Chromium reduction in Pseudomonas putida.

Reduction of hexavalent chromium (chromate) to less-toxic trivalent chromium was studied by using cell suspensions and cell-free supernatant fluids from Pseudomonas putida PRS2000. Chromate reductase activity was associated with soluble protein and not with the membrane fraction. The crude enzyme activity was heat labile and showed a Km of 40 microM CrO4(2-). Neither sulfate nor nitrate affected chromate reduction either in vitro or with intact cells.     

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.     

Reduction and precipitation of chromate by mixed culture sulphate-reducing bacterial biofilms

The ability of sulphate-reducing bacterial biofilms to reduce hexavalent chromium (Cr(VI)) to insoluble Cr(III), a process of environmental and biotechnological significance, was investigated. The reduction of chromate to insoluble form has been quantified and the effects of chromate on the carbon source utilization and sulphate-reducing activity of the bacterial biofilms evaluated. Using lactate as the carbon/energy source and in the presence of sulphate, reduction of 500 micromol l-1 Cr(VI) was monitored over a 48-h period where 88% of the total chromium was removed from solution. Mass balance calculations showed that ca 80% of the total chromium was precipitated out of solution with the bacterial biofilm retaining less than 10% of the chromium. Only ca 12% of the chromate added was not reduced to insoluble form. Although Cr(VI) did not have a significant effect on C source utilization, sulphate reduction was severely inhibited by 500 micromol-1 Cr(VI) and only ca 10% of the sulphate reducing activity detected in control biofilms occurred in the presence of Cr(VI). Low levels of sulphide were also produced in the presence of chromate, with control biofilms producing over 10-times more sulphide than Cr(VI)-exposed biofilms. Sulphide- or other chemically-mediated Cr(VI) reduction was not detected. The biological mechanism of Cr(VI) reduction is likely to be similar to that found in other sulphate-reducing bacteria.     

Membrane-associated chromate reductase activity from Enterobacter cloacae.

Washed cells of Enterobacter cloacae HO1 reduced hexavalent chromium (chromate: CrO4(2-) anaerobically. Chromate reductase activity was preferentially associated with the membrane fraction of the cells. Right-side-out membrane vesicles prepared from E. cloacae cells showed high chromate reductase activities when ascorbate-reduced phenazine methosulfate was added as an electron donor.     

Isolation and Characterization of an Enterobacter cloacae Strain That Reduces Hexavalent Chromium under Anaerobic Conditions

An Enterobacter cloacae strain (HO1) capable of reducing hexavalent chromium (chromate) was isolated from activated sludge. This bacterium was resistant to chromate under both aerobic and anaerobic conditions. Only the anaerobic culture of the E. cloacae isolate showed chromate reduction. In the anaerobic culture, yellow turned white with chromate and the turbidity increased as the reduction proceeded, suggesting that insoluble chromium hydroxide was formed. E. cloacae is likely to utilize toxic chromate as an electron acceptor anaerobically because (i) the anaerobic growth of E. cloacae HO1 accompanied the decrease of toxic chromate in culture medium, (ii) the chromate-reducing activity was rapidly inhibited by oxygen, and (iii) the reduction occurred more rapidly in glycerol- or acetate-grown cells than in glucose-grown cells. The chromate reduction in E. cloacae HO1 was observed at pH 6.0 to 8.5 (optimum pH, 7.0) and at 10 to 40°C (optimum, 30°C).     

Chromate metabolism in liver microsomes

The carcinogenicity and mutagenicity of various chromium compounds have been found to be markedly dependent on the oxidation state of the metal. The carcinogen chromate was reduced to chromium(III) by rat liver microsomes in vitro. Metabolism of chromate by microsomal enzymes occurred only in the presence of either NADPH or NADH as cofactor. The chromium(III) generated upon metabolism formed a complex with the NADP⁺ cofactor. Significant binding of chromium to DNA occurred only when chromate was incubated in the presence of microsomes and NADPH. Specific inhibitors of the mixed function oxidase enzymes, 2′-AMP, metyrapone, and carbon monoxide, inhibited the rate of reduction of chromate by microsomes and NADPH. The possible relationship of metabolism of chromate and its interaction with nucleic acids to its carcinogenicity and mutagenicity is discussed.     

Detection of the mutagenic activity of lead chromate using a battery of microbial tests.

