Prenatal Lead (Pb) Exposure and Peripheral Blood DNA Methylation (5mC) and Hydroxymethylation (5hmC) in Mexican Adolescents from the ELEMENT Birth Cohort

BACKGROUND: Gestational lead (Pb) exposure can adversely affect offspring health through multiple mechanisms, including epigenomic alterations via DNA methylation (5mC) and hydroxymethylation (5hmC), an intermediate in oxidative demethylation. Most current methods do not distinguish between 5mC and 5hmC, limiting insights into their individual roles. OBJECTIVE: Our study sought to identify the association of trimester-specific (T1, T2, T3) prenatal Pb exposure with 5mC and 5hmC levels at multiple cytosine-phosphate-guanine sites within gene regions previously associated with prenatal Pb (HCN2, NINJ2, RAB5A, TPPP) in whole blood leu-kocytes of children ages 11-18 years of age. METHODS: Participants from the Early Life Exposure in Mexico to Environmental Toxicants (ELEMENT) birth cohorts were selected (n = 144) for pyrosequencing analysis following oxidative or standard sodium bisulfite treatment. This workflow directly quantifies total methylation (5mC+5hmC) and 5mC only; 5hmC is estimated by subtraction. RESULTS: Participants were 51% male, and mean maternal blood lead levels (BLL) were 6.43 +/- 5.16 mu g/dL in Trimester 1 (T1), 5.66 +/- 5.21 mu g/dL in Trimester 2 (T2), and 5.86 +/- 4.34 mu g/dL in Trimester 3 (T3). In addition, 5hmC levels were calculated for HCN2 (mean +/- standard deviation(SD), 2.08 +/- 4.18%), NINJ2 (G/C: 2.01 +/- 5.95; GG: 0.90 +/- 3.97), RAB5A (0.66 +/- 0.80%), and TPPP (1.11 +/- 6.67%). Furthermore, 5mC levels were meas-ured in HCN2 (81.3 +/- 9.63%), NINJ2 (heterozygotes: 38.6 +/- 7.39%; GG homozygotes: 67.3 +/- 9.83%), RAB5A (1.41 +/- 1.21%), and TPPP (92.5 +/- 8.03%). Several significant associations between BLLs and 5mC/5hmC were identified: T1 BLLs with 5mC in HCN2 (beta = +/- 0.37, p = 0.03) and 5hmC in NINJ2 (beta = 0.49, p = 0.003); T2 BLLs with 5mC in HCN2 (beta = 0.37, p = 0.03) and 5hmC in NINJ2 (beta= 0.27, p = 0.008); and T3 BLLs with 5mC in HCN2 (beta= 0.50, p = 0.01) and NINJ2 (beta= +/- 0.35, p = 0.004) and 5hmC in NINJ2 (beta = 0.45, p < 0.001). NINJ2 5mC was negatively cor-related with gene expression (Pearson r = +/- 0.5, p-value = 0.005), whereas 5hmC was positively correlated (r = 0.4, p-value = 0.04). DISCUSSION: These findings suggest there is variable 5hmC in human whole blood and that prenatal Pb exposure is associated with gene-specific 5mC and 5hmC levels at adolescence, providing evidence to consider 5hmC as a regulatory mechanism that is responsive to environmental exposures.