Biomonitoring of chromium(VI) deposited in pulmonary tissues: Pilot studies of a magnetic resonance imaging technique in a post-mortem rodent model

The biomonitoring of individuals exposed to chromium(VI) by inhalation is often based on determinations of chromium in body fluids such as blood, plasma or urine, or on assessments of DNA damage in non-lung surrogate tissues such as peripheral blood lymphocytes. These techniques are of some use as biomarkers of internal exposure or biological effect, mainly in the case of soluble chromium(VI) compounds, but they provide at best only indirect information about chromium(VI) concentrations in the main target organ of interest – the lung. An urgent need exists for a non-invasive technique to permit the visualization and quantification of chromium(VI) in the lung of exposed humans. This study details the development of a lung imaging technique based on the detection of paramagnetic chromium using magnetic resonance imaging (MRI). The intracellular reductive conversion of chromium(VI) is a crucial bioactivation step in its carcinogenicity, and the MRI method described here relies on the conversion of non-paramagnetic (MRI ‘silent’) chromium(VI) to detectable paramagnetic species such as chromium(III). Initial studies with chromium(III) revealed that a range of 2.5–5 μg chromium(III) instilled in rat lung is considered to be the lower limit of detection of this method. It was possible to demonstrate the presence of 30 μg chromium(VI) in our post-mortem rat model. The ultimate objective of this work is to determine whether this technique has applicability to the biomonitoring of chromium(VI) inhalation exposures that result in internalized lung doses in human subjects.     

One- and Two-Dimensional Electron Paramagnetic Resonance Imaging in Skin

EPR imaging with modulated field gradients provides the possibility for obtaining an EPR spectrum in a selected volume We demonstrate the feasibility of X-band (9.5GHz) electron paramagnetic resonance (EPR) imaging in skin biopsies of hairless mice. One- (ID) and two-dimensional (2D) EPR images of the persistent free radical di-tertiary-butyl-nitroxide are measured. At a microwave frequency of 9.5 GHz (X-band), 2D images are obtained in skin biopsies with an actual point distinction resolution of 25 μm. In a biological model system. 2D images are measured at L-band frequency (2.0 GHz) with a pixel resolution of 61 μm. and a theoretical spatial resolution of 12.5 μm. In combination with the spin labeling and spin trapping technique. EPR imaging is the most direct approach to analyzing spatial distribution of physico-chemical properties in skin, such as membrane fluidity and polarity. as well as detection of free radicals.     

Role of physiological antioxidants in chromium(VI)-induced cellular injury.

Chromium(VI) compounds are well known to be potent toxic and carcinogenic agents. Because chromium(VI) is easily taken up by cells and is subsequently reduced to the trivalent form, the formation of chromium(III) or other intermediate oxidation states such as chromium(V) and (IV) is believed to play a role in the adverse biological effects of chromium(VI) compounds. Recent in vitro studies have shown that this reduction process generates free radical species such as active oxygen radicals. Furthermore, physiological antioxidants are reported to modify the genotoxic and toxic effects of chromate. This article reviewed the recent in vitro and in vivo studies of the effects of antioxidants including active oxygen scavengers; glutathione; vitamins B2, E, and C, on chromate-induced injury such as DNA lesions; lipid peroxidation; enzyme inhibition; cytotoxicity; mutation; and so on. In addition, the mechanism of action of these antioxidants was discussed with respect to the formation of active oxygen radicals and paramagnetic chromium such as chromium(V) and (III). Such studies may help elucidate the mechanism of chromium(VI) toxicity as well as the mechanism of protection.     

In vitro metabolism and permeation studies in rat jejunum: organic chromium compared to inorganic chromium.

The purpose of these studies was to compare the in vitro absorption of two inorganic chromium(III) compounds: chromium chloride and chromium nitrate, with organic chromium(III)-picolinate; and to investigate if any in vitro metabolism of chromium(VI) takes place. The in vitro metabolism studies showed that chromium (VI) was reduced by artificial gastric juice. The reduction followed first order kinetics with a half-life of 23 min. The studies also showed that the chromium picolinate complex was stable in artificial gastric juice for 4 hours. By the rat everted gut sac technique, chromium chloride, chromium nitrate and chromium picolinate penetrated the rat jejunum with 165 +/- 59, 160 +/- 26 and 127 +/- 36 ng chromium per g rat jejunum, respectively, whereas the permeability coefficients (Papp) were 0.7 +/- 0.3, 1.0 +/- 0.4, and 9.6 +/- 2.2 microns/min, respectively. Absorption studies on pig intestine in Ussing chambers showed a nearly total adsorption of chromium(III) by the chambers, resulting in unreliable data.     

