Chromium incorporated in RNA and DNA

The objective of this study was to examine the effect of Cr(III) (chromium chloride) and Cr(VI) (potassium dichromate) on RNA and DNA-chromium adducts formation in isolated nucleic acids and isolated pig lymphocytes. The incubation of cells with potassium dichromate and chromium chloride at concentrations of 10 and 100 microM results in binding of a 1.2-1.9 fold greater number of chromium atoms to nuclear DNA than to total cellular RNA. The incubation of total cellular RNA and nuclear DNA isolated from lymphocytes with CrCl3 and K2Cr2O7 yielded a binding of 1.1-1.6 fold more of Cr atoms to RNA than to DNA. The number of chromium atoms bound to nucleic acids is higher after incubation with K2Cr2O7 than with CrCl3 in both experimental systems.     

Genotoxicity of chromium compounds. A review

This article reviews approximately 700 results reported in the literature with 32 chromium compounds assayed in 130 short-term tests, using different targets and/or genetic end-points. The large majority of the results obtained with Cr(VI) compounds were positive, as a function of Cr(VI) solubility and bioavailability to target cells. On the other hand, Cr(III) compounds, although even more reactive than Cr(VI) with purified nucleic acids, did not induce genotoxic effects in the majority of studies using intact cells. Coupled with the findings of metabolic studies, the large data-base generated in short-term test systems provides useful information for predicting and interpreting the peculiar patterns of Cr(VI) carcinogenicity.     

Incision of trivalent chromium [Cr(III)]-induced DNA damage by Bacillus caldotenax UvrABC endonuclease

Some hexavalent chromium [Cr(VI)]-containing compounds are lung carcinogens. Once within cells, Cr(VI) is reduced to trivalent chromium [Cr(III)] which displays an affinity for both DNA bases and the phosphate backbone. A diverse array of genetic lesions is produced by Cr including Cr-DNA monoadducts, DNA interstrand crosslinks (ICLs), DNA-Cr-protein crosslinks (DPCs), abasic sites, DNA strand breaks and oxidized bases. Despite the large amount of information available on the genotoxicity of Cr, little is known regarding the molecular mechanisms involved in the removal of these lesions from damaged DNA. Recent work indicates that nucleotide excision repair (NER) is involved in the processing of Cr-DNA adducts in human and rodent cells. In order to better understand this process at the molecular level and begin to identify the Cr-DNA adducts processed by NER, the incision of CrCl(3) [Cr(III)]-damaged plasmid DNA was studied using a thermal-resistant UvrABC NER endonuclease from Bacillus caldotenax (Bca). Treatment of plasmid DNA with Cr(III) (as CrCl(3)) increased DNA binding as a function of dose. For example, at a Cr(III) concentration of 1 microM we observed approximately 2 Cr(III)-DNA adducts per plasmid. At this same concentration of Cr(III) we found that approximately 17% of the plasmid DNA contained ICLs ( approximately 0.2 ICLs/plasmid). When plasmid DNA treated with Cr(III) (1 microM) was incubated with Bca UvrABC we observed approximately 0.8 incisions/plasmid. The formation of endonuclease IV-sensitive abasic lesions or Fpg-sensitive oxidized DNA bases was not detected suggesting that the incision of Cr(III)-damaged plasmid DNA by UvrABC was not related to the generation of oxidized DNA damage. Taken together, our data suggest that a sub-fraction of Cr(III)-DNA adducts is recognized and processed by the prokaryotic NER machinery and that ICLs are not necessarily the sole lesions generated by Cr(III) that are substrates for NER.     

