Carcinogenic Chromium(VI) Induces Cross-Linking of Vitamin C to DNA in Vitro and in Human Lung A549 Cells

Abstract

Reductive activation of carcinogenic Cr(VI) is required for the induction of DNA damage and mutations. Here, we examined the formation of Cr-DNA adducts in the reactions of Cr(VI) with its dominant biological reducer, vitamin C (ascorbate). Reductive conversion of Cr(VI) to Cr(III) by ascorbate produced stable Cr-DNA adducts, of which approximately 25% constituted ascorbate-Cr(III)-DNA cross-links. No evidence was found for the involvement of Cr(V) or Cr(IV) intermediates in the formation of either binary or ternary adducts. The cross-linking reaction was consistent with the attack of DNA by transient Cr(III)-ascorbate complexes. The yield of Cr(III)-DNA adducts was similar on dsDNA and AGT, ACT, or CT oligonucleotides and was strongly inhibited by Mg(2+), suggesting predominant coordination of Cr(III) to DNA phosphate oxygens. We also detected cross-linking of ascorbate to DNA in Cr(VI)-exposed human lung A549 cells that were preincubated with dehydroascorbic acid to create normal levels of intracellular ascorbate. Ascorbate-Cr-DNA cross-links accounted for approximately 6% of the total Cr-DNA adducts in A549 cells. Shuttle-vector experiments showed that ascorbate-Cr-DNA cross-links were mutagenic in human cells. Our results demonstrate that in addition to reduction of Cr(VI) to DNA-reactive Cr(III), vitamin C contributes to the genotoxicity of Cr(VI) via a direct chemical modification of DNA. The absence of Asc in A549 and other human cultured cells indicates that cells maintained under the usual in vitro conditions lack the most important reducing agent for Cr(VI) and would primarily display slow thiol-dependent activation of Cr(VI).

Cr3+ Binding to DNA Backbone Phosphate and Bases: Slow Ligand Exchange Rates and Metal Hydrolysis

“…The inorganic phosphate was previously shown to dissociate Cr 3+ /DNA binding. 13,37 On the other hand, we recently used phosphate to mask other metal ions, leaving Cr 3+ to be the only active metal for the Ce13d…”

Depletion of Intracellular Ascorbate by the Carcinogenic Metals Nickel and Cobalt Results in the Induction of Hypoxic Stress

“…Measurement of Ascorbate Levels-HPLC measurements of total intracellular vitamin C levels were performed by HPLC as described previously (28).…”

Kinetics of oxidation of Cr(III)-organic complexes by H2O2

“…Following reduction of Cr(VI), Cr(III) may be immobilised in sparingly soluble hydroxide precipitates and in solid solution with Cr(OH) 3 at circumneutral or higher pH (Zachara et al, 2004), or may remain mobile as dissolved Cr(III) species. Numerous investigators have provided evidence for the existence of Cr(III)-organic complexes under environmental conditions (Harris, 1977;Nakayama et al, 1981;Ahern et al, 1985;Davis et al, 1994;Kaczynskl et al, 1994;Fukushima et al, 1995;Zhitkovich et al, 1995Zhitkovich et al, , 1996Mattuck and Nikolaidis, 1996;Sule and Ingle, 1996;Walsh and O'Halloran, 1996;Quievryn et al, 2002;Howe and Loeppe, 2003;Puzon et al, 1997Puzon et al, , 2005Puzon et al, , 2008. Organo -Cr(III) species increase the mobilisation of chromium and play a relevant role either in phyto-remediation strategies by increasing Cr uptake by plants (Erenoglu et al, 2007) or in microbial bioremediation of Cr(VI) contaminated sites by increasing the risk of Cr(III) migration in groundwater (Puzon et al, 2005).…”