Chromatin dynamics underlying latent responses to xenobiotics

Jonathan Moggs * and Rémi Terranova *
Preclinical Safety, Translational Medicine, Novartis Institutes for BioMedical Research, Basel, Switzerland

Received 28th November 2017 , Accepted 26th February 2018

First published on 28th February 2018


Pleiotropic xenobiotics can trigger dynamic alterations in mammalian chromatin structure and function but many of these are likely non-adverse and simply reflect short-term changes in DNA transactions underlying normal homeostatic, adaptive and protective cellular responses. However, it is plausible that a subset of xenobiotic-induced perturbations of somatic tissue or germline epigenomes result in delayed-onset and long-lasting adverse effects, in particular if they occur during critical stages of growth and development. These could include reprogramming, dedifferentiation, uncontrolled growth, and cumulative toxicity effects through molecular memory of prior xenobiotic exposures or altered susceptibility to subsequent xenobiotic exposures. Here we discuss the current evidence for epigenetic mechanisms underlying latent responses to xenobiotics, and the potential for identifying molecular epigenetic changes that are prodromal to overt morphologic or functional toxicity phenotypes.


Epigenetic memory of chromatin states

Epigenetic processes regulate all genome functions (including DNA replication, DNA repair, DNA recombination, gene transcription and cell cycle) and require the coordinated interplay of local and long-range chromatin interactions within the three-dimensional space of the nucleus, including DNA methylation, histone post-translational modifications, non-coding RNAs and nucleosome remodelling.1,2 Key epigenetic regulators, such as the Polycomb and Trithorax group proteins, were initially characterized as cellular memory systems, maintaining transcriptional programs after the initial gene regulatory factors had disappeared from the embryos, a phenomenon that is best exemplified by the colinear developmental regulation of Hox gene clusters.3,4 Epigenetic mechanisms have been extensively studied in developmental and cellular differentiation systems but it is increasingly evident that the dynamic regulation of chromatin structure and function also takes place in proliferating and post-mitotic adult tissue compartments.

A plethora of cellular processes (including X-chromosome inactivation, genomic imprinting and the control of cell-cycle and identity) are dynamically regulated at the chromatin level through interactions with endogenous and exogenous environmental cues. Ultimately, coordinated epigenetic mechanisms control the accessibility transcription factors to key regulatory genome loci (e.g. enhancers and insulators) and these regions are characterized by cell-type-specific open chromatin structures and can be efficiently mapped using nuclease- or transposase-based assays coupled with deep sequencing.5–7 This cis-regulatory landscape, or cistrome, represents a complex repertoire of docking stations for multiple regulatory transcription factors, interacting over long-distances, and ultimately enabling the integration of extracellular signaling pathways with intracellular cell fate information through the generation of cell-type-specific transcriptional responses.8,9 The cistrome has also been shown to be strongly enriched for disease-relevant genetic variants which systematically perturb transcription factor recognition sequences, frequently alter allelic chromatin states, and modify gene regulatory networks.10–13 Mapping functional enhancers is providing a rich source of mechanism-based biomarkers for enhanced disease characterization, diagnosis, and therapy.14 Thus, characterizing the potential for xenobiotic-induced chromatin perturbations at the level of enhancer structure and function represents an important opportunity for gaining novel insights into mechanisms of toxicity.

The kinetics of epigenome modifications can span an extremely broad dynamic range. In somatic tissues, transient epigenetic changes (from minutes to days) are exemplified by metabolic and circadian regulation, whilst more stable changes (from weeks to months or years) are associated with the maintenance of lineage identity. Multigenerational epigenetic changes mediated via the germline have the potential to last for several years or even decades.15,16 Notably, transient chromatin events at early stages of development have the potential to indelibly program adult phenotypes,17,18 thus highlighting the interplay between plasticity of chromatin and cellular memory systems. The epigenetic landscape is also strongly influenced by a broad range of environmental changes, including diet, stress, temperature changes, and exposure to chemicals and therapeutic drugs, thus representing a dynamic adaptive interface between the genome and the environment. This is particularly well exemplified by phenotypic discordance for a wide range of traits in monozygotic twins that inherently share identical genetic material and early-life environmental exposure conditions.19,20

Impact of xenobiotic-induced epigenomic perturbations for safety assessment

Over the past 10 years there has been extensive debate within the toxicological sciences community (including industry and regulatory agency stakeholders) regarding the relevance of epigenetic mechanisms and biomarkers for safety assessment.21–33 Whilst the current consensus is that more knowledge and experience is required prior to the routine incorporation of epigenetic evaluations into safety assessment,23 the increasing body of literature on diverse epigenetic mechanisms of toxicity, combined with the current trend towards therapeutic targeting of chromatin regulatory proteins,2,34–38 provides a strong stimulus for careful consideration of potentially unique safety assessment concerns associated with xenobiotic epigenome modulators, in particular with respect to delayed-onset and long-lasting toxicity phenotypes.

