One-electron reduction of chromate by NADPH-dependent glutathione reductase

Electron spin resonance (ESR) measurements provide evidence for the formation of Cr(V) intermediates in the enzymatic reduction of Cr(VI) by glutathione reductase (GSSG-R) in the presence of NADPH, indicating an initial single-electron transfer step in the reduction mechanism. Depending on the pH, at least two different Cr(V) species are generated which are relatively long-lived. In addition, we have detected the hydroxyl (.OH) radical formation during the GSSG-R catalyzed reduction of Cr(VI) by spin trapping, employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) as spin traps. Superoxide dismutase (SOD) causes only a minor effect on the .OH radical and Cr(V) formation, indicating that the O2- is not significantly involved in the reaction mechanism. Catalase enhances the Cr(V) formation and substantially inhibits the .OH radical formation, indicating the involvement of hydrogen peroxide (H2O2) in the reaction mechanism. Addition of H2O2 suppresses Cr(V) and enhances the .OH radical formation. Measurements involving N-ethylmaleimide show that the Cr(V) species, produced enzymatically by the reduction of Cr(VI) by GSSG-R, react with H2O2 to generate .OH radicals, which might participate in the initiation of Cr(VI) carcinogenicity.     

ESR Spin Trapping Detection of Hydroxyl Radicals in the Reactions of Cr(V) Complexes with Hydrogen Peroxide

Electron spin resonance (ESR) measurments provide direct evidence for the involvement of Cr(V) in the reduction of Cr(VI) by NAD(P)H. Addition of hydrogen peroxide (H₂O₂) to NAD(P)H-Cr(VI) reaction mixtures suppresses the Cr(V) signal and generates hydroxyl (OH) radicals (as detected via spin trapping), suggesting that Cr(V) reacts with H₂O₂ to generate the OH radicals. Reaction between H₂O₂ and a Cr(V)-glutathione complex. and between H₂O₂ and several Cr(V)-cdrboxylato complexes also produces OH radicals. These results suggest that Cr(V) complexes catalyze the generation of OH radicals from H₂O₂, and that OH radicals might play a significant role in the mechanism of Cr(VI) cytotoxicity.     

Deferoxamine inhibition of Cr(V)-mediated radical generation and deoxyguanine hydroxylation: ESR and HPLC evidence.

Electron spin resonance (ESR) and high-performance liquid chromatography (HPLC) techniques were utilized to investigate the effect of deferoxamine on free radical generation in the reaction of Cr(V) with H2O2 and organic hydroperoxides. ESR measurements demonstrated that deferoxamine can efficiently reduce the concentration of the Cr(V) intermediate as formed in the reduction of Cr(VI) by NAD(P)H or a flavoenzyme glutathione reductase/NADH. ESR spin trapping studies showed that deferoxamine also inhibits Cr(V)-mediated .OH radical generation from H2O2, as well as Cr(V)-mediated alkyl and alkoxy radical formation from t-butyl hydroperoxide and cumene hydroperoxide. HPLC measurements showed that .OH radicals generated by the Cr(VI)/flavoenzyme/NAD(P)H enzymatic system react with 2'-deoxyguanine to form 8-hydroxy-2'-deoxyguanine (8-OHdG), a DNA damage marker. Deferoxamine effectly inhibited the formation of 8-OHdG also.     

ESR Spin Trapping Detection of Hydroxyl Radicals in the Reactions of Cr(V) Complexes with Hydrogen Peroxide

Electron spin resonance (ESR) measurments provide direct evidence for the involvement of Cr(V) in the reduction of Cr(VI) by NAD(P)H. Addition of hydrogen peroxide (H₂O₂) to NAD(P)H-Cr(VI) reaction mixtures suppresses the Cr(V) signal and generates hydroxyl (OH) radicals (as detected via spin trapping), suggesting that Cr(V) reacts with H₂O₂ to generate the OH radicals. Reaction between H₂O₂ and a Cr(V)-glutathione complex. and between H₂O₂ and several Cr(V)-cdrboxylato complexes also produces OH radicals. These results suggest that Cr(V) complexes catalyze the generation of OH radicals from H₂O₂, and that OH radicals might play a significant role in the mechanism of Cr(VI) cytotoxicity.     

