Selectivity of Polycyclic Inhibitors for Human ... - ACS Publications

Tulane Univeristy School of Medicine, New Orleans, Louisiana 70112-2699, and. Department of Chemistry, Tulane University, New Orleans, Louisiana 70118...
0 downloads 0 Views 211KB Size
1048

Chem. Res. Toxicol. 1998, 11, 1048-1056

Selectivity of Polycyclic Inhibitors for Human Cytochrome P450s 1A1, 1A2, and 1B1 Tsutomu Shimada,*,†,‡ Hiroshi Yamazaki,‡ Maryam Foroozesh,§,| Nancy E. Hopkins,§,⊥ William L. Alworth,§ and F. Peter Guengerich*,† Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, Osaka Prefectural Institute of Public Health, Osaka 537, Japan, Chemistry Department, Xavier Univeristy of Louisiana, New Orleans, Louisiana 70125-1098, Department of Pharmacology, Tulane Univeristy School of Medicine, New Orleans, Louisiana 70112-2699, and Department of Chemistry, Tulane University, New Orleans, Louisiana 70118-5645 Received April 29, 1998

Human cytochrome P450s 1A1, 1A2, and 1B1 are known to have overlapping substrate specificities. All are regulated in part by the Ah locus; P450 1A2 is expressed essentially only in liver, but P450s 1A1 and 1B1 are both expressed in many extrahepatic tissues. Twentyfive polycyclic hydrocarbons, many containing acetylenic side chains, were examined as inhibitors of the three enzymes using 7-ethoxyresorufin O-deethylation as the enzyme assay in all cases. Several compounds were inhibitory at low nanomolar concentrations. 1-(1Propynyl)pyrene and 2-(1-propynyl)phenanthrene nearly completely inhibited P450 1A1 at concentrations at which no P450 1B1 inhibition was observed. 2-Ethynylpyrene and R-naphthoflavone (7,8-benzoflavone) nearly completely inhibited P450 1B1 at concentrations at which no P450 1A1 inhibition was noted. All four of the above compounds also inhibited P450 1A2. Several polycyclic hydrocarbons devoid of acetylenic groups were also inhibitory with respect to all three P450s. Some of the acetylenic compounds examined showed enhanced inhibition following preincubation with the P450s in the presence of cofactors NADPH and O2. However, of seven compounds (five acetylenes) tested with P450 1B1, only two [2-ethynylpyrene and 4-(1-propynyl)biphenyl] showed such evidence for mechanism-based inactivation. We conclude that (i) several polycyclic hydrocarbons and their oxidation products are very inhibitory with respect to human P450s 1A1, 1A2, and 1B1; (ii) of these inhibitors only some are mechanismbased inactivators; and (iii) some of the inhibitors are potentially useful for distinguishing between human P450s 1A1 and 1B1.

Introduction P450 enzymes catalyze a great variety of oxidations of various drugs, carcinogens, and protoxicants (1, 2). Historically, there has been considerable interest in what are now termed the family 1 P450s (3). Much of the initial interest developed from the field of chemical carcinogenesis and particularly studies with polycyclic aromatic hydrocarbons, e.g., benzo[a]pyrene (4, 5). Many experimental studies have been done on the inducibility of the enzymes involved in the metabolism of these procarcinogens (6), and epidemiologists have devoted much effort to understanding the relationship between the inducibility of P450 1A1 and human cancer (7-9). In studies with experimental animals, induction of what * Address correspondence to either F. Peter Guengerich, Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146 [telephone, (615) 322-2261; fax, (615) 322-3141; e-mail, [email protected]. vanderbilt.edu] or Dr. Tsutomu Shimada, Osaka Prefectural Institute of Public Health, 3-69, Nakamichi 1-Chome, Higashinari-ku, Osaka 537, Japan [telephone, 81-6-972-1321; fax, 81-6-972-2393; e-mail, [email protected]]. † Vanderbilt University School of Medicine. ‡ Osaka Prefectural Institute of Public Health. § Tulane University. | Xavier University of Louisiana. ⊥ Tulane University School of Medicine.

are probably family 1 P450s has also been known to exert a protective effect against tumor induction by certain chemicals (10). Much of the early work on induction of P450s and their activities by polycyclic aromatic hydrocarbons and related chemicals is now understood in the context of the three individual P450s in the family: 1A1, 1A2, and 1B1 (11). P450s 1A1 and 1A2 are found in all mammals and have been well-characterized in rodent models and humans (3). The other family member, P450 1B1, was discovered more recently, primarily through work by Jefcoate with rat adrenal glands (12) and mouse 10T1/2 cells (13) and by Greenlee and Sutter with human keratinocytes (14, 15). P450 1B1 has been the subject of considerable interest due to its regulation by the Ah locus and the demonstration of catalytic activities with 17β-estradiol (16, 17) and polycyclic hydrocarbons and other carcinogens (12, 18). In humans, P450 1A1 and 1A2 share 80% amino acid sequence identity and are ∼40% identical with P450 1B1 (19, 20). P450 1A2 is expressed essentially only in the liver (21), but P450s 1A1 and 1B1 are expressed together in several extrahepatic tissues (18). One of the inherent problems in research involving these P450s is distinguishing between them, since no truly specific catalytic assay is known for any of them. Antibodies raised to one

S0893-228x(98)00090-3 CCC: $15.00 © 1998 American Chemical Society Published on Web 08/05/1998

Human P450 1A and 1B Inhibitors Chart 1. Structures of Chemicals That Were Used

Chem. Res. Toxicol., Vol. 11, No. 9, 1998 1049

potential inhibitors, with a particular goal of identifying inhibitors that would discriminate between P450s 1A1 and 1B1.

Experimental Procedures

of the proteins recognize all three (22). One approach is the production of peptide-based antibodies (or crossabsorbed antibodies), but even these are limited in that they cannot be used in experiments with cells. Another approach is to utilize chemical inhibitors; however, preliminary experiments with R-naphthoflavone indicated that a concentration of 10 µM nearly abolished the catalytic activities of P450s 1A1, 1A2, and 1B1 (22). Vinylic and acetylenic compounds have long been recognized as mechanism-based inhibitors of P450s (2331), and we considered these as potentially selective inhibitors of P450s 1A1, 1A2, and 1B1. The generally accepted mechanism of inhibition by the acetylenes (R1CtCR2) involves the formation of an FeOCR1dC(•/+)R2 complex, where a radical or a positive charge is localized on carbon, depending on the formal charge on the Fe atom (26). A pyrrolic nitrogen of the P450 heme can attack the •/+ center to yield a covalent porphyrin adduct, in which the heme is no longer functional. Alternatively, the R1 group (which can be H) can migrate to the neighboring carbon atom to yield a ketene (Od CdCR1R2) which can react with nucleophilic protein sites in the P450 (27). The oxidation of acetylenes has also been associated with the covalent cross-linking of fragments of the heme porphyrin to apoprotein (32), although it is not clear that the acetylene itself is involved in the cross-link. Acetylenes and propynes (CtCCH3) have been attached to many P450 ligands with the goal of developing selective inhibitors and active site labels (30, 33-35). Our earlier studies on the inhibition of rat P450 1A enzymes (36) were extended to some work with the human enzymes (37). Of the compounds examined in the initial studies, 2-ethynylnaphthalene and 4-propynylbiphenyl appeared to have some potential (Chart 1). However, we observed notable differences in patterns of inhibition by 2-ethynylnaphthalene between human liver microsomes and a reconstituted enzyme system containing recombinant proteins (37). We developed new recombinant systems in which human P450s 1A1, 1A2, and 1B1 were expressed along with human NADPH-P450 reductase in bacterial membranes (38-40) and used this system to evaluate the potential usefulness of 17 acetylenic polycyclic aromatic hydrocarbons and eight other

