Introduction
Bisphenol A diglycidyl ether (BADGE) is synthesized through the reaction of one mole of bisphenol A (BPA) and two moles of epichlorohydrin [1]. It has 100 times lower estrogenic potency compared to BPA, which is a well-known endocrine disruptor [2]. It is widely used in epoxy resin production, especially for protective coatings and civil engineering applications [1]. Food and drink cans coated with BADGE-based epoxy resins can leach BADGE into consumables, resulting in human exposure. Based on consumer exposure data, the calculated annual intake of BADGE from canned food is 6 -10 μg/person/day [3]. Given the estimated daily intake for a 60-kg adult of 0.098-0.16 mg/kg of body weight, the higher value is used as the estimated daily intake level.
BADGE, when intact in vitro [4-6] and in vivo systems [7,8], is rapidly hydrolyzed to form the corresponding bis-diol, which is not observed in BPA. When 14C-BADGE was orally administered to mice, it was metabolized and excreted mostly (being over 88%) within two days [7]. In a phase I in vitro liver metabolism study, BADGE was rapidly hydrolyzed and resulting in the formation of hydrolysis derivatives [9], and further hydroxylation and carboxylation, which have been detected in urine [10-12], in serum [13-15], and in adipose tissue [15]. Because BADGE is unlikely to be transformed into BPA, it has not been widely considered a potential inducer of systemic toxicity.
However, BADGE (10 μM) induced minimal proliferation (<2 fold) in the E-Screen assay [16, 17], and BADGE (200 μM) induced morphological changes in Caco-2 cells and cellular detachment from the substrate [18]. In addition, the hydrolysis and chlorohydroxy products of BADGE exhibited proliferation in the T47D breast cancer cell line from 10-14 to 10-4 M, without binding to estrogen receptor alpha (ERα) [19]. Additionally, up-regulation of luteinizing hormone and disruption of steroidogenic enzyme expression were observed in a mouse testicular cell line after exposure to the hydrolysis derivative of BADGE [20]. Orally administered BADGE at 750 mg/kg/day may reduce sperm count and motility while increasing sperm abnormalities in adult male rats [21]. A decrease of spermatid numbers in the seminiferous tubules and relatively lower testosterone levels [22], and an increase of anogenital distance (AGD) and a down-regulation of clusterin mRNA expression in epididymis [23] in adult male offspring were also observed in perinatal exposure to BADGE at 375 mg/kg/day. A significant dose-dependent decrease in clusterin mRNA and protein levels was observed in male offspring perinatally exposed to BADGE at 50, 200, and 400 mg/kg/day [24]. In accordance with laboratory studies, serum BADGE and its hydrolysis derivative levels in adult male workers were positively associated with follicle stimulating hormones and negatively associated with estradiol [14]. These findings suggest that BADGE may disrupt steroidogenesis, potentially affecting gonadal development, although direct evidence is limited.
Epigenetic changes induced by environmental factors have been considered a sensitive marker of environmental effects [25]. DNA methylation is an essential epigenetic modification in which a methyl group (-CH3) is enzymatically attached to cytosine residues, primarily within CpG dinucleotides, playing a key role in gene expression regulation [26]. Studies have shown that the alterations in DNA methylation are associated with abnormal male reproduction and infertility [27, 28]. Although it has been suggested that BADGE may cause male reproductive damage [20-22] and be associated with steroid hormones in adult men [14], information regarding its potential impact on testicular development remains limited. Examination of epigenetics changes in the gonad may provide insights into the disruption of steroidogenesis caused by BADGE exposure.
In addition, Danio rerio, commonly known as zebrafish, is a suitable model for assessing epigenetic alterations induced by environmental chemical exposure, given that its genome exhibits approximately 70% homology to human genome [29]. The use of zebrafish offers several advantages over other model organisms, including ease of husbandry and maintenance, high reproductive output, external fertilization, and a short life cycle with rapid generation times [30]. It is suggested that examination of DNA methylation in zebrafish may help uncover the underlying mechanism in male-mediated reproductive defects resulting from BADGE exposure.