The potential mutagenicity of the carcinogen lead chromate was tested by the following battery of microbial tests: the Escherichia coli PolA+/PolA- survival test; the Salmonella/microsome His+ reversion assay; the E. coli Trp+ reversion test as a plate assay; the E. coli Gal+ forward mutation test; and the Saccharomyces cerevisiae assay for mitotic recombination. Lead chromate is mutagenic in Salmonella and in Saccharomyces and is thus identified as a microbial mutagen by this battery. Metabolic activation by rat liver homogenate (S9) is not required for the mutagenic activity of lead chromate. The most statistically significant, positive result is found with a supplementary assay, the E. coli fluctuation test. To determine whether the lead ion and/or the chromate ion were responsible for the mutagenicity observed, lead chloride and chromium trioxide (chromic acid) were also tested. In E. coli fluctuation test, the ranges of maximal mutagenicity for chromium trioxide and lead chromate overlap at the concentration 10(-5)M, whereas lead chloride shows no mutagenicity and little lethality at concentrations up to 10(-3)M. Thus, it appears that the chromate ion is responsible for the mutagenicity of lead chromate.     

Chromate (VI) uptake by and interactions with cyanobacteria

Summary The short-term accumulation of chromate by the cyanobacteriaAnabaena variabilis andSynechococcus PCC 6301 has been described as consisting of a rapid and relatively low level of biosorption of chromate to the cell walls; no energy-dependent uptake was detected. This biosorption was dependent on chromate concentration and could be described by a Freundlich adsorption isotherm for both cyanobacterial species studied. Decreasing the external pH increased the chromate accumulation by both species. Over a longer time period with growth it was shown thatA. variabilis was capable of reducing chromate (VI) to chromium (III) and then accumulating the chromium (III).Synechococcus PCC 6301 showed no further interaction with chromate concentrations over the same time period after the initial biosorption.     

Purification and characterization of a highly active chromate reductase from endophytic Bacillus sp. DGV19 of Albizzia lebbeck (L.) Benth. actively involved in phytoremediation of tannery effluent-contaminated sites

Phytoremediation using timber-yielding tree species is considered to be the most efficient method for chromium/tannery effluent-contaminated sites. In this study, we have chosen Albizzia lebbeck, a chromium hyperaccumulator plant, and studied one of its chromium detoxification processes operated by its endophytic bacterial assemblage. Out of the four different groups of endophytic bacteria comprising Pseudomonas, Rhizobium, Bacillus, and Salinicoccus identified from A. lebbeck employed in phytoremediation of tannery effluent-contaminated soil, Bacillus predominated with three species, which exhibited not only remarkable chromium accumulation ability but also high chromium reductase activity. A chromate reductase was purified to homogeneity from the most efficient chromium accumulator, Bacillus sp. DGV 019, and the purified 34.2-kD enzyme was observed to be stable at temperatures from 20°C to 60°C. The enzyme was active over a wide range of pH values (4.0–9.0). Furthermore, the enzyme activity was enhanced with the electron donors NADH, followed by NADPH, not affected by glutathione and ascorbic acid. Cu²⁺ enhanced the activity of the purified enzyme but was inhibited by Zn²⁺ and etheylenediamine tetraacetic acid (EDTA). In conclusion, due to its versatile adaptability the chromate reductase can be used for chromium remediation.     

Mutagenic activity of copper(II) chromate and dichromate complexes with polypyridines

Copper(II) chromate and dichromate complexes with 2,2'-bipyridyl and 1,10-phenathroline were tested for their mutagenic activity in the standard Ames test. All of six tested complexes exhibited markedly lower mutagenic activity than the reference compounds--potassium dichromate and sodium chromate. The blockage of Cr(VI) reduction capability in the presence of the complex Cu2+ ion and the competition between copper and chromium ions in the interaction with cellular components are discussed in the light of the results of our previous chemical study.     

Effect of ascorbic acid on DNA damage, cytotoxicity, glutathione reductase, and formation of paramagnetic chromium in Chinese hamster V-79 cells treated with sodium chromate(VI).

The effect of pretreatment with ascorbic acid (vitamin C) on chromate-induced DNA damage, cytotoxicity, and enzyme inhibition as well as on the cellular reduction of chromium(VI) was investigated using Chinese hamster V-79 cells. Cellular pretreatment with nontoxic levels of 1 mM ascorbic acid for 24 h prior to exposure resulted in a significant increase (1.7-fold) in cellular levels of this vitamin. Alkaline elution assays demonstrated that this pretreatment decreased cellular levels of Na2CrO4-induced alkali-labile sites while the numbers of DNA-protein crosslinks produced by chromate increased. In colony-forming assays, pretreatment with ascorbic acid enhanced the cytotoxicity of chromate. However, the inhibition of glutathione reductase attributed to Na2CrO4 was attenuated by this pretreatment. Under the same experimental condition, the uptake of chromate in pretreated cells was found to increase. ESR studies revealed that cellular pretreatment with ascorbic acid reduced the level of chromium(V) intermediate and increased the level of chromium(III) complex, indicating that cellular reduction of chromium(VI) to chromium(III) was accelerated by this vitamin. These results suggest that ascorbic acid decreases chromate-induced alkali-labile sites and chromium inhibition of glutathione reductase, but it enhances DNA-protein cross-links and cytotoxicity caused by this metal through its ability to directly reduce chromium(VI).     

