Attenuation of Amiodarone-Induced Pulmonary Fibrosis by Vitamin E Is Associated with Suppression of Transforming Growth Factor- 1 Gene Expression but Not Prevention of Mitochondrial Dysfunction

Amiodarone (AM) is an efficacious antidysrhythmic agent that can cause numerous adverse effects, including potentially lifethreatening pulmonary fibrosis. The current study was undertaken to investigate potential protective mechanisms of vitamin E against AM-induced pulmonary toxicity (AIPT) in the hamster. Three weeks after intratracheal administration of AM (1.83 mol), increased pulmonary hydroxyproline content and histological damage were observed, indicative of fibrosis. These effects were preceded by increased pulmonary levels of transforming growth factor (TGF)1 mRNA at 1 week post-AM, which remained elevated 3 weeks post-AM. Dietary supplementation with vitamin E resulted in rapid pulmonary accumulation of the vitamin, and prevention of AM-induced increases in TGF1, hydroxyproline, and histological damage. Although dietary supplementation also markedly elevated lung mitochondrial vitamin E content, it did not attenuate AM-induced inhibition of mitochondrial respiration or disruption of mitochondrial membrane potential in vitro, or lung mitochondrial respiratory inhibition resulting from in vivo AM administration. These results suggest that vitamin E reduces the extent of pulmonary damage after AM administration via downregulating TGF1 overexpression but that it does not modify AM-induced mitochondrial dysfunction, a potential initiating event in AIPT. Pulmonary toxicity is an adverse effect of great concern in patients on amiodarone (AM) pharmacotherapy (Pollak, 1999). This is primarily due to the potential for development of pulmonary fibrosis, a condition for which there is currently no effective treatment and for which patient prognosis is poor (Phan, 1995). The pathogenesis of AM-induced pulmonary toxicity (AIPT) has not been elucidated and may involve numerous processes (Massey et al., 1995; Reasor and Kacew, 1996). Several in vitro studies have demonstrated deleterious effects of AM on mitochondrial structure and function (Fromenty et al., 1990; Yasuda et al., 1996; Card et al., 1998). Furthermore, AM and its primary metabolite N-desethylamiodarone (DEA) cause disruption of mitochondrial membrane potential and decrease cellular ATP levels before cell death in freshly isolated hamster lung cells, with DEA being more potent than AM (Bolt et al., 2001a). Thus, mitochondrial dysfunction induced by AM and DEA may be critical to initiation of AM pulmonary cytotoxicity that precedes fibrosis. Vitamin E has been reported to decrease AM-induced cytotoxicity in cultured pulmonary (Futamura, 1996) and nonpulmonary cells (Kachel et al., 1990; Ruch et al., 1991), whereas other antioxidant treatments were ineffective. Additionally, cell type-selective protection against AM-induced cytotoxicity by vitamin E in isolated hamster lung cells has been observed (Bolt et al., 2001b). In the in vivo hamster model of AIPT, we showed that dietary vitamin E supplementation substantially reduced the extent of pulmonary collagen deposition and histological damage after intratracheal AM administration (Card et al., 1999). To date, the mechanism(s) of protection of vitamin E against AIPT has not been determined. Recognized primarily for its free radical scavenging and chain-breaking antioxidant properties, vitamin E has reThis research was supported by Operating Grant MT-13257 from the Canadian Institutes of Health Research. J.W.C. was the recipient of a Canadian Institutes of Health Research Doctoral Research Award and the Procter and Gamble Graduate Student Fellowship from the Society of Toxicology. Portions of this work have been presented in abstract form under the following citations: Card JW, Racz WJ, Brien JF, Margolin SB, and Massey TE (2001) Anti-fibrotic studies in the hamster model of amiodarone-induced pulmonary fibrosis. Proc Soc Toxicol Canada; and Card JW, Steenbakkers MJ, Beard KM, Racz WJ, Brien JF, Bennett BM, and Massey TE (2000) Dietary vitamin E supplementation does not prevent lung mitochondrial dysfunction induced by in vitro amiodarone and N-desethylamiodarone. Toxicologist 54 (1-S):319. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.043208. ABBREVIATIONS: AM, amiodarone; AIPT, AM-induced pulmonary toxicity; DEA, N-desethylamiodarone; TGF, transforming growth factor; RCR, respiratory control ratio; ADP:O, ratio of adenosine diphosphate to oxygen. 0022-3565/03/3041-277–283$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 304, No. 1 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 43208/1030956 JPET 304:277–283, 2003 Printed in U.S.A.