Evidence for stimulated glutathione synthesis by phenobarbital pretreatment during an oxidative challenge in isolated hepatocytes.
Hepatocytes isolated from phenobarbital-pretreated and naive male Sprague-Dawley rats were preincubated with 80 microM N, N-bis (2-chloroethyl)-N-nitrosourea and subsequently exposed to varying concentrations of menadione. We observed that the reduced glutathione levels of the hepatocytes isolated from the sodium phenobarbital(PB)-pretreated, but not the naive rats, recovered to near-control levels after exposure to 200 microM menadione. Since this recovery occurred in the presence of N, N-bis (2-chloroethyl)-N-nitrosourea (an inhibitor of glutathione reductase), we hypothesized that this represented a PB-mediated increase in de novo synthesis of glutathione. To test this hypothesis and to further assess the possible contribution of glutathione reductase in the recovery of the glutathione levels, we preincubated hepatocytes isolated from PB-pretreated and naive rats with 2 mM buthionine sulfoximine, with or without N, N-bis (2-chloroethyl)-N-nitrosourea. Following exposure to menadione, samples were periodically removed for glutathione assessment. Consistent with our hypothesis, the addition of buthionine sulfoximine abrogated the ability of the PB-pretreated hepatocytes to restore glutathione levels following a menadione challenge. Buthionine sulfoximine in combination with N, N-bis (2-chloroethyl)-N-nitrosourea completely abolished hepatocellular glutathione homeostasis for all of the concentrations of menadione employed. The findings from this investigation underscore the importance of phenobarbital-mediated increases in glutathione synthesis, as well as the enhanced levels of glutathione reductase, in maintaining the pool of reduced glutathione and ultimately mitigating the consequences of oxidative stress. In addition, these findings suggest that PB pretreatment increases the reserve capacity of the hepatocyte for glutathione synthesis via a hitherto undescribed hormetic mechanism, a reserve expressed fully only on an oxidative stress of sufficient magnitude.
Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson 39216-4505.
W S
WS
Utley
H M
HM
Mehendale
D000818:Animals; D019328:Buthionine Sulfoximine; D002330:Carmustine / Q000494:pharmacology; D005978:Glutathione / Q000096:biosynthesis*; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors / Q000378:metabolism; D006706:Homeostasis / Q000187:drug effects; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000494:pharmacology; D010084:Oxidation-Reduction; D010634:Phenobarbital / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D014343:Trypan Blue; D014812:Vitamin K / Q000494:pharmacology
D014812:Vitamin K; D008717:Methionine Sulfoximine; D019328:Buthionine Sulfoximine; D007770:L-Lactate Dehydrogenase; D005980:Glutathione Reductase; D005978:Glutathione; D014343:Trypan Blue; D002330:Carmustine; D010634:Phenobarbital