Transgenerational Hazards Evade Traditional Safety Thresholds: <i>ryr1b</i> Hypermethylation Mediates Azoxystrobin-Induced Transgenerational Developmental Toxicity in Zebrafish ( <i>Danio rerio</i> ) at Subthreshold Exposure

Accumulating evidence has demonstrated that exposure to environmental pollutants causes abnormalities in unexposed offspring through epigenetic transgenerational inheritance. In this study, subthreshold azoxystrobin exposure (1, 10, 100 μg/L, 4 days) caused no observable abnormalities in F0 embryos, whereas unexposed F1 and F2 generations exhibited developmental abnormalities, with effects independent of parental gender, demonstrating azoxystrobin exhibited transgenerational developmental toxicity at subthreshold exposure. Moreover, reduced protein levels of DNA methyltransferases and altered genomic DNA methylation patterns were detected in F1 and F2 embryos. Further research found that the <i>ryr1b</i> gene exhibited concurrent suppression of gene expression and DNA hypermethylation in F1 and F2 generations, with identical DNA methylation modification sites located in intron regions, indicating that the DNA hypermethylation of <i>ryr1b</i> was stably inherited transgenerationally and mediated epigenetic regulation of gene expression. Through constructing the interaction networks of differentially expressed genes with consistent expression trends across two generations, it was found <i>ryr1b</i> occupied the central position in the gene interaction network, and triggered cascade reactions through calcium signaling pathways. These pathways modulated excitation-contraction coupling (ECC)-associated gene expression in cardiomyocytes, ultimately driving cardiac functional deficits and developmental abnormalities in offspring embryos. This study provides critical insights into epigenetic mechanisms underlying pollutant-induced multigenerational toxicity.