Simultaneous measurements of glutathione and activated sulphate (PAPS) synthesis rates and the effects of selective inhibition of glutathione conjugation or sulphation of acetaminophen.
The aim of the present study was to examine the effects of the hepatotoxic drug acetaminophen (AA) on the synthesis rates of glutathione (GSH), activated sulphate (PAPS; adenosine 3'-phosphate 5'-phosphosulphate) and the AA metabolites AA-GSH and AA-sulphate after selective inhibition of GSH biosynthesis or sulphation in isolated rat hepatocytes. Selective inhibition of the two interdependent metabolic pathways was accomplished by buthionine sulphoximine (BSO) and 2,6-dichloro-4-nitrophenol (DCNP). The synthesis rates of GSH and PAPS were determined simultaneously by a previously described method based on trapping of radioactivity (35S) in the pre-labelled GSH and PAPS pools. Pre-incubation with 10 mM BSO for 30 min depleted GSH by 38% (P < 0.05) and PAPS by 27% (P < 0.05). The depletion resulted in increased PAPS synthesis at low, non-toxic [5-19 nmol/(10(6) cells.min)] (P < 0.05) and at high, toxic [7-30 nmol/10(6) cells.min)] (P < 0.05) AA concentrations. In both cases sulphur is diverted from GSH biosynthesis to sulphoxidation and PAPS synthesis, thereby maintaining the PAPS pool and preserving the sulphation capacity. This corresponds to the finding that AA sulphation was unaffected by BSO irrespective of AA concentration [6 vs 5 and 20 vs 17 nmol/(10(6) cells.hr), respectively]. Even though the GSH synthesis was halved after BSO pre-incubation, the GSH conjugating capacity of AA was well preserved. Incubation with 200 microM DCNP and 5 mM AA diminished PAPS synthesis from 24 to 10 nmol/(10(6) cells.min) (P < 0.02) and reduced AA-sulphate synthesis by 67% compared to experiments without DCNP incubation [4.8 vs 14.7 nmol/(10(6) cells.hr)] (P < 0.05). GSH and AA-GSH synthesis rates did not change compared to control experiments in which sulphation was not inhibited [1165 vs 1487 nmol/(10(6) cells.min), respectively] and [1.7 vs 1.7 nmol/(10(6) cells.hr), respectively]. This indicates that increased sulphur availability due to decreased PAPS synthesis is unable to raise the cysteine pool and stimulate the gamma-glutamyl cycle and GSH synthesis.
Dalhoff
K
K
Department of Medicine A, Rigshospitalet, Copenhagen, Denmark.
Poulsen
H E
HE
D000082:Acetaminophen / Q000737:chemistry*; D000818:Animals; D019328:Buthionine Sulfoximine; D002478:Cells, Cultured; D005260:Female; D005978:Glutathione / Q000096:biosynthesis / Q000737:chemistry* / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000494:pharmacology; D009596:Nitrophenols / Q000494:pharmacology; D010724:Phosphoadenosine Phosphosulfate / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D013431:Sulfates / Q000032:analysis / Q000737:chemistry*; D015238:Sulfotransferases / Q000378:metabolism
D009596:Nitrophenols; D013431:Sulfates; D008717:Methionine Sulfoximine; D000082:Acetaminophen; D010724:Phosphoadenosine Phosphosulfate; D019328:Buthionine Sulfoximine; C015802:2,6-dichloro-4-nitrophenol; D015238:Sulfotransferases; D005978:Glutathione