The consequences of nitrofurantoin-induced oxidative stress in isolated rat hepatocytes: evaluation of pathobiochemical alterations

Chemico-Biolo9ical Interactions Chemico-Biological Interactions 93 (1994) 91 - 102

ELSEVIER

The consequences of nitrofurantoin-induced oxidative stress in isolated rat hepatocytes: evaluation of pathobiochemical alterations Sabine Klee*, M a r t i n C. Nfirnberger, Fritz R. U n g e m a c h Institute of Pharmacology and Toxicology, Faculty of Veterinary Medicine. Free University of Berlin, Koserstr. 20, 14195 Berlin, Germany

Received I1 August 1993; revision received 19 November 1993; accepted 24 November 1993

Abstract Oxidative stress was induced in isolated rat hepatocytes by incubation with nitrofurantoin in the absence and presence of the GSSG reductase inhibitor BCNU. In both cases nitrofurantoin markedly reduced glutathione but exerted cytotoxicity as measured by LDH release and loss of intracellular potassium only in BCNU pretreated cells. The onset of cytotoxicity was accompanied by an increase of lipid peroxidatfon. Oxidation of protein thiols, however, could not be detected in the early phase of cell damage. The cytoprotective activity of N-acetylcysteine >dithiothreitol = deferoxamine revealed the substantial importance of glutathione for cellular defence and the sensitivity of not yet identified thiol-dependent targets of oxidative stress. Keywords: Cytoprotective agents; GSSG reductase inhibition; Isolated hepatocytes; Nitrofurantoin; Oxidative stress; Thiol oxidation

1. Introduction

Oxidative stress is a pathophysiological phenomenon based on excessive formation of reactive oxygen species during e.g., cytochrome P450-dependent metabolism Abbreviations: BCNU, 1,3-bis(2-chloroethyl)-l-nitrosourea; DFA, deferoxamine; DMFA, dimethylformamide; DTNB, 5,5'-dithio-bis(2-nitrobenzoic acid): DTT, dithiothreitol; GSH, reduced glutathione; GSSG, oxidized glutathione; HEPES, N-(2-hydroxyethyl)piperazine-N'-2-ethane-sulfonic acid: LDH, lactate dehydrogenase; MDA, malondialdehyde; NAC, N-acetylcysteine; NFT, nitrofurantoin; Quin-2AM, Quin-2-tetra(acetoxy-methyl)ester. *Corresponding author. 0009-2797/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0009-2797(93)03254-R

