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.     

Chromium-induced production of reactive oxygen species,DNA single-strand breaks,nitric oxide production,and lactate dehydrogenase leakage in J774A.1 cell cultures

The involvement of oxidative stress in the toxicity of chromium (VI) and chromium (III) has been proposed. We have therefore examined the effects of these cations on the production of superoxide anion, nitric oxide (NO), and DNA single strand breaks (SSB) in J774A.1 macrophage cells in culture as well as the effects on lactate dehydrogenase (LDH) leakage and cell viability. Following a 48 hour incubation, over twofold increases in superoxide anion and NO production were observed at concentrations of approximately 0.30 and 50 μM for Cr (VI) and Cr (III), respectively. The patterns of cell viability and LDH leakage paralleled superoxide anion and NO production for Cr (VI) and Cr (III). A 50% decrease in viability was observed at approximately the concentrations that produced a twofold increase in superoxide and NO production. Concentration-dependent increases in DNA-SSB were observed after incubation with Cr (III) with maximum increases occurring at a concentration of approximately 60 μM. Cr (VI) had no effect on the incidence of DNA-SSB at any of the tested concentrations. The results indicate that Cr (VI) and Cr (III) are toxic to the J774A.1 cell line, and the toxicity may be due at least in part to an oxidative stress induced by the production of reactive oxygen species. © 1996 John Wiley & Sons, Inc.     

Evaluation of the speciation status of aluminium(III) ions in isolated osteoarthritic knee-joint synovial fluid

High field 1H NMR spectroscopy demonstrated that the equilibration of added Al(III) ions in osteoarthritic (OA) knee-joint synovial fluid (SF) resulted in its complexation by citrate and, to a much lesser extent, tyrosine and histidine. The ability of these ligands, together with inorganic phosphate, to compete for the available Al(III) in terms of (1) thermodynamic equilibrium constants for the formation of their complexes and (2) their SF concentrations was probed through the use of computer speciation calculations, which considered low-molecular-mass binary and ternary Al(III) species, the predominant Al(III) plasma transport protein transferrin, and also relevant hydrolysis and precipitation processes. It was found that, at relatively low added Al(III) concentrations, citrate species were more favoured, whilst phosphate species became dominant at higher levels. The significance of these findings with regard to the in vivo corrosion of aluminium-containing metal alloy joint prostheses (e.g., TiAlV alloys) is discussed.     

Biphasic kinetics in the reaction between amino acids or glutathione and the chromium acetate cluster, [Cr3O(OAc)6]+

Kinetics for the breakdown of the trinuclear chromium acetate cluster with a series of monoprotic and diprotic amino acid ligands and with glutathione in aqueous media have been investigated spectrophotometrically at pH 3.5-5.5 and in a temperature range of 45-60 degrees C. Under pseudo-first-order conditions, reactions with these ligands exhibited biphasic kinetic behavior that can be accounted for by a consecutive two-step reaction, A-->B-->C, where A is assumed to be a forced ion pair, B an intermediate and C is the product; experimental data fit to a biexponential equation for the transformation. Rates for k(short), k(long), and k(obs) were determined by manual extrapolation of absorbance data or curve-fitting routines; associated activation parameters for each step of the reaction were calculated using the Eyring equation. Rates for the first and second steps of the reaction are on the order of approximately 10(-4) and approximately 10(-5)s(-1), respectively. The large negative values of DeltaS++ and smaller DeltaH++ in the first step indicate an associative step, while high positive values of DeltaS(double dagger) in the second step indicate dissociation. To account for the results mechanistically, the results are interpreted to be a first step of ligand exchange with a pseudo-axial aqua ligand, followed by a dissociative step involving acetate or oxo ligand displacement. The dissociative step is the rate determining step, with k(obs) approximately k(long). The results demonstrate reaction pathways that are available to the Cr(III) metal centers that may be physiologically relevant in the ligand-rich environment of biological systems. Under general conditions Cr(III) clusters may be expected to be broken down, unless some unique biological environment stabilizes the cluster. The present study has application to the processes related to Cr(III) transport and excretion, to potential mechanisms of Cr(III) action in a biological setting, and to the pharmacokinetics of Cr(III) supplements for animal and human consumption.     

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.     

