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.     

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.     

ESR characterization and metallokinetic analysis of Cr(V) in the blood of rats given carcinogen chromate(VI) compounds.

It has been shown that bio-trace metal elements are related to many diseases and the aging process. For many years, carcinogen hexavalent chromium (VI) has been known to be toxic to animals, but its dynamic toxicological mechanism is not sufficiently elucidated. Bioinorganic chemistry in terms of metallokinetic analysis of beneficial or toxic metal ions and their complexes is an important investigation for understanding their biochemical and physiological roles. We have tried to examine the real-time behavior of paramagnetic metal ions and complexes in animals, in which electron spin resonance (ESR) was capable of measuring paramagnetic species in chemical and biological systems. On the basis of our previous results on stable nitroxide spin probes, we have developed the in vivo blood circulation monitoring-electron spin resonance (BCM-ESR) method to analyze time-dependent ESR signal changes due to paramagnetic metal ions and their complexes in real time. When K2Cr2O7 or Na2Cr2O7 in saline was intravenously administered to rats, two ESR signals due to pentavalent chromium(V) were detectable in the circulating blood of rats. Cr(V) detected in the blood was indicated to be in the CrO(O4) and CrO(S2O2) coordination modes after the study on model complexes. From the changes of ESR signal intensities due to Cr(V) in the blood, the metallokinetic parameters were obtained using the pharmacokinetic analysis and the curve-fitting methods. The obtained results are important for understanding carcinogen chromate in terms of the formation of Cr(V) in animals. In addition, we propose the BCM-ESR method, which is useful to analyze the disposition of paramagnetic metal species in the blood of living animals.     

Chromium (V) and hydroxyl radical formation during the glutathione reductase-catalyzed reduction of chromium (VI)

Electron spin resonance measurements provide evidence for the formation of long-lived Cr(V) intermediates in the reduction of Cr(VI) by glutathione reductase in the presence of NADPH and for the hydroxyl radical formation during the glutathione reductase catalyzed reduction of Cr(VI). Hydrogen peroxide suppresses Cr(V) and enhances the formation of hydroxyl radicals. Thus Cr(V) intermediates catalyze generation of hydroxyl radicals from hydrogen peroxide through a Fenton-like reaction. Thus the mechanism of Cr(VI) toxicity might involve the interaction between macromolecules and the hydroxyl radicals.     

Evidence for the generation of hydroxyl radicals from a chromium(V) intermediate isolated from the reaction of chromate with glutathione.

The formation of hydroxyl radicals from a chromium(V) complex isolated from the reaction of glutathione with chromate has been demonstrated in spin trapping experiments using dimethylsulfoxide and 3,5-dibromo-4-nitrosobenzene sulfonate. Mechanisms for the formation of radicals in such systems are discussed. These results help to explain the ability of solutions containing chromate and glutathione to cause strand breaks in DNA.     

Comparison of in vitro Cr(VI) reduction by CFEs of chromate resistant bacteria isolated from chromate contaminated soil

Chromate resistant and reducing strains were isolated from chromium contaminated soil and identified as Bacillus sp. (KCH2 and KCH3), Leucobacter sp. (KCH4) and Exiguobacterium sp. (KCH5). KCH3 and KCH4 showed higher Cr(VI) tolerance (2 mM) and Cr(VI) reduction (1.5 mM) than KCH5 (1.5 mM and 0.75 mM, respectively). Cr(VI) reduction by CFEs of KCH3 and KCH4 showed NAD(P)H dependence, optimum activity at pH 5.5, low K(m) (45-55 microM) and substrate inhibition by Cr(VI) (>75 microM), whereas that of KCH5 showed NADH dependence, pH optimum at 6.0, high K(m) (200 microM) and no inhibition by Cr(VI). Cr(VI) reduction was optimum at 35 degrees C for CFEs of KCH3 and KCH5 and 30 degrees C for that of KCH3. Cr(VI) reduction by CFEs of all the strains were inhibited by Hg(2+) and enhanced by Cu(2+). Activity enhancement by Cu(2+) was more predominant (290%) for KCH4. The characterization of Cr(VI) reduction by CFEs of chromate resistant isolates of different genera is useful for development of Cr(VI) bioremediation.     

