Pergamon Biochemical Pharmacology, Vol. 48, No. 7, pp. 1483-1492, 1994. Copyright t~) 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0006-2952/94 $7.00 + 0.00 0006-2952(94)00306-8 EFFECT OF 5-NITROINDOLE ON ADENYLATE ENERGY CHARGE, OXIDATIVE PHOSPHORYLATION, AND LIPID PEROXIDATION IN RAT HEPATOCYTES MARTA DUBIN,* PATRICIA H. CARRIZO,J" ANA M. BISCARDI, SILVIA H. FERNANDEZ VILLAMIL and ANDRI~S O. M. STOPPANI*~ Centro de Investigaciones Bioenerg6ticas, Facultad de Medicina, Paraguay 2155, (1121)-Buenos Aires, Argentina (Received 14 February 1994; accepted 12 May 1994) Abstract--5-Nitroindole (NI), a mutagenic nitroarene, was assayed for cytotoxic effects on rat hepatocytes. After incubation with 25-100/~M NI, the adenylate energy charge of the hepatocytes decreased significantly as a result of the decrease in ATP and the increase in AMP. ATP depletion correlated well with the effects of NI on mitochondriai electron transfer and energy transduction in hepatocytes. Thus, NI (a) inhibited the antimycin-sensitive hepatocyte respiration; (b) inhibited NADH oxidation by disrupted hepatocyte mitochondria; (c) inhibited L-malate-L-glutamate oxidation by ADP- supplemented mitochondria; (d) in the absence of ADP, stimulated the same substrates and also succinate oxidation by mitochondria; (e) released the latent ATPase activity of mitochondrial F~F0- ATP synthase; (f) shifted the redox level of reduced cytochromes (c + cl) and b towards the oxidized state; (g) inhibited NADH oxidation by disrupted mitochondria in the vicinity of the NADH- dehydrogenase flavoprotein; (h) inhibited Ca2+ uptake by mitochondria using L-malate-L-glutamate as an energy source; (i) inhibited valinomycin-induced, endogenously energized K + uptake, with little effect on the ATP-induced uptake; and (j) inhibited the MgATP-dependent contraction of Ca2+-swollen mitochondria. NI inhibited lipid peroxidation in hepatocytes and also in substrate-supplemented liver microsomes and mitochondria, thus ruling out hydroperoxides as a cause of NI cytotoxicity. Long-term incubation with NI produced loss of hepatocyte viability, as indicated by lactate dehydrogenase leakage. Key words: 5-nitroindole; nitrofuran compounds; hepatocytes; adenylate energy charge; oxidative phosphorylation; lipid peroxidation Heterocyclic nitroarenes are widely used as chemo- therapeutic agents. In a series of nitroarenes tested for their mutagenic activity on Salmonella, NI§ proved to be one of the most effective [1-4]. Since no information was available on the action of this compound on eukaryotic cells, it seemed of interest to assay NI in rat hepatocytes, a procedure that constitutes a powerful tool in mechanistic studies on the metabolic action of xenobiotics [5]. Cytotoxicity was investigated using several biochemical parameters, namely (a) the ATP level in hepatocytes [6], (b) the adenylate energy charge [7], (c) electron transfer; (d) lipid peroxidation [8-10], and (e) release of LDH [11]. Moreover, the following parameters * Career Investigator, Consejo Nacional de Invest- igaciones Cientificas y T6cnicas, Argentina. t Research Fellow, Consejo Nacional de Investigaciones Cientificas y T6cnicas, Argentina. :~ Corresponding author. Tel. and FAX: 54-1-961-6521. § Abbreviations and chemical terms: NI, 5-nitroindole; LDH, lactate dehydrogenase; DMFA, N,N-dimethyl- formamide; TCA, trichloroacetic acid; MGM, L-malate-L- glutamate-malonate; t-BuOOH, tert-butylhydroperoxide; TBA, thiobarbituric acid; MDA, malondialdehyde; G6P, glucose-6-phosphate;G6PD, glucose-6-phosphate dehydro- genase; NF, (5-nitrofurfurylidene)amino; nifurtimox, tetrahydro-3- methyl- 4- [NF]- 2/-/- 1,4- thiazine- 1,1- dioxide; NF-pyrazole, 1-[NF]-pyrazole; NF-triazole, 4-[NF]-I,2,4- triazole; and DETAPAC, diethylenetriaminepentaacetic acid. were investigated in isolated mitochondria: (a) electron transfer and energy coupling; (b) Ca 2+ and K + uptake, and (c) FIF0-ATP synthase ATPase activity. The adenylate energy charge [the ratio (ATP + 0.5 ADP)/(ATP + ADP + AMP)] [71 dep- ends essentially on the intracellular ATP level, a limiting factor for cell viability [6]. Lipid peroxidation is one of the basic mechanisms of cell damage [8- 10], and alteration of mitochondrial permeability by lipid peroxidation products may cause ATP depletion [10]. Finally, irreversible injury renders the hepa- tocyte plasma membrane unable to prevent the leakage of cytosolic molecules, particularly protein molecules, thus contributing to cell death [6, 12]. Studies on structure-activity relationships indicate that the nitro group may be responsible for the biological activity of nitro compounds [13-16]. Redox-cycling reactions of these compounds may produce oxygen radicals that are responsible for their toxicity [16-18]. Accordingly, the effects of NI were compared with those of nitrofuran compounds characterized by their capability of producing oxygen radicals, such as the antichagasic compound nifurtimox [16-18]. MATERIALSAND METHODS Animals. Male Wistar rats (220-250 g) were used in the experiments. Animals were fed a Purina-like 1483