A CYTOCHROME P4502B FORM IS THE MAJOR BIOACTIVATION ENZYME FOR THE PYRROLIZIDINE ALKALOID SENECIONINE IN GUINEA-PIG

1. We have purified three P450s from the liver of the phenobarbital (P B)-treated guinea pig in order to evaluate the role of these enzymes i n pyrrolizidine alkaloid (PA) metabolism. 2. PB treatment of guinea pi g increased the hepatic microsomal conversion of the PA senecionine (S N) to the pyrrolic metabolite 7-dihydro-7-hydroxy-1-hydroxymethyl-5H-p yrrolizine (DHP), an activation product, and SN N-oxide, a detoxificat ion product by 224 and 70% respectively. 3. Reconstitution of a PR-ind ucible guinea pig P4502B isoform ((M(r) = 57512 by MALDI-TOF mass spec trometry) in a reconstituted system metabolized SN to DHP and SN N-oxi de at rates of 1.98 and 1.45 min(-1) respectively. A second purified g uinea pig P450, a 2C-type isoform (M(r) = 56496 by MALDI-TOF mass spec trometry), produced SN N-oxide from SN at the rate of 13.3 min(-1) but catalyzed little DHP formation. The third guinea pig P450, an apparen t 3A type (M(r) = 54-56 000 by SDS-PAGE), lost its catalytic activity towards SN during the final purification process. 4. Immunoinhibition of microsomal SN metabolism by rabbit antibodies raised against the gu inea pig P4502B, 2C and 3A isoforms indicated that the 2B played the m ost important role (> 70% of the total metabolism) in bioactivation of SN in both the untreated or PR-treated guinea pig, whereas 2C and 3A seemed to exhibit little (around 13%) PA metabolism. P4502B, along wit h flavin-containing monooxygenase, also contributed to the detoxificat ion of SN in both the untreated (34%) and PB-treated (40%) guinea pig. 5. This study suggests that the putative P4502B form plays the most i mportant role in SN bioactivation in guinea pig.