Microbial reduction of Cr(VI) in the presence of chromate conversion coating constituents

The reduction of Cr(VI) by the metal-reducing bacterium Shewanella oneidensis MR-1 was evaluated, to determine the potential for exploiting Cr(VI) bioreduction as a means of treating chromate conversion coating (CCC) waste streams. Inclusion of Cr(VI) at concentrations ≥1 mM inhibited aerobic growth of S. oneidensis, but that organism was able to reduce Cr(VI) at a concentration of up to 1 mM under anaerobic, nongrowth conditions. S. oneidensis reduced Cr(VI) in the presence of common CCC constituents, with the exception of ferricyanide, when these CCC constituents were included at concentrations typical of CCC waste streams. Ferricyanide inhibited neither aerobic growth nor metabolism under aerobic, nitrate- or iron-reducing conditions, suggesting that the ferricyanide-depended inhibition of Cr(VI) reduction is not due to broad metabolic inhibition, but is specific to Cr(VI) reduction. Results indicate that under some conditions, the activities of metal-reducing bacteria, such as S. oneidensis, could be exploited for the removal of Cr(VI) from CCC waste streams under appropriate conditions.     

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.     

Kinetics and mechanism for reduction of anticancer-active tetrachloroam(m)ine platinum(IV) compounds by glutathione

trans -[PtCl₄(NH₃)(thiazole)] (1), trans-[PtCl₄(cha)(NH₃)] (2), cis-[PtCl₄(cha)(NH₃)] (3) (cha =cyclohexylamine), and cis-[PtCl₄(NH₃)₂] (4) has been investigatedat 25 °C in a 1.0 M aqueous medium at pH 2.0–5.0 (1) and 4.5–6.8 (2–4) using stopped-flow spectrophotometry. The redox reactions follow the second-order rate law , where k is a pH-dependent rate constant and [GSH]_tot the total concentration of glutathione. The reduction takes place via parallel reactions between the platinum(IV) complexes and the various protolytic species of glutathione. The pH dependence of the redox kinetics is ascribed to displacement of these protolytic equilibria. The thiolate species GS⁻ is the major reductant under the reaction conditions used. The second-order rate constants for reduction of compounds 1–4 by GS⁻ are (1.43±0.01)×10⁷, (3.86±0.03)×10⁶, (1.83±0.01)×10⁶, and (1.18±0.01)×10⁶M⁻¹s⁻¹, respectively. Rate constants for reduction of 1 by the protonated species GSH are more than five orders of magnitude smaller. The mechanism for the reductive elimination reactions of the Pt(IV) compounds is proposed to involve an attack by glutathione on one of the mutually trans coordinated chloride ligands, leading to two-electron transfer via a chloride-bridged activated complex. The kinetics results together with literature data indicate that platinum(IV) complexes with a trans Cl-Pt-Cl axis are reduced rapidly by glutathione as well as by ascorbate. In agreement with this observation, cytotoxicity profiles for such complexes are very similar to those for the corresponding platinum(II) product complexes. The rapid reduction within 1 s of the platinum(IV) compounds with a trans Cl-Pt-Cl axis to their platinum(II) analogs does not seem to support the strategy of using kinetic inertness as a parameter to increase anticancer activity, at least for this class of compounds. Received: 8 December 1999 / Accepted: 15 February 2000     

The DNA cleavage induced by a chromium(V) complex and by chromate and glutathione is mediated by activated oxygen species

