Consequences of acrylonitrile metabolism in rat hepatocytes: Effects on lipid peroxidation and viability of the cells

ENVIRONMENTAL RESEARCH 46, 133-141 (1988)

Consequences of Acrylonitrile Metabolism in Rat Hepatocytes: Effects on Lipid Peroxidation and Viability of the Cells J A N A NERUDOV•, *'1 IVAN G U T , * AND HEIKKI S A V O L A I N E N t

*Institute of Hygiene and Epidemiology, Srobdrova 48, 100 42 Prague 10, Czechoslovakia, and ?Institute of Occupational Health Sciences, University of Lausanne, Switzerland Received January 26, 1987 Acrylonitrile caused thiobarbituric acid-positive reactants time- and concentrationdependently in isolated hepatocytes. This effect was markedly enhanced by gassing of the medium with 95% oxygen-5% CO2 gas mixture. Glycidonitrile, an acrylonitrile metabolite, proved more potent in this respect than the parent acrylonitrile or its end metabolite, cyanide anion. The latter decreased greatly the viability of isolated liver cells but caused thiobarbituric acid-positive reactants only in the presence of diethylmaleate. Acrylonitrile caused also a decrease in the concentration of nonprotein sulfhydryl groups but the oxidation of glutathione (GSH) to GSSG (oxidized glutathione) was not the major mechanism. This might indicate the consumption of GSH in the glutathione S-transferase catalyzed reactions. In contrast to cyanide anion-induced effects acrylonitrile did not affect markedly the viability of hepatocytes. © 1988AcademicPress, Inc.

INTRODUCTION Acute toxicity of acrylonitrile (ACN) has been associated with glutathione (GSH) depletion with its consequences in the cellular redox equilibrium and with the inhibition of cytochrome oxidase by cyanide anion, the end metabolite of ACN (Brieger et al., 1952; Nerudov(t et al., 1981; Gut et al., 1975, 1981). ACN has been found to bind covalently in cell protein (Ahmed et al., 1982; Gut et al., 1981) and to initiate lipid peroxidation in liver microsomes (Ivanov and Alshanski, 1982) and in isolated hepatocytes (Nerudov~i, 1983). Lipid peroxidation by CC14, paracetamol, adriamycin, or paraquat as a mechanism of their hepatotoxicity has been the topic of numerous papers (Stacey et al., 1982; Mold6us, 1978; Myers et al., 1977; Shu et al., 1979; Steffen et al., 1980; Muliawan et al., 1980). The impairment of cellular antioxidative defenses may render cells more susceptible to the peroxidative damage (Younes and Siegers, 1984). GSH consumed in glutathione S-transferase catalyzed reactions depletes its stores in cells in the conjugation reaction aimed at the removal of the toxic peroxidation products and xenobiotic electrophiles. This causes smaller pools for the glutathione peroxidase-supported inactivation of hydrogen peroxide thus propagating the toxic effects of, e.g., hypoxia (Savolainen, 1982). The latter is a probable occurrence in the acrylonitrile toxicity as C N - is a potent inhibitor of cellular respiration. It appeared interesting to us to probe more closely these mechanisms in isolated

1 To whom correspondence should be addressed. 133

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NERUDOV~, GUT, AND SAVOLAINEN

hepatocytes where, e.g., oxygen concentration, cellular GSH, and substrate concentration of ACN metabolites can be easily manipulated (Hole6ek and KopeckS,, 1981; Stacey et al., 1982; Geiger et al., 1983). MATERIALS AND METHODS

Crude collagenase (540 PZS units/g) and bovine serum albumin were obtained from OSOL, Prague. Hepes and 2-thiobarbituric acid were obtained from Serva. EGTA was purchased from BDH Chemicals, sulfanilamide from Fluka, and ammonium amidosulfonate, N-(1-naphtyl)ethylene-diammonium dichloride, Nethylmaleimide, o-phthalaldehyde, and diethylmaleate (DEM) from Merck. Male Wistar rats, 200-250 g, were given free access to food and water before the isolation of hepatocytes (Mold6us et al., 1978). The isolated cells (2 x 106/ml) were suspended in Krebs-Henseleit buffer supplemented with 12.6 mM Hepes, pH 7.4. All incubations were carried out at 37°C using rotating 50-ml roundbottom bottles in a final volume of 20 ml. Air or carbogen (95% 0 2, 5% CO2) mixtures were flown in the vials. The cells were preincubated for 10 min before the addition of the xenobiotics. The reactions were terminated at appropriate intervals by sampling the incubation mixture and mixing the aliquots with the reagents of the specific assay in question or by separating the ceils as pellet from the supernatant by centrifugation. The viability of cells was ascertained by the trypan blue exclusion test and by the release of lactate dehydrogenase (LDH) in the medium (Mold6us et al., 1978). The nonprotein sulfhydryl groups and GSH were analyzed according to Saville (1958) and oxidized glutathione (GSSG) as described by Hissin and Hilf (1976). Malonaldehyde was determined using 2-thiobarbituric acid as a reagent (Buege and Aust, 1978). The oxygen consumption was estimated micropolarographically using an integrated unit in a Shimadzu spectrophotometer. Carbogen was used as the feed gas, and aliquots of cell suspension (2 x 10 6 cells) were transferred in the reaction vessel and diluted by gentle magnetic stirring to 6 ml with the abovementioned Krebs-Henseleit buffer. The incubations were carried out at 37°C. Under these conditions, the consumption of Oz was linear up to 5 min. RESULTS

