An in vitro study of the microsomal metabolism and cellular toxicity of phenytoin, sorbinil and mianserin.
1. The cytotoxicity of metabolites generated from phenytoin, sorbinil and mianserin by human and mouse liver microsomes was assessed by co-incubation with human mononuclear leucocytes as target cells. Cytotoxicity was determined by trypan blue dye exclusion. 2. Phenytoin and sorbinil were metabolised by NADPH-dependent murine microsomal enzymes to cytotoxic metabolites. Cytotoxicity produced by both drugs was significantly enhanced by the epoxide hydrolase inhibitor trichloropropane oxide (TCPO). No significant cytotoxicity was observed in the presence of human liver microsomes. 3. Mianserin was metabolised by both human and mouse liver microsomes to a cytotoxin. Cytotoxicity was greater in the presence of human liver microsomes (13.7 +/- 2.2%; mean +/- s.d. for four livers, compared with 6.0 +/- 2.4%, mean +/- s.d., n = 4, with mouse liver microsomes), and was unaffected by pretreatment with TCPO. 4. Stable metabolites were quantified by radiometric high performance liquid chromatography. Phenytoin and sorbinil were metabolised to 5-(p-hydroxyphenyl)-5-phenyl-hydantoin (0.3-0.5% of incubated radioactivity) and 2-hydroxysorbinil (0.4-2.7% of incubated radioactivity), respectively, by both human and mouse liver microsomes. 5. Mianserin was metabolised to 8-hydroxymianserin and desmethylmianserin by both human and mouse liver microsomes. Desmethylmianserin was the major product in incubations with human liver microsomes (32.3 +/- 12%, mean +/- s.d. for four livers), whereas 8-hydroxymianserin was the predominant metabolite generated by mouse liver microsomes (25.9 +/- 1.5%, mean +/- s.d., n = 4). 6. Generation of electrophilic metabolites was assessed by determination of the amount of radiolabelled material which became irreversibly bound to protein. Only mouse liver microsomes activated phenytoin to a chemically reactive metabolite, whereas both mouse and human liver microsomes generated reactive metabolites from sorbinil and mianserin. 7. These studies show that drug cytotoxicity can be mediated by low concentrations (circa microM) of metabolites generated by NADPH-dependent hepatic microsomal enzymes; however demonstration of cytotoxicity in vitro has not been established as a means of predicting in vivo toxicity.
Riley
R J
RJ
Department of Pharmacology and Therapeutics, University of Liverpool.
Maggs
J L
JL
Lambert
C
C
Kitteringham
N R
NR
Park
B K
BK
D000328:Adult; D000818:Animals; D001711:Biotransformation; D002470:Cell Survival / Q000187:drug effects; D004851:Epoxide Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000378:metabolism* / Q000633:toxicity; D048289:Imidazolidines; D066298:In Vitro Techniques; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D008297:Male; D008803:Mianserin / Q000378:metabolism* / Q000633:toxicity; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology* / Q000378:metabolism; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D010672:Phenytoin / Q000378:metabolism* / Q000633:toxicity
D007093:Imidazoles; D048289:Imidazolidines; D008803:Mianserin; D009249:NADP; D010672:Phenytoin; D005982:Glutathione Transferase; D004851:Epoxide Hydrolases; C026411:sorbinil