Furthermore, the potential for long-lasting adverse consequences of unintended environmental xenobiotic-induced effects is gaining significant societal attention39 (https://live.newscientist.com/exhibitor-press-releases/what-research-is-phe-doing-in-epigenetics; https://cfpub.epa.gov/ncea/risk/recordisplay.cfm?deid=308271; https://www.toxicology.org/events/shm/cct/Toxicoepigenetics.asp), especially where there is a risk of adverse outcomes based on exposure to environmental contaminants during pregnancy and early life.40

It will be important to further explore the degree to which xenobiotics may perturb the epigenome through both direct and/or indirect mechanisms, and the degree to which such perturbations result in adverse or beneficial phenotypes. We highlight below some important insights that have been gained through xenobiotics that directly target epigenetic regulatory proteins and also through genetic modification of cellular and animal models, with an emphasis on the potential for latent effects.

Experience from xenobiotics that directly target epigenetic modifiers

Whilst most xenobiotics are not likely to directly target chromatin regulatory proteins, they would still be expected to ultimately modulate signalling to chromatin to some extent in exposed cells, but many of these epigenetic effects would likely be non-adverse and simply reflect short-term changes in DNA transactions (e.g. replication, recombination, repair, transcription, cell cycle) underlying normal homeostatic, adaptive and protective cellular responses. Nevertheless, an increasingly diverse range of environmental toxicants have been reported to be associated with epigenetic alterations.41–43 In some cases, a mechanistic basis for direct targeting of epigenetic modifiers by toxicants has been elucidated. For example, Nickel(II) has been reported to inhibit TET-mediated 5-methylcytosine oxidation through displacement of the cofactor Fe(II).44 In contrast, redox-active quinones appear to stimulate TET-dependent DNA demethylation via an iron-regulated mechanism.45,46 Exposure to xenobiotics that directly target transcriptional and chromatin regulatory proteins is plausibly associated with unique safety concerns that warrant special attention. In particular, a number of potentially unique mechanism-based safety concerns have recently been highlighted in the context of therapeutic epigenetic modifiers including cardiovascular toxicity, CNS toxicity, perturbation of stem cell homeostasis, secondary carcinogenesis and multigenerational effects.31

There is some evidence to suggest that acute exposure to targeted epigenetic modifiers can result in adverse outcomes through short-term alterations of genome functions or stability. For example, the treatment of cancer patients with therapeutic HDAC inhibitors has been associated with delayed cardiac repolarization and in rare cases resulted in a fatal ventricular tachyarrhythmia. Non-clinical in vivo mechanistic studies have revealed that HDAC inhibitor-induced prolongation of cardiac repolarization can occur within 24 hours following a single-dose and may be mediated in part by transcriptional changes of genes required for ion channel trafficking and localization to the sarcolemma.47 Similar observations of delayed and transcription change-related cardiac dysfunction were also made in human induced pluripotent stem cell-derived cardiomyocytes.48

Since a number of chromatin regulatory proteins are intimately associated with the maintenance of genome stability,49–53 it is plausible that their direct pharmacologic modulation might result in impaired genome integrity in the absence of any intrinsic potential for direct DNA damage. Whilst there is currently only very limited evidence to support this notion, it is noteworthy that RNA interference-based modulation of BRD4 alters AURORA B expression and ultimately genome stability in primary human cells.54 Likewise, the HDAC inhibitor Trichostatin A was reported to induce genotoxic effects in a cultured human lymphoblastoid cell line.55 The potential for xenobiotic-induced epigenetic changes to alter genome stability and the ability of current genetic toxicity assays to detect such events warrant further investigation.