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of endogenous and exogenous substrates [ 1], electron transport at the mitochondrial respiratory chain [2], inflammatory respiratory burst [3], and biotransformation of prooxidative as well as redoxcycling xenobiotics [4]. The ability to redoxcycle with oxygen was shown for quinone compounds like menadione [5] or the antineoplastic agents daunomycin and Adriamycin [6], for bipyridyl herbicides like diquat and paraquat [7], and also for nitrofurans like nitrofurantoin [8]. Increasing evidence has been obtained on the clinical importance of oxidative stress. For example, the deleterious effects of reperfusion on ischaemic hearts were associated with enhanced formation of reactive oxygen species [1]. Chronic inflammations are accompanied by lasting exposure of the inflamed area to 'oxygen radicals' generated by activated leukocytes [9]. The mutagenic effects of reactive oxygen species are supposed to contribute to the pathogenesis of cancer [10]. The molecular mechanisms by which oxidative stress induces cytotoxicity are still a matter of controversy. A well-known consequence of oxidative stress is lipid peroxidation [11]. Iron ion complexes, such as perferryl or ferryl, and particularly hydroxyl radicals are supposed to be the initiating agents [10-12]. Due to their high reactivity hydroxyl radicals easily abstract hydrogen atoms from polyunsaturated fatty acids. Hydroxyl radicals are formed during an Fe ÷+- or Cu+-catalyzed reaction from superoxide anion radicals and hydrogen peroxide. Hence, metal ion sequestration is an important antioxidant preventive [10]. Oxidative cell injury is usually accompanied by pertubations of thiol homeostasis [13], i.e., depletion of soluble thiols, mainly glutathione (GSH), and protein-bound thiols, the latter, however, being equivocal due to analytical problems [14]. The crucial role of glutathione for cellular defence is generally accepted [15], and GSHdepleted cells have been widely used as a valuable experimental system to study cytotoxicity [7,16,17]. Disturbances in intraceilular calcium ion homeostasis were supposed to be fundamental for cytotoxicity [18,19]. Several redoxcycling agents like diquat [20] and quinones [21] proved to affect intraceilular Ca ++ compartmentalization raising the question, whether Ca +* mediates the deleterious effects of oxidative stress. Ca +* activates some catabolic enzymes like proteases, endonucleases, and phospholipases which might contribute to cellular damage [22]. Ca +* seems to be essential for mitochondrial functions [23] and cytoskeleton organization [24] which both also depend on an intact thiol homeostasis [15]. Another intriguing aspect of reactive oxygen toxicity is the ability of superoxide anion as well as redoxcycling xenobiotics like Adriamycin, diquat, and nitrofurantoin to release iron from ferritin thereby promoting the generation of hydroxyl radicals and iron oxygen complexes [25]. The aim of this study was to demonstrate the effects of oxidative stress induced by nitrofurantoin on isolated rat hepatocytes and the use of thiol restoring and iron chelating agents for cellular protection. 2. Materials and methods 2. I. Animals

Hepatocytes were isolated from male Wistar rats (220-250 g) bred at Zentrale

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Versuchstierzucht (Bundesgesundheitsamt, Berlin, Germany). They were fed a laboratory chow ad libitum and housed at 21°C on a 12 h light/dark cycle. Food but not water was withdrawn 18 h before the experiments started.

2.2. Chemicals Nitrofurantoin (NFT), dithiothreitol (DTT), deferoxamine (DFA), N-acetylcysteine (NAC), N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonicacid (HEPES), Quin2-tetra(acetoxymethyl)ester (quin-2-AM) were obtained from Sigma (M~inchen, Germany). 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was purchased from Bristol (Miinchen, Germany). Other chemicals were of analytical purity grade and commercially available. 2.3. Hepatocyte isolation and incubation Hepatocytes were isolated by collagenase perfusion of the liver as described by Ungemach [26]. Cells were suspended in Krebs-Henseleit buffer pH 7.4 supplemented with 20 mmol/1 HEPES and stored at 4°C until use. Cell density was adjusted to 70 mg/ml wet weight after addition of the respective drugs. Initial viability was always higher than 80% as determined by trypan blue exclusion. Aliquots of suspension were placed in round-bottomed flasks and heated to 37°C in a gyratory water bath shaker under humidified air. After the first sample was drawn (0 min) the experiments were started by addition of NFT dissolved in dimethylformamide (DMFA) resulting in a final concentration of 500/zmol/1 NFT and 1% solvent. Controls received vehicle only. Solutions and probes containing NFT were carefully protected from light throughout the experiments. In order to inhibit GSSG reductase cold hepatocyte suspensions were administered 75 #mol/1 BCNU in 0.5% DMFA (final concentration), heated to 37°C and incubated for further 15 min before NFT was added. Controls were treated in the same way with BCNU and solvent instead of NFT. Dithiothreitol, N-acetylcysteine and deferoxamine dissolved in Krebs-Henseleit buffer were added at 0 (DTT, NAC, DFA) and 60 min (DTT, DFA) to give final concentrations of 1 mmol/1, 1.25 mmol/1, and 100/zmol/1, respectively. 2.4. Analytical methods 2.4.1.Lactate dehydrogenase (LDH) release. The activity of LDH was measured in the supernatant of cell suspensions (20 × g) using a standard test kit (Boehringer Mannheim, Germany). 2.4.2. Intracellular potassium. An aliquot of cell suspension (1 ml) was washed twice with K+-free 0.15 mmol/i Tris-HCl pH 7.4, and precipitated with 0.1 mol/l perchloric acid. [K ÷] was measured in the supernatant by flame photometry and expressed as mmol K÷/1 cellular water. According to Ungemach intracellular water can be calculated from the protein content of cells using a factor of proportionality of 2.4 for rat hepatocytes [26]. 2.4.3. Malondialdehyde (MDA)formation. Products of lipid peroxidation were measured in 0.1 ml aliquots of cell suspension by the thiobarbituric acid method according to Ohkawa [27] and expressed as nmol malondialdehyde formed/mg cells (wet weight).