Generation of reactive oxygen species in the enzymatic reduction of PbCrO4 and related DNA damage

Free radical reactions are believed to play an important role in the mechanism of Cr(VI)-induced carcinogenesis. Most studies concerning the role of free radical reactions have been limited to soluble Cr(VI). Various studies have shown that solubility is an important factor contributing to the carcinogenic potential of Cr(VI) compounds. Here, we report that reduction of insoluble PbCrO4 by glutathione reductase in the presence of NADPH as a cofactor generated hydroxyl radicals (.OH) and caused DNA damage. The .OH radicals were detected by electron spin resonance (ESR) using 5,5-dimethyl-N-oxide as a spin trap. Addition of catalase, a specific H2O2 scavenger, inhibited the .OH radical generation, indicating the involvement of H2O2 in the mechanism of Cr(VI)-induced .OH generation. Catalase reduced .OH radicals measured by electron spin resonance and reduced DNA strand breaks, indicating .OH radicals are involved in the damage measured. The H2O2 formation was measured by change in fluorescence of scopoletin in the presence of horseradish peroxidase. Molecular oxygen was used in the system as measured by oxygen consumption assay. Chelation of PbCrO4 impaired the generation of .OH radical. The results obtained from this study show that reduction of insoluble PbCrO4 by glutathione reductase/NADPH generates .OH radicals. The mechanism of .OH generation involves reduction of molecular oxygen to H2O2, which generates .OH radicals through a Fenton-like reaction. The .OH radicals generated by PbCrO4 caused DNA strand breakage.     

One-electron reduction of carcinogen chromate by microsomes, mitochondria, and Escherichia coli: identification of Cr(V) and .OH radical.

Earlier studies have shown that a long-lived Cr(V) species is produced during the reduction of chromate (Cr(VI] by microsomes/NADPH, mitochondria, and other cellular constituents and that this Cr(V) species plays a significant role in the mechanism of Cr(VI) toxicity. The present work indicates that this species is a Cr(V) complex involving the diol moieties of NADPH as the ligand. Additionally, ESR spin trapping investigations show that the hydroxyl (.OH) radical is also generated in the reduction process. Hydrogen peroxide (H2O2) enhances the .OH generation but suppresses the Cr(V)-NADPH complex formation. Catalase decreases the .OH radical generation and enhances the Cr(V)-NADPH formation. Measurements under anaerobic atmosphere show decreased .OH radical generation, indicating that during the cellular Cr(VI) reduction process molecular oxygen is reduced to H2O2, which reacts with the Cr(V)-NADPH complex to generate the .OH radical via a Fenton-like mechanism.     

Mechanism of chromium(VI) carcinogenesis

Since chromium(VI) is unreactive toward DNA under physiological conditions in vitro, the ability of carcinogenic chromium(VI) compounds to damage DNA depends on the presence of cellular redox components that reduce chromium(VI) to reactive species capable of interacting with DNA. We have examined the role of glutathione and hydrogen peroxide in chromium(VI)-induced DNA damage in vitro. Upon reaction with chromium(VI), glutathione produced chromium(V) and glutathione thiyl radical reactive intermediates, whereas hydrogen peroxide produced chromium(V) and hydroxyl radical. Reaction of DNA with chromium(VI) in the presence of glutathione resulted in binding of chromium and glutathione to DNA with little or no DNA strand breakage. Reaction of DNA with chromium(VI) in the presence of hydrogen peroxide produced the 8-hydroxydeoxy-guanosine adduct and extensive DNA strand breakage in the absence of significant Cr-DNA adduct formation. These results suggest that the nature of chromium(VI)-induced DNA damage will be strongly dependent on reactive intermediates such as chromium(V), glutathione thiyl radical, and hydroxyl radical, produced by cellular components active in chromium(VI) metabolism. In order to assess the ability of chromium(VI)-induced DNA damage to affect the normal template function of DNA, we investigated the effects of chromium(VI) on steady-state mRNA levels of various genes in chick embryo liver in vivo, and compared the effects to the levels of DNA damage observed. Chromium(VI) induced DNA-protein and DNA interstrand cross-links in chick embryo liver in vivo and suppressed the induction of 5-aminolevulinic acid synthase and cytochrome P-450 mRNA expression by porphyrinogenic drugs. In contrast, chromium(VI) increased the basal levels of expression of these two inducible genes, but had little or no effect on the expression of the constitutive albumin, β-actin, and conalbumin genes. Comparison of the time course of formation and repair of DNA damage with that of changes in gene expression suggests that chromium(VI) may form a mono-adduct prior to formation of DNA cross-links, and that chromium(VI)-induced DNA lesions may target certain classes of genes and lead to changes in their expression.     

