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Arch Toxicol (1990) 64: 169-176

Toxicology 9 Springer-Verlag 1990

Reduction of hexavalent chromium by ascorbic acid and glutathione with special reference to the rat lung Yasutomo Suzukil and Kazuo Fukuda z I Department of Occupational Diseases and 2 Department of Experimental Toxicology, National Institute of Industrial Health, 21-1, Nagao 6-chome, Tama-ku, Kawasaki 214, Japan Received August 2, 1989/Received after revision November 16, 1989/Accepted November 20, 1989

Abstract. The reduction of 20 ~tM hexavalent chromium [chromium(VI)] by L-ascorbic acid (AsA) (0.06-2 raM) and/or glutathione (GSH) (2-15 mM) in buffer solutions, cell-free bronchoalveolar lavage fluids or soluble fractions of rat lungs was investigated at physiological pH (37* C). The reduction in AsA solution was pseudo-first-order in a single phase with respect to chromium(VI), but that in GSH solution showed a two-phase process. The half-life of chromium(IV) ranged from seconds to hours. The reducing ability of AsA was markedly higher than that of GSH. Coexistence of equimolar GSH with AsA accelerated the reduction rate slightly, in comparison with that in the corresponding AsA solution. Lavage fluids containing 0.06 mM AsA showed pH-dependent reactions similar to those of the corresponding AsA solutions. The lungsoluble fractions reduced chromium(VI) in a process composed of phase I and phase II, characterized by the reducing ability of AsA-GSH cooperation and of AsA alone, respectively. Reduction in the former was 30-40% more rapid than in the latter. The biological half-life of chromium(VI) in the lung was estimated to be 0.6 min, on the basis of the reducing activity in the first phase. However, the apparent biological half-life of chromium(VI) was about 2 min in rat lungs after intratracheal injection of chromate, involving depletion of AsA, but no significant changes in GSH. The difference is discussed in terms of AsA-induced initiative reduction in the alveolar lining fluid and subsequent obstructive effects of the resulting trivalent species on trans-membrane permeability of chromate anions. These results suggest that AsA is more reactive than GSH in the reduction of chromium(VI) in the rat lung and that the extracellular AsA in the alveolar lining fluid plays an important role in antioxidant defense against inhaled chromium(VI) compounds. Key words: Hexavalent chromium - Reduction Ascorbic acid - Glutathione - Rat lung - Bronchoalveolar lavage fluid

Offprint requests to: Y. Suzuki

Introduction

The toxicity of chromium is strongly dependent on its valence state; hexavalent chromium is more toxic than the trivalent species [chromium(III)] (Tandon 1982). In inhalation exposure to chromate compounds, an increased risk of respiratory cancer in man has been documented epidemiologically (L6onard and Lauwerys 1980; Norseth 1981; Franchini et al. 1983; Yassi and Nieboer 1988). Chromate anions penetrate biological membranes easily (Gray and Sterling 1950; Levis et al. 1978) and are subjected to subsequent reduction to chromium(III) (Levis et al. 1978; Kitagawa et al. 1982; Levis and Bianchi 1982). This intracellular reduction is widely believed to be closely involved in the toxic effects of the metal anions (Levis and Bianchi 1982). The reduction of chromium(VI) is caused by microsomal electron transport systems (Gruber and Jennette 1978; Garcia and Jennette 1981; Mikalsen et al. 1989) involving NADPH and NADH, certain heme proteins and flavoproteins (Connett and Wetterhahn 1983), ascorbic acid (Samitz 1970; Korallus et al. 1984; Connett and Wetterhahn 1985), glutathione (Wiegand et al. 1984a; Connett and Wetterhahn 1985) and other cellular thiols (Connett and Wetterhahn 1985). The epithelial lining fluid of the human lower respiratory tract contains high levels of reduced glutathione (Cantin et al. 1987). In contrast, there are considerable amounts of AsA in the alveolar lining fluid of the rat (Willis and Kratzing 1974, 1976) and the guinea pig (Willis and Kratzing 1976). Recently, it has been shown that bronchoalveolar lavage fluids of rats possess AsA-related ability for reduction of chromium(VI) (Suzuki 1988). Thus these reductants have been suggested to play important roles in the reduction of chromium(VI) in the parenchyma of the lung and also in the alveolar lining fluid. The extracellular reducing ability is considered to be of substantial importance for antioxidant defense in the lung against exogenous and endogenous oxidants (Cantin et al. 1987; Suzuki 1988) as well as the atmospheric oxygen tension (Willis and Kratzing 1976). The purpose of this study was to obtain an insight into kinetic discrimination between AsA and GSH in the biological reduction of chromium(VI), especially in the lung.

