Elsevier

Toxicology Letters

Volume 98, Issues 1–2, 1 September 1998, Pages 41-50
Toxicology Letters

Amiodarone-induced disruption of hamster lung and liver mitochondrial function: lack of association with thiobarbituric acid-reactive substance production

https://doi.org/10.1016/S0378-4274(98)00097-6Get rights and content

Abstract

Amiodarone (AM) is an efficacious antidysrhythmic agent that is limited clinically by numerous adverse effects. Of greatest concern is AM-induced pulmonary toxicity (AIPT) due to the potential for mortality. Mitochondrial alterations and free radicals have been implicated in the etiology of AM-induced toxicities, including AIPT. Isolated hamster lung and liver mitochondria were assessed for AM-induced effects on respiration, membrane potential, and lipid peroxidation. AM (50–400 μM) stimulated state 4 (resting) respiration at complexes I and II of tightly coupled lung mitochondria, with higher concentrations (200 and 400 μM) resulting in a subsequent inhibition. This biphasic effect of AM (200 μM) was also observed with isolated liver mitochondria. Only inhibition of respiration was observed with AM (50–400 μM) in less tightly coupled lung mitochondria. Based on safranine fluorescence, 200 μM AM decreased lung mitochondrial membrane potential (p<0.05), while a concentration-dependent (50–200 μM) decrease of membrane potential was observed with liver mitochondria exposed to AM (p<0.05). Formation of thiobarbituric acid-reactive substances (TBARS) was not altered by AM (50–400 μM) in incubations lasting up to 1 h. These results indicate that lipid peroxidation, as indicated by levels of TBARS, does not play a role in AM-induced alterations in mitochondrial respiration and membrane potential.

Introduction

Amiodarone (AM), an iodinated benzofuran derivative, is a potent antidysrhythmic agent, effective in the treatment of many ventricular and supraventricular dysrhythmias (Mason, 1987, Kennedy, 1990, Singh, 1996). Clinical use of AM is limited because of the potential for development of numerous adverse effects. Of greatest concern is AM-induced pulmonary toxicity (AIPT), due to the potential for mortality (Mason, 1987, Vrobel et al., 1989). However, hepatotoxicity and other adverse effects are also of clinical importance (Lewis et al., 1989, Figge and Figge, 1990).

The mitochondrion has been identified as a potential target organelle in AM-induced toxicities. AM decreases membrane potential, ATP production, and β-oxidation of fatty acids in mouse liver and/or rat heart mitochondria (Guerreiro et al., 1986b, Fromenty et al., 1990a, Fromenty et al., 1990b), and has a biphasic effect on mitochondrial resting respiration (Fromenty et al., 1990b). Accumulation of AM in mitochondria has been demonstrated in vitro (Hostetler et al., 1988, Fromenty et al., 1990b) and in vivo (Pirovino et al., 1988), while structural effects on mitochondria have also been reported. Human lymphocytes exposed to AM demonstrated intact cell membranes, but their mitochondria had disorganized cristae and were swollen (Yasuda et al., 1996). Furthermore, isolated rat heart mitochondria incubated with AM displayed ruptured inner membranes, extended cristae and increased matrix volume (Guerreiro et al., 1986a).

It has been proposed that free radicals play a role in the etiology of AM-induced toxicities, including hepatic (Vereckei et al., 1993) and pulmonary (Bennett et al., 1987). The formation of an AM or metabolite radical under certain conditions has been demonstrated (Li and Chignell, 1987, Vereckei et al., 1993), and might initiate or contribute to AM-induced cytotoxicity via free radical-mediated damage to cellular membranes. Formation of a drug or metabolite radical might occur in situ at the level of the mitochondrion, as a result of electron leakage from the enzymes of the respiratory chain or the mitochondrial cytochrome P450 mono-oxygenase system (Kubow et al., 1985, Davis, 1995). Ensuing peroxidative damage to mitochondrial structure and respiratory function could then occur, thereby initiating or contributing to AM-induced cytotoxicity.

The present study was undertaken to compare the effects of AM on mitochondria from lung, arguably the most important target of AM toxicity, to those on hepatic mitochondria, and to determine if the effects are associated with lipid peroxidation, which could be initiated by an AM or metabolite radical.

Section snippets

Chemicals

Chemicals were obtained from suppliers as follows: amiodarone hydrochloride, rotenone (95–98%), adenosine 5′-diphosphate (ADP, free acid), β-nicotinamide adenine dinucleotide phosphate (β-NADPH reduced form, tetrasodium salt), l-glutamate (monosodium salt), l(−) malate (monosodium salt), ethylenediamine tetraacetic acid (EDTA, disodium salt, dihydrate), succinate (disodium salt, hexahydrate), d-mannitol, ethylene glycol-bis-(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid (EGTA), 3-[N

Effect of AM on mitochondrial oxygen consumption

Calculated RCR and ADP:O ratio values of untreated hamster lung and liver mitochondria are summarized in Table 1. When RCRs were calculated, the results revealed two distinct groups. Mitochondria with RCR values>2.50 were considered to be tightly coupled, while those with RCR values<2.50 were considered non-tightly coupled. The RCR and ADP:O ratios for complex I of tightly coupled lung mitochondria were higher than those for non-tightly coupled lung mitochondria (p<0.05). Statistical analysis

Discussion

The present study utilized isolated hamster lung and liver mitochondria to examine effects of AM on parameters indicative of altered mitochondrial membrane integrity and respiratory function. The golden Syrian hamster has been used as a model for studying AIPT in our laboratory (Daniels et al., 1989, Rafeiro et al., 1994, Leeder et al., 1996), and those of other investigators (Cantor et al., 1984, Blake and Reasor, 1995), and was therefore utilized in the present study. The literature indicates

Acknowledgements

This work was supported by the Medical Research Council of Canada, Grant No. MT-13257, and by an Ontario Thoracic Society Block Term Grant to Queen's University. J.W. Card is the recipient of an Ontario Graduate Scholarship. The authors would like to thank Luna Al-Khalili for assistance with mitochondrial lipid peroxidation experiments, and Graeme Smith and Michael Bolt for assistance with animal surgery.

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