Hydroxyl radical formation and lipid peroxidation enhancement by chromium

Chromium VI compounds have been shown to be carcinogenic in occupationally exposed humans, and to be genotoxic, mutagenic, and carcinogenic in a variety of experimental systems. In contrast, most chromium III compounds are relatively nontoxic, noncarcinogenic, and nonmutagenic. Reduction of Cr⁶⁺ leads to reactive intermediates, such as Cr⁵⁺, Cr⁴⁺, or other radical species. The molecular mechanism for the intracellular Cr⁶⁺ reduction has been the focus of recent studies, but the details are still not understood. Our study was initiated to compare the effect of Cr⁶⁺-hydroxyl radical formation and Cr⁶⁺-induced lipid peroxidation vs those of Cr³⁺. Electron spin responance measurements provide evidence for the formation of long-lived Cr⁵⁺ intermediates in the reduction of Cr⁶⁺ by glutathione reductase in the presence of NADPH and for the hydroxyl radical formation during the glutathione reductase catalyzed reduction of Cr⁶⁺. Hydrogen peroxide suppresses Cr⁵⁺ and enhances the formation of hydroxyl radical. Thus, Cr⁵⁺ intermediates catalyze generation of hydroxyl radicals from hydrogen peroxide through a Fenton-like reaction. Comparative effects of Cr⁶⁺ and Cr³⁺ on the development of lipid peroxidation were studied by using rat heart homogenate. Heart homogenate was incubated with different concentrations of Cr⁶⁺ compounds at 22°C for 60 min. Lipid peroxidation was determined as thiobarbituric acid reacting materiels (TBA-RM). The results confirm that Cr⁶⁺ induces lipid peroxidation in the rat heart homogenate. These observations might suggest a possible causative role of lipid peroxidation in Cr⁶⁺ toxicity. This enhancement of lipid peroxidation is modified by the addition of some metal chelators and antioxidants. Thus, strategies for combating Cr⁶⁺ toxicity should take into account the role of the hydroxy radicals, and hence, steps for blocking its chain propagation and preventing the formation of lipid peroxides.     

The role of superoxide radical in chromium (VI)-generated hydroxyl radical: the Cr(VI) Haber-Weiss cycle.

To understand the role of the superoxide (O-2) radical in chromate-related genotoxicity, we investigated whether Cr(VI) can catalyze the Haber-Weiss cycle in vitro: O-2 + Cr(VI)----Cr(V) + O2 Cr(V) + H2O2----Cr(VI) + .OH + OH-. ESR and spin trapping techniques were utilized to monitor the O-2 (produced using xanthine/xanthine oxidase), .OH, and Cr(V) species. Superoxide dismutase as well as catalase inhibited the .OH radical radical formation, attesting to the direct involvement of O-2 and H2O2 in the process. ESR measurements also provided direct evidence for the formation of Cr(V). Kinetic measurements were consistent with the role of Cr(V) and H2O2 as intermediates in .OH formation. These results indicate that in cellular media, especially during chromate phagocytosis, the O-2 radical can become a significant source of .OH radicals and hence a significant factor in the biochemical mechanism of cellular damage due to Cr(VI) exposure.     

NAD(P)H-dependent chromium (VI) reductase of Pseudomonas ambigua G-1: a Cr(V) intermediate is formed during the reduction of Cr(VI) to Cr(III).

An NAD(P)H-dependent Cr(VI) reductase (molecular weight = 65,000) was purified from a Cr(VI)-resistant bacterium, Pseudomonas ambigua G-1. Stoichiometric analysis of the enzymatic reaction showed that the enzyme catalyzed the reduction of 1 mol of Cr(VI) to Cr(III) while consuming 3 mol of NADH as an electron donor. Chromium(VI) was reduced to Cr(V) by one equivalent NADH molecule in the absence of the enzyme. Electron spin resonance analysis showed that Cr(V) species (g = 1.979) was formed during the enzymatic reduction. The amount of Cr(V) species formed was about 10 times larger than that of the nonezymatic reduction. These findings show that the Cr(VI) reductase reduced Cr(VI) to Cr(III) with at least two reaction steps via Cr(V) as an intermediate.     