Incubation of hepatocytes with 1 mM ACN with air as feed gas caused increased formation of thiobarbituric acid-positive material after 120 min (Fig. 1A) while viability was not different from control cells (Fig. 1B). With carbogen as feed gas, the thiobarbituric acid (TBA)-positive products appeared sooner and were sixfold greater in concentration than using air and were 20-fold greater than in the absence of ACN (Fig. 1A). The viability of the ACN-incubated hepatocytes under carbogen was markedly decreased in 3 hr of incubation but, in comparison to TBA-positive material, the effect was delayed at least 60 min (Fig. 1B). GSH concentration (Fig. 2A) decreased very rapidly in the cells incubated with 1 mN ACN under air and at an even higher rate under the carbogen mixture. The GSH depletion preceded the appearance of the TBA-positive products by 2 hr under air and by 1 hr under carbogen. It is notable that GSSG was not accumu-

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lated in the medium nor by the increased concentration of 02 even if GSH was consumed (Fig. 2B). ACN added at final concentrations of 0.1, 1, or 10 mM did not immediately inhibit the cellular Oz consumption while cells preincubated with 1 mM ACN for 3 hr showed a 30% decrease in the oxygen consumption (Table 1). Cellular hypoxia in terms of oxygen consumption appeared thus only after an interval, probably because of the time needed for the biotransformation reactions of ACN to produce the more toxic metabolites and decrease in the GSH pool. Incubation of hepatocytes with 1 mM KCN under air also caused the formation of TBA-positive products (Fig. 3). This effect by 1 mM ACN was, however, stronger. The effect of ACN on viability was weak while KCN decreased it greatly (Fig. 3). This might show that the TBA-positive products had lesser effects on the viability of cells than a direct inhibition of respiration. Thiosulfate, the well-known antidote of cyanide poisoning, was equally efficient in preventing the effect of KCN or ACN on the formation of TBA-positive products. It also increased the viability of cells after KCN dosage (Fig. 3), which supports the idea that the inhibition of cytochrome oxidase by C N - is the proximate causing the cell death. One millimolar KCN did not induce the formation of TBA-positive products,

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nor was there any effect on viability. Simultaneous depletion of tissue sulfhydryls by diethyl maleate with initial 0.1 mM KCN concentration had a strong potentiating effect on lipid peroxidation, but there was no effect on cellular viability (Fig. 4). The enhanced lipid peroxidation with 1 mM ACN occurred at least an hour later than with 0.1 mM K C N and DEM and viability was similar as with controls. The combination of diethyl maleate and 0.1 mM KCN could therefore resemble a longer incubation with 1 mM ACN where a decrease in GSH pool is rapid, but accumulation of C N - from ACN biotransformation, with resultant effects on cytochrome oxidase activity, proceeds more slowly. Glycidonitrile (GN), the primary oxidation product of ACN, proved to be the TABLE 1 EFFECT OF ACRYLONITRILE (1 mM) ON OXYGEN CONSUMPTION BY HEPATOCYTES I N

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FIG. 3. Effect of acrylonitrile, KCN, and thiosulfate on formation of thiobarbituric acid-positive material (A), on the exclusion capacity of trypan blue (B), and on the latency of lactate dehydrogenase (C). Hepatocytes were incubated under air with 1 mM acrylonitrile (ACN), 1 mM KCN, and with 1 mM KCN and 2.5 mM sodium thiosulfate. Results represent the means of three experiments.

most potential inducer of the TBA-positive products (Fig. 5). They were generated at 1 mM GN much faster and reached about the same levels as with 10 times greater ACN concentration (Fig. 5). A further comparison of GN, ACN, and CC14 suggests that ACN belongs to a category of chemicals causing the formation of TBA-positive products after depletion of GSH without an immediate damage of the cells while CC14does so directly simultaneously decreasing the viability of cells. DISCUSSION

