Role of mitochondrial membrane permeability transition in N-nitrosofenfluramine-induced cell injury in rat hepatocytes

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Abstract

The role of mitochondrial membrane permeability transition in N-nitrosofenfluramine-induced cell injury was studied in mitochondria and hepatocytes isolated from rat liver. Mitochondrial permeability transition has been proposed as a common final pathway in acute cell death through mitochondrial dysfunction. In isolated mitochondria, N-nitrosofenfluramine (0.25 to 1.0 mM) in the presence of Ca2+ (50 μM) elicited a concentration-dependent induction of mitochondrial swelling dependent on mitochondrial permeability transition and the release of cytochrome c, both of which were prevented by pretreatment with a specific inhibitor of mitochondrial permeability transition, cyclosporin A (0.2 μM). The effects of N-nitrosofenfluramine on mitochondria were more potent than those of fenfluramine, which is a sympathomimetic amine with anorectic action. The pretreatment of isolated hepatocytes with cyclosporin A (2 μM) partially but not completely prevented N-nitrosofenfluramine (0.6 mM; a low toxic dose)-induced cell death, loss of cellular ATP, formation of cell blebs and decrease in mitochondrial membrane potential. These results suggest that the onset of N-nitrosofenfluramine-induced cytotoxicity is linked to mitochondrial failure dependent upon induction of mitochondrial permeability transition accompanied by mitochondrial depolarization, the release of cytochrome c and depletion of intracellular ATP through uncoupling of oxidative phosphorylation.

Introduction

Dietary supplements containing natural herbal products are available worldwide and are believed to be harmless and safe with few side effects as compared with conventional drugs. However, some supplements on the market have been found to have adverse effects, which may have been directly or indirectly involved in the deaths of some individuals. Recently, there has been an increase in clinical reports on organ and tissue injuries caused by dietary supplements, accompanying the concomitant increase in their consumption. For example, from 2000 to 2002 in Japan, more than 800 clinical cases of serious liver dysfunction with necrosis and marked elevation of serum alanine and asparate aminotransferase levels were reported in patients taking Chinese dietary supplements, marketed as Chaso-genpi, Onshido-genpi-kouno and Sennomoto-kouno, for weight loss (Adachi et al., 2003, Kanda et al., 2003, Kawata et al., 2003, Lau et al., 2004). In addition, Kawaguchi et al. (2004) reported that one of these supplements caused focal hepatonecrosis, which was accompanied by a decrease in the number of mitochondria and extensive destruction of the cristae. Based on constituent analyses using mass spectroscopy coupled with gas chromatography/high-performance liquid chromatography (HPLC) and/or nuclear magnetic resonance, two synthetic products, N-ethyl-α-methyl-3-(trifluoromethyl)-benzeneethanamine (fenfluramine) and its N-nitroso derivative (N-nitrosofenfluramine), were commonly detected in these products along with various herbal components. Fenfluramine, which is a halogenated derivative of amphetamine, has been used as an appetite suppressant in the treatment of obesity, since it is known that the sympathomimetic amine has an anorectic action mediated through activation of the serotonergic pathway in the brain (Rowland and Carlton, 1986). However, the use of fenfluramine to treat obesity has been prohibited since 1977, because of the associated primary pulmonary hypertension (Brenot et al., 1993, Thomas et al., 1995), valvular heart disease (Connolly et al., 1997), and neurotoxicity (Ricaurte et al., 1991). Consequently, the pharmacologic and toxicologic effects of fenfluramine have been studied in vivo and in vitro, whereas little is known about the effects of N-nitrosofenfluramine, which is unapproved and illegal as a food and drug additive.

The results of a previous study (Nakagawa et al., 2005) showed that mitochondria are the target organelles of N-nitrosofenfluramine, which elicits toxicity in isolated rat hepatocytes through mitochondrial dysfunction related to oxidative phosphorylation and/or membrane potential at an early stage and subsequently lipid peroxidation at a later stage. A number of observations indicated that mitochondria are a primary target of chemical-induced injury, and that their dysfunction ultimately leads to cell death due to an insufficient supply of ATP (Kehrer et al., 1990, Gunter and Pfeiffer, 1990, Nakagawa et al., 1995). The induction of mitochondrial permeability transition, an abrupt increase in the permeability of the inner mitochondrial membrane to small molecular-weight solutes, has been proposed to be an important common pathway causing cellular injury. A direct consequence of mitochondrial permeability transition is that mitochondria are no longer able to maintain energy generation (Hunter and Haworth, 1979, Kass et al., 1992, Nieminen et al., 1995). It has been reported that the induction of mitochondrial permeability transition through the opening of high-conductance pores, which are specifically blocked by cyclosporin A, induces mitochondrial depolarization, release of intramitochondrial ions and metabolic intermediates, uncoupling of oxidative phosphorylation with ATP depletion, mitochondrial swelling and ultimately cell death (Bosser and Gores, 1995, Zoratti and Szabo, 1995). In the present study, we investigated 1) the effects of N-nitrosofenfluramine and fenfluramine on mitochondrial permeability transition in isolated liver mitochondria; and 2) the possible role of mitochondrial permeability transition in N-nitrosofenfluramine-induced cell death in isolated hepatocytes. The mechanism of the toxic effects on N-nitrosofenfluramine is discussed.

Section snippets

Materials

The chemical compounds used were obtained from the following companies: (±)-fenfluramine hydrochloride, (±)-N-nitrosofenfluramine, (purities > 97% and > 99%, respectively; Fig. 1) and collagenase from Wako Pure Chemical Industries Ltd. (Osaka, Japan); cyclosporin A, cytochrome c, HEPES, rhodamine 123 and bovine serum albumin from Sigma Chemical Co. (St. Louis, MO). All other chemicals were of the highest purity commercially available.

Isolation and incubation of hepatocytes

Male F344/DuCrj (200–240 g) rats were obtained from Clea Japan

Effects of N-nitrosofenfluramine on mitochondrial permeability transition

The rapid depletion of intracellular ATP and a decrease in mitochondrial membrane potential (ΔΨ) caused by N-nitrosofenfluramine in isolated rat hepatocytes (Nakagawa et al., 2005) suggest that the compound may induce mitochondrial permeability transition. To investigate this mechanism, N-nitrosofenfluramine was added to a mitochondrial suspension, and the swelling of mitochondria due to mitochondrial permeability transition was measured spectrophotometrically. Incubation of mitochondria with N

Discussion

The results of a previous study indicated that the exposure of hepatocytes to N-nitrosofenfluramine results in acute cell death and that the cytotoxicity is associated with ATP depletion through impairment of mitochondrial function related to mitochondrial membrane potential and/or oxidative phosphorylation (Nakagawa et al., 2005). The results obtained in this study show that in isolated liver mitochondria, N-nitrosofenfluramine elicits a concentration-dependent induction of mitochondrial

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