Effect of N-acetylcysteine and deferoxamine on endogenous antioxidant defense system gene expression in a rat hepatocyte model of cocaine cytotoxicity.
In the present study we investigated on cultures of hepatocytes from phenobarbital-pretreated rats, the effect of the antioxidants, 0.5 mM N-acetylcysteine (NAC) or 1.5 mM deferoxamine (DFO), previously incubated for 24 h and coincubated with cocaine (0-1000 microM) for another 24 h. Cocaine cytotoxicity was monitored by either the lysis of the cell membranes or apoptosis. Lysis of the cell membranes was evidenced by lactate dehydrogenase leakage, apoptosis was observed by detecting a hypodiploid peak (<2C) in DNA histograms obtained by flow cytometry, peroxide production was quantified with 2', 7'-dichlorodihydrofluorescein diacetate and gene expression of the antioxidant enzymes: Mn- and Cu,Zn-superoxide dismutases, catalase and glutathione peroxidase were measured by Northern blot analysis. NAC and DFO significantly decreased the extent of lysis of cell membranes and apoptosis, and the antiapoptotic effect was parallel to peroxide generation. By the effect of NAC and DFO, significant increases were detected in the levels of mRNA of catalase, manganese superoxide dismutase and glutathione peroxidase. From these results we conclude that NAC or DFO, when incubated in the presence of cocaine, exerted a protective effect against cocaine toxicity at the level of both lysis of the membranes and apoptosis. This protective effect, in the case of NAC, was directed towards an increase in antioxidant enzyme expression, and in the case of DFO against reactive oxygen species generation.
Zaragoza
A
A
Instituto de Bioquímica (CSIC-UCM), Facultad de Farmacia, Universidad Complutense, Plaza de Ramón y Cajal sn, 28040, Madrid, Spain.
Díez-Fernández
C
C
Alvarez
A M
AM
Andrés
D
D
Cascales
M
M
D000111:Acetylcysteine / Q000494:pharmacology*; D000818:Animals; D000975:Antioxidants / Q000378:metabolism* / Q000494:pharmacology; D002374:Catalase / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D003042:Cocaine / Q000633:toxicity; D003676:Deferoxamine / Q000494:pharmacology*; D004171:Diploidy; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005979:Glutathione Peroxidase / Q000235:genetics / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000032:analysis; D008099:Liver / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D008297:Male; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism
D000975:Antioxidants; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; D002374:Catalase; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase; D003042:Cocaine; D003676:Deferoxamine; D000111:Acetylcysteine