Tissue-specific changes in glutathione and cysteine after buthionine sulfoximine treatment of rats and the potential for artifacts in thiol levels resulting from tissue preparation.
L-Buthionine-S,R-sulfoximine (BSO), a potent inhibitor of gamma-glutamylcysteine synthetase, is commonly used as an experimental tool for the specific depletion of glutathione. Since cysteine is a key precursor for glutathione biosynthesis, we investigated the possibility that BSO might also affect the free cysteine pool in rat liver and kidney tissues in vivo. Male CD(SD)BR rats (150-200 g) were injected ip with various doses of BSO (0.25-4.0 mmol/kg), and glutathione and cysteine were measured in liver and kidney using HPLC with electrochemical detection and/or spectroscopic techniques. No hepatotoxicity or nephrotoxicity was observed at the highest BSO dose (4.0 mmol/kg) used. BSO caused the expected decreases of hepatic and renal glutathione at all doses, although glutathione depletion was more rapid, was achieved at a lower BSO dose, and was more sustained in kidney than in liver. Hepatic cysteine levels nearly doubled 20 min after BSO treatment (1.0 mmol/kg, ip), but were not significantly different from control at later time points. In contrast, renal cysteine was significantly depleted from 20 min to 25 hr postinjection with a time course closely paralleling that of renal glutathione depletion. These changes are discussed in the context of models for inter- and intraorgan transport of glutathione and cysteine. We also provide evidence that an artifact, most likely the gamma-glutamyltranspeptidase (GGT)-initiated breakdown of glutathione, leads to a rapid postmortem increase of cysteine levels in liver and particularly in kidney of rats. Simultaneous decreases in GSH levels can be demonstrated in kidney. This artifact needs to be minimized in toxicological studies of glutathione and cysteine in kidney and other GGT-rich organs, as the measured levels of these thiols may not reflect the true concentrations occurring in vivo.
Standeven
A M
AM
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire.
Wetterhahn
K E
KE
D000818:Animals; D000963:Antimetabolites / Q000008:administration & dosage / Q000494:pharmacology* / Q000633:toxicity; D019328:Buthionine Sulfoximine; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005978:Glutathione / Q000378:metabolism*; D007274:Injections, Intraperitoneal; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008717:Methionine Sulfoximine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000633:toxicity; D051381:Rats; D011919:Rats, Inbred Strains; D013053:Spectrophotometry / Q000379:methods; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013997:Time Factors
D000963:Antimetabolites; D013438:Sulfhydryl Compounds; D008717:Methionine Sulfoximine; D019328:Buthionine Sulfoximine; D005978:Glutathione; D003545:Cysteine