Pesticide pollution is a global environmental concern. Exposure during critical developmental periods can disrupt epigenetic programming, potentially causing long-term and transgenerational effects—even in unexposed offspring. Despite growing interest in environmental epigenetics, key questions remain about how toxicant-induced DNA methylation changes affect gene expression and phenotype. This thesis aimed to contribute to our understanding of pesticide effects on genome-wide DNA methylation, gene expression, and phenotype, using a multiomics approach combining Reduced Representation Bisulfite Sequencing (RRBS) and RNA sequencing (RNA-seq). Xenopus tropicalis was used as a model species, providing insights with implications for human health—due to its high genetic synteny with mammals—and amphibian populations, which are experiencing global declines driven by pollution and other anthropogenic pressures.

Early developmental exposure to the anti-androgenic herbicide linuron induced transgenerational epigenetic alterations in the unexposed second-offspring generation (F2)—transmitted through the paternal germline (Papers I-II). Alterations in DNA methylation were observed in several regions (differentially methylated regions, DMRs) in the brain, testis, and pancreas of male F2 frogs. Phenotypic changes in reproductive and metabolic traits correlated with methylation patterns in functionally relevant genes. In the brain, DMRs in genes involved in growth (igfbp4) and thyroid signaling (dio1, tg) correlated with body size, weight, and hind limb length. In the testis, methylation of germ cell development genes piwil1, spo11, and tdrd9 correlated with germ cell nest counts. In the pancreas, altered methylation of the type 2 diabetes biomarker adcy5 correlated with plasma glucose levels. DMRs were also found in regulators of DNA methylation, including dnmt3a. These findings suggest that linuron-induced DNA methylation alterations may mediate the observed transgenerational phenotypic effects.

Toxicogenomic effects of pesticide exposure during peripubertal periods critical for sexual maturation were also investigated. The azole fungicides propiconazole and imazalil caused significant alterations in the juvenile’s methylome and transcriptome (Papers III-IV). RRBS revealed widespread DNA methylation changes across all analyzed tissues. Propiconazole exposure induced stronger gene expression responses in the female brain, with gene set enrichment analysis (GSEA) showing sex-specific disruptions in nervous system development and gonadotropin-releasing hormone signaling. Imazalil more strongly affected the male liver, causing disruptions in cell cycle and chromatin organization pathways, while immune response pathways were disturbed in females, and metabolic pathways were affected in both sexes. Integration of methylome and transcriptome data using Spearman’s correlations revealed networks of key CpG sites (cytosine followed by guanine) whose methylation levels correlated with expression of functionally related genes, suggesting a mechanistic link between epigenetic disruption and altered gene expression. However, functional validation is needed to corroborate these findings. This thesis deepens our understanding of how environmentally relevant pesticide exposures induce epigenetic changes that may shape molecular and phenotypic traits. It also highlights the value of multiomics and toxicogenomic approaches in environmental risk assessment.