Intracellular Reduction Of Cr Research Articles

Interactions between chromium species and DNA in vitro and their potential role in the toxicity of hexavalent chromium.

Epidemiological and animal studies have supported the carcinogenicity of hexavalent chromium [Cr(VI)]; however, molecular changes responsible for the induction of cancer by Cr(VI) are not entirely understood. Numerous mechanistic studies suggested the role of oxidative stress and genotoxicity in Cr(VI)-mediated carcinogenesis; however, specific types of DNA damage have not yet been conclusively attributed to specific chromium species or other reactive byproducts generated in biological systems exposed to Cr(VI). Due to the remarkably complex chemistry and biological effects of chromium species generated through the intracellular reduction of Cr(VI), their relevance for Cr(VI)-mediated carcinogenesis has not yet been fully elucidated and continues to be a subject of ongoing discussions in the field. In this report, we describe a complex world of chromium species and their reactivity with DNA and other biologically relevant molecules in vitro to inform a more complete understanding of Cr(VI)-mediated toxicity. In addition, we discuss previous results in the context of in vitro models and analytical methods to reconcile some conflicting findings on the biological role of chromium species.

Intracellular uptake and reduction of hexavalent chromium by the cyanobacterium Synechocystis sp. PUPCCC 62

A hexavalent chromium-tolerant cyanobacterial strain isolated from the Satluj River, Ludhiana, India was identified as Synechocystis sp. strain PUPCCC 62 based on the 16S rRNA gene and phycocyanin cpcBA-intergenic spacer sequence analysis. Cr IC50 for the organism was determined to be 100 μmol L−1. The organism removed nearly four times more Cr(VI) from imidazole-HCl buffer (200 mmol L−1, pH 6.0) (250 nmol Cr(VI) mg−1 protein) compared with basal medium (59 nmol Cr(VI) mg−1 protein) in 8 h. Temperature of 28 °C, pH of 6.0, and biomass load of 200 μg protein mL−1 were found to be the optimum parameters for Cr(VI) bioremoval from the buffer solution. Cr(VI) bioremoval by the cyanobacterium was light/photosynthesis dependent. Along with Cr(VI) bioremoval, its reduction to Cr(III) was also demonstrated. In a 6-h period, the amount of Cr(VI) in the solution decreased to 7.3 μmol and the amount of Cr(III) increased to 2.7 μmol from initial amount of 10 μmol Cr(VI). Both Cr(VI) and Cr(III) were observed in the solution only and not on the surface or inside the cells. It has been demonstrated that the organism reduced Cr(VI) to Cr(III) intracellularly and excreted it outside the cells. This is the first report on intracellular reduction of Cr(VI) by a cyanobacterium.

Hexavalent Chromium Reduction by Bacteria from Tannery Effluent

Chromium is generated from several industrial processes. It occurs in different oxidation states, but Cr(III) and Cr(VI) are the most common ones. Cr(VI) is a toxic, soluble environmental contaminant. Some bacteria are able to reduce hexavalent chromium to the insoluble and less toxic Cr(III), and thus chromate bioremediation is of considerable interest. An indigenous chromium-reducing bacterial strain, Rb-2, isolated from a tannery water sample, was identified as Ochrobactrum intermedium, on the basis of 16S rRNA gene sequencing. The influence of factors like temperature of incubation, initial concentration of Cr, mobility of bacteria, and different carbon sources were studied to test the ability of the bacterium to reduce Cr(VI) under variable environmental conditions. The ability of the bacterial strain to reduce hexavalent chromium in artificial and industrial sewage water was evaluated. It was observed that the mechanism of resistance to metal was not due to the change in the permeability barrier of the cell membrane, and the enzyme activity was found to be inductive. Intracellular reduction of Cr(VI) was proven by reductase assay using cell-free extract. Scanning electron microscopy revealed chromium precipitates on bacterial cell surfaces, and transmission electron microscopy showed the outer as well as inner distribution of Cr(VI). This bacterial strain can be useful for Cr(VI) detoxification under a wide range of environmental conditions.