Therefore, this study evaluated the epigenetic effects induced by BADGE using whole genome bisulfide sequencing (WGBS) on male zebrafish gonads. To our knowledge, this is the first study to use WGBS to examine the disruption of steroidogenesis in zebrafish exposed to BADGE. Understanding these epigenetic effects could provide valuable insights into BADGE's role as an endocrine disruptor.
Materials and Methods
Chemicals
BADGE (CAS No. 1675-54-3) and dimethyl sulfoxide (DMSO, CAS No. 67-68-5) were purchased from Sigma-Aldrich (St. Louis, MO, USA). DMSO (0.001% v/v) was used as the solvent.
Fish Husbandry and BADGE Exposure
Adult male zebrafish (3–4 months old) were obtained from a commercial vendor (Gwansune System, Osan, Korea). Fish were maintained at 26 ± 1 °C under a 14:10 h light-dark cycle and fed Aquatox Fish Diet flakes (Zeigler, PA, USA) twice per day, supplemented with brine shrimp. After acclimation, fish were divided into three replicates (five fish per 2L beaker). BADGE was dissolved in DMSO to prepare a stock working solution. The final concentrations of DMSO and BADGE (10 μM, 3.4 mg/L) did not exceed 0.1%, ensuring no effect on any endpoint measured. The exposure concentration of BADGE was determined based on a tolerance daily intake of 0.15 mg/kg bw/day, which was established by European Food Safety Authority [31], and a dose did not induce cytotoxic effects in Caco-2 cells for 24 h [18]. Half of the exposure media were renewed daily, and water quality (dissolved oxygen, pH, conductivity, and temperature) was routinely monitored. No mortality occurred during the exposure. After 21 days of exposure, males (n=15/group) were anesthetized and dissected on ice in each group. The experimental duration was determined based on the OECD test guideline 230 [32]. Testes were immediately separated and rinsed in saline (0.9% NaCl), and immediately frozen in liquid nitrogen. The collected tissues were kept individually in the tubes and kept in -80 °C until being further processed. The experiments were carried out by Central Bio Inc., and all procedures were approved by the Institutional Animal Care and Use Committee (IACUC), in compliance with institutional guidelines and regulations.
Whole-Genome Bisulfite Sequencing
Genomic DNA was extracted from frozen gonads (n=10/group) using a Qiagen DNA isolation kit (Cat. No. 69504) according to the manufacturer’s protocol. DNA yield was measured using a Nanodrop spectrophotometer (Thermo Electron Corporation, USA) and stored at -80°C until being further processed. Samples were pooled to minimize biological variation and due to the limited amount of DNA available.
Bisulfite conversion using the pooled DNA samples was performed to convert unmethylated cytosines to uracils. The Adapter step is a highly efficient, template-independent reaction that performs simultaneous tailing and ligation of a truncated adapter to the 3' ends. In the Extension step, truncated adapter 1 is incorporated through a primer extension reaction. The Ligation step selectively adds truncated adapter 2 to the bottom strand only. During the Indexing PCR step, full-length adapters are incorporated for single or dual indexing, while also increasing yield. Bead-based clean-ups remove oligonucleotides and small fragments, and adjust the enzymatic buffer composition. Sequencing was conducted on a HiSeq X platform (Macrogen, Korea).
Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis
The functions “enrich GO” and “enrich KEGG” were applied with default parameters to identify significantly enriched GO terms and KEGG pathways, with a threshold of p < 0.05. For GO enrichment, differentially expressed genes between control group and BADGE treated group, as well as down-regulated genes with hypermethylated regions (hyper-down) and up-regulated genes with hypomethylated regions (hypo-up), were used as input data.
