Introduction

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Porphyrinogenic xenobiotics are compounds that interfere with heme biosynthesis, resulting in an accumulation of porphyrins and other heme precursors and producing a condition known as experimental porphyria (De Matteis and Marks, 1996). A variety of these agents, including dihydropyridines, dihydroquinolines, and sydnones, cause mechanism-based inactivation of selected hepatic cytochrome P450 (P450)¹ isozymes, which is a critical event in their porphyrinogenicity (De Matteis and Marks, 1996; McNamee and Marks, 1996; Riddick et al., 1989; Coffman et al., 1982; Ortiz de Montellano et al., 1981a; Riddick et al., 1990; Marks et al., 1988). Mechanism-based P450 inactivation can involve three distinct mechanisms: (a) N-alkylation of the heme moiety, resulting in N-alkylprotoprophyrin IX (N-alkylPP) formation, (b) alkylation of the apoprotein, or (c) conversion of the heme moiety to products that covalently bind the heme apoprotein (Halpert et al., 1994). N-AlkylPPs (fig. 1) are potent inhibitors of the terminal enzyme in heme biosynthesis (ferrochelatase, EC 4.99.1) (Marks et al., 1985; McCluskey et al., 1986; McCluskey et al., 1988; Ortiz de Montellano et al., 1980; Ortiz de Montellano et al., 1981c; Tephly et al., 1979) and only when mechanism-based P450 inactivation is accompanied by N-alkylPP formation does inhibition of ferrochelatase occur, leading to elevation of hepatic protoporphyrin IX (De Matteis and Marks, 1996).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Structure of an N-alkylprotoporphyrin IX. The NA regioisomer is shown. For N-ethylPP, R = -CH2-CH3. For N-vinylPP, R = -CH=CH2.

Development of the potent anti-arthritic compound 3-[(arylthio)ethyl]sydnone (TTMS) was abandoned when it was shown to cause hepatic protoporphyrin IX accumulation in dogs and rodents (Stejskal et al., 1975). These observations were attributed to mechanism-based inactivation of P450 with concurrent N-vinylPP formation, inhibition of ferrochelatase, and an induction of aminolevulinic acid synthase activity, all of which are hallmarks of experimental porphyria (Sutherland et al., 1986; Ortiz de Montellano and Grab, 1986). Studies in our laboratory indicated that P4501A, 3A, 2B1, 2C6, and 2C11 were targeted for mechanism-based inactivation by TTMS (McNamee and Marks, 1996). Because only those isozymes undergoing N-alkylation would contribute to the porphyrinogenicity of TTMS, our next studies were directed to determining the P450 isozyme or isozymes responsible for N-vinylPP production. This goal was pursued by comparing selective P450 isozyme induction and inhibition with hepatic N-vinylPP production in rats. In untreated (UT) rats, co-administration of troleandomycin (TAO), a selective P4503A inhibitor (Chang et al., 1994), with TTMS resulted in a 66% decrease in N-vinylPP formation (McNamee and Marks, 1996). Conversely, induction of P4501A with NF did not affect N-vinylPP production. On the basis of the above induction and inhibition studies, P4503A was identified as the major source of N-vinylPP production, and P4501A was eliminated as a possible source of N-vinylPP.

Assuming that P4503A contributed approximately 66% to total N-vinylPP formation, the remaining 34% was considered to originate from the other P450 isozymes that are mechanistically inactivated by TTMS, namely P450 2B1, 2C6, and/or 2C11. Limitations in the selectivity of P450 inducers and inhibitors (Halpert, 1995; Roos and Mahnke, 1996), in addition to the poor inducibility of P4502C6 and 2C11 (Guengerich et al., 1982; Waxman et al., 1985; Shimada et al., 1989), prompted the exploration of alternative methods to determine which of these isozymes were quantitatively important for the production of N-vinylPP. We decided on an alternative research plan, which takes advantage of the marked gender difference in P450 isozyme expression in rodents (Cooper et al., 1993; Ghosal et al., 1996; Gonzalez et al., 1986; Waxman, 1988; Zaphiropoulos et al., 1989). Male rat livers contain large amounts of P4503A2 and 2C11, whereas only trace amounts of these isozymes are present in female rat liver (Cooper et al., 1993; Ghosal et al., 1996). Because of these gender differences in P450 isozyme profiles, and because P4503A2 was determined to be a major source of N-vinylPP in male rats, we anticipated marked differences between male and female rats in N-vinylPP production in response to TTMS administration. Our first objective was to use these gender differences in P450 isozyme expression to determine the relative contributions of P4502C11 and 3A2 to the production of N-vinylPP.

