Reaction of Chromium(VI) with Ascorbate Produces Chromium(V), Chromium(IV), and Carbon-Based Radicals

Stearns, Diane M.; Wetterhahn, Karen E.

doi:10.1021/tx00038a016

Cited by 179 publications

(162 citation statements)

References 63 publications

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“…2B), which is consistent with previous results and is due to the slow rate of Cr(VI) reduction catalyzed by NADPH [39]. To confirm the authenticity of the Cr(V) signal, parallel studies were conducted with isotopically pure 53 Cr (stable isotope) as Na 2 53 CrO 4 . Because 53 Cr has a nuclear spin of I = 3/2, the single ESR line of Cr(V) was split into four lines (separation of 17.4 G) (Fig.…”

Section: Generation Of Cr(v)supporting

confidence: 89%

“…The reduction of Cr(VI) to Cr(V) was observed and confirmed with isotopically pure 53 In addition, it was discovered that some commercial sources of catalase have Cr(VI)-reducing contaminants which could confound interpretation of results in Cr-related studies.…”

Section: Introductionmentioning

confidence: 79%

“…Cr(V), a d 1 paramagnetic species [53,54], can be directly detected by ESR spectroscopy by its sharp line spectrum (g = 1.979) at conventional X-band frequency. Previous studies demonstrated that human lung and liver microsomes generate Cr(V) during anaerobic Cr(VI) reduction [39].…”

Section: Generation Of Cr(v)mentioning

confidence: 99%

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Reduction of hexavalent chromium by human cytochrome b5: Generation of hydroxyl radical and superoxide

Borthiry

Antholine

Kalyanaraman

et al. 2007

Free Radical Biology and Medicine

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The reduction of hexavalent chromium, Cr(VI), can generate reactive Cr intermediates and various types of oxidative stress. The potential role of human microsomal enzymes in free radical generation was examined using reconstituted proteoliposomes (PLs) containing purified cytochrome b 5 and NADPH:P450 reductase. Under aerobic conditions, the PLs reduced Cr(VI) to Cr(V) which was confirmed by ESR using isotopically pure 53 Cr(VI). When 5-Diethoxyphos-phoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) was included as a spin trap, a very prominent signal for the hydroxyl radical (HO • ) adduct was observed as well as a smaller signal for the superoxide (O 2 •− ) adduct. These adducts were observed even at very low Cr(VI) concentrations (10 μM). NADPH, Cr(VI), O 2 and the PLs were all required for significant HO • generation. Superoxide dismutase eliminated the O 2 • − adduct and resulted in a 30% increase in the HO • adduct. Catalase largely diminished the HO • adduct signal indicating its dependence on H 2 O 2 . Some sources of catalase were found to have Cr(VI)-reducing contaminants which could confound results, but a source of catalase free of these contaminants was used for these studies. Exogenous H 2 O 2 was not needed, indicating that it was generated by the PLs. Adding exogenous H 2 O 2 , however, did increase the amount of DEPMPO/ HO • adduct. The inclusion of formate yielded the carbon dioxide radical adduct of DEPMPO, and experiments with dimethylsulfoxide (DMSO) plus the spin trap α-phenyl-N-tert-butylnitrone (PBN) yielded the methoxy and methyl radical adducts of PBN, confirming the generation of HO • . Quantification of the various species over time was consistent with a stoichiometric excess of HO • relative to the net amount of Cr(VI) reduced. This also represents the first demonstration of a role for cytochrome b 5 in the generation of HO • . Overall, the simultaneous generation of Cr(V) and H 2 O 2 by the PLs and the resulting generation of HO • at low Cr(VI) concentrations could have important implications for Cr(VI) toxicity.

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Section: Generation Of Cr(v)supporting

confidence: 89%

Section: Introductionmentioning

confidence: 79%

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Reduction of hexavalent chromium by human cytochrome b5: Generation of hydroxyl radical and superoxide

Borthiry

Antholine

Kalyanaraman

et al. 2007

Free Radical Biology and Medicine

View full text Add to dashboard Cite

show abstract

“…as Cr(V)O 4 3− ) is a d 1 paramagnetic species [44,45] that can be directly detected by ESR spectroscopy by its sharp line spectrum (g = 1.979) at conventional X-band frequency. As initial controls, Cr(VI) (as Na 2 CrO 4 ) was incubated with cell culture media in the absence of cells (Fig.…”