A comparison of the in vitro genotoxicity of tri- and hexavalent chromium

Chromium can be found in the environment in two main valence states: hexavalent (Cr(VI)) and trivalent (Cr(III)). Cr(VI) salts are well known human carcinogens, but the results from in vitro studies are often conflicting. Cr(VI) primarily enters the cells and undergoes metabolic reduction; however, the ultimate product of this reduction, Cr(III) predominates within the cell. In the present work, we compared the effects of tri- and hexavalent chromium on the DNA damage and repair in human lymphocytes using the alkaline single cell gel electrophoresis (comet assay). Potassium dichromate induced DNA damage in the lymphocytes, measured as the increase in comet tail moment. The effect was dose-dependent. Treated cells were able to recover within a 120-min incubation. Cr(III) caused greater DNA migration than Cr(VI). The lymphocytes did not show measurable DNA repair. Vitamin C at 50 microM reduced the extent of DNA migration. This was either due to a decrease in DNA strand breaks and/or alkali labile sites induced by Cr(VI) or to the formation of DNA crosslinks by Cr(VI) in the presence of vitamin C. Vitamin C, however, did not modify the effects of Cr(III). Catalase, an enzyme inactivating hydrogen peroxide, decreased the extent of DNA damage induced by Cr(VI) but not the one induced by Cr(III). Lymphocytes exposed to Cr(VI) and treated with endonuclease III, which recognizes oxidized pyrimidines, displayed greater extent of DNA damage than those not treated with the enzyme. Such an effect was not observed when Cr(III) was tested. The results obtained suggest that reactive oxygen species and hydrogen peroxide may be involved in the formation of DNA lesions by hexavalent chromium. The comet assay did not indicate the involvement of oxidative mechanisms in the DNA-damaging activity of trivalent chromium and we speculate that its binding to cellular ligands may play a role in its genotoxicity.     

Genotoxicity of Tri- and Hexavalent Chromium Compounds In Vivo and Their Modes of Action on DNA Damage In Vitro

Chromium occurs mostly in tri- and hexavalent states in the environment. Hexavalent chromium [Cr(VI)] compounds are extensively used in diverse industries, and trivalent chromium [Cr(III)] salts are used as micronutrients and dietary supplements. In the present work, we report that they both induce genetic mutations in yeast cells. They both also cause DNA damage in both yeast and Jurkat cells and the effect of Cr(III) is greater than that of Cr(VI). We further show that Cr(III) and Cr(VI) cause DNA damage through different mechanisms. Cr(VI) intercalates DNA and Cr(III) interferes base pair stacking. Based on our results, we conclude that Cr(III) can directly cause genotoxicity in vivo.     

A comparative study of calf thymus DNA binding to Cr(III) and Cr(VI) ions. Evidence for the guanine N-7-chromium-phosphate chelate formation

Chromium(VI) salts are well known to be mutagens and carcinogens and to easily cross the cell membranes. Because they are powerful oxidizing agents, Cr(VI) reacts with intracellular materials to reduce to trivalent form, which binds DNA. This study was designed to investigate the interaction of calf thymus DNA with Cr(VI) and Cr(III) in aqueous solution at pH 6.5-7.5, using Cr(VI)/DNA(P) molar ratios (r) of 1:20 to 2:1 and Cr(III)/DNA(P) molar ratios (r) of 1:80 to 1:2. UV-visible and Fourier transform infrared (FTIR) difference spectroscopic methods were used to determine the metal ion-binding sites, binding constants, and the effect of cation complexation on DNA secondary structure. Spectroscopic results showed no interaction of Cr(VI) with DNA at low anion concentrations (r = 1:20 to 1:1), whereas some perturbations of DNA bases and backbone phosphate were observed at very high Cr(VI) contents (r > 1) with overall binding constant of K = 508 M(-1). Cr(III) chelates DNA via guanine N-7 and the nearest PO(2) group with overall binding constant of K = 3.15 x 10(3) M(-1). Evidence for cation chelate formation comes from major shiftings and intensity variations of the guanine band at 1717 and the phosphate asymmetric stretching vibration at 1222 cm(-1). At low Cr(III) concentration (r = 1:40), the number of Cr(III) ions bound to DNA were 6-7 cations/500 base pairs, and this increased to 30-35 cations/500 base pairs at high metal ion content (r = 1:4). DNA condensation occurred at high cation concentration (r = 1:10). No major alteration of DNA conformation was observed, and the biopolymer remained in the B family structure upon chromium complexation.     