The number of strand breaks induced by the combination of chromate and glutathione (GSH) in PM2 DNA was effectively reduced upon addition of the hydroxyl radical scavengers dimethyl sulphoxide (DMSO), formate and benzoate. Administration of catalase also led to a depression of DNA degradation whereas superoxide dismutase (SOD) had very little influence. Essentially the same results were obtained in experiments employing a chromium(V) complex Na4(GSH)4Cr.8H20, which is an intermediate chromium species isolated from the reduction of chromate by glutathione. DNA cleavage was dependent on the presence of iron (FeCl3). When compared with the number of breaks produced by FeCl3 and GSH alone, chromate stimulated the generation of single-strand breaks. These findings suggest that hydroxyl radicals are one ultimate DNA cleaving agent in both reactions. A reaction scheme for the production of hydroxyl radicals is proposed.     

Isolation and characterization of Cr(VI) reducing Cellulomonas spp. from subsurface soils: implications for long-term chromate reduction

Microbial enrichments from Cr(VI) contaminated and uncontaminated US Department of Energy Hanford Site sediments produced Cr(VI) reducing consortia when grown in the presence of Cr(VI) with acetate, D-xylose or glycerol as a carbon and energy source. Eight of the nine isolates from the consortia were Gram positive and four of these were identified by 16S rRNA sequence homology and membrane fatty acid composition as belonging to the genus Cellulomonas. Two strains, ES6 and WS01, were further examined for their ability to reduce Cr(VI) under growth and non-growth conditions. During fermentative growth on D-xylose, ES6 and WS01 decreased aqueous Cr(VI) concentrations from 0.04 mM Cr(VI) to below the detection limit (0.002 mM Cr(VI)) in less than three days and retained their ability to reduce Cr(VI) even after four months of incubation. Washed ES6 and WS01 cells also reduced Cr(VI) under non-growth conditions for over four months, both with and without the presence of an exogenous electron donor. K-edge XANES spectroscopy confirmed the reduction of Cr(VI) to Cr(III). The ability to reduce Cr(VI) after growth had stopped and in the absence of an external electron donor, suggests that stimulation of these types of organisms may lead to effective long-term, in situ passive reactive barriers for Cr(VI) removal. Our results indicate that Cr(VI) reduction by indigenous Cellulomonas spp. may be a potential method of in situ bioremediation of Cr(VI) contaminated sediment and groundwater.     

Hexavalent chromate reduction by alkaliphilic Amphibacillus sp. KSUCr3 is mediated by copper-dependent membrane-associated Cr(VI) reductase

The present study was aimed to localize and characterize hexavalent chromate [Cr(VI)] reductase activity of the extreme alkaliphilic Amphibacillus sp. KSUCr3 (optimal growth pH 10.5). The resting cells were able to reduce about 62 % of the toxic heavy metal Cr(VI) at initial concentration of 200 μM within 30 min. Cell permeabilization resulted in decrease of Cr(VI) reduction in comparison to untreated cells. Enzymatic assays of different sub-cellular fractions of Amphibacillus sp. KSUCr3 demonstrated that the Cr(VI) reductase was mainly associated with the membranous fraction and expressed constitutively. In vitro studies of the crude enzyme indicated that copper ion was essential for Cr(VI) reductase activity. In addition, Ca2? and Mn2? slightly stimulated the chromate reductase activity. Glucose was the best external electron donor, showing enhancement of the enzyme activity by about 3.5-fold. The K (m) and V (max) determined for chromate reductase activity in the membranous fraction were 23.8 μM Cr(VI) and 72 μmol/min/mg of protein, respectively. Cr(VI) reductase activity was maximum at 40 °C and pH 7.0 and it was significantly inhibited in the presence of disulfide reducers (2-mercaptoethanol), ion chelating agent (EDTA), and respiratory inhibitors (CN and Azide). Complete reduction of 100 and 200 μM of Cr(VI) by membrane associated enzyme were observed within 40 and 180 min, respectively. However, it should be noted that biochemical characterization has been done with crude enzyme only, and that final conclusion can only be drawn with the purified enzyme.     

