Humic acid induced genotoxicity in human peripheral blood lymphocytes using comet and sister chromatid exchange assay

Humic acid (HA) in well water used by the inhabitants for drinking is one of the possible etiological factors for blackfoot disease (BFD). Moreover, within BFD endemic areas cancers occur at significantly higher rates than in areas free of BFD. In this study, the genotoxic potential of HA is assessed using human peripheral blood lymphocytes. The cells were exposed to HA (0-200 μg/mL for 2 h), and the induction of DNA primary damage in cellular DNA was evaluated by single-cell gel electrophoresis (comet assay). HA-induced DNA damage was decreased by superoxide (O₂^-), hydrogen peroxide (H₂O₂), and reactive oxygen species (ROS) scavengers (superoxide dismutase, catalase, and Trolox), and nitric oxide (NO) synthase inhibitors (N^G-nitro-l-arginine methyl ester and N^G-methyl-l-arginine). Moreover, formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III (Endo III), known to catalyze the excision of oxidized bases, increase the amount of DNA migration in HA-treated cells. Pretreatment of the cells with both the Ca²⁺-chelator BAPTA and EGTA completely inhibited HA-induced DNA damage, indicating that HA-induced changes in Ca²⁺-homeostasis are the predominant pathways for the HA induction of genotoxicity. Furthermore, sister chromatid exchange was found in the HA-treated lymphocytes. Our findings suggest that HA can induce oxidative DNA damage and genotoxicity in human lymphocytes.