Effects of iron,copper, and chromate ions on the oxidative degradation of cellulose model compounds

Iron(II) and iron(III) ions promote the degradation of the cellulose model 1,5-anhydrocellobiitol by oxygen or hydrogen peroxide; copper and chromate ions have marked and different effects on the iron catalysis. With starch, iron promotes the hydrogen peroxide-induced reaction and copper and chromate ions further enhance the reaction rate. The tensile strength of paper board is reduced by the action of hydrogen peroxide and iron(II) salts, and mixtures of copper, chromate, and arsenate salts (CCA, a timber preservative) also promote degradation in the presence or absence of iron ions. The oxidation of 1,5-anhydrocellobiitol by oxygen in the presence of iron ions is strongly inhibited by CCA and by cetyltrimethylammonium chloride, and is accelerated by phenols and related compounds.     

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.     

Purification to homogeneity and characterization of a novel Pseudomonas putida chromate reductase

Cr(VI) (chromate) is a widespread environmental contaminant. Bacterial chromate reductases can convert soluble and toxic chromate to the insoluble and less toxic Cr(III). Bioremediation can therefore be effective in removing chromate from the environment, especially if the bacterial propensity for such removal is enhanced by genetic and biochemical engineering. To clone the chromate reductase-encoding gene, we purified to homogeneity (>600-fold purification) and characterized a novel soluble chromate reductase from Pseudomonas putida, using ammonium sulfate precipitation (55 to 70%), anion-exchange chromatography (DEAE Sepharose CL-6B), chromatofocusing (Polybuffer exchanger 94), and gel filtration (Superose 12 HR 10/30). The enzyme activity was dependent on NADH or NADPH; the temperature and pH optima for chromate reduction were 80 degrees C and 5, respectively; and the K(m) was 374 microM, with a V(max) of 1.72 micromol/min/mg of protein. Sulfate inhibited the enzyme activity noncompetitively. The reductase activity remained virtually unaltered after 30 min of exposure to 50 degrees C; even exposure to higher temperatures did not immediately inactivate the enzyme. X-ray absorption near-edge-structure spectra showed quantitative conversion of chromate to Cr(III) during the enzyme reaction.     

Mechanism of hydroxylation of aromatic compounds: II. Evidence for the involvement of superoxide anions in enzymatic hydroxylations

The mechanism of hydroxylation reactions catalyzed by m-hydroxybenzoate-4-hydroxylase and anthranilate hydroxylase from Aspergillus niger was investigated using superoxide dismutase from ovine erythrocytes. Inclusion of superoxide dismutase in the assay mixtures of the two enzymes resulted in complete inhibition of the hydroxylation reaction, indicating the possible involvement of superoxide anions (O₂⁻) in these reactions.     

The iron-containing superoxide dismutase of Ralstonia metallidurans CH34

The iron-containing superoxide dismutase (Fe-SOD) of Ralstonia metallidurans CH34 was purified and characterised as a homodimer of 2 x 21500 Da containing one iron atom per monomer and exhibiting all the characteristics of the prokaryotic Fe-SODs except for a higher isoelectric point. The protein was 2-fold overexpressed in the presence of selenite, zinc or paraquat. R. metallidurans CH34 was suggested to contain a gene encoding for a manganese-containing SOD located in the inducible chromate resistance operon. Whatever the culture conditions used in this study, including the presence of chromate, only a Fe-SOD, genetically distinct from the putative Mn-SOD, was detected. This Fe-SOD seems to be the only active superoxide dismutase expressed in R. metallidurans CH34.     

Polycyclic aromatic hydrocarbon quinone-mediated oxidation reduction cycling catalyzed by a human placental NADPH-linked carbonyl reductase.

Polycyclic aromatic hydrocarbon quinones, hydroquinones, and glutathionyl adducts of quinones undergo oxidation-reduction (redox) cycling in the presence of NADPH and the NADPH-linked human placental carbonyl reductase. K-region and non-K-region o-quinones and their glutathione adducts are the best substrates of this enzyme; they are reduced to hydroquinones. Under aerobic conditions, the hydroquinones are autoxidized with the formation of potentially hazardous semiquinones and the superoxide anion. Because of these reactions it is unlikely that polycyclic aromatic hydrocarbon quinones or their glutathione adducts are inert products of detoxication in tissues that contain the carbonyl reductase or another enzyme with similar substrate specificity. If superoxide dismutase is added to reaction mixtures containing the carbonyl reductase and quinones, it inhibits redox cycling. Presumably this results from destruction of the superoxide anion which acts as a chain propagator in these reactions.     