The data shown above indicate that ACN (a) greatly depleted GSH in hepatocytes, (b) induced, after some delay, lipid peroxidation, and (c) its effects, espe-

NERUDOV~,GUT, AND SAVOLAINEN

138

cially the latter, where markedly potentiated by increased oxygen tension. This suggested an important role of oxygen, or rather oxygen radicals, in these events. GSH depletion was apparently due to its conjugation with ACN (cyanoethylation) and GN, catalyzed by GSH S-transferases (Hole~ek and Kopecky, 1981). The oxidation of GSH to GSSG was not involved in GSH depletion. (It is proper to mention here that carbogen accelerated dying of hepatocytes caused by ACN, but the effect became evident in the third hour of incubation at least an hour later than significant increase of lipid peroxidation.) Therefore, we believe that GSH depletion by cyanoethylation was so rapid that it effectively prevented GSH oxidation to GSSG, although increased oxygen tension provided conditions for this event. The high concentration of molecular oxygen in the cells could lead to its incomplete reduction to superoxide anion radical (02) instead of water in the cytochrome oxidase catalyzed reaction (for review see Savolainen, 1982). Only 5% of the 02 consumption in hepatocytes under normoxic conditions give rise to superoxide anion (Jones, 1982). Superoxide anion is dismutated to H202 and 02 by superoxide dismutase, and hydrogen peroxide is inactivated to water primarily by glutathione peroxidase catalyzed reaction. Catalase seems to play a lesser role because of its restricted cellular localization in the peroxisomes (Kaplowitz and Ookthens, 1985). Under conditions of rapid GSH depletion by ACN or its metabolite GN (Geiger et al., 1983) it may be expected that glutathione peroxidase cannot handle the excessively generated H202 because of low GSH concentration. If oxygen concentration is increased, more H202 could be formed despite competition of CN -

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with molecular oxygen for the active site of cytochrome oxidase. ACN may, therefore, induce increased concentration of oxygen radicals by facilitating their formation (through cyanide) and inhibiting their degradation (via GSH depletion medicated inhibition of GSH peroxidase). Lipid peroxidation as studied by the formation of TBA-positive material probably plays a minor role in the cell death caused by KCN. In ACN poisoning at least two toxic events predestine the formation of TBA-positive products: depletion of GSH (90% in 60 min) and formation of CN- via GN. GN is formed in hepatocytes and accumulates in time with reported maximum at 2 hr (Geiger et al., 1983). One-millimolar ACN-induced lipid peroxidation seemed of minor importance in the cellular death; greatly increased TBA-positive material by excessive oxygen concentration or increased ACN concentration (10 mM) were accompanied by decreased viability of the cells. In the sequence of rapid GSH depletion and slower lipid peroxidation and markedly delayed cellular death, the first two events alone or in combination are apparently not the immediate causes of cellular death, but may trigger events which act in that way. With respect to lipid peroxidation and cellular death one more point should be mentioned: lipid peroxidation may start where ACN is metabolized to GN (in microsomes, where increased lipid peroxidation by ACN was demonstrated by Silver and Szabo, 1982; Ivanov and Alshanski, 1982) and plasma membranes (whose damage is demonstrated by the trypan blue exclusion test and lactate dehydrogenase release into the medium) may be affected later. This aspect might also help to explain why lipid peroxidation markedly precedes the symptoms commonly used for indication of cellular death.