Kinetics of Cr(III) and Cr(VI) removal from water by two floating macrophytes

The aim of this work was to compare Cr(III) and Cr(VI) removal kinetics from water by Pistia stratiotes and Salvinia herzogii. The accumulation in plant tissues and the effects of both Cr forms on plant growth were also evaluated. Plants were exposed to 2 and 6 mg L^?1 of Cr(III) or Cr(VI) during 30 days. At the end of the experiment, Cr(VI) removal percentages were significantly lower than those obtained for Cr(III) for both macrophytes. Cr(III) removal kinetics involved a fast and a slow component. The fast component was primarily responsible for Cr(III) removal while Cr(VI) removal kinetics involved only a slow process. Cr accumulated principally in the roots. In the Cr(VI) treatments a higher translocation from roots to aerial parts than in Cr(III) treatments was observed. Both macrophytes demonstrated a high ability to remove Cr(III) but not Cr(VI). Cr(III) inhibited the growth at the highest studied concentration of both macrophytes while Cr(VI) caused senescence. These results have important implications in the use of constructed wetlands for secondary industrial wastewater treatment. Common primary treatments of effluents containing Cr(VI) consists in its reduction to Cr(III). Cr(III) concentrations in these effluents are normally below the highest studied concentrations in this work.     

Effect of 60 degrees head-down tilt on peripheral gas mixing in the human lung.

The phase III slope of sulfur hexafluoride (SF6) in a single-breath washout (SBW) is greater than that of helium (He) under normal gravity (i.e., 1G), thus resulting in a positive SF6-He slope difference. In microgravity (microG), SF6-He slope difference is smaller because of a greater fall in the phase III slope of SF6 than He. We sought to determine whether increasing thoracic fluid volume using 60 degrees head-down tilt (HDT) in 1G would produce a similar effect to microG on phase III slopes of SF6 and He. Single-breath vital capacity (SBW) and multiple-breath washout (MBW) tests were performed before, during, and 60 min after 1 h of HDT. Compared with baseline (SF6 1.050 +/- 0.182%/l, He 0.670 +/- 0.172%/l), the SBW phase III slopes for both SF6 and He tended to decrease during HDT, reaching nadir at 30 min (SF6 0.609 +/- 0.211%/l, He 0.248 +/- 0.138%/l; P = 0.08 and P = 0.06, respectively). In contrast to microG, the magnitude of the phase III slope decrease was similar for both SF6 and He; therefore, no change in SF6-He slope difference was observed. MBW analysis revealed a decrease in normalized phase III slopes at all time points during HDT, for both SF6 (P < 0.01) and He (P < 0.01). This decrease was due to changes in the acinar, and not the conductive, component of the normalized phase III slope. These findings support the notion that changes in thoracic fluid volume alter ventilation distribution in the lung periphery but also demonstrate that the effect during HDT does not wholly mimic that observed in microG.     

Chromium(III) Oxidation Coupled with Microbially Mediated Mn(II) Oxidation

Abstract

Reductive immobilization of Cr(VI) has been widely explored as a cost-effective approach for Cr-contaminated site remediation. In soils containing manganese oxides, however, the immobilized form of chromium, i.e., Cr(III), could potentially be reoxidized. In this study, batch experiments were conducted to assess whether there were any microbial processes that could accelerate Cr(III) oxidation in aerobic, manganese-containing systems. The results showed that in the presence of at least one species of manganese oxidizers, Pseudomonas putida, Cr(III) oxidation took place at low concentrations of Cr(III). About 30–50% of added Cr(III) (10–200 μ M) was oxidized to Cr(VI) within five days in the systems with P. putida and biogenic Mn oxides. The rate of Cr(III) oxidation was approximately proportional to the initial concentration of Cr(III) up to 100 μ M, but the growth of P. putida was partially inhibited by Cr(III) at 200 μ M and totally stopped when it reached 500 μ M. Cr(III) oxidation was dependent upon the biogenic formation of Mn oxides, though the oxidation rate was not directly proportional to the amount of Mn oxides formed. Chromium(III) oxidation took place through a catalytic pathway, in which the microbes mediated Mn(II) oxidation to form Mn-oxides, and Cr(III) was subsequently oxidized by the biogenic Mn-oxides.     

