Lead exposure triggers DNA damage in TK6 cells via alteration of DNA repair gene expression and hypermethylation of Ku80 promoter region

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Abstract
Lead (Pb) is a widespread environmental metal with recognized genotoxicity, yet whether it impairs DNA damage repair via epigenetic regulation remains unclear. Here, human lymphoblastoid TK6 cells were exposed to lead acetate at 0, 120, 240, or 480 µM for 24 h. We quantified cell viability, oxidative stress indicators (ROS, MDA, SOD, CAT), DNA damage markers (single-cell gel electrophoresis/Comet Tail DNA% and γ-H2AX foci), cell-cycle distribution, apoptosis, and the mRNA and protein levels of DNA repair genes (RAD51, CHEK2, BRCA1, Ku80, MSH2, LIG4). We further measured DNMT and TET expression and determined methylation of the Ku80 promoter by pyrosequencing. Our results showed that lead decreased cell viability, increased ROS levels and MDA while reduced SOD and CAT activity; Lead caused dose-dependent increases in DNA single-and double-strand breaks with percentage of Comet tail DNA and the number of γ-H2AX foci. G0/G1-phase arrest and apoptosis also rose with dose. DNA repair genes showed a biphasic response-elevated at lower doses and suppressed at higher doses-at both mRNA and protein levels. Themethylation level in promoter region of Ku80 increased with dose and coincided with higher DNMT1 and lower TET2 expression. Collectively, Pb exposure induces oxidative stress and DNA damage in TK6 cells; higher lead concentration inhibited DNA repair capacity, potentially via DNMT1/TET2 mediated hypermethylation of the Ku80 promoter.

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