Interdependent genotoxic mechanisms of monomethylarsonous acid: Role of ROS-induced DNA damage and poly(ADP-ribose) polymerase-1 inhibition in the malignant transformation of urothelial cells

摘要

Exposure of human bladder urothelial cells (UROtsa) to 50 nM of the arsenic metabolite, monomethylarsonous acid (MMA^III), for 12 weeks results in irreversible malignant transformation. The ability of continuous, low-level MMA^III exposure to cause an increase in genotoxic potential by inhibiting repair processes necessary to maintain genomic stability is unknown. Following genomic insult within cellular systems poly(ADP-ribose) polymerase-1 (PARP-1), a zinc finger protein, is rapidly activated and recruited to sites of DNA strand breaks. When UROtsa cells are continuously exposed to 50 nM MMA^III, PARP-1 activity does not increase despite the increase in MMA^III-induced DNA single-strand breaks through 12 weeks of exposure. When UROtsa cells are removed from continuous MMA^III exposure (2 weeks), PARP-1 activity increases coinciding with a subsequent decrease in DNA damage levels. Paradoxically, PARP-1 mRNA expression and protein levels are elevated in the presence of continuous MMA^III indicating a possible mechanism to compensate for the inhibition of PARP-1 activity in the presence of MMA^III. The zinc finger domains of PARP-1 contain vicinal sulfhydryl groups which may act as a potential site for MMA^III to bind, displace zinc ion, and render PARP-1 inactive. Mass spectrometry analysis demonstrates the ability of MMA^III to bind a synthetic peptide representing the zinc-finger domain of PARP-1, and displace zinc from the peptide in a dose-dependent manner. In the presence of continuous MMA^III exposure, continuous 4-week zinc supplementation restored PARP-1 activity levels and reduced the genotoxicity associated with MMA^III. Zinc supplementation did not produce an overall increase in PARP-1 protein levels, decrease the levels of MMA^III-induced reactive oxygen species, or alter Cu-Zn superoxide dismutase levels. Overall, these results present two potential interdependent mechanisms in which MMA^III may increase the susceptibility of UROtsa cells to genotoxic insult and/or malignant transformation: elevated levels of MMA^III-induced DNA damage through the production of reactive oxygen species, and the direct MMA^III-induced inhibition of PARP-1.