Reduction of hexavalent chromium by human cytochrome b(5): Generation of hydroxyl radical and superoxide

Department of Pharmacology and Toxicology , Medical College of Wisconsin, Milwaukee, Wisconsin, United States
Free Radical Biology and Medicine (Impact Factor: 5.74). 04/2007; 42(6):738-55; discussion 735-7. DOI: 10.1016/j.freeradbiomed.2006.10.055
Source: PubMed


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-diethoxyphosphoryl-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 muM). 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 dimethyl sulfoxide (DMSO) plus the spin trap alpha-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.

9 Reads
  • Source
    • "First, we chose H 2 O 2 /UV DEPMPO spin trap system to evaluate the effects of PteFt on hydroxyl radical signal intensity. This system was previously used to detect hydroxyl radical formation [35]. PteFt was observed to reduce the DEPMPO/OH $ adduct signal intensity in a dose and time dependent fashion. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Ferritins are nanoscale globular protein cages encapsulating a ferric core. They widely exist in animals, plants, and microbes, playing indispensable roles in iron homeostasis. Interestingly, our study clearly demonstrates that ferritin has an enzyme-mimic activity derived from its ferric nanocore but not the protein cage. Further study revealed that the mimic-enzyme activity of ferritin is more thermally stable and pH-tolerant compared with horseradish peroxidase. Considering the abundance of ferritin in numerous organisms, this finding may indicate a new role of ferritin in antioxidant and detoxification metabolisms. In addition, as a natural protein-caged nanoparticle with an enzyme-mimic activity, ferritin is readily conjugated with biomolecules to construct nanobiosensors, thus holds promising potential for facile and biocompatible labeling for sensitive and robust bioassays in biomedical applications.
    Analytical Chemistry 09/2011; 83(22):8611-6. DOI:10.1021/ac202049q · 5.64 Impact Factor
  • Source
    • "Electronic supplementary material The online version of this article (doi:10.1007/s11248-010-9426-1) contains supplementary material, which is available to authorized users. toxins and drugs (Akhtar et al. 2005; Yamazaki et al. 2002; Borthiry et al. 2007; Kurian et al. 2006). The modulation of P450 activity by cytochrome b 5 is reported to be both substrate-and P450-specific, with evidence of both stimulation and inhibition of substrate metabolism (Yamazaki et al. 2002; Schenkman and Jansson 2003). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Microsomal cytochrome b5 is a ubiquitous, 15.2 kDa haemoprotein implicated in a number of cellular processes such as fatty acid desaturation, drug metabolism, steroid hormone biosynthesis and methaemoglobin reduction. As a consequence of these functions this protein has been considered essential for life. Most of the ascribed functions of cytochrome b5, however, stem from in vitro studies and for this reason we have carried out a germline deletion of this enzyme. We have unexpectedly found that cytochrome b5 null mice were viable and fertile, with pups being born at expected Mendelian ratios. However, a number of intriguing phenotypes were identified, including altered drug metabolism, methaemoglobinemia and disrupted steroid hormone homeostasis. In addition to these previously identified roles for this protein, cytochrome b5 null mice displayed skin defects closely resembling those observed in autosomal recessive congenital ichthyosis and retardation of neonatal development, indicating that this protein, possibly as a consequence of its role in the de novo biosynthesis of unsaturated fatty acids, plays a central role in skin development and neonatal nutrition. Results from fatty acid profile analysis of several tissues suggest that cytochrome b5 plays a role controlling saturated/unsaturated homeostasis. These data demonstrate that regional concentrations of unsaturated fatty acids are controlled by endogenous metabolic pathways and not by diet alone. Electronic supplementary material The online version of this article (doi:10.1007/s11248-010-9426-1) contains supplementary material, which is available to authorized users.
    Transgenic Research 06/2011; 20(3):491-502. DOI:10.1007/s11248-010-9426-1 · 2.32 Impact Factor
  • Source
    • "Recently, we reported an important role for glutathione in the protection against Cr 6+ -induced oxidative stress in goldfish (Lushchak et al., 2008). These data are in the line with other studies where roles for glutathione and glutathione reductase (Raghunathan et al., 2007; Shi and Dalal, 1989, 1990), cytochrome b 5 (Borthiry et al., 2007), and ascorbate (Kaczmarek et al., 2007) in chromium detoxification were ascribed. Although Cr 6+ is believed to be more toxic, Cr 3+ also exhibits toxicity (Blasiak and Kowalik, 2000) and can be carcinogenic and induce DNA damage (Bagchi et al., 2002 and therein; Qi et al., 2000). "
    [Show abstract] [Hide abstract]
    ABSTRACT: In the environment chromium is found mainly in two valence states—hexavalent chromium (Cr6+) and trivalent chromium (Cr3+). The present study evaluates the effects of Cr3+ exposure on goldfish by analyzing parameters of oxidative stress and antioxidant defense in liver and kidney of fish given 96 h exposures to Cr3+ concentrations of 1, 2.5, 5 or 10 mg/l in aquarium water. Cr3+ exposure did not alter two parameters of oxidative stress—protein carbonyl content and lipid peroxide concentrations in either organ. However, Cr3+ exposure did decrease total glutathione concentration in liver by 34–69% and in kidney to 36–49% of the respective control values. Oxidized GSSG content fell by similar percentages so that the ratio [GSSG]/[total glutathione] remained constant at all Cr3+ exposure levels except in liver under the highest, 10 mg/l, exposure level. In liver, exposure to 1–5 mg/l Cr3+ led to a decrease in the activity of superoxide dismutase (SOD) by 29–36%, and at 10 mg/l Cr3+ the reduction was 54%, whereas in kidney ∼30% reductions in SOD activity were seen at concentrations 1 and 10 mg/l Cr3+. Catalase activity was not significantly affected by 1–5 mg/l Cr3+, but was reduced by 57 and 42% in liver and kidney, respectively. Chromium exposure also reduced the activity of glutathione-S-transferase in both organs by 17–50% but did not affect glutathione reductase or glucose-6-phosphate dehydrogenase activities. A comparison of Cr3+ effects on goldfish liver and kidney metabolism indicates that the trivalent ion induces stronger oxidative stress than Cr6+ at the same concentrations.
    Aquatic toxicology (Amsterdam, Netherlands) 06/2009; 93(1-93):45-52. DOI:10.1016/j.aquatox.2009.03.007 · 3.45 Impact Factor
Show more


9 Reads
Available from