Problems in the biological monitoring of chromium(VI) exposed individuals

The usefulness of currently available techniques for the biological monitoring of chromium(VI) exposed individuals is reviewed. Chromium levels in body fluids, such as urine and blood plasma, are reliable markers of exposure to chromium in oxidation states (VI) and (III) and provide a measure of the internalized dose of chromium. These markers are sufficiently sensitive to be useful in most occupational settings encountered today. In contrast, the majority of cytogenetic surveillance studies among chromium platers, ferrochromium workers and stainless steel welders using the manual metal arc (MMA) method have yielded negative or inconclusive results. As a marker for genotoxicity, the number of sister chromatid exchanges in blood lymphocytes proved to be relatively insensitive towards exposure to chromium(VI). There were however significant increases in rare chromosome aberrations among MMA stainless steel welders, although the reported levels of all aberrations combined were similar to those observed among control groups of many other studies. The relative lack of success of cytogenetic surveillance studies using blood lymphocytes is surprising in view of the strong genotoxicity of chromium(VI). A possible explanation comes from recent studies which showed that the differences in chromium lymphocyte levels between exposed and controls were disproportionately small. Another factor which complicates attempts to correlate genotoxic effects in lymphocytes with the processes giving rise to cancers of the respiratory system is the toxicokinetics of inhaled chromium(VI). Only small fractions of the total inhaled dose are distributed in the body while the bulk of chromium(VI) deposited in the lungs remains there for very long periods of time. The vast majority of lymphocytes will therefore come into contact with chromium(VI) not while travelling through the supporting tissues of the lungs but during their migration through the blood. There they take up chromium(VI) that has leached from the lungs. Blood lymphocytes therefore seem to be inappropriate for the monitoring of the biologically effective dose, and of early biological effects arising from exposure to chromium(VI). Thus there is an urgent need to develop techniques which would allow the non-invasive monitoring of internalized doses of chromium in the lung.     

Problems in the biological monitoring of chromium(VI) exposed individuals

The usefulness of currently available techniques for the biological monitoring of chromium(VI) exposed individuals is reviewed. Chromium levels in body fluids, such as urine and blood plasma, are reliable markers of exposure to chromium in oxidation states (VI) and (III) and provide a measure of the internalized dose of chromium. These markers are sufficiently sensitive to be useful in most occupational settings encountered today. In contrast, the majority of cytogenetic surveillance studies among chromium platers, ferrochromium workers and stainless steel welders using the manual metal arc (MMA) method have yielded negative or inconclusive results. As a marker for genotoxicity, the number of sister chromatid exchanges in blood lymphocytes proved to be relatively insensitive towards exposure to chromium(VI). There were however significant increases in rare chromosome aberrations among MMA stainless steel welders, although the reported levels of all aberrations combined were similar to those observed among control groups of many other studies. The relative lack of success of cytogenetic surveillance studies using blood lymphocytes is surprising in view of the strong genotoxicity of chromium(VI). A possible explanation comes from recent studies which showed that the differences in chromium lymphocyte levels between exposed and controls were disproportionately small. Another factor which complicates attempts to correlate genotoxic effects in lymphocytes with the processes giving rise to cancers of the respiratory system is the toxicokinetics of inhaled chromium(VI). Only small fractions of the total inhaled dose are distributed in the body while the bulk of chromium(VI) deposited in the lungs remains there for very long periods of time. The vast majority of lymphocytes will therefore come into contact with chromium(VI) not while travelling through the supporting tissues of the lungs but during their migration through the blood. There they take up chromium(VI) that has leached from the lungs. Blood lymphocytes therefore seem to be inappropriate for the monitoring of the biologically effective dose, and of early biological effects arising from exposure to chromium(VI). Thus there is an urgent need to develop techniques which would allow the non-invasive monitoring of internalized doses of chromium in the lung.     