170

Materials and methods

Apparatus and analytical methods The analyses of AsA and chromium(VI) were carried out by anion-exchange high-performance liquid chromatography (HPLC) (FPLC system with Mono Q HR 5/5, Pharmacia, Uppsala, Sweden) as reported previously (Suzuki and Fukuda 1989). GSH in samples was separated by the same I-IPLC, but with a different buffer system. The starting buffer (buffer A) was 20 mM Bis-tris propane {l,3-bis[tris(hydroxymethyl)methylamino]propane} (BTP) buffer, pH 8.0. The eluting solution (buffer B) was buffer A plus 0.5 M sodium chloride. Gradients of sodium chloride from 100% buffer A to 100% buffer B were substantially the same as those used in the AsA analysis described above. A flow-rate of 2.0 ml/min was used and the sample volume was 10 ~tl. The column was operated at 30" C. GSH separated was determined by fluorometric analysis using o-phthalaldehyde (Wako, Osaka, Japan), after Keller and Menzel (1985), with a few technical modifications.

Reagents Sodium chromate tetrahydrate of analytical reagent grade was purchased from J. T. Baker (Phillipsburg, NJ, USA) and a stock solution of 2 mM chromium(VI) was prepared with distilled water. L-Ascorbic acid and GSH (both reduced form) of reagent grade were purchased from Sigma (St Louis, MO, USA). BTP buffers, 20 mM and 0.1 M, pH 7.4-7.8, were prepared using distilled water. Stock solutions of AsA and GSH ( 2 0 - 9 0 raM) were prepared daily by dissolving the reagents in BTP buffer (pH 7.4), and kept ice-cold. Standard solutions for the determination of AsA, GSH and chromium(VI) in the samples for reduction experiments were prepared in the same manner. In the determination of GSH and chromium(VI) in the lungs, standard solutions prepared with 10 mM EDTA and 5% sodium carbonate solutions (Suzuki and Fukuda 1989) were used, respectively, to obtain higher stability. In the results from a preliminary stability test using four different buffers of 0.1 M, pH 7.4, namely, BTP, HEPES [N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid], TRIS and phosphate, the stability of AsA was the highest in BTP buffer, followed in decreasing order by HEPES >phosphate >TRIS, at 37* C. The stability of GSH was higher than that of AsA in the corresponding buffers, and showed the same buffer-dependent order of decrease as that of AsA. BTP buffer was therefore used for the preparation of the samples for reduction studies.

Preparation of AsA and~or GSH test solutions Dilute solutions of AsA (0.06-2 mM) or GSH ( 2 - 1 5 mM) and mixed solutions of~equimolar AsA and GSH (0.06-2 mM) for reduction studies were prepared by addition of the stock solutions into BTP buffer. The GSH solutions above 3 mM were prepared with 0.1 M BTP buffer. The other solutions of the reductant(s) were prepared with 20 mM BTP buffer. In preparing high levels of the reductant(s), buffers of higher pH values were used to obtain the final pH of 7.4

Preparation of pulmonary samples Bronchoalveolar lavage fluid.

Male adult rats (400-420 g) of the Sprague-Dawley strain (Clea Japan, Tokyo, Japan), fed on a commercial pelleted diet (CE-2, Clea Japan) and sterilized tap water ad lib were used. After blood had been taken from the hearts of Nembutal-anesthetized animals, the lungs were lavaged in situ with BTP buffer (pH 7.4 or 6.6) containing 0.9% sodium chloride (saline-BTP buffer), in the same manner as that described previously (Suzuki 1988), with a few modifications.