In vivo nephrotoxicity induced in mice by chromium(VI)

The role of glutathione (GSH) and chromium (V) in chromium (VI)-induced nephrotoxicity in mice was investigated at 24 h after K2Cr(VI)2O7 ip injection. Nephrotoxicity was assessed by measurements of relative kidney weight and serum urea nitrogen. Cr(VI) nephrotoxicity was accompanied by decreased renal GSH and glutathione reductase (GSSG-R) levels. Pretreatment with buthionine sulfoximine, an inhibitor of GSH biosynthesis, enhanced Cr(VI)-induced nephrotoxicity, and remarkably diminished kidney GSH and GSSG-R levels. In contrast, pretreatment with glutathione methyl ester, a GSH-supplying agent, prevented Cr(VI) from exerting a harmful effect on mouse kidney and restored kidney GSH level. Administration of a Cr(V) compound, K3Cr(V)O8, induced much higher toxicity in mouse kidney than Cr(VI), but it failed to diminish renal GSH level. Another Cr(V) compound, Cr(V)-GSH complex, and Cr(III) nitrate did not cause a nephrotoxic effect in mice. The mechanism of Cr(VI)-induced nephrotoxicity was explained using GSH and Cr(V).     

The role of hydroxyl radical as a messenger in Cr(VI)-induced p53 activation

The present study investigates whether reactive oxygen species (ROS)are involved in p53 activation, and if they are, which species isresponsible for the activation. Our hypothesis is that hydroxyl radical(·OH) functions as a messenger for the activation of this tumorsuppressor protein. Human lung epithelial cells (A549) were used totest this hypothesis. Cr(VI) was employed as the source of ROS due toits ability to generate a whole spectrum of ROS inside the cell. Cr(VI)is able to activate p53 by increasing the protein levels and enhancingboth the DNA binding activity and transactivation ability of theprotein. Increased cellular levels of superoxide radicals(O₂·), hydrogen peroxide(H₂O₂), and ·OH radicals were detected on theaddition of Cr(VI) to the cells. Superoxide dismutase, by enhancing theproduction of H₂O₂ from O₂·radicals, increased p53 activity. Catalase, anH₂O₂ scavenger, eliminated ·OH radicalgeneration and inhibited p53 activation. Sodium formate and aspirin,·OH radical scavengers, also suppressed p53 activation. Deferoxamine,a metal chelator, inhibited p53 activation by chelating Cr(V) to makeit incapable of generating radicals from H₂O₂.NADPH, which accelerated the one-electron reduction of Cr(VI) to Cr(V)and increased ·OH radical generation, dramatically enhanced p53activation. Thus ·OH radical generated from Cr(VI) reduction in A549cells is responsible for Cr(VI)-induced p53 activation.

     

Formation of the paramagnetic complex [Cr(OH)5O2]5- in the reaction between chromium(VI) oxide, and hydrogen peroxide or superoxide anion radicals studied by EPR spectroscopy.

Hydrogen peroxide or superoxide anion radicals form a paramagnetic complex in the reaction with chromium(VI) oxide in an alkaline water solution at room temperature. The complex [Cr(OH)5O2]5- with the g-value equal to 1.9734 is believed to contain hydroxyl groups derived from the alkaline solution and dioxygen derived from hydrogen peroxide or superoxide anion radicals.     

In vivo reduction of chromium (VI) and its related free radical generation

Chromium (VI) compounds are widely recognized as human carcinogens. Extensive studies in vitro and in model systems indicate that the reactive intermediate, Cr (V), generated by cellular reduction of Cr (VI), is likely the candidate for the ultimate carcinogenic form of chromium compounds. Here we review our current understanding of the in vivo reduction of Cr (VI) and its related free radical generation. Our results demonstrate that Cr (V) is indeed generated from the reduction of Cr (VI) in vivo, and that Cr (V) thus formed can mediate the generation of free radicals. Cr (V) and its related free radicals are very likely to be involved in the mechanism of Cr (VI)induced toxicity and carcinogenesis. These studies also illustrate that in vivo EPR spectroscopy and magnetic resonance imaging can be very useful and powerful tools for studying paramagnetic metal ions in chemical and biochemical reactions occurring in intact animals.     

An investigation into the genotoxic species generated during reduction of chromium(VI) by catechol(amine)s

Abstract

Chromium(VI) is a common occupational carcinogen.¹ The major carcinogenic and mutagenic species are proposed to be Cr(V) and Cr(IV) intermediates formed during the reduction of Cr(VI) to stable Cr(III) compounds,² although indirect evidence suggests that reactive oxygen species (ROS) may also be important.³ The reductions of Cr(VI) by some biological reductants (e.g. ascorbate) have been studied previously, and genotoxic Cr(IV/V) species have been detected.⁴ Another potential reductant in vivo is protein-bound DOPA, which is present on oxidised proteins at low steady-state concentrations prior to enzymatic breakdown.⁵ Recently, we have shown, by EPR spectroscopy, that the reactions of Cr(VI) with model DOPA compounds (catechol(amine)s), and with oxidised proteins themselves, generate several reactive intermediates, including Cr(V) complexes and organic radicals.⁶ Previous studies have proposed that ROS may also be produced during catechol(amine) oxidation.⁷ Here we describe studies of the interaction of DNA with the reactive species produced during the reductions of K₂Cr₂O₇ by catechol(amine)s.     

