Phenobarbital-induced cytosolic cytoprotective mechanisms that offset increases in NADPH cytochrome P450 reductase activity in menadione-mediated cytotoxicity.
Hepatocytes isolated from phenobarbital (PB)-pretreated and naive male Sprague-Dawley rats were incubated with menadione under one of three oxygen conditions (0, 21, or 95% oxygen) for 3 hr. During this time, samples were drawn and assayed for lactate dehydrogenase release and trypan blue exclusion as indices of cytotoxicity. Neither parameter indicated any significant difference in menadione-induced cytotoxicity between naive and PB-pretreated hepatocytes. Likewise, no difference was observed between hepatocytes incubated in 21% versus 95% O2. Consistent with the oxyradical hypothesis of menadione-induced cytotoxicity, hepatocytes incubated under 0% O2 (95:5; N2:CO2) did not exhibit any menadione cytotoxicity. Hepatic microsomes prepared from PB-pretreated rats exhibited a threefold increase in NADPH cytochrome P450 reductase activity over those of controls. Menadione-stimulated superoxide (O2-) production was twofold higher in PB pretreated versus naive liver microsomes. However, PB pretreatment failed to produce an increase in O2- production in intact hepatocytes or in hepatocytes disrupted by sonication. The failure of PB pretreatment to increase menadione-induced cytotoxicity and superoxide production in either intact or sonicated hepatocytes suggests that a concomitant cytoprotective mechanism is induced as well. The data further indicate that the cytoprotective elements are located in a nonmicrosomal fraction of the cell. In support of this, we observed PB-induced increases in glutathione levels, glutathione reductase, and DT-diaphorase activities. These findings indicate that PB-induced enhancements of the hepatocellular cytoprotective mechanisms collectively compensate for the increased redox cycling mechanism, resulting in a mitigation of the anticipated increased hepatocellular cytotoxicity of menadione.
Utley
W S
WS
Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson 39216-4505.
Mehendale
H M
HM
D000818:Animals; D003600:Cytosol / Q000378:metabolism; D004347:Drug Interactions; D005980:Glutathione Reductase / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D008297:Male; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism / Q000494:pharmacology; D010634:Phenobarbital / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D013481:Superoxides / Q000378:metabolism; D014812:Vitamin K / Q000633:toxicity*
D013481:Superoxides; D014812:Vitamin K; D007770:L-Lactate Dehydrogenase; D009251:NADPH-Ferrihemoprotein Reductase; D005980:Glutathione Reductase; D010100:Oxygen; D010634:Phenobarbital