Interactions of Chromium (III) and Chromium (VI) with Bovine Serum Albumin Studied by UV Spectroscopy,Circular Dichroism,and Fluorimetry

In the present work, the interactions of bovine serum albumin (BSA) with chromium (III) chloride, potassium dichromate, and chromate were studied by fluorescence, circular dichroism, and UV–vis absorbance spectroscopy. Fluorescence quenching of BSA by chromium (III) was found to be a dynamic process in the beginning, turning static at later stages. Spectroscopic data show that both dichromate and chromate bind in similar electrostatic fashion to BSA and does not follow the fluorescence quenching observation for chromium (III).     

Chromium resistance strategies and toxicity: what makes <Emphasis Type="Italic">Ochrobactrum tritici</Emphasis> 5bvl1 a strain highly resistant

Large-scale industrial use of chromium (Cr) resulted in widespread environmental contamination with hexavalent chromium (Cr(VI)). The ability of microorganisms to survive in these environments and detoxify chromate requires the presence of specific resistance systems. Several Cr(VI) resistant species, belonging to a variety of genera, have been isolated in recent years. Ochrobactrum tritici strain 5bvl1 is a model for a highly Cr(VI)-resistant and reducing microorganism, with different strategies to cope with chromium. The strain contains the transposon-located (TnOtChr) chromate resistance genes chrB, chrA, chrC, chrF. The chrB and chrA genes were found to be essential for the establishment of high resistance but not chrC or chrF genes. Other mechanisms involved in chromium resistance in this strain were related to strategies such as specific or unspecific Cr(VI) reduction, free-radical detoxifying activities, and repairing DNA damage. Expression of the chrB, chrC or chrF genes was related to increased resistance to superoxide-generating agents. Genetic analyses also showed that, the ruvB gene is related to chromium resistance in O. tritici 5bvl1. The RuvABC complex probably does not form when ruvB gene is interrupted, and the repair of DNA damage induced by chromium is prevented. Aerobic or anaerobic chromate reductase activity and other unspecific mechanisms for chromium reduction have been identified in different bacteria. In the strain O. tritici 5bvl1, several unspecific mechanisms were found. Dichromate and chromate have different effects on the physiology of the chromium resistant strains and dichromate seems to be more toxic. Toxicity of Cr(VI) was evaluated by following growth, reduction, respiration, glucose uptake assays and by comparing cell morphology.     

The in vitro transformation of a rat liver epithelial cell line with chromium

The transformation of a rat liver epithelial cell line under a wide range of doses of chromium was determined by anchorage-independent growth and tumor formation in syngeneic animals. Chronic exposure to low concentrations and brief exposure to high concentrations of hexavalent chromium (K₂CrO₄) transformed the cells, but one dose (1 mM K₂CrO₄, 2h) was clearly optimal in this regard. The cytotoxicity, effects on cell cycle, rates of chromium uptake, and mutagenic activity under the different treatment conditions were evaluated. The results showed that cells could adapt to the presence of chromium under certain treatment conditions, but this was not the case for the optimal transforming dose. Cells treated with chromium above the optimal transforming dose showed evidence of a transient G2 arrest, whereas all lower levels of treatment did not. A low level continuous exposure to chromate was mutagenic, whereas high level short exposures, including the optimal transforming dose, were not. An increase in the amount of protein complexed with isolated nucleic acids was detected in cells following treatment with the optimal transforming dose of chromate. The results indicate that the effects of chromium on this in vitro system vary with dose; and the identification of those events relevant to metal carcinogenesis will require consideration of treatment conditions.     

Reduction of chromium(VI) in Chinese hamster V-79 cells

The cellular reduction of chromate(VI) was studied by electron spin resonance spectrometry. Incubation of Chinese hamster V-79 cells with Na2CrO4 resulted in the formation of both chromium(V) and chromium(III) complex in a manner dependent on time (30 min-2 h) and concentration (50-500 microM). Following removal of extracellular chromate, the level of chromium(V) complex decreased quickly during the first hour but more slowly for the next hour, whereas the level of chromium(III) remained unchanged, indicating that chromium(III) is the ultimate ion of this metal in cells. Alkaline elution studies demonstrated that treatment of cells with Na2CrO4 induced DNA single-strand breaks that decreased quickly and DNA-protein crosslinks that persisted for 2 h after removal of this metal. These results suggest that the cellular levels of chromium(V) and chromium(III) may be associated with the formation of DNA damage induced by chromium (VI).     