Chemicals. 4-Acetylbiphenyl, diisopropylamine, n-butyllithium in hexane solution, diethyl chlorophosphate, and ethyl iodide were purchased from Aldrich Chemical Co. (Milwaukee, WI). Tetrahydrofuran (THF) was distilled from sodium benzophenone dianion under a N2 atmosphere immediately before it was used; other solvents were reagent grade. 7-Ethoxyresorufin and resorufin were purchased from Sigma Chemical Co. (St. Louis, MO). Catalase and superoxide dismutase were obtained from Sigma; the former enzyme was dialyzed twice overnight against 50 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM EDTA before being used to remove thymol. Other chemicals were from the same sources as described previously (37, 41). Ethynylaryl and propynylaryl compounds were synthesized as described previously (37). 4-(1-Butynyl)biphenyl was synthesized from the corresponding methyl ketone, 4-acetylbiphenyl (20 mmol, 3.9 g), by a modification of the methods used previously (37). A solution of lithium diisopropylamide (1.1 equiv) was prepared at 0 °C under a N2 atmosphere in freshly distilled THF. The solution was cooled to -78 °C, and 4-acetylbiphenyl, dissolved in freshly distilled THF, was added dropwise. After 1 h, 1.2 equiv of diethyl chlorophosphate was added, and the reaction mixture was allowed to warm to room temperature over the course of 2-3 h. This reaction mixture was then transferred via a double-ended needle (with N2 pressure) to a second flask containing 2.5 equiv of freshly prepared lithium diisopropylamide solution at -78 °C under a N2 atmosphere. Ethyl iodide (1.2 equiv) was then added via a syringe, and this final reaction mixture was allowed to warm slowly to room temperature. Petroleum ether was added, and the organic and aqueous layers were separated. The organic phase was washed with cold aqueous 10% HCl, washed with H2O, and dried over MgSO4. The crude 4-(1-butynyl)biphenyl (1.6 g, 40% overall yield) was purified by flash chromatography on a silica gel column eluted with petroleum ether/heptane (2:1, v/v). The fractions containing the purified product were pooled (0.87 g) and judged to be >98% pure by GC/MS (relative abundance in parentheses): m/z 207 (MH+, 23), 206 (MH+ - 1, 100), 191 (M - 15, 100). Enzymes. Bacterial “bicistronic” P450 1A1, 1A2, and 1B1 systems were prepared as described (38, 40). Briefly, plasmids for the expression of P450 1A1, 1A2, or 1B1 and human NADPH-P450 reductase (using a single promoter) were introduced into Escherichia coli DH5R cells by a heat shock procedure, and the transformants were selected in Luria-Bertani medium containing 100 µg of ampicillin/mL. Bacterial membranes were prepared and suspended in 10 mM Tris-HCl buffer (pH 7.4) containing 1.0 mM EDTA and 20% (v/v) glycerol (42). Human P450s 1A2 and 1B1 were purified from membranes of E. coli in which (monocistronic) human P450 1A2 and P450 1B1 plasmids (expressing only the P450) had been introduced as described previously (40, 43). Rabbit NADPH-P450 reductase was purified as described previously (44, 45). Quantification of Recombinant Protein Expression. Hemoprotein expression was quantified by difference spectroscopy. Fe2+•CO versus Fe2+ difference spectra were measured using a modified Aminco DW2-OLIS spectrophotometer (OnLine Instrument Systems, Bogart, GA) as described previously (45, 46). Enzyme Assays. Standard incubation mixtures for the assay of 7-ethoxyresorufin O-deethylation consisted of human P450 enzymes (usually membranes containing both P450 and NADPH-P450 reductase, 5 pmol for P450 1A1 and 20 pmol for P450 1A2 and 1B1) with a substrate concentration of 10 µM in a final volume of 1.0 mL of 100 mM potassium phosphate buffer

1050 Chem. Res. Toxicol., Vol. 11, No. 9, 1998 (pH 7.4) containing an NADPH-generating system consisting of 0.5 mM NADP+, 5 mM glucose 6-phosphate, and 0.5 unit of glucose-6-phosphate dehydrogenase/mL (45). Purified P450 1A2 (20-50 pmol) was reconstituted with a 2-fold molar excess of NADPH-P450 reductase and L-R-dilauroyl-sn-glycero-3-phosphocholine as described (45). In the case of purified P450 1B1 (20 nM), NADPH-P450 reductase (40 nM) and 5 µg of a phospholipid mixture of l-R-dilauroyl-sn-glycero-3-phosphocholine, L-R-dioleoyl-sn-glycero-3-phosphocholine, and bovine brain phosphatidylserine (1:1:1, w/w/w)/mL were added for the reconstitution, and sodium cholate (final concentration of 0.01%, w/v) was also included, as described previously (40). Formation of resorufin from 7-ethoxyresorufin was determined fluorimetrically as described, using either a fixed time/extraction or continuous assay (47, 48). When inhibitors were tested, they were either added to the incubation system with the substrate (prior to the NADPHgenerating sytem) or, when indicated, added to a system containing all components (except NADPH) and then incubated with NADPH for a fixed time before the addition of substrate. All inhibitors and 7-ethoxyresorufin were dissolved as stock solutions (10 mM) in (CH3)2SO and diluted to working solutions with CH3CN; the final concentration of organic solvent was e1% (v/v) in all cases. Measurement of the Disappearance of Inhibitors. Bacterial membranes containing human P450 1B1 and NADPHP450 reductase (25 nM P450) were suspended in 10 mL of 0.10 M potassium phosphate buffer (in amber glass vials) containing 25 nM inhibitor and 250 µg of bovine erythorcyte catalase/mL, at 37 °C. Reactions were initiated by the addition of the usual NADPH-generating system, and 1.0 mL aliquots were withdrawn at 1.0 min intervals and mixed with 2.0 mL of CH2Cl2. The layers were separated by centrifugation (2000g for 10 min), and 1.6 mL of each organic layer was withdrawn and dried under N2. The residues were dissolved in 100 µL of CH3OH, and 50 µL of each was injected onto a 6.2 mm × 80 mm octadecylsilane HPLC column (3 µm, Zorbax, MacModd, Chadds Ford, PA). Comparison of UV peak areas was made with aliquots recovered from each system prior to the addition of NADPH (and corrected for dilution). HPLC involved isocratic solvent systems containing either 19 or 28% CH3OH (v/v) in 5 mM aqueous potassium phosphate buffer (pH 7.0) (flow rate of 3.0 mL/min). The % CH3OH and wavelength (λmax) used for each inhibitor were as follows: anthracene (28%, 253 nm), benz[a]anthracene (19%, 288 nm), 2-ethynylanthracene (28%, 272 nm), 2-ethynylphenanthrene (28%, 265 nm), 2-ethynylpyrene (19%, 263 nm), R-naphthoflavone (28%, 279 nm), phenanthrene (28%, 251 nm), 4-(1-propynyl)biphenyl (28%, 272 nm), 2-(1-propynyl)phenanthrene (19%, 269 nm), 1-(1-propynyl)pyrene (19%, 359 nm), 4-(1-propynyl)pyrene (19%, 347 nm), and pyrene (28%, 334 nm).

