the epigenetic status of the promoter in the first component and expression of the eGFP signal from the second construct. The functionality of this system was demonstrated using targeted DNA methylation (TDM), a method by which the transfection of a methylated genomic fragment with the same sequence as the target locus (here the promoter of the first component) is used as hemi-methylated docking site for DNMT1 during DNA replication and that progressively leads to the silencing of the promoter through cell division. Using TDM and the two-component system transfected in human bone marrow-derived mesenchymal stem cells (MSC), the following were shown: (1) methylation silencing of *Trip10* (thyroid hormone receptor interactor 10) promoter during the differentiation of the MSC to neurons and to osteocytes [399], (2) concurrent methylation silencing of *Hic1* (hypermethylated in cancer 1) and *Rassf1a* (ras-associated family protein isoforms 1A) is sufficient to induce malignant transformation of MSCs [453], and (3) screening a library of 169 compounds, mostly procainamide derivatives (active reactive oxide metabolite N-acetyl procainamide (NAPA)), for DNA demethylase activities using the *Trip10* system in the MCF7 human breast cancer cell line [452]. With the latter demonstration of suitability to a (limited) chemical applicability domain, this is a promising platform to screen chemicals for DNA methylation capability.

While the previous systems were developed by random insertion of reporter constructs, the reporter for genomic methylation (RGM) system is a CRISPR/Cas-guided insertion model [24,400], and can potentially allow for the monitoring of endogenous decreases or increases in DNA methylation at single-cell resolution using microscopy or flow cytometry. The reporter system is based on a minimal promoter region that includes the conserved elements between human and mouse of the imprinted gene Snrpn (small nuclear ribonucleoprotein polypeptide). Snrpn was chosen because unlike many other genes, Snrpn is expressed in most if not all tissues regardless of cellular differentiation. This system relies on the demonstration that methylation from sequences adjacent to the insertion sites spreads into the promoter that drives the expression of a GFP. Using the CRISPR/Cas gene editing technology, the RGM system was integrated into endogenous promoters (unmethylated *Gapdh*, methylated *Dazl*) and super enhancer regions (*Sox2*, miR-290), and the RGM system adopted the methylation characteristics of these adjacent unmethylated or methylated genomic regions, demonstrating its potential to monitor methylation. The system can also monitor the demethylation process, as demonstrated by an increased GFP signal following the insertion of the methylated RGM system into the unmethylated *Gapdh* promoter. The functionality of the RGM system was also demonstrated under in vivo conditions in chimeric embryos. Whilst the RGM system is promising for use in screening strategies to identify putative epigenetic regulators [400], application for chemical screening has not been reported to date.

## 8.5. Global Genome Changes in Histone Modifications and DNA Methylation

Indexes of GGDm and GG histone modifications can be measured by multiple methods depending on the availability of samples and of instruments. High-performance liquid chromatography and capillary electrophoresis [454,455] methodologies with various mass spectrometry detection and quantification systems [456,457] are the gold standards because they provide absolute measures of GGDm and histone modifications. The strengths of these methodologies reside in the provision of highly accurate data and addition of assay modifications to simultaneously measure other modified bases of epigenetic relevance [458–460].

Whole-genome bisulphite sequencing (WGBS) and reduced-representation bisulphite sequencing (RRBS; [461]) are based on the bisulphite reaction of unmethylated cytosines and next-generation sequencing, and in addition to providing indexes of GGDm, they inform about sequence-specific changes in DNA methylation for further validation [462,463]. Alternative techniques to measure GGD methylation include the pyrosequencing-based luminometric methylation assay (LUMA) method [464], antibody driven techniques followed by blotting, flow cytometry analyses [394], or commercially ELISA kits from multiple
companies. Additional methodological resources with respect to DNA methylation assays are documented in Wang and Petronis [465], Esteller [466], and Tost [467].

Liquid chromatography-mass spectrometry (LC-MS) and capillary electrophoresis (CE) systems are antibody free systems and highly accurate in reporting absolute changes in global genome abundance of many HPTM and DNA covalent modifications. Many histone modifications (mono, di, or trimethylation) can be measured within the same sample [468]. Similarly, cytosine modifications (5mC, 5hmC, 5fC, and 5caC) can also be measured within the same sample [469–471]. Given that such assays can be performed from paraffin embedded samples [468], or from in vitro and in vivo samples, this permits verification that in vitro screening observations can correlate with in vivo outcomes. In contrast to locus-specific measurements, a major advantage of measuring global epigenomic changes is that it is applicable to any cell types and chemical treatments, but without providing locus-specific information.

Cytosine extension assays were first described in 1999 [472], are based on parallel use of methylation-sensitive (HpaII, Acil, BssHII) and insensitive (MspI) isoschizomer restriction endonucleases that leave a 5'-guanine overhang after DNA cleavage, and on their extension using DNA polymerase and labelled dCTP. The wide distribution of the CCGG sequence [8% of CpG sites in the human genome [473]], and measurement of incorporated labelled-C reported as ratio of the isoschizomers products, provide an indicator of the global cytosine-methylation level in a cell or tissue sample. The type of labelled dCTP (radioactive, fluorescent, HRP, biotin labelled) gave rise to a family of assays based upon the same principle [reviewed in [394]]. The luminometric methylation assay (LUMA) developed by Karimi et al. [474] is similar but based on pyrosequencing.

In addition to Western blot and enzyme-linked immunosorbent assay (ELISA), commercially available tools can be used to measure global genome changes in histone modifications from cellular extracts. These may include the AlphaLISA/Alphascreen (PerkinElmer, Waltham, MA, USA) [475,476] and the LanthaScreen systems (Life Technologies, Carlsbad, CA, USA), for which the assay specificity is dependent on the antibody quality [477]. The latter group applied this technology to the high-throughput analyses of H3 modifications, including H3K4me2, H3K4me3, H3K9me2, H3K9 acetylation, H3S10 phosphorylation, and H3K27me3. Machleidt et al. [477] confirmed that hypoxic stress, induced by the iron chelator deferoxamine, increases K4 methylation levels, and that the methyltransferase G9a inhibitor, UNC-0638, decreases K9me2 levels with little effects on other modifications. The K9 acetylation assay was also used to screen effects of the 43 compounds of the Enzo Epigenetics Library (Enzo Life Sciences International, Plymouth Meeting, PA, USA) and identified the modulators for type I/II HDACs but not SIRTs. Other similar assays measure histone acetylation [478], and lysine acetyltransferases [479].

## 9. Markers of Specific Key Events for Consideration in Multiplex DNA Methylation Assays

The list of genes listed in Table 3, together with those discussed here, showed deregulated expression by DNA methylation (either hypo or hypermethylation) during carcinogenesis. These genes are relevant to the key events of the IATA leading to cancer development [10] and are explored in more detail. Some of these genes may be considered for the development of multiplex DNA methylation assays, so that more than one gene can be monitored simultaneously from many samples at reasonable cost (Oku et al. in preparation).

### 9.1. Epigenetic Impairment of Detoxification and DNA Repair Pathway; MGMT, BRCA1, and GSTP1

Promoter hypermethylation and gene silencing of the DNA mismatch repair gene hMLH1, the DNA alkyl-repair gene O(6)-methylguanine-DNA methyltransferase (MGMT), the detoxifier glutathione S-transferase P1 (GSTP1) and the familial breast cancer gene BRCA1 were reported to create predispositions to four specific genetic lesions: microsatellite instability, G to A transitions, steroid-related adducts and double-strand breaks in DNA,
respectively [480]. Commercially available DNA methylation tests, including two of these genes (*Gstp1*, *Mgmt*), were recently examined for (a) associations between their targeted genomic location with corresponding methylation profiles from The Cancer Genome Atlas, (b) for links with changes in gene expression, and other relevant characteristics [210]. They report that nine assays (*Gstp1*, *Apc*, *Rassf1*, *Ndrg4*, *Bmp3*, two *Sept9* biomarkers, *Shox2*, and *Mgmt*) have been included in one or more clinical guidelines [210]. *Mgmt* is the most clinically advanced epigenetic biomarker to predict the response to temozolomide and carmustine in gliomas [425]. On the basis that defective detoxification and DNA repair mechanisms can be at the origin of carcinogenesis, assays for *Gstp1*, *Mgmt*, and for the tumour suppressor gene *Brca1* (as discussed below), will provide useful data and are worth further consideration in the IATA.

### 9.1.1. BRCA1, H2AX, and H4K20

A systematic review by Vos et al. [481] reports that *Brca1* promoter hypermethylation is rare in breast and ovarian carcinomas of *Brca1* germline mutation carriers but is relatively more frequent in sporadic cases. In sporadic breast carcinomas and in sporadic ovarian carcinomas, *Brca1* promoter methylation was found in 5.8–35.7% and in 12.3 to 22.5% of the cases, respectively. However, most studies (19/21, 90.5%) were judged as having either a high or unclear risk of bias regarding the methylation analyses due to absence or unclear information on blinding, thresholds and controls [481]. *Brca1* and *Mgmt* promoter hypermethylation are not limited to breast cancer but are observed in other malignancies such as lung adenocarcinoma [482]. Interestingly, sodium arsenite exposure was shown to increase *Brca1* promoter methylation in the human breast cancer cell line MCF7 in vitro [313], supporting that it may be a useful marker.

