Introduction

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Several xenobiotics interfere with heme biosynthesis, and result in the accumulation of porphyrins and other heme precursors producing a condition known as experimental porphyria (De Matteis and Marks, 1996). These xenobiotics include dihydropyridines, dihydroquinolines, and sydnones, which cause mechanism-based inactivation of selected hepatic cytochrome P450 isozymes, loss of iron from the heme moiety, and formation of N-alkylprotoporphyrins (N-alkylPPs)¹ by covalent binding of an alkyl group to one of the nitrogen atoms of protoporphyrin IX. This results in the formation of four biological regioisomers of N-methylprotoporphyrin IX (N-methylPP; Ortiz de Montellano et al., 1981). A mixture of N-alkylPP regioisomers produced chemically are termed synthetic N-alkylPPs to distinguish them from biologically derived N-alkylPPs. N-alkylPPs such as N-methylPP are potent inhibitors of ferrochelatase (FC) and result in the accumulation of protoporphyrin IX (Tephly et al., 1979; Ortiz de Montellano et al., 1980).

Although there are differences in the potency of various N-alkylPPs as inhibitors of FC, the four regioisomers of synthetic N-methylPP have been shown to inhibit chick embryo and rat liver FC with equal potency (Ortiz de Montellano et al., 1980). This comparison of regioisomer potency was carried out using crude chick embryo or rat liver enzyme preparations, and this work may have been compromised because of the possibility of the binding of the N-alkylPPs to constituents in the crude protein preparations other than FC. The first objective of this study was, therefore, to examine the potency of N-methylPP as an inhibitor of purified recombinant chicken FC, and compare the potency with that previously observed using crude chick embryo liver FC.

Due to sequence variations between the FC of different species, it is possible that differences exist between the active sites of human FC and the FCs of other species. If this is true, there may also be differences in the potencies of N-methylPPs as inhibitors of human FC and the FCs of other animal species, in particular, the chicken, which has been widely used to test the porphyrinogenicity of chemicals. If such differences in potency exist, then extrapolating porphyrinogenicity data from animal test species to humans would be problematic. Thus, the second objective of this study was to compare the potency of N-methylPP between purified recombinant human FC and purified recombinant chicken FC preparations.

Materials and Methods

Sources of Compounds. Protoporphyrin IX and mesoporphyrin IX were obtained from Porphyrin Products (Logan, UT). Iodomethane and iodoethane were obtained from Sigma Chemical Co. (St. Louis, MO). The HisTrap Kit and low-molecular-weight protein calibration kit were obtained from Amersham Pharmacia Biotech (Baie d'Urfé, PQ).

Preparation and Purification of N-Methylprotoporphyrin. N-methylPP was synthesized according to a procedure developed by De Matteis et al. (1980). Protoporphyrin IX dimethyl ester (3.2 mg) was reacted with methyl iodide (2 ml) for 4 h at 108°C in a sealed reaction vessel. The crude mixture of N-methylPP regioisomers was purified by thin layer chromatography as described previously (Kimmett et al., 1992). The N-methylPP dimethyl esters were converted to the free acid form by hydrolysis in 300 µl of 6.0 N hydrochloric acid overnight, in the dark, at room temperature (Ortiz de Montellano et al., 1979). After removal of the hydrochloric acid under a stream of nitrogen, the N-methylPP was dissolved in 95% ethanol for use in the FC activity assay.

Purification of Human and Chicken FC. Recombinant human FC was expressed in E. coli and purified as described previously (Burden et al., 1999). The cloning, expression, and purification of recombinant chicken FC has been described previously (Day et al., 1998). The purification of FC was confirmed using SDS-polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining to identify a single band at the appropriate molecular mass and by means of the FC assay described below.

Determination of FC Inhibitory Activity of N-MethylPP. Aliquots of purified FC (0.9 ml) and N-methylPP (0.1 ml) were added to the sidearm of Thunberg tubes. The body of the Thunberg tubes contained mesoporphyrin IX (120 nmol), 1% w/v Tween 80 (0.3 ml), 95% ethanol (0.3 ml), 0.2 M Tris-HCl buffer pH 8.2 (1.5 ml), 0.2 M dithioerythritol (60 µl), and 1.0 mM ferrous sulfate (120 µl). The assay was conducted as described previously (Porra and Jones, 1963; Cole et al., 1979). The EC₅₀ values for the inhibition of FC by N-methylPP were derived using curve-fitting analysis using GraphPad Prism 3.0. The EC₅₀ value for each experiment was determined separately, and a Student's t test was performed on the means of the EC₅₀ determinations (P < .05). Protein was measured by the method of Lowry et al. (1951).