The dihydropyridine, 4-ethylDDC, is another classical porphyrinogenic drug for which the mechanism involves the N-alkylation of heme, resulting in the production of N-ethylPPs (Riddick et al., 1990; Ortiz de Montellano et al., 1981a; Ortiz de Montellano et al., 1981b; De Matteis et al., 1980; Tephly et al., 1979). 4-EthylDDC causes mechanism-based inactivation of rat P4501A, 2C6, 2C11, and 3A (Kimmett et al., 1994; Riddick et al., 1989; Tephly et al., 1986; Correia et al., 1987). Our second objective was to determine which of these isozymes, or combination of isozymes, were the sources of N-ethylPP. The experimental procedure employed was to use selective inducers/inhibitors of P450 isozymes, and to compare N-ethylPP formation in response to 4-ethylDDC administration between male and female rats.

	Materials and Methods

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Reagents and Chemicals. TTMS and 4-ethylDDC were obtained from Color Your Enzyme (Bath, Ontario, Canada). Solvents (methanol, dichloromethane, and acetone) were purchased from VWR Canada (Mississauga, Ontario, Canada). Inhibitors (NF and TAO) and inducers (NF, DEX, and PB) were purchased from Sigma Chemical Co. (St. Louis, MO).

Treatment of Animals. For the inducer and inhibitor studies, male Sprague-Dawley rats (200-250g) were obtained from Charles River Canada, Inc. (St. Constant, Quebec, Canada). For the gender studies, male and female rats (150-200 g) were obtained. All rats received Ralston Purina Laboratory Chow (no. 5001; Ren's Feed and Supplies, Ltd., Oakville, Ontario, Canada) and water ad libitum and were housed under controlled conditions (22^oC, 14-hr/10-hr light/dark cycle). Animals were allowed a 1-week environmental adjustment period.

Induction Study. Rats received the appropriate inducer, dissolved in 0.5 ml vehicle, intraperitoneally once daily for either 3 days (NF) or 4 days (DEX and PB). The vehicle for NF (40 mg/kg) and DEX (100 mg/kg) was DMSO:corn oil (1:7 v/v), and for PB (80 mg/kg) it was deionized water. Control animals received DMSO:corn oil (1:7 v/v) once daily for four days.

Inhibitor Studies. The inhibitor TAO (450 mg/kg) was co-administered with TTMS (111.3 mg/kg) or 4-ethylDDC (400 mg/kg) 24 hr after the final dose of DEX or vehicle. -Naphthoflavone (NF) (100 mg/kg) was co-administered with 4-ethylDDC (400 mg/kg) 24 hr after the final dose of NF or vehicle.

Isolation, Purification, and Separation of N-AlkylPP. Four hours after administration of porphyrinogenic compounds, in the presence or absence of inhibitor, rats were decapitated and their livers perfused in situ with 100 ml ice-cold 1.15% potassium chloride solution. Livers were individually weighed and homogenized in ice-cold 5% H₂SO₄:methanol (v/v; 100 ml/liver), and stored in the dark at 4^oC for 24 hr. The resulting N-alkylPP dimethyl ester mixture was filtered, diluted with an equal volume of deionized water, and then extracted with dichloromethane (2 × 30 ml) in a separatory funnel. After successive washes with 5% sodium bicarbonate (80 ml) and deionized water (2 × 80 ml) the dichloromethane solution was dried over anhydrous sodium sulfate. After removal of the sodium sulfate by filtration, zinc acetate (25 µmol) in methanol (2 ml) was added to form the Zn N-alkylPP dimethyl ester, and the solution was evaporated to dryness. The residue was dissolved in dichloromethane and applied to an Analtech silica gel G TLC plate (2,000 µm; Newark, DE) and developed in dichloromethane:methanol (13:2) for 60 min. A single green band (R_f 0.68-0.74) that fluoresced red under long-wavelength UV light was eluted from the plate with acetone and evaporated to dryness. The residue was dissolved in dichloromethane, applied to a second Analtech silica gel G plate (1000 µm) and developed in dichloromethane:acetone (5:1) for 45-50 min. Two green bands, at R_f 0.69-0.72 and R_f 0.75-0.77, that fluoresced red under long wavelength UV light were eluted together, with acetone. The electronic absorption spectrum was determined using an Hewlett-Packard 8451A diode array spectrophotometer (Mississauga, Ontario, Canada). The concentration of the Zn N-alkylprotoporphyrin dimethyl ester was determined using the molar extinction coefficient for Zn N-methylPP dimethyl ester ( = 124,000 m¹cm¹ at 432 nm) (Ortiz de Montellano et al., 1981b).