The relevance of monitoring *Brca1* promoter methylation is better appreciated knowing the function of BRCA1 in DNA repair. DNA double-strand breaks (DSB) can occur as a consequence of replication fork collapse, or following exposure to chemicals or ionizing radiation, or can also occur transiently during DNA repair processes [179]. Broken DNA sites are recognised by the MRE11–RAD50–NBS1 (MRN) complex, which recruits the Ataxia telangiectasia mutated (ATM) and related protein kinases, leading to the phosphorylation of serine-139 in the carboxyl-terminal tail of the H2AX protein. The accumulation of the phosphorylated H2AX forms regions called γH2AX. These γH2AX regions mark the site of DNA damage and provides a nucleation site for the formation of damage response and repair complexes. BRCA1 and BRCA2 are involved in the homologous recombination (HR) error-free repair process of DNA double-strand breaks (DSB). HR can occur during the S-phase and G2 phase of the cell cycle due to the presence of homologous DNA in the sister chromatid [180,483]. In contrast, the nonhomologous DNA end-joining (NHEJ) error-prone DNA repair process occurs in all phases of the cell cycle and recruits p53 Binding Protein 1 (53BP1) to DNA damage sites and blocks the HR process [180]. Reduction in BRCA1 abundance favours the error-prone NHEJ pathway, supporting the predominance of the error-prone NHEJ pathway following *Brca1* gene silencing [180]. Many factors can direct DNA repair toward the HR or NHEJ pathway, including the level of methylation of histone H4 lysine 20 (H4K20) that changes across the cell cycle [180,483]. H4K20me2 is abundant during G1 phase and diluted 2-fold during S phase as new unmodified histones are deposited onto nascent DNA. H4K20me2 levels are subsequently restored during the G2 phase. Unmethylated H4K20 is required for BRCA1 (through BARD1, its obligate binding partner) to conduct the HR pathway [483], and to oppose the 53BP1 function that requires H4K20me2 [179,483]. H4K20 is monomethylated (H4K20me) by KMT5A, and di (H4K20me2), and trimethylated (H4K20me3) by KMT5B/C, demethylation of H4K20me2/3 is conducted by KDM4A (alias JMJD2A). It may be speculated that in addition to *Brca1* silencing by promoter DNA methylation, interference with the above histone methylase and demethylase activities can contribute to DNA repair deficiencies.
### 9.1.2. GSTP1

There are nine classes of glutathione S-transferases that can be cytosolic, membrane bound microsomal, or mitochondrial. The function of GST is classically to eliminate by-products of oxidative stress and electrophilic xenobiotics by catalyzing their binding to the tripeptide glutathione (GSH), but also GST can act as intracellular transport proteins for various hormones/proteins, or as modulators of signaling pathways (e.g., JNK/c-JUN, MAPK) [484]. Overexpression of GSTP1 is found to occur in many cancer types [484,485] and in foci as a marker of preneoplastic lesions in numerous tissues in rodents and human [486–488], and it is indicative of cellular stress and antioxidant response as observed in an arsenic exposed population [324]. Reduction in GST activity is also of great relevance to carcinogenesis. Gene deletion, single nucleotide polymorphism, and epigenetic silencing, all cause reduction in GST activities that are positively associated with increased DNA damage and incidence of various cancers [484,485]. *Gstp1* is one of the nine genes for which commercially available methylation assays were developed and integrated in clinical guidelines [210]. The promoter of *Gstp1* includes a hypomethylated region at the preneoplastic stage, and later during carcinogenesis these sites get methylated to silence GSTP1 expression, as observed with human hepatocarcinoma [488,489]. Ongoing controversies in the *Gstp1* methylation literature concern the adjacent methylated and unmethylated regions of the promoter, methylation progression during carcinogenesis, as well as differences in technologies and assay designs [490]. Only well-validated *Gstp1* assays should be used (e.g., pyrosequencing [490]) to further test *Gstp1* methylation as a marker in chemical testing strategies.

### 9.2. *Cell Cycle Regulation Breakdown, the Ink4b/Arf/Ink4a Locus*

DNA damage, aging, and cellular stress (occurrence of reactive oxygen species, oncogene activation, telomere erosion, stalled replication forks [181]), are detected by gene pathways (e.g., telomerase, p53, RB) regulating the transcription of the *Ink4b/Arf/Ink4a* locus, which produces cyclin-dependent kinase inhibitors inducing cell cycle arrest and contributing to senescence and apoptosis to prevent carcinogenesis. In the absence of such a key event relationship, chemical induction of proliferation can contribute to carcinogenesis by permitting the fixation of mutations (and epimutations) in subsequent cellular generations. This section summarises how the *Ink4b/Arf/Ink4a* locus can be epigenetically silenced, and how it can be reported as a measurable key event in the IATA.

Progression through the cell cycle is mediated at the G1, G2, and mitosis checkpoints of the cell cycle by cyclins, cyclin-dependent kinases, and their inhibitors such as p14<sup>Arf</sup> (p19<sup>Arf</sup> in mouse), p15<sup>Ink4b</sup>, p16<sup>Ink4a</sup>, p21<sup>Cip1/Waf1</sup>, and p27<sup>Kip1</sup>. The *Ink4b/Arf/Ink4a* locus is an important carcinogenic target as it transcribes four genes mediating activity of the p53 and pRB pathways. The cyclin-dependent kinase inhibitors (CDKI) p14<sup>Arf</sup> (p19<sup>Arf</sup>), p15<sup>Ink4b</sup>, p16<sup>Ink4a</sup>, and the long non-coding RNA ANRIL (antisense non-coding RNA in the *INK4* locus) are four genes transcribed from the *Ink4b/Arf/Ink4a* locus, considered by some as the most frequently mutated locus (MYC amplifications and *Cdkn2a/b* deletions in 14% of 3131 cancer samples [491]), while p16<sup>Ink4a</sup> is epigenetically silenced in more than 70% of cases, depending on cancer types [492,493]. Upon detection of an oncogenic signal and aging, p16<sup>Ink4a</sup> is transcribed from the tumor suppressor gene *Cdkn2a* from the *Arf/Ink4a* locus to arrest the cell cycle at the G1 checkpoint [181]. P14<sup>Arf</sup> is also transcribed from *Cdkn2a*, but *Arf* express exon 1α and alternate reading frame of exon 2, whereas *Ink4a* express exon 1β, 2 and 3. P14<sup>Arf</sup> (p19<sup>Arf</sup> in mouse) arrest the cell cycle at G1 and G2 phase of the cell cycle and inhibits MDM2 (HDM2 in human) and thus stabilizes p53 [494,495]. P15<sup>Ink4b</sup> is transcribed from *Cdkn2b* to arrest the cell cycle either at G1 or G2 [496,497]. The long non-coding RNA ANRIL can reduce p15<sup>Ink4b</sup> expression by recruiting the polycomb repressive complex PRC2 [498].

In the G1 phase of the cell cycle, the RB tumour suppressor proteins are bound to the transcription factor E2F preventing it from inducing transcription of genes necessary to advance in the cell cycle. To move from the G1 to the S phase, cyclin D and cyclin-dependent
kinase CDK4/6 form the CDK4/6-cyclin D active complex that can phosphorylate RB, which then releases E2F to induce gene transcription and proteins necessary for cell cycle progression. Under cellular stresses and senescence (replicative, oncogene-induced), p16<sup>Ink4a</sup> binds CDK4/6 and prevents the formation of the CDK4/6-cyclin D complex, this prevents RB phosphorylation and the release of E2F, and consequently inhibits cell cycle progression. During carcinogenesis the *Ink4b/Arf/Ink4a* locus and cell cycle checkpoints can be deregulated by mutation, homozygous or heterozygous gene loss, or by epigenetic anomalies acting either at the enhancer element upstream of the entire locus or at gene-specific promoters, or by actions of the lncRNA ANRIL [492,499].

Given both the importance and complexity of the *Ink4b/Arf/Ink4a* locus, more than a single assay type together with long-term assays may be required to assess its functionality. p16<sup>Ink4a</sup> alone, or in association with p14<sup>Arf</sup> [494] or with p15<sup>Ink4b</sup> [500] can be epigenetically silenced. In either case, this will lead to the absence of functional *Ink4b/Arf/Ink4a* locus and permit cell cycle progression in the presence of unrepaired genomic and/or other epigenetic anomalies. p14<sup>Arf</sup> and p16<sup>Ink4a</sup> are barely detectable in normal cells and can be silenced in cancers, consequently measuring differences in protein expression may not differentiate both situations, but epigenetic silencing of these genes should permit to distinguish normal from epigenetically abnormal tumorigenic cells. The expression and silencing of the *Ink4b/Arf/Ink4a* locus require the interplay of numerous epigenetic enzymes [126,181,492,495], with gene silencing occurring prior to the detection of DNA methylation [501]. Indeed, after growing primary human mammary epithelial cells in serum-free medium for 2–4 weeks, colonies emerge with silenced p16<sup>INK4A</sup>, but it is in the following weeks that the promoter shows gradual de novo DNA methylation and histone remodelling (H3K9 deacetylation and dimethylation) to consolidate its suppression [501]. In other cases, high abundance of p16<sup>Ink4a</sup> can be found in cells with ongoing senescence or subjected to various stressors, as well as in some cell lines and some cancers when the RB negative feedback exerted on p16<sup>INK4A</sup> is inhibited as in the case of human papillomavirus (HPV) infection [181,495,502]. The p16<sup>Ink4a</sup>-RB pathway can be deregulated by HPV infections that release the oncoproteins E6/E7 that bind pRB and prevent cell cycle arrest as well as the negative feedback mechanism regulating p16<sup>Ink4a</sup> expression [181,503]. In this case, the measurement of an indicator of cell proliferation (e.g., Ki67) or inflammation should permit distinguishing between senescent and oncogenic dividing cells with both showing increased p16<sup>Ink4a</sup> expression [181]. Finally, species differences are important to note in the investigation of the *Ink4b/Arf/Ink4a* locus; cell cycle progression and culture-induced growth arrest are more regulated by p14<sup>Arf</sup> (p19<sup>Arf</sup>) in the mouse but by p16<sup>Ink4a</sup> in human [504,505]. Further, ANRIL exons do not exist in the mouse [499]. Overall, despite its complexity, given the importance of the *Ink4b/Arf/Ink4a* locus and that it is found to be frequently epigenetically silenced during carcinogenesis, investigating the DNA methylation "hot spots" of the *Ink4b/Arf/Ink4a* locus [501] may generate informative biomarkers.

### 9.3. Inflammation/Immune Response Disruption

The immunosurveillance system eliminates abnormal cells to prevent cancer development, although some transformed cells can evade immune detection to create cancers [506]. An excellent review was recently published explaining how tumour cells can evade immune detection [507]. The impact of the multiple forms of cellular stress (proteotoxic, oxidative, endoplasmic reticulum, genotoxic, and metabolic stress, and hypoxia) on activation and inhibition of the components of the immunosurveillance system were recently reviewed [181]. Tissues exposed to chemicals can generate immune-stimulating signals due to cellular stress and toxicity, and cells that reach senescence produce the senescence associated secretory phenotype (SASP) stimulating the immune system to expedite their elimination, and extracellular matrix and growth factors to facilitate tissue regeneration. Persistent senescence contributes to chronic inflammation, diseases of aging, and tumour formation [508,509]. In contrast, tumorigenic cells emanating from this environment might generate immune-suppressive signals to avoid immune detection. The duration
of treatment and timing of sampling might influence the transformation process and the abundance of cells sending immune-stimulating (stressed or senescent cells) or immuno-suppressive signals. The diverse signals emanating from such an environment complicates the identification of the relevant ones as predictive of adverse effects. Chemical hazard assessment considers mechanisms that reduce the efficiency of the immune system (mostly in animals), and those adopted by transforming cells to avoid or escape immune detection. While immune-stimulating signals are important in toxicological assessment, the adverse outcome in carcinogenesis is the detection of immunosuppressive signals from tumorigenic cells. The latter therefore guides the selection of in vitro epigenetic assays presented here. Important challenges in assay selection are on the one hand the consideration of the complexity of the immune system that involves numerous cell type interactions and differentiation in vivo, and on the other hand the international pressure to phase out animal testing and to develop in vitro chemical testing procedures [510]. While there are epigenetic mechanisms that can be targeted to suggest a reduction in the efficiency of the immune system under in vivo conditions [511–513], the current section discusses some epigenetic measurements that can support the acquisition of immune evasion properties during the in vitro transformation of tumorigenic cells.