Chromium-induced excretion of urinary lipid metabolites,DNA damage,nitric oxide production,and generation of reactive oxygen species in Sprague-Dawley rats

Chromium and its salts induce cytotoxicity and mutagenesis, and vitamin E has been reported to attenuate chromate-induced cytotoxicity. These observations suggest that chromium produces reactive oxygen species which may mediate many of the untoward effects of chromium. We have therefore examined and compared the effects of Cr(III) (chromium chloride hexahydrate) and Cr(VI) (sodium dichromate) following single oral doses (0.50 ld₅₀) on the production of reactive oxygen species by peritoneal macrophages, and hepatic mitochondria and microsomes in rats. The effects of Cr(III) and Cr(VI) on hepatic mitochondrial and microsomal lipid peroxidation and enhanced excretion of urinary lipid metabolites as well as the incidence of hepatic nuclear DNA damage and nitric oxide (NO) production were also examined. Increases in lipid peroxidation of 1.8- and 2.2-fold occurred in hepatic mitochondria and microsomes, respectively, 48 hr after the oral administration of 25 mg Cr(VI)/kg, while increases of 1.2- and 1.4-fold, respectively, were observed after 895 mg Cr(III)/kg. The urinary excretion of malondialdehyde (MDA), formaldehyde (FA), acetaldehyde (ACT) and acetone (ACON) were determined at 0–96 hr after Cr administration. Between 48 and 72 hr post-treatment, maximal excretion of the four urinary lipid metabolites was observed with increases of 1.5- to 5.4-fold in Cr(VI) treated rats. Peritoneal macrophages from Cr(VI) treated animals 48 hr after treatment resulted in 1.4- and 3.6-fold increases in chemiluminescence and iodonitrotetrazolium reduction, indicating enhanced production of Superoxide anion, while macrophages from Cr(III) treated animals showed negligible increases. Increases in DNA single strand breaks of 1.7-fold and 1.5-fold were observed following administration of Cr(VI) and Cr(III), respectively, at 48 hr post-treatment. Enhanced production of NO by peritoneal exudate cells (primarily macrophages) was monitored following Cr(VI) administration at both 24 and 48 hr post-treatment with enhanced production of NO being observed at both timepoints. The results indicate that both Cr(VI) and Cr(III) induce an oxidative stress at equitoxic doses, while Cr(VI) induces greater oxidative stress in rats as compared with Cr(III) treated animals.     

Use of synchrotron- and plasma-based spectroscopic techniques to determine the uptake and biotransformation of chromium(III) and chromium(VI) by Parkinsonia aculeata

In this study, a combination of inductively coupled plasma optical emission spectroscopy and X-ray absorption spectroscopy (XAS) was used to study the uptake and speciation of chromium in Parkinsonia aculeata, commonly known as Mexican Palo Verde. Plants were treated for 14 days in a modified Hoagland solution containing chromium(III) or chromium(VI) at several concentrations. The results showed that plants treated with 70 mg Cr(III) L(-1) and 30 mg Cr(VI) L(-1) had similar Cr concentrations in leaves (～200 mg kg(-1) dry weight, DW). The results also showed that neither Cr(III) nor Cr(VI) affected the uptake of phosphorus and sulfur. However, the concentration of calcium in the stems of plants treated with Cr(VI) at 40 mg L(-1) (about 6000 mg Ca kg(-1) DW) was significantly higher compared to the Ca concentration (about 3000 mg kg(-1) DW) found in the stems of plants treated with 150 mg Cr(III) L(-1). However, no differences were observed in potassium and magnesium concentrations. The iron concentration (about 1000 mg kg(-1) DW) in roots treated with 40 mg Cr(VI) L(-1) was similar to the iron concentration found in the roots of plants treated with 110 mg Cr(III) L(-1). The XAS data showed that Cr(VI) was reduced to Cr(III) in/on the plant roots and transported as Cr(III) to the stems and leaves. The XAS studies also showed that Cr(III) within plants was present as an octahedral complex.     

