Effects of cysteine and acetaminophen on the syntheses of glutathione and adenosine 3'-phosphate 5'-phosphosulfate in isolated rat hepatocytes.
The aim of the present study was to introduce and validate a radioactive tracer method in which adenosine 3'-phosphate 5'-phosphosulfate (PAPS) and glutathione (GSH) are measured simultaneously in isolated hepatocytes. PAPS and GSH are co-substrates in sulphation and GSH conjugation, and both are dependent on sulphur deriving from cysteine. The effect of cysteine on the syntheses was investigated at non-toxic and toxic concentrations of the hepatotoxic drug acetaminophen (AA). Administration of AA trapped radioactivity (35S) in the pre-labelled PAPS and GSH pools by formation of the metabolites, AA-sulphate and AA-GSH. Turnover rates were determined from the decline of AA-sulphate and AA-GSH specific activity. Syntheses of PAPS and GSH were calculated by multiplying the rates with the concentrations of the respective co-substrates. Increasing AA concentration from non-toxic to toxic levels resulted in increased median PAPS and GSH syntheses (8 to 11 and 311 to 2218 nmol/10(6) cells/min, respectively) (P less than 0.05). Addition of cysteine did not alter median PAPS synthesis (5 to 3 nmol/10(6) cells/min) but decreased median GSH synthesis (666 to 261 nmol/10(6) cells/min) (P less than 0.05) in experiments with non-toxic AA concentrations. In experiments with toxic AA concentrations opposite effects of cysteine were seen, i.e. median PAPS synthesis was reduced (3 to 2 nmol/10(6) cells/min) (P less than 0.05) while median GSH synthesis was unchanged (23 to 16 nmol/10(6) cells/min). The present method provides a tool in which two important detoxification pathways can be measured simultaneously and the data suggest that the two pathways are regulated by substrate availability.
Dalhoff
K
K
Department of Medicine A, Rigshospitalet, Copenhagen, Denmark.
Poulsen
H E
HE
D000082:Acetaminophen / Q000494:pharmacology*; D000818:Animals; D003545:Cysteine / Q000494:pharmacology*; D005260:Female; D005978:Glutathione / Q000096:biosynthesis*; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D010724:Phosphoadenosine Phosphosulfate / Q000096:biosynthesis*; D051381:Rats; D011919:Rats, Inbred Strains
D000082:Acetaminophen; D010724:Phosphoadenosine Phosphosulfate; D005978:Glutathione; D003545:Cysteine