Protein degradation by peroxide catalyzed by chromium (III): role of coordinated ligand

In order to understand the role of coordinated ligands in controlling the biotoxicity of chromium (III), interactions of three types of chromium (III) complexes viz. trans-diaquo [1,2 bis (salicyledeneamino) ethane chromium (III) perchlorate, [(Cr(salen)(H(2)O)(2)](ClO(4)); tris (ethylenediamine) chromium (III) chloride, [Cr(en)(3)]Cl(3), and monosodium ethylene diamine tetraacetato monoaquo chromiate (III), [Cr(EDTA)(H(2)O)]Na with BSA has been investigated. Spectroscopic and equilibrium dialysis studies show that the two cationic complexes Cr(salen)(H(2)O)(+)(2) and Cr(en)(3+)(3) bind to the protein with a protein-metal ratio of 1:8 and 1:4. The anionic complex Cr(EDTA)(H(2)O)(-) binds to the protein with a protein-metal ratio of 1:2. The binding constant K(b) as estimated from the fluorescence quenching studies has been found to be 7.6 +/- 0.4 x 10(3) M(-1), 3.1 +/- 0.2 x 10(2) M(-1), and 1.8 +/- 0.2 x 10(2) M(-1) for Cr(salen)(H(2)O)(+)(2), Cr(en)(3+)(3), and Cr(EDTA)(H(2)O)(-) respectively indicating that the thermodynamic stability of protein-chromium complex is Cr(salen)(H(2)O)(+)(2) > Cr(en)(3+)(3) approximately Cr(EDTA)(H(2)O)(-). The complexes Cr(salen)(H(2)O)(+)(2) and Cr(EDTA)(H(2)O)(-) in the presence of hydrogen peroxide have been found to bring about protein degradation, whereas Cr(en)(3+)(3) does not bring about any protein damage. This clearly shows that the nature of the chromium (III) complex plays a major role in the biotoxicity of chromium (III).     

Competition studies in horse spleen ferritin probed by a kinetically inert inhibitor, [Cr(TREN)(H2O)(OH)]2+, and a highly luminescent Tb(III) reagent

The ability of ferritin as an Fe(II) detoxifier and Fe(III) storage protein is limited by its ability to recognize and incorporate Fe(II), which is then oxidized and mineralized at internal protein sites. The Cr(III) amine complex [Cr(N(CH(2)CH(2)NH(2))(3)(H(2)O)(OH)](2+) [abbreviated as Cr(TREN)] is a kinetically inert inhibitor of iron incorporation and mineralization in ferritin. Unlike other inhibitors, Cr(TREN) can only exchange its two aqua/hydroxy ligands. Competition studies between Cr(TREN) and Tb(III) binding have been performed in horse spleen ferritin (HoSF) to probe uptake of Fe(II). From these studies, we propose that Cr(TREN) inhibits Fe(II) uptake by obstructing the routes of metal uptake and by disrupting the early recognition events at the protein surface that precede metal ion uptake. Using an improved luminescence approach to quantify Tb(III) binding to the protein, we demonstrate that Tb(III) cannot interfere with Cr(TREN) binding to ferritin, but that Cr(TREN) dramatically inhibits Tb(III) binding. We show that bound Tb(III) serves as a reliable reporter for Cr(TREN) binding, as the latter efficiently quenches the Tb(III) luminescence via inter-ion energy transfer. Two types of Cr(TREN) binding sites were successfully distinguished from these competition experiments. A common Tb(III)/Cr(TREN) site was identified with stoichiometry of approximately 0.6 equivalents of metal cation per ferritin subunit. We propose that the sites along the three-fold channels and the ferroxidase sites are common binding sites for Tb(III) and Cr(TREN). The remaining Cr(TREN) (2.4 equivalents of metal ions/subunit) does not compete with Tb(III) but rather blocks Tb(III) access into the cavity and decreases the protein's affinity for Tb(III).     

Carcinogenic chromium(VI) induces cross-linking of vitamin C to DNA in vitro and in human lung A549 cells