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2.4.4. Glutathione. Intracellular G S H was determined with 5,5'-dithio-bis(2nitrobenzoic acid) (DTNB) as described by Kaka~ and Vejdelek [28]. 2.4.5. Protein sulfhydryl groups. Reduced protein thiols were determined by reacting perchloric acid-precipitated cellular protein with D T N B as described previously [29]. Protein thiols were expressed as % o f initial value (0 min) since considerable variations were observed between individual hepatocyte preparations (ranging from 100 to 150 nmol protein-SH/mg protein). 2.4.6. Protein. A modification o f the biuret method was employed for the determination of cellular protein using bovine albumin as standard [30]. 2.4.7. Cytosolic calcium concentration. Cytosolic free calcium was analyzed fluorimetrically using quin-2-AM as indicator. For further details see [31]. Since N F T interfered with the fluorescence signal o f the quin-2/Ca ++ complex, 150 instead of 100/~mol/l quin-2-AM were used. Prior to analysis the NFT-containing incubation medium was removed by centrifugation (20 × g) and replaced by KrebsHenseleit buffer thereby overcoming the quenching effects o f NFT. 2.4.8. Statistical analysis. D a t a are presented as means ± S.E.M. Significant differences (P <0.05) between means were calculated by Dunnett's t-test for multiple comparisons [32] with the exception o f protein thiols which were analyzed by the U-test o f M a n n and Whitney [33]. 3. Results 3.1. NFT-treated hepatocytes in the absence and presence o f B C N U 3.1.1. Viability. N F T affected cellular viability after 60 min o f incubation, thereby raising extracellular L D H activity for about 50% in the absence and for more than 300% in the presence o f B C N U (Fig. 1). e

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Fig. 1. Influence of NFT-induced oxidative stress on LDH release. Hepatocytes were preincubated for 15 min with either 0.5% DMFA or 75 t~mol/1BCNU and subsequently treated with 1% DMFA (control) or 500/,Lmol/l NFT. II, control (1.5% DMFA); D, 500/zmol/l NFT; 0, 75 #mol/l BCNU + 500 #mol/I NFT. The first sample (0 rain) was drawn prior to NFT addition. Data represent the mean + S.E.M. of 8 (control), 17 (NFT) and 20 (BCNU + NFT) experiments. *Significantly different from the corresponding value at 0 min (P < 0.05).

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Fig. 2. Influence of NFT-induced oxidative stress on intracellular potassium. Results were obtained from the same experiments as described in the legend of Fig. 1. Identical symbols were used. Data represent the mean + S.E.M. of 8 (control), 17 (NFT) and 20 (BCNU + NFT) experiments. *Significantly different from the corresponding value at 0 min (P < 0.05). I n t r a c e l l u l a r p o t a s s i u m decreased o n l y in the presence o f B C N U f r o m 108 to 24 m m o l / l cellular w a t e r (Fig. 2). F a i l u r e o f K ÷ a c c u m u l a t i o n preceded L D H release for at least 1 h, i n d i c a t i n g a n i m p a i r m e n t o f K + h o m e o s t a s i s i n d e p e n d e n t o f severe plasma membrane damage. Vehicle (Figs. 1 a n d 2), as well as B C N U a l o n e (data n o t shown), did n o t significantly affect h e p a t o c e l l u l a r viability. 3.1.2. I n t r a c e l l u l a r g l u t a t h i o n e . G S H was in the r a n g e o f 5 - 6 mmol/1 in c o n t r o l s (Fig. 3). In N F T - t r e a t e d cells G S H decreased f r o m 5.3 to 3.3 mmol/1 in the a b s e n c e o f B C N U a n d f r o m 4.3 to 0.7 m m o l / l in the presence o f B C N U (Fig. 3). C o n s u m p -

