Effect of inhibitors on the biotransformation of tamoxifen by female rat and mouse liver slices and homogenates.
The metabolism of tamoxifen was studied in female Sprague-Dawley rat and mouse liver slices and homogenates, and the three principal tamoxifen metabolites, 4-hydroxytamoxifen, N-desmethyl-tamoxifen and tamoxifen N-oxide, were identified by HPLC using authentic standards. It was not possible to identify any of the minor metabolites such as the epoxides using this technique. The N-oxide metabolite only appeared when NADPH was added to the system; this is because the production of tamoxifen N-oxide is primarily mediated by microsomal flavin monooxygenase (FMO) which is NADPH dependent. However, this metabolite did appear in incubations with mouse liver slices only, because they are rich in flavin monooxygenases (FMOs). It did not appear in female rat or mouse liver homogenates, because the NADPH present is destroyed during homogenisation, therefore it was necessary to add NADPH to the system to produce the N-oxide metabolite. The purpose of this study was to investigate the effect of inhibitors on the biotransformation of tamoxifen by female rat and mouse liver slices and homogenates. Female rat liver slices and homogenates were incubated with the following inhibitors (1 mM): cimetidine, ascorbate, sodium azide and reduced glutathione. Cimetidine, a general P-450 inhibitor, inhibited the production of the N-desmethyl metabolite by about 80%; this is in agreement with the action of the other inhibitors. Reduced glutathione, ascorbate and sodium azide are mainly peroxidase inhibitors, so therefore from these novel and interesting results it was possible to suggest that peroxidases play a role in the metabolism of tamoxifen. This observation was also strengthened when the production of the N-desmethyl metabolite increased when horseradish peroxidase was added to the incubate. The production of 4-hydroxytamoxifen was reduced and the N-oxide metabolite was completely inhibited in the presence of peroxidase inhibitors. When rat liver homogenates was incubated with superoxide dismutase (SOD) and catalase, it was observed that the N-desmethyl metabolite disappeared completely at 60 min and the N-oxide and 4-hydroxy metabolites were completely inhibited. However, this phenomenon was only observed when SOD and catalase were preincubated for 30 min with the rat liver homogenate at 37 degrees C; without preincubation the production of these metabolites was unaffected. Finally, the effect of long incubation periods (300 min) on the production of metabolites was examined. It was found that there was a reduction in the concentration of metabolite produced after 60 min which was due to enzyme and co-factor degradation.
Jordan
C G
CG
Department of Pharmaceutical Chemistry, School of Pharmacy, University of Bradford, West Yorkshire, UK.
Brown
K
K
Beedham
C
C
Brown
J E
JE
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D002927:Cimetidine / Q000494:pharmacology; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D005978:Glutathione / Q000494:pharmacology; D066298:In Vitro Techniques; D008099:Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D051379:Mice; D009249:NADP / Q000494:pharmacology; D051381:Rats; D019810:Sodium Azide / Q000494:pharmacology; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000378:metabolism*; D013997:Time Factors
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D013629:Tamoxifen; C016601:afimoxifene; D009249:NADP; C035830:tamoxifen N-oxide; D002927:Cimetidine; D019810:Sodium Azide; D005978:Glutathione; C028787:N-desmethyltamoxifen; D001205:Ascorbic Acid