Oxidative activation of the human carcinogen chromate by arsenite: A model for synergistic metal activation leading to oxidative DNA damage

Human exposure to toxic metals and metalloids in the environment seldom occurs from a single pure compound. Most environmental exposure profiles are heterogeneous with co-exposure occurring coincident with multiple toxic metal species. This co-exposure to metals and metalloids in complex mixtures can result in a synergistic, additive or even depletive toxic response. The complexity of interactions presented by metal mixtures presents a need for convenient and sensitive methods to determine potential toxic responses from such co-exposure. We have studied the reaction between the two commonly associated toxic metals of chromate, Cr(VI), and arsenite, As(III), with regards to the ability of As(III) to reductively activate Cr(VI) to generate oxidative stress and DNA damage. Using a DCF-based fluorescent dye assay we have demonstrated that the redox reaction between As(III) and Cr(VI) yields high valent intermediates of chromium, Cr(V), that are highly oxidizing. This induction of oxidizing potential was dose dependent and did not occur with As(III) or Cr(VI) alone or, with the other major oxidation state of arsenic, arsenate, As(V). The mechanism of oxidation of DCFH to the fluorescent species, DCF, in this reaction was through a direct, metal-based oxidation since addition of radical scavengers did not significantly decrease oxidation of the dye in this system. The addition of a ligand that stabilizes the high valent Cr(V) oxidation state, 2-ethyl-2-hydroxybutyric acid (EHBA), to the chromate and arsenite mixture resulted in an enhancement of DCF fluorescence. The DCF fluorescence observed with the Cr(VI) and As(III) mixture was also found to correlate with oxidative DNA damage as measured by a plasmid nicking assay. These data show how metal-metal interactions in environmental mixtures could result in the synergistic induction of oxidative stress and DNA damage. Further, these data demonstrate the utility of the DCF fluorescence assay as a sensitive method for screening synergistic redox interactions in metal mixtures.