Reductive activation of carcinogenic Cr(VI) is required for the induction of DNA damage and mutations. Here, we examined the formation of Cr-DNA adducts in the reactions of Cr(VI) with its dominant biological reducer, vitamin C (ascorbate). Reductive conversion of Cr(VI) to Cr(III) by ascorbate produced stable Cr-DNA adducts, of which approximately 25% constituted ascorbate-Cr(III)-DNA cross-links. No evidence was found for the involvement of Cr(V) or Cr(IV) intermediates in the formation of either binary or ternary adducts. The cross-linking reaction was consistent with the attack of DNA by transient Cr(III)-ascorbate complexes. The yield of Cr(III)-DNA adducts was similar on dsDNA and AGT, ACT, or CT oligonucleotides and was strongly inhibited by Mg(2+), suggesting predominant coordination of Cr(III) to DNA phosphate oxygens. We also detected cross-linking of ascorbate to DNA in Cr(VI)-exposed human lung A549 cells that were preincubated with dehydroascorbic acid to create normal levels of intracellular ascorbate. Ascorbate-Cr-DNA cross-links accounted for approximately 6% of the total Cr-DNA adducts in A549 cells. Shuttle-vector experiments showed that ascorbate-Cr-DNA cross-links were mutagenic in human cells. Our results demonstrate that in addition to reduction of Cr(VI) to DNA-reactive Cr(III), vitamin C contributes to the genotoxicity of Cr(VI) via a direct chemical modification of DNA. The absence of Asc in A549 and other human cultured cells indicates that cells maintained under the usual in vitro conditions lack the most important reducing agent for Cr(VI) and would primarily display slow thiol-dependent activation of Cr(VI).     

Pyridine-2,6-bis(thiocarboxylic acid) Produced by <Emphasis Type="BoldItalic">Pseudomonas stutzeri</Emphasis> KC Reduces Chromium(VI) and Precipitates Mercury,Cadmium, Lead and Arsenic

Interactions of the Pseudomonas stutzeri KC siderophore pyridine-2,6-bis(thiocarboxylic acid) (pdtc) with chromium(VI), mercury(II), cadmium(II), lead(II), and arsenic(III) are described. Pdtc was found to reduce Cr(VI) to Cr(III) in both bacterial cultures and in abiotic reactions with chemically synthesized pdtc. Cr(III) subsequently formed complexes with pdtc and pdtc hydrolysis products, and their presence was confirmed using electrospray ionization-mass spectrometry (ESI-MS). Cr(III):pdtc complexes were found to slowly release Cr(III) as chromium sulfide and possibly Cr(III) oxides. Pdtc also formed poorly soluble complexes with Hg, Cd, Pb, and As(III). Hydrolysis of those complexes led to the formation of their respective metal sulfides as confirmed by energy dispersive X-ray spectroscopy (EDS) elemental analysis. The pdtc-producing strain P. stutzeri KC showed higher tolerance to most of these metals as compared to a pdtc-negative mutant. A novel role of pdtc is postulated as its involvement in providing an extracellular pool of thiols that are used for redox processes in detoxification of the bacterial extracellular environment. These redox processes can be mediated by transition metal:pdtc complexes.     

Bacterial Reduction of Cr(VI) and Fe(III) in In Vitro Conditions

ABSTRACT Chemical reduction of Cr(VI) can be a strategy to detoxify toxic metals in oxidized states, whereas reduction of Fe(III) could enhance the availability of Fe in the form of Fe(II) to boost plant growth. However, it creates another problem of chemical sludge disposal. Hence, microbial conversion of Cr(VI) to Cr(III) and Fe(III) to Fe(II) is preferred over the chemical method. Out of 11 bacterial strains isolated from the rhizospheric zone of Typha latifolia growing on fly ash dump sites, four isolates were selected for the reduction of Cr(VI) and Fe(III) and were identified as Micrococcus roseus NBRFT2 (MTCC 9018), Bacillus endophyticus NBRFT4 (MTCC 9021), Paenibacillus macerans NBRFT5 (MTCC 8912), and Bacillus pumilus NBRFT9 (MTCC 8913). These strains were individually tested for survival at different concentrations of Cr(VI) and Fe(III), pH, and temperature, and then, their ability for reduction of both metals was evaluated at optimum pH 8.0 and temperature 35°C. The results indicated that NBRFT5 was able to reduce the maximum amount, 99% Cr(VI) and 98% Fe(III). Other strains also reduced these metals to different levels, but less than NBRFT5. Hence, these strains may be used for decontamination of metal-contaminated sites, particularly with Cr(VI) and Fe(III) through the reduction process.     

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.     