The bronchoalveolar lavage fluids were then recovered. As alveolar lining fluids are weakly acidic, e. g. pH 6.6 in living cats (Scarpelli 1977) and 6.9 in the lungs of anesthetized rabbits (Nielson et al. 1981), the reduction in lavage fluids was investigated at pH 6.6 as well as 7.4. The pH of the recovered lavage fluids was readjusted to 7.4 or 6.6 by addition of dilute solution of sodium hydroxide. Cell-free lavage fluids were obtained after centrifugation at 500 g for 5 min at 4* C. On the basis of AsA measurements of these fluids, the AsA levels were adjusted to 0.06 mM by addition of the buffers with corresponding pH value. Solublefraction. After blood had been taken in the manner described above, the lungs were excised. Homogenates of the tissues (0.41.2 g/20 ml BTP buffer, pH 7.4) were prepared and then centrifuged as described previously (Suzuki and Fukuda 1989). The soluble fractions were separated and analyzed for AsA. On the basis of the analytical data, the soluble fractions were diluted by addition of BTP buffer to reach 0.06, 0.08 and 0.12 mM AsA (higher levels were technically difficult). These operations were performed as quickly as possible under ice-cold conditions. The preparations were considered to contain GSH approximately equimolar with AsA and other tissue components related to chromium(VI) reduction, as described in Results.

Reduction conditions and calculation of half-life The above-described test samples of AsA and/or GSH, lavage fluids and soluble fractions were pre-incubated at 37* C until they reached temperature equilibrium (for 2 min), followed by addition of the stock solution of chromium(V/) to reach 20/.tM in all samples, on a magnetic stirrer. The same incubation conditions were maintained and the time-related decreases in chromium(VI) were followed. If necessary, AsA and GSH were also analyzed. The half-life (T) of chromium(VI) in each sample was calculated by the following equation: T = IA 9 In 2 where k is the rate constant of the first-order kinetic equation.

Determination of the normal levels of lung AsA and GSH In the determination of the normal AsA levels in rat lungs, the precipitates obtained by the first centrifugation were re-used for preparing additional soluble components. These samples obtained by the first and second separations were analyzed for AsA. The measurements of AsA were totalled for each lung. In the determination of GSH, the lung tissues were treated by the same procedure, using 10 mM EDTA solution, for higher stability.

In vivo experiments Ethyl ether-anesthetized animals of the same strain as described above were injected intratracheally with 0.6 ml saline solution of sodium chromate (1.2 ~tmol chromium(VI)/animal). Control animals were injected with saline solution of the same volume. Blood was taken from the heart at 4, 10 and 18 min after injection, and then the lung, liver, kidneys and spleen were removed. The lung tissues were homogenized in BTP buffer (pH 7.4), 10 mM EDTA solution or 5% sodium carbonate solution (Suzuki and Fukuda 1989). The soluble samples obtained after centrifugation of these homogenates were used for the analyses of AsA, GSH and chromium(VI), respectively. Aliquots of tissues of the lungs and the other organs, and of blood were digested in nitric acid with addition of hydrogen peroxide on a hot plate. The residues were dissolved in dilute nitric acid and analyzed for total chromium using an electrothermal atomic absorption spectrometer (Zeeman 5000, Perkin-Elmer, Norwalk, CT, USA).

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Fig. 7. Biological reduction of chromium(VI) (a) and concomitant changes in AsA and GSH levels (b) in the lungs of rats after intratracheal injection of chromate. The experimental animals were injected with 0.6 ml saline solution containing 2 mM sodium chromate. The control animals were injected with saline solution of the same volume. Each value represents the mean • SD for four or five animals. C, control levels

Table 2. Chromium levels in the lung, other organs and blood of rats after intratracheal injection of sodiu m chromate a

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Fig. 6, Relationships between the initial concentrations of AsA and the average half-life values of chromium(VI) in buffer solutions of AsA (1), mixed solutions of equimolar AsA and GSH (2) and soluble fractions in phase I (3). The original data are shwon in Figs. 2 and 5, and in Table 1. T, half-life (min); A, declination constant; B, constant; C, initial concentration of AsA (mM)