Results Screens of Inhibition of 7-Ethoxyresorufin O-Deethylation by Polycyclic Chemicals. Preliminary studies indicated that 2-(1-propynyl)phenanthrene and 1-(1-propynyl)pyrene were very effective in inhibiting the catalytic activity of P450 1A1 when they were added at concentrations as low as 10 nM (Figure 1). A total of 24 chemicals were then examined for their abilities to inhibit bicistronic P450 1A1-, 1A2-, and 1B1-dependent 7-ethoxyresorufin O-deethylation activities at 100 nM concentrations (Figure 2), including R-naphthoflavone, a selective inhibitor of P450 1 subfamily enzymes (22, 49). P450 1A1 has about 25- and 5-fold higher activities for 7-ethoxyresorufin O-deethylation than P450 1A2 and 1B1, respectively (22). Of the chemicals tested, 1-(1-propynyl)pyrene and 2-(1-propynyl)phenanthrene were the most potent in inhibiting 7-ethoxyresorufin O-deethylation catalyzed by P450 1A1, followed by 4-(1-propynyl)biphenyl and benz-

Shimada et al.

Figure 1. Effects of inhibitors on 7-ethoxyresorufin O-deethylation activities by 10 nM 1-(1-propynyl)pyrene (0) and 10 nM 2-(1-propynyl)phenanthrene (2) in a system containing 5 nM bicistronic P450 1A1 E. coli membranes. 7-Ethoxyresorufin O-deethylation was also determined in the absence of chemicals (O).

[a]anthracene. Pyrene, 1-(1-propynyl)pyrene, benz[a]anthracene, and R-naphthoflavone inhibited P450 1A2 very strongly. P450 1B1 activity was most potently inhibited by pyrene, 2-ethynylpyrene, 2-ethynylanthracene, benz[a]anthracene, 2-ethynylphenanthrene, and R-naphthoflavone. In general, P450 1A1 was more sensitive to the chemicals containing a propynyl group, while P450 1B1 was generally more inhibited by the chemicals with ethynyl groups. The concentration dependence of chemicals for inhibiting 7-ethoxyresorufin O-deethylation catalyzed by bacterial membranes containing human P450 1A1, 1A2, or 1B1 in the presence of NADPH-P450 reductase was determined with 16 chemicals (Figure 3), with a view toward selecting compounds that would distinguish betweeen P450s 1A1 and 1B1. These two P450s are often expressed together in extrahepatic tissues but not in liver, while P450 1A2 is expressed only in liver (18, 21). The chemicals showing the most selectivity between P450s 1A1 and 1B1 were 1-(1-propynyl)pyrene and 2-(1-propynyl)phenanthrene (both in favor of P450 1A1) and 2-ethynylpyrene and R-naphthoflavone (both in favor of P450 1B1). That is, 40 nM 1-(1-propynyl)pyrene or 80 nM 2-(1-propynyl)pyrene inhibited >90% of the P450 1A1 activity and e10% of the P450 1B1 activity. Conversely, 80 nM 2-ethynylpyrene or 20 nM R-naphthoflavone inhibited P450 1B1 activity nearly completely but did not inhibit P450 1A1. Interestingly, although 4-(1-propynyl)biphenyl inhibited all three enzymes, the homologue 4-(1-butynyl)biphenyl was not very inhibitory. Apparent IC50 values were estimated from the plots shown in Figure 3 and are presented in Table 1. In some cases, the IC50 values approximate the P450 concentrations, and a quadratic equation was used to estimate IC50 with the assumption that a single molecule of inhibitor (I) binds per P450 and

IC50 = Ki = Kd )

([P450] - [P450‚I])([I] - [P450‚I]) [P450‚I]

where I is the inhibitor and no provision is made for inhibition by a mechanism other than competitive. Biotransformation of Inhibitors by P450 1B1. One of the main goals of this study was to identify

Human P450 1A and 1B Inhibitors

Chem. Res. Toxicol., Vol. 11, No. 9, 1998 1051

Figure 2. Effects of chemicals on 7-ethoxyresorufin O-deethylation catalyzed by P450 1A1 (A), P450 1A2 (B), and P450 1B1 (C) in bicistronic E. coli membranes. Control activities (means of triplicate determinations) in the absence of chemicals were 24, 4.5, and 1.1 nmol of resorufin formed min-1 nmol of P450-1 for P450 1A1, P450 1A2, and P450 1B1, respectively. P450 concentrations for determination of catalytic activities were 5, 20, and 20 nM for P450 1A1, P450 1A2, and P450 1B1, respectively, and the substrate concentration was 10 µM in all cases. The concentration of each chemical was 100 nM; results are presented as means of results of duplicate experiments, which varied by e15%.

Figure 3. Concentration-dependent inhibiton of 7-ethoxyresorufin O-deethylation activities catalyzed by P450s 1A1 (O), 1A2 (b), and 1B1 (9) in E. coli membranes. Experimental procedures were like those described in the legend to Figure 1, except for the concentrations of chemicals added to the incubation mixtures.

potentially useful selective inhibitors of P450 1A1 versus P450 1B1. The preliminary results (Figures 2 and 3) indicated that several of the polycyclic compounds under consideration are very effective inhibitors of these P450s. The oxidation of numerous polycyclic aromatic hydrocar-

bons by rodent and human P450 1A1 has been welldocumented (50-52), but less information is available about human P450 1B1. Because the inhibition of a low concentration of P450 1B1 was observed with relatively low concentrations (nanomolar) of some of the inhibitors

1052 Chem. Res. Toxicol., Vol. 11, No. 9, 1998

Shimada et al.

