Diclofenac Toxicity to Hepatocytes: A Role for Drug Metabolism in Cell Toxicity1
- 1Unidad de Hepatologı́a Experimental, Centro de Investigación, Hospital Universitario “La Fe”, Valencia, Spain (R.J., M.J.G.-L., R.B., J.V.C.); 2Departamento de Bioquı́mica, Facultad de Medicina, Universidad de Valencia, Valencia, Spain (R.B., R.J., J.V.C.); and 3Departamento de Parasitologı́a y Biologı́a Celular, Facultad de Ciencias Biológicas, Universidad de Valencia, Valencia, Spain (X.P.)
Abstract
Diclofenac, a 2-arylacetic acid, nonsteroidal anti-inflammatory drug, has been reported to cause adverse hepatic effects in certain individuals. To discriminate among possible mechanisms of hepatotoxicity, we examined the effects of diclofenac on human and rat hepatocytes and hepatic cell lines (HepG2, FaO), investigated the major biochemical events in the course of diclofenac cytotoxicity (calcium homeostasis, lipid peroxidation, and mitochondrial dysfunction), and investigated whether cytotoxicity could be related to drug metabolism by cytochrome P-450. Acute diclofenac-induced toxicity in hepatocytes was preluded by a decrease in ATP levels, whereas no significant oxidative stress (decrease in glutathione and lipid peroxidation) or increase in intracellular calcium concentration could be observed at early incubation stages. Diclofenac was more cytotoxic to drug metabolizing cells (rat and human primary cultured hepatocytes) than to nonmetabolizing cell lines (HepG2, FaO). Despite the fact that diclofenac itself was effective in impairing ATP synthesis by mitochondria, we found evidence that toxicity was also related to drug metabolism and was reduced by the addition of cytochrome P-450 inhibitors (proadifen and ketoconazole) to culture medium. The in vitro cytotoxicity correlated well with the formation by hepatocytes of 5-hydroxydiclofenac and, in particular,N,5-dihydroxydiclofenac, a minor metabolite first characterized in this article. Hepatic microsomes showed the ability to both oxidize 5-hydroxydiclofenac toN,5-dihydroxydiclofenac and back reduce the latter to 5-hydroxydiclofenac with the consumption of NADPH. The experimental results suggest that the toxic effect of diclofenac on hepatocytes may be caused by drug-induced mitochondrial impairment, together with a futile consumption of NADPH.
Footnotes
-
Send reprint requests to: Dr. José V. Castell, Unidad de Hepatologı́a Experimental. Centro de Investigación, Hospital Universitario “La Fe”, SVS. Avda. Campanar 21, E-46009 Valencia, Spain. E-mail:Jose.Castell{at}uv.es
-
↵1 This study was supported, in part, by the BIOMED II Research Program of the European Union and the Fondo de Investigaciones Sanitarias, Spanish Ministry of Health. R.B. was recipient of a predoctoral fellowship from the Consellerı́a de Educación y Ciencia, Generalitat Valenciana.
- Abbreviations:
- BHT
- buthylated hydroxytoluene
- BSO
- l-buthionine-[S,R]-sulfoximine
- CYP
- cytochrome P-450
- MDA
- malondialdehyde
- DEM
- maleic acid diethyl ester
- MTT
- 3-[4,5-dimethylthiazol-2-yl]-3,5-diphenylformazan
- PBS
- phosphate-buffered saline
- 4′-OHdic
- 4′-hydroxydiclofenac
- 5-OHdic
- 5-hydroxydiclofenac
- N
- 5-(OH)2dic,N,5-dihydroxydiclofenac
- [Ca++]i
- intracellular calcium
- GSH
- glutathione
- HPLC-MS
- high-performance liquid chromatography-mass spectrometry
-
- Received April 8, 1998.
- Accepted July 21, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
