It has now been more than a half-century since Roy Hertz in 1958 first proposed the notion that certain chemicals, particularly those used in livestock feed at the time, could contaminate food sources and bioaccumulate in humans mimicking the activity of hormones (Gassner et al., 1958). As the number of potential chemicals that could exert these effects has grown, so too has the interest in this area to the point that this is now a major topic of active research. The dangers of many endocrine disrupting chemicals (EDCs) studied to date have largely been investigated utilizing animal models. A primary example is the multi-phased Toxicant Exposures and Responses by Genomic and Epigenomic Regulators of Transcription (TaRGET) program established to determine the contribution of environmental exposures to disease pathogenesis as a function of epigenome perturbation in mouse models (Wang et al., 2018). While the use of animal models has been quite informative for determining the various systemic or organ/tissue-specific disease-related effects associated with exposure to different EDCs, the molecular mechanisms underlying these phenomena remain poorly understood. Thus, questions persist regarding (i) the mechanisms by which exposure of cells to EDCs results in the initial formation of epimutations defined here as any change in epigenomic programming, (ii) potential cell-type specific differential susceptibility to epimutagenesis induced by different EDCs, (iii) the potential involvement of relevant hormone receptors and/or genomic hormone response elements in EDC-induced disruption of the epigenome, and (iv) the ability of an EDC-induced epimutated state to persist inter- or transgenerationally despite generational epigenetic reprogramming (Diaz-Castillo et al., 2019).

Questions about molecular mechanisms responsible for EDC-exposure based disruption of normal epigenetic programming have been difficult to interpret in whole animal models due, in part, to the inherent complexity of the intact animal including potential interactions among multiple different organs, tissues, and cell types at the paracrine, metabolic and/or systemic levels. To circumvent this challenge, we opted to expose homogeneous populations of specific cell types in culture to doses of the EDC, Bisphenol S (BPS), that are below the maximum safe limit previously set by the EPA for exposure of humans to the similar estrogen mimetic, Bisphenol A (BPA). In this way, we hoped to learn more about the manner in which direct exposure to a specific EDC induces epimutations and differential gene expression in different individual cell types including somatic cells, pluripotent cells, and germ cells. This approach was designed to distinguish the direct effects of an EDC exposure in vitro from indirect effects that may accrue as the result of an EDC exposure on one cell type inducing a secondary effect on a neighboring or related cell type in vivo.

Previous in vitro studies have demonstrated direct susceptibility of cultured cell types to EDC-induced epimutagenesis, however, those studies were focused on immortalized cancer cell lines that do not necessarily model key cell types involved in normal initial incursion or subsequent intragenerational propagation or inter- or transgenerational transmission of environmentally induced epimutations in vivo (Deb et al., 2016; Goodman et al., 2014; Huang et al., 2019; Karaman and Ozden, 2019; Senyildiz et al., 2017). We chose to examine direct exposure of three key types of cells relevant to environmental exposures and subsequent propagation and transmission of induced epimutations in vivo – somatic cell types known to be responsive to endocrine signaling (Sertoli cells and granulosa cells), pluripotent cells mimicking the preimplantation embryo (iPSCs), and germline cells (PGCLCs).

While there have been reports that have clearly indicated the ability of EDCs to disrupt classical endocrine signaling (Henley and Korach, 2010; Kelce et al., 1995; Swedenborg et al., 2009; vom Saal and Hughes, 2005; You et al., 1998), there exist other reports indicating EDCs can also disrupt non-classical endocrine signaling via nuclear receptors (Ozgyin et al., 2015; Myers et al., 2011), G protein-coupled receptors (Thomas and Dong, 2006), and calcium channel signaling (Brenker et al., 2018). This has further complicated our understanding of the mechanism(s) by which EDC exposure initially induces epimutations, and has particularly confounded insight into the susceptibility of germ cells to EDC-induced epimutagenesis given that germ cells are reported to lack expression of classical endocrine receptors (Meccariello et al., 2014). This is relevant to the effort to understand mechanisms underlying transgenerational epigenetic inheritance of EDC-induced epimutations (Anway et al., 2006; Guerrero-Bosagna et al., 2012; Nilsson et al., 2008) which clearly implicates transmission via the germ line.