Extrapolation of the biological half-life in the lung As shown in Fig. 6, straight lines could be drawn between logarithms of the initial AsA levels and of the half-life values of chromium(VI) obtained in the above-described samples. As shown by the approximately similar value for the declination constant "A", these linear lines are parallel to each other. As for the soluble fractions in phase I, an extension line (the dotted line) following the short linear line would represent half-life values in soluble fractions at higher levels of the reductants than the adjusted levels. As described above, the cytoplasmic levels of AsA and GSH may be approximately 2 raM. The half-life of chromium(VI) in the soluble fractions of these high reductant levels could be extrapolated to be about 0.6 min on the

Time after injection (min) 4 10 18

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Other organs e (~tmol/organs)

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a The experimental animals were injected intratracheally with 0.6 ml 2 mM sodium chromate saline solution. The control animals were injected with saline solution of the same volume b Differences between the mean levels in the experimental and control groups of four or five animals c The liver, kidneys and spleen

basis of the extended linear relationship. This would be comparable to the biological half-life of chromium(VI) in the rat lung cytoplasm, as discussed later.

In vivo reduction after intratracheal injection of chromate As shown in Fig. 7 a, the reduction of chromium(VI) in the lungs of rats given an intratracheal injection of chromate was rapid for the initial few minutes, but slow thereafter, showing no sign of a pseudo-first-order reaction. About 20% of the initial chromate burden remained unreduced in the lungs even at 18 min after injection. The biological half-life of chromium(VI) determined by the fast decreasing pattern was about 2 min, which was rather longer than

174 the extrapolated half-life value in the cytoplasmic reducing systems. This difference will be discussed later. As shown in Fig. 7 b, about 30% of the control level of AsA was depleted during the reduction process. However, no significant losses of GSH were observed. Table 2 reveals that only small amounts of chromium were released from the lungs during the reduction process. The amounts of chromium deposited in the other organs (liver, kidneys and spleen) were only 10% of the initial burden, consistent with the small decreases of total chromium in the lungs. The increased blood levels of total chromium were also slight. These results indicate that most of the chromate injected into the lungs was quickly reduced, followed by immediate deposition of the resulting trivalent species and that the release of chromium from the lungs, probably in the hexavalent form, was less than 10% of the initial burden.

Discussion The reduction of chromium(VI) by AsA and/or GSH in buffer solutions and in soluble fractions of rat lungs was investigated at physiological pH. Connett and Wetterhahn (1985) have investigated in vitro reduction of chromium(VI) by cellular thiols and carboxylic acids, and concluded that in the reduction of chromate in the cell cytoplasm, the significant reactive species are expected to be glutathione, cysteine and ascorbate. The present results (Figs. 2 and 3, Table 1) indicate that AsA is more reactive than GSH under physiological conditions and suggest that the former is predominantly involved in the reduction of chromium(VI) in biological systems, especially in the rat lung, where approximately equimolar AsA and GSH exist. The reduction of chromate by low-molecular-weight reductants (e. g. cysteine, ascorbate, glutathione) belongs theoretically to second-order kinetics. However, Baldea and Munteanu (1980) have demonstrated that the reaction between chromate and AsA is first-order with respect to both ascorbate and chromate at weakly acidic pH. Connett and Wetterhahn (1983) have confirmed that the reaction is first-order at pH 7.4 (1 M TRIS-HC1) and 25~ The present reduction processes can obviously be regarded as pseudo-first-order reactions with respect to chromium(VI), except for the slight deviation from the strict behavior in the samples with lower reductant levels. The reason seems to be that in comparison with chromate, the reductant levels in the samples used here were so high that no retardation of the reduction caused by the partial depletion of the reductant(s) was apparent. This would be the case with pulmonary reduction of inhaled chromate compounds, provided that the inhaled levels are not extremely high and that the reductant levels are normal. The available data for pulmonary AsA and GSH levels suggest that the cytoplasmic levels of these reductants in the rat lung would be approximately 2 mM, as described above. Then, from the linear relationship between the reductant levels and the half-life values in phase I (Fig. 6), the half-life of chromium(VI) in the soluble fractions at the physiological reductant levels was extrapolated, resulting in a value of about 0.6 rain. Furthermore, it is proposed that