Cr(III) exerts stronger structural effects than Cr(VI) on the human erythrocyte membrane and molecular models

Chromium exists in many oxidation states, of which only the hexavalent Cr(VI) and the trivalent Cr(III) ions are stable under environmental conditions. It is generally reported that Cr(VI) is highly toxic while Cr(III) is relatively innocuous, although others have reported just the opposite. On the other hand, despite the many studies on chromium toxicity, and particularly after the knowledge that Cr(VI) anions readily enter the erythrocytes where they are reduced to Cr(III), there are practically no reports on the structural effects induced by chromium compounds on the erythrocyte membrane. With the aim to better understand the molecular mechanisms of the interaction of Cr(III) and Cr(VI) with cell membranes, CrCl(3), and K(2)CrO(4) were incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM), and molecular models of the erythrocyte membrane. These consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylcholine (DMPE), phospholipid classes present in the outer and inner monolayers of the erythrocyte membrane, respectively. The capacity of Cr(III) and Cr(VI) to perturb the bilayer structures of DMPC and DMPE was evaluated by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, and intact human erythrocytes were observed with scanning electron microscopy (SEM). In all these systems, it was found that Cr(III) induced considerably higher structural perturbations than Cr(VI).     

Incision of trivalent chromium [Cr(III)]-induced DNA damage by Bacillus caldotenax UvrABC endonuclease

Some hexavalent chromium [Cr(VI)]-containing compounds are lung carcinogens. Once within cells, Cr(VI) is reduced to trivalent chromium [Cr(III)] which displays an affinity for both DNA bases and the phosphate backbone. A diverse array of genetic lesions is produced by Cr including Cr–DNA monoadducts, DNA interstrand crosslinks (ICLs), DNA–Cr–protein crosslinks (DPCs), abasic sites, DNA strand breaks and oxidized bases. Despite the large amount of information available on the genotoxicity of Cr, little is known regarding the molecular mechanisms involved in the removal of these lesions from damaged DNA. Recent work indicates that nucleotide excision repair (NER) is involved in the processing of Cr–DNA adducts in human and rodent cells. In order to better understand this process at the molecular level and begin to identify the Cr–DNA adducts processed by NER, the incision of CrCl₃ [Cr(III)]-damaged plasmid DNA was studied using a thermal-resistant UvrABC NER endonuclease from Bacillus caldotenax (Bca). Treatment of plasmid DNA with Cr(III) (as CrCl₃) increased DNA binding as a function of dose. For example, at a Cr(III) concentration of 1 μM we observed 2 Cr(III)–DNA adducts per plasmid. At this same concentration of Cr(III) we found that 17% of the plasmid DNA contained ICLs (0.2 ICLs/plasmid). When plasmid DNA treated with Cr(III) (1 μM) was incubated with Bca UvrABC we observed 0.8 incisions/plasmid. The formation of endonuclease IV-sensitive abasic lesions or Fpg-sensitive oxidized DNA bases was not detected suggesting that the incision of Cr(III)-damaged plasmid DNA by UvrABC was not related to the generation of oxidized DNA damage. Taken together, our data suggest that a sub-fraction of Cr(III)–DNA adducts is recognized and processed by the prokaryotic NER machinery and that ICLs are not necessarily the sole lesions generated by Cr(III) that are substrates for NER.     

Critical role of chromium (Cr)-DNA interactions in the formation of Cr-induced polymerase arresting lesions

The genotoxicity associated with the metabolic reduction of hexavalent chromium [Cr(VI)] is complex and can impede DNA polymerase-mediated replication in vitro. The exact biochemical nature of Cr-induced polymerase arresting lesions (PALs) is not understood, but is believed to involve the formation of Cr-DNA interstrand cross-links (ICLs). The aim of this investigation was to determine the dependence of direct Cr-DNA interactions on the development of PALs in DNA treated with trivalent Cr [Cr(III)] or with Cr(VI) in the presence of ascorbic acid (Asc), a major intracellular reductant, using an in vitro, acellular system. The formation of Cr-DNA adducts, ICLs, and PALs was maximal at Asc:Cr(VI) molar ratios of 0.5-2, but gradually decreased at higher ratios. EDTA, a Cr(III) chelator, significantly decreased Cr-DNA binding and ICL and PAL formation. Co-treatment of DNA with Cr(VI)/Asc and mannitol, a Cr(V) chelator, selectively inhibited the formation of mono/bifunctional DNA adducts and PALs produced by Cr(VI) reduction, but had no effect on Cr(III)-DNA binding or Cr(III)-induced polymerase arrest. Blocking Cr-DNA phosphate interaction by preincubation of DNA with MgCl(2) abrogated DNA binding and ICL and PAL production. DNA strand breaks and abasic sites may lead to the in vitro arrest of DNA polymerases; however, we failed to detect significant increases in the frequency of these lesions following Cr(VI)/Asc treatment. These data indicate that the bifunctional adduction of Cr to DNA phosphates (ICLs) constitutes a major PAL. Furthermore, the generation of DNA strand breaks and abasic sites by Cr(VI) reduction is insufficient to explain PALs observed in vitro.     