Another mechanism by which targeted epigenetic modifiers might cause adverse alterations in genome function is through the perturbation of expression from integrated virus-derived DNA sequences. This is exemplified by the observation that therapeutic DNMT and HDAC inhibitors can trigger widespread transcriptional activation of endogenous retroviruses that are normally silenced by epigenetic modifications,56 although this phenomenon has yet to be associated with an adverse phenotype. However, latent infections by herpes viruses or human immunodeficiency virus also involves epigenetic silencing of their genomes,57 and the potential for targeted epigenetic modifiers to stimulate viral infection through reactivation of such latent viruses is supported by observations in cellular models. For example, the histone H3 lysine 9 methyltransferase G9a and related molecule GLP (G9a-like protein) contribute to the maintenance of transcriptional quiescence for latent HIV-1 provirus and chemical probe or chemogenetic inhibition of G9a or GLP reactivated expression of HIV-1 from latently infected cells.58,59 Similarly, the maintenance of Epstein–Barr virus latency was shown to be disrupted by an inhibitor of the histone H3 lysine 9 methyltransferase Suv39h1.60 Likewise, other epigenetic repressive pathways such as the Polycomb Repressive Complex 2 (PRC2) mediated H3 lysine 27 methylation play a role in virus latency.61–63

Whilst it is plausible that xenobiotic-mediated targeting of the epigenome could result in delayed-onset and long-lasting adverse effects, in particular in sensitive cell types (e.g. stem andprogenitor cells) or during critical stages of growth and development, there are relatively few known examples of such effects. Delayed-onset pharmacodynamic effects have been reported in cancer cell lines and in vivo rodent xenograft tumor models for catalytic inhibitors of EZH2 and DOT1L,64,65 and thus inhibitors of histone modifying enzymes might conceivably result in similar delayed-onset effects in adult tissues. Transgenerational (paternal and maternal) inheritance effects have also been reported in animals exposed to valproic acid, an anti-epileptic drug that can inhibit histone deacetylase activity.66 The rapid emergence, and wide availability, of potent chemical probes that specifically target a variety of chromatin regulatory proteins67–69 will enable more extensive and rigorous assessments of whether xenobiotic exposure could potentially induce delayed-onset and long-lasting adverse effects through perturbations of the epigenome. To date, supporting evidence for these phenomena have predominantly been derived from observations following targeted epigenome modifications in genome engineered cellular and animal models as described below.

Experience from targeted epigenome modifications in genome engineered cellular and animal models

Recent advances in genome engineering enable in vitro and in vivo toxicity phenotyping for a broad repertoire of modifications in genes encoding epigenetic regulatory proteins including systemic and inducible knock-down, knock-out and knock-in of gene functions.70 A major advantage of these approaches is specific modification of the intended target(s), in contrast to xenobiotic epigenetic modifiers that exhibit varying degrees of target selectivity and often uncharacterized off-target profiles. Cell- and tissue-specific genome modifications can also be deployed without the limitations of individual absorption, distribution, metabolism and excretion properties of xenobiotics.

A plethora of theoretical safety concerns associated with targeting chromatin regulatory proteins have been identified through toxicity phenotyping of engineered cellular and animal models, and also through analysis of genetic associations with disease in humans.31 Germline genetic deletion of chromatin regulatory proteins frequently results in developmental defects and in many cases embryonic lethality. However, conditional genetic modification of epigenetic regulators in adult tissue and specific cell lineages is generally deemed to more accurately reflect potential safety liabilities that may arise from xenobiotic-mediated targeting of epigenome modifiers. In particular, the inducible and reversible transgenic RNAi-mediated knockdown71 of chromatin regulatory proteins in mice offers a powerful in vivo approach for toxicity phenotyping in a setting where dose and duration of the targeted epigenome modification can be controlled. This is exemplified by RNAi-mediated suppression of the BET protein Brd4 in adult mice which resulted in epidermal hyperplasia, alopecia, and stem cell depletion in the small intestine.72 It is noteworthy that whilst the time of onset of these phenotypes ranged from days to weeks, all exhibited full reversibility following cessation of RNAi expression.

One of the most noteworthy examples of a targeted epigenome modifications in somatic tissues resulting in significantly delayed-onset toxicity is a fatal neurodegeneration phenotype associated with genetically-engineered PRC2 deficiency in mouse striatal neurons.73 Early (2 to 6 weeks) epigenetic reprogramming of neuronal cell type-specific molecular pathways via altered gene expression preceded late-onset (3 to 6 months) activation of cell death pathways, overt degenerative pathology and attenuated motor activity and balance. Importantly this study revealed a profound temporal delay between short-term molecular reprogramming (epigenomic and transcriptomic effects) and late-onset morphologic and functional deficits, a scenario that if reproduced with a xenobiotic would present significant challenges to current safety assessment paradigms.