Intracellular chromium reduction

Two steps are involved in the uptake of Cr(VI): (1) the diffusion of the anion CrO4(2-) 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.     

Gas chromatographic determination of 3-butene-1,2-diol in urine samples after 1,3-butadiene exposure

1,3-Butadiene is an important industrial chemical and a common environmental contaminant. Because of its suspected carcinogenicity butadiene-related research has gained high activity. The obvious lack of knowledge so far has been that a biomonitoring method that can detect at least one of the metabolites of butadiene from body fluids or excretas does not exist. In this communication we describe a robust and simple analytical method which can be applied for biomonitoring purposes. We have developed a method that can detect 3-butene-1,2-diol in urine samples of rats inhalation-exposed to various concentrations of 1,3-butadiene. The method is based on liquid–liquid extraction and subsequent gas chromatographic analysis. The extraction efficiency of 3-butene-1,2-diol at a concentration of 2.2 μg/ml was 95% (SD=±3%, n=3) and was achieved by using sodium chloride saturation and isopropanol as an extracting solvent. The standard deviation of the gas chromatographic analysis was ±2% (n=12), the limit of detection was 0.08 μg/ml, the limit of quantitation was 0.11 μg/ml (SD=±4.8%, n=3) and the analysis was observed to be linear from 0.11 to 486 μg/ml (R=0.9987). Animals exposed to 1,3-butadiene showed a linear excretion of 3-butene-1,2-diol into urine as a function of butadiene exposure. During the exposure saturation of metabolism or accumulation of 1,3-butadiene or 3-butene-1,2-diol into the body was not observed in any exposure levels used.     

DNA fragmentation, DNA-protein crosslinks, P postlabeled nucleotidic modifications, and 8-hydroxy-2′-deoxyguanosine in the lung but not in the liver of rats receiving intratracheal instillations of chromium(VI). Chemoprevention by oral N-acetylcysteine

An in vivo study was carried out with the objectives of evaluating (a) the localization of DNA lesions resulting from exposure to chromium(VI) by the respiratory route, (b) the molecular nature of DNA alterations, and (c) modulation of DNA damage by a known chemopreventive agent. To this purpose, Sprague-Dawley rats received intratracheal instillations of sodium dichromate (0.25 mg/kg body weight) for three consecutive days, and the day after the last treatment lung and liver were removed for DNA purification. The results showed a selective localization of DNA lesions in the lung but not in the liver, which can be ascribed to toxicokinetics and metabolic characteristics of chromium(VI). DNA alterations included DNA-protein crosslinks, DNA fragmentation, nucleotidic modifications, and 8-hydroxy-2′-deoxyguanosine. The last two endpoints were evaluated, for the first time in chromium toxicology, by means of ³²P postlabeling procedures. This methodology was adapted to the detection of the DNA damage produced by those reactive oxygen species which result from the intracellular reduction of chromium(VI). The oral administration of the thiol N-acetylcysteine completely prevented any induction of DNA lesions in lung cells.     

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).     

Synthesis and characterization of new porphyrazine-Gd(III) conjugates as multimodal MR contrast agents

Magnetic resonance imaging (MRI) has long been used clinically and experimentally as a diagnostic tool to obtain three-dimensional, high-resolution images of deep tissues. These images are enhanced by the administration of contrast agents such as paramagnetic Gd(III) complexes. Herein, we describe the preparation of a series of multimodal imaging agents in which paramagnetic Gd(III) complexes are conjugated to a fluorescent tetrapyrrole, namely, a porphyrazine (pz). Zinc metalated pzs conjugated to one, four, or eight paramagnetic Gd(III) complexes are reported. Among these conjugates, Zn-Pz-8Gd(III) exhibits an ionic relaxivity four times that of the monomeric Gd(III) agent, presumably because of increased molecular weight and a molecular relaxivity that is approximately thirty times larger, while retaining the intense electronic absorption and emission of the unmodified pz. Unlike current clinical MR agents, Zn-Pz-1Gd(III) is taken up by cells. This probe demonstrates intracellular fluorescence by confocal microscopy and provides significant contrast enhancement in MR images, as well as marked phototoxicity in assays of cellular viability. These results suggest that pz agents possess a new potential for use in cancer imaging by both MRI and near-infrared (NIR) fluorescence, while acting as a platform for photodynamic therapy.     