Quantitative Real-Time PCR Analysis
Total RNA was extracted from gonads (n=5/group) using a Qiagen RNA isolation kit (Cat. No. 74004) and reverse-transcribed to cDNA using PrimeScript™ RT Master Mix (Takara, Japan; RR036A). The mRNA expression of identified genes and steroidogenic enzymes was analyzed via qRT-PCR. Relative expression levels were determined using the ΔΔCt method, normalized to the average of glyceraldehyde-3-phosphate dehydrogenase (gapdh), β-actin, and ribosomal protein lateral stalk subunit 0 (rplp0). Primer sequences are listed in Table 1.
Results and Discussion
Following a 21-day exposure to BADGE, WGBS analysis was performed to assess DNA methylation in the gonads of zebrafish,. Approximately 55 Gb (ranging from 54 to 57 Gb) of raw data were generated from both the control and BADGE-treated groups. Bisulfite conversion was 99.7% for all samples. About 97% of bases had quality scores greater than Q20, and 93% scored greater than Q30. The mean mapping rate was about 52%. Methylation in the CpG context was 79.6% in the control group and 78.7% in the BADGE-treated group. Cytosines in CHG and CHH contexts were approximately 0.5% methylated, respectively. Of these CpG methylated sites, 2,673 sites were differentially methylated (DM) between the control and BADGE-treated groups, with 1,311 DM hypomethylated sites and 1,362 DM hypermethylated sites. Total DM and hyper/hypomethylation varied among chromosomes (Figure 1). Chromosome 24 had the fewest hyper/hypomethylated sites (29 regions and 26 regions, respectively), while chromosome 4 had the greatest number of hyper/hypomethylated sites (94 regions and 110 regions, respectively).
Of the 2,673 DM sites, 1,533 were annotated to zebrafish genes. The functions and pathway enrichment of the DM sites were evaluated using the DAVID website (https://david.ncifcrf.gov/list.jsp). GO analysis further classified the DM sites into cellular components, molecular functions, and biological processes. Six KEGG pathways were enriched (p < 0.05 and q < 0.01) for genes containing DM sites: the phosphatidylinositol signaling system (17 genes), inositol phosphate metabolism (14 genes), cardiac muscle contraction (13 genes), insulin resistance (16 genes), insulin signaling pathway (19 genes), and FoxO signaling pathway (18 genes) (Table 2 and Table S1). Among them, phosphatidylinositol signaling system, insulin resistance, insulin signaling pathway, and forkhead box O (FoxO) signaling pathway are related with phosphatidylinositol 3-kinases (PI3K)/protein kinase B (AKT) pathway, which is the most crucial signaling pathways in cellular metabolism [35]. In the testis, the PI3K/AKT pathway plays a role in spermatogenesis and Sertoli cell development [36-38]. Studies on the mechanism of BADGE-induced PI3K/AKT dysregulation with testicular toxicity are still limited. However, exposure to BPA (20 mg/kg every 2 days for 2 months), a precursor of BADGE, induced germ cell loss, and decreased sperm viability, motility, and density [39]. They suggested that BPA-induced testicular toxicity might result from the dysregulation of a DPY30-mediated H3K4me3 epigenetic modification, which regulates the PI3K/AKT pathway. In addition, although unrelated to reproduction, exposure to BADGE and BPA interfered with the PI3K/AKT pathway and resulted in the reduction of vincristine cytotoxicity in lymphoblstic leukemia cells [40]. Based on these previous studies, BADGE-induced testicular disorders might be associated with the alterations in the PI3K/AKT pathway.
Based on KEGG pathway enrichment analysis, the most frequently identified genes, including phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma (pik3cg) and phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit beta (pik3cb), were selected. Additionally, phosphoinositide-3-kinase regulatory subunit 3b (gamma) (pik3r3b), insulin receptor substrate 1 (irs1), and insulin receptor A (irsra) were selected due to their common presence in the insulin signaling pathway and insulin resistance. Among the selected genes, differentially methylated sites with a methylation change exceeding 5% and a p-value less than 0.05 were annotated within 1,000 bp. In the BADGE-treated group, irs1 and pik3cg exhibited hypomethylation, while irsra, pik3r3b, and pik3cb showed hypermethylation compared to the control group (Figure 2).
