Porphyrinogenic xenobiotic-induced N-alkylprotoporphyrin IX formation in single cDNA-expressed rat and human P450 enzymes: Detection using ferrochelatase as a bioassay and by fluorometry.

摘要

The porphyrinogenicity of certain xenobiotics depends on mechanism-based inactivation of selected P450 enzymes, with subsequent formation of N-alkylprotoporphyrins IX (N-alkylPPs). To investigate the porphyrinogenicity of select xenobiotics in humans, and to assess the validity of an animal model for predicting human xenobiotic-induced porphyria, the overall goal of this thesis was to develop a ferrochelatase (FC)-bioassay for the detection and quantification of N-alkylPPs, and to compare the sensitivity of this method with fluorometry. Subsidiary goals were: (1) To compare the formation of N-alkylPPs in rat P450 enzymes with their human orthologues, and (2) To compare the regioisomers of biological N-methylPP as inhibitors of FC.; The first FC-bioassay utilized purified recombinant human FC and although this method appeared initially to be promising, the purified protein was unstable and an alternative method was investigated. We have demonstrated that although chick embryo hepatic mitochondrial FC is less sensitive than human FC to inhibition by N-alkylPPs it provides a stable source of FC, and can detect as little as 7.2 × 10−3 nmol N-alkylPP. Moreover, this bioassay could distinguish between FC-inhibitory N-alkylPPs and those that are not. This bioassay was more sensitive than UV-Visible spectrophotometry combined with TLC, but less sensitive than fluorometry.; Using fluorometry to detect N-alkylPP formation following mechanism-based inactivation of cDNA-expressed single rat P450 enzymes, N-alkylPP formation was found following interaction of three porphyrinogenic xenobiotics with CYP1A2, 2B1, 2C6, 2C11 and 3A2. When the results were compared to previous results with human orthologues, some results did not correspond, indicating that when xenobiotic-porphyrinogencity depends upon interaction with P450 resulting in N-alkylPP formation, animal studies should be augmented with human P450 preparations.; N-alkylPP formation was compared in microsomes from human lymphoblastoid cell lines and baculovirus-infected insect cells and it was concluded that mechanism-based inactivation and N-alkylPP formation after porphyrinogenic xenobiotic administration should be compared in microsomes from the same cell system.; The N_B regioisomer of N-methylPP was approximately 15-times more potent as an inhibitor of FC than was the N_A regioisomer. The N_D regioisomer was approximately 40-times more potent an inhibitor of FC than was the N_C regioisomer. The greater inhibitory activity of the N_B and N_D, rather than the N_A and N_C regioisomers, indicates that the normal mechanism for FC-catalyzed iron insertion has preference for a B-D ring tilt than an A-C ring tilt.