Online column preconcentration for speciation and selective determination of Cr(III) in natural water samples using flow injection with chemiluminescence detection

A simple, rapid, sensitive and inexpensive approach is described in this work based on a combination of solid-phase extraction of 8-hydroxyquinoline (8HQ), for speciation and preconcentration of Cr(III) and Cr(VI) in river water, and the direct determination of these species using a flow injection system with chemiluminescence detection (FI–CL) and a 4-diethylamino phenyl hydrazine (DEAPH)–hydrogen peroxide system. At different pH, the two forms of chromium [Cr(III) and Cr(VI)] have different exchange capacities for 8HQ, therefore two columns were constructed; the pH of column 1 was adjusted to pH 3 for retaining Cr(III) and column 2 was adjusted to pH 1 for retaining of Cr(VI). The sorbed Cr(III) and Cr(VI) species were eluted from columns using 3.0 ml of 0.1 N of HCl and 3.0 ml of 0.1 N of NaOH, respectively. The flow injection–chemiluminescence (FI–CL) method is based on light emitted due to the oxidation of DEAPH by the H₂O₂ in the presence of Cr(III), which catalyzes the reaction. The flow cell is a transparent coiled tube made from glass (2.0 × 4.0, inner and outer diameter) and located close to the photodetector. The flow parameters: flow rate, sample volume, flow cell length, and distance to the CL detector were studied and optimized. Under optimum flow conditions, the Cr(III) concentration can be determined over the range 5–350 μg L⁻¹ with a limit of detection of 1.2 μg L⁻¹, as the Cr(III) concentration is proportional to the intensity of the CL signal. The relative standard deviations (%) for 10 and 50 μg L⁻¹ Cr(III) were 1.2% and 3.2%, respectively. The effects of Al(III), Cd(II), Zn(II), Hg(II), Pb(II), Co(II), Cu(II), Ni(II), Mn(II), Ca(II), and Fe(III) were investigated. The proposed method is highly selective and sensitive, enabling a rapid determination of the Cr(III) amount in the presence of other interfering metals. Finally, the FI–CL method was examined in five river water samples with excellent recoveries.     

Simultaneous speciation of chromium by spectrophotometry and multicomponent analysis

Abstract

A simple, fast and sensitive spectrophotometric method for the simultaneous determination of Cr(III) and Cr(VI) in effluents and contaminated waters using a UV-visible spectrophotometer, which operates with an advanced software for multicomponent analysis, is proposed. The method consists in the complexation of Cr (III) with EDTA and reaction of Cr(VI) with diphenylcarbazide (DPC). Variables, such as pH and colour stability time, were studied. The effect of concomitant ions on the simultaneous Cr(III) and Cr(VI) determination was also investigated. The sums of the chromium species concentrations obtained by the proposed method were compared with the total chromium concentrations found by electrothermal atomic absorption spectrometry. Recoveries of the chromium species between 75 and 136% were obtained for spiked samples. The linear working range for Cr(III) was 0.5-30 mg L^?1, while for Cr(VI) was 0.005-0.30 mg L^?1. The detection limits were 0.3 mg L^?1 for Cr(III) and 0.003 mg L^?1 for Cr(VI) while the quantification limits were 1.0 mg L^?1 for Cr(III) and 0.01 mg L^?1 for Cr(VI).     

Chromium distribution in shoots of macrophyte Callitriche cophocarpa Sendtn.

The aim of the study was the analysis of Cr distribution in shoots of the macrophyte Callitriche cophocarpa by means of two X-ray-based techniques: micro X-ray fluorescence (μXRF) and electron probe X-ray microanalysis (EPXMA). Plants were treated with 100 μM (5.2 mg l^?1) chromium solutions for 7 days. Cr was introduced independently at two speciations as Cr(III) and Cr(VI), known for their diverse physicochemical properties and different influence on living organisms. A comparative analysis of Cr(III)-treated plants by EPXMA and μXRF demonstrated high deposition of Cr in epidermal glands/hairs localized on leaves and stems of the plant shoots. Cr in Cr(III)-treated plants was recorded solely in glands/hairs, and the element was not present in any other structures. On the other hand, Cr in Cr(VI)-treated group of plants was rather found in vascular bundles. Moreover, the concentration of Cr in Cr(VI)-treated plants was significantly lower than in plants incubated in Cr(III) solution. The results obtained in this work suggest differences in chromium uptake, transport and accumulation dependent on the oxidative state of the element.     

Cr(III)-mediated crosslinks of glutathione or amino acids to the DNA phosphate backbone are mutagenic in human cells.

