Crosstalk regulation between gut microbiota and DNA methylation: Sex-specific effects of intestinal metabolic activities induced by prothioconazole and prothioconazole-desthio in zebrafish

The large-scale production and widespread use of the triazole fungicide prothioconazole (PTC) have led to its frequent detection in environmental samples. However, the effects of PTC and its metabolite prothioconazole-desthio (dPTC) on the aquatic organism intestinal metabolic activities remain insufficiently studied. In this study, adult male and female zebrafish were exposed to PTC and dPTC at environmental-related doses (0.5 and 10 μg/L) for 30 days to investigate their sex-specific effects on intestinal injury. PTC and dPTC induced intestinal mucosal injury, with more severe effects in females. Compared to the control group, female zebrafish treated with PTC or dPTC exhibited a significant decrease in intestinal goblet cells (0.67-, 0.64-fold), damage to intestinal villi, and an increase in macrophages (2.21-, 2.79-fold). 16S rRNA sequencing, targeted metabolomics, and molecular docking analysis showed that significant reductions in bacterial metabolites, including betaine, choline, and methylthioadenosine, caused abnormal methyl transfer in one-carbon metabolism, mediating gut microbiota dysbiosis. The fundamental causes of this difference are rooted in three interconnected biological mechanisms, which together amplify toxicity in females while conferring relative resistance in males. Changes in DNA methylation-related transcription levels further confirmed that the interaction between intestinal metabolic activity and epigenetic regulation had a cascade effect on intestinal injury. PTC and dPTC have sex-specific effects on zebrafish intestinal health through one-carbon metabolism via the betaine-homocysteine methyltransferase -mediated pathway, emphasizing the importance of considering microbiota and sex dependence in ecological risk assessment.