Mechanism of DNA cleavage induced by sodium chromate(VI) in the presence of hydrogen peroxide

Reactivities of chromium compounds with DNA were investigated by the DNA sequencing technique using 32P 5'-end-labeled DNA fragments, and the reaction mechanism was investigated by ESR spectroscopy. Incubation of double-stranded DNA with sodium chromate(VI) plus hydrogen peroxide or potassium tetraperoxochromate(V) led to the cleavage at the position of every base, particularly of guanine. Even without piperidine, the formation of oligonucleotides was observed, suggesting the breakage of the deoxyribose-phosphate backbone. ESR studies using hydroxyl radical traps demonstrated that hydroxyl radical is generated both during the reaction of sodium chromate(VI) with hydrogen peroxide and the decomposition of potassium tetraperoxochromate(V), and that hydroxyl radical reacts significantly not only with mononucleotides but also with deoxyribose 5-phosphate. ESR studies using a singlet oxygen trap demonstrated that singlet oxygen is also generated both by the same reaction and decomposition, and reacts significantly with deoxyguanylate, but scarcely reacts with other mononucleotides. Furthermore, ESR studies suggested that tetraperoxochromate(V) is formed by the reaction of sodium chromate(VI) with hydrogen peroxide. These results indicate that sodium chromate(VI) reacts with hydrogen peroxide to form tetraperoxochromate(V), leading to the production of the hydroxyl radical, which causes every base alteration and deoxyribose-phosphate backbone breakage. In addition, sodium chromate(VI) plus hydrogen peroxide generates singlet oxygen, which subsequently oxidizes the guanine residue. The mechanism by which both hydroxyl radical and singlet oxygen are generated during the reaction of sodium chromate(VI) with hydrogen peroxide was presented. Finally, the possibility that this reaction may be one of the primary reactions of carcinogenesis induced by chromate(VI) is discussed.     

On the role of superoxide radical in the mechanism of action of galactose oxidase

The inhibition of galactose oxidase by superoxide dismutase is a function of the method of assay, nature of substrate, and composition of incubation and assay mixtures, as well as the concentration of dismutase. A reasonable level of inhibition is attained only when superoxide dismutase is present prior to the onset of catalysis although this effect is not observed under all conditions tried. Peroxidase activates galactose oxidase and blocks its interaction with either superoxide dismutase or catalase. These results further obscure the possible role of superoxide radical in the galactose oxidase reaction.     

Mechanism of chromate reduction by the Escherichia coli protein, NfsA, and the role of different chromate reductases in minimizing oxidative stress during chromate reduction

Chromate [Cr(VI)] is a serious environmental pollutant, which is amenable to bacterial bioremediation. NfsA, the major oxygen-insensitive nitroreductase of Escherichia coli, is a flavoprotein that is able to reduce chromate to less soluble and less toxic Cr(III). We show that this process involves single-electron transfer, giving rise to a flavin semiquinone form of NfsA and Cr(V) as intermediates, which redox cycle, generating more reactive oxygen species (ROS) than a divalent chromate reducer, YieF. However, NfsA generates less ROS than a known one-electron chromate reducer, lipoyl dehydrogenase (LpDH), suggesting that NfsA employs a mixture of uni- and di-valent electron transfer steps. The presence of YieF, ChrR (another chromate reductase we previously characterized), or NfsA in an LpDH-catalysed chromate reduction reaction decreased ROS generation by c. 65, 40, or 20%, respectively, suggesting that these enzymes can pre-empt ROS generation by LpDH. We previously showed that ChrR protects Pseudomonas putida against chromate toxicity; here we show that NfsA or YieF overproduction can also increase the tolerance of E. coli to this compound.     