Hexavalent chromium removal in vitro and from industrial wastes, using chromate-resistant strains of filamentous fungi indigenous to contaminated wastes

Two chromate-resistant filamentous fungi, strains H13 and Ed8, were selected from seven independent fungal isolates indigenous to Cr(VI)-contaminated soil because of their ability to decrease hexavalent chromium levels in the growth medium. Morphophysiological studies identified strain H13 as a Penicillium sp. isolate and Ed8 as an Aspergillus sp. isolate. When incubated in minimal medium with glucose as a carbon source and in the presence of 50 microg/mL Cr(VI), these strains caused complete disappearance of Cr(VI) in the growth medium after about 72 h of incubation. Total chromium concentration in growth medium was constant during culture growth, and no accumulation of chromium in fungal biomass was observed. Quantitative determinations of oxidized and reduced chromium species during the reduction process revealed stoichiometric conversion of Cr(VI) to Cr(III). A decrease in Cr(VI) levels from industrial wastes was also induced by Ed8 or H13 biomass. These results indicate that chromate-resistant filamentous fungi with Cr(VI)-reducing capability could be useful for the removal of Cr(VI) contamination.     

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).     

Efficiency of Penicillium chrysogenum PTCC 5037 in reducing low concentration of chromium hexavalent in a chromium electroplating plant wastewater

The effectiveness of Penicillium chrysogenum was evaluated for reducing Cr(VI) from the wastewater of a chromium electroplating plant. Statistically-based experimental designs were applied to optimize the condition for reducing Cr(VI) to Cr(III). By applying Plackett-Burman factorial design and central composite design as the optimization step, attempts were made to identify optimal values of the three factors that bringing about maximum microorganism activity and therefore maximum hexavalent chromium(VI) bioreduction. It was found that each gram of P. chrysogenum of dry biomass condition could reduce 66 mg of Cr(VI) to Cr(III) in the wastewater of the chromium electroplating plant.     

Effects of chromium(VI) and chromium(III) on energy charge and oxygen consumption in rat thymocytes

The cytotoxic effects of chromium compounds in two oxidation states have been studied in rat thymocytes. endogenous nucleotide levels and oxygen consumption were examined as relevant parameters of the physiological state of the cell. Incubation of rat thymocytes with Cr(VI) produced a marked unbalance of endogenous purine nucleotide pool and a parallel decrease in oxygen consumption. A close correlation between the reduction of oxygen consumption and ATP level in rat thymocytes treated with increasing concentrations of Cr(VI) has been found. In rat thymocytes permeabilized with digitonin and in isolated rat liver mitochondria both Cr(VI) and Cr(III) showed, at different range of concentrations, a marked inhibition of maximal oxygen consumption rate (uncoupled respiration). The effects observed were depending on chromium oxidation state and on different mitochondrial sites of substrate oxidation.     

EPR detection of paramagnetic chromium in liver of fish (Anguilla anguilla) treated with dichromate(VI) and associated oxidative stress responses—Contribution to elucidation of toxicity mechanisms

The impact of chromium (Cr) on fish health has been the subject of numerous investigations, establishing a wide spectrum of toxicity, attributed particularly to the hexavalent form [Cr(VI)]. However, reports on the simultaneous assessment of Cr toxicity in fish and its toxico-kinetics, namely involving metal speciation, are scarce. Therefore, keeping in view the understanding of the mechanisms of Cr(VI) toxicity, this work intended to detect the formation of paramagnetic Cr species in liver of Anguilla anguilla following short-term dichromate(VI) intraperitoneal treatment (up to 180 min), assessing simultaneously the pro-oxidant properties. The formation of Cr(V) and Cr(III) was examined by electron paramagnetic resonance (EPR), as an innovative approach in the context of fish toxicology, and related with the levels of total Cr. Cr(V) was successfully detected and quantified by EPR spectrometry, showing a transient occurrence, mostly between 15 and 90 min post-injection, with a peak at 30 min. The limitations of EPR methodology towards the detection and quantification of Cr(III) were confirmed. Although Cr(VI) exposure induced the antioxidant system in the eel's liver, the oxidative deterioration of lipids was not prevented. Overall, the results suggested that Cr(V), as a short-lived species, did not appear to be directly and primarily responsible for the cellular damaging effects observed, since stress responses persisted up to the end of exposure regardless Cr(V) drastic decay. Though further research is needed, ROS mediated pathways (suggested by superoxide dismutase and catalase activity induction) and formation of Cr(III) complexes emerged as the most plausible mechanisms involved in Cr(VI) toxicity.     