Table 1. Inhibition Constants and Half-Lives for Inhibitors

inhibitor pyrene 2-ethynylpyrene 1-(1-propynyl)pyrene 4-(1-propynyl)pyrene anthracene 2-ethynylanthracene benz[a]anthracene phenanthrene 2-ethynylphenanthrene 2-(1-propynyl)phenanthrene 4-(1-propynyl)biphenyl R-naphthoflavone a

P450 P450 P450 P450 1A1 1A2 1B1 1B1 IC50 (nM) IC50 (nM) IC50 (nM) t1/2 (min)c 41a 150a 3b 160a 17b 80a 70a 2b 11b 60a

7b 60a 37b 50a 75b 120b 2b 9b 140a 140a 58b 6b

2b 30a 90a 140a 140a 75b 14b 94b 70a 180a 16b 5b

e0.5 e0.5 e0.5 e0.5 e0.5 e0.5 e0.5 1.0 e0.5 e0.5 1.0 2.3

b

Estimated from Figure 1 by interpolation. Estimated using a quadratic equation. c Estimated with 25 nM P450 1B1 and an initial concentration of 25 nM of each chemical.

under consideration (Figure 3), we measured the disappearance of 25 nM concentrations of each in the presence of 25 nM P450 1B1. Most of the chemicals were completely converted to uncharacterized, more polar products with a t1/2 of e0.5 min, with phenanthrene and 4-(1propynyl)biphenyl being somewhat more stable (Table 1). R-Naphthoflavone was removed with an estimated t1/2 of 2.3 min. Dependence of Inhibition on Enzymatic Transformation. A general method of testing for mechanismbased P450 inactivation involves examination of the effect of preincubation with the inhibitor, NADPH, and O2 prior to addition of a (noninhibitory) substrate (37, 53). Historically, we have carried out such preincubations for 10 min at 37 °C (37). However, in the case of both the bicistronic P450 1B1 membrane system and reconstituted P450 1B1/NADPH-P450 reductase mixtures formed with purified components, we found a considerable loss of activity after preincubation for 10 min with NADPH at 37 °C in the absence of any inhibitor (∼40%). This loss was also observed with the bicistronic P450 1A2 membrane system and is presumably due to generation of reactive oxygen species, since the loss could be blocked by the addition of catalase (800 units/mL) (54). However, neither catalase, bovine erythrocyte superoxide dismutase (0.18 µM), nor a mixture of the two was completely effective in blocking the loss in the P450 1B1 system. We did find that losses of P450 1B1 activity were much less significant when the preincubation was carried out at 25 °C for 2 min, conditions used by Roberts et al. (33). 1-(1-Propynyl)pyrene inhibited P450s 1A1, 1A2, and 1B1 (Figure 3). The inhibition of P450s 1A1 and 1A2 was enhanced by preincubation (with NADPH) for 2 min, but no enhancement (or loss of inhibition) was seen in the case of P450 1B1 (Figure 4). The inhibition of P450 1A1 was shown to be concentration-dependent (Figure 4) and also time-dependent. In other work (not shown), the preincubation time was varied and a t1/2 (for inhibition) of ∼1.2 min (kinact ∼ 0.6 min-1) was estimated at a 1-(1-propynyl)pyrene concentration of 13 nM. More studies were done on the effects of preincubation (Figure 5). 2-(1-Propynyl)phenanthrene is a selective inhibitor of P450 1A1 (Figure 3). The inhibition was enhanced by preincubation with NADPH (Figure 5A). However, no such enhancement of P450 1B1 inhibition by incubation was observed with pyrene, benz[a]an-

threne, 2-ethynylanthracene, 2-ethynylphenanthrene, or 2-ethynylpyrene at the concentrations examined. Results of further examination of the effects of a 2 min preincubation at 25 °C with NADPH are shown (Figure 6). Evidence for mechanism-based inactivation was seen for P450 1A1 with 1-(1-propynyl)pyrene, 2-ethynylpyrene, 2-(1-propynyl)phenanthrene, and 4-(1-propynyl)biphenyl. P450 1A2 showed such an inhibition with 1-(1-propynyl)phenanthrene and 4-(1-propynyl)biphenyl. Of the seven inhibitors of P450 1B1 examined, only 2-ethynylpyrene and 4-(1-propynyl)biphenyl showed evidence for mechanism-based inactivation.

Discussion A goal of this work was the characterization of a series of potential polycyclic inhibitors of human P450s 1A1, 1A2, and 1B1. One of the particular aims was finding chemicals that could differentially inhibit human P450s 1A1 and 1B1. To avoid differences in the systems used to make comparisons, a bicistronic expression system (38) was used to produce bacterial membranes containing human NADPH-P450 reductase and each of the P450s and a single reaction was examined (7-ethoxyresorufin O-deethylation), which has been documented to be an activity catalyzed at reasonable rates by all three enzymes (22). We found that many of the polycyclics examined showed strong inhibition of the P450s at very low concentrations (Figure 3 and Table 1). Some of these discriminated between P450s 1A1 and 1B1, with 1-(1propynyl)pyrene and 2-(1-propynyl)phenanthrene preferentially inhibiting P450 1A1 and 2-ethynylpyrene and R-naphthoflavone preferentially inhibiting P450 1B1 (Figure 3). The mechanism of inhibition is more complex than we anticipated when we began this work using the acetylenic compounds (Chart 1). In many cases, the “parent” polycylic hydrocarbons appear to be as inhibitory as their acetylenic derivatives (Figure 2), i.e., pyrene and phenanthrene. Also, in many cases, the oxidation products of the polycyclic hydrocarbons must be approximately as inhibitory as the parent molecules, since disappearance of the parent molecules happened quickly, within the time frame of the assay. For instance, consider the t1/2 values for P450 1B1 (Table 1) and the results presented in Figures 3-5. Some of the inhibitors appear to be mechanism-based, particularly in the cases of P450s 1A1 and 1A2 (Figure 6). However, few of the P450 1B1 inhibitors showed evidence of mechanism-based inactivation (Figures 5 and 6). In cases where P450s are inhibited by acetylenic substrates, both heme destruction and protein modification have been observed (24, 25, 27, 29, 31, 34, 35, 37, 55, 56). With the compounds under consideration here, we are probably seeing both phenomena with even a single compound. For instance, incubation of the P450 1A2 system with NADPH and pyrene, benz[a]anthracene, or 4-(1-propynyl)pyrene did not lead to any detectable loss of spectrally determined P450 under conditions where inhibition was extensive and NADPH-dependent (Figures 1 and 6), but under similar conditions, 20% of the COdetectable heme of P450 1A2 was lost upon incubation with 2-ethynylpyrene (results not shown). Evidence for similar mixtures of destructive phenomena was obtained earlier when purified recombinant human P450s 1A1 and 1A2 were incubated with 2-ethynylnaphthalene at higher