Chromium(VI) resistance and removal by Acinetobacter haemolyticus

The present work highlighted the studies on Cr(VI) reduction by cells of Acinetobacter haemolyticus (A. haemolyticus). The strain tolerated 90 mg Cr(VI) l−1 in LB broth compared to only 30 mg Cr(VI) l−1 in LB agar. From the FTIR analysis, the Cr(III) species formed was also most likely to form complexes with carboxyl, hydroxyl, and amide groups from the bacteria. A TEM study showed the absence of precipitates on the cell wall region of the bacteria. Instead, microprecipitates were observed in the cytoplasmic region of the cells, suggesting the transportation of Cr(VI) into the cells. Intracellular reduction of Cr(VI) was supported by a reductase test using soluble crude cell-free extracts. The specific reductase activity obtained was 0.52 µg Cr(VI) reduced per mg of protein an hour at pH 7.2 and 37°C. Our results indicated that A. haemolyticus can be used as a promising microorganism for Cr(VI) reduction from industrial wastewaters.

Effect of Cr(V) on reproductive organ morphology and sperm parameters: an experimental study in mice.

BackgroundCr(V) species are formed during the intracellular reduction of Cr(VI), a ubiquitous environmental pollutant. In this study, the acute toxicity of a physiologically stable Cr(V) compound, [CrV-BT]2- (BT = bis(hydroxyethyl)aminotris(hydroxymethyl)methane) was investigated in the male reproductive system of sexually mature 60-day-old male ICR-CD1 mice.MethodsEight-week-old animals were subcutaneously injected daily with a dose of ca 8 μmol of Cr/mouse, during 5 days. The control group was injected with 0.5 mL of BT buffer. Testis and epididymis morphology was evaluated using light and transmission electron microscopy. Epididymal sperm counts, motility and acrosome integrity were also assayed using standard methods.ResultsSeminiferous epithelium abnormalities were detected in the CrV-BT experimental group, including intraepithelial vacuolation, and remarkable degeneration of Sertoli cells, spermatocytes and spermatids. The premature release of germ cells into the tubular lumen was also evident. Histological evaluation of epididymal compartments revealed apparently normal features. However, the epididymal epithelium presented vacuolation. [CrV-BT]2- induced a reduction in sperm acrosome integrity. However, sperm motility and density were not significantly affected.ConclusionThis in vivo study using a Cr(V) compound, provides evidence for the potential reproductive hazards caused on male reproductive system by species containing chromium in intermediate oxidation states.

Time-dependent uptake, distribution and biotransformation of chromium(VI) in individual and bulk human lung cells: application of synchrotron radiation techniques

Chromium(VI) is a human carcinogen, primarily affecting the respiratory tract probably via active transport into cells, followed by the reduction to Cr(III) with the formation of DNA-damaging intermediates. Distribution of Cr and endogenous elements within A549 human lung adenocarcinoma epithelial cells, following treatment with Cr(VI) (100 microM, 20 min or 4 h) were studied by synchrotron-radiation-induced X-ray emission (SRIXE) of single freeze-dried cells. After the 20-min treatment, Cr was confined to a small area of the cytoplasm and strongly co-localized with S, Cl, K, and Ca. After the 4-h treatment, Cr was distributed throughout the cell, with higher concentrations in the nucleus and the cytoplasmic membrane. This time-dependence corresponded to approximately 100% or 0% clonogenic survival of the cells following the 20-min or 4-h treatments, respectively, and could potentially be explained by a new cellular protective mechanism. Such processes may also be important in reducing the potential hazards of Cr(III) dietary supplements, for which there is emerging evidence that they exert their anti-diabetic effects via biological oxidation to Cr(VI). The predominance of Cr(III) was confirmed by micro-XANES spectroscopy of intracellular Cr hotspots. X-ray absorption spectroscopy (XANES and EXAFS, using freeze-dried cells after the 0-4-h treatments) was used to gain insight into the chemical structures of Cr(III) complexes formed during the intracellular reduction of Cr(VI). The polynuclear nature of such complexes (probably with a combination of carboxylato and hydroxo bridging groups and O-donor atoms of small peptides or proteins) was established by XAFS data analyses.

Synthesis and Characterization of a Chromium(V) cis-Dioxo Bis(1,10-phenanthroline) Complex and Crystal and Molecular Structures of Its Chromium(III) Precursor