An electron spin resonance (ESR) study on the mechanism of ascorbyl radical production by metal-binding proteins

The mechanism of ascorbate oxidation by metal-binding proteins (ceruloplasmin, albumin and transferrin) was investigated in vitro and in isolated plasma by the measurement of the ascorbyl free radicals (AFR) by electron spin resonance (ESR). In plasma of 13 healthy volunteers, a spontaneous and variable pro-duction of AFR was detected, which was increased by a 10 M ascorbate overloading; however, this increase was not correlated to the intensity of the spontaneous AFR signal. The addition of Cu and ceruloplasmin to plasma increased the ESR signal, while the addition of transferrin decreased the signal intensity in a dose-dependent manner. In vitro, we demonstrated that ascorbate was oxidized by human serum albumin and by ceruloplasmin, and that this oxidase-like activity was lost by trypsin or heat treatment of these proteins. These two proteins positively interacted in the oxidation of ascorbate, since addition of crude albumin to a solution of ascorbate and ceruloplasmin increased the intensity of ESR signal in a dose-dependent manner. The treatment of albumin by a metal chelator (DDTC) abolished these positive inter-actions. The respective roles of copper and iron in ascorbate oxidation were studied and showed a dose-dependent effect of these ions on ascorbate oxidation. The role of iron was confirmed by the inhibiting effect of metal-free transferrin on iron-dependent ascorbate oxidation. Concerted actions between iron carrying albumin and copper carrying ceruloplasmin appear responsible for the production of AFR in vitro and in vivo. © Rapid Science 1998     

Elucidation of the mechanism of selenoprotein glutathione peroxidase (GPx)-catalyzed hydrogen peroxide reduction by two glutathione molecules: a density functional study

The mechanism of the hydrogen peroxide reduction by two molecules of glutathione catalyzed by the selenoprotein glutatione peroxidase (GPx) has been computationally studied. It has been shown that the first elementary reaction of this process, (E-SeH) + H(2)O(2) --> (E-SeOH) + H(2)O (1), proceeds via a stepwise pathway with the overall barrier of 17.1 kcal/mol, which is in good agreement with the experimental barrier of 14.9 kcal/mol. During reaction 1, the Gln83 residue has been found to play a key role as a proton acceptor, which is consistent with experiments. The second elementary reaction, (E-SeOH) + GSH --> (E-Se-SG) + HOH (2), proceeds with the barrier of 17.9 kcal/mol. The last elementary reaction, (E-Se-SG) + GSH --> (E-SeH) + GS-SG (3), is initiated with the coordination of the second glutathione molecule. The calculations clearly suggest that the amide backbone of the Gly50 residue directly participates in this reaction and the presence of two water molecules is absolutely vital for the reaction to occur. This reaction proceeds with the barrier of 21.5 kcal/mol and is suggested to be a rate-determining step of the entire GPx-catalyzed reaction H(2)O(2) + 2GSH --> GS-SG + 2H(2)O. The results discussed in the present study provide intricate details of every step of the catalytic mechanism of the GPx enzyme and are in good general agreement with experimental findings and suggestions.     

Reactions of Cr(III) tetraphenylporphyrin complex with superoxide ion: ESR evidence for the formation of a new superoxide adduct of Cr(IV) porphyrinate and its reactive character

The electron spin resonance (ESR) spectrum of the reaction product of superoxide ion, O₂⁻, with chloro-5,10,15,20-tetraphenylporphyrinatochromium(III) [Cr(III)(TPP)Cl] shows strong hyperfine interactions with the metal nucleus and the metal ligand, indicating the formation of a superoxide adduct, Cr(IV)(TPP)(Cl)(O₂⁻). The formation of this superoxide adduct was also confirmed by UV-Vis spectroscopy. The reactive character of this superoxide adduct was investigated by ESR spectrometry. It was found that Cr(IV)(TPP)(Cl)(O₂⁻) can oxidize t-butylamine and triphenylphosphine to give the corresponding radical species, respectively, but not pyridine, 4-cyanopyridine or imidazole. These results indicate that the reactive character of Cr(IV)(TPP)- (Cl)(O₂⁻) resembles that of the free superoxide ion.     