Effects of oxygen stress on chromate reduction in Enterobacter cloacae strain HO1

Enterobacter cloacae strain HO1 was able to reduce toxic hexavalent chromium (chromate) anaerobically. The reduction of chromate by E. cloacae cells was sensitive to oxygen stress. Cultures under continuous aeration showed no chromate reduction. However, when released from the oxygen stress, the cultures readily resumed chromate reduction.     

The cytotoxicity and genotoxicity of hexavalent chromium in Steller sea lion lung fibroblasts compared to human lung fibroblasts

In this study we directly compared soluble and particulate chromate cytotoxicity and genotoxicity in human (Homo sapiens) and sea lion (Eumetopias jubatus) lung fibroblasts. Our results show that hexavalent chromium induces increased cell death and chromosome damage in both human and sea lion cells with increasing intracellular chromium ion levels. The data further indicate that both sodium chromate and lead chromate are less cytotoxic and genotoxic to sea lion cells than human cells, based on an administered dose. Differences in chromium ion uptake explained some but not all of the reduced amounts of sodium chromate-induced cell death. By contrast, uptake differences could explain the differences in sodium chromate-induced chromosome damage and particulate chromate-induced toxicity. Altogether they indicate that while hexavalent chromium induces similar toxic effects in sea lion and human cells, there are different mechanisms underlying the toxic outcomes.     

Chromate reduction is expedited by bacteria engineered to produce the compatible solute trehalose

The toxicity and solubility of chromium(VI) can be decreased by certain microbes that reduce chromium(VI) to chromium(III). However, these bacteria do not escape unscathed from this process. Chromium(VI) reduction damages the essential macromolecules of living systems. Trehalose protects organisms from chemical stress but has not been tested in the context of bioremediation. We engineered bacteria to produce trehalose and found that they then reduced 1 mM chromium(VI) to chromium(III), whereas wild-type cells were only able to reduce half that amount. Thus, by providing bacteria with a biochemical defense against the side-effects of chromate reduction may be a new approach to cleaning up sites that are contaminated with high levels of chromate.     

Extra-cellular chromate-reducing activity of the yeast cultures

This paper reports on the experimental data supporting an essential role of extra-cellular reduction in chromate detoxification by baker’s and non-conventional yeasts. A decrease of chromate content in the yeast culture coincides with an increase of Cr(III) content in extra-cellular liquid. At these conditions, cell-bound chromium level was insignificant and a dominant part of extra-cellular Cr(III) species was detected in the reaction with chromazurol S only after mineralization of the cell-free samples. This phenomenon of chromium “disappearance” can be explained by the formation of Cr(III) stable complexes with extra-cellular yeast-secreted components which are “inaccessible” in the reaction with chromazurol S without mineralization. It was shown that increasing sucrose concentration in a growth medium resulted in an increase of chromate reduction. A strong inhibition of chromate reduction by 0.25 mM sodium azide, a respiration inhibitor and a protonophore, testifies that extra-cellular chromate detoxification depends on energetic status of the yeast cells. It was shown that Cr(III)-biochelates produced in extra-cellular medium are of a different chemical nature and can be separated into at least two components by ion-exchange chromatography on anionit Dowex 1x10. A total yield of the isolated Cr(III)-biocomplexes is approximately 65 % (from initial level of chromate) with a relative molar ratio 8:5.     

Cr(VI) reduction in a chromate-resistant strain of Candida maltosa isolated from the leather industry

A Cr(VI)-resistant yeast was isolated from tanning liquors from a leather factory in Leon, Guanajuato, Mexico. Based on morphological and physiological analyses and the D1/D2 domain sequence of the 26S rDNA, the yeast was identified as Candida maltosa. Resistance of the strain to high Cr(VI) concentrations and its ability to chemically reduce chromium was studied. When compared to the three laboratory yeasts Candida albicans, Saccharomyces cerevisiae and Yarrowia lipolytica, the C. maltosa strain was found to tolerate chromate concentrations as high as 100 micro g/ml. In addition to this phenotypic trait, the C. maltosa strain showed ability to reduce Cr(VI). Chromate reduction occurred both in intact cells (grown in culture medium or in soil containing chromate) as well as in cell-free extracts. NADH-dependent chromate reductase activity was found associated with soluble protein and, to a lesser extent, with the membrane fraction.