Tumorigenic cells can escape immune detection through a variety of mechanisms such as the maintenance of an immunosuppressive tumour microenvironment by producing IL-4, -6, -10, -13, TGFβ, and VEGF, by the loss of the expression of a variety of tumour-associated antigens, or by loss of antigen-presenting machinery-related genes, either through genomic instability or epigenetic reprogramming [514,515]. Seeding information suggesting a role for DNA methylation in contributing to immune evasion are provided by numerous observations following treatment with DNA methyltransferase inhibitors (DNMTi). DNMTi treatment can increase tumour cell immunogenicity by: (1) re-activating expression of major histocompatibility complex (MHC) components to increase neoantigen presentation, (2) re-activating expression of silenced immune checkpoint (IC) and IC ligands (ICL), (3) increasing the expression of natural killer group 2 D receptor ligands (NKG2DL), (4) or inducing expression of cancer testis antigens (CTAG). Although less related to the immune evasion strategy, it should be noted that treatment with DNMTi can contribute to the elimination of tumour cells by re-activation of hypermethylated tumor suppressor genes, by activating retrotransposons and retroviral elements leading to dsRNA and cytosolic DNA that activate interferon responses [50,368,516]. Regulators of immune evasion, like other genes are subjected to many transcriptional (transcription factors, epigenetic regulators), translational (e.g., miRNAs), and post-translational regulatory mechanisms, offering multiple complementary investigative approaches (e.g., genomic, epigenomic, immunohistochemistry (IHC)). IHC is being used extensively for clinical purposes, but antibody specificity and assay variability introduce difficulties in data interpretation. Considering that immune evasion must be prolonged from the initial stage of transformation until later cancer progression, combined with immune evasion and suppression assays (Corsini et al. in preparation), DNA methylation can be promising robust assays indicative of persistent and long-term changes. The development of multiplex assays for the measurement of DNA methylation changes at multiple loci would offer the advantage of deriving a better coverage of the multiple mechanisms of immune evasion. More details supporting the consideration of developing epigenetic assays related to MHC, CTAG, IC, ICL, and NKG2DL are provided in the following three sections. Note that such approaches are relevant to chemical hazard assessments only when studying target tumorigenic cells, and not tumor samples that include combination of infiltrating immune cells with tumorigenic cells that can express the same molecules, and thus would dilute and complicate data interpretation.

### 9.3.1. Major Histocompatibility Complex-I and -II

Chemicals that reduce the abundance of cell surface antigens are of concern. To eliminate proteins from invading agents or non-functional endogenous proteins (mutated,
misfolded, etc.), the proteasomes degrade proteins into peptides that can be recycled or brought to the cell surface and presented as antigens by components of the major histocompatibility complex (MHC). Components of the MHC-I complex (e.g., human leucocyte antigen HLA-A, -B, and -C) are expressed on nucleated cells and tumour cells, and components of the MHC-II complex (e.g., HLA-DR, -DP, and -DQ) are expressed on cells of the immune system (e.g., dendritic cells, macrophage, B cells) but can also be induced on some tumour cells or normal cell types in inflammatory conditions [517,518]. It is through these mechanisms that abnormal antigenic peptides originating from invading agents or abnormal proteins can be presented by MHC-I at the cell surface and elicit an immune response leading to the elimination of the affected cells by cytotoxic CD8⁺ T cells. The expression of the MHC-I complex can be regulated directly by cytokines such as interferon gamma (INFγ), or indirectly through the MHC-I regulating proteins NLRC5 (nucleotide-binding domain and leucine-rich repeats containing (NLR) family, caspase activation and recruitment domain (CARD) domain containing 5) and CIITA (Class II transactivator), whereas genes from the MHC-II complex are regulated through CIITA [517,519]. Genes from both MHC-I and -II antigen-presenting machinery can either be epigenetically modulated [515,519] or rendered defective by mutations [520]. In both cases, these mechanisms reduce abundance of antigens at cell surface and contribute to immune evasion of the tumorigenic cells.

NLRC5 is a major regulator of the expression of the MHC-I gene complex, including HLA-A, HLA-B, HLA-C, β2m, LMP2, LMP7 and TAP1, which are necessary for the presentation of neoantigens to CD8⁺ T cells resulting in the elimination of cancer cells [521]. The investigation of 21 tumor types [522] and a review by the same laboratory [515] revealed that NLRC5 is the main tumorigenic target of the MHC-I pathway displaying genetic and epigenetic alterations. The DNA methylation level of the *Nlrc5* promoter varies across cancers, but it is the most methylated among MHC-I genes, and its expression is responsive to DNMTi treatment. Consequently, DNA methylation represents the main mechanism of *Nlrc5* regulation, followed by copy number loss, and somatic mutations [515]. The gene *Nlrc5* deserves consideration in chemical hazard assessment.

Some of the NLRC5 target genes of the MHC-I complex are also modulated by epi-
genetic mechanisms. HLA-A expression is affected by methylation but not HLA-B and
-C. Indeed, the investigation of peripheral blood mononuclear cells from healthy donors
revealed allelic lineage-specific methylation patterns within the *Hla-a* promoter where
increased DNA methylation levels correlated with reduced HLA-A expression, and in-
creased expression following DNMTi exposure [523]. Additional antigen-processing and
-presentation molecules that can be regulated by DNA methylation include B2M (beta-2
microglobulin), CALR (calreticulin, a reticulum endoplasmic protein), CD58 (alias LFA-3,
lymphocyte function-associated antigen 3), PSMB8, and PSMB9 (Proteasome subunit beta
type-8, -9), which were found upregulated following DNMTi exposure in colon and ovarian
cancer cell lines [524].

In contrast to HLA expressions which are generally associated with immunogenicity,
the HLA-G (isoform G3) expression in colorectal cancer is suggested as a mechanism
to escape immune recognition and destruction [525]. The placenta avoids immune reac-
tion through the production of HIF1α-mediated expression of HLA-G and PD-L1 (see
Section 9.3.2) [526]. In human cancers the expression of HLA-G is also mediated through
HIF1α [527,528]. While exposure to DNMTi increases the expression of HLA-G [528], the
exact contribution of DNA methylation in the regulation of HLA-G expression remains
controversial [525].

Drugs and environmental contaminants have been shown to contribute to immune deficiency. Substances inducing oxidative stress such as sodium arsenite (≥1 µM NaAs₂O₃), or the antioxidant dimethyl fumarate (≥25 µM DMF; autoimmune disease drug) and tert-butylhydroquinone (≥25 µM tBHQ) reduce INFγ-induced (100 ng/mL) MHC-II (HLA-DR) surface protein expression in HeLa and in U118 cell lines and consequently reduce the efficiency of the immune system [529]. The effects of arsenite are mediated by the inhibition
of the oxidative stress sensor KEAP1 (which also mediates NRF2-dependent antioxidant gene expression), and inhibition of H4K16 histone acetyltransferase MYST1, both involved in INFγ pathway inducing MHC-II surface protein expression. The antioxidant DMF is a broad regulator of INFγ-induced MHC-II and chemokine expression acting through multiple pathways [529].

Overall, developing and validating multiplex epigenetic assays to monitor functions of the antigen presenting machinery, including epigenetic silencing of *Nlrc5* and MHC-I-related genes (e.g., *Hla-a*) and expression of HLA-G, can be useful to predict predisposition to immune evasion.

### 9.3.2. T Cell Immune Checkpoints, NK Cell NKG2D Receptor, and Their Ligands

To avoid over stimulation of the immune system and the destruction of normal cells, cell surface proteins are expressed on normal cells to interact with T-cell surface proteins to reduce potential T-cell overactivity. As a mechanism of immune evasion, tumorigenic cells can produce cell surface proteins called immune checkpoint ligands (ICL) (e.g., B7/CD80/86, PD-L1/2, GALECTIN-9, LSECTIN, NECTIN-2, VSIG-3) that interact with proteins called immune checkpoints (IC) (e.g., CTLA-4, PD-1, TIM-3, LAG-3, TIGIT, VISTA, BTLA) located at the surface of T-cells to inactivate them [530]. In clinical practice, interaction between IC and ICL are targeted by immunotherapies to prevent immune evasion. To prevent side effects of unnecessary immunotherapy, immunohistochemistry (IHC) analysis of biopsies is performed to identify the presence of IC and ICL. However, tumor heterogeneity, antibody and assay variability affect the accuracy of IHC analyses [531]. The roles of DNA methylation and histone modification in immune checkpoint regulation (PD-L1, PD-1, CTLA-4, TIM-3, LAG3 and TIGIT) have been reviewed [530], and as expected, DNA hypomethylation and decreases in the abundance of repressive marks (H3K9me3, H3K27me3) are associated with increased expression of these ICL and IC.

