Integrated analysis of the blood metallome and DNA methylation, gene expression, and protein levels of immune-associated genes in residents from mining region

Environmental exposure to toxic and essential metals can disrupt host immune function through mechanisms involving epigenetic, transcriptional, and post-transcriptional regulation. Although numerous studies have investigated these regulatory layers separately, integrative analyses across molecular levels in relation to metallome is missing. In this study, we performed a targeted multi-omics analysis of six immune-associated genes (NFKB1, CDKN2A, IGF2, H19, ESR1, and APOA5) and corresponding proteins in healthy residents from a long-term mining region (MRR, n = 46) and a non-mining region (NMR, n = 48). Transcriptome data were generated by mRNA sequencing, while DNA methylation data were obtained using targeted bisulfite sequencing by analyzing previously identified differentially methylated positions. Plasma protein levels were measured by enzyme-linked immunosorbent assay, and plasma metal concentrations were quantified using inductively coupled plasma mass spectrometry. We observed significantly higher plasma levels of NFKB1 and CDKN2A proteins, along with lower ESR1 transcript levels, in residents of the mining region compared to the non-mining region. NFKB1 protein levels were associated with both promoter methylation and residence in mining region, suggesting a regulatory cascade from DNA methylation to protein expression. IGF2 protein levels were higher in males and showed positive associations with age and the cumulative Z-score of essential metal mixture burden. Our results show that long-term residence in mining regions is associated with changes in NFKB1 at both the DNA methylation and protein levels, which may serve as a sensitive biomarker of metal exposure.