Mechanism of mitochondrial dysfunction and cytotoxicity induced by tropolones in isolated rat hepatocytes.
The mechanism of mitochondrial dysfunction and toxicity induced by the tropolones, beta-thujaplicin (4-isopropyl tropolone), tropolone and tropone, has been studied in freshly isolated rat hepatocytes. Incubation of hepatocytes with beta-thujaplicin (1-4 mM) elicited a concentration and time-dependent cell killing. The toxicity was accompanied by losses of cellular ATP, total adenine nucleotides and glutathione, independently of lipid peroxidation and protein thiol oxidation. The beta-thujaplicin-induced cytotoxicity was enhanced by the pretreatment of hepatocyte suspensions with EDTA (4 mM), a hydrophilic chelator, or by incubation in Ca2+ and Mg2+-deficient Krebs-Henseleit buffer. The partition coefficient of beta-thujaplicin, which formed complex with the divalent cations in Krebs-Henseleit buffer, in n-octanol/buffer was increased either in the presence of EDTA or absence of divalent cations. Comparison of toxic effects based on cell viability and adenine nucleotide levels showed that beta-thujaplicin was more toxic than tropolone or tropone in Krebs-Henseleit buffer containing EDTA (4 mM). The addition of beta-thujaplicin to isolated hepatic mitochondria reduced state 3 respiration with NAD+-linked substrate (pyruvate plus malate) and/or with an FAD-linked substrate (succinate plus rotenone), whereas state 3 respiration of ascorbate plus tetramethyl-p-phenylenediamine (cytochrome oxidase-linked respiration) was not significantly affected by beta-thujaplicin. Further, the addition of these tropolones caused a concentration-dependent increase in the rate of state 4 oxygen consumption, indicating an uncoupling effect. These results indicate that beta-thujaplicin- and tropolone-induced cytotoxicity are associated with an acute ATP depletion via mitochondrial dysfunction related to oxidative phosphorylation and that the induction of cytotoxicity is affected by EDTA or divalent cations.
Nakagawa
Y
Y
Department of Toxicology, Tokyo Metropolitan Research Laboratory of Public Health, Japan. yoshio@tokyo-eiken.go.jp
Tayama
K
K
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002413:Cations, Divalent; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004347:Drug Interactions; D004492:Edetic Acid / Q000494:pharmacology; D007502:Iron Chelating Agents / Q000378:metabolism / Q000633:toxicity; D008099:Liver / Q000166:cytology / Q000187:drug effects*; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000502:physiology; D039821:Monoterpenes; D051381:Rats; D011916:Rats, Inbred F344; D014334:Tropolone / Q000031:analogs & derivatives* / Q000378:metabolism / Q000633:toxicity*
D002413:Cations, Divalent; D002614:Chelating Agents; D007502:Iron Chelating Agents; D039821:Monoterpenes; D014334:Tropolone; D004492:Edetic Acid; C003730:tropone; D008274:Magnesium; D002118:Calcium; C009479:beta-thujaplicin