PD-L1 (programmed death ligand 1, alias CD274) is an important ICL that activates the inhibitory PD-1 receptor on T cells compromising T-cell functions and contributing to immune evasion. Exposure to DNMT inhibitor increased PD-L1 expression in melanoma cell lines [532], in non-small cell lung cancer cell lines [533], in breast, colon, and ovarian carcinoma cell lines [534]. These data suggest that DNA methylation of the *Pd-l1* promoter prevents its expression, but there are contrasting levels of DNA methylation depending on cell lines and tissue types, which raise the need for careful selection of in vitro experimental strategies in the development of a *Pd-l1* assay that is suitable for the NGTxC IATA. The culture of human breast cancer cell lines (MCF7 and BT-549) for more than 7 days in cancer stem cell medium promotes the formation of tumourspheres associated with increased expression of mesenchymal markers (VIMENTIN, N-CADHERIN, SNAIL) as well as PD-L1 [535], supporting that mechanisms predisposing to immune evasion can be monitored in relatively short experiments. During this process, DNA in the promoter of *Pd-l1* gets hypomethylated in MCF7 cells, but the level of DNA methylation in BT-549 remains statistically unaffected, being already at a low level of methylation. Decreases in the abundance of repressive histone marks (H3K9me3, H3K27me3) have been observed in the promoter of *Pd-l1* in tumourspheres from both cell lines [535]. Similar to the BT-549 cell line, the promoter of *Pd-l1* is also unmethylated in pairs of normal tissues and in colorectal [536] and breast cancer samples [537]. Refinement in the analyses of melanomas suggests that the *Pd-l1* gene expression is regulated by two specific CpG sites in the promoter [538]. By comparing 12 melanoma cell lines showing either constitutive or inducible PD-L1 expression, Chatterjee et al. [532] demonstrated that global genome DNA hypomethylation, particularly in intergenic regions and repeated elements, promote constitutive PD-L1 expression, and hence immune evasion. Overall, these experiments suggest that DNA methylation is one of the mechanisms that regulates PD-L1 expression, and in some cases, the promoter is the influential locus but in others, distant regulatory intergenic and intronic loci needs further investigation. A DNA methylation biomarker based on 269 CpG sites was recently proposed to predict success of PD-1/PD-L1 inhibition therapy [539]. Other ICL
genes for which promoter methylation regulates their expression include *Ctla4* (cytotoxic T lymphocyte-associated protein 4) [531]. Further investigation of the epigenetic mechanism leading to the expression of PD-L1 and CTLA4 and immune evasion is beneficial for both clinical and chemical safety.

While IC/ICL interactions attenuate T cell functions, in contrast, natural killer (NK) cells can be activated to kill cancer cells through the interaction of their receptor NKG2D (natural killer group 2, member D) with their family of ligands (NKG2DL) produced by cancer cells. The NKG2D receptor is also present in many T cell groups (e.g., γδT, natural killer T (NKT), CD8⁺ T cells) as costimulatory receptor but not directly mediating cytotoxicity [540]. In humans, the NKG2D ligands include the MHC class I-related chain A or B (MICA/B), and UL16-binding proteins 1 to 6 (ULBP1-6, alias retinoic acid early transcripts 1 (RAE-1)) that become expressed in infected or tumorigenic cells ([540], see also Esteban et al. in preparation). Human/murine species differences in the type of NKG2DL should be noted for species extrapolation [541]. Cellular stress (e.g., abnormal proliferation, heat shock, DNA damage, and infections) activates signaling pathways leading to the expression of NKG2DL at the cell surface. However, cells can evade immune detection by proteolytic shedding of the cell surface ligands MICA and MICB by the action of disulfide isomerase (ERp5) and several proteases of the matrix metalloproteinases (MMPs) and ADAMs (a disintegrin and metalloproteinases) enzyme families. It was recently demonstrated that the oncoprotein MITF (Melanocyte-Inducing Transcription Factor) regulates the expression of ADAM10 that cleaves MICA/B allowing cells to bypass NK cell surveillance [542]. Antibodies targeting MICA/B prevent proteolytic shedding and maintain immunoactivity [543]. Epigenetic mechanisms can also contribute to silencing of NKG2D ligand as a mechanism of immune evasion. The HDAC inhibitor valproic acid (24 h, ≥2.5 mM) increases the abundance of NKG2D ligand (MICA, MICB and ULBP-2) mRNAs, surface protein expression, and shedding from pancreatic carcinoma (Panc89) and prostate carcinoma (PC3) cell lines [544]. Others, through the investigation of 10 NKG2D ligands in 7 hepatocarcinoma cell lines found that many ligands were downregulated compared to normal liver samples, which could serve as an immune evasion mechanism [545]. The latter found that among the NKG2D ligands, ULBP1 is both necessary and sufficient to regulate NK cell-mediated cytotoxicity. The silencing mechanisms involved EZH2-induced recruitment of DNMT3A to methylate the *Ulbp1* promoter, consequently, treatment with an EZH2 inhibitor (GSK343) or DNMT inhibitor (5aCdR) increased the expression of several NKG2D ligands [545]. In isocitrate dehydrogenase (IDH) mutant glioma cells (a gain-of-function mutation catalyzing the production of 2-hydroxyglutarate (2HG)), 2HG inhibits α-ketoglutarate-dependent dioxygenases and consequently reduces TET demethylase activities and increases genomic DNA methylation. As a result, the level of promoter methylation for *Micb*, *Ulbp1* and *Ulbp3* is higher in gliomas with mutated IDH than with wild type IDH, and the two primary transcriptionally silenced NKG2D ligands ULBP1 and ULBP3 were responsive to DNMTi [546]. Collectively, these data suggest that histone acetylation and methylation, as well as DNA methylation, are mechanisms involved in NKG2D ligand silencing and immune evasion, and ULBP1/2/3, and MICA/B (in human but not mouse) can be priority markers for further development and validation.

### 9.3.3. Cancer-Testis Antigen Gene Families as Epigenetic Markers of Carcinogenicity

This section describes how exposure to carcinogens that induce DNA hypomethylation can also induce the expression of cancer-testis antigen genes (CTAGs), supporting potential adverse effects of DNA hypomethylation and thus the use of CTAGs as marker of cancer predisposition. CTAGs belong to a large group of tumour-associated antigens (more than 100 gene families listed in http://www.cta.lncc.br, accessed on 6 October 2021) expressed in transformed cells and in numerous cancers but generally not in normal tissues except during spermatogenesis in the testis and in the placenta (some expressed at <1% of testis levels in pancreas, liver, spleen) [547]. A large number of these genes are located on the X and Y chromosomes, but others are distributed throughout the genome. DNMTi treatment
upregulates many CTAGs common to both colon and ovarian cancer [524]. The most frequently expressed CTAGs are NY-ESO-1, SSX-2, SSX-4, MAGE-A1, and MAGE-A3 [547]. The melanoma antigen gene (*Mage*) family originates from more than 60 genes generating two families of MAGE proteins (Type I and II) among which the Type-I (MAGE-A, -B, -C subfamilies) are expressed in cancers through epigenetic deregulation [548]. Type-I MAGE promotes cancer cell survival in various ways: (1) by interacting with ubiquitin system to promote degradation of tumor suppressor proteins (e.g., p53, AMPKα1, ZNF382), (2) by inhibiting cyclin degradation, (3) by acting as transcription regulators [548], (4) by repressing, in the case of other CTAGs (PRAME), retinoic acid signaling thereby inhibiting differentiation [549]. It appears that the carcinogenic roles of these CTAGs outweigh their immunogenicity in the tumorigenic environment. The blood–testis barrier and the lack of HLA class I expression on the surface of germ cells may provide protection from attack by the immune system [547], but the growth of CTAG-expressing tumour cells suggests an immune-suppressive tumour environment perhaps involving mutations or silencing of MHC-I components [520,549]. Despite this, the presence of these antigens in cancer cells promotes the design of epigenetic cancer immunotherapies by which treatment with DNA hypomethylating drugs promote their expression and increases the antigenicity of the tumour [50,547].

DNA hypomethylation is an important mechanism regulating the expression of CTAGs with some events promoting carcinogenesis [50,548,550]. In addition to epigenetic drugs [50], exposure to some carcinogens (*Helicobacter pylori* [551], methylcholanthrene [552], smoking [553,554]) induces expression of CTAGs. Consequently, for adequate consideration of CTAGs epigenetics in chemical hazard testing strategies, time-course analyses are needed following exposure to carcinogenic substances for primary cell transformation, global genome DNA hypomethylation, and CTAGs DNA hypomethylation and expression.

## 9.4. *The Cytoskeleton; Relevance to Global Genome Epigenetic Marks, E-Cadherin, MYO10*

During carcinogenesis, normal cells undergo changes in morphology as they progress through stages of hyperplasia, metaplasia, anaplasia, dysplasia, neoplasia, epithelial-to-mesenchymal transition (EMT [555]), and metastasis. The dysplastic stage (change in morphology, abnormal mitosis, disorganised cell proliferation with loss of cell polarity, cellular and/or structural atypia) may represent the decisive carcinogenic progression step. Progression across these histological stages requires molecular adaptations, pathway signaling, and epigenetic mechanisms conducive to cytoskeleton modifications, changes in cell-to-cell adhesion, and interactions between the cells and the extracellular matrix [556,557]. These mechanisms involve various structures (e.g., intercellular desmosome, gap, tight, and adherens junctions; extracellular matrix hemidesmosomes and focal contact), interacting protein families (e.g., integrins, actins, connexins, claudins, occludin, catenins, cadherins, fibronectin), and signaling pathways (TGFβ-SMAD3, WNT-β CATENIN, and NOTCH) inducing the expression of transcription factors (e.g., ZEB1, ZEB2, SNAIL, SLUG, TWIST) to downregulate or upregulate the expression of epithelial (e.g., E-CADHERIN, CLAUDINS, OCCLUDING), and mesenchymal markers (e.g., N-CADHERIN, VIMENTIN, FIBRONECTIN). There is an increasing number of studies on different cancers highlighting epigenetic modifications and changes in gene expression that have already occurred by the stage of dysplasia [558–562], and later during EMT [557]. Therefore, monitoring epigenetic deregulation may assist in better predicting the potential adversity of cellular transformation processes. Below we highlight three examples where pre-existing global genome changes in DNA methylation or histone modifications can create epigenetically permissive conditions for oncogenic activation of gene regulatory elements by transcription factors. Epigenetic mechanisms regulating E-CADHERIN expression are discussed. Finally, the epigenetic system (KMT3A, EZH2, WDR5, and PRMT2) can also directly methylate components of the cytoskeleton to maintain genomic stability [563,564]. Collectively, these
mechanisms suggest that deregulation of the epigenetic system may impact cell morphology/cytoskeleton and cell function during early and late key events of carcinogenesis.

Neoplastic cells spontaneously develop an epigenetic state [565] to invade the normal surrounding stroma in a cohesive group of cells, with the “leader” cells being distinct from the cells behind referred to as the “follower” cells [565,566], particularly based on the expression of the filopodia protein Myosin-X (MYO10). Such patterns of distinct cell types participating in invasion supports the polyclonal nature of tumours, as opposed to the idea of expansion of a single clone to form a tumour [567]. Investigating breast [565] and lung cancer cells [567], invasive “leader” cells were shown to be phenotypically and epigenetically different from the non-invasive “follower” cell population. Summerbell et al. [567] demonstrate that leader cells activate the NOTCH pathway by increasing the expression of JAGGED1 (JAG1, a canonical ligand of the cell surface receptor NOTCH [568]), which then induces the expression of the filopodia protein MYO10. Filopodia are membrane protrusions involved in cell adhesion, migration, and invasion in normal and cancer cells. The induced expression of MYO10 by JAG1 is possible only when the promoter of *Myo10* is hypomethylated [567]. Moreover, as these phenotypically and epigenetically different cell types were adjacent to a larger cell population, only imaging systems and flow cytometry analyses could distinguish leader cells from other cell types, on the basis of MYO10 expression.

