Identification of Human Hepatic Cytochrome P450 Sources of N-alkylprotoporphyrin IX after Interaction with Porphyrinogenic Xenobiotics, Implications for Detection of Xenobiotic-Induced Porphyria in Humans

Abstract

ABSTRACT:Porphyrinogenicity of certain xenobiotics depends upon mechanism-based inactivation of specific cytochrome P450 (P450) enzymes, followed by formation of N-alkylprotoporphyrin IX (N-alkylPP). Examination of the porphyrinogenicity of xenobiotics in animals and extrapolation of the results to humans is associated with ambiguity due, in part, to differences between P450 enzymes. The goal of this study was to develop an in vitro test for the detection of N-alkylPPs, produced in human liver after administration of xenobiotics found to be porphyrinogenic in animals. This goal was achieved using fluorometry to detect N-alkylPP formation following mechanism-based inactivation by porphyrinogenic xenobiotics of single cDNA-expressed human P450 enzymes in microsomes prepared from baculovirus-infected insect cells ( Xenobiotics which interfere with control of heme biosynthesis and allow porphyrins to accumulate are referred to as porphyrinogenic. The porphyrinogenic effects of several xenobiotics (e.g., TTMS 1 , 4-ethyl DDC, and AIA) depend on their ability to cause mechanismbased inactivation of P450 enzymes (Ortiz de Montellano et al., 1981a;Ortiz de Montellano and Grab, 1986; Oritz de Montellano and Mico, 1981c). These xenobiotics, upon selective P450 enzyme mediated biotransformation, form reactive intermediates at the P450 active site. The reactive intermediates interact with the heme moiety resulting in N-alkylation of a pyrrole ring (Marks et al., 1988;Ortiz de Montellano and Correia, 1995). P450 inactivation and dissociation of N-alkylheme is followed by loss of iron, yielding N-alkylprotoporphyrin IX (N-alkylPP), some of which are potent inhibitors of ferrochelatase (EC 4.99.1.1) (De Matteis et al., 1980;Cole et al., 1981). Ferrochelatase inhibition results in decreased heme production and less control over the rate-limiting enzyme aminolevulinic acid synthase (EC 2.3.1.37; aminolevulinic acid synthase). Increased aminolevulinic acid synthase activity results in porphyrin accumulation and porphyria (De Matteis and Marks, 1996). The N-alkylPP formed after mechanism-based inactivation of P450 by AIA, viz. N-AIAPP 2 , does not inhibit ferrochelatase. The porphyrinogenic effect of AIA is explained as follows: since P450 can be reconstituted after undergoing mechanism-based inactivation by AIA as a result of the exchange of the N-alkylated heme moiety (N-AIAPP) for a fresh heme molecule, AIA functions as a heme-destructive catalyst that depletes regulatory free heme (Ortiz de Montellano et al., 1985).The porphyrinogenicity of TTMS, 4-ethyl DDC, and AIA, has been shown in animals to be dependent on mechanism-based inactivation of specific P450 enzymes (Marks et al., 1988). Our previous in vivo studies have elucidated the important rat liver P450 enzymes responsible for N-alkylPP formation. CYP3A2 is the major source of Nvinylprotoporphyrin IX (N-vinylPP) after the administration of TTMS to rats whereas CYP2C11 is the major source of N-ethylprotoporphyrin IX (N-ethylPP) and N-AIAprotoporphyrin IX (N-AIAPP) after...

Comparison of the Formation of N-Alkylprotoporphyrin IX after Interaction of Porphyrinogenic Xenobiotics with Single cDNA-Expressed Human P450 Enzymes in Microsomes Prepared from Baculovirus-Infected Insect Cells and Human Lymphoblastoid Cell Lines+

“…The hypothesis to be tested was that N-alkylPP formation following interaction of porphyrinogenic xenobiotics with single cDNA-expressed human P450 enzymes in microsomes from HLCL would Table 1). The LLD for N-alkylPP detection by fluorometry was found previously in our laboratory to be 0.04 nmol/2 ml of DCM (Lavigne et al, 2002). This LLD would apply equally to studies of N-alkylPP formation determined by fluorometry in microsomes from HLCL and BIICL.…”

“…Some of the results obtained were unexpected. Thus, while mechanism-based inactivation of CYP2D6, 2C9, and 3A4 was not observed after interaction of AIA and NADPH with microsomes prepared from HLCL (McNamee et al, 1997), considerable amounts of N-AIAPP were formed after interaction of AIA and NADPH with Supersomes (Lavigne et al, 2002). A possible explanation for these unexpected results was the difference between the microsomes obtained from HLCL and BIICL (e.g., a difference in the P450 to P450 reductase ratio).…”

“…The DCM solution was evaporated to dryness under a stream of nitrogen, and the Zn N-alkylPP dimethyl ester was redissolved in DCM (2.0 ml). The amount of Zn N-alkylPP dimethyl ester formed was subsequently estimated via fluorometry using an excitation wavelength of 432 nm and measuring the peak height of the major emission band at 650 to 660 nm (Lavigne et al, 2002). Peak heights obtained from control experiments in which NADPH was omitted were subtracted.…”

“…In view of the fact that these microsomes contained considerably higher amounts of P450 than the microsomes from the lymphoblastoid cells, we elected to use Supersomes to enhance the potential for formation of N-alkylPP from the heme moiety of P450. When Supersomes (containing CYP1A2, 2C9, 2D6, or 3A4) were incubated with TTMS, 4-ethylDDC, and AIA, plus NADPH, N-alkylPP formation was quantitated using the sensitive technique of fluorometry (Lavigne et al, 2002). Some of the results obtained were unexpected.…”

“…It was anticipated that N-ethylPP would be formed since N-ethylPP formation was observed with Supersomes containing either CYP1A2 (ratio of P450:NADPH-P450 reductase, 1:10.4) or 2C9 (ratio of P450:NADPH-P450 reductase, 1:0.7) after 4-ethylDDC treatment. However, following incubation less than 0.04 nmol of N-ethylPP was formed where 0.04 nmol is the lower limit of detection (LLD) (Lavigne et al, 2002), and this unexpected result was explained as follows: although human liver microsomes (HG56) contained elevated levels of CYP1A2 and 2C9, CYP3A4 also constitutes 12.5% of the total P450. Correia et al (1987) have shown that 4-ethylDDC caused mechanism-based inactivation of rat CYP3A2, which is not accompanied by N-ethylPP formation.…”