Human P450 1A and 1B Inhibitors

Chem. Res. Toxicol., Vol. 11, No. 9, 1998 1053

Figure 4. Effects of preincubation (2 min) on inhibition of P450 1A1 (A)-, P450 1A2 (B)-, and P450 1B1 (C)-dependent 7-ethoxyresorufin O-deethylation activities by 1-(1-propynyl)pyrene. P450 concentrations were 5 nM for P450 1A1 and 25 nM for P450s 1A2 and 1B1. The temperature was 25 °C. At t ) 0 in panel A, either no inhibitor (O) or 12.5 (4) or 25 nM (0) 1-(1-propynyl)pyrene was added to an incubation mixture containing 10 µM 7-ethoxyresorufin, and the formation of resorufin was determined. The P450 system was also incubated with 1-(1-propynyl)pyrene and NADPH in the absence of 7-ethoxyresorufin, and after incubation for 2 min (t ) 2 min), the substrate was added [(b) no inhibitor, (2) 12.5 nM 1-(1-propynyl)pyrene, and (9) 25 nM 1-(1-propynyl)pyrene]. In panels B and C, results from similar experiments that were carried out to examine the effects of preincubation in the absence (O or b) or presence of 500 nM 1-(1-propynyl)pyrene are shown (4 or 2). The substrate 7-ethoxyresorufin was added at either t ) 0 (white symbols) or t ) 2 min (black symbols).

Figure 5. Effects of preincubation with inhibitors on 7-ethoxyresorufin O-deethylation activities of P450s 1A1 and 1B1. All experiments were carried out at 25 °C, and resorufin formation was measured continuously. E. coli membranes containing P450 (5 nM P450 1A1 or 20 nM P450 1B1) and NADPH-P450 reductase were incubated with an NADPH-generating system and the indicated inhibitor; 7-ethoxyresorufin (10 µM) was added at either 0 or 2 min: (A) P450 1A1 with either no inhibitor (none) or 25 or 50 nM 2-(1-propynyl)phenanthrene, (B) P450 1B1 with either no inhibitor (none) or 50 or 250 nM benz[a]anthracene, and (C) P450 1B1 with either no inhibitor (none) or 100 or 500 nM 2-ethynylphenanthrene.

concentrations (37). The only detectable unbound product was determined to be the expected 2-naphthylacetic acid (26), and the partition ratios for human P450s 1A1 and 1A2 were determined to be 4 and 23, respectively (ratio of the rate of 2-naphthylacetic acid formation to first-order P450 destruction) (53, 57). 2-Naphthylacetic acid was not inhibitory with respect to human P450 1A1, 1A2, or 1B1 at concentrations of up to 200 µM (results

Figure 6. Effects of preincubation with NADPH on inhibition of 7-ethoxyresorufin O-deethylation activity by chemicals: (A) P450 1A1, (B) P450 1A2, and (C) P450 1B1. The experimental design was like that in Figure 5, with all preincubations carried out at 25 °C for 2 min in the absence (0) or presence (9) of NADPH with the indicated concentration of each chemical.

not shown), ruling out a mechanism involving formation of this product as a stable, tight-binding inhibitor (57). In this work, 4-(1-propynyl)biphenyl appears to be a mechanism-based inactivator of human P450s 1A1, 1A2,

1054 Chem. Res. Toxicol., Vol. 11, No. 9, 1998

and 1B1 (Figures 3 and 6). We found partition ratios of 2 and 3 for human P450s 1A1 and 1A2, respectively, based upon the measurement of the reaction product 2-methyl-2-(4-biphenyl)acetic acid.1 Under conditions in which 90% of the P450 1A1 catalytic activity was lost, 60% of heme was destroyed; 80% of the P450 1A2 activity was inhibited, but only 30% of the heme spectrum was lost (37). With both human P450s 1A1 and 1A2, repeated efforts at labeling the protein never resulted in g0.2 molecule of (label from) 2-ethynylnaphthalene bound to the protein,1 in contrast to the situation with rat P450 2B1 (34). Some of the bound label may be unstable, or alternatively, the fraction of enzyme inhibition not accounted for may simply be the result of non-mechanismbased inhibition. Nevertheless, we conclude that with many of the acetylenic compounds under consideration here we are seeing varying mixtures of competitive inhibition, heme destruction, and covalent modification of these three human P450s. The nonacetylenic compounds (and their oxidation products) are postulated to be acting only as competitive inhibitors (Figure 1). The estimated IC50 values, which should be no less than Ki, were estimated using quadratic fits and in some cases are on the order of 10-8 to 10-9 M (Table 1). Although these values may seem low for P450 ligands, they are not unprecedented and sub-micromolar Ki values have been reported for P450 2D6 [quinidine (58, 59)], P450 3A4 [ketoconazole (60)], P450 51 [lanosterol 14R-demethylation inhibitors (61)], and P450 19 [R-naphthoflavone (62) and selected aromatase inhibitors (63)]. Roberts et al. (56) reported a Ki of 138 nM for inhibition of rat P450 2B1 by 9-ethynylphenanthrene. Voorman and Aust (64) reported an IC50 of 38 nM for 3,3′,4,4′,5,5′hexabromobiphenyl when rat P450 1A2 was present at 50 nM in an assay, probably indicating a Ki of e20 nM. In the same study, 3-methylcholanthrene showed an even lower IC50. A Kd of 10 nM for binding of benzo[a]pyrene to rat P450 1A1 was reported by Jefcoate and his associates (65), and a series of phenolic derivatives of the compound showed Kd values of 3-25 nM (66). Similar tight binding of 3-methylcholanthrene to P450 1A2 (purified from livers of 3-methylcholanthrene-treated rabbits) has also been reported (67). In our earlier study (37), low Ki values were also reported for the competitive inhibition of rat P450s 1A1 and 1A2 by some of the current group of acetylenic polycyclic hydrocarbons in liver microsomes. A corollary of these results is that some highly inhibitory polycyclic aromatic hydrocarbons derived from animals treated with these compounds remain tightly bound and modify activity (68). This issue is circumvented with the recombinant systems. In conclusion, we report strong inhibition of human P450s 1A1, 1A2, and 1B1 by a number of polycyclic hydrocarbon derivatives. The mechanisms of inhibition appear to be diverse and not mechanism-based in some cases. Of particular note is the identification of a set of four compounds that may be used to discriminate between human P450s 1A1 and 1B1: 2-(1-propynyl)pyrene, 2-(1-propynyl)phenanthrene, 2-ethynylpyrene, and R-naphthoflavone (Chart 1 and Figure 3). Finally, we emphasize that these patterns may not necessarily be expected to be seen with the apparent homologues in experimental animals. Indeed, human P450 1B1 catalyzes estradiol 1 Z. Guo, L. C. Bell-Parikh, and F. P. Guengerich, unpublished results.