	Results and Discussion

Top Abstract Introduction Results and Discussion References

The total activity found in the 100,000g supernatant (3.5 ml) of human FC was 2760 nmol mesoheme formed/10 min, and the specific activity was found to be 238 nmol mesoheme formed/mg protein/10 min. On purification on the HisTrap column, the first three 1-ml fractions were pooled and had a total activity of 64.2 nmol mesoheme formed/10 min. The specific activity increased to 3622 nmol mesoheme formed/mg protein/10 min (15-fold purification). In the case of chicken FC, the total activity found in the 100,000g supernatant was 2486 nmol mesoheme formed/10 min, and the specific activity was found to be 214 nmol mesoheme formed/mg protein/10 min. After purification with the HisTrap column, the first three 1-ml fractions were pooled and had a total activity of 99.6 nmol mesoheme formed/10 min. The specific activity increased to 2849 nmol mesoheme formed/mg protein/10 min (12-fold purification). Aliquots from 1-ml eluates collected from the HisTrap column were applied to a SDS gel, which was stained with Coomassie Brilliant Blue. A prominent band was shown to be present with a molecular mass slightly greater than 43 kDa. This was inferred to be FC because human FC has been shown to have a molecular mass of 43 kDa (Dailey et al., 1994a). This inference was confirmed as FC activity resided exclusively in this fraction. Previously, the specific enzyme activity measured in homogenates of chick embryo liver cells grown in monolayer cell cultures was 2.5 to 3 nmol mesoheme/mg protein/10 min (Cole et al., 1982; McCluskey et al., 1989). The purity of both human and chicken FC preparations obtained from E. coli is thus approximately 1000 times higher than that observed previously in the crude chick embryo FC preparation.

The first objective of this study was to examine the potency of N-methylPP as an inhibitor of purified chicken FC and compare the potency with that observed previously in crude chick embryo liver FC. The results of inhibition of purified chicken FC by a range of doses of N-methylPP are shown in Fig. 1, and an EC₅₀ value of 2.07 × 10³ nmol/mg protein was found. This result was not statistically significantly different from the EC₅₀ value of 2.9 × 10³ nmol/mg protein reported previously in our laboratory for the inhibition of chick embryo liver FC by N-methylPP (Ortiz de Montellano et al., 1980). Therefore, possible interaction between N-methylPP and impurities in a crude FC preparation from chick embryo liver cell culture do not appear to have played an important role, as it is clear that the relative purity of the enzyme preparation has no measurable effect on potency determination for synthetic N-methylPP.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Comparison of the inhibition of purified chicken FC () and purified human FC () by different concentrations of N-methylPP. Each point represents the mean (± S.D.) of results obtained from at least three separate experiments.

Differences exist in the amino acid sequences of FCs among animals commonly used for testing xenobiotics for porphyrinogenicity, and between the FCs of these animals and humans. These differences may be reflected in differences among the active sites of the various FCs. Thus, differences may exist in the potency of N-methylPP as an inhibitor of FC. Because such variations may cause differences in xenobiotic-induced porphyrinogenicity among animal species and humans, it may complicate the extrapolation of data from animal test models to humans. The second objective of this study was to compare the potency of N-methylPP between purified human FC and purified chicken FC preparations. A comparison between the potency of N-methylPP as an inhibitor of purified human FC and purified chicken FC is shown in Fig. 1. The EC₅₀ value for purified human FC was 1.7 × 10⁶ nmol/mg protein, whereas the EC₅₀ value for purified chicken FC was 2.07 × 10³ nmol/mg protein. Thus, N-methylPP was approximately 1218 times more potent as an inhibitor of human FC as compared with chicken FC. These results show that human FC is much more sensitive to inhibition by N-methylPP than is chicken FC, and suggests that results obtained using chickens, 17-day old chick embryos, and the chick embryo liver cell culture system (McCluskey et al., 1989) may underestimate the porphyrinogenicity of drugs that owe their activity to N-methylPP formation. Human FC is also much more sensitive to inhibition by N-methylPP than is rat FC because previous results show that the EC₅₀ value of N-methylPP as an inhibitor of rat liver mitochondrial FC was 4 × 10³ nmol/mg protein (Ortiz de Montellano et al., 1980).

The above results suggest that an improved method for testing xenobiotics, such as dihydropyridines, sydnones, or dihydroquinolines, which owe their porphyrinogenicity in test animals to mechanism-based inactivation of cytochrome P450 and to FC inhibitory N-alkylPP formation, is the following: The xenobiotics would be added to human liver microsomes and mechanism-based inactivation and N-alkylPP formation assessed. This would be followed by assessing FC inhibitory activity of the N-alkylPPs in human FC preparations.