Statistical Analysis. For the induction study (fig. 3), a randomized design one-way analysis of variance with a Newman-Keul's post hoc test was used to determine if means were significantly different (p < 0.05). For the male-female rat gender studies (fig. 2) an unpaired Student's t test was used to determine if mean values of male and females were significantly different (p < 0.05). For the inhibitor studies with TAO or NF, an unpaired Student's t test was used to determine if the mean data of inhibitor-treated rats was significantly different (p < 0.05) from the control animals receiving no inhibitor.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   N-VinylPP and N-ethylPP formation in male and female rat liver after administration of TTMS and 4-ethylDDC, respectively. Each bar represents the mean (± SD) of determinations from four rats. **, significantly different (p < 0.01) from female rats, as determined by an unpaired Student's t test. *, significantly different (p < 0.05) from female rats, as determined by an unpaired Student's t test.

	Results and Discussion

Top Abstract Introduction Materials & Methods Results & Discussion References

N-VinylPP Production in Male and Female Rats. Previously, when TAO was co-administered with TTMS, N-vinylPP formation was reduced to 34% of control in UT rats, showing that P4503A was the major source of N-vinylPP formation (McNamee and Marks, 1996). It remains to be determined which other P450 isozyme(s) contributed to the formation of N-vinylPP. We hypothesized that, if P4503A2 and 2C11 were major sources of N-vinylPP, female rats would produce much less N-vinylPP than males, because they effectively lack these isozymes (Cooper et al., 1993; Ghosal et al., 1996).

Selective Inhibition of P4503A and N-EthylPP Production. Because selective inhibition of P4503A with TAO reduced N-vinylPP production in male rats (McNamee and Marks, 1996), we hypothesized that the same might be true for N-ethylPP production after administration of 4-ethylDDC. Thus, our first experiments were directed to testing the effects of TAO administration on N-ethylPP production in UT- and DEX-pretreated rats after 4-ethylDDC administration. TAO was unable to significantly effect N-ethylPP production in both UT- and DEX-pretreated rats (data not shown). These results indicate that, unlike TTMS, P4503A does not contribute significantly to N-alkylPP production. The fact that the P4503A isozyme is not an important source of N-ethylPP supports previous results obtained by Correia et al. (Correia et al., 1987). These workers suggested that mechanism-based inactivation of P4503A by 4-ethylDDC is accompanied by conversion of the prosthetic heme to products that irreversibly bind to the apoprotein, rather than to N-ethylPP formation.

Induction Study and N-EthylPP Production. Previous studies have shown that, in addition to P4503A, 4-ethylDDC elicits mechanism-based inactivation of rat P450 1A1/2, 2C6, and 2C11 (Riddick et al., 1990; Riddick et al., 1989; Tephly et al., 1986; Correia et al., 1987). To determine which of these isozyme candidates were sources of the N-ethylPP, the effect of several selective P450 inducers, namely NF, PB, and DEX, on N-ethylPP production was determined, and the results are shown in fig. 3. In untreated rats, P450 1A1/2 represents only 3%-4% of the total P450 content, whereas, in NF-treated animals, the proportions of these isozymes increase to 65%-75% of the total P450 in rat liver (Waxman et al., 1985; Guengerich et al., 1982). Considering that NF selectively induces P450 1A1/2 (Waxman et al., 1985), and was the only inducer in our study that increased the amount of N-ethylPP produced, it was inferred that P450 1A1/2 was/were quantitatively important sources of the N-ethylPP in BNF-treated rats. These results support previous studies (Coffman et al., 1982) indicating that induction of P450 1A1/2 with 3-methylcholanthrene increased N-ethylPP formation in rats after administration of 4-ethylDDC. DEX, which increases the functional activity of P4503A 4.2-fold (Wrighton et al., 1985), did not significantly increase the production of N-ethylPP. This observation is consistent with our results (data not shown) demonstrating the inability of TAO-mediated 3A inhibition to decrease N-ethylPP production, and with Correia's results (Correia et al., 1987) indicating that 4-ethylDDC elicits mechanism-based inactivation of P4503A, resulting in apoprotein alkylation rather than N-alkylation of heme and N-ethylPP formation. PB was also unable to significantly affect the production of N-ethylPP, suggesting that the P450 isozymes which it induces (mainly 2B1/2 (Waxman et al., 1985; Swinney et al., 1987)) were not quantitatively important sources of N-ethylPP.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Effect of pretreatment of rats with NF, DEX, and PB on total N-ethylPP production in rat liver after administration of 4-ethylDDC. Each bar represents the mean (± SD) of determinations from four rats. *, significantly different (p <0.05) from untreated rats, as determined by a randomized design one-way analysis of variance with the Newman-Keul's post hoc test.

