Reductive metabolism of Cr(VI) by cysteine leads to the formation of binary and ternary Cr--DNA adducts in the absence of oxidative DNA damage.
Carcinogenic chromium(VI) compounds require reduction for the induction of genotoxicity. In this work, we examined a spectrum of DNA damage produced in Cr(VI)-cysteine reactions at neutral pH. Cr(VI) reduction followed single-component kinetics and led to a significant oxidation of 2',7'-dichlorofluoroscein (DCFH). The presence of residual Fe and/or Cu resulted in an increased level of oxidation of DCFH, and the removal of adventitious metals required rigorous purification of cysteine. DNA breakage and abasic sites were not detected, suggesting that DNA is much less susceptible to oxidation than DCFH. Cr(VI) reduction led to the extensive formation of Cr-DNA adducts and Cys-Cr-DNA and interstrand DNA-DNA cross-links. Cr-DNA binding resulted in unwinding of supercoiled DNA and a greater stability of the DNA duplex to denaturation. Ionically bound Cr comprised 40-60% of the total DNA-bound Cr, while the remaining Cr-DNA complexes represented stable Cr-DNA adducts that exhibited significant resistance to dissociation by EDTA. The yield of Cr-DNA adducts was strongly influenced by the nature of the buffer that was used. Phosphate buffer completely blocked Cr-DNA binding, whereas adduct formation in organic buffers was largely dependent on the extent of buffer ionization. The level of formation of Cr-DNA adducts was several times higher at pH 6 which resulted from lower levels of buffer ionization and diminished competition from hydroxyl ions. Yield of a number of Cr-DNA and Cys-DNA adducts increased linearly as a function of Cr(VI) concentration, whereas formation of interstrand DNA cross-links exhibited exponential dose dependence. Approximately 60-90 min was required to convert a Cr-DNA monoadduct into a DNA-DNA cross-link. Prolonged incubations at alkaline conditions led to a selective cleavage of cross-linked DNA. The alkali sensitivity of Cr-adducted DNA suggests that results of alkaline elution analysis of DNA damage in cells require a more cautious interpretation. Overall, a spectrum of DNA damage derived from Cr(VI)-cysteine reactions was similar to that found in exposed cells.
Zhitkovich
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Department of Pathology and Laboratory Medicine, Brown University, Box G-B511, Providence, Rhode Island 02912, USA. Anatoly.Zhitkovich@brown.edu
Shrager
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S
Messer
J
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D000818:Animals; D002021:Buffers; D002412:Cations; D002417:Cattle; D002840:Chromates / Q000737:chemistry / Q000633:toxicity; D017608:Chromium Compounds / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D018736:DNA Adducts / Q000096:biosynthesis* / Q000737:chemistry; D004249:DNA Damage; D004278:DNA, Superhelical / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D004355:Drug Stability; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D019163:Reducing Agents / Q000378:metabolism
D002021:Buffers; D002412:Cations; D002840:Chromates; D017608:Chromium Compounds; D018736:DNA Adducts; D004278:DNA, Superhelical; D005452:Fluoresceins; D005456:Fluorescent Dyes; D019163:Reducing Agents; C065013:2',7'-dichlorodihydrofluorescein; D003545:Cysteine