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It can also be doubted whether the administration of hyperbaric oxygen to poisoning victims would be of real benefit as the cellular respiration is compromised and, in rats, the administration of sodium nitrite (forming methemoglobin and thus partially decreased blood oxygen-binding capacity, but at the same time effective binding formed cyanide (Ghiringheli, 1954)) decreased ACN acute toxicity. In conclusion, molecular oxygen is a very important variable in the planning and execution of toxicity studies by isolated hepatocytes in vitro. Especially, a direct comparison of lipid peroxidation and cellular viability is a task qualifying many reservations and restrictions. REFERENCES Ahmed, A. E., Farooqui, M. Y. H., Upreti, R. K., and E1-Shabrawy, O. (1982). Distribution and covalent interactions of [14C]acrylonitrilein the rat. Toxicology 23, 15%175. Brieger, H., Rieders, F., and Hodes, W. B. (1952). Acrylonitrile: Spectrophotometric determination, acute toxicity, and mechanism of action. A M A Arch. Ind. Hyg. Occup. Med. 6, 128-140. Buege, J. A., and Aust, S. D. (1978). Microsomal lipid peroxidation. In "Methods in Enzymology" (S. Fleischer and L. Packer, Eds.), Vol. LII, pp. 302-310. Academic Press, New York. Geiger, L. E., Hogy, L. L., and Guengerich P. (1983). Metabolism of acrylonitrile by isolated rat hepatocytes. Cancer Res. 43, 3080-3087. Ghiringheli, L. (1954). Acrylonitrile: Toxicity and mechanism of action. Med. Lav. 45, 305-312. Gut, I., KopeckS, J., and Nerudov~i, J. (1981). Relationship between acrylonitrile biotransformation, pharmacokinetics and acute toxicity. M e d Lav. 3, 131-136. Gut, I., Nerudov~i, J., Kopeck3~, J., and Hole6ek, V. (1975). Acrylonitrile biotransformation in rats, mice, and Chinese hamsters as influenced by the route of administration and by phenobarbital, SKF-525-A, cysteine, dimercaprol, or thiosulphate. Arch. Toxicol. 33, 151-161. Hissin, P. J., and Hilf, R. (1976). A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal. Biochem. 74, 214-226. Hole6ek, V., and Kopeck3~, J. (1981). Conjugation of glutathione with acrylonitrile and glycidonitrile. In "Industrial and Environmental Xenobiotics" (I. Gut et al., Eds.), pp. 239-244. SpringerVerlag, Berlin. Ivanov, V. V., and Alshanski, A. M. (1982). Komponenty GAMK-orgitcheskoy sistemy i perekisnoe okislenie lipidov pri ostroy ekzogennoy intoxikacii akrylonitrilom. Bull. Exp. Biol. Med. XCIV, 40--43. Jones, D. P. (1982). Intracellular catalase function: Analysis of catalytic activity by product formation in isolated liver cells. Arch. Biochem. Biophys. 214, 806-814. Kaplowitz, N., and Ookthens, T. Y. Aw.M. (1985). The regulation of hepatic glutathione. Annu. Rev. Toxicol. 25, 715-744. Mold6us, P. (1978). Paracetamol metabolism and toxicity in isolated hepatocytes from rat and mouse. Biochem. Pharmacol. 27, 2859-2863. Mold6us, P., H6gberg, J., and Orrenius, S. (1978). Isolation and use of liver cells. In "Methods in Enzymology" (S. Fleischer and L. Packer, Eds.), Vol. LI1, pp. 60-71. Academic Press, New York. Muliawan, H., Scheulen, M. E., and Kappus, H. (1980). Acute adriamycin treatment of rats does not increase ethane expiration. Res. Commun. Chem. Path. Pharmacol. 30, 509-519. Myers, C. E., McGuire, W. P., Liss, R. H., Ifrim, I., Grotzinger, K., and Young, R. C. (1977). The role of lipid peroxidation in cardiac toxicity and tumor response. Science 197, 165-167. Nerudov~, J. (1983) Isolovan6 hepatocyty jako toxikologick~ model. Acta Hyg. Epidemiol. Microbiol. (Pffloha) 1,183-192. Nerudov~i, J., Gut, I., and KopeckS, J. (1981). Cyanide effect in acute acrylonitrile poisoning in mice. In "Industrial and Environmental Xenobiotics" (I. Gut et al., Eds.), pp. 245-250. SpringerVerlag, Berlin. Saville, B. (1958). A scheme for colorimetric determination of microgram amounts of thiols. Analyst 83, 670-672.

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Savolainen, H. (1982). Neurotoxicity of industrial chemicals and contaminants: Aspects of biochemical mechanisms and effects. Arch. Toxicol. Suppl. 5, 71-83. Shu, H., Talcott, R. E., Rice, S. A., and Wei, E. T. (1979). Lipid peroxidation and paraquat toxicity. Biochem. Pharmacol. 28, 327-331. Silver, E. H., and Szabo, S. (1982). Possible role of lipid peroxidation in the action of acrylonitrile on the adrenals, liver and gastrointestinal tract. Res. Comm. Chem. Pathol. Pharmacol. 36, 33--43. Stacey, N. H., Ottenw~ilder, H., and Kappus, H. (1982). CC14-inducedlipid peroxidation in isolated rat hepatocytes with different oxygen concentrations. Toxicol. Appl. Pharmacol. 62, 421-427. Steffen, C., Muliawan, H., and Kappus, H. (1980). Lack of in vivo lipid peroxidation in experimental paraquat poisoning. Naunyn Schmiedeberg's Arch. Pharmacol. 310, 241-243. Younes, M., and Siegers, C. P. (1984). Interrelation between lipid peroxidation and other hepatotoxic events. Biochem. Pharmacol, 39, 2001-2003.