微生物铬转化和抗性机制与生物修复研究进展

铬(Chromium,Cr)是过渡金属元素,在自然界中以六价[CrO_4~(2-),Cr_2O_7~(2-),Cr(Ⅵ)]和三价[Cr(OH)_3,Cr(Ⅲ)]为主。很多微生物在长期铬胁迫的条件下,进化出了一系列铬转化和抗性机制。微生物对铬的转化包括Cr(Ⅵ)的还原和Cr(Ⅲ)的氧化。微生物的Cr(Ⅵ)还原可以将毒性强的六价铬转化为毒性弱或无毒的三价铬,这类微生物有较强的土壤和水体铬污染治理潜力。Cr(Ⅲ)的氧化也在铬的生物地球化学循环过程中起着至关重要的作用。除了Cr(Ⅵ)的还原,微生物对铬的抗性机制还有:(1)减少摄入;(2)外排;(3)清除胞内氧化压力;(4)DNA修复。本文主要介绍微生物的铬转化和抗性机制,以及其在铬污染生物修复中应用的最新研究进展。     

The interaction of chromium with nucleic acids

Native and denatured calf thymus DNA, and homopolyribonucleotides were compared with respect to chromium and protein binding after an in vitro incubation with rat liver microsomes, NADPH, and chromium(VI) or chromium(III). A significant amount of chromium bound to DNA when chromium(VI) was incubated with the native or the denatured form of DNA in the presence of microsomes and NADPH. For both native and denatured DNA the amount of protein bound to DNA increased with the amount of chromium bound to DNA. Denatured DNA had much higher amounts of chromium and protein bound than native DNA. There was no interaction between chromium(VI) and either form of DNA in the absence of the complete microsomal reducing system. The binding of chrornium(III) to native or denatured DNA was small and relatively unaffected by the presence of microsomes and NADPH. The binding of chromium and protein to polyriboadenylic acid (poly(A)), polyribocytidylic acid (poly(C), polyri-boguanylic acid (poly(G)) and polyribouridylic acid (poly(U)) was determined after incubation with chromium(VI) in the presence of microsomes and NADPH. The magnitude of chromium and protein binding to the ribo-polymers was found to be poly(G) ? poly(A) ? poly(C) ? poly(U). These results suggest that the metabolism of chromium(VI) is necessary in order for chromium to interact significantly with nucleic acids. The metabolically-produced chromium preferentially binds to the base guanine and results in DNA-protein cross-links. These findings are discussed with respect to the proposed scheme for the carcinogenicity of chromium(VI). Keywords: DNA-protein cross-links — Chromium-guanine interaction-Microsomal reduction of chromate     

Human nucleotide excision repair efficiently removes chromium-DNA phosphate adducts and protects cells against chromate toxicity

Intracellular reduction of carcinogenic Cr(VI) leads to the extensive formation of Cr(III)-DNA phosphate adducts. Repair mechanisms for chromium and other DNA phosphate-based adducts are currently unknown in human cells. We found that nucleotide excision repair (NER)-proficient human cells rapidly removed chromium-DNA adducts, with an average t((1/2)) of 7.1 h, whereas NER-deficient XP-A, XP-C, and XP-F cells were severely compromised in their ability to repair chromium-DNA lesions. Activation of NER in Cr(VI)-treated human fibroblasts or lung epithelial H460 cells was manifested by XPC-dependent binding of the XPA protein to the nuclear matrix, which was also observed in UV light-treated (but not oxidant-stressed) cells. Intracellular replication of chromium-modified plasmids demonstrated increased mutagenicity of binary Cr(III)-DNA and ternary cysteine-Cr(III)-DNA adducts in cells with inactive NER. NER deficiency created by the loss of XPA in fibroblasts or by knockdown of this protein by stable expression of small interfering RNA in H460 cells increased apoptosis and clonogenic death by Cr(VI), providing genetic evidence for the role of monofunctional chromium-DNA adducts in the toxic effects of this metal. The rate of NER of chromium-DNA adducts under saturating conditions was calculated to be approximately 50,000 lesions/min/cell. Because chromium-DNA adducts cause only small changes in the DNA helix, rapid repair of these modifications in human cells indicates that the presence of major structural distortions in DNA is not required for the efficient detection of the damaged sites by NER proteins in vivo.     