the extrapolated value would be comparable with the biological half-life of chromium(VI) in the cytoplasmic reduction systems. This proposition is based on the assumption that the samples used here contained most of the possible cytoplasmic components related chemically and enzymatically to the reduction. This is supported by the following findings. First, the fact that the reduction in the soluble fractions in phase I (Fig. 5) was more rapid than that in the corresponding mixed solutions of AsA and GSH (Table 1) indicates that other cellular reducing components were contained in the samples, though their reducing ability was minor. Second, the instability of GSH seen in the soluble fractions (Fig. 1) suggests that GSH-related cellular enzymes, which may possibly affect the reduction by GSH, also existed in the soluble fractions. Several investigations (Gruber and Jennette 1978; Garcia and Jennette 1981) have demonstrated the enzymatical chromium(VI)-reducing ability of microsomes with NADPH and HADH. However, it is unclear to what extent microsomes actually contribute to the intracellular reduction of chromium(VI) under normal aerobic conditions, especially in oxygen-abundant tissues such as the lung, since the oxygen tension is obviously critical for microsomal chromium(VI) reduction (Mikalsen et al. 1989). The reducing activities of the bronchoalveolar lavage fluids were similar to those of the AsA solutions of the same reductant level at each pH (Fig. 4). These findings support the previous conclusion that the alveolar lining layer of the rat has reducing ability induced exclusively by AsA (Suzuki 1988). Cantin et al. (1987) have shown that the epithelial lining fluid of the human lower respiratory tract contains large amounts of reduced glutathione and suggested that this reductant is an important component of the antioxic defenses in the alveolus. However, in our preliminary analysis, GSH was not detected in the bronchoalveolar lavage fluids from normal rats. Further investigations are required to determine whether GSH exists in the alveolar lining fluid of the rat. Even if GSH does exist in the fluid, it would show only minor effects on the AsAinduced chromium(VI) reduction in the lining fluid, because of its lower reducing ability. The blood level of chromium was highest immediately after intratracheal injection and decreased thereafter (Table 2). This changing pattern consists with earlier results obtained in rats following short-term inhalation of chromium(VI) aerosols (Suzuki et al. 1984) and mist (Adachi et al. 1983), but conflicts with the observations (Wiegand et al. 1984b) that the maximum concentration of chromium in the blood of rabbits following intratracheal instillation of 51Cr(VI) appeared later. This may be caused by possible difference in lung ability for the reduction of chromium(VI) between the rat and rabbit. The present results suggest that the lung ability would be related to the synthesis and metabolism of AsA, which involve species differences (Chatterjee 1973; Rucker et al. 1980; Ginter 1980). The biological half-life of chromium(VI) (about 2 min) in the lungs following intratracheal injection of chromate seems to be somewhat longer than the predicted half-life value in the cytoplasmic reduction systems. This suggests that the greater part of the chromate anions injected might have been reduced directly by the extracellular AsA in the

175

alveolar lining fluid before penetration into the epithelial cells. This possibility is supported by the fact that AsA was simultaneously depleted in the reduction process (Fig. 7 b), and also by the following kinetic considerations. The extracellular AsA in rat lungs has been reported to be within the range 12% (Suzuki 1988) to 30% (Willis and Kratzing 1976) of total lung AsA. Therefore, the extracellular fluid levels of AsA would be diluted to 0 . 4 - 1 mM by intratracheal injection of 0.6 ml chromate solution. No other effective reductants apart from AsA exist in the lining fluids, as suggested by the results shown in Fig. 4. Then, according to the relationship shown in Fig. 6 (linear line 1), the half-life values of chromium(VI) in the dilute extracellular fluid are estimated to be 4 - 2 min, comparable to the in vivo results. In the alveoli under physiological conditions, the lower pH values of the alveolar lining fluid (Scarpelli 1977; Nielson et al. 1981) might result in more rapid reduction. Although the extrapolation of these findings to the human situation would be crucial, no available data of AsA levels inhuman alveolar lining fluid have been found. Unlike the situation with in vitro reduction, in vivo reduction left considerable amounts of unreduced chromate in the lungs (Fig. 7a), although large amounts of AsA existed without oxidation (Fig. 7 b). In the earlier period after injection, the rate-determining step in the reduction of chromium(VI) is probably the initiating reaction with extracellular AsA. The resulting chromium(III) associates with membrane constituents of the epithelial cells and macrophages as well as with the components of the lining fluid. Interactions may occur with proteins, followed by their denaturation (Clark 1959), and probably with phospholipids, as chromium(III) interacts with nucleotide molecules through their phosphate moieties (DePamphilis and Cleland 1973; Legg 1978; Campomar et al. 1986). The low reduction rates (Fig. 7a) suggest that after the complete depletion of extracellular AsA, the rate-determining step may be membrane permeability for the chromate anions, and not their reduction rate in the cytoplasm. This permeability is likely to be suppressed as a result of the interaction of chromium(HI) with the membrane constituents. On the basis of the previous results obtained following acute exposure of rats to dichromate aerosols (Suzuki et al. 1984), the burden used here was much higher, corresponding to about 60% of the lethal level. At Such an excessive burden as the present exposure level, remarkable permeability changes would occur. More studies will be required to clarify the important role of extracellular reduction by AsA in the antioxidant defenses against inhaled particles of chromium(VI)-containing materials and possible interactions of the resulting trivalent species in the alveoli. Acknowledgements. The authors thank Mrs S. Kurimori and Mr S. Kishidaof this institute for technical assistance.