The mRNA expression of the selected genes was examined to evaluate the relationship between DNA methylation and gene expression following BADGE exposure in the zebrafish gonad (Figure 3). The irs1 mRNA expression was significantly increased in BADGE treated group compared to the control (p=0.023), which may be associated with the hypomethylation of its respective CpG sites. Irs1 is the first identified member of the insulin receptor substrates family and is found in various tissues, including muscle, adipose tissue and liver, which are insulin-responsive tissues [41]. In ovarian granulosa cells, phosphorylation of IRS1 plays a role the transmitting follicle-stimulating hormone (FSH) to active PI3K [42]. Specifically, in the presence of both FSH and insulin-like growth factor 1 (IGF1), protein kinase A activates protein phosphatase 1 (PP1), which dephosphorylates Ser/Thr residues on IRS1, thereby facilitating IRS1 phosphorylation by IGF1 receptor. The phosphorylated IRS1 then enhances the PI3K/AKT pathway, driving a synergistic gene response to IGF1 and FSH [43]. Although statistical significance was not observed, the up-regulation of pik3cg, which is involved in the PI3K pathway, might be induced by the increase in irs1, resulting in the activation of the PI3K/AKT pathway. Nevertheless, adult male rats with orally administered BPA (0.005 – 500 μg/kg bw/day) showed a dose-dependent decrease of insulin signaling molecules including insulin receptor, IRS-1 and PI-3 kinase in testis [44]. The inconsistent results with our study might be attributed to the differences in the chemical used and exposure dosage. Nevertheless, it seems that BADGE could disrupt insulin signaling transduction and thereby induce male reproductive disorders, and in that process, IRS1 might be a key mediator of steroidogenesis in the testis.
In addition, in contrast to the irs1 and pik3cg, the mRNA expression of hyper-methylated genes such as insra, pik3r3b, and pik3cb was increased in BADGE treated group compared to the control group (Figure 3). In general, DNA methylation influences gene expression by recruiting proteins that promote gene repression or by preventing transcription factors from binding to DNA [28]. However, in some cases, genes that undergo hypermethylation may retain their expression levels or even become upregulated [45, 46]. One possible explanation is that certain transcription factors preferentially bind to methylated CpG sites, leading to gene activation rather than suppression. Genes with unmethylated CpGs in the promoter region may not produce functional transcripts [47-50]. Additionally, local methylation patterns can play a more significant role than overall methylation status. Even if a genomic region is hypermethylated, localized demethylation at critical regulatory sites can allow gene transcription to continue [51, 52]. Furthermore, the density and distribution of methylation influence gene expression. A low level of methylation in promoter regions may still permit transcription machinery to bind, and sparsely methylated genes can be activated by enhancers despite the presence of global methylation [51, 53, 54]. It has also been shown that certain genes with unmethylated CpG islands in the promoter regions are unable to produce functional transcripts because RNA polymerase II is not recruited [55]. Thus, the simultaneous increase in both gene expression and CpG methylation levels is uncommon but can occur. Since these complexities suggested that a toxicant like BADGE might influence gene regulation in unexpected ways, it is needed to evaluate the specific conditions under which hypermethylated genes induced by BADGE exposure may still exhibit increased mRNA expression.