The DNA hypomethylation of the *Myo10* promoter is one of three examples suggesting that the pre-existing global genome epigenetic background provides plasticity to epithelial transformation by creating epigenetically permissive conditions for oncogenic transcription factor activities. A second example involves enzymes regulating the abundance of H3K36me2. Using a mouse model of pancreatic ductal adenocarcinoma and CRISPR/Cas9 sgRNA to screen epigenetic modifiers, Yuan et al. [569] identified the nuclear receptor binding SET domain protein 2 (NSD2) as the top gene significantly enriched in a subpopulation of cells showing the epithelial marker E-CADHERIN (ECAD+). NSD2 is a histone methyl-transferase that dimethylates H3K36 to H3K36me2. In contrast, lysine-specific demethylase 2A (KDM2A) was overrepresented in the ECAD-subpopulation. KDM2A is a demethylase that preferentially targets H3K36me2. Monitoring activities of EMT-transcription factors (TF) and cytoskeleton markers, it was determined that mechanisms regulating the abundance of H3K36me2 underlie the capacity of TF to alter the EMT and the reverse process mesenchymal-to-epithelial transition (MET) [569]. The third example involves the SETD1A/KMT2F that methylates H3K4 (me1, me2, and me3; [570]) and promotes gastric cancer tumorigenesis by enhancing glycolysis [571], and by contributing to EMT regulation [572]. The transcription factor SNAIL controls EMT by downregulating ECAD expression and favours cancer cell's EMT [573,574]. In gastric cancer cells, Wu et al. [572] found that SETD1A/KMT2F is necessary to reprogramme the *Snail* promoter by H3K4 methylation thereby regulating its expression and indirectly EMT [572].

ECAD, a gene product of *Cdh1*, is a growth and invasion suppressor in some cancers [575] but may also have bimodal oncogenic roles (reduction and increase in ECAD promote invasion, and metastasis, respectively) in other cancers [576,577]. ECAD is a member of one of the nine families of cadherins (calcium-dependent cell adhesion molecules) that are localized at intercellular adherens junctions. Cadherins possess extra- and intracellular domains that interact with catenins, providing a link to the actin cytoskeleton and with the WNT/β-CATENIN, PI3K/AKT, HIPPO, and NFκB signaling pathways [575]. Loss of ECAD with increased abundance of N-CADHERIN (*Cdh2*) occurs during EMT [578]. ECAD expression was reported to be silenced by DNA hypermethylation of its *Cdh1* promoter and by TF activation [575]. The transcription factors SNAIL, TWIST, and ZEB1/2 are known to bind the enhancer boxes (E-box) of *Cdh1*, attracting epigenetic complexes and silencing its expression [575], reaching 30% of hepatocellular carcinoma cases with silenced *Cdh1* [579]. Repression of *Cdh1* transcription preceded the subsequent acquisition of methylated CpG sites [580]. In addition to *Cdh1*, other cytoskeleton-related genes are silenced by DNA hypermethylation, e.g., Connexin26 in rat hepatocellular carcinomas is
induced by choline-deficient diets [581], and *Cdh13* hypermethylation (CADHERIN-13) correlates negatively with hormone receptor status in breast cancers [582]. The role of ncRNAs in regulating ECAD expression was also previously reviewed [575].

The epigenetic system has "chromatocytoskeletal" activities, modifying the chromatin to alter the function of DNA loci, but also directly targeting the cytoskeleton to regulate the structure and function of microtubules and actin filaments [563,564]. SETD2/KMT3A establishes the H3K36me3 marks on the chromatin, and the α-TUBULIN K40me3 mark on microtubules required for proper chromosome segregation and genomic stability [563]. EZH2, WDR5, and PRMT2 as other chromatin HMT also methylates cytoskeleton components [563].

## 9.5. Senescence Bypass and Telomerase Reverse Transcriptase (TERT) Regulation

Cellular senescence is a state of "irreversible" growth arrest leading to quiescence and cell death. Normally, the prevalence of senescence increases with aging and protects against the development of cancer cells [583,584]. There are different forms and origins of senescence: replication-induced senescence observed with aging and associated with telomere shortening with increasing number of cell divisions; oncogene-induced senescence due to oncogene activation; and senescence induced by exposure to substances when sufficient cellular injury and DNA damage are created. However, senescence can be reversible as a rare event. Senescence bypass or the ability of a cell to evade senescence and to achieve replicative immortality usually by activation of the telomerase enzyme are essential steps toward cancer development [56] (Vaccari et al. in preparation).

Telomeres are terminal structures at the end of chromosomes including kilobases-long double-stranded DNA tandem repeats (TTAGGG) followed by a 3' single-stranded-DNA that forms a telomere loop (t-loop) covered by SHELTERIN-protein complexes [585]. The telomeres and the SHELTERIN components permit DNA replication by preventing chromosomal ends from being mistaken as sites of DNA damage. However, each cell division is associated with telomere attrition that impose a limit in the number of cell divisions that a cell type can accommodate [586,587]. Telomeres that are eroded activate the p53 and RB tumor suppressor pathways (including activation of the *Ink4a/Arf* locus) inducing senescence and cell death, which prevent cancer development. If these pathways are defective when telomeres are eroding or if SHELTERIN components (a six-protein complex) are lost [585], this can lead to a telomere crisis by which DNA repair is activated. When proliferation continues this leads to telomere ligation among chromosomes, dicentric chromosome formation, genomic instability, aneuploidy, chymotrypsis, kataegis, and cancers [586–589].

The telomerase is a ribonucleoprotein multicomponent enzyme that maintains the telomeres. Activity of the telomerase is undetectable in most normal cells and tissues limiting the cell's lifespan; however, telomerase activity is present in cancer cells providing replicative immortality. This telomerase enzymatic complex includes a range of associated proteins, RNAs, and a rate-limiting component which is the telomerase reverse transcriptase (TERT) [421,587], and TERT expression appeared early during tumorigenesis in vivo [590]. Dogan and Forsyth [421] report methylation and mutation of the *Tert* promoter in 53% and 31%, respectively, of TERT expressing cancer cell lines.

The epigenetic mechanisms (DNA methylation, histone modifications, and non-coding RNAs) regulating the expression of TERT were recently reviewed [421,591]. A role for DNA methylation in regulating TERT expression was initially controversial; however, it became clear that the TERT Hypermethylated Oncological Region (*Thor*) located from -649 to -217 nucleotides from the transcription start site can regulate the expression of TERT by DNA methylation [421,592]. In contrast to the usual relationship of hypermethylation that silences gene expression, hypermethylation of the *Tert* promoter increases TERT expression by preventing binding of the transcriptional repressors WT1 and CTCF [421]. As expected, abundance of H3K4me1/me3 and H3K27ac increase, and H3K27me3 and H3K9me3 decrease with augmentation and diminution in TERT expression, respectively.
Additionally, a number of ncRNAs (let-7g-3p, and 17 miRNAs) are involved in regulating TERT mRNA abundance [421]. Telomere length can be regulated by the expression of the telomerase but also by the Alternative Lengthening of Telomeres (ALT) pathway under the influence of the long non-coding RNA TERRA (Telomeric Repeat-containing RNA) [593–595]. The subtelomeric regions represent transition regions between the telomere and the chromosome-specific region. Human subtelomeres are CpG rich and include promoters for TERRA which expression is repressed by DNA methylation. It has been recently proposed that TERRA expression contribute to DNA replication stress and DNA damage in the telomeres which activate their repair and elongation through the ALT and break-induced replication mechanisms [594]. In the context of chemical carcinogen testing strategy using DNA methylation, it would appear that an increase in telomere length favouring replicative immortality might by associated with hypermethylation of *Thor*, or hypomethylation of *Terra* promoters.

While these epigenetic mechanisms can contribute to telomere length and replicative immortality, a series of genetic mechanisms can lead to the overexpression of the telomerase gene to maintain telomere lengths [596]. This includes (1) point mutations in the promoter that lead to de novo transcription factor binding; (2) linking TERT to active regulatory elements elsewhere in the genome; (3) insertions of viral enhancers upstream of the gene; (4) increased dosage through chromosomal amplification, and (5) the 'alternative lengthening of telomeres' (ALT) pathway in which telomeres are lengthened through homologous recombination, mediated by loss-of-function mutations in the *Atrx* and *Daxx* genes. Overall, activation of the telomerase activity by genetic or epigenetic events can be used to predict chemical-induced carcinogenesis.

## 9.6. Angiogenesis and Thrombospondin-1

The further development of a tumour is dependent on angiogenesis, the development of blood capillaries to support tumour growth. This process is initiated when the tumour reaches 1–2 mm [597,598]. Thrombospondin-1 (THBS1) plays multiple roles in carcinogenesis, one being as an inhibitor of angiogenesis [599], therefore mechanisms that decrease the expression of THBS1 favour angiogenesis. DNA hypermethylation of the *Thbs1* promoter was shown in 50% of brain tumours and reported to regulate its expression in two cell lines (SW1783, T98G) derived from glial brain tumours [600]. The mechanisms leading to the repression of THBS1 appear to be cell type specific, involving the RAS-PI3 KINASE-RHO-ROCK-MYC cascade or the p53 and pRB pathways in transformed epithelial cells or fibroblasts, respectively [601]. Consequently, DNA methylation of the *Thbs1* promoter might provide an epigenetic approach to detect initiation of angiogenesis as a progressive step in carcinogenesis.

## 9.7. The Homeobox (HOX) Genes

The expressions of homeobox (*Hox*) genes are deregulated in cancers where they act as oncogenes by sustaining cell proliferation and controlling cell differentiation [602,603]. Le Boiteux et al. [604] recently shed light on the role of epigenetic marks in regulating the expression of *Hox* genes in brain cancer. The *Hox* genes are divided into four clusters (A, B, C, D) located on different chromosomes from which transcribed transcription factors and non-coding RNAs are normally involved in embryonic development. Le Boiteux et al. [604] report that the DNA sequence of *Hox* gene clusters are hypermethylated and silenced (except for *Hoxd1* and *Hoxd-As1*) in normal brain samples, but among the 57 HOX transcripts (39 sense and 18 antisense) a median of 37 transcripts was deregulated in the HOX clusters from the isocitrate dehydrogenase (IDH) wild type glioma samples. (Note the IDH mutation status is considered in the WHO classification of glioblastoma with the wild type form present in 95% of glioblastoma [605] (IDH in Sections 2.3 and 9.3.2). They observed that a subset of *Hox* gene transcription start sites escapes DNA hypermethylation and was associated with loss of H3K27me3 but enrichment in H3K4me3 and H3K9ac. Among these epigenetic changes, the loss of H3K27me3 along the four HOX clusters best
predicted the cluster transcriptional activities [604]. Epigenetic deregulation of *Hox* clusters
can identify the mechanisms to explain changes in expression of numerous transcripts.