References Adachi S, Yoshimura H, Miyayama R, Katayama H, Takemoto K, Kawai H (1983) Effects of chromium compounds on the respiratory system. Part 2. Difference between water-soluble hexavalent compounds and trivalent compounds. Jpn J Ind Health 25: 149-154 Baldea I, Munteanu L (1980) The kinetics of the oxidation of ascorbic acid by chromate. Studia Univ Babes-Bolyai Chemia 25: 24-31 Campomar JA, Fiol JJ, Terron A, Moreno V (1986) Chromium(III) interactions with nucleotides. II. Inorg Chim Acta 124: 75- 81 Cantin AM, North SL, Hubbard RC, Crystal RG (1987) Normal alveolar epithelial lining fluid contains high levels of glutathione. J Appl Physio163" 152- 157 Chatterjee IB (1973) Evolution and the biosynthesis of ascorbic'acid. Science 182: 1271-1272 Clark JH (1959) The denaturation of proteins by chromium salts. A M A Arch Ind Health 20:117-123 Connett PH, Wetterhahn KE (1983) Metabolism of the carcinogen chromate by cellular constituents. In: Structure and bonding. Inorganic elements in biochemistry. Springer-Verlag, Berlin, 54:93-124 Connett PH, Wetterhahn KE (1985) In vitro reaction of the carcinogen chromate with cellular thiols and carboxylic acids. J Am Chem Soc 107:4282-4288 DePamphilis ML, Cleland WW (1973) Preparation and properties of chromium(IlI)-nucleotide complexes for use in the study of enzyme mechanisms. Biochemistry 12:3714-3724 Franchini I, Magnani F, Mutti A (1983) Mortality experience among chromeplating workers. Scand J Work Environ Health 9.247-252 Garcia JD, Jennette KW (1981) Electron-transport cytochrome P-450 system is involved in the microsomal metabolism of the carcinogen chromate. J Inorg Biochem 14:281-295 Ginter E (1980) Endogenous ascorbic acid synthesis and recommended dietary allowances for vitamin C. Am J Clin Nutr 33: 1448-1449 Gray SJ, Sterling K (1950) The tagging of red cells and plasma proteins with radioactive chromium. J Clin Invest 29: 1604-1613 Gruber JE, Jennette KW (1978) Metabolism of the carcinogen chromate by rat liver microsomes. Biochem Biophys Res Commun 82: 700706 Hornig D (1975) Distribution of ascorbic acid, metabolites and analogues in man and animals. In: King CG, Burns JJ (ed) Second conference on vitamin C. Ann NY Acad Sci 258: 103-I 18 Keeling PL, Smith LL (1982) Relevance of NADPH depletion and mixed disulphide formation in rat lung to the mechanism of cell damage following paraquat administration. Biochem Pharmacol 31: 32433249 Keller DA, Menzel DB (1985) Picom01e analysis of glutathione, glutathione disulfide, glutathione S-sulfonate, and cysteine S-sulfonate by high-performance liquid chromatography. Anal Biochem 151: 418423 Kitagawa S, Seki H, Kametani F, Sakurai H (1982) Uptake of hexavalent chromium by bovine erythrocytes and its interaction with cytoplasmic components; the role of glutathione. Chem Biol Interact 40: 265-274 Korallus U, Harzdorf C, Lewalter J (I 984) Experimental bases for ascorbic acid therapy of poisoning by hexavalent chromium compounds. Int Arch Occup Environ Health 53: 247-256 Legg JI (1978) Substitution-inert metal ions as probes of biological function. Coordination Chem Rev 25: 103-132 L6onard A, Lauwerys RR (1980) Carcinogenicity and mutagenicity of chromium. Mutat Res 76: 227-239 Levis AG, Bianchi V (1982) Mutagenic and cytogenetic effects of chromium compounds. In: Lang~d S (ed) Biological and environmental aspects of chromium. Elsevier Biomedical Press, Amsterdam, New York, Oxford, pp 171-208