The first structurally characterized Cr(V) dioxo complex, cis-[CrV(O)2(phen)2](BF4) (2, phen=1,10-phenanthroline) has been synthesized by the oxidation of a related Cr(III) complex, cis-[Cr(III)(phen)2(OH2)2](NO3)3.2.5H2O (1, characterized by X-ray crystallography), with NaOCl in aqueous solutions in the presence of excess NaBF4, and its purity has been confirmed by electrospray mass spectrometry (ESMS), EPR spectroscopy, and analytical techniques. Previously reported methods for the generation of Cr(V)-phen complexes, such as the oxidation of 1 with PbO2 or PhIO, have been shown by ESMS to lead to mixtures of Cr(III), Cr(V), Cr(VI), and in some cases Cr(IV) species, 3. Species 3 was assigned as [CrIV(O)(OH)(phen)2]+, based on ESMS and X-ray absorption spectroscopy measurements. A distorted octahedral structure for 2 (CrO, 1.63 A; Cr-N, 2.04 and 2.16 A) was established by multiple-scattering (MS) modeling of XAFS spectra (solid, 10 K). The validity of the model was verified by a good agreement between the results of MS XAFS fitting and X-ray crystallography for 1 (distorted octahedron; Cr-O, 1.95 A; Cr-N, 2.06 A). Unlike for the well-studied Cr(V) 2-hydroxycarboxylato complexes, 2 was equally or more stable in aqueous media (hours at pH=1-13 and 25 degrees C) compared with polar aprotic solvents. A stable Cr(III)-Cr(VI) dimer, [Cr(III)(Cr(VI)O4)(phen)2]+ (detected by ESMS), is formed during the decomposition of 2 in nonaqueous media. Comparative studies of the oxidation of 1 by NaOCl or PbO2 have shown that [Cr(V)(O)2(phen)2]+ was the active species responsible for the previously reported oxidative DNA damage, bacterial mutagenicity, and increased incidence of micronuclei in mammalian cells, caused by the oxidation products of 1 with PbO2. Efficient oxidation of 1 to a genotoxic species, [Cr(V)(O)2(phen)2]+, in neutral aqueous media by a biological oxidant, hypochlorite, supports the hypothesis on a significant role of reoxidation of Cr(III) complexes, formed during the intracellular reduction of Cr(VI), in Cr(VI)-induced carcinogenicity. Similar oxidation reactions may contribute to the reported adverse effects of a popular nutritional supplement, Cr(III) picolinate.

Guanine and 7,8-dihydro-8-oxo-guanine-specific oxidation in DNA by chromium(V).

The hexavalent oxidation state of chromium [Cr(VI)] is a well-established human carcinogen, although the mechanism of cancer induction is currently unknown. Intracellular reduction of Cr(VI) forms Cr(V), which is thought to play a fundamental role in the mechanism of DNA damage by this carcinogen. Two separate pathways of DNA damage, an oxidative pathway and a metal-binding pathway, have been proposed to account for the lesions observed in cell systems. We have used a model Cr(V) complex, N,N-ethylenebis(salicylidene-animato)oxochromium(V) [Cr(V)-Salen], to investigate the oxidative pathway of DNA damage and to elucidate the lesions generated from this oxidation process. Reaction of Cr(V)-Salen with synthetic oligonucleotides produced guanine-specific lesions that were not 8-oxo-2'-deoxyguanosine, based on the inability of iridium(IV) to further oxidize these sites. Oxidation products were identified using a 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-G) containing oligonucleotide to increase the yields of product for identification by electrospray ionization mass spectrometry. The guanine-based lesions observed by mass spectrometry corresponded to the lesions guanidinohydantoin and spiroiminodihydantoin. The effects of these Cr(V)-Salen-induced lesions on DNA replication fidelity was assayed using a polymerase-based misincorporation assay. These lesions produced G --> T transversion mutations and polymerase stops at levels greater than those observed for 8-oxo-G. These data suggest a model by which chromate can cause DNA damage leading to mutations and cancer.

Competition between 1,2-Diol and 2-Hydroxy Acid Coordination in Cr(V)-Quinic Acid Complexes: Implications for Stabilization of Cr(V) Intermediates of Relevance to Cr(VI)-Induced Carcinogenesis

For the speciation of Cr(V) intermediates formed during the intracellular reduction of Cr(VI) to be understood, the intramolecular competition between 1,2-diol and 2-hydroxy acid coordination to Cr(V) as a function of pH has been studied in quinic acid complexes. The Cr(V)-2-hydroxy acid complex, K[Cr(O)(qaH3)2]·H2O (qaH5 = 1R,3R,4R,5R-1,3,4,5-tetrahydroxycyclohexanecarboxylic acid, I), has been isolated and characterized. In aqueous solutions at pH values <4.0, K[Cr(O)(qaH3)2]·H2O gives two EPR signals (giso = 1.9787, Aiso = 17.2 × 10-4 cm-1; giso = 1.9791, Aiso = 16.4 × 10-4 cm-1). The relative intensities of the signals are independent of [qaH5]/[Cr(V)], and of increasing [qaH5] and [Cr(V)] at constant [qaH5]/[Cr(V)] and pH values. These signals are consistent with those found with well-characterized Cr(V)-2-hydroxy acid complexes and are assigned to two geometric isomers of the [Cr(O)(O1,O7-qaH3)2]- linkage isomer. Both the 2-hydroxy acid (O1,O7) and vic-diol (cis-O3,O4; trans-O4,O5) groups of qaH5 ar...

