Tolbutamide hydroxylation by hepatic microsomes from Atlantic salmon (Salmo salar L.).
Metabolic transformations of two substrates for human cytochrome P450 (CYP450) 2C9, tolbutamide and diclofenac, were investigated in hepatic microsomes from Atlantic salmon (Salmo salar L.). Tolbutamide hydroxylation followed Michaelis-Menten kinetics. Mean apparent Michaelis-Menten constant (K(m)) and maximum reaction velocity (V(max)) values for 4-hydroxytolbutamide (TBOH) formation were 0.09 ± 0.031 mM and 49.5 ± 6.03 pmol/min/mg, respectively. Addition of sulfaphenazole, an inhibitor for mammalian CYP2C9, in a range from 1 to 200 μM decreased formation of TBOH in a concentration-dependent manner, but not to 50%. Neither fluconazole, an inhibitor of human CYP2C9, nor ketoconazole, inhibitor of CYP1A and CYP3A in fish, affected TBOH formation. In contrast ellipticine, an inhibitor of CYP1A in fish inhibited TBOH formation with the IC(50) value of 12.1 μM. The rate of TBOH formation was competitively inhibited by 100 μM of sesamin in the incubations, but the degree of inhibition did not increase with increased sesamin concentration. Ethoxyresorufin hydroxylase (EROD) activity was inhibited by tolbutamide in a non-competitive manner (inhibition constant K(i) = 218 μM). Our data suggest that tolbutamide is metabolized by salmon microsomes with formation of TBOH. CYP1A might be involved in this reaction as suggested by decreased TBOH formation in the presence of ellipticine and decreased EROD activity in the presence of tolbutamide. Incubation of diclofenac with the microsomes yielded no metabolite formation, suggesting that salmon does not possess diclofenac-metabolizing activity.
Department of Food Science, BioCenter, Swedish University of Agricultural Sciences, Uppsala, Sweden.
AnnaLotta Schiller
AS
Vestergren
Vladimir
V
Zlabek
Jana
J
Pickova
Galia
G
Zamaratskaia
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000378:metabolism; D001711:Biotransformation; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D065692:Cytochrome P-450 CYP3A Inhibitors; D004008:Diclofenac / Q000378:metabolism; D004149:Dioxoles / Q000494:pharmacology; D004611:Ellipticines / Q000494:pharmacology; D029941:Fish Proteins / Q000378:metabolism; D015725:Fluconazole / Q000494:pharmacology; D006900:Hydroxylation; D007654:Ketoconazole / Q000494:pharmacology; D007700:Kinetics; D017705:Lignans / Q000494:pharmacology; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D010078:Oxazines / Q000378:metabolism; D020317:Salmo salar; D013426:Sulfaphenazole / Q000494:pharmacology; D014044:Tolbutamide / Q000378:metabolism* / Q000494:pharmacology
D065692:Cytochrome P-450 CYP3A Inhibitors; D004149:Dioxoles; D004611:Ellipticines; D029941:Fish Proteins; D017705:Lignans; D010078:Oxazines; D013426:Sulfaphenazole; C034192:ellipticine; D004008:Diclofenac; C007649:ethoxyresorufin; D015725:Fluconazole; D014044:Tolbutamide; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D007654:Ketoconazole; C054125:sesamin