Importantly, some adverse phenotypes associated with epigenetic perturbations in post-mitotic organs have been shown to be reversible. This is exemplified by a mouse model for Rett syndrome, an autism spectrum disorder associated with mutant copies of the MeCP2 gene in neurons, in which advanced neurological symptoms were phenotypically reversed in both immature and mature adult animals, following activation of MeCP2 expression.74 Thus, it is not yet clear whether delayed onset and/or long-lasting toxicity associated with targeted epigenome modifications in tissue compartments that are post-mitotic is of any greater concern than for mitotic tissues.

However, caution needs to be taken when extrapolating from phenotypes observed following genetic modification of epigenetic regulatory proteins to xenobiotics associated with targeted epigenome modifications since they can differ significantly in the resultant influence on potency and duration of target modulation effects. In particular, most epigenetic regulatory proteins are intimately associated with large multi-subunit protein complexes that may be perturbed in a distinct manner by genetic versus xenobiotic modulation. Furthermore, genome engineering has an intrinsic potential to disrupt chromatin structure and function which is a potentially major confounding factor when assessing phenotypes associated with epigenetic regulatory proteins. In this respect it is noteworthy that new chemical genetics technologies are now emerging that use small molecules to degrade proteins in a selective manner and offer innovative research tools to dynamically and reversibly characterize in vivo phenotypes associated to the modulation of potential therapeutic targets in conditions that share similar pharmacokinetic properties of typical small-molecule drugs.75–77

Application of epigenomic endpoints to characterise mechanisms and biomarkers associated with carcinogenesis

In contrast to the scarce examples of targeted epigenetic modifiers that result in delayed-onset and long-lasting adverse effects, several distinct classes of xenobiotics have been shown to elicit epigenetic changes during the early phases of both genotoxic and non-genotoxic carcinogenesis in rodents including altered DNA methylation, histone modifications, non-coding RNAs and chromatin accessibility.14,78,79 A key role for epigenetic changes during carcinogenesis is supported by the observation that mutations in epigenetic regulatory proteins and aberrant expression of stem cell reprogramming genes are associated with cancer etiology and progression. Furthermore, epigenetic changes have been suggested to be amongst the earliest events during non-genotoxic carcinogenesis.80,81 Indeed, both inflammatory responses to tissue injury and chronic exposure to environmental factors have been proposed as mechanisms for inducing cancer-predisposing epigenetic changes in vulnerable populations of somatic stem cells, transition zones and progenitor compartments.

These emerging concepts in human cancer epigenetics have been leveraged to explore early mechanism-based biomarkers of xenobiotic-induced non-genotoxic carcinogenesis, in particular for xenobiotics that directly targeting nuclear receptors such as CAR and PPARalpha.82–89

Tumor promoting doses of phenobarbital have been extensively studied in the context of investigating the kinetics of epigenetic changes associated with rodent non-genotoxic hepatocarcinogenesis. Phenobarbital induces both short-term and long-lasting changes in the mouse liver DNA methylation within regulatory regions of CAR and beta-catenin gene targets, including dynamic and reciprocal changes in levels of both 5-methylcytosine and 5-hydroxymethylcytosine.90 A profound example of delayed onset molecular epigenetic changes that are prodromal to a long-term adverse phenotype is illustrated by early phenobarbital induced loss of 5-hydroxymethylcytosine (following ∼12 weeks of dosing) at specific gene loci that predicts a mouse liver tumor phenotype that is associated with hypermethylated CpG islands.91 Furthermore, the early phenobarbital-induced induction of epigenetically imprinted long non-coding RNA biomarkers has been proposed to reflect dedifferentiation/reprogramming of hepatocytes towards a stem-cell like state,92 a notion that is consistent with the molecular hallmarks of cancer.81

Another example of a mechanism through which xenobiotics might indirectly result in altered tumor growth-promoting potential is via altered intermediary metabolism. The levels and availability of cofactors, substrates, and inhibitors of chromatin-modifying enzymes are intimately associated with intermediary metabolism.93 This is exemplified by S-adenosylmethionine as a cofactor for histone and DNA methyltransferases, alpha-ketoglutarate as a substrate for histone and DNA demethylases, and competitive inhibitors of chromatin modifying enzymes such as S-adenosyl homocysteine, fumarate and succinate.93 One of the best characterized connections between metabolic changes, epigenetics and carcinogenesis is represented by gain of function mutations in isocitrate dehydrogenase genes IDH1 and IDH2 leading to accumulation of the (R) 2-hydroxyglutarate (2HG) metabolite, inhibition of TET-family DNA and JmjC-family histone demethylase enzymes, and accumulation of aberrant gene regulatory events that result in pathogenesis.94–97 Importantly, selective inhibition of mutant IDH enzymes leads to dynamic reversal of histone and DNA hypermethylation signatures, highlighting the plasticity of the epigenetic landscape and associated therapeutic opportunities.98–100