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Fig. 3. Influence of NFT-induced oxidative stress on GSH content of isolated hepatocytes. Results were obtained from the same experiments as described in the legend of Fig. I. Identical symbols were used. Data are the mean ± S.E.M. of 8 (control), 17 (NFT) and 20 (BCNU + NFT) experiments. *Significantly different from the corresponding value at 0 min (P < 0.05).

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tion of 2 mmol/l GSH occured after addition of NFT in the absence of BCNU. In the presence of BCNU, however, 3.6 mmol/1 was consumed, thus pointing out excess consumption of GSH due to inhibition of GSSG reductase. GSH depletion preceded the loss of viability. This phenomenon was most evident in BCNU-pretreated cells which lost 50% of GSH within 15 min after addition of NFT (Fig. 3). 3.1.3. Protein sulJhydryl groups. Protein thiols were not affected in control and NFT-treated hepatocytes (not shown). Administration of BCNU + NFT induced a significant loss (25%) after 180 min of incubation (not shown). 3.1.4. Formation ofmalondialdehyde. In control cells endogenous lipid peroxidation remained on a constant level of 0.05 nmol MDA/mg cells throughout the incubation. After administration of NFT malondialdehyde formation was higher, but not significantly different from controls (not shown). BCNU + NFT rapidly induced lipid peroxidation after 30 min resulting in an almost 10 fold increase of malondialdehyde at 180 min (Fig. 5). 3.1.5. Cytosolic free Ca ++. Neither NFT nor BCNU + NFT affected cytosolic Ca ++ concentrations for 2 h of incubation (not shown).

3.2. Protective effects of dithiothreitol on hepatocytes treated with BCNU + N F T 3.2.1. Viability. As shown by LDH release 1 mmol/l DTT delayed the hepatocellular loss of viability induced by BCNU + NFT by at least 1 h when applied concomitantly with the nitrofuran (Fig. 4). DTT administered lh later was somewhat more effective by postponing and diminishing the leakage of LDH (not shown). After 2 h of incubation, however, DTT failed to protect the cells as shown by increasing LDH activities in the supernatants of DTT-supplemented suspensions (Fig. 4). 200

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Fig. 4. Cytoprotection by dithiothreitol, N-acetylcysteine and deferoxamine in hepatocytes exposed to NFT-induced oxidative stress. Isolated hepatocytes were preincubated for 15 min with 75 ~mol/l BCNU and subsequently treated with 500 ~mol/1 NFT alone (I); or additionally I mmol/1 DTT (12); 1.25 mmol/l NAC (O) and 100 t~mol/l DFA (<}); respectively. Cytoprotection was determined by the reduction of LDH release in the presence of either of these agents. Data represent the mean ~ S.E.M. of 20 (BCNU + NFT alone), 6 (DTT), 5 (NAC) and 7 (DFA) experiments. *Significantly different from hepatocytes treated with BCNU + NFT alone (P < 0.05).

