The role of Zearalenone in epigenetic modifications of candidate genes in Nellore heifers

Mycotoxins are toxic secondary metabolites produced by fungi that proliferate in stored feeds such as grains and silage. Zearalenone (ZEN) is a frequently detected mycotoxin with well-known estrogenic activity. This study aimed to evaluate the effects of ingesting ZEN-contaminated feed on the methylation and hydroxymethylation of CpG islands located in the candidate genes CYP1A1, CYP21A2, and CYP1B1 in Nellore heifers. Twenty heifers were confined for 12 weeks during the finishing phase and assigned to two groups: a control group (non-contaminated feed) and a ZEN-treated group (approximately 300 ppb). After slaughter, liver samples were collected, and genomic DNA was extracted. DNA was treated with T4-β-glucosyltransferase and digested with the restriction enzymes MspI and HpaII. CpG island methylation and hydroxymethylation were quantified by real-time PCR. Statistical analyses were performed using analysis of variance followed by t-tests, and p-values were adjusted for multiple comparisons using the false discovery rate (FDR). No significant differences were observed in methylation or hydroxymethylation levels of CYP1A1 between groups after FDR adjustment. In contrast, animals exposed to ZEN-contaminated feed exhibited increased DNA methylation in the CpG island of CYP1B1, and this effect remained statistically significant after FDR correction. For CYP21A2, differences in hydroxymethylation observed in the unadjusted analysis were attenuated after FDR adjustment and are therefore interpreted as a trend. These findings indicate that ZEN exposure induces gene-specific epigenetic modulation in the liver of Nellore heifers, with a robust effect observed for CYP1B1. The results highlight the importance of monitoring ZEN contamination in cattle feed and support the need for further studies integrating epigenetic, transcriptomic, and endocrine analyses to clarify the biological implications of mycotoxin exposure in beef cattle.