TY - JOUR
T1 - Formation and reduction of glutathione-protein mixed disulfides during oxidative stress. A study with isolated hepatocytes and menadione (2-methyl-1,4-naphthoquinone)
AU - Bellomo, G.
AU - Mirabelli, F.
AU - DiMonte, D.
AU - Richelmi, P.
AU - Thor, H.
AU - Orrenius, C.
AU - Orrenius, S.
N1 - Funding Information:
Ack~o~le~ge~en~-Theex pertt echnicala ssistanceof Mr Paolo Crin6 is gratefullya cknowledgedT. his study was supported by grants from Consiglio Nazionale delle Richerche( Italy) andt he SwedishM edicalR esearchC oun-cil (project No. 03X-2471).
PY - 1987/4/15
Y1 - 1987/4/15
N2 - Incubation of isolated rat hepatocytes with menadione (2-methyl-1,4-naphthoquinone) resulted in a dose-dependent depletion of intracellular reduced glutathione (GSH), most of which was oxidized to glutathione disulfide (GSSG). Menadione metabolism was also associated with a dose- and time-dependent inhibition of glutathione reductase, impairing the regeneration of GSH from GSSG produced during menadione-induced oxidative stress. Inhibition of glutathione reductase by pretreatment of hepatocytes with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) greatly potentiated both GSH depletion and GSSG formation during the metabolism of low concentrations of menadione. Concomitant with GSH oxidation, mixed disulfides between glutathione and protein thiols were formed. The amount of mixed disulfides produced and the kinetics of their formation were dependent on both the intracellular GSH/GSSG ratio and the activity of glutathione reductase. The mixed disulfides were mainly recovered in the cytosolic fraction and, to a lesser extent, in the microsomal and mitochodrial fractions. The removal of glutathione from protein mixed disulfides formed in hepatocytes exposed to oxidative stress was dependent on GSH and/or cysteine and appeared to occur predominantly via a thioldisulfide exchange mechanism. However, incubation of the microsomal fraction from menadione-treated hepatocytes with purified glutathione reductase in the presence of NADPH also resulted in the reduction of a significant portion of the glutathione-protein mixed disulfides present in this fraction. Our results suggest that the formation of glutathione-protein mixed disulfides occurs as a result of increased GSSG formation and inhibition of glutathione reductase activity during menadione metabolism in hepatocytes.
AB - Incubation of isolated rat hepatocytes with menadione (2-methyl-1,4-naphthoquinone) resulted in a dose-dependent depletion of intracellular reduced glutathione (GSH), most of which was oxidized to glutathione disulfide (GSSG). Menadione metabolism was also associated with a dose- and time-dependent inhibition of glutathione reductase, impairing the regeneration of GSH from GSSG produced during menadione-induced oxidative stress. Inhibition of glutathione reductase by pretreatment of hepatocytes with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) greatly potentiated both GSH depletion and GSSG formation during the metabolism of low concentrations of menadione. Concomitant with GSH oxidation, mixed disulfides between glutathione and protein thiols were formed. The amount of mixed disulfides produced and the kinetics of their formation were dependent on both the intracellular GSH/GSSG ratio and the activity of glutathione reductase. The mixed disulfides were mainly recovered in the cytosolic fraction and, to a lesser extent, in the microsomal and mitochodrial fractions. The removal of glutathione from protein mixed disulfides formed in hepatocytes exposed to oxidative stress was dependent on GSH and/or cysteine and appeared to occur predominantly via a thioldisulfide exchange mechanism. However, incubation of the microsomal fraction from menadione-treated hepatocytes with purified glutathione reductase in the presence of NADPH also resulted in the reduction of a significant portion of the glutathione-protein mixed disulfides present in this fraction. Our results suggest that the formation of glutathione-protein mixed disulfides occurs as a result of increased GSSG formation and inhibition of glutathione reductase activity during menadione metabolism in hepatocytes.
UR - http://www.scopus.com/inward/record.url?scp=0023257674&partnerID=8YFLogxK
U2 - 10.1016/0006-2952(87)90087-6
DO - 10.1016/0006-2952(87)90087-6
M3 - Article
SN - 0006-2952
VL - 36
SP - 1313
EP - 1320
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
IS - 8
ER -