Permeability, cytotoxicity, and genotoxicity of chromium (V) and chromium (VI) complexes in V79 Chinese hamster lung cells.

The genotoxicity of Cr(V) complexes in mammalian cells (V79 Chinese hamster lung cells) has been studied for the first time using the in vitro micronucleus assay. Two complexes were investigated, [CrO(ehba)2]-, which undergoes ligand-exchange and disproportionation reactions in the cell growth medium, and [CrO(mampa)]-, which is chemically inert in the medium for the duration of the exposure period. Results of in vitro micronucleus assays show that both complexes are genotoxic and exhibit similar potencies to that of [Cr2O7]2-. The permeabilities of the Cr(V) complexes were also investigated for the first time using particle-induced X-ray emission (PIXE) analysis of individual cells. The Cr uptake increased in the order: [Cr(phen)2-(H2O)2]3+ < [CrO(ehba)2]- < [CrO(mampa)]- < [Cr2O7]2-. Clonal assays showed that Cr(VI) exhibits an expectedly higher cytotoxicity than the Cr(V) complexes. While the genotoxicities of the Cr(V) and Cr(VI) complexes increase according to their permeabilities, the genotoxicities of the Cr(V) complexes are equal to, if not greater than, that of Cr(VI) in terms of the amount of Cr entering the cell. This supports other evidence that Cr(V), produced as a metabolic intermediate from the intracellular reduction of Cr(VI), may be important in Cr-induced cancers.

Induction of mutagenic DNA damage by chromium(VI) and glutathione

Certain chromium (Cr) compounds are known to be carcinogenic in humans and mutagenic in cell culture. However, the mechanism of Cr mutagenesis is not well understood. It appears that intracellular reduction of Cr by agents such as glutathione plays a role in the induction of DNA damage. We have used a simian virus 40-based shuttle vector to investigate the relationship between chromium-induced DNA damage and Cr mutagenicity. The treatment of the plasmid pZ189 with Cr(VI) plus glutathione (GSH) induced DNA strand breaks and reduced the plasmid biological activity, whereas Cr(III) treatment with or without GSH did not give rise to such DNA damage. When Cr(VI)/GSH- or Cr(III)/GSH-treated pZ189 was replicated in mammalian cells, a dose-dependent increase in mutant frequency was observed with Cr(VI)/GSH-treated pZ189, but not with Cr(III)/GSH-treated plasmid. About 43% of the mutants from Cr(VI)/GSH-treated pZ189 were deletion mutants. The remainder were base substitution mutants, mostly GC → AT transitions and GC → TA transversions. This pattern of mutagenesis is similar to that observed with other agents that cause oxidative DNA damage such as ionizing radiation and H2O2. These results support the hypothesis that Cr mutagenesis can be induced by the generation of reactive oxygen intermediates during the reduction of Cr(VI) by glutathione. © 1996 Wiley-Liss, Inc.

Induction of mutagenic DNA damage by chromium (VI) and glutathione.

Certain chromium (Cr) compounds are known to be carcinogenic in humans and mutagenic in cell culture. However, the mechanism of Cr mutagenesis is not well understood. It appears that intracellular reduction of Cr by agents such as glutathione plays a role in the induction of DNA damage. We have used a simian virus 40-based shuttle vector to investigate the relationship between chromium-induced DNA damage and Cr mutagenicity. The treatment of the plasmid pZ189 with Cr(VI) plus glutathione (GSH) induced DNA strand breaks and reduced the plasmid biological activity, whereas Cr(III) treatment with or without GSH did not give rise to such DNA damage. When Cr(VI)/GSH- or Cr(III)/GSH-treated pZ189 was replicated in mammalian cells, a dose-dependent increase in mutant frequency was observed with Cr(VI)/GSH-treated pZ189, but not with Cr(III)/GSH-treated plasmid. About 43% of the mutants from Cr(VI)/GSH-treated pZ189 were deletion mutants. The remainder were base substitution mutants, mostly GC-->AT transitions and GC-->TA transversions. This pattern of mutagenesis is similar to that observed with other agents that cause oxidative DNA damage such as ionizing radiation and H2O2. These results support the hypothesis that Cr mutagenesis can be induced by the generation of reactive oxygen intermediates during the reduction of Cr(VI) by glutathione.

