Journal of Pharmacological and Toxicological Methods
Original articlesFormation of N-alkylprotoporphyrin IX after interaction of porphyrinogenic xenobiotics with rat liver microsomes
Introduction
Several potent porphyrinogenic xenobiotics disrupt the heme biosynthetic pathway through mechanism-based cytochrome P450 (CYP) inactivation. During this process, these xenobiotics are converted within the CYP active site to highly reactive species that can alkylate one of the four pyrrole nitrogens of the heme moiety (for reviews see Marks et al. 1988, Ortiz de Montellano & Correia 1996). The modified heme, referred to as N-alkylprotoporphyrin IX (N-alkylPP; Fig. 1), dissociates from the CYP apoprotein and is a potent inhibitor of the terminal enzyme in heme biosynthesis, ferrochelatase Marks et al. 1985, McCluskey et al. 1986. Ferrochelatase inhibition leads to decreased heme production, elevation of aminolevulinic acid synthase (ALA-S) activity, accumulation of porphyrins and other heme precursors, and eventually porphyria (for a review see De Matteis & Marks, 1996).
3-[(arylthio)ethyl]sydnone (TTMS) (Fig. 2a) and 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethylDCC) (Fig. 2b) are potent porphyrinogenic xenobiotics capable of eliciting mechanism-based CYP inactivation, N-alkylPP formation, and subsequent ferrochelatase inhibition in rats Sutherland et al. 1986, Ortiz de Montellano & Grab 1986, Tephly et al. 1979, De Matteis et al. 1980, Riddick et al. 1990. Allylisopropylacetamide (AIA) (Fig. 2c) also elicits N-alkylPP formation; however, the heme adduct, N-AIA protoporphyrin IX (N-AIAPP) is unable to inhibit ferrochelatase. Dissociation of N-AIAPP from the CYP enzyme leaves the apoprotein intact and capable of reconstitution with heme (Bornheim et al., 1987). Continued mechanism-based inactivation and heme reconstitution leads to heme depletion and porphyria De Matteis et al. 1980, Ortiz de Montellano & Mico 1981b. 1-Aminobenzotriazole (ABT) (Fig. 2d) is a nonselective mechanism-based CYP inactivator that produces an abnormal porphyrin, N′N′aryl bridged protoporphyrin IX (NNAPP), but does not elicit experimental porphyria Ortiz de Montellano & Mathews 1981a, Ortiz de Montellano et al. 1981c. Previous studies in our laboratory have shown that the important rat CYP isozymes responsible for N-alkylPP formation, and thus the critical mediators of toxicity are different for TTMS, 4-ethylDDC, AIA, and ABT Wong et al. 1999, Wong et al. 1998, McNamee & Marks 1996. CYP3A is the major source of N-vinylPP after administration of TTMS to rats whereas CYP2C11 is the major source of N-ethylPP and N-AIAPP from 4-ethylDDC and AIA, respectively. ABT, on the other hand, showed no preference for a particular CYP isozyme for N-alkylation and NNAPP formation. Considering the involvement of specific CYP isozymes in xenobiotic-induced porphyria, species differences in CYP isozyme expression are particularly relevant when using animal models to predict the potential porphyrinogenicity of compounds in humans. During routine toxicity testing, when drug-induced porphyria in animals is found to be dependent on N-alkylPP formation from a CYP isozyme, it is necessary to determine whether this isozyme or a closely related isozyme found in humans also produces N-alkylPPs. Thus, a thorough understanding of the human and animal CYP isozymes responsible for N-alkylPP formation is necessary in order to establish whether porphyria elicited in animals by mechanism-based CYP inactivation, accompanied by N-alkylPP formation, is likely to be a problem in humans.