176 Levis AG, Bianchi V, Tamino G, Pegoraro B (1978) Cytotoxic effects of hexavalent and trivalent chromium on mammalian cells in vitro. Br J Cancer 37." 386- 396 Mikalsen A, Alexander J, Ryberg D (1989) Microsomal metabolism of hexavalent chromium. Inhibitory effect of oxygen and involvement of cytochrome P-450. Chem Biol Interact 69:175-192 Nielsou DW, Goerke J, Clements JA (1981 ) Alveolar subphase pH in the lungs of anesthetized rabbits. Proc Natl Acad Sci USA 78: 71197123 Norseth T (1981) The carcinogenicity of chromium. Environ Health Perspect 40: 121-130 Rucker RB, Dubick MA, Mouritsen J (1980) Hypothetical calculations of ascorbic acid synthesis based on estimates in vitro. Am J Clin Nutr 33:961-964 Samitz MH (1970) Ascorbic acid in the prevention and treatment of toxic effects from chromates. Acta Derm Venereol (Stockholm) 50: 59 - 64 Scarpelli EM (1977) The surfactant system of the lung. Int Anesthesiol Clin 15:19-60 Stubbs DW, Griffin JF (1973) The influence of dietary protein on gulonolactone hydrolase, gulonate NADP oxidoreductase, and tissue ascorbate in male and female rats. Proc Soc Exp Biol Med 144: 199-202 Suzuki Y (1988) Reduction of hexavalent chromium by ascorbic acid in rat lung lavage fluid. Arch Toxicol 62:116 - 122

Suzuki Y, Fukuda K (1989) Anion-exchange high-performance liquid chromatographic determination of ascorbic acid and hexavalent chromium in rat lung preparations after treatment with sodium chromate in vitro and in vivo. J Chromatogr 489:283 -290 Suzuki Y, Homma K, Minami M, Yoshikawa H (1984) Distribution of chromium in rats exposed to hexavalent chromium and trivalent chromium aerosols. Ind Health 22:261-277 Tandon SK (1982) Organ toxicity of chromium in animals. In: Lang~d S (ed) Biological and environmental aspects of chromium. Elsevier Biomedical Press, Amsterdam, New York, Oxford, pp 209-220 Wiegand H J, Ottenw~ilder H, Bolt HM (1984 a) The reduction of chromium(VI) to chromium(Ill) by glutathione: an intracellular redox pathway in the metabolism of the carcinogen chromate. Toxicology 33: 341-348 Wiegand H J, Ottenw~ilder H, Bolt HM (1984 b) Disposition of intratracheally administered chromium(I/l) and chromium(VI) in rabbits. Toxicol Lett 22:273-276 Willis RJ, Kratzing CC (1974) Ascorbic acid in rat lung. Biochem Biophys Res Commun 59: 1250-1253 Willis ILl, Kratzing CC (1976) Extraeellular ascorbic acid in lung. Binchim Biophys Acta 444: 108-117 Yassi A, Nieboer E (1988) Carcinogenicity of chromium compounds. In: Nriagu JO, Nieboer E (ed) Chromium in the natural and human environments. John Wiley & Sons, New York, Chichester, Brisbane, Toronto, Singapore, pp 443-495

Reduction of hexavalent chromium by ascorbic acid and glutathione with special reference to the rat lung.

The reduction of 20 microM hexavalent chromium [chromium(VI)] by L-ascorbic acid (AsA) (0.06-2 mM) and/or glutathione (GSH) (2-15 mM) in buffer soluti...
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