A cytochrome P4502B form is the major bioactivation enzyme for the pyrrolizidine alkaloid senecionine in guinea pig

Abstract

1. We have purified three P450s from the liver of the phenobarbital (PB)-treated guinea pig in order to evaluate the role of these enzymes in pyrrolizidine alkaloid (PA) metabolism.

2. PB treatment of guinea pig increased the hepatic microsomal conversion of the PA senecionine (SN) to the pyrrolic metabolite (±)6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H/-pyrrolizine (DHP), an activation product, and SN N-oxide, a detoxification product by 224 and 70% respectively.

3. Reconstitution of a PB-inducible guinea pig P4502B isoform (M_r = 57512 by MALDI-TOF mass spectrometry) in a reconstituted system metabolized SN to DHP and SN N-oxide at rates of 1.98 and 1.45 min^-1 respectively. A second purified guinea pig P450, a 2C-type isoform (M_r = 56 496 by MALDI-TOF mass spectrometry), 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 apparent 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 guinea pig P4502B, 2C and 3A isoforms indicated that the 2B played the most important role (>70% of the total metabolism) in bioactivation of SN in both the untreated or PB-treated guinea pig, whereas 2C and 3A seemed to exhibit little (around 13%) PA metabolism. P4502B, along with flavin-containing monooxygenase, also contributed to the detoxification 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 important role in SN bioactivation in guinea pig.