gestodene and senecionine

The roles of cytochrome CYP3A and CYP2B isozymes in the bioactivation and detoxification of the pyrrolizidine alkaloid (PA) senecionine (SN) have been investigated in vitro with sheep and hamster hepatic microsomes. Our results show that the rate of SN activation measured by (+/-)-6, 7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) formation greatly exceeded the rate of SN N-oxide formation (detoxification) in hamsters. In contrast, SN N-oxide, a detoxification product, was the major metabolite in sheep with much lower DHP production. Immunoinhibition studies with anti-sheep CYP3A and CYP2B antibodies show that members of CYP3A subfamily play the major role in the conversion of PA to pyrrolic metabolites in both species (over 90% in sheep; 68% in hamster). These enzymes also contribute 38.8 and 41. 3% of SN N-oxidation in sheep and hamsters, respectively. In contrast, CYP2B isoforms have a limited capacity toward DHP formation in both species (47% in sheep; 32% in hamster), while these enzymes catalyzed only 24.6 and 35.4% SN N-oxidation in sheep and hamster, respectively. Using triacetyloleandomycin (TAO) and gestodene, two highly selective chemical inhibitors of CYP3A isoforms, our data show that 90% of DHP formation was inhibited by either inhibitor in sheep. Gestodene appeared to be more efficient than TAO in the inhibition of DHP production in hamsters. Testosterone 6beta-hydroxylase activity, a functional marker of CYP3A, was significantly inhibited by TAO and gestodene in sheep liver microsomes and by gestodene (100 microM) in hamster liver microsomes. These results suggest that CYP3A isozymes have important roles in bioactivation and detoxification of PA in both species, whereas CYP2B subfamily members are less efficient in biotransformation of PA.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Biotransformation; Cricetinae; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Enzyme Inhibitors; Immunochemistry; In Vitro Techniques; Inactivation, Metabolic; Isoenzymes; Male; Microsomes, Liver; Norpregnenes; Oxidoreductases, N-Demethylating; Pyrrolizidine Alkaloids; Sheep; Testosterone; Troleandomycin

Studies were carried out to investigate the metabolism of senecionine by human liver microsomes and the role of human cytochrome P450IIIA4 in this process. Human liver microsomes metabolized senecionine to two major products, (+/-)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) and senecionine N-oxide. The rates of product formation (DHP and senecionine N-oxide) varied widely with the microsomal samples tested. There was a 30-fold difference in DHP formation and a 25-fold difference in N-oxidation between the poorest metabolizer and the highest metabolizer of senecionine. The conversion of senecionine to DHP and senecionine N-oxide in human liver microsomes was markedly inhibited by the mechanism-based inactivators of P450IIIA4, gestodene and triacetyloleandomycin. Anti-P450IIIA4 IgG, at a concentration of 1 mg/nmol of P450, was found to inhibit completely the formation of DHP and senecionine N-oxide in human liver microsomes (HL101) having low activity toward senecionine. At 5 mg IgG/nmol P450, anti-P450IIIA4 inhibited 90 and 84% respectively of the formation of DHP and senecionine N-oxide in liver microsomes (HL110) with the highest activity toward senecionine. The formation of DHP or senecionine N-oxide was highly correlated with the amount of P450IIIA4 measured in the microsomes using polyclonal anti-P450IIIA4 IgG. The rate of DHP production also had a strong correlation with the rate of senecionine N-oxide formation (r = 0.999) and with the rate of nifedipine oxidation (r = 0.998). Our present studies provide evidence that P450IIIA4 is the major enzyme catalyzing the bioactivation (DHP formation) and detoxication (senecionine N-oxide formation) of senecionine in human liver.

Topics: Antineoplastic Agents, Phytogenic; Carcinogens; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Humans; Immunoglobulin G; Kinetics; Liver; Microsomes, Liver; Mixed Function Oxygenases; Monocrotaline; Norpregnenes; Oxidation-Reduction; Pyrrolizidine Alkaloids; Troleandomycin