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


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Abstract

Exposure of human bladder urothelial cells (UROtsa) to 50 nM of the arsenic metabolite, monomethylarsonous acid (MMAIII), for 12 weeks results in irreversible malignant transformation. The ability of continuous, low-level MMAIII 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 MMAIII, PARP-1 activity does not increase despite the increase in MMAIII-induced DNA single-strand breaks through 12 weeks of exposure. When UROtsa cells are removed from continuous MMAIII 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 MMAIII indicating a possible mechanism to compensate for the inhibition of PARP-1 activity in the presence of MMAIII. The zinc finger domains of PARP-1 contain vicinal sulfhydryl groups which may act as a potential site for MMAIII to bind, displace zinc ion, and render PARP-1 inactive. Mass spectrometry analysis demonstrates the ability of MMAIII 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 MMAIII exposure, continuous 4-week zinc supplementation restored PARP-1 activity levels and reduced the genotoxicity associated with MMAIII. Zinc supplementation did not produce an overall increase in PARP-1 protein levels, decrease the levels of MMAIII-induced reactive oxygen species, or alter Cu–Zn superoxide dismutase levels. Overall, these results present two potential interdependent mechanisms in which MMAIII may increase the susceptibility of UROtsa cells to genotoxic insult and/or malignant transformation: elevated levels of MMAIII-induced DNA damage through the production of reactive oxygen species, and the direct MMAIII-induced inhibition of PARP-1.

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