The accumulation of intracellular fumarate levels has been shown to lead to inhibition of alpha-ketoglutarate dependent TET dioxygenases which in turn result in altered DNA demethylation of genes associated with regulating epithelial-to-mesenchymal transition.101,102 These observations have been proposed to have potential safety implications for the therapeutic use of fumarates in the clinic.103 Consistent with such a hypothesis is the association renal cell cancer with human mutations in fumarate hydrates and the observation of kidney tubular adenomas/carcinomas in dimethyl fumarate mouse and rat 2-year carcinogenicity studies (EPAR Tecfidera assessment report EMA/800904/2013 Corr. 1: http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Public_assessment_report/human/002601/WC500162070.pdf).

Together these examples clearly illustrate the potential for delayed-onset prodromal molecular epigenetic changes as predictors of long-term adverse (tumorigenic) phenotypes associated with xenobiotic exposure. In contrast, vitamin C, a cofactor for several chromatin-modifying dioxygenases,104–106 has recently been shown to regulate hematopoietic stem cell function and suppress leukemogenesis through modulation of TET2 activity, thus raising the possibility of innovative treatment strategies for leukemia that combine specific epigenome-modifying metabolites or diets with standard of care cancer treatments.107

Opportunities for characterising epigenetic mechanisms and biomarkers associated with multi-generational effects

A more extreme scenario where molecular epigenetic changes may be prodromal to overt morphologic or functional toxicity phenotypes involves multi-(inter- and trans-) generational effects that are mediated via perturbation of the germline epigenome.

Major epigenomic reprogramming events occur during germline development and early embryogenesis, including dynamic genome-wide and locus-specific remodelling of cellular chromatin landscapes, ultimately supporting successful embryonic development and programming of adult phenotypes. Genomic imprinting and X-inactivation have long represented the sole paradigms of germline programming events associated with adult tissue homeostasis. However, it is becoming clear that a broader landscape of genomic loci could be epigenetically (re-)programmed to inform successive generational phenotypic adaptations.

Phenotypically, a wealth of research now strongly suggest that a parent's environment can cause changes that can be passed to future generations, a phenomenon known as transgenerational epigenetic inheritance108 (TEI). Several studies suggest the existence of TEI in humans (including the Dutch hunger winter survivors and the Överkalix studies).109–112 In preclinical rodent models TEI was also reported in a diverse range of parental exposure paradigms.16,113 For instance, a father's diet was shown to affect metabolic phenotypes in the next generation;114 paternal stress or fear conditioning was shown to be transmitted to offspring115,116 and a growing number of toxins were reported to induce effects on various offspring phenotypes.117–120

The fundamental molecular mechanisms underlying transgenerational epigenetic inheritance, such as the nature of molecular signals and mechanisms that inform the germline in one generation and are subsequently transmitted to (beneficially or adversely) influence the phenotype of successive generations, are largely elusive121 but may include specific genomic changes in DNA methylation,122 histone modifications,123 and ncRNAs expression.124,125

Importantly, not all heritable epigenetic changes lead to adverse outcomes as exemplified by a protective toxicity phenotype that was observed in rats following cross-generational adaptation to chemically-induced liver fibrosis.126 Likewise, paternal exposure of mice to nicotine prior to reproduction induced a broad protective response to multiple xenobiotics in male offspring.119 Together, these studies highlight the diverse range of phenotypic effects that can be associated with the descendants fitness and raise important questions regarding the potential specificity of the transmitted phenotypic response from a given environmental cue.119 However, it is not yet clear whether there will be significant differences between species regarding sensitivity to xenobiotic-induced heritable epigenomic perturbations and associated phenotypic effects.