The concept of germline transmission of EDC-induced epimutations is further confounded by the known epigenetic reprogramming events that occur in the preimplantation embryo and developing fetal and neonatal germ line. Thus, regardless of how EDC-induced epimutations become initially manifest in the germ line, it is not clear how they subsequently persist and are transmitted inter- or transgenerationally given the large portion of the epigenome that undergoes genome-wide erasure and resetting of epigenetic programming during each generation (Cantone and Fisher, 2013; Lee et al., 2014; Santos et al., 2002; Sanz et al., 2010). In vitro cell culture systems afford the opportunity to focus on individual cell types independently, including somatic cells which are likely initially exposed to most environmental disruptive influences, pluripotent cells which represent the preimplantation embryo in which the first major reprogramming event occurs during normal development, and early germline cells where the second major reprogramming event is initiated in primordial germ cells (PGCs). Importantly, in addition to the potential to study each of these cell types independently, it is possible to induce transitions in cell fate in vitro that model those that occur during normal development, thereby recapitulating normal reprogramming events in a way that can facilitate high resolution studies of the fate of EDC-induced epimutations once they have been induced in any of these cell types.

Here we describe our study of the relative susceptibility to induction of epimutations by direct exposure of four different cell types maintained in culture – somatic Sertoli and granulosa cells, pluripotent iPSCs, and germline PGCLCs – to BPS, followed by our analysis of the fate of epimutations initially induced in mouse iPSCs that are then induced to transition into PGCLCs. We found that there are cell-type specific differences in susceptibility to epimutagenesis and associated dysregulation of gene expression patterns following exposure of these different cell types to a similar dose of BPS below the maximum safe limit established by the EPA for exposure of human cells to BPA (Ribeiro et al., 2019). We further found that BPS induction of epimutations in both pluripotent and somatic cell types that express relevant estrogen receptors (ERs), as well as in germ cells which do not express ERs, suggests disruption of both canonical and non-canonical endocrine signaling. Most interestingly, we found that when iPSCs exposed to BPS were then induced to undergo a major transition in cell fate and related epigenetic reprogramming to form PGCLCs, a similar prevalence of epimutations and DEGs was retained, but, surprisingly, very few (<10%) specific epimutations or DEGs were conserved during this process. This suggests that the initial EDC exposure induces disruption of the chromatin landscape that subsequent epigenetic reprogramming is unable to fully restore. Thus, reprogramming during a major cell fate transition appears to correct many of the initially induced epimutations, but also appears to induce many de novo epimutations, and this imbalance may persist across multiple generations which may contribute to continued transgenerational epigenetic inheritance of EDC-induced phenotypes during succeeding generations.

While nearly 20 years of research on the effects of exposure of live animals to various EDCs and other environmental disruptive influences has clearly established the potential to perturb the epigenome in ways that can dysregulate normal gene expression patterns and predispose the development of disease states, the molecular mechanisms underlying these phenomena have remained largely undefined. Thus, the manner in which environmental exposures introduce biochemical changes in epigenetic programming, how such disruptions can be propagated within a tissue or throughout the soma of an exposed individual or enter that individual’s germ line, or how these disruptions predispose disease states in that individual, or how a resulting prevalence of epimutations can be transmitted to multiple subsequent generations despite generational epigenetic reprogramming, are all mechanistic questions that have remained unanswered despite more than 5000 publications on this topic since the early 2000s. The vast majority of those publications have described experiments in animal models – typically rodents. While informative, such live animal studies require months to years to complete, are cost-, labor-, and animal-intensive, and have not yielded substantial insight into the molecular mechanisms underlying the deleterious effects of environmentally induced epimutations.

