Integrated Analysis of Buccal‐Cell microRNAs Reveals Molecular Signatures Associated With Chronic Pesticide Exposure in Agricultural Workers

Chronic pesticide exposure has been associated with oxidative stress, inflammation, and genomic instability, and microRNAs (miRNAs) may reflect regulatory responses occurring between exposure and adverse outcomes. This cross-sectional biomonitoring study evaluated selected miRNAs as molecular response indicators associated with chronic pesticide exposure and genomic instability in 137 participants classified as putatively non-exposed (n = 34), indirectly exposed (n = 41), or directly exposed agricultural workers (n = 62). Buccal epithelial cells were analyzed for micronucleus frequency and expression of miR-21-5p, miR-34a-5p, miR-146a-5p, miR-155-5p, miR-126-3p, and miR-222-3p by RT-qPCR. Hierarchical clustering, principal component analysis (PCA), logistic regression, ROC analysis, and repeated 10-fold cross-validation were performed. Directly exposed workers showed significantly increased expression of miR-21-5p, miR-34a-5p, miR-146a-5p, miR-155-5p, and miR-222-3p, whereas miR-126-3p was significantly reduced (all p < 0.001). Micronucleus frequency increased progressively across the three groups (2.1 ± 0.4, 4.6 ± 0.7, and 7.8 ± 1.2 MN/2000 cells; p < 0.001). PCA explained 78.1% of the total variance and distinguished the exposure groups. The integrated six-miRNA signature achieved an AUC of 0.92 (95% CI: 0.88-0.97) and an optimism-corrected AUC of 0.89 after internal validation. These findings indicate that chronic pesticide exposure is associated with coordinated miRNA dysregulation and increased genomic instability. The six-miRNA signature may complement established occupational biomonitoring approaches as an indicator of intermediate molecular responses, but it should not be interpreted as a direct measure of pesticide dose or clinical effect.