Purification to Homogeneity and Characterization of a Novel Pseudomonas putida Chromate Reductase

Cr(VI) (chromate) is a widespread environmental contaminant. Bacterial chromate reductases can convert soluble and toxic chromate to the insoluble and less toxic Cr(III). Bioremediation can therefore be effective in removing chromate from the environment, especially if the bacterial propensity for such removal is enhanced by genetic and biochemical engineering. To clone the chromate reductase-encoding gene, we purified to homogeneity (>600-fold purification) and characterized a novel soluble chromate reductase from Pseudomonas putida, using ammonium sulfate precipitation (55 to 70%), anion-exchange chromatography (DEAE Sepharose CL-6B), chromatofocusing (Polybuffer exchanger 94), and gel filtration (Superose 12 HR 10/30). The enzyme activity was dependent on NADH or NADPH; the temperature and pH optima for chromate reduction were 80°C and 5, respectively; and the K_m was 374 μM, with a V_max of 1.72 μmol/min/mg of protein. Sulfate inhibited the enzyme activity noncompetitively. The reductase activity remained virtually unaltered after 30 min of exposure to 50°C; even exposure to higher temperatures did not immediately inactivate the enzyme. X-ray absorption near-edge-structure spectra showed quantitative conversion of chromate to Cr(III) during the enzyme reaction.     

Ultrastructural cytochemistry of biogenic amines in nervous tissue: methodologic improvements.

A new fixation method has been developed for localizing biogenic amines in nervous tissue. The method is a modification of the chromaffin reaction in which all fixation steps are buffered with mixtures of sodium chromate and potassium dichromate. In this way the fixation and cytochemical reaction are carried out almost simultaneously. Using the rat vas deferens as a model tissue, it was found that the preservation of electron dense (chromaffin) cores in the vesicles of adrenergic nerve terminals depended on several factors: a short primary fixation using low concentrations of aldehydes, the presence of the chromate/dichromate buffer during all fixation steps and, finally, a long incubation period in a slightly acidic (pH 6.0) solution of this buffer before postfixation in osmium tetroxide. Using this method it was possible to identify not only small and large dense-cored vesicles as storage sites for amines but also a tubular reticulum (neuronal endoplasmic reticulum), the latter especially in nerve terminals of mesenteric arteries and iris. Biogenic amines were also visualized in sympathetic ganglion cells and in the central nervous system e.g., supraependymal nerve terminals, tissues that up to now proved the most difficult in terms of amine localization. In all the tissues examined the cytochemical reaction was highly selective and present in well preserved tissue, which is a significant advance over previously available techniques. It therefore offers new opportunities for further studies on the role of biogenic amines as neurotransmitters.     

Autoinactivation of xanthine oxidase: the role of superoxide radical and hydrogen peroxide.

Xanthine oxidase suffers autoinactivation in the course of catalyzing the oxidation of acetaldehyde. When no special efforts were made to maintain a high pO2 in these reaction mixtures catalase protected the xanthine oxidase, but superoxide dismutase did not. However, when oxygen depletion was slowed or prevented by working at lower concentrations of xanthine oxidase, at lower temperatures or by vigorous agitation under an atmosphere of 100% oxygen, superoxide dismutase or catalase protected markedly when added separately and protected almost completely when added together. This result correlates with the greater production of O2-, relative to H2O2, by xanthine oxidase, at elevated pO2. Since histidine also provided some protection and the high levels of acetaldehyde used would have precluded any significant effect of OH., we conclude that singlet oxygen, or something with similar reactivity, was generated from O2- plus H2O2 and contributed significantly to the observed autoinactivation.     

Alterations in the prooxidant and antioxidant status of human leukemic T-lymphocyte MOLT4 cells treated with potassium chromate

The involvement of reactive oxygen species in chromate-induced genotoxicity has been postulated. Because intracellular antioxidants help in eliminating the reactive species of oxygen, we have investigated both the prooxidant and antioxidant status of human leukemic T-lymphocyte MOLT4 cells exposed to nontoxic levels of chromium(VI) in culture. The cells treated with 0 200 M potassium chromate in a salts/glucose medium for 2 h were found to contain significantly lower levels of both small molecular weight and macromolecular antioxidants. In particular, the levels of glutathione and ascorbate were found to decrease with increased doses of chromate exposure in a dose-dependent manner. As little as 10 M chromate was found to decrease these small molecular weight antioxidants significantly (p<0.01). The macromolecular antioxidants, such as glutathione peroxidase, catalase, glutathione reductase, glucose-6-phosphate dehydrogenase and superoxide dismutase were also significantly (p<0.01) decreased by exposing the cells to as little as 10 M chromate. Concomitantly there was a dose-dependent increase in intracellular H₂O₂ accumulation in cells exposed to chromium(VI). These results indicate that chromate-induced genotoxicity may be due, at least in part, to decreased levels of intracellular antioxidants in conjunction with an increased production of the reactive oxygen species.     