Uptake of chromium(III) and chromium(VI) compounds in the yeast cell structure

The study presented in this article investigated the influence of different Cr(III) and Cr(VI) compounds in the cultivation medium on the uptake and localization of chromium in the cell structure of the yeast Candida intermedia. The morphology of the yeast cell surface was observed by the scanning electron microscopy. Results demonstrated that the growth inhibitory concentration of Cr(III) in the cultivation medium induced changes in the yeast cell shape and affected the budding pattern, while inhibitory concentration of Cr(VI) did not cause any visible effects on morphological properties of the yeast cells. The amount of total accumulated chromium in yeast cells and the distribution of chromium between the yeast cell walls and spheroplasts were determined by atomic absorption spectroscopy. No significant differences were found neither in total chromium accumulation nor in the distribution of chromium in yeast cell walls and spheroplasts between the two of Cr(VI) compounds. Conversely, substantial differences between Cr(III) compounds were demonstrated in the total uptake as well as the localization of chromium in yeast cells.     

Enhanced exopolymer production and chromium stabilization in Pseudomonas putida unsaturated biofilms

Chromium-contaminated soils threaten surface and groundwater quality at many industrial sites. In vadose zones, indigenous bacteria can reduce Cr(VI) to Cr(III), but the subsequent fate of Cr(III) and the roles of bacterial biofilms are relatively unknown. To investigate, we cultured Pseudomonas putida, a model organism for vadose zone bioremediation, as unsaturated biofilms on membranes overlaying iron-deficient solid media either containing molecular dichromate from potassium dichromate (Cr-only treatment) or with deposits of solid, dichromate-coated hematite (Fe+Cr treatment) to simulate vadose zone conditions. Controls included iron-deficient solid medium and an Fe-only treatment using solid hematite deposits. Under iron-deficient conditions, chromium exposure resulted in lower cell yield and lower amounts of cellular protein and carbohydrate, but providing iron in the form of hematite overcame these toxic effects of Cr. For the Cr and Fe+Cr treatments, Cr(VI) was completely reduced to Cr(III) that accumulated on biofilm cells and extracellular polymeric substances (EPSs). Chromium exposure resulted in elevated extracellular carbohydrates, protein, DNA, and EPS sugars that were relatively enriched in N-acetyl-glucosamine, rhamnose, glucose, and mannose. The proportions of EPS protein and carbohydrate relative to intracellular pools suggested Cr toxicity-mediated cell lysis as the origin. However, DNA accumulated extracellularly in amounts far greater than expected from cell lysis, and Cr was liberated when extracted EPS was treated with DNase. These results demonstrate that Cr accumulation in unsaturated biofilms occurs with enzymatic reduction of Cr(VI), cellular lysis, cellular association, and extracellular DNA binding of Cr(III), which altogether can facilitate localized biotic stabilization of Cr in contaminated vadose zones.     