In vitro model systems have proven to be very useful tools for deciphering molecular mechanisms related to normal development and homeostasis, or disruptions of those processes predisposing onset of disease. Examples include cell culture systems used to study developmental processes such as X-chromosome inactivation in mammals (Almeida et al., 2017; Patrat et al., 2009) or early embryogenesis (Bao et al., 2022; Lau et al., 2022), or to elucidate the cellular and molecular etiology of many different diseases based on the now popular ‘disease-in-a-dish’ approach (Davaapil et al., 2020; Song et al., 2023). While in vitro cell culture systems are, by nature, devoid of a majority of the physiological complexities present within the intact organism in vivo, this provides an actual advantage in that it facilitates deconvolution of those complexities while unequivocally focusing on only certain, specific aspects or cell types. However, this ultimately warrants validation of results discerned from studies of in vitro models to ensure they also reflect functions ongoing in the more complex and heterogeneous environment of the intact animal in vivo.

We assembled an in vitro system in which we combined the three cell types normally involved in (i) the initial exposure to disruptive environmental effects (somatic cells), (ii) the first phase of generational epigenetic reprogramming in the preimplantation embryo (pluripotent cells), and (iii) the second phase of generational reprogramming in the developing germ line (germ cells). This system allowed us to gain insights into multiple mechanistic aspects of environmentally induced epimutagenesis including the initial induction of epimutations by exposure of cells to the EDC, BPS, cell-type specific differences in susceptibility to BPS-induced epimutagenesis, factors that appear to contribute to that differential susceptibility, and the mechanism by which an epimutated state may persist across multiple generations despite generational reprogramming.

Here we have shown that exposure of homogeneous populations of cells maintained in culture to an EDC such as BPS can induce epimutagenesis and dysregulation of gene expression, and that this occurs in a partially additive dose-dependent manner, although the quantitative effect appears to plateau at higher doses. This afforded us the opportunity to test the relative effects of direct exposure of different specific cell types to a similar dose of BPS with no confounding effects imposed by interactions with other cell types or systemic effects of the sort that normally occur in the in vivo context. We found that somatic, pluripotent, and germ cell types do indeed show differences in direct susceptibility to induction of epimutations following exposure to BPS, both in terms of the quantity of epimutations induced and qualities of the resulting epimutations such as a predominance of hyper- versus hypomethylation, and the genomic locations where such epimutations tend to occur. Our results suggest that these differences reflect distinctions in the normal status of each cell type at the time of exposure. Thus, somatic, pluripotent, and germ cell types differ in the normal status of genome-wide patterns of accessible versus inaccessible chromatin, global DNA methylation, and expression of relevant endocrine receptors. Interestingly, our data suggest that all of these parameters can influence cell-type specific susceptibility to induction of epimutations by exposure to BPS, and that different combinations of these variables ultimately determine the quantity and quality of epimutations induced by exposure to BPS. We note that while our exposure dose of 1 µM of BPS was below that deemed safe by the EPA for exposure of humans to the similar EDC, BPA, that same dose may exert greater effects when used to expose cells in culture in the absence of any sort of mitigating metabolic effects that may accrue in intact animals or humans. Indeed, the potential to quantify the epimutagenic effects of different doses of an EDC on different cell types as shown in our study could be used in future studies to assess the relative effects of a specific dose of an EDC on a specific cell type when that cell type is exposed either in a homogeneous culture or within an intact animal.

Endogenous fetal germline cells (e.g. PGCs and prospermatogonia in males) normally display lower levels of global DNA methylation and higher levels of chromatin accessibility than pluripotent or somatic cell types (Hajkova, 2011), yet PGCLCs developed fewer epimutations following exposure to BPS than the other cell types. This appears to be explained by the fact that germline cells do not express either estrogen receptor, whereas pluripotent and somatic cell types express one or both of the two estrogen receptors, apparently rendering them more susceptible to induction of epimutations following exposure to the estrogen mimetic, BPS. This is consistent with the notion that EDCs impose disruptive effects by interfering with canonical endocrine signaling pathways, and that notion is further supported by our observation that, in the cell types that express one or both estrogen receptors, we observed a prevalence of BPS-induced epimutations in genomic regions containing nearby EREs, whereas in PGCLCs, which do not express either estrogen receptor, we did not observe a strong correlation between the locations of BPS-induced epimutations and genomic EREs.