Carcinogenic Cr(VI) compounds were previously found to induce amino acid/glutathione-Cr(III)-DNA crosslinks with the site of adduction on the phosphate backbone. Utilizing the pSP189 shuttle vector plasmid we found that these ternary DNA adducts were mutagenic in human fibroblasts. The Cr(III)-glutathione adduct was the most potent in this assay, followed by Cr(III)-His and Cr(III)-Cys adducts. Binary Cr(III)-DNA complexes were only weakly mutagenic, inducing a significant response only at a 10 times higher number of adducts compared with Cr(III)-glutathione. Single base substitutions at the G:C base pairs were the predominant type of mutations for all Cr(III) adducts. Cr(III), Cr(III)-Cys and Cr(III)-His adducts induced G:C-->A:T transitions and G:C-->T:A transversions with almost equal frequency, whereas the Cr(III)-glutathione mutational spectrum was dominated by G:C-->T:A transversions. Adduct-induced mutations were targeted toward G:C base pairs with either A or G in the 3' position to the mutated G, while spontaneous mutations occurred mostly at G:C base pairs with a 3' A. No correlation was found between the sites of DNA adduction and positions of base substitution, as adducts were formed randomly on DNA with no base specificity. The observed mutagenicity of Cr(III)-induced phosphotriesters demonstrates the importance of a Cr(III)-dependent pathway in Cr(VI) carcinogenicity.     

DNA oxidatively damaged by chromium(III) and H(2)O(2) is protected by the antioxidants melatonin, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine, resveratrol and uric acid

Chromium (Cr) compounds are widely used industrial chemicals and well known carcinogens. Cr(III) was earlier found to induce oxidative damage as documented by examining the levels of 8-hydroxydeoxyguanosine (8-OH-dG), an index for DNA damage, in isolated calf thymus DNA incubated with CrCl(3) and H(2)O(2). In the present in vitro study, we compared the ability of the free radical scavengers melatonin, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK), resveratrol and uric acid to reduce DNA damage induced by Cr(III). Each of these scavengers markedly reduced the DNA damage in a concentration-dependent manner. The concentrations that reduced 8-OH-dG formation by 50% (IC(50)) were 0.10 microM for both resveratrol and melatonin, and 0.27 microM for AFMK. However, the efficacy of the fourth endogenous antioxidant, i.e. uric acid, in terms of its inhibition of DNA damage in the same in vitro system was about 60--150 times less effective than the other scavengers; the IC(50) for uric acid was 15.24 microM. These findings suggest that three of the four antioxidants tested in these studies may have utility in protecting against the environmental pollutant Cr and that the protective effects of these free radical scavengers against Cr(III)-induced carcinogenesis may relate to their direct hydroxyl radical scavenging ability. In the present study, the formation of 8-OH-dG was likely due to a Cr(III)-mediated Fenton-type reaction that generates hydroxyl radicals, which in turn damage DNA. Once formed, 8-OH-dG can mutate eventually leading to cancer; thus the implication is that these antioxidants may reduce the incidence of Cr-related cancers.     

Unusual reactivity in a commercial chromium supplement compared to baseline DNA cleavage with synthetic chromium complexes

Commercially available chromium supplements were tested for their DNA cleavage ability compared with synthetic chromium(III) complexes, including chromium(III) tris-picolinate [Cr(pic)3], basic chromium acetate [Cr3O(OAc)6]+, model complexes, and recently patented Cr-complexes for use in supplements or therapy. Four different supplements (P1-P4) were tested for their DNA cleaving activity in the presence and the absence of H2O2, dithiothreitol (DTT) or ascorbate. One supplement, P1, showed nicking of DNA in the absence of oxidant or reductant at 120 microM metal concentration. Different lot numbers of P1 were also tested for DNA cleavage activity with similar results. Commercial supplements containing Cr(pic)3 nicked DNA at 120 microM metal concentrations in the presence of 5 mM ascorbate or with excess hydrogen peroxide, analogous to reactions with synthetic Cr(pic)3 reported elsewhere. Another chromium (non-Cr(pic)3) supplement, P2, behaves in a comparable manner to simple Cr(III) salts in the DNA nicking assay. Chromium(III) malonate [Cr(mal)2] and chromium(III) acetate [Cr(OAc)] can nick DNA in the presence of ascorbate or hydrogen peroxide, respectively, only at higher metal concentrations. The Cr(III) complexes of histidine, succinate or N-acetyl-L-glutamate do not nick DNA to a significant degree.