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DTT did not inhibit the loss of K ÷ induced by BCNU + NFT. Intracellular [K ÷] was usually higher, but not significantly different in the presence of DTT (not shown). 3.2.2. Glutathione. When DTT was administered together with N F T it significantly diminished GSH consumption induced by N F T + BCNU by about 1 mmol/l after 1 h. This difference was no longer observed after 2 h of incubation (not shown). No effect on GSH was found when DTT was added 60 min later. 3.2.3. Protein sulfhydryl groups. In the presence of DTT protein thiols were not depleted in the late phase of cellular injury (not shown). 3.2.4. Formation ofmalondialdehyde. Up to 3 h, DTT-treated hepatocytes were effectively protected from lipid peroxidation. Subsequently MDA formation rapidly increased (Fig. 5). This effect was independent of the time of application (not shown). 3.3. Protective effects of N-acetylcysteine on hepatocytes treated with B C N U + N F T 3.3.1. Viability. N-acetylcysteine proved to be the most effective substance in the defence of oxidative stress-induced cell damage. Even after 4 h of incubation, when DTT-treated cells no longer resisted the oxidative damage, 1.25 mmol/l NAC nearly completely abolished release of LDH (Fig. 4). Potassium homeostasis was hardly affected in the presence of N-acetylcysteine. After 4 h of incubation, < 10% of intracellular K ÷ was lost compared to 80% in unprotected cells (not shown). 3.3.2. Glutathione. N-acetylcysteine was the only substance which prevented the NFT-induced loss of glutathione. Hepatocytes treated with NAC initially increased GSH from 3.5 mmol/l at 0 minutes to 5.0 mmol/l at 60 min. During the next three hours of incubation < 1 mmol/l GSH was lost (not shown). 0,4

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time of incubation (min) Fig. 5. Influence of dithiothreitol, N-acetylcysteine and deferoxamine on lipid peroxidation in hepatocytes exposed to NFT-induced oxidative stress. Results were obtained from the same experiments as described in the legend of Fig. 4. The same symbols were used. Data represent the mean ± S.E.M. of 20 (BCNU + N F T alone), 6 (DTT), 5 (NAC) and 7 (DFA) experiments. *Significantly different from hepatocytes treated with BCNU + N F T alone (P < 0.05).

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Low molecular thiols can interfere with GSH in the DTNB assay [32], raising the question, whether N-acetylcysteine partly masked the decrease of GSH induced by NFT. In this case, however, addition of NAC is supposed to shift up the curve of GSH, but not to affect its typical slope. 3.3.3. Protein sulfhydryl groups. Protein thiol groups were completely preserved in the presence of N-acetylcysteine (data not shown). 3.3.4. Formation of malondialdehyde. N-acetylcysteine also completely inhibited malondialdehyde formation induced by BCNU + NFT (Fig. 5).

3.4. Protective effects of deferoxamine on hepato~Ttes treated with BCNU + NFT 3.4.1. Viability. The loss of viability was somewhat more effectively delayed by 100 #mol/1 deferoxamine than by 1 mmol/1 DTT (Fig. 4), as measured by LDH release. In contrast to DTT-supplemented cells there was hardly any difference between hepatocytes treated with the iron chelator at 0 min (Fig. 4) and 1 h of incubation (not shown). Deferoxamine also significantly reduced K + leakage. After 4h of incubation about 50% of K + was lost in the presence of DFA (not shown), compared to about 80% in its absence (Fig. 2). The loss of K + did not start before 2 h of incubation (not shown). 3.4.2. Glutathione. Deferoxamine did not interfere with the NFT-induced depletion of GSH in BCNU-pretreated hepatocytes (not shown). 3.4.3. Formation ofmalondialdehyde. Deferoxamine suppressed lipid peroxidation comparable to N-acetylcysteine (Fig. 5). During the first hour of incubation malondialdehyde tended to increase in suspensions receiving deferoxamine after l h, but returned to initial levels subsequently (not shown). 4. Discussion