Generation of PM2 DNA breaks in the course of reduction of chromium(VI) by glutathione

The carcinogen chromate is efficiently taken up and reduced to chromium(III) compounds by various biological systems. To test the possible DNA damage induced in the course of chromium(VI) reduction, we used a combination of chromate with the reductant glutathione (GSH) as well as a green complex of chromium(V), which is formed in the reaction of chromate with GSH. The combination of chromate and glutathione was found to cause single-strand breaks in supercoiled circular DNA of the bacteriophage PM2. The green chromium(V) complex Na4(GSH)4Cr(V).8H2O, prepared from chromate and glutathione, also cleaved supercoiled PM2 DNA. No DNA-degrading effects were observed with either chromate or the final product of the reaction with GSH, a purple anionic chromium(III) GSH complex. The nature of the buffering agents revealed a strong influence on the extent of DNA strand breaks produced by chromate and GSH. A variation of the GSH concentration in the reaction with chromate and PM2 DNA, performed in sodium phosphate-buffered solutions showed an initial increase in the number of strand breaks at GSH concentrations up to 1 mM followed by a decline at higher GSH concentrations. Since neither chromate, when administered individually, nor the final product of chromium(VI) reduction, the purple chromium(III) GSH complex, produced any detectable DNA cleavage, the critical steps leading to DNA strand breaks occur in the course of the conversion of chromium(VI) to chromium(III) by GSH, the most abundant intracellular low molecular thiol. Moreover, the demonstration that DNA cleavage is induced in the presence of the chromium(V) complex identifies chromium(V) as the oxidation state of the metal, which is involved in the steps leading to DNA-damaging effects of chromate.     

A comparative study of EPR spin trapping and cytochrome c reduction techniques for the measurement of superoxide anions

Superoxide anions (O₂^.−) generated by the reaction of xanthine with xanthine oxidase were measured by the reduction of cytochrome c and by electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the relative sensitivities of these two techniques for the measurement of O₂^.−. Mixtures of xanthine, xanthine oxidase, DMPO generated two adducts, a transient DMPO-OOH and a smaller but longer-lived DMPO-OH. Both adducts were inhibited by superoxide dismutase (SOD), demonstrating they originated from O₂^.−, and were also significantly decreased when the experiments were performed using unchelated buffers, suggesting that metal ion impurities in unchelated buffers alter the formation or degradation of DMPO-adducts. O₂^.−, generated by concentrations of xanthine as low as 0.05 μM, were detectable using EPR spin trapping. In contrast, mixtures of xanthine, xanthine oxidase, and cytochrome c measured spectrophotometrically at 550 nm demonstrated that concentrations of xanthine above 1 μM were required to produce measurable levels of reduced cytochrome c. These studies demonstrate that spin trapping using DMPO was at least 20-fold more sensitive than the reduction of cytochrome c for the measurement of superoxide anions. However, at levels of superoxide generation where cytochrome c provides a linear measurement of production, EPR spin trapping may underestimate radical production, probably due to degradation of DMPO radical adducts.     