Previously, we have used microsomes prepared from human lymphoblastoid cell lines containing single cDNAexpressed human CYP isozymes (Gentest Corp, Woburn, MA) to determine which human CYP isozymes are susceptible to mechanism-based inactivation after administration of several porphyrinogenic drugs (McNamee et al., 1997). Because hepatic porphyrin accumulation will result only after N-alkylation of the CYP heme moiety, it is important to determine which of the CYP isozymes targeted for mechanism-based inactivation also elicit N-alkylPP formation. Our long-term objective is to be able to use these human lymphoblastoid microsomes to determine which CYP isozymes are involved in N-alkylPP formation and thus to develop a direct test of xenobiotic porphyrinogenicity in humans. In order to achieve this long-term goal, several preliminary studies are required. Previous studies in which N-alkylPPs have been isolated from liver have involved in vivo administration of porphyrinogenic xenobiotics to rats. The first objective of this study was to determine if N-alkyl-PPs are formed on interaction of porphyrinogenic xenobiotics with rat liver microsomes in vitro. In order to determine whether isolation of N-alkylPPs from in vitro preparations containing human hepatic CYP isozymes was feasible for future study, the second objective was to determine the amount of N-alkylPPs formed per nmol of CYP.
Section snippets
Reagents and chemicals
TTMS, 4-ethylDDC, and ABT were obtained from Colour Your Enzyme (Bath, Ontario, Canada). AIA was obtained as a gift from Hoffman-La Roche Ltd. (Vaudreuil, Quebec, Canada). Solvents (methanol, dichloromethane, and acetone) were purchased from VWR Canada (Mississauga, Ontario, Canada). Inhibitors (TAO) and inducers (DEX, and PB) were purchased from Sigma Chemical Co. (St. Louis, MO).
Treatment of animals
Male Sprague Dawley rats (250 to 300 g) were obtained from Charles River Canada, Inc. (St. Constant, Quebec,
N-alkylPP formation after incubation of rat hepatic microsomes with TTMS, 4-ethylDDC, AIA, or ABT
Because the heme moieties of selective CYP isozymes are converted to N-alkylPPs, microsomes were isolated from rats which were pretreated with inducers that selectively increase CYP isozyme levels. Thus, expression of CYP3A, CYP1A1/2, or CYP2B1/2 was increased by administering DEX, βNF, or PB to rats to increase N-alkylPP formation elicited by TTMS, 4-ethylDDC, and AIA, respectively McNamee et al. 1997, Wong et al. 1998, Wong et al. 1999. Total CYP content in the induced rats was determined to
Acknowledgements
Supported by the Medical Research Council of Canada.
References (23)
- et al.
Induction of the male-specific cytochrome P450 3A2 in female rats by phenytoin
Arch Biochem Biophys
(1996) - et al.
N-alkylprotoporphyrin IX Formation in 3,5-Dicarbethoxy-1,4-dihydrocollidine-treated Rats. Transfer of the alkyl group from the substrate to the porphyrin
J Biol Chem
(1981) - et al.
Porphyrinogenic activity and ferrochelatase-inhibitory activity of sydnones in chick embryo liver cells
FEBS Lett
(1986) - et al.
Inactivation of multiple hepatic cytochrome P-450 isozymes in rats by allylisopropylacetamidemechanistic implications
Mol Pharmacol
(1987) - et al.
Substrate-dependent irreversible inactivation of cytochrome P-450conversion of its haem moiety into modified porphyrins
Ciba Foundation Symposium
(1980) - et al.
Cytochrome P450 and its interactions with the heme biosynthetic pathway
Can J Physiol Pharmacol
(1996) - et al.
Suicidal destruction of cytochrome P-450 and reduction of ferrochelatase activity by 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine and its analogues in chick embryo liver cells
Mol Pharmacol
(1985) - et al.
Disruption of hepatic heme biosynthesis after interaction of xenobiotics with cytochrome P-450
FASEB J
(1988) - et al.
Ferrochelatase-inhibitory activity and N-alkylprotoporphyrin formation with analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) containing extended 4-alkyl groupsimplications for the active site of ferrochelatase
Mol Pharmacol
(1986) - et al.
Cytochrome P4503A is the major source of N-vinylprotoporphyrin IX formation after administration of 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone to untreated and dexamethasone-pretreated rats
Drug Metab Dispos
(1996)
cDNA-expressed human cytochrome P450 isozymes. Inactivation by porphyrinogenic xenobiotics
Drug Metab Dispos
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