N-Nitrosofenfluramine induces cytotoxicity via mitochondrial dysfunction and oxidative stress in isolated rat hepatocytes.
The cytotoxic effects of fenfluramine, an appetite suppressant, and its N-nitroso derivative, N-nitrosofenfluramine, have been studied in freshly isolated rat hepatocytes and isolated hepatic mitochondria. Exposure of hepatocytes to N-nitrosofenfluramine caused not only concentration (0.25-1.0 mmol L(-1)) and time (0-3 h)-dependent cell death accompanied by the loss of cellular ATP, adenine nucleotide pools, reduced glutathione (GSH), and protein thiols, but also the accumulation of oxidized glutathione and malondialdehyde (MDA), indicating lipid peroxidation. There was a time lag for the onset of the accumulation of MDA after the rapid depletion of ATP. Supplementation of the hepatocyte suspensions with N-acetylcysteine (4 mmol L(-1)), a precursor of intracellular GSH, partially inhibited N-nitrosofenfluramine (1 mmol L(-1))-induced cytotoxicity. In comparative effects based on cell viability and rhodamine 123 retention, an index of mitochondrial membrane potential, fenfluramine was less toxic than N-nitrosofenfluramine. In mitochondria isolated from rat liver, N-nitrosofenfluramine caused an increase in the rate of state-4 oxygen consumption, indicating an uncoupling effect, and a decrease in the rate of state-3 oxygen consumption in a concentration-dependent manner. These results indicate that (a) mitochondria are target organelles for N-nitrosofenfluramine, which elicits cytotoxicity through mitochondrial dysfunction related to membrane potential and/or oxidative phosphorylation at an early stage and subsequently lipid peroxidation at a later stage; and (b) the toxicity of N-nitrosofenfluramine is greater than that of fenfluramine, suggesting participation of the nitroso group in the toxicity.
Nakagawa
Yoshio
Y
Division of Pharmacology, Tokyo Metropolitan Institute of Public Health, 3-24-1, Hyakunin-cho, Shinjuku-ku, Tokyo, 169-0073, Japan. Yoshio_1_Nakagawa@member.metro.tokyo.jp
Suzuki
Toshinari
T
Kamimura
Hisashi
H
Nagai
Fumiko
F
D000227:Adenine Nucleotides / Q000378:metabolism; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005277:Fenfluramine / Q000031:analogs & derivatives* / Q000633:toxicity; D005978:Glutathione / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008315:Malondialdehyde / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D018384:Oxidative Stress / Q000187:drug effects*; D051381:Rats; D011916:Rats, Inbred F344; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013997:Time Factors
D000227:Adenine Nucleotides; D013438:Sulfhydryl Compounds; C493137:nitrosofenfluramine; D005277:Fenfluramine; D008315:Malondialdehyde; D005978:Glutathione