Importance of hydroxyl radical in the vanadium-stimulated oxidation of NADH

Vanadium compounds are known to stimulate the oxidation of NAD(P)H, but the mechanism remains unclear. This reaction was studied spectrophotometrically and by electron spin resonance spectroscopy (ESR) using vanadium in the reduced state (+4, vanadyl) and the oxidized state (+5, vanadate). In 25 mM sodium phosphate buffer at pH 7.4, vanadyl was slightly more effective in stimulating NADH oxidation than was vanadate. Addition of a superoxide generating system, xanthine/xanthine oxidase, resulted in a marked increase in NADH oxidation by vanadyl, and to a lesser extent, by vanadate. Decreasing the pH with superoxide present increased NADH oxidation for both vanadate and vanadyl. Addition of hydrogen peroxide to the reaction mixture did not change the NADH oxidation by vanadate, regardless of concentration or pH. With vanadyl however, addition of hydrogen peroxide greatly enhanced NADH oxidation which further increased with lower pH. Use of the spin trap DMPO in reaction mixtures containing vanadyl and hydrogen peroxide or a superoxide generating system resulted in the detection by ESR of hydroxyl. In each case, the hydroxyl radical signal intensity increased with vanadium concentration. Catalase was able to inhibit the formation of the DMPO--OH adduct formed by vanadate plus superoxide. These results show that the ability of vanadium to act in a Fenton-type reaction is an important process in the vanadium-stimulated oxidation of NADH.     

Cyanide catalyzes the oxidation of alpha-hydroxyaldehydes and related compounds: monitored as the reduction of dioxygen, cytochrome c, and nitroblue tetrazolium

Cyanide catalyzed the oxidation of α-hydroxycarbonyls and of related compounds. In the cases of glyceraldehyde 3-phosphate and of dihydroxyacetone phosphate the tautomeric enediol was the obligatory intermediate which reacted with cyanide yielding the active reductant. Cytochrome c, nitroblue tetrazolium, and dioxygen were all reduced by this reductant. In the case of dioxygen the product was the superoxide radical which could then secondarily reduce cytochrome c or nitroblue tetrazolium. In air-equilibrated reaction mixtures, at 25 °C, approximately 35% of cytochrome c reduction and 95% of nitroblue tetrazolium reduction was mediated by superoxide, as judged from susceptibilities to inhibition by superoxide dismutase. Since the oxidations observed were univalent, carbon-centered radicals appear to be necessary intermediates, and their secondary reactions generated a multiplicity of products, seen as smears on thin-layer chromatograms. Free cyanide must be regenerated during these secondary reactions, since cyanide functioned catalytically in the overall process. A partial mechanism has been proposed in explanation of these observations.     

Toxicity to rainbow trout and minnows of some substances known to be present in waste water discharged to rivers

Toxicity to fish (rainbow trout or minnows) of solutions of several pure substances has been measured under controlled conditions. The substances (sodium arsenite, sodium arsenate, sodium picrate, sodium dinitrophenate, zinc sulphate, potassium chromate, potassium dichromate, ammonium chloride, and ammonium sulphate) were dissolved in distilled water, in Watford tap water, or in mixtures of distilled water and tap water.     

The chromate-inducible chrBACF operon from the transposable element TnOtChr confers resistance to chromium(VI) and superoxide

Large-scale industrial use of chromium(VI) has resulted in widespread contamination with carcinogenic chromium(VI). The abilities of microorganisms to survive in these environments and to detoxify chromate require the presence of specific resistance systems. Here we report identification of the transposon-located (TnOtChr) chromate resistance genes from the highly tolerant strain Ochrobactrum tritici 5bvl1 surviving chromate concentrations of >50 mM. The 7,189-bp-long TnOtChr of the mixed Tn21/Tn3 transposon subfamily contains a group of chrB, chrA, chrC, and chrF genes situated between divergently transcribed resolvase and transposase genes. The chrB and chrA genes, but not chrF or chrC, were essential for establishment of high resistance in chromium-sensitive O. tritici. The chr promoter was strongly induced by chromate or dichromate, but it was completely unresponsive to Cr(III), oxidants, sulfate, or other oxyanions. Plasmid reporter experiments identified ChrB as a chromate-sensing regulator of chr expression. Induction of the chr operon suppressed accumulation of cellular Cr through the activity of a chromate efflux pump encoded by chrA. Expression of chrB, chrC, or chrF in an Escherichia coli sodA sodB double mutant restored its aerobic growth in minimal medium and conferred resistance to superoxide-generating agents menadione and paraquat. Nitroblue tetrazolium staining on native gels showed that ChrC protein had superoxide dismutase activity. TnOtChr appears to represent a mobile genetic system for the distribution of the chromate-regulated resistance operon. The presence of three genes protecting against superoxide toxicity should provide an additional survival advantage to TnOtChr-containing cells in the environments with multiple redox-active contaminants.