mission of our society. It is because of the relentless effort of a small number of EMGS members who have kept the germ cell mutagenesis flame alive that we are experiencing now a renaissance of the field. Challenges still lay ahead. First and foremost, germ cell mutagenesis is “threatened” by the current push to limit, reduce and replace in vivo studies and the increased empha- sis on in vitro and computational approaches to assess the toxicological properties of chemicals. Under this paradigm, germ cells risk may be marginalized as in vitro germ cell systems are still in their infancy, with a consequent reliance on somatic systems to predict effects in germ cells. Further- more, even if in vitro germ cell systems would be fully developed, they would just provide demonstration that a chemical can affect the germ cell genome while contribut- ing little to the ultimate goal of germ cell mutagenesis: the demonstration of the heritability of environmentally induced de novo mutations. We now have the tools and are closer than ever before to investigating the best possible experimental system: ourselves. The continuing decline in the costs of NGS approaches and the availability of new technologies that are directly applicable to humans and that enable the detection of rare mutations among heterogeneous populations of cells (Salk et al. 2018) makes it inevitable that many more agents will be demonstrated to induce mutations in human germ cells and increase adverse health effects in the offspring. These error-reduced sequencing technologies can be applied in any species/tissue and are poised to revolutionize genotoxicity testing in general (Salk and Kennedy 2020). Such technologies provide more opportunities for any laboratory in the world to con- duct germ cell mutagenicity assessment. It is our hope that with the GHS strategy, and the European Union regu- lation for the Registration, Evaluation, Authorisation and Environmental and Molecular Mutagenesis. DOI 10.1002/em 50 Marchetti et al. 10982280, 2020, 1, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1002/em .22327 by H ealth C anada, W iley O nline L ibrary on [15/03/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense Restriction of Chemicals requiring germ cell mutation assess- ment, in parallel with our increasing knowledge of the major prevalence of human genetic disorders that result from de novo mutations and the impact of even small increases in mutation rates on population health, more laboratories around the world will begin to contribute data to increase our under- standing of candidate human germ cell mutagens. We predict that we will soon have data demonstrating an increase in de novomutations in the offspring of exposed par- ents. A great challenge is to be sufficiently prepared to inter- pret the coming outpouring of data and, together with the regulatory community, develop a framework for managing, communicating and mitigating the risk of exposure to human germ cell mutagens. As indicated by DeMarini (2012), it is time for the applied genetic toxicology community to formu- late a plan to respond to the demonstration of environmentally induced de novo mutations in the human population. We add our voices to his call for the establishment of an international body that is tasked with the mandate of reviewing the avail- able data and formally declare whether there is sufficient evi- dence for supporting the existence of human germ cell mutagens and evaluate the risk of exposure to these agents for the health of human populations. ACKNOWLEDGMENTS We thank Drs Marc Beal and Vinita Chauhan of Health Canada for providing comments on the manuscript before it was submitted. Funding for this work was provided by Health Canada’s Chemicals Management Plan and the Genomics Research and Development Initiative. AUTHORCONTRIBUTIONS All authors contributed to the writing of the manuscript and all authors agree to its publication. 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Harnessing genomics to identify environ- mental determinants of heritable disease. Mutat Res 752:6–9. Yauk CL, Aardema MJ, Benthem J, Bishop JB, Dearfield KL, DeMarini DM, Dubrova YE, Honma M, Lupski JR, Marchetti F, et al. 2015a. Approaches for identifying germ cell mutagens: Report of the 2013 IWGT workshop on germ cell assays. Mutat Res 783:36–54. Yauk CL, Lambert IB, Meek ME, Douglas GR, Marchetti F. 2015b. Development of the adverse outcome pathway "alkylation of DNA in male premeiotic germ cells leading to heritable mutations" using the OECD’s users’ handbook supplement. Environ Mol Mutagen 56:724–750. Accepted by— R. Heflich Environmental and Molecular Mutagenesis. DOI 10.1002/em 54 Marchetti et al. 10982280, 2020, 1, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1002/em .22327 by H ealth C anada, W iley O nline L ibrary on [15/03/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense A Return to the Origin of the EMGS: Rejuvenating the Quest for Human Germ Cell Mutagens and Determining the Risk to Future... ROOTS OF EMGS IN GERM CELL MUTAGENESIS IMPACT OF GERM CELL MUTATIONS THE ENIGMA OF THE EXISTENCE OF HUMAN GERM CELL MUTAGENS ARE HUMAN GERM CELLS FUNDAMENTALLY DIFFERENT FROM RODENT GERM CELLS? TECHNICAL LIMITATIONS OF AVAILABLE GERM CELL MUTATION TESTS RISK ASSESSMENT OF GERM CELL MUTATIONS OVER THE NEXT 50YEARS OF THE EMGS THE FUTURE: NEXT GENERATION SEQUENCING THE NEXT 50YEARS: A CALL TO ACTION ACKNOWLEDGMENTS AUTHOR CONTRIBUTIONS REFERENCES