H. Ottenwälder

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(VI) at 370 nm. At stoichiometric conditions (molar ratio Cr(VI)/GSH of 1:3) the reduction was strongly dependent on the solutions 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 Na2CrO4 (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 Na2 51CrO4 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.

Fast uptake kinetics in vitro of 51Cr (VI) by red blood cells of man and rat

Fast transport kinetics of 51Cr (VI) into red blood cells (RBCs) in vitro were studied. No significant species differences were found between RBCs of man and rat. The uptake of 51Cr (VI) by RBCs in whole blood was composed of two different first order processes of different velocities (apparent t1/2 of 22.7 s and 10.4 min for man and 6.9 s and 10.1 min for rat, respectively). However, even after longer time periods a fixed portion of approximately 15% of the administered dose remained in the plasma and did not penetrate into RBCs Over the entire concentration range studied (10 μM–50 mM), the fast initial uptake followed Michaelis-Menten kinetics. The maximal capacity of this Cr(VI) transport into RBCs of man and rat was 3.1×108 CrO42− ions × cell−1 × min−1 and 2.5×108 CrO4−2 ions × cell−1 × min−1, respectively. It is likely that Cr(VI) is transported into RBCs via a physiological anion carrier (“band-3-protein”).

Differential effect of N-acetylcysteine on excretion of the metals Hg, Cd, Pb and Au

Male Wistar rats were subacutely treated with sublethal doses of HgCl2, CdCl2, Pb(NO3)2, or Na-aurothiomalate. The metal preparations contained trace doses of radioactive nuclide. Based on the doses given and on the radioactivity excretion in urine and faeces the body burden was determined. After the metal treatment periods, some of the animals received N-acetylcysteine (up to 100 mg/kg daily, on 6 consecutive days, i.p.), and the effect of this potential chelator on metal excretion was monitored. The excretion of Hg (after dosing with HgCl2) was not influenced by N-acetylcysteine. The elimination of Cd in urine (after dosing with CdCl2) was increased by a factor of four. Also, the elimination of Pb [after dosing with Pb(NO3)2] was gradually increased (in faeces and urine) by increasing doses of N-acetylcysteine. After dosing with Na-aurothiomalate, the excretion of Au in urine was increased to about 30%. The data suggest some activity of N-acetylcysteine in facilitating excretion of Pb, Cd or Au, but not of Hg.

Recent advances in biological monitoring of hexavalent chromium compounds

Intratracheal instillation of 51CrCl3 in anaesthetized rabbits resulted in partial absorption. In blood, the absorbed material was entirely confined to the plasma compartment. By contrast, after similar application of Na251CrO4 the bulk of blood radioactivity was present in red blood cells (RBC). It is suggested that Cr (VI) may enter the body not reduced via the lung and may be deposited in RBC for the cells lifetime (approximately 110 d). Since inhalation of Cr (VI)-containing aerosols or particles is the main occupational exposure route for man, it is concluded that the chromium content of RBC could be used as a selective biological indicator for exposures to (carcinogenic) hexavalent chromium. The new theoretical concept was confirmed in a pilot study with workers of a dye pigment plant. Elevated chromium content of whole blood evoked by exposure to chromate particles correlated strongly with increased chromium values in RBC (correlation coefficient: 0.86).

Detection of diepoxybutane-induced DNA-DNA crosslinks by cesium trifluoracetate (CsTFA) density-gradient centrifugation

The purpose of our study was to establish the suitability of cesium trifluoroacetate (CsTFA) isopycnic centrifugation as a method for the detection and quantification of DNA-DNA crosslinks (DDC). Rodent liver DNA treated with different concentrations of diepoxybutane and liver DNA of mice and rats exposed to high concentrations of butadiene were investigated

Disposition of intratracheally administered chromium(III) and chromium(VI) in rabbits

Intratracheal instillation of 51CrCl3 in anaesthetized rabbits resulted in partial absorption. In blood, the absorbed material was entirely confined to the plasma compartment. Only trace amounts were deposited in liver and kidney. By contrast, after similar application of Na(2)51CrO4 the bulk of blood radioactivity was present in erythrocytes. Substantial deposition occurred in liver and kidneys. It is concluded that Cr(VI) may enter the body unreduced via the lung and is partly deposited in cells over a prolonged period of time.

The Formation of Glutathione-Chromium Complexes and Their Possible Role in Chromium Disposition

The reduction of Cr(VI) to Cr(III) by glutathione (GSH) in vitro resulted in the formation of two different GSH-chromium complexes with an approximate molecular-weight of 10,000 and 5000, respectively. Both complexes exhibited strong fluorescence. The molar ratio between oxidized glutathione (GSSG) and chromium was calculated by determining the chromium content of the two isolated complex species by flameless Atomic Absorption Spectrometry (AAS).

Bioavailability and metabolism of hexavalent chromium compounds

Intratracheal instillation of 51CrCl3 in anaesthetized rabbits resulted in partial absorption. In blood, the absorbed material was entirely confined to the plasma compartment. Only trace amounts were deposited in liver and kidney. By contrast, after similar application of Na, 51CrO4 the bulk of blood radioactivity was present in red blood cells (RBC). Substantial deposition occurred in liver and kidneys. It is concluded that Cr(VI) may enter the body unreduced via the lung and is partially deposited in cells over a prolonged period of time. Since chromium was accumulated in liver after administration of Cr(VI) we investigated the intracellular disposition of Cr(VI) in the isolated perfused liver. No significant sex differences in chromium distribution were observed. At the end of the experiments (1 h), 60% of the applied dose (312μg Cr/liver) was located in the cytosol, whilst 14% was in the mitochondria, 9% in the microsomal pellet and 2% was associated with the nuclei. Gel chromatography of the cytosoli...

Vinyl acetate: DNA-binding assay in vivo

The question of carcinogenicity of vinyl acetate led us to perform a DNA binding assay in vivo in rats, using 14C-labelled vinyl acetate. After administration of [14C]vinyl acetate to male and female Fischer-344 rats, either orally or by inhalation, no specific hepatic DNA adducts, known to occur after administration of labelled vinyl halides or vinyl carbamates, could be detected in hepatic tissues.

Nick translation studies on DNA strand breaks in pBR322 plasmid induced by different chromium species.

Single strand breaks are DNA defects caused by various chemicals. DNA strand breaks induced in vitro by chromium(VI) during the reduction of this chromium species with glutathione or hydrogen peroxide were examined. Using DNA agarose gel electrophoresis and a nick translation assay, strand breaks were detected only when chromium(VI) was reduced by hydrogen peroxide. The reduction of chromium(VI) by an excess of glutathione led to no alteration in the DNA agarose gel electrophoresis pattern of the double-stranded plasmid pBR322 DNA and in the nick translation assay, indicating that no strand breaks had occurred under these conditions. No strand breaks could be detected during the reduction by hydrogen peroxide after the addition of superoxide dismutase. This indicates that hydroxyl radicals from peroxochromium complexes may be a relevant reactive species involved in chromate genotoxicity.