Nevertheless, we did observe induction of epimutations in PGCLCs exposed to BPS, suggesting that while cell types expressing relevant endocrine receptors may display elevated susceptibility to EDC-induced epimutagenesis presumably based on disruption of canonical endocrine signaling, this is not the only mechanism by which EDCs can induce epimutations. Indeed, our results support the suggestion that there may be at least two types of epimutations induced by exposure to EDCs – one that is independent of canonical endocrine signaling and so occurs in all cell types regardless of expression of relevant endocrine receptors or the genomic location of relevant HREs, and another that does involve disruption of canonical endocrine signaling and so is elevated in cell types expressing relevant endocrine receptors and predisposes the induction of epimutations in genomic regions near relevant HREs. Beyond this, a comparison of the quantities of BPS-induced epimutations in pluripotent and somatic cell types, which both express one or both estrogen receptors, but which differ with respect to elevated genome-wide chromatin accessibility in pluripotent cells compared to much more limited chromatin accessibility in somatic cell types (Cantone and Fisher, 2013; Santos et al., 2002), suggests that chromatin accessibility also contributes to susceptibility to EDC-induced epimutagenesis.

One of our most intriguing findings was that, in addition to estrogen-receptor expressing somatic and pluripotent cell types developing more epimutations than non-estrogen-receptor expressing germ cells in response to exposure to BPS, the occurrence of epimutations in the former (somatic and pluripotent) cell types was predominantly in enhancer regions whereas that in the latter (germ) cell type was predominantly in gene promoters. As noted above, there was a significant increase in the occurrence of BPS-induced epimutations near EREs in somatic and pluripotent cell types compared to germ cells. A genome-wide analysis confirmed that ERE half-sites occur more frequently in enhancer regions than in promoter regions (Table 3 and Figure 3—figure supplement 4) supporting our suggestion that the higher prevalence of BPS-induced epimutations we observed in enhancer regions in somatic and pluripotent cell types was due to disruption of canonical hormone signaling.

Our finding that PGCLCs, which do not express endocrine receptors, still accrued epimutations following exposure to BPS indicates that in addition to inducing epimutations by disrupting canonical endocrine signaling pathways, exposure to EDCs can induce epimutations via mechanisms that do not involve disruption of such pathways. This notion is further supported by the occurrence of BPS-induced epimutations in genomic regions that do not include nearby EREs, even though a prevalence of the epimutations induced in PGCLCs were located in gene promoters. Pathway analysis of the genes in which promoter-region epimutations were induced by exposure of PGCLCs to BPS suggests an apparent endocrine-signaling independent group of genes that can become disrupted by exposure to an EDC via non-canonical signaling which may occur through the PI3K/AKT pathway via G-protein-coupled receptors. While we observed expression of a number of G-protein-coupled receptors known to bind to either 17β-estradiol or BPA, further studies will be required to determine if any of these contribute to susceptibility to BPS epimutagenesis.

As expected, disruption of normal epigenetic programming induced by exposure of cells to BPS also led to dysregulation of gene expression. Thus, RNA-seq analysis detected differentially expressed genes in all cell types exposed to BPS. Surprisingly, the greatest number of DEGs was detected in PGCLCs which was the cell type that developed the lowest overall number of epimutations in response to exposure to BPS. However, PGCLCs showed a higher prevalence of BPS-induced epimutations in gene promoter regions than was observed in any of the other cell types, so it appears that promoter-region epimutations impose the most direct impact on gene expression. As noted above, fetal germ cells normally display the lowest level of global DNA methylation of any cell type at any developmental stage. This is characteristic of the epigenetic ground state that accrues uniquely in developing germ cells (Hajkova, 2011), and is believed to reflect a generally more accessible chromatin state genome-wide in germ cells than in other cell types. Thus, it may be that the epigenetic ground state in developing germ cells renders this cell type uniquely susceptible to epimutagenesis in gene promoter regions, thereby predisposing a high level of dysregulation of gene expression in response to exposure to an EDC.