Shimada et al.

4-hydroxylation, but the rodent P450 1B1 proteins do not (17, 40, 69). Mouse P450 1B1 apparently oxidizes 7,12dimethylbenz[a]anthracene much more effectively than the human enzyme (69). Considerable differences in the selectivity of acetylenic polycyclic aromatic hydrocarbons for inhibiting rat, rabbit, and human P450 1A2 have already been cited (37).

Acknowledgment. We thank A. Parikh and Dr. E. M. J. Gillam for preparing the bicistronic expression constructs, C. G. Turvy for preparing NADPH-P450 reductase, and L. C. Bell-Parikh and Dr. Z. Guo for providing the information about covalent binding of radiolabeled inhibitors to P450s 1A1 and 1A2. This research was supported in part by U.S. Public Health Service Grants R35 CA44353 and P30 ES00267 (F.P.G.) and R01 CA38192 (W.L.A.) and by grants from the Ministry of Education, Science, and Culture of Japan (T.S. and H.Y.).

References (1) Porter, T. D., and Coon, M. J. (1991) Cytochrome P-450: multiplicity of isoforms, substrates, and catalytic and regulatory mechanisms. J. Biol. Chem. 266, 13469-13472. (2) Ortiz de Montellano, P. R. (1995) Cytochrome P450: Structure, Mechanism, and Biochemistry, 2nd ed., Plenum Press, New York. (3) Nelson, D. R., Koymans, L., Kamataki, T., Stegeman, J. J., Feyereisen, R., Waxman, D. J., Waterman, M. R., Gotoh, O., Coon, M. J., Estabrook, R. W., Gunsalus, I. C., and Nebert, D. W. (1996) P450 superfamily: update on new sequences, gene mapping, accession numbers, and nomenclature. Pharmacogenetics 6, 1-42. (4) Heidelberger, C. (1975) Chemical carcinogenesis. Annu. Rev. Biochem. 44, 79-121. (5) Dipple, A., Michejda, C. J., and Weisburger, E. K. (1985) Metabolism of chemical carcinogens. Pharmacol. Ther. 27, 265296. (6) Conney, A. H. (1982) Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons: G. H. A. Clowes Memorial Lecture. Cancer Res. 42, 48754917. (7) Kellerman, G., Shaw, C. R., and Luyten-Kellerman, M. (1973) Aryl hydrocarbon hydroxylase inducibility and bronchogenic carcinoma. N. Engl. J. Med. 298, 934-937. (8) Kouri, R. E., McKinney, C. E., Slomiany, D. J., Snodgrass, D. R., Wray, N. P., and McLemore, T. L. (1982) Positive correlation between high aryl hydrocarbon hydroxylase activity and primary lung cancer as analyzed in cryopreserved lymphocytes. Cancer Res. 42, 5030-5037. (9) Goto, I., Yoneda, S., Yamamoto, M., and Kawajiri, K. (1996) Prognostic significance of germ line polymorphisms of the CYP1A1 and glutathione S-transferase genes in patients with non-small cell lung cancer. Cancer Res. 56, 3725-3730. (10) Richardson, H. L., Stier, A. R., and Borsos-Nachtnebel, E. (1952) Liver tumor inhibition and adrenal histologic responses in rats to which 3′-methyl-4-dimethylaminoazobenzene and 20-methylcholanthrene were simultaneously administered. Cancer Res. 12, 356-361. (11) Conney, A. H., Miller, E. C., and Miller, J. A. (1956) The metabolism of methylated aminoazo dyes. V. Evidence for induction of enzyme synthesis in the rat by 3-methylcholanthrene. Cancer Res. 16, 450-459. (12) Bhattacharyya, K. K., Brake, P. B., Eltom, S. E., Otto, S. A., and Jefcoate, C. R. (1995) Identification of a rat adrenal cytochrome P450 active in polycyclic hydrocarbon metabolism as rat CYP1B1: demonstration of a unique tissue-specific pattern of hormonal and aryl hydrocarbon receptor-linked regulation. J. Biol. Chem. 270, 11595-11602. (13) Savas, U ¨ ., Bhattacharyya, K. K., Christou, M., Alexander, D. L., and Jefcoate, C. R. (1994) Mouse cytochrome P450EF, representative of a new 1B subfamily of cytochrome P450s. Cloning, sequence determination, and tissue expression. J. Biol. Chem. 269, 14905-14911. (14) Sutter, T. R., Guzman, K., Dold, K. M., and Greenlee, W. F. (1991) Targets for dioxin: genes for plasminogen activator inhibitor-2 and interleukin-1β. Science 254, 415-418. (15) Sutter, T. R., Tang, Y. M., Hayes, C. L., Wo, Y. Y. P., Jabs, E. W., Li, X., Yin, H., Cody, C. W., and Greenlee, W. F. (1994) Complete

Human P450 1A and 1B Inhibitors

(16)

(17)

(18)

(19)

(20)

(21)

(22)

(23)

(24)

(25)

(26)

(27)

(28)

(29)

(30)

(31)

(32)

(33)

(34)

(35)

(36)

