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Title
| - Non-Oxidative Mechanisms Are Responsible for the Induction of Mutagenesis byReduction of Cr(VI) with Cysteine: Role of Ternary DNA Adducts inCr(III)-Dependent Mutagenesis
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Abstract
| - Intracellular reduction of carcinogenic Cr(VI) generates Cr−DNA adducts formed throughthe coordination of Cr(III) to DNA phosphates (phosphotriester-type adduct). Here, we examined the roleof Cr(III)−DNA adducts in mutagenesis induced by metabolism of Cr(VI) with cysteine. Reduction ofCr(VI) caused a strong oxidation of 2‘,7‘-dichlorofluoroscin (DCFH) and extensive Cr−DNA binding butno DNA breakage. Cr−DNA adducts induced unwinding of supercoiled plasmids and structural distortionsin the DNA helix as detected by decreased ethidium bromide binding. Propagation of Cr-treated pSP189plasmids in human fibroblasts led to a dose-dependent formation of the supF mutants and inhibition ofreplication. Blocking of Cr(III)−DNA binding by occupation of DNA phosphates with Mg2+ or bysequestration of Cr(III) by inorganic phosphate or EDTA eliminated mutagenic responses and restored anormal yield of replicated plasmids. Dissociation of Cr(III) from DNA by a phosphate-based reversalprocedure returned mutation frequency to background levels. The mutagenic responses at the differentphases of the reduction reaction were unrelated to the amount of reduced Cr(VI) but reflected the numberand the spectrum of Cr(III)−DNA adducts that were formed. Ternary cysteine−Cr(III)−DNA adductswere approximately 4−5 times more mutagenic than binary Cr(III)−DNA adducts. Although intermediatereaction products (CrV/IV, thiyl radicals) were capable of oxidizing DCFH, they were insufficiently reactiveto damage DNA. Single-base substitutions at G/C pairs were the predominant type of Cr-induced mutations.The majority of mutations occurred at the sites where G had adjacent purine in the 3‘ or 5‘ position.Overall, our results present the first evidence that Cr(III)−DNA adducts play the dominant role in themutagenicity caused by the metabolism of Cr(VI) by a biological reducing agent.
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