Effect of Rhamnolipids on Chromium-Contaminated Kaolinite

Hexavalent chromium Cr(VI) is a common environmental pollutant that is treated by its reduction to the trivalent form Cr(III). The latter can be re-oxidized to the toxic form, Cr(VI), under specific conditions. A study was conducted on the removal of Cr(III) to eliminate the hazard imposed by its presence in soil as there has been some evidence that organic compounds can decrease its sorption. The effect of addition of negatively-charged biosurfactants (rhamnolipids) on chromium contaminated kaolinite was studied. Results showed that the rhamnolipids have the capability of extracting 25% portion of the stable form of chromium, Cr(III), from the kaolinite, under optimal conditions. The removal of hexavalent chromium was also enhanced compared to water by a factor of 2 using a solution of rhamnolipids. Results from the sequential extraction procedure showed that rhamnolipids remove Cr(III) mainly from the carbonate and oxide/hydroxide portions of the kaolinite. The rhamnolipids had also the capability of reducing close to 100% of the extracted Cr(VI) to Cr(III) over a period of 24 days. This study indicated that rhamnolipids could be beneficial for the removal or long–term conversion of chromium Cr(VI) to Cr(III).     

Modulation of chromium(VI) toxicity by organic and inorganic sulfur species in yeasts from industrial wastes

Two chromium(VI) resistant yeast strains (Candida sp. and Rhodosporidium sp.) were isolated from industrial wastes. Four different yeasts, three from the Industrial Yeast Collection and one of pharmaceutical origin, were also studied in relation to chromate toxicity and its alleviation by sulfur species. The growth of yeasts from industrial wastes was inhibited by 50% by high concentrations of Cr(VI): Candida sp. by 4 mM Cr(VI) and Rhodosporidium sp. by 10 mM Cr(VI) in Sabouraud Broth medium. The other Cr(VI)-sensitive yeasts were inhibited by 0.1 mM Cr(VI). The general mechanism of chromium resistance in Candida sp. and Rhodosporidium sp. was due to reduced uptake of chromium, but not to biological reduction from Cr(VI) to Cr(III). In Cr(VI)-sensitive yeasts, chromium was accumulated as much as 10-fold, as in Saccharomyces cerevisiae. Cr(VI) toxicity in Candida sp. was modulated from Cr(VI)-resistance to Cr(VI)-hypersensitivity depending on the addition of methionine, cysteine, sulfate and djenkolic acid. If Candida sp. was grown in the presence of S-amino acids, especially methionine, it was more resistant than if the sulfur source was sulfate. When sulfate transport was enhanced by addition of djenkolic acid, Candida sp. became hypersensitive. Rhosporidium sp. was always resistant to Cr(VI) because sulfate transport was inefficient and it assimilated sulfur as S-amino acids. Cr(VI)-sensitive yeasts required larger amounts of S-amino acids, especially methionine, to tolerate Cr(VI) toxicity. Cysteine was toxic for C.famata 6016 above 50 microM.     

Role of Bacillus subtilis Error Prevention Oxidized Guanine System in Counteracting Hexavalent Chromium-Promoted Oxidative DNA Damage

Chromium pollution is potentially detrimental to bacterial soil communities, compromising carbon and nitrogen cycles that are essential for life on earth. It has been proposed that intracellular reduction of hexavalent chromium [Cr(VI)] to trivalent chromium [Cr(III)] may cause bacterial death by a mechanism that involves reactive oxygen species (ROS)-induced DNA damage; the molecular basis of the phenomenon was investigated in this work. Here, we report that Bacillus subtilis cells lacking a functional error prevention oxidized guanine (GO) system were significantly more sensitive to Cr(VI) treatment than cells of the wild-type (WT) strain, suggesting that oxidative damage to DNA is involved in the deleterious effects of the oxyanion. In agreement with this suggestion, Cr(VI) dramatically increased the ROS concentration and induced mutagenesis in a GO-deficient B. subtilis strain. Alkaline gel electrophoresis (AGE) analysis of chromosomal DNA of WT and ΔGO mutant strains subjected to Cr(VI) treatment revealed that the DNA of the ΔGO strain was more susceptible to DNA glycosylase Fpg attack, suggesting that chromium genotoxicity is associated with 7,8-dihydro-8-oxodeoxyguanosine (8-oxo-G) lesions. In support of this notion, specific monoclonal antibodies detected the accumulation of 8-oxo-G lesions in the chromosomes of B. subtilis cells subjected to Cr(VI) treatment. We conclude that Cr(VI) promotes mutagenesis and cell death in B. subtilis by a mechanism that involves radical oxygen attack of DNA, generating 8-oxo-G, and that such effects are counteracted by the prevention and repair GO system.