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.     

Causes of DNA single-strand breaks during reduction of chromate by glutathione in vitro and in cells

Carcinogenic chromates induce DNA single-strand breaks (SSB) that are detectable by conventional alkali-based assays. However, the extent of direct breakage has been uncertain because excision repair and hydrolysis of Cr-DNA adducts at alkaline pH also generate SSB. We examined mechanisms of SSB production during chromate reduction by glutathione (GSH) and assessed the significance of these lesions in cells using genetic approaches. Cr(VI) reduction was biphasic and the formation of SSB occurred exclusively during the slow reaction phase. Catalase or iron chelators completely blocked DNA breakage, as did the use of GSH purified by a modified Chelex procedure. Thus, the direct intermediates of GSH-chromate reactions were unable to cause SSB unless activated by H2O2. SSB repair-deficient XRCC1(-/-) and proficient XRCC1+ EM9 cells had identical survival at doses causing up to 60% clonogenic death and accumulation of 1 mM Cr(VI). However, XRCC1(-/-) cells displayed higher lethality in the more toxic range and the depletion of GSH made them hypersensitive even to moderate doses. Elevation of cellular catalase or GSH levels eliminated survival differences between XRCC1(-/-) and XRCC1+ cells. In summary, formation of toxic SSB in cells occurs at relatively high chromate doses, requires H2O2, and is suppressed by high GSH concentrations.     

Bacterial mechanisms for Cr(VI) resistance and reduction: an overview and recent advances

Chromium pollution is increasing incessantly due to continuing industrialization. Of various oxidation states, Cr⁶⁺ is very toxic due to its carcinogenic and mutagenic nature. It also has deleterious effects on different microorganisms as well as on plants. Many species of bacteria thriving in the Cr⁶⁺-contaminated environments have evolved novel strategies to cope with Cr⁶⁺ toxicity. Generally, decreased uptake or exclusion of Cr⁶⁺ compounds through the membranes, biosorption, and the upregulation of genes associated with oxidative stress response are some of the resistance mechanisms in bacterial cells to overcome the Cr⁶⁺ stress. In addition, bacterial Cr⁶⁺ reduction into Cr³⁺ is also a mechanism of specific significance as it transforms toxic and mobile chromium derivatives into reduced species which are innocuous and immobile. Ecologically, the bacterial trait of reductive immobilization of Cr⁶⁺ derivatives is of great advantage in bioremediation. The present review is an effort to underline the bacterial resistance and reducing mechanisms to Cr⁶⁺ compounds with recent development in order to garner a broad perspective.     

Formation of glutathione-conjugated semiquinones by the reaction of quinones with glutathione: an ESR study

The nonenzymatic reaction of the cytotoxic compounds menadione (2-methyl-1,4-naphthoquinone) and 1,4-naphthoquinone (a reactive metabolite of 1-naphthol) with reducing agents such as NADPH and glutathione led to the formation of semiquinone-free radicals, which were detected with electron spin resonance spectroscopy. In the presence of glutathione as a reducing agent, menadione and 1,4-naphthoquinone underwent net one-electron reduction and conjugation with glutathione. At higher concentrations of glutathione, 1,4-naphthoquinone formed the semiquinones of both the monoconjugate and the diconjugate. The naphthoquinone-glutathione conjugates should redox cycle in a manner already known for the menadione conjugate. The semiquinone intermediates could be detected only under a nitrogen atmosphere and are probably the primary oxygen-reactive species responsible for the redox cycling of menadione- and naphthoquinone-glutathione conjugates.