A compromised hepatocyte model [7] was used to study pathobiochemical mechanisms of oxidative stress. Rat hepatocytes were pretreated with BCNU, an inhibitor of GSSG reductase [34], to induce an irreversible loss of intracellular GSH under conditions of its enhanced use [17]. This was achieved by administration of the chemotherapeutic drug nitrofurantoin which was supposed to generate reactive oxygen species by redoxcycling with molecular oxygen [35]. In fact, GSH consumption by NFT was shown in experimental systems of lung and liver [35-37]. Pretreatment of hepatocytes with BCNU was expected to rapidly exhaust the hepatocellular GSH-dependent defence towards NFT, thereby facilitating the elucidation of mechanistic aspects of oxidative cell damage. The only remarkable effect of N F T alone was a decrease of intracellular GSH from 5.3 to 3.3 mmoi/1 (Fig. 3) indicating formation ofsuperoxide anion radicals and hydrogen peroxide by redoxcycling and subsequent detoxication at the expense of GSH. GSH is oxidized to its disulfide GSSG [15], which is extruded from the cells or reduced by GSSG reductase to restore GSH [38]. Nitrofurans have been shown to inhibit GSSG reductase [39], thereby probably stimulating efflux of GSSG. This might explain the irreversible loss of 2 mmol/l GSH in NFT-treated hepatocytes

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(Fig. 3). Plasma membrane leakage could not account for GSH depletion since it was accomplished prior to the increase of LDH release (Fig. 1). NFT slightly enhanced lipid peroxidation after lh of incubation (not shown). Just as GSH consumption lipid peroxidation is an indicator of free radical reactions [45]. In summary, N F T induced oxidative stress in hepatocytes but failed to affect cellular viability (Figs. 1 and 2), the total bulk of reduced protein thiols, and cytosolic Ca ++ (not shown). Inhibition of the GSH/GSSG redoxcycle by BCNU enhanced the sensitivity of hepatocytes towards NFT. Potassium homeostasis was one of the first targets of oxidative stress revealing a significant decrease of intracellular [K +] at lh and complete breakdown after 4h of incubation (Fig. 2). This was accompanied by an increase of malondialdehyde formation (Fig. 5) suggesting that lipid peroxidation affected plasma membrane enzymes like Na+,K+-ATPase. Severe membrane damage, however, as indicated by LDH release was evident only lh later (Fig. 1). In the late phase of cellular injury a loss of reduced protein thiols was detected in hepatocytes treated with BCNU + N F T (not shown). This might be attributed to sustained generation of 'oxygen radicals' which could not be detoxicated due to the lack of GSH (Fig. 3), and therefore attacked reactive sulfhydryl groups of proteins. GSH, however, was quite rapidly consumed suggesting that protein thiol oxidation should have started earlier. As mentioned above the sensitivity of the DTNB assay for thiols has been questioned previously and minor changes of this parameter were supposed not to be reliably detected [14]. Recently, thiol oxidation was related to disturbances of Ca ++ homeostasis by several hepatotoxins, e.g., acetaminophen [40], tert-butylhydroperoxide [41] and menadione [421. The underlying mechanisms were supposed to be inactivation of Ca++-translocating ATPases as well as inhibition of mitochondrial Ca ++ sequestration [41] either by modification of specific protein thiols or by an imbalance of the mitochondrial glutathione redox system [2]. Although NFT unequivocally interfered with soluble and protein-bound thiols, it failed to alter cytosolic Ca ++ in the absence as well as in the presence of BCNU (not shown). These results do not definitely rule out disorders of thiol and Ca ++ homeostasis in the early phase of oxidative stress-induced cell damage, but rather indicate that the total pool of protein-bound thiols as well as cytosolic [Ca ++] are hardly sensitive towards oxidative attack. Data obtained from dithiothreitoi-supplemented cells also pointed to participation of thiol modification in NFT-induced cell damage. DTT (1 mmol/l) given concomitantly with N F T to BCNU-pretreated cells delayed the rise of LDH release for at least 1 h (Fig. 4). When DTT was applied 1 h later LDH leakage was significantly reduced even at 3 h of incubation (not shown) indicating a transient reversibility of early biochemical changes in oxidative stress. DTT prevented from the loss of protein thiols in the late phase of cellular injury (not shown) and reduced lipid peroxidation up to 3 h of incubation (Fig. 5). These effects can be attributed to direct reaction of DTT with 'oxygen radicals' as well as to reduction of oxidized sulfhydryl groups by DTT [9] thereby recovering some GSH from GSH-protein mixed disulfides or from GSSG [29]. It is therefore reasonable to assume that DTT protected some protein sulfhydryl groups in the early phase of oxidative stress which were critical for

