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.

Early life exposure to endocrine disrupting chemicals (EDCs) has been suggested to adversely affect reproductive health in humans and wildlife. Here, we characterize endocrine and adverse effects on the reproductive system after juvenile exposure to propiconazole (PROP) or imazalil (IMZ), two common azole fungicides with complex endocrine modes of action. Using the frog Xenopus tropicalis, two short-term (2-weeks) studies were conducted. I: Juveniles (2 weeks post metamorphosis (PM)) were exposed to 0, 17 or 178 µg PROP/L. II: Juveniles (6 weeks PM) were exposed to 0, 1, 12 or 154 µg IMZ/L. Histological analysis of the gonads revealed an increase in the number of dark spermatogonial stem cells (SSCs)/testis area, and in the ratio secondary spermatogonia: dark SSCs were increased in all IMZ groups compared to control. Key genes in gametogenesis, retinoic acid and sex steroid pathways were also analysed in the gonads. Testicular levels of 3β-hsd, ddx4 were increased and cyp19 and id4 levels were decreased in the IMZ groups. In PROP exposed males, increased testicular aldh1a2 levels were detected, but no histological effects observed. Although no effects on ovarian histology were detected, ovarian levels of esr1, rsbn1 were increased in PROP groups, and esr1 levels were decreased in IMZ groups. In conclusion, juvenile azole exposure disrupted testicular expression of key genes in retinoic acid (PROP) and sex steroid pathways and in gametogenesis (IMZ). Our results further show that exposure to environmental concentrations of IMZ disrupted spermatogenesis in the juvenile testis, which is a cause for concern as it may lead to impaired fertility. Testicular levels of id4, ddx4 and the id4:ddx4 ratio were associated with the number of dark SSCs and secondary spermatogonia suggesting that they may serve as a molecular markers for disrupted spermatogenesis.

The herbicide linuron can cause endocrine disrupting effects in Xenopus tropicalis frogs, including offspring that were never exposed to the contaminant. The mechanisms by which these effects are transmitted across generations need to be further investigated. Here, we examined transgenerational alterations of brain and testis DNA methylation profiles paternally inherited from grandfathers developmentally exposed to an environmentally relevant concentration of linuron. Reduced representation bisulfite sequencing (RRBS) revealed numerous differentially methylated regions (DMRs) in brain (3060 DMRs) and testis (2551 DMRs) of the adult male F2 generation. Key genes in the brain involved in somatotropic (igfbp4) and thyrotropic signaling (dio1 and tg) were differentially methylated and correlated with phenotypical alterations in body size, weight, hind limb length and plasma glucose levels, indicating that these methylation changes could be potential mediators of the transgenerational effects of linuron. Testis DMRs were found in genes essential for spermatogenesis, meiosis and germ cell development (piwil1, spo11 and tdrd9) and their methylation levels were correlated with the number of germ cells nests per seminiferous tubule, an endpoint of disrupted spermatogenesis. DMRs were also identified in several genes central for the machinery that regulates the epigenetic landscape including DNA methylation (dnmt3a and mbd2) and histone acetylation (hdac8, ep300, elp3, kat5 and kat14), which may at least partly drive the linuron-induced transgenerational effects. The results from this genome-wide DNA methylation profiling contribute to better understanding of potential transgenerational epigenetic inheritance mechanisms in amphibians.

The unsustainable use of manmade chemicals poses significant threats to biodiversity and human health. Emerging evidence highlights the potential of certain chemicals to cause transgenerational impacts on metabolic health. Here, we investigate male transmitted epigenetic transgenerational effects of the anti-androgenic herbicide linuron in the pancreas of Xenopus tropicalis frogs, and their association with metabolic phenotypes. Reduced representation bisulfite sequencing (RRBS) was used to assess genome-wide DNA methylation patterns in the pancreas of adult male F2 generation ancestrally exposed to environmentally relevant linuron levels (44 ± 4.7 μg/L). We identified 1117 differentially methylated regions (DMRs) distributed across the X. tropicalis genome, revealing potential regulatory mechanisms underlying metabolic disturbances. DMRs were identified in genes crucial for pancreatic function, including calcium signalling (clstn2, cacna1d and cadps2), genes associated with type 2 diabetes (tcf7l2 and adcy5) and a biomarker for pancreatic ductal adenocarcinoma (plec). Correlation analysis revealed associations between DNA methylation levels in these genes and metabolic phenotypes, indicating epigenetic regulation of glucose metabolism. Moreover, differential methylation in genes related to histone modifications suggests alterations in the epigenetic machinery. These findings underscore the long-term consequences of environmental contamination on pancreatic function and raise concerns about the health risks associated with transgenerational effects of pesticides.

The extensive use of agricultural pesticides has caused widespread environmental contamination, raising concerns about their adverse effects. The fungicide imazalil is known to cause endocrine-disrupting hepatotoxic effects in vertebrates, but its epigenetic mechanisms remain understudied. In this multiomics study, we investigate the epigenetic and transcriptomic effects of imazalil on the liver of Xenopus tropicalis juveniles (aged 6.5 weeks post-metamorphosis) after exposure to an environmentally relevant concentration of imazalil (12.3 µg/L) for two weeks. Thousands of differentially methylated cytosines (DMCs) were observed in both sexes, while significant gene expression changes were primarily detected in males. Gene Set Enrichment Analysis (GSEA) highlighted disruption in energy metabolism in both sexes, while sex-specific effects included downregulation of cell cycle and chromatin organization pathways in males and upregulation of immune response pathways in females. Integrative analysis identified strong correlations between methylation levels in key CpGs and the expression of multiple genes involved in cell cycle regulation, suggesting that epigenetic mechanisms may mediate the hepatotoxic effects of imazalil. This study underscores the toxicogenomic effects of imazalil exposure on hepatic function, highlighting potential consequences for human health given the genetic similarity between X. tropicalis and humans, as well as the ecotoxicological impacts on vulnerable amphibian populations.

Amphibian populations face significant threats of extinction, including environmental pollution. This multiomics study investigated the toxicogenomic effects of the endocrine-disrupting fungicide propiconazole on juvenile Xenopus tropicalis by combining RNA sequencing (RNA-seq) and genome-wide DNA methylation (RRBS) analysis to examine transcriptomic and epigenomic changes in brain and liver after 16 days exposure to an environmentally relevant concentration (17.3 µg/L). RNA-seq revealed sex-specific gene expression changes in the brain. Gene set enrichment and over-representation analyses identified key affected pathways crucial to brain function, including nervous system development, glutamate signaling, voltage-gated calcium channels and GnRH signaling. Interestingly, the expression direction in these pathways differed between males and females. Genome-wide DNA methylation analysis revealed significant sex- and tissue-specific alterations. Integrative network analysis identified key CpG sites strongly correlated with the expression of genes critical for brain function, suggesting a role for DNA methylation in mediating propiconazole's neurotoxic effects. Notably, specific CpGs were strongly correlated with multiple genes with related functions. These findings highlight the potential of propiconazole to disrupt vital biological processes, with implications for both amphibian and human health due to the significant genomic similarities between X. tropicalis and mammals.