To estimate the influence of BADGE on steroidogenesis, the mRNA expression of steroidogenic enzymes including steroidogenic acute regulatory (star), and cytochrome P450 family 11 subfamily A member 1 (cyp11a1), cytochrome P450 family 17 subfamily A member 1 (cyp17a1), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2 (hsd3b2), and hydroxysteroid 17-beta dehydrogenase 1 (hsd17b1), as well as estrogen receptor 1 (esr1), were measured. The mRNA expression of star, cyp11a1, and cyp17a1 was significantly increased in BADGE-treated group compared to the control (p=0.032, p=0.018, and p=0.020, respectively [Figure 4]). Despite statistical significance not being observed, BADGE also increased the mRNA expression of hsd3b2 and hsd17b1 compared to the control group. These results are consistent with the previous study that the hydrolyzed form of BADGE (1 μM) increased steroidogenesis enzymes such as Star and 3β-HSD in mouse testicular Leydig cells [20]. In addition, BPA (1 μM) increased the expression of CYP11A1 and CYP19 in mouse Leydig cells [56]. They suggested that the decrease of sex-hormone ratio (testosterone/estradiol) following BPA exposure compared to the control might be associated with the up-regulation of CYP enzymes in vitro and in vivo. However, A decrease in luteinizing hormone receptor and HSD17B3 expression was observed in Leydig cells isolated from adult male rats after maternal exposure to BPA (2.5 and 25 μg/kg/day) [57]. Although environmental disrupting chemicals can directly affect sex hormone balance through modulating the expression of steroidogenic enzymes, there is evidence that the activation of insulin receptor might influence steroid hormone synthesis through enhancing StAR protein expression in human ovarian cells [58].
StAR has an important role in the transportation of cholesterol from outer to inner mitochondrial membrane, and regulates the production of steroid hormone biosynthesis [58, 59]. In addition, CYP enzymes are located on the endoplasmic reticulum network, and play a role in catalyzes the final stage of steroid biosynthesis [60]. Among the CYP genes, CYP11A1 and CYP17A1 genes play a key role in steroid synthesis and production, cholesterol, and drug metabolism. CYP11A1 gene is mainly involved in androgen metabolism and cholesterol side-chain cleavage, and CYP17A1 gene is involved in the biosynthesis of androgens as a 17-20 lyase in the gonads. In addition, HSD3B2 has a role in the conversion from pregnenolone to progesterone and from dehydroepiandrosterone to androstenedione through dehydrogenase and isomerase reactions [61]. HSD17B1 plays a crucial role in estrogen and testosterone synthesis [62]. Although there is evidence that several upstream regions participate in the regulation of reproductive development, it seems that BADGE directly affects the activity of steroidogenic enzymes.
It is thought that BADGE has estrogenic properties that can induce biological actions. In this regard, the level of ESR1 in the male testis was examined. The expression of esr1 in the zebrafish gonad tended to increase following BADGE exposure compared to the control; however, statistical significance was not observed (p=0.132; Figure 4). Although previous in vitro study suggested that estrogenic activity following exposure to BADGE hydrolysis and chlorohydroxy derivatives (10-14 to 10-4 M) was not associated with ERα in the T47D breast cancer cell line [19]. However, Maternal exposure to BPA (2.5 and 25 μg/kg/day) significantly increased ESR1 protein levels in Leydig cells isolated from adult male rats [57]. Because the elevation of ESRs following BPA exposure might interfere with the tissue differentiation in prepubertal Leydig cells [63], BADGE might modulate the ESRs and thereby induce reproductive defects.
Conclusions
Studies have reported that BADGE had no effect on reproductive or endocrine toxicity [64, 65]; however, it has also been shown to exhibit estrogenic effects in vitro [16, 17, 19, 20] and in vivo studies [21-24]. Based on our results, BADGE may directly disrupt steroidogenesis, while DNA methylation of genes related to the insulin signaling molecules, including irs1, could also involve in the reproductive development of adult zebrafish gonads. However, due to the limitations of small sample size, it is hard to establish a definitive relationship between BADGE exposure and epigenetic changes in steroidogenesis. Further studies are needed to elucidate whether BADGE-induced DNA methylation in zebrafish gonads influences the steroidogenesis by interfering with the PI3K/AKT pathway.
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