# 10. Transcriptomic Biomarkers and Genome-Wide Signatures

## 10.1. Transcriptomic Biomarkers

Transcriptomic analyses revealing differential expression of epigenetic driver genes might provide epigenetic markers of early carcinogenic events. This approach would be similar to the transcriptomic TGx-DDI [428,606,607] and TGx-HDACi [432] biomarker assays discussed in Section 5.3. In contrast, a transcriptomic response of epigenetic driver genes might be induced only when there is sufficient tissue-specific magnitude of “cellular stress” that limits the availability of cofactors and intermediate metabolites (SAM, acetate, phosphate) necessary for epigenetic machinery functioning. Consequently, duration of exposure, timing of measurement, and tissue/cell type, may differ from those of the TGx-DDI which is based on a unifying cell response. Not all exposed tissues develop cancers and the epigenetic response of a target tissue developing a cancer differs from a non-target tissue [608]. Exposure to chemicals (GTxC or NGTxC) can induce epigenetic effects either as adaptive or as toxic responses, or as early pro-carcinogenic steps [609]. Therefore, given this diversity of mechanisms [610], the discovery at a specific time-course of an epigenetic signature for NGTxC may require investigation in more than one cell type. Overall, a complementary data source of multigene biomarkers based on the epigenetic genes that are drivers of carcinogenic transformation in human cells [179] would be an important weight of evidence component, whether the molecular initial events are due to GTx or NGTx mechanisms, or a combination of both.

## 10.2. Transcription Factors, Enhancers, and Other List of Endpoints for Targeted NGS Assay Development

A large number of genes and their components can potentially be epigenetically reprogrammed and thereby contribute to cell transformation. DNA methylation changes can occur in lists of enhancers, super-enhancers (https://academic.oup.com/nar/article/44/D1/D164/2502575, accessed on 23 August 2021), promoters, associated with changes in gene expression of tumour suppressor genes, oncogenes, imprinted genes (http://geneimprint.com/site/genes-by-species, accessed on 16 July 2021). Consideration must also be given to the transcriptome of stem cell transcription factors (e.g., Yamanaka reprogramming factors OCT4, SOX2, KLF4, cMYC [611], and NANOG) that induce epigenetic remodelling and cellular differentiation. Epithelial-to-mesenchymal transition factors (http://dbemt.bioinfo-minzhao.org/tutorial.cgi, accessed on 23 August 2021), non-coding RNAs (http://rnacentral.org [612,613], accessed on 23 August 2021), and homeobox genes, can contribute to chemical-induced cell transformation and carcinogenesis. Note that in some cases, methylated cancer biomarkers demonstrate increased expression, not reduced expression (SHOX2, OTX1, and ONECUT2 [210]). Recent approaches to generate multiplex assays based on gene expression data [614] could be generated based on RASL-Seq methodology [615], or the TempO-Seq methodology [614,616].

# 11. Technical Considerations for Improving the Regulatory Value of Epigenetic Data

## 11.1. DNA Methylation Assay Robustness and Performance Comparisons

A simple chart, or algorithm, has been developed to facilitate the selection of methods for analyses of DNAm based on needs, costs, and assay robustness [617]. However, the diversity and numbers of techniques to measure epigenetic endpoints have been expanding. A caveat regarding available commercial assays is that when questionable performance is noted, it is often problematic to clearly investigate and correct given proprietary issues. For the purposes of the NGTxC IATA, it will be essential to show transferability and reproducibility of test methods selected.

Different methods targeting the same endpoint were demonstrated to generate different results, for example, effects of phenobarbital on L1 DNAm could not be detected by
methylation-specific PCR (MS-PCR) but were detected with pyrosequencing [201]. A group of 18 laboratories compared a series of 10 clinically relevant methods to measure absolute and relative DNAm abundance in 27 predefined genomic regions as well as global genome DNAm [48]. They found that amplicon bisulphite next-generation sequencing (ABS) and bisulphite pyrosequencing as the best methodologies (pyrosequencing technical correlation within lab (r = 0.996) and across lab (r = 0.98)) [48]. They observed that relative methods (MethyLight, MS-PCR) can report methylation in opposite direction, while methylation-specific melting curve analysis (MS-MCA), and methylation-sensitive high-resolution melting (MS-HRM), detected fewer expected differences in methylation. They also indicated that MS-MCA, MS-HRM, and MS-PCR should only be used for qualitative comparisons of fully methylated regions (not for heterogeneously methylated regions). Only ABS and pyrosequencing were able to cope with low amount or fragmented DNA. High-performance LC-MS found to be the best method for global DNAm, while immune-quantification was not reliable, repetitive DNA elements did not correlate well with expected differences in global DNAm. Others demonstrated the reliability of pyrosequencing and MALDI-MS analyses compared to other techniques (Methylight, MS-PCR, bisulphite-seq) in measuring DNA methylation of the *Gstp1* promoter [490]. NGS permitted to develop methods (e.g., simultaneous targeted methylation sequencing (sTM-Seq)) to measure multiple DNAm sites in multiple samples simultaneously [618] which can be instrumental in screening series of targeted genes for epigenetic disruption.

Beck [212] reviewed studies that compared the performance of 6 methods to measure genome-wide DNA methylation, five are sequencing (seq) based and one is array-based. MethylC-seq, reduced-representation bisulphite sequencing (RRBS), and the Infinium-27K bead-array, all use sodium bisulphite converted DNA, whereas methylated DNA immunoprecipitation sequencing (MeDIPseq), methylated DNA capture by affinity purification (MethylCap-seq) and methylated DNA binding domain sequencing (MBDseq), rely on capture of methylated DNA by a monoclonal antibody or by the recombinant methyl-binding domains of MECP2 or MBD2, respectively. The methods using sodium bisulphite conversion have high resolution (1 bp) while the capture methods have low resolution (≥100 bp). The overall concordance across methods was high (84–100%), and it was concluded that all evaluated methods are capable of producing accurate high-content data [212].

The performances of "rapid multiplexed" reduced-representation bisulphite sequencing (rmRRBS) performed on the Illumina HiSeq2500 sequencing platform were compared to analyses on the Illumina Infinium BeadChip Human Methylation450 (450K), and MethylationEPIC (850K) platform [211]. Preferences were expressed for NGS-based rmRRBS, but consideration should be given to experimental needs; (1) rmRRBS requires only 60–200 ng DNA, whereas the Infinium BeadChip arrays require 500 ng–1 µg and whole-genome bisulphite sequencing (WGBS) requires 3 µg of DNA; (2) rmRRBS allows for genotyping and the detection of single nucleotide polymorphisms (SNP), which incidentally were found to influence allele-specific methylation of the *H19* imprinted region [211]; (3) rmRRBS offers greater flexibility with the potential to investigate sites that are not interrogated by Infinium arrays; (4) rmRRBS covers more CpG loci and greater abundance of "CpG shores" and "open sea regions". However, with abundant DNA samples, Infinium arrays were reported to provide consistency in both genomic coverage and methylation estimates. In contrast to array-based approaches, many methodological details can induce NGS inconsistencies in methylation analyses across experiments [211]. These include number of reads covering each CpG sites, consistency of the enzymatic digestion and fragment size, fragment ligation to adapters, bisulphite conversion, PCR amplification, successful sequence alignment, and DNA input quantity and quality. Finally, the bioinformatics step, including CpG site grouping of various size rather than investigating individual CpG sites will influence the results based on the heterogeneity of the methylation levels across sites and density of CpG sites. Examples of other methods exist, including EpiTYPER® (a mass spectrometry-based bisulphite sequencing method that enables region-specific quantitative DNA methylation analysis; evaluated by [48]), Illumina's VeraCode GoldenGate customiz-
able assays, and newer techniques (PacBio, single-molecule real-time (SMRT) sequencing, nanopore sequencing), all of which are still to be thoroughly evaluated, together with recent bioinformatics applications [619].

Overall, pyrosequencing and NGS-based techniques offer versatile approaches for investigating DNA methylation, with due consideration of the technical differences and limitations within and across studies. Different parameters (such as “window” selection process, CpG density, methylation threshold cut-off; see for example the video from Dr. Simon Andrew at Babraham Institute: https://www.youtube.com/watch?v=MWZ8dBpOpkM, accessed on 19 July 2021) can be selected to identify differently methylated regions (DMR) that can influence the interpretation of methylation calls as biological observations. Of note is the supplementary Data 1 section in [48], which provides validated DNA methylation protocols.

## 11.2. Methodology to Distinguish 5mC from 5hmC

5mC, its oxidised derivative 5hmC, and histone post-translational modifications contribute to the regulation of chromatin structure and function [620,621]. The various methods that use sodium bisulphite to identify CpG methylation sites have been instrumental in revealing the importance of DNAm, but these methodologies cannot distinguish 5mC from 5hmC. The following discussion summarises the importance of 5hmC and the methodologies to separate both forms of cytosines.

