Bisphenol A diglycidyl ether-induced DNA methylation abnormalities may disrupt testis development in adult male zebrafish

Bisphenol A diglycidyl ether (BADGE) is commonly used to stabilize products synthesized from epichlorohydrin and bisphenol A. Although recent studies suggest that BADGE may adversely affect the male reproductive system, its underlying mechanisms remain unclear. This study investigates the impact of BADGE exposure on steroidogenesis via DNA methylation changes in adult zebrafish gonads. Adult male zebrafish were exposed to BADGE (10 μM) for 21 days (n = 15 per group). Genomic DNA and mRNA were extracted from the testes. Whole-genome bisulfite sequencing revealed differentially methylated (DM) regions, and the expression levels of genes associated with these DM sites and steroidogenesis were analyzed using quantitative reverse transcription polymerase chain reaction. Among the 2,673 DM sites (1,311 hypomethylated and 1,362 hypermethylated), 1,533 were successfully annotated. Pathway enrichment analysis showed that DM sites were associated with the phosphatidylinositol signaling system, inositol phosphate metabolism, cardiac muscle contraction, insulin resistance, insulin signaling, and the forkhead box O signaling pathway. Notably, the gene expression of insulin receptor substrate 1 (irs1) was significantly upregulated in the BADGE-treated group. In addition, the mRNA expression of steroidogenic enzymes, including steroidogenic acute regulatory protein, cytochrome P450 family 17 subfamily A member 1, and cytochrome P450 family 11 subfamily A member 1, was significantly increased in BADGE-treated group compared to the control group. These findings suggest that while BADGE may directly influence steroidogenesis, DNA methylation of insulin signaling-related molecules, including irs1, may also contribute to this process.