cDNA sequence of a human dioxin-inducible mRNA identifies a new gene subfamily of cytochrome P450 that maps to chromosome 2. J. Biol. Chem. 269, 13092-13099. Liehr, J. G., Ricci, M. J., Jefcoate, C. R., Hannigan, E. V., Hokanson, J. A., and Zhu, B. T. (1995) 4-Hydroxylation of estradiol by human uterine myometrium and myoma microsomes: implications for the mechanism of uterine tumorigenesis. Proc. Natl. Acad. Sci. U.S.A. 92, 9220-9224. Hayes, C. L., Spink, D. C., Spink, B. C., Cao, J. Q., Walker, N. J., and Sutter, T. R. (1996) 17β-Estradiol hydroxylation catalyzed by human cytochrome P450 1B1. Proc. Natl. Acad. Sci. U.S.A. 93, 9776-9781. Shimada, T., Hayes, C. L., Yamazaki, H., Amin, S., Hecht, S. S., Guengerich, F. P., and Sutter, T. R. (1996) Activation of chemically diverse procarcinogens by human cytochrome P450 1B1. Cancer Res. 56, 2979-2984. Quattrochi, L. C., Okino, S. T., Pendurthi, U. R., and Tukey, R. H. (1985) Cloning and isolation of human cytochrome P-450 cDNAs homologous to dioxin-inducible rabbit mRNAs encoding P-450 4 and P-450 6. DNA 4, 395-400. Jaiswal, A. K., Gonzalez, F. J., and Nebert, D. W. (1985) Human dioxin-inducible cytochrome P1-450: complementary DNA and amino acid sequence. Science 228, 80-83. Guengerich, F. P. (1995) Human cytochrome P450 enzymes. In Cytochrome P450 (Ortiz de Montellano, P. R., Ed.) 2nd ed., pp 473-535, Plenum Press, New York. Shimada, T., Gillam, E. M. J., Sutter, T. R., Strickland, P. T., Guengerich, F. P., and Yamazaki, H. (1997) Roles of recombinant human cytochrome P450 1B1 in the oxidation of xenobiotic chemicals. Drug Metab. Dispos. 25, 617-622. Ortiz de Montellano, P. R., and Kunze, K. L. (1980) Self-catalyzed inactivation of hepatic cytochrome P-450 by ethynyl substrates. J. Biol. Chem. 255, 5578-5585. Gan, L. S. L., Acebo, A. L., and Alworth, W. L. (1984) 1-Ethynylpyrene, a suicide inhibitor of cytochrome P-450 dependent benzo[a]pyrene hydroxylase activity in liver microsomes. Biochemistry 23, 3827-3836. Ortiz de Montellano, P. R., Kunze, K. L., Yost, G. S., and Mico, B. A. (1979) Self-catalyzed destruction of cytochrome P-450: covalent binding of ethynyl sterols to prosthetic heme. Proc. Natl. Acad. Sci. U.S.A. 76, 746-749. Komives, E. A., and Ortiz de Montellano, P. R. (1987) Mechanism of oxidation of π bonds by cytochrome P-450: electronic requirements of the transition state in the turnover of phenylacetylenes. J. Biol. Chem. 262, 9793-9802. CaJacob, C. A., Chan, W. K., Shephard, E., and Ortiz de Montellano, P. R. (1988) The catalytic site of rat hepatic lauric acid ω-hydroxylase: protein versus prosthetic heme alkylation in the ω-hydroxylation of acetylenic fatty acids. J. Biol. Chem. 263, 18640-18649. Viaje, A., Lu, J. Y. L., Hopkins, N. E., Nettikumara, A. N., DiGiovanni, J., Alworth, W. L., and Slaga, T. J. (1990) Inhibition of the binding of 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene to DNA in mouse skin epidermis by 1-ethynylpyrene. Carcinogenesis 11, 1139-1143. Yun, C.-H., Hammons, G. J., Jones, G., Martin, M. V., Hopkins, N. E., Alworth, W. L., and Guengerich, F. P. (1992) Modification of cytochrome P450 1A2 enzymes by the mechanism-based inactivator 2-ethynylnaphthalene and the photoaffinity label 4-azidobiphenyl. Biochemistry 31, 10556-10563. Hopkins, N. E., Foroozesh, M. K., and Alworth, W. L. (1992) Suicide inhibitors of cytochrome P450 1A1 and P450 2B1. Biochem. Pharmacol. 44, 787-796. Roberts, E. S., Ballou, D. P., Hopkins, N. E., Alworth, W. L., and Hollenberg, P. F. (1995) Mechanistic studies of 9-ethynylphenanthrene-inactivated cytochrome P450 2B1. Arch. Biochem. Biophys. 323, 303-312. Guengerich, F. P. (1986) Covalent binding to apoprotein is a major fate of heme in a variety of reactions in which cytochrome P-450 is destroyed. Biochem. Biophys. Res. Commun. 138, 193-198. Roberts, E. S., Pernecky, S. J., Alworth, W. L., and Hollenberg, P. F. (1996) A role for threonine 302 in the mechanism-based inactivation of P450 2B4 by 2-ethynylnaphthalene. Arch. Biochem. Biophys. 331, 170-176. Roberts, E. S., Hopkins, N. E., Alworth, W. L., and Hollenberg, P. F. (1993) Mechanism-based inactivation of cytochrome P450 2B1 by 2-ethynylnaphthalene: identification of an active-site peptide. Chem. Res. Toxicol. 6, 470-479. Ortiz de Montellano, P. R., and Reich, N. O. (1986) Inhibition of cytochrome P-450 enzymes. In Cytochrome P-450 (Ortiz de Montellano, P. R., Ed.) pp 273-314, Plenum Press, New York. Hammons, G. J., Alworth, W. L., Hopkins, N. E., Guengerich, F. P., and Kadlubar, F. F. (1989) 2-Ethynylnaphthalene as a

Chem. Res. Toxicol., Vol. 11, No. 9, 1998 1055

(37)

(38)

(39)

(40)

(41)

(42)

(43)

(44)

(45)

(46)

(47)

(48)

(49)

(50)

(51)

(52)

(53)

(54)

(55)

(56)