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the manifestation of cytotoxicity. A long-lasting cytoprotection was not achieved by D T T possibly due to dimerization in aqueous solutions. The onset of cytotoxicity was also delayed by iron chelation with deferoxamine. D F A previously was shown to inhibit oxidative stress-induced lipid peroxidation and cytotoxicity in vivo and in vitro [43,44]. D F A (100 #mol/l) completely inhibited lipid peroxidation induced by B C N U + N F T (Fig. 5). Protection from the loss of viability (Fig. 4) was comparable to D T T administered after 1 h o f incubation (not shown). The effects o f deferoxamine are based mainly on the prevention of Fe ÷÷catalyzed generation o f hydroxyl radicals [10] which are probably the most effective initiators o f lipid peroxidation. G S H consumption was not reduced in DFA-treated hepatocytes (not shown) indicating that hardly any intermediates o f lipid peroxidation, e.g., lipid hydroperoxides, were detoxicated by G S H peroxidase. Lipid peroxidation unequivocally contributed to the oxidative cell damage, but obviously not before G S H depletion. Thus, two targets o f oxidative stress were revealed by DTTand DFA-supplementation which were transiently protected either by thiolreduction or by iron chelation. In contrast to DTT, D F A was working independent of the time of application indicating that iron chelation was beneficial not before l h of incubation with N F T while thiol-reduction was effective immediately after the addition of NFT. Complete protection of hepatocytes was achieved by addition of 1.25 mmol/l Nacetylcysteine (Fig. 5), a precursor o f G S H which was shown to augment G S H synthesis in isolated hepatocytes [28,45]. It proved to be a useful antidote, e.g., in acetaminophen poisoning even when the reactive metabolite was no longer present [46]. In the presence o f N A C intracellular G S H was maintained in the range o f control cells (not shown). N A C also completely inhibited lipid peroxidation (Fig. 5) and preserved K ÷ homeostasis (not shown). Whether these effects were mediated by G S H alone or by L-cysteine, the deacetylated form o f N A C being an effective thiolreducing agent itself [46], might be a subject o f further investigations.

References I A. Bast, G.R.M.N. Haenen and C.J.A. Doelman, Oxidants and antioxidants: state of the art, Am. J. Med., 91(3C) (1991) 2. 2 D.J. Reed, Status of calcium and thiols in hepatocellular injury by oxidative stress, Sem. Liver Disease, 10 (1990) 285. 3 C.G. Cochrane, Cellular injury by oxidants, Am. J. Med., 91(3C) (1991) 23. 4 H. Kappus, Overview of enzyme systems involved in bioreduction of drugs and in redox cycling, Biochem. Pharmacol., 35 (1986) 1. 5 H.G. Shertzer, L. Lastbom, M. Sainsbury and P. Moldeus, Menadione-mediated membrane fluidity alterations and oxidative damage in rat hepatocytes, Biochem. Pharmacol., 43 (1992) 2135. 6 J.H. Peters, G. Ross Gordon and D. Kashiwase, Redox activities of antitumor anthracyclines determined by microsomal oxygen consumption and assays for superoxide anion and hydroxyl radical generation, Biochem. Pharmacol., 35 (1986) 1309. 7 M.S. Sandy, P. Moldeus, D. Ross and M.T. Smith, Role of redox cycling and lipid peroxidation in bipyridyl herbicide cytotoxicity, Biochem. Pharmacol., 35 (1986) 3095. 8 A. Adam, L.L. Smith and G.M. Cohen, An evaluation of the redox cycling potencies of paraquat and nitrofurantoin in microsomal and lung slice systems, Biochem. Pharmacol., 40 (1990) 1533.

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