Intracellular chromium reduction

Two steps are involved in the uptake of Cr(VI): (1) the diffusion of the anion CrO 2− 4 through a facilitated transport system, presumably the non-specific anion carrier and (2) the intracellular reduction of Cr(VI) to Cr(III). The intracellular reduction of Cr(VI), keeping the cytoplasmic concentration of Cr(VI) low, facilitates accumulation of chromate from extracellular medium into the cell. In the present paper, a direct demonstration of intracellular chromium reduction is provided by means of electron paramagnetic (spin) resonance (EPR) spectroscopy. Incubation of metabolically active rat thymocytes with chromate originates a signal which can be attributed to a paramagnetic species of chromium, Cr(V) or Cr(III). The EPR signal is originated by intracellular reduction of chromium since: (1) it is observed only when cells are incubated with chromate, (2) it is present even after extensive washings of the cells in a chromium-free medium; (3) it is abolished when cells are incubated with drugs able to reduce the glutathione pool, i.e., diethylmaleate or phorone; and (4) it is abolished when cells are incubated in the presence of a specific inhibitor of the anion carrier, 4-acetamido-4′-isothiocyanatostilbene-2-2′-disulfonic acid.

The reduction of chromium (VI) to chromium (III) by glutathione: An intracellular redox pathway in the metabolism of the carcinogen chromate

The capacity of glutathione (GSH) to reduce Cr(VI) to Cr(III) in vitro was investigated. The reaction was determined spectrophotometrically by following the absorption of Cr(VI0 at 370 nm. At stoichiometric conditions (molar ratio Cr(VI)/GSH of 1:3) the reduction was strongly dependent on the solution's pH. It was much slower at pH 7.4 than at pH values below 5. An excess of GSH (100- or 1000-fold) accelerated the reaction. In any case, 3 GSH molecules were required to reduce 1 molecule of chromate. Incubation of human red blood cells (RBC) with an excess of of Na 2CrO 4 (10 mM) decreased the GSH content of the cells to 10% of the original amount. This depletion of GSH was similar to that obtained when RBC were incubated with 62 mM diethylmaleate (DEM), a well known GSH depleting agent. Sephadex G-100 chromatography of lysates from human RBC incubated with radioactive chromate ( 51Cr(VI)) showed a strong affinity of 51Cr for hemoglobin: 97% of the applied dose was bound to hemoglobin whilst only minor amounts of 51Cr were found in the low-molecular fractions. However, incubations of prepared lysates (as opposed to intact cells) with 10 mM Na 2 51CrO 4 markedly raised the chromium content of low-molecular fractions (probably GSH-Cr-complexes), probably indicative of a role of GSH in the intra-cellular reduction of Cr(VI) to Cr(III), the latter being regarded as the ultimately toxic species of this metal.

Effects of trivalent and hexavalent chromium on the physicochemical properties of mammalian cell nucleic acids and synthetic polynucleotides

The effects of trivalent (chromium chloride) and hexavalent (potassium dichromate) chromium have been studied on the nucleic acids of cultured mammalian cells (BHK hamster fibroblast line), commercial DNA and RNA, and synthetic polynucleotides of known base composition. Modifications of UV absorption spectra and alterations of thermal denaturation and renaturation patterns have been observed by directly treating purified nucleic acids, as well as by examining nucleic acids extracted from cells treated with chromium compounds. Cr(III) interacts with nucleic acid bases, mostly guanine and cytosine, but also with phosphate groups, leading to deprotonation of bases as well as intramolecular cross-links, sandwich complexes between bases and chelation between bases and phosphates. Such interactions destabilize the DNA structure. On the contrary, stabilization of RNA, due to intramolecular metal bonds between nitrogen bases in GC-rich regions, is mainly produced. The kind of interaction of Cr(III) with nucleic acids is not significantly different when intact BHK cells are treated. Cr(VI) interacts similarly with DNA and RNA giving instead different effects when purified nucleic acids or intact cells are treated. Treatment of purified DNA produces breakages in the polynucleotide chain due to the oxidizing power of Cr(VI). In intact cell treatments, changes in the properties of DNA are observed. These could result from the combined action of Cr(III), produced by the intracellular reduction of Cr(VI) and the oxidizing activity of residual Cr(VI). The relevance of Cr(VI) and Cr(III) interactions to the mechanisms of chromium (carcino)genic action is summarized. It is stressed that Cr(VI), if not completely reduced to Cr(III) by extracellular and endoplasmic constituents, can reach the cell nucleus and directly interact with DNA.