As previously mentioned, the TET enzymes oxidize 5mC into 5hmC which is a demethylation intermediate and an epigenetic mark with functional roles [94]. As an intermediate, 5hmC can lead to active and passive demethylation. The active process involves further oxidation by the TET enzymes of 5hmC to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) that are then actively removed by thymine-DNA glycosylase and the base excision repair mechanism. 5hmC also leads to passive demethylation because 5hmC is not recognized by UHRF1, which prevents the formation of the DNMT1/UHRF1 complex on the hemimethylated DNA, and therefore the complex cannot restore 5mC on the nascent strand at the replication fork. 5hmC has biological roles, despite the fact that it is approximately 10-fold less abundant than 5mC. It is more abundant in the brain and embryonic stem cells than in other tissues and is preferentially distributed in euchromatin at functional regions of the genome in enhancers, promoters and gene bodies [94]. Its roles are mediated by many binding proteins “readers”. These include, (1) the protein HMCES (5-hydroxymethylcytosine binding, embryonic stem cell-specific protein), which is a DNA lesion protein that senses abasic sites in single-stranded DNA at the replication fork and shields the lesion from error-prone DNA repair processing, thus preserving genome integrity [622]. (2) The methyl binding protein MBD3 (methyl binding domain-3) preferentially binds 5hmC and attracts the DNA demethylase TET1 required for the maintenance of 5hmC, or alternatively MBD3 binds the NuRD complex regulating gene expression in cell differentiation and cancers [623]. (3) UHRF2 (Ubiquitin-like, containing PHD and RING finger domains 2), which preferentially binds 5hmC, but unlike UHRF1, UHRF2 also binds 5mC and contributes to methylated H3K9. UHRF2 has ubiquitination but also oncogenic or tumour suppressive roles depending on cancer cell types [623]. (4) As a final example, 5-methylcyctosine binding protein 2 (MeCP2) is abundant in post-mitotic neurons where it contributes to gene repression and brain functions [624]. MeCP2 has a high affinity for 5mC but low affinity for 5hmC, consequently the accumulation of 5hmC in transcribed genes replaces the high-affinity 5mC binding sites for MeCP2, thus decreasing occupancy and repressive effects of MeCP2 [625]. Overall, 5hmC is an important epigenetic mark with roles in neuronal physiology, cell differentiation, and diseases of aging including cancer [625]. However, it is only relatively recent that techniques were developed to distinguish sites with 5mC from 5hmC [626], and the number of technical approaches is still increasing [627].

Considering (1) that 5mC and 5hmC have different biological roles, (2) that the activities of the dioxygenase TET enzymes are sensitive to numerous metabolic factors (Vit-C,
pH, intermediate metabolites) possibly modified by chemical exposures, (3) that TET1 and TET2 are downregulated in many cancers, and (4) that there is generally a global loss of 5hmC in cancers [94], perhaps adequate measurements of both 5mC and 5hmC could generate early markers of chemically induced carcinogenesis. A number of techniques have been developed to examine the position of modified C within DNA sequences, and Liu and collaborators [627] present a summary of these approaches. All techniques involve the comparison of the known in silico DNA sequence to the sequences read in two different aliquots of the same samples; one to infer the 5mC position the other the 5hmC. One approach involves analyzing an aliquot treated with only sodium bisulphite (BS) and comparing the results from a second aliquot receiving two treatments: an oxidizing treatment with $KRuO_4$ that transforms 5hmC to 5fC and then a SB treatment (referred to as oxBS technique [626]). In the first aliquot, the BS treatment converts cytosines (C) to uracil (U) but leaves 5mC and 5hmC intact, therefore following amplification of BS-treated samples C, 5mC and 5hmC become T, C, and U. In the second aliquot, following the $KRuO_4$, BS, and amplification treatment, the original C, 5mC and 5hmC, become C, 5mC and 5fC, then U, 5mC, and U, and finally T, C, and U, respectively. The comparison of the final sequence in both aliquots permits to infer the position of all modified C. Another approach protects 5hmC by glucosylation prior to TET enzyme mediated oxidation and then followed by BS treatment (TAB-Seq; [628]). A combination of these techniques but replacing SB by a borane reduction treatment generates the technique called TET-assisted pyridine borane sequencing (TAPS), or TAPSβ when a β-glucosyltransferase step is included to protect the 5hmC, or finally, CAPS when the initial oxidation is performed chemically ($KRuO_4$) [627]. The borane reduction, as a replacement to BS, does not affect C and avoids issues of a harsh BS reaction and variable conversion efficiencies of C. Tierling et al. [629] recently compared the performance of commercially available BS kits, and proposed a revised BS protocol to discriminate 5mC from 5hmC. A combination of the above techniques can infer the position of C, 5mC and 5hmC, but given the low amount of 5hmC and rates of false positive calls (although limited) among approaches, the NGS strategy requires a higher number of reads to assess abundance of 5hmC [626]. The increasing number of investigations of modified C using alternative techniques (LC/MS-MS [470], biological settings [469–471], and multiplex methodology [618], should collectively facilitate the reduction in ambiguity in the interpretation of the role of DNA methylation in carcinogenic processes.

### 11.3. Extrapolation of Experimental Models to Human

In the absence of clear human data, experimental animal and cell culture models are essential in toxicology but a perfect model does not exist, and a careful examination of species differences and in vitro confounding factors must be considered. The case of the rodent cancer bioassay highlights difficulties in extrapolating findings to humans [630], considering, for example PPARα and CAR-mediated carcinogenicity ([341,631,632], further explored in Esteban et al. (in preparation)). Animal experimental models offer several advantages; they can support suspected epidemiological adverse outcomes and can generate awareness of potential health issues. Despite improvement of in vitro models, and of in silico tools such as quantitative structure–activity relationship (QSAR) and toxicokinetic models [633,634], currently, chemical hazard assessment practices are still dependent on in vivo data to confirm adverse effects of mixtures, data-poor chemicals, and metabolites, to generate guiding kinetic parameters for dose–response differences on absorption, distribution, metabolism, and excretion of substances, and identify tissues where substances accumulate and become toxicity-targets. Moreover, developmental and long-term effects cannot be predicted by current in vitro or in silico methods [206,635]. On the toxico-epigenetic discovery side, models from bacteria to mammals have been instrumental in elucidating epigenetic mechanisms [31,636,637], such as the requirement of H3K9 methylation to direct *de novo* DNA methylation by DNMT3A/B [33,224]. Data derived from *C. elegans* and *Daphnia* [638], and a growing library on zebrafish (*Danio rerio*) [639] are informative and represent reductions and refinements in the uses of animals. However, interspecies epigenetic differences, in-
cluding the distribution of epigenetic marks and repeated sequences that exert regulatory roles, differ across genomes of human, non-human primates [225,226], and other laboratory models [227], impeding direct extrapolation of findings [3]. Pending suitable alternatives, while in vivo models remain fundamental in characterizing chemical hazards for complex NGTxC endpoints, the relevance of in vivo and in vitro toxicology and epigenetic models to human health risk assessment requires careful species-specific mechanistic considerations.

### 11.4. *Considerations in the Extrapolation from In Vitro Cultures, Cell Lines and Cell Types*

The use of cell lines has many limitations in supporting early epigenetic events in carcinogenesis. Particularly as many commonly used cell lines are already at an advanced transformation stage, and additional chemical-induced transformations may be dependent on pre-existing genetic and epigenetic anomalies. Even when a cell line originates from a biopsy of "normal" tissues adjacent to tumours, these cells are epigenetically abnormal [640] (see Figure 6). Cell lines have overcome senescence and have proliferated in vitro as a result of induced or spontaneous transformation involving genetic and epigenetic changes [641]. Many cell lines undergo genetic drift that may induce interlaboratory variability [642], and reproducibility issues through time. Epigenetics and gene expression profiles differ depending on the gene initially mutated that drives the early carcinogenic process [232], hence these activated pathways may affect cellular responses when testing chemicals. Consequently, data related to effects of chemical exposure using cell lines may reflect anomalies in this cell type and may therefore not be relevant to chemical-induced mechanisms in normal cells. Despite these many limitations, cell lines, whether "normal" in origin, or transformed, remain useful tools for the investigation of specific questions associated with later stages of carcinogenesis, especially when they can be used to independently confirm results.

Horvath et al. [643] indicate that in vitro cultures are under artificial conditions frequently characterised by high glucose, abundant growth factors, high oxygen partial pressure (20% O₂ in incubator air compared to physiological level of 1% to 14% dependent on tissues), mechanical resistance exerted by culture flasks (plastics, glass), inappropriate extracellular matrix, absence of multi-cell type interactions, large concentrations of serum frequently from a species different from the investigated cells, and with unknown constituents such as ascorbic acid (Vit-C) concentrations. Human cells are unable to synthesize Vit-C and dioxygenase enzymes are dependent on Vit-C as cofactor. Some of these enzymes include TET1-3 (oxidizing 5mC to 5hmC to 5fC to 5caC), JMJC domain-containing histone demethylases, prolyl and asparagine hydroxylases regulating HIF1 stability and normoxic condition [97]. A concentration-response study of Vit-C in culture medium with the human colon cancer HCT116 cell line showed re-expression of p21 (CDKN1A) at 50 µM, increased abundance of 5hmC at concentrations up to 100 µM with no effects on 5mC by LC/MS-MS, and cytotoxicity above 1 mM [644]. Vit-C median human plasma concentration of 53 µM (range of 16–89 µM) has been reported [645]. Amounts of Vit-C present in human and rat tissues can vary greatly (≈2 and 8-fold more abundant in rat plasma and testes, respectively, than in man [646]). The epigenetic system can change rapidly under in vitro conditions. For example, mouse embryonic fibroblasts (historically named "embryonic" but in fact derived from fetus) show erasure of global 5hmC within three days (partially rescued by addition of Vit-C), followed by gains of 5mC in specific gene promoters within seven days of culture initiation [100]. Whilst optimal culture conditions may differ between species, they need to be optimised for specific, reproducible epigenetic investigations, both for initial screening and subsequent mechanistic studies.

Human primary 3D culture model systems that are closer to the human tissue environment are encouraged [643], but the successful development of systems for regulatory purposes is more difficult than standard in vitro tests. The comparison of 2D and 3D in vitro cultures suggests that there is less of a gap between 3D data and in vivo conditions than with usual 2D systems [647]. Cell line data showed that the expression of the lncRNA HOTAIR in breast cancer cells (MDA-MB-231 and Hs578T cell lines) is lower in 2D than
in 3D systems, and that the 3D culture method generates a different isoform [648]. The use of pooled human primary cells from multiple donors to create 3D cultures that are metabolically competent with normal karyotype and intact DNA repair system is gaining momentum to replace cell line analyses. For example, using such a hepatocyte 3D-culture system, 5-day exposure to valproic acid at a non-toxic but steatotic 15 mM concentration, followed by three-day withdrawal, led to persistent differentially methylated regions in 31 genes [649], with persistent disruption of energy metabolism [650]. The culture of pools from multiple donors of human primary cells might be a promising avenue for epigenetic testing, provided data reproducibility.

Finally, some epigenetic changes are associated with phases of the cell cycle, or with proliferation rate and seeding density, and may bias effects due to chemical exposure. The H3K27me3 mark (but with less impact on H3K9me3 or on H3K14ac) in breast cancer subtypes can be influenced by the proliferation rate, as determined by correlation with proliferation rate marker Ki67, or by the analyses of cell samples synchronized at the G2-M versus G1-S phase of the cell cycle [34]. Seeding HepG2 cells at low density (21,000 to 42,000 cells/cm²) compared to high density (63,000 cells/cm²) led to a transient 10% drop in methylation of *AluYb8* after 120–144 h of culture, but such an effect was gene and cell type dependent [176]. It is therefore important to document cell culture conditions and to remain vigilant in the interpretation of data.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10.3390/ijms222010969/s1.