NF Inhibition of P450 1A1/2 and N-EthylPP Production. Because selective induction of P450 1A1/2 increased the amount of N-ethylPP after 4-ethylDDC administration, we hypothesized that selective inhibition of these isozymes should correspondingly decrease production of N-ethylPP. NF is a potent and relatively selective inhibitor of P450 1A1/2 (Chang et al., 1994) and, when administered in conjunction with NF, significantly decreased the production of N-ethylPP from 2.49 ± 0.419 nmol/g liver to 0.927 ± 0.600 nmol/g liver, confirming that P450 1A1/2 were important sources of the N-ethylPP in NF-treated rats. However, when NF was administered to non-induced rats concurrently with 4-ethylDDC, N-ethylPP formation was not decreased, compared with rats receiving no inhibitor (results not shown). This observation suggests that P4501A/2 are not major sources of N-ethylPP in non-induced rats, which might be attributed to the relatively low levels of these isozymes, approximately 5%, present in untreated rat liver, compared with 65%-70% in NF-treated rats (Waxman et al., 1985). Additionally, the possibility that competing P450 isozymes in the untreated rat liver may have a higher catalytic efficiency than P4501A1/2 could account for the apparently low contributions of P450 1A1/2 to N-ethylPP formation in untreated rats.

N-EthylPP Production in Male and Female Rats. To examine the roles of P450 2C6 and 2C11 in N-ethylPP formation, 4-ethylDDC was administered to male and female rats, and N-ethylPP was extracted and quantified. Female rats produced 0.081 nmol N-ethylPP/g wet weight liver, which was 22% of the amount produced by male rats, namely 0.3675 nmol N-ethylPP/g wet weight liver (fig. 2). Considering that P4501A1/2 and P4503A2 were excluded as possible sources of the N-ethylPP, and that female rats lack the male-specific isozyme P4502C11 (Morgan et al., 1985; Waxman, 1984), the increased N-ethylPP production seen in male compared with female rats is most likely due to the contributions of P4502C11 in male rat liver. The relatively smaller amount of N-ethylPP that was produced in the female rat liver most likely originated from P4502C6, because 2C11 is absent. Because P4502C6 exists in similar levels in both sexes, we interpreted the N-ethylPP production in females to approximately represent the contribution of P4502C6 to the total N-ethylPP production in males. Thus we conclude that P4502C6 contributes approximately 22% to the total N-ethylPP production, whereas P4502C11 is most likely the source of the remaining 78% in males.

Comparison of 4-EthylDDC to TTMS. Although both TTMS and 4-ethylDDC elicit mechanism-based inactivation of P450 isozymes 1A1/2, 2C6, 2C11, and 3A, our data shows that different P450 isozymes are quantitatively important for N-alkylPP formation from each of these xenobiotics. The heme moiety of P4502C11 is an important source of both N-ethylPP formation after 4-ethylDDC administration and N-vinylPP formation after TTMS administration. P4503A is a major source of N-vinylPP formation, but does not contribute to N-ethylPP formation. In contrast, the heme moiety of P4502C6 is an important contributor to N-ethylPP but not N-vinylPP formation.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Mechanism of heme N-alkylation by oxidative activation of 4-ethylDDC. Adapted from Ortiz de Montellano (1989), Augusto et al. (1982), and Lee et al. (1988).

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Mechanism of heme N-alkylation by oxidative activation of TTMS, as proposed by Ortiz de Montellano and Grab (Grab et al., 1988). indicates the carbon atoms which eventually form the vinyl group, attached to one of the four pyrrole nitrogens in heme.