Section: Cr(v) Generated By Mediummentioning

confidence: 99%

“…2 are due to Cr(V), analogous studies were conducted with isotopically pure 53 Cr (stable isotope) as Na 2 53 CrO 4 . Because 53 Cr has a nuclear spin of I = 3/2, Cr(V) signals will be split into four lines (separation of 17.4 G), typical for the hyperfine coupling for 53 Cr(V) [44]. With 53 Cr, both the g = 1.979 and g = 1.985 components of the Cr(V) signal ( …”

Section: Cr(v) Generated By Cellsmentioning

confidence: 99%

Reductive activation of hexavalent chromium by human lung epithelial cells: Generation of Cr(V) and Cr(V)-thiol species

Borthiry

Antholine

Myers

et al. 2008

Journal of Inorganic Biochemistry

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Chromium(VI) compounds (e.g. chromates) are cytotoxic, mutagenic, and potentially carcinogenic. The reduction of Cr(VI) can yield reactive intermediates such as Cr(V) and reactive oxygen species. Bronchial epithelial cells are the primary site of pulmonary exposure to inhaled Cr(VI) and are the primary cells from which Cr(VI)-associated human cancers arise. BEAS-2B cells were used here as a model of normal human bronchial epithelium for studies on the reductive activation of Cr(VI). Cells incubated with Na 2 CrO 4 exhibited two Cr(V) ESR signals, g = 1.979 and 1.985, which persisted for at least one hour. The g = 1.979 signal is similar to that generated in vitro by human microsomes and by proteoliposomes containing P450 reductase and cytochrome b 5 . Unlike many cells in culture, these cells continued to express P450 reductase and cytochrome b 5 . Studies with the non-selective thiol oxidant diamide indicated that the g = 1.985 signal was thiol-dependent whereas the g = 1.979 signal was not. Pretreatment with phenazine methosulfate eliminated both Cr(V) signals suggesting that Cr(V) generation is largely NAD(P)H-dependent. ESR spectra indicated that a portion of the Cr (VI) was rapidly reduced to Cr(III). Cells incubated with an insoluble chromate, ZnCrO 4 , also generated both Cr(V) signals, whereas Cr(V) was not detected with insoluble PbCrO 4 . In clonogenic assays, the cells were very sensitive to Na 2 CrO 4 and ZnCrO 4 , but considerably less sensitive to PbCrO 4 .

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Arrest of replication by mammalian DNA polymerases α and β caused by chromium-DNA lesions

1998

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We have previously shown that trivalent chromium, and hexavalent chromium in the presence of one of its primary in vivo reductants, ascorbate, can bind to DNA and form interstrand crosslinks capable of obstructing replication. This effect was demonstrated in vitro by using Sequenase Version 2.0 T7 DNA polymerase; its parent enzyme, the unmodified T7 DNA polymerase; and Escherichia coli polymerase I large (Klenow) fragment; and it was demonstrated ex vivo by using Taq polymerase and DNA from chromium-treated human lung cells as template. This study was performed to determine whether DNA-bound chromium affects mammalian DNA polymerases in the same manner. Two mammalian enzymes, DNA polymerase alpha and DNA polymerase beta, were used. DNA polymerase alpha is a processive enzyme believed to be the primary lagging-stand synthetase, whereas DNA polymerase beta is a non-processive enzyme believed to function in DNA repair by filling single stranded gaps one base at a time. DNA polymerase arrest assays were performed with each of these enzymes to replicate DNA with toxicologically relevant levels of chromium adducts produced by either trivalent chromium or hexavalent chromium and ascorbate. Both enzymes responded to chromium-DNA damage by arresting replication, and the arrests increased in a dose-dependent manner. Furthermore, the guanine-specific pattern of arrests produced when an exonuclease-free preparation of DNA polymerase beta was used corresponded exactly to the arrest patterns produced in vitro by the exonuclease-free enzyme Sequenase and ex vivo by Taq polymerase. These results suggest that replication arrest may be a common response of polymerases to DNA-chromium lesions and provide a plausible mechanism for the inhibition of DNA synthesis and S-phase cell-cycle delay that occurs in mammalian cells treated with genotoxic chromium compounds.

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Reaction of Chromium(VI) with Ascorbate Produces Chromium(V), Chromium(IV), and Carbon-Based Radicals

Cited by 179 publications

References 63 publications

Reduction of hexavalent chromium by human cytochrome b5: Generation of hydroxyl radical and superoxide

Reduction of hexavalent chromium by human cytochrome b5: Generation of hydroxyl radical and superoxide

Reductive activation of hexavalent chromium by human lung epithelial cells: Generation of Cr(V) and Cr(V)-thiol species

Arrest of replication by mammalian DNA polymerases α and β caused by chromium-DNA lesions

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