Evaluation of in vitro Cr(VI) reduction potential in cytosolic extracts of three indigenous Bacillus sp. isolated from Cr(VI) polluted industrial landfill

Three efficient Cr(VI) reducing bacterial strains were isolated from Cr(VI) polluted landfill and characterized for in vitro Cr(VI) reduction. Phylogenetic analysis using 16S rRNA gene sequencing revealed that the newly isolated strains G1DM20, G1DM22 and G1DM64 were closely related to Bacillus cereus, Bacillus fusiformis and Bacillus sphaericus, respectively. The suspended cultures of all Bacillus sp. exhibited more than 85% reduction of 1000 microM Cr(VI) within 30 h. The suspended culture of Bacillus sp. G1DM22 exhibited an ability for continuous reduction of 100 microM Cr(VI) up to seven consecutive inputs. Assays with the permeabilized cells and cell-free extracts from each of Bacillus sp. demonstrated that the hexavalent chromate reductase activity was mainly associated with the soluble fraction of cells and expressed constitutively. The Cr(VI) reduction by the cell-free extracts of Bacillus sp. G1DM20 and G1DM22 was maximum at 30 degrees C and pH 7 whereas, Bacillus sp. G1DM64 exhibited maximum Cr(VI) reduction at pH 6. Addition of 1mM NADH enhanced the Cr(VI) reductase activity in the cell-free extracts of all three isolates. Amongst all three isolates tested, crude cell-free extracts of Bacillus sp. G1DM22 exhibited the fastest Cr(VI) reduction rate with complete reduction of 100 microM Cr(VI) within 100 min. The apparent K(m) and V(max) of the chromate reductase activity in Bacillus sp. G1DM22 were determined to be 200 microM Cr(VI) and 5.5 micromol/min/mg protein, respectively. The Cr(VI) reductase activity in cell-free extracts of all the isolates was stable in presence of different metal ions tested except Hg(2+) and Ag(+).     

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

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 Na₂CrO₄ exhibited two Cr(V) ESR signals, g = 1.979 and 1.985, which persisted for at least 1 h. The g = 1.979 signal is similar to that generated in vitro by human microsomes and by proteoliposomes containing P450 reductase and cytochrome b₅. Unlike many cells in culture, these cells continued to express P450 reductase and cytochrome b₅. 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, ZnCrO₄, also generated both Cr(V) signals, whereas Cr(V) was not detected with insoluble PbCrO₄. In clonogenic assays, the cells were very sensitive to Na₂CrO₄ and ZnCrO₄, but considerably less sensitive to PbCrO₄.     

Reactions of Low Valent Transition Metal Complexes with Hydrogen Peroxide. Are they “Fenton-Like” or not? 4. The Case of Fe(II)L,L = Edta; Hedta and Tcma

The question whether hydroxyl free radicals are formed in the reactions of divalent iron complexes Fe(II)L; L = edta; hedta; tcma (tcma = l-acetato-l,4,7-triazacyclononane) with hydrogen peroxide in neutral and slightly acidic solutions was studied by using the β elimination reaction as an assay for the formation of hydroxyl free radicals, OH. The results show that at pH<5.5 the iron(II)peroxide intermediate complex decomposes rapidly to yield free hydroxyl radicals for L=edta and hedta. This is in contrast to the mechanism of the corresponding Fe(II)nta peroxide complex, which probably decomposes to form Fe(IV)nta which then reacts with organic substrates to yield aliphatic free radicals. Thus, the non-participating ligand L has an appreciable effect on the mechanism of reaction of the metal center with hydrogen peroxide. Blank experiments using ionizing radiation as the source of CH₂CR(CH₃)OH, R = H or CH₃ radicals indicate that when L = tcma intermediates of the type LFe^III-CH₂CR(CH₃)OH_aq are formed, but their major mode of decomposition is not the β elimination reaction. Thus, the present assay for the formation of hydroxyl free radicals by the Fenton Reaction does not fit the latter system.