mechanism-based inactivator of the cytochrome P-450 catalyzed N-oxidation of 2-naphthylamine. Chem. Res. Toxicol. 2, 367-374. Foroozesh, M., Primrose, G., Guo, Z., Bell, L. C., Guengerich, F. P., and Alworth, W. L. (1997) Propynylaryl acetylenes as mechanism-based inhibitors of cytochrome P450 1A1, 1A2, and 2B1 enzymes. Chem. Res. Toxicol. 10, 91-102. Parikh, A., Gillam, E. M. J., and Guengerich, F. P. (1997) Drug metabolism by Escherichia coli expressing human cytochromes P450. Nat. Biotechnol. 15, 784-788. Josephy, P. D., Evans, D. H., Parikh, A., and Guengerich, F. P. (1998) Expression of active human cytochrome P450 1A2, NADPHcytochrome P450 reductase, and N-acetyltransferase in Escherichia coli: metabolic activation of aromatic amine mutagens. Chem. Res. Toxicol. 11, 70-74. Shimada, T., Wunsch, R. M., Hanna, I. H., Sutter, T. R., Guengerich, F. P., and Gillam, E. M. J. (1998) Recombinant human cytochrome P450 1B1 expression in Escherichia coli. Arch. Biochem. Biophys. (in press). Shimada, T., Iwasaki, M., Martin, M. V., and Guengerich, F. P. (1989) Human liver microsomal cytochrome P-450 enzymes involved in the bioactivation of procarcinogens detected by umu gene response in Salmonella typhimurium TA1535/pSK1002. Cancer Res. 49, 3218-3228. Gillam, E. M. J., Baba, T., Kim, B.-R., Ohmori, S., and Guengerich, F. P. (1993) Expression of modified human cytochrome P450 3A4 in Escherichia coli and purification and reconstitution of the enzyme. Arch. Biochem. Biophys. 305, 123-131. Sandhu, P., Guo, Z., Baba, T., Martin, M. V., Tukey, R. H., and Guengerich, F. P. (1994) Expression of modified human cytochrome P450 1A2 in Escherichia coli: stabilization, purification, spectral characterization, and catalytic activities of the enzyme. Arch. Biochem. Biophys. 309, 168-177. Yasukochi, Y., and Masters, B. S. S. (1976) Some properties of a detergent-solubilized NADPH-cytochrome c (cytochrome P-450) reductase purified by biospecific affinity chromatography. J. Biol. Chem. 251, 5337-5344. Guengerich, F. P. (1994) Analysis and characterization of enzymes. In Principles and Methods of Toxicology (Hayes, A. W., Ed.) 3rd ed., pp 1259-1313, Raven Press, New York. Omura, T., and Sato, R. (1964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. 239, 2370-2378. Burke, M. D., and Prough, R. A. (1978) Fluorimetric and chromatographic methods for measuring microsomal biphenyl hydroxylation. Methods Enzymol. 52, 399-407. Lubet, R. A., Mayer, R. T., Cameron, J. W., Nims, R. W., Burke, M. D., Wolff, T., and Guengerich, F. P. (1985) Dealkylation of pentoxyresorufin: a rapid and sensitive assay for measuring induction of cytochrome(s) P-450 by phenobarbital and other xenobiotics in the rat. Arch. Biochem. Biophys. 238, 43-48. Correia, M. A. (1995) Rat and human liver cytochromes P450. Substrate and inhibitor specificities and functional markers. In Cytochrome P450 (Ortiz de Montellano, P. R., Ed.) 2nd ed., pp 607-630, Plenum Press, New York. Wood, A. W., Levin, W., Lu, A. Y. H., Yagi, H., Hernandez, O., Jerina, D. M., and Conney, A. H. (1976) Metabolism of benzo[a]pyrene and benzo[a]pyrene derivatives to mutagenic products by highly purified hepatic microsomal enzymes. J. Biol. Chem. 251, 4882-4890. Bauer, E., Guo, Z., Ueng, Y.-F., Bell, L. C., and Guengerich, F. P. (1995) Oxidation of benzo[a]pyrene by recombinant human cytochrome P450 enzymes. Chem. Res. Toxicol. 8, 136-142. Shou, M., Korzekwa, K. R., Crespi, C. L., Gonzalez, F. J., and Gelboin, H. V. (1994) The role of 12 cDNA-expressed human, rodent, and rabbit cytochromes P450 in the metabolism of benzo[a]pyrene and benzo[a]pyrene trans-7,8-dihydrodiol. Mol. Carcinog. 10, 159-168. Halpert, J. R., and Guengerich, F. P. (1997) Enzyme inhibition and stimulation. In Biotransformation (Guengerich, F. P., Ed.) Vol. 3 of Comprehensive Toxicology (Sipes, I. G., McQueen, C. A., and Gandolfi, A. J., Eds.) pp 21-35, Elsevier Science Ltd., Oxford, U.K. Guengerich, F. P. (1978) Destruction of heme and hemoproteins mediated by liver microsomal reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase. Biochemistry 17, 3633-3639. Guengerich, F. P. (1990) Mechanism-based inactivation of human liver cytochrome P-450 IIIA4 by gestodene. Chem. Res. Toxicol. 3, 363-371. Roberts, E. S., Hopkins, N. E., Zaluzec, E. J., Gage, D. A., Alworth, W. L., and Hollenberg, P. F. (1995) Mechanism-based inactivation of cytochrome P450 2B1 by 9-ethynylphenanthrene. Arch. Biochem. Biophys. 323, 295-302.

1056 Chem. Res. Toxicol., Vol. 11, No. 9, 1998 (57) Silverman, R. B. (1995) Mechanism-based enzyme inactivators. Methods Enzymol. 249, 240-283. (58) Otton, S. V., Inaba, T., Mahon, W. A., and Kalow, W. (1982) In vitro metabolism of sparteine by human liver: competitive inhibition by debrisoquine. Can. J. Physiol. Pharmacol. 60, 102105. (59) Guengerich, F. P., Mu¨ller-Enoch, D., and Blair, I. A. (1986) Oxidation of quinidine by human liver cytochrome P-450. Mol. Pharmacol. 30, 287-295. (60) Baldwin, S. J., Bloomer, J. C., Smith, G. J., Ayrton, A. D., Clarke, S. E., and Chenery, R. J. (1995) Ketoconazole and sulphaphenazole as the respective selective inhibitors of P4503A and 2C9. Xenobiotica 25, 261-270. (61) Swinney, D. C., So, O. Y., Watson, D. M., Berry, P. W., Webb, A. S., Kertesz, D. J., Shelton, E. J., Burton, P. M., and Walker, K. A. M. (1994) Selective inhibition of mammalian lanosterol 14Rdemethylase by RS-21607 in vitro and in vivo. Biochemistry 33, 4702-4713. (62) Kellis, J. T., Jr., and Vickery, L. E. (1984) Inhibition of human estrogen synthetase (aromatase) by flavones. Science 225, 10321034. (63) Vanden Bossche, H., Moereels, H., and Koymans, L. M. H. (1994) Aromatase inhibitorssmechanisms for non-steroidal inhibitors. Breast Cancer Res. Treat. 30, 43-55. (64) Voorman, R., and Aust, S. D. (1987) Specific binding of polyhalogenated aromatic hydrocarbon inducers of cytochrome P-450d

Shimada et al. to the cytochrome and inhibition of its estradiol 2-hydroxylase activity. Toxicol. Appl. Pharmacol. 90, 69-78. (65) Marcus, C. B., Turner, C. R., and Jefcoate, C. R. (1985) Binding of benzo[a]pyrene by purified cytochrome P-450c. Biochemistry 24, 5115-5123. (66) Turner, C. R., Marcus, C. B., and Jefcoate, C. R. (1985) Selectivity in the binding of hydroxylated benzo[a]pyrene derivatives to purified cytochrome P-450c. Biochemistry 24, 5124-5130. (67) Imai, Y., Hashimoto-Yutsudo, C., Satake, H., Girardin, A., and Sato, R. (1980) Multiple forms of cytochrome P-450 purified from liver microsomes of phenobarbital- and 3-methylcholanthrenepretreated rabbits. I. Resolution, purification, and molecular properties. J. Biochem. 88, 489-503. (68) Miller, D. M., Aust, A. E., Voorman, R., and Aust, S. D. (1988) Inhibition of 2-aminofluorene mutagenesis in bacteria by inducers of cytochrome P-450d. Carcinogenesis 9, 327-329. (69) Savas, U ¨ ., Carstens, C. P., and Jefcoate, C. R. (1997) Recombinant mouse CYP1B1 expressed in Escherichia coli exhibits selective binding by polycyclic hydrocarbons and metabolism which parallels C3H10T1/2 cell microsomes, but differs from human recombinant CYP1B1. Arch. Biochem. Biophys. 347, 181-192.

TX980090+