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.     

Mapping oxidative DNA damage at nucleotide level

DNA damage induced by reactive oxygen species (ROS) is considered an important intermediate in the pathogenesis of human conditions such as cancer and aging. By developing an oxidative-induced DNA damage mapping version of the Ligation-mediated polymerase chain reaction (LMPCR) technique, we investigated the in vivo and in vitro frequencies of DNA base modifications caused by ROS in the human p53 and PGK1 gene. Intact human male fibroblasts were exposed to 50 mM H₂O₂, or purified genomic DNA was treated with 5 mM H₂O₂, 100 μM Ascorbate, and 50 μM, 100 μM, or 100 μM of Cu(II), Fe(III), or Cr(VI) respectively. The damage pattern generated in vivo was nearly identical to the in vitro Cu(II) or Fe(III) damage patterns; damage was non-random with guanine bases heavily damaged. Cr(VI) generated an in vitro damage pattern similar to the other metal ions, although several unique thymine positions were damaged. Also, extra nuclear sites are a major contributor of metal ions (or metal-like ligands). These data show that the local probability of H₂O₂-mediated DNA damage is determined by the primary DNA sequence, with chromatin structure having a limited effect. The data suggest a model in which DNA-metal ion binding domains can accommodate different metalions. LMPCR's unique aspect is a blunt-end ligation of an asymmetric double-stranded linker, permitting exponential PCR amplification. An important factor limiting the sensitivity of LMPCR is the representation of target gene DNA relative to non-targeted genes; therefore, we recently developed a method to eliminate excess non-targeted genomic DNA. Restriction enzyme-digested genomic DNA is size fractionated by Continuous Elution Electrophoresis (CEE), capturing the target sequence of interest. The amount of target DNA in the starting material for LMPCR is enriched, resulting in a stronger amplification signal. CEE provided a 24-fold increase in the signal strength attributable to strand breaks plus modified bases created by ROS in the human p53 and PGK1 genes, detected by LMPCR. We are currently taking advantage of the enhanced sensitivity of target gene-enriched LMPCR to map DNA damage induced in human breast epithelial cells exposed to non-cytotoxic concentrations of H₂O₂.     

Safety of trivalent chromium complexes: no evidence for DNA damage in human HaCaT keratinocytes

Several studies have demonstrated beneficial effects of supplemental trivalent Cr in subjects with reduced insulin sensitivity with no documented signs of toxicity. However, recent studies have questioned the safety of supplemental trivalent Cr complexes. The objective of this study was to evaluate the cytotoxic and genotoxic potential of the Cr(III) complexes (histidinate, picolinate, and chloride) used as nutrient supplements compared with Cr(VI) dichromate. The cytotoxic and genotoxic effects of the Cr complexes were assessed in human HaCaT keratinocytes. The concentrations of Cr required to decrease cell viability were assessed by determining the ability of a keratinocyte cell line (HaCaT) to reduce tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. DNA damage using the Comet assay and the production of 8-hydroxy-2′-deoxyguanosine were also determined with and without hydrogen peroxide-induced stress. The LC50 for human cultured HaCaT keratinocytes was 50 μM for hexavalent sodium dichromate and more than 120-fold higher for Cr chloride (6 mM) and Cr histidinate (10 mM). For Cr picolinate at saturating concentration (120 μM) the LC50 was not attained. High Cr(III) concentrations, 250 μM Cr as Cr chloride and Cr histidinate and 120 μM Cr picolinate (highest amount soluble in the system), not only did not result in oxidative DNA damage but exhibited protective antioxidant effects when cells were exposed to hydrogen peroxide-induced oxidative stress. These data further support the low toxicity of trivalent Cr complexes used in nutrient supplements.