Disruption of mitochondrial function and cellular ATP levels by amiodarone and N-desethylamiodarone in initiation of amiodarone-induced pulmonary cytotoxicity.

Amiodarone (AM), a potent antidysrhythmic agent, can cause potentially life-threatening pulmonary fibrosis. In the present investigation of mechanisms of initiation of AM lung toxicity, we found that 100 microM AM decreased mitochondrial membrane potential in intact hamster lung alveolar macrophages and preparations enriched in isolated alveolar type II cells and nonciliated bronchiolar epithelial (Clara) cells, following 2 h of incubation. This was followed by a drop in cellular ATP content (by 32--77%) at 4 to 6 h, and 30 to 55% loss of viability at 24 h. Supplementation of incubation media with 5.0 mM glucose or 2.0 mM niacin did not reduce AM-induced ATP depletion or cell death in macrophages, and the mitochondrial permeability transition inhibitor cyclosporin A (1.0 microM) did not affect AM cytotoxicity. At 50 microM, the AM metabolite N-desethylamiodarone (DEA) produced effects similar to those of AM, but more rapidly and extensively, with the Clara cell-enriched preparation being particularly susceptible. In isolated whole lung mitochondria, DEA was accumulated to a greater extent than AM. Both AM and DEA inhibited complex I- and complex II-supported respiration, but DEA inhibited complex II to a greater degree than AM. These results demonstrate that AM and DEA disrupt mitochondrial membrane potential prior to ATP depletion and subsequent lung cell death, that DEA is more potent than AM, and that the mitochondrial permeability transition is not involved in mitochondrial perturbation by AM. This suggests that AM- and DEA-induced perturbations of mitochondrial function may initiate AM-induced pulmonary toxicity.

The role of mitochondrial injury in bromobenzene and furosemide induced hepatotoxicity.

Bromobenzene (BB) and furosemide (FS) are two hepatotoxicants whose bioactivation to reactive intermediates is crucial to the development of liver injury. However, the events which lead to hepatocellular toxicity following metabolite formation and covalent binding to cellular macromolecules remain unknown. The present study was undertaken to investigate the effect of administered BB and FS on mitochondrial total glutathione (GSH+GSSG, henceforth referred to as glutathione) content and respiratory function as potential initiating mechanisms of the hepatotoxicity of these compounds in the mouse. Bromobenzene (2 g/kg i.p.) significantly decreased mitochondrial glutathione to 48% of control at 3 h post administration, and to 41% at 4 h. This decrease in mitochondrial glutathione was subsequent to a significant decrease in cytosolic glutathione to 64 and 28% of control at 1 and 2 h, respectively. Oxygen consumption supported by complex I (glutamate-supported) of the respiratory chain was not inhibited by BB until 4 h, where state 3 (active) respiration was reduced to 16% of control. This resulted in a decreased respiratory control ratio (RCR) for complex I-supported respiration. Complex II (succinate)-supported state 3 and state 4 respiration were unaffected by BB until 4 h, at which time they were reduced to 57 and 48% of control, respectively. However, the similar reductions in state 3 and state 4 respiratory rates did not alter the corresponding RCR for complex II. Overt hepatic injury was detected at 4 h, with plasma alanine aminotransferase (ALT) activity increasing significantly at this time point. In contrast to the effects of BB, FS administration (400 mg/kg i.p.) did not alter mitochondrial or cytosolic glutathione, and had no effect on respiration supported by complex I or II for up to 5 h following dosing. However, ALT activity was significantly increased 5 h following FS administration. These results suggest that inhibition of mitochondrial respiratory function coinciding with a decrease in mitochondrial glutathione content may be crucial to the initiation of BB-induced hepatotoxicity, while such events are not required for the initiation of FS-induced hepatotoxicity.

Effects of dietary vitamin E supplementation on pulmonary morphology and collagen deposition in amiodarone- and vehicle-treated hamsters.

Amiodarone (AM) is a potent antidysrhythmic agent that is limited in clinical use by its adverse effects, including potentially life-threatening AM-induced pulmonary toxicity (AIPT). The present study tested the ability of dietary supplementation with vitamin E (500 IU d,1-alpha-tocopherol acetate/kg chow) to protect against pulmonary damage following intratracheal administration of AM (1.83 micromol) to the male golden Syrian hamster. At 21 days post-dosing, animals treated with AM had increased lung hydroxyproline content and histological disease index values compared to control (P < 0.05), which were indicative of fibrosis. Dietary vitamin E supplementation for 6 weeks resulted in a 234% increase in lung vitamin E content at the time of AM dosing, and maintenance on the diet prevented AM-induced elevation of hydroxyproline content and disease index 21 days post-dosing. Dietary vitamin E supplementation also decreased hydroxyproline content and disease index values in hamsters treated intratracheally with distilled water, the AM vehicle. These results demonstrate a protective role for vitamin E in an in vivo model of AIPT, and suggest that this antioxidant may have non-specific antifibrotic effects in the lung.

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

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 microM) stimulated state 4 (resting) respiration at complexes I and II of tightly coupled lung mitochondria, with higher concentrations (200 and 400 microM) resulting in a subsequent inhibition. This biphasic effect of AM (200 microM) was also observed with isolated liver mitochondria. Only inhibition of respiration was observed with AM (50-400 microM) in less tightly coupled lung mitochondria. Based on safranine fluorescence, 200 microM AM decreased lung mitochondrial membrane potential (p < 0.05), while a concentration-dependent (50-200 microM) 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 microM) 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.