Finally, our in vitro system allowed us to follow the fate of BPS-induced epimutations through a major transition in cell fate from pluripotent to germline cells during which a portion of the normal generational germline epigenetic reprogramming process is known to occur (Kurimoto and Saitou, 2018). Thus, when we induced iPSCs previously exposed to 1 μM BPS to differentiate into PGCLCs, we found that a substantial prevalence of epimutations persisted during this transition. However, very few of the specific DMCs or the associated DEGs detected in the BPS-exposed iPSCs persisted in the subsequently derived PGCLCs. Specifically, only 3.7% of DMCs and 8.4% of DEGs were conserved between the BPS-exposed iPSCs and the subsequently derived PGCLCs. This suggests that exposure of cells to EDCs has the potential to disrupt epigenetic programming in a way that then interferes with subsequent generational reprogramming that normally accompanies either germline to pluripotent or pluripotent to germline transitions in cell fate. Recent in vivo studies have suggested that exposure of gestating female mice to the EDC tributyltin results in disruption not simply of the pattern of epigenetic modifications but also of the underlying chromatin landscape (Chamorro-García et al., 2021; Chang et al., 2022). In this context, our results are consistent with the suggestion that exposure of cells to EDCs can disrupt the underlying chromatin landscape (e.g. the pattern of A and B chromatin compartments) such that when the ‘normal’ reprogramming machinery then acts on this disrupted landscape it corrects many of the originally induced epimutations, but simultaneously induces many novel epimutations de novo. This would predispose ongoing abnormalities in the underlying chromatin landscape that would, in turn, lead to the recurring correction of many existing epimutations in concert with the genesis of many novel epimutations during each subsequent generation.

We note that, with the exception of our analysis of granulosa cells, all of our studies were carried out in ‘male’ XY-bearing cells. It remains possible that XX-bearing cells might differ from XY-bearing cells in the way(s) in which they respond to exposure to an EDC. However, the similarity we observed between responses of XX granulosa cells and XY Sertoli cells suggest this may not be the case – at least in cell types in which dosage compensation has been established by X-chromosome inactivation.

Taken together, our results demonstrate the utility of an in vitro cell culture approach for pursuing molecular mechanisms underlying environmentally induced disruption of normal epigenetic programming manifest as the formation of epimutations and dysregulated gene expression. This approach clearly affords unique potential to reveal mechanisms responsible for the initial induction of epimutations in response to direct exposure of cells to disruptive effects such as EDCs, as well as offering potential means to elucidate mechanisms by which environmentally induced epimutations are then propagated within the exposed individual and then to that individual’s descendants. Knowledge of these mechanisms will afford the best opportunity to understand how these defects may, in the future, be better diagnosed, treated, and/or prevented. With the ever-expanding catalog of potentially hazardous man-made compounds permeating our environment, it is increasingly important that we recognize the potential dangers such compounds may impose and maximize our ability to protect ourselves from those dangers.

Methylation beadchip, EM-seq, and RNA-seq data discussed in this publication have been uploaded and can be obtained from the SRA BioProject PRJNA1026145 and GEO accession GSE252723.

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Author details

1. Jake D Lehle

   Department of Neuroscience, Developmental and Regenerative Biology, The University of Texas at San Antonio, San Antonio, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0833-7068

2. Yu-Huey Lin

   Department of Neuroscience, Developmental and Regenerative Biology, The University of Texas at San Antonio, San Antonio, United States

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0009-0003-1321-0680

3. Amanda Gomez

   Department of Neuroscience, Developmental and Regenerative Biology, The University of Texas at San Antonio, San Antonio, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

4. Laura Chavez

   Department of Neuroscience, Developmental and Regenerative Biology, The University of Texas at San Antonio, San Antonio, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. John R McCarrey

   Department of Neuroscience, Developmental and Regenerative Biology, The University of Texas at San Antonio, San Antonio, United States

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Investigation, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   john.mccarrey@utsa.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5784-9318

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