**Author Contributions:** Conceptualization, D.D., P.V., A.J., M.C.A., N.E., and M.N.J.; writing—original draft, D.D.; writing—reviewing and editing, D.D., M.N.J., and P.V.; editing, A.J., M.C.A., N.E.; figures, D.D., N.E., and M.N.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors are grateful to Robyn Hocking (Reference Librarian, Health Library, Health Canada and Public Health Agency of Canada), to Jochen vom Brocke for discussion of assays, and to Tim Gant, Jianli Jiao, and Jane J. Pappas for carefully reviewing this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Disclaimer:** The statements, opinions and conclusions expressed herein do not necessarily represent the views or positions of the author's institutions.

**Abbreviations**

<table><tr><td>5aCdR</td><td>5-aza-2'-deoxycytidine.</td></tr><tr><td>5hmC</td><td>5-hydroxymethylcytosine.</td></tr><tr><td>5hmC</td><td>hydroxymethyl cytosine.</td></tr><tr><td>5mC</td><td>5-methylcytosine.</td></tr><tr><td>AE</td><td>adverse event.</td></tr><tr><td>ALT</td><td>alternative lengthening of telomeres pathway.</td></tr><tr><td>Alu</td><td>arthrobacter luteus DNA repeats.</td></tr><tr><td>AML</td><td>acute myeloid leukemia.</td></tr><tr><td>AOP</td><td>adverse outcome pathway.</td></tr><tr><td>APC</td><td>adenomatous polyposis coli protein.</td></tr><tr><td>B[a]P</td><td>benzo[a]pyrene.</td></tr><tr><td>BCAT1</td><td>branched-chain-amino-acid aminotransferase, cytosolic.</td></tr><tr><td>BEAS-2B</td><td>human bronchial epithelial SV-40 immortalised cell line.</td></tr><tr><td>BMD</td><td>benchmark-doses.</td></tr><tr><td>BMP3</td><td>bone morphogenic protein 3.</td></tr><tr><td>BPA</td><td>bisphenol-A.</td></tr><tr><td>CAN</td><td>copy number alterations.</td></tr><tr><td>CAR</td><td>constitutive androstane receptor.</td></tr></table>
<table>
  <tr>
    <td>cGAS</td>
    <td>cyclic GMP-AMP synthase.</td>
  </tr>
  <tr>
    <td>CTA</td>
    <td>cell transformation assay.</td>
  </tr>
  <tr>
    <td>CTAG</td>
    <td>cancer-testis antigens.</td>
  </tr>
  <tr>
    <td>CTCF</td>
    <td>CCCTC-binding factor.</td>
  </tr>
  <tr>
    <td>DNAm</td>
    <td>DNA methylation.</td>
  </tr>
  <tr>
    <td>DNMT1, 2, 3a, 3b, 3L</td>
    <td>DNA methyltransferases.</td>
  </tr>
  <tr>
    <td>Ecad</td>
    <td>E-cadherin.</td>
  </tr>
  <tr>
    <td>EMT</td>
    <td>epithelial to mesenchymal transition.</td>
  </tr>
  <tr>
    <td>GGD</td>
    <td>global genome DNA.</td>
  </tr>
  <tr>
    <td>GGDHo</td>
    <td>global genome DNA hypomethylation.</td>
  </tr>
  <tr>
    <td>GGDm</td>
    <td>global genome DNA methylation.</td>
  </tr>
  <tr>
    <td>GSH</td>
    <td>glutathione.</td>
  </tr>
  <tr>
    <td>GSTP1</td>
    <td>glutathione S-transferase Pi1.</td>
  </tr>
  <tr>
    <td>GTxC</td>
    <td>genotoxic carcinogens.</td>
  </tr>
  <tr>
    <td>HA</td>
    <td>hazard assessment.</td>
  </tr>
  <tr>
    <td>HaCaT</td>
    <td>human keratinocyte immortalized cell line.</td>
  </tr>
  <tr>
    <td>HBE</td>
    <td>human bronchial epithelial cell line.</td>
  </tr>
  <tr>
    <td>HCA</td>
    <td>high-content analysis.</td>
  </tr>
  <tr>
    <td>HDAC</td>
    <td>histone deacetylase.</td>
  </tr>
  <tr>
    <td>HDM</td>
    <td>histone demethylase.</td>
  </tr>
  <tr>
    <td>hESC</td>
    <td>human embryonic stem cells.</td>
  </tr>
  <tr>
    <td>HMEC</td>
    <td>human mammary epithelial cells.</td>
  </tr>
  <tr>
    <td>HMT</td>
    <td>histone methyltransferase.</td>
  </tr>
  <tr>
    <td>HPTM</td>
    <td>histone post-translational modifications.</td>
  </tr>
  <tr>
    <td>HTS</td>
    <td>high-throughput screening.</td>
  </tr>
  <tr>
    <td>HUC1</td>
    <td>human urothelial immortalised cell line.</td>
  </tr>
  <tr>
    <td>IARC</td>
    <td>International Agency for the Research on Cancer.</td>
  </tr>
  <tr>
    <td>IATA</td>
    <td>integrated approach to the testing and assessment.</td>
  </tr>
  <tr>
    <td>IDH</td>
    <td>isocitrate dehydrogenase.</td>
  </tr>
  <tr>
    <td>IKZF1</td>
    <td>DNA-binding protein Ikaros.</td>
  </tr>
  <tr>
    <td>KDM</td>
    <td>lysine demethylase.</td>
  </tr>
  <tr>
    <td>KE</td>
    <td>key event.</td>
  </tr>
  <tr>
    <td>KER</td>
    <td>key event relationship.</td>
  </tr>
  <tr>
    <td>KMT</td>
    <td>lysine methyltransferase.</td>
  </tr>
  <tr>
    <td>L-02</td>
    <td>human hepatic cell line.</td>
  </tr>
  <tr>
    <td>Line</td>
    <td>long interspersed nuclear element.</td>
  </tr>
  <tr>
    <td>LncRNA</td>
    <td>long non-coding RNA.</td>
  </tr>
  <tr>
    <td>LOAELs</td>
    <td>lowest-observable-adverse-effect levels.</td>
  </tr>
  <tr>
    <td>LTR</td>
    <td>long terminal repeat.</td>
  </tr>
  <tr>
    <td>MCF7</td>
    <td>breast cancer cell line #7 from the Michigan Cancer Foundation.</td>
  </tr>
  <tr>
    <td>mESC</td>
    <td>mouse embryonic stem cells.</td>
  </tr>
  <tr>
    <td>MGMT</td>
    <td>methylated DNA-protein-cysteine methyltransferase.</td>
  </tr>
  <tr>
    <td>MHC</td>
    <td>major histocompatibility complex.</td>
  </tr>
  <tr>
    <td>MIE</td>
    <td>molecular initiating event.</td>
  </tr>
  <tr>
    <td>MS</td>
    <td>mass spectrometry.</td>
  </tr>
  <tr>
    <td>NAM</td>
    <td>novel approach methodologies.</td>
  </tr>
  <tr>
    <td>ncRNA</td>
    <td>non-coding RNA.</td>
  </tr>
  <tr>
    <td>NDRG4</td>
    <td>N-myc downregulated gene 4.</td>
  </tr>
  <tr>
    <td>NGS</td>
    <td>next-generation sequencing.</td>
  </tr>
  <tr>
    <td>NGTxC</td>
    <td>non-genotoxic carcinogens.</td>
  </tr>
  <tr>
    <td>NOAELs</td>
    <td>no-observable-adverse-effect levels.</td>
  </tr>
  <tr>
    <td>OECD</td>
    <td>Organization for Economic Cooperation and Development.</td>
  </tr>
  <tr>
    <td>OGG1</td>
    <td>8-oxoguanine glycosylase.</td>
  </tr>
  <tr>
    <td>ONECUT2</td>
    <td>one cut domain family member 2.</td>
  </tr>
  <tr>
    <td>OTX1</td>
    <td>homeobox protein OTX1.</td>
  </tr>
  <tr>
    <td>PBMC</td>
    <td>peripheral blood mononuclear cells.</td>
  </tr>
  <tr>
    <td>PCB</td>
    <td>polychlorinated biphenyls.</td>
  </tr>
  <tr>
    <td>PCNA</td>
    <td>proliferative cell nuclear antigen.</td>
  </tr>
  <tr>
    <td>PHD</td>
    <td>prolyl hydroxylase dioxygenase.</td>
  </tr>
</table>
<table><tr><td>PND</td><td>postnatal day.</td></tr><tr><td>PoD</td><td>point of departure.</td></tr><tr><td>RA</td><td>risk assessment.</td></tr><tr><td>RASSF1</td><td>ras association domain-containing protein 1.</td></tr><tr><td>RCB</td><td>two-year rodent cancer bioassay.</td></tr><tr><td>RRBS</td><td>reduced-representation bisulphite sequencing.</td></tr><tr><td>SAHA</td><td>suberoylanilide hydroxamic acid.</td></tr><tr><td>SAM</td><td>s-adenosylmethionine.</td></tr><tr><td>09-Sep</td><td>septin-9.</td></tr><tr><td>SHOX2</td><td>short stature homeobox protein 2.</td></tr><tr><td>Sine</td><td>short interspersed nuclear element.</td></tr><tr><td>SUV39H1</td><td>suppressor of variegation 3–9 homolog 1; catalyzes H3K9me3.</td></tr><tr><td>TCA</td><td>tricarboxylic acid cycle.</td></tr><tr><td>TCDD</td><td>2,3,7,8-tetrachlorodibenzo-p-dioxin.</td></tr><tr><td>TDG</td><td>thymine-DNA glycosylase.</td></tr><tr><td>TET-1, -2, -3</td><td>ten eleven translocation enzymes.</td></tr><tr><td>TSG</td><td>tumour suppressor gene.</td></tr><tr><td>TSS</td><td>transcription start sites.</td></tr><tr><td>TWIST1</td><td>twist-related protein 1.</td></tr><tr><td>UHRF1</td><td>ubiquitin-like PHD and RING finger domain 1.</td></tr><tr><td>WGBS</td><td>whole-genome bisulphite sequencing.</td></tr><tr><td>WoE</td><td>weight of evidence.</td></tr></table>

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