Amiodarone (AM) is a potent antidysrhythmic agent which can cause potentially life-threatening pulmonary fibrosis, and N-desethylamiodarone (DEA) is a metabolite of AM that may contribute to the toxicity of AM in vivo. Recent evidence has implicated the involvement of the renin-angiotensin system (RAS) in the initiation and progression of amiodarone-induced pulmonary toxicity. In cultured HPL1A human peripheral lung epithelial cells, we found AM to be converted to DEA minimally (< 2%) after 24 h of incubation, indicating that the HPL1A cell culture model can be used to study the effects of AM and DEA independently. Apoptotic cell death was assessed by annexin-V-FITC (ann-V) staining and by terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate nick-end labeling (TUNEL), while necrotic cell death was determined by propidium iodide (PI) staining. The percentage of PI positive cells increased over six-fold after 24 h treatment with 20 μM AM (80.8%) compared to control (12.0%), and doubled after 24 h treatment with 3.5 μM DEA (20.4%) compared to control (10.8%). The percentage of ann-V positive cells decreased from 8.26% (control) to 1.56% following 24 h treatment with 10 μM AM and more than doubled after 24 h incubation with 3.5 μM DEA (22.0%) compared to control (9.86%) (p<0.05). Treatment for 24 h with 5.0 μM DEA caused the percentage of TUNEL positive cells to increase from 4.21% (control) to 26.7% (p<0.05). Vitamin E (5 – 20 μM) did not protect against AM or DEA cytotoxicity, as determined by ann-V and PI dual staining. Angiotensin II (100 pM – 1 μM) alone or in combination with AM or DEA did not alter cytotoxicity. Furthermore, the angiotensin converting enzyme inhibitor captopril did not protect against AM or DEA cytotoxicity. In conclusion, in vitro, AM activates primarily necrotic pathways, whereas DEA activates both necrotic and apoptotic pathways, and the RAS does not seem to be involved in AM or DEA cytotoxicity in HPL1A cells. Multiple mechanisms may contribute to the initiation of lung damage observed clinically, due to actions of both AM and its metabolite DEA. Keywords: amiodarone, desethylamiodarone, vitamin E, renin-angiotensin system / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2009-11-26 13:57:09.65

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