The possibility that ancestral experiences are written in our epigenome has profound implications for human health and disease, and has captured public awareness through media representation and extensive debates on both ethical and legal implications.39 However, there is no strong evidence to date that such effects occur in humans. Thus, whether or not exposure of humans to defined xenobiotic compounds has the potential to adversely impact the health of future generations remains an open question for the toxicological sciences community. Thus elucidating the molecular basis and the prevalence of transgenerational epigenetic inheritance across a wide range of xenobiotic cues is a priority area for future research.

Perspectives and future directions

There is increasing evidence that a subset of xenobiotic-induced perturbations of somatic tissue or germline epigenomes can result in delayed-onset and long-lasting adverse effects, including cases where molecular epigenetic changes are prodromal to overt morphologic or functional toxicity phenotypes (Fig. 1).
image file: c7tx00317j-f1.tif
Fig. 1 Illustrative overview of latent phenotypic responses associated with xenobiotic-induced modulation of transcriptional regulators. Different xenobiotic exposure paradigms are illustrated (A), which are associated with a range of molecular, cellular and physiological phenotypic responses (B). Xenobiotic exposure may indirectly (signalling to chromatin) or directly affect chromatin effectors (e.g. histone or DNA modifiers, non-coding RNAs or nucleosome remodeling) or (specific tissue-specific transcription factors, ultimately affecting the genetic regulation of cellular function, potentially in a strain- species- or inter-individual-dependent manner). Physio-pathological phenotypic manifestation of xenobiotic-induced chromatin alterations may be acute, latent and/or long-lasting, potentially affecting the somatic tissue over long periods of time, including across generations (C). Phenotypic time-courses are currently difficult to predict and will need case-by-case empirical assessments.

Xenobiotic-induced epigenetic changes leading to reprogramming, dedifferentiation and uncontrolled growth have been identified during drug-induced non-genotoxic carcinogenesis and critical windows of sensitivity to xenobiotic-induced epigenetic perturbations have been identified during fetal and early life environmental exposures. The ability of the epigenome to serve as a molecular memory of prior environmental xenobiotic exposures is an important area for future investigations, in particular due to the high potential for discovering novel biomarkers of xenobiotic exposure. This notion is supported by the observation that long-lasting blood cell gene locus specific DNA methylation biomarkers associate with prior smoking history in humans.127–129 The memory of such epigenetic modifications might ultimately contribute to cumulative toxicity effects or altered susceptibility to toxicity. The latter phenomenon is precedented by long-lasting epigenetic mechanisms underlying innate immune responses that are mediated by Toll-like receptor (TLR)-4. TLR4-mediated stimulation of mouse macrophages by bacterial lipopolysaccharide results in differential epigenetic reprogramming of inflammatory response genes versus anti-microbial genes, thus avoiding excessive inflammation upon re-exposure without compromising an effective secondary immune response.130,131

The notion that a broader range of xenobiotics may lead to latent toxic effects through epigenomic perturbations could be further explored in a targeted manner by focusing on toxicants for which latent phenotypic effects were established during an era when a potential role for epigenetic mechanisms could not be fully explored. For example, neonatal exposure of rats to the hormonally active xenobiotics o,p′-DDT [1,1,1-trichloro-2-(o-chlorophenyl-2-(chlorophenyl)ethane)] or methoxychlor has been reported to result in latent but permanent increases in hepatic monoamine oxidase activity,132 an observation that warrants re-evaluation at the level of tissue- and gene locus-specific chromatin modifications.

Given the complexity and centrality of epigenetics for regulating genome functions, there are no current standardized approaches for derisking xenobiotic-induced perturbations of the epigenome. However, for xenobiotics that are known to directly target chromatin regulatory proteins, either through their intrinsic design or empirical mechanistic determination, there is a clear need to carefully consider customized toxicology study designs that take into account mechanism-based knowledge on uniquely sensitive gene loci, cells and tissues. Furthermore, it will also be important to characterise the time course and reversibility of such xenobiotic-induced epigenetic modifications, including the potential for altered sensitivity to rechallenge with the same or a distinct xenobiotic.

A key challenge in the field of safety epigenetics is the need to define the dynamic range of epigenomic normality in different cells, tissues, strains and species so that adequate baseline data are available for providing context to xenobiotic-induced epigenetic changes. Whilst a wealth of genome-wide atlases of epigenome modifications have been generated for human cells and tissues, relatively little data is available for the rodent and non-rodent animal species that are routinely used in toxicology studies. Closing this knowledge gap is an important goal since it would provide novel opportunities for assessing the human relevance of xenobiotic-induced epigenetic alterations.

Conflicts of interest

There are no conflicts to declare.

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