Human cytochrome P450 (P450) 1B1 is found mainly in extrahepatic tissues and is overexpressed in a variety of human tumors. Metabolic activation of 17β-estradiol (E2) to 4-hydroxy E2 by P450 1B1 has been postulated to be a factor in mammary carcinogenesis. The inhibition of recombinant human P450 1B1 by 2,4,3′,5′-tetramethoxystilbene (TMS) was investigated using either bacterial membranes from a human P450/NADPH-P450 reductase bicistronic expression system or using purified enzymes. TMS showed potent and selective inhibition of the ethoxyresorufin O-deethylation (EROD) activity of P450 1B1 with an IC50 value of 6 nm. TMS exhibited 50-fold selectivity for P450 1B1 over P450 1A1 (IC50 = 300 nm) and 500-fold selectivity for P450 1B1 over P450 1A2 (IC50 = 3 μm). The inhibitory effects of TMS on EROD activity of human liver microsomes were determined. TMS inhibited EROD activity of human liver microsomes at the same concentration as with recombinant human P450 1A2. TMS also strongly inhibited 4- and 2-hydroxylation of E2 by P450 1B1-expressing membranes or purified P450 1B1. TMS was a competitive inhibitor of P450 1B1 with a Ki of 3 nm. The inhibition by TMS was not mechanism-based, and the loss of activity was not blocked by the trapping agents glutathione, N-acetylcysteine, or dithiothreitol. Using purified histidine-tagged P450 1B1, the binding kinetic analysis was performed with TMS, yielding a Kd of 3 μm. The activation of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline in an Escherichia coli lac-based mutagenicity tester system containing functional human P450 1B1 was strongly inhibited by TMS. Our results indicate that TMS is a very selective and potent competitive inhibitor of P450 1B1. TMS is selective for inhibiting P450 1B1 among other human P450s including 1A1, 1A2, and 3A4 and warrants consideration as a candidate for preventing mammary tumor formation by E2 in humans.

Human P4503 1B1 is a major E2 hydroxylase and is involved in the metabolic activation of several polycyclic aromatic hydrocarbon carcinogens, including benzo(a)pyrene, dibenzo(a,l)pyrene, 7,12-dimethylbenz(a)anthracene, and 5-methylchrysene (1). P450 1B1 is mainly found in extrahepatic steroidogenic tissues such as the ovary, testis, and adrenal gland and in steroid-responsive tissues such as breast, uterus, and prostate. P450 1B1 has been reported to be induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in experimental animals and in human breast cancer cells (2, 3, 4).

Metabolic activation of E2 has been suggested to be a major factor in mammary carcinogenesis. E2 is usually metabolized by P450 1A1 and P450 1B1 to the 2-hydroxylated and 4-hydroxylated forms. P450 1B1 participates in E2 hydroxylation at the C4 position, whereas P450 1A1 mainly hydroxylates E2 at the C2 position (5, 6). Many reports have suggested that the 4-hydroxylation of E2 by P450 1B1 is important for mammary carcinogenesis, because 4-hydroxy E2 is a carcinogenic metabolite. Treatment with 4-hydroxy E2 but not 2-hydroxy E2 induced kidney tumors in Syrian hamsters (7, 8). Because of the postulated significant role of P450 1B1 in the carcinogenicity of E2, selective inhibition of P450 1B1 may prevent formation of mammary tumors.

Resveratrol has been studied to determine its role as a cancer chemopreventive agent, and we reported that resveratrol can inhibit human P450 1A1 in a concentration-dependent manner (9). However, the IC50 value for P450 1A1 inhibition (23 mm) by resveratrol is not very low compared with other well-known P450 family 1 inhibitors such as α-naphthoflavone and 7-hydroxyflavone. To find more potent and selective P450 family 1 inhibitors among resveratrol analogues, several hydroxystilbene compounds obtained from natural sources were evaluated for selective inhibition of P450 family 1 activity. Among them, rhapontigenin exhibited a potent and selective inhibition of human P450 1A1 with an IC50 value of 0.4 mm. Rhapontigenin is a potent mechanism-based inactivator of P450 1A1 (Ki = 0.09 mm and kinactivation = 0.06 min−1) as well as a competitive inhibitor (Ki = 0.21 mm; Ref. 10). We found that the selectivity and potency of inhibition against P450s by trans-stilbene compounds were sensitive to the substitution patterns on the trans-stilbene template. We designed and prepared a series of compounds having dimethoxy groups on the 3 and 5 positions of the phenyl ring 1 instead of the corresponding dihydroxyl groups of the natural stilbenes such as rhapontigenin or 2,4,3′,5′-tetrahydroxystilbene. Substitution of dihydroxyl for dimethoxy groups may increase the lipophilicity and the binding to the active sites of P450 family 1 enzymes. Several structural changes were performed on phenyl ring 2 to obtain a more selective inhibitor of P450 1B1. TMS, a methoxy derivative of 2,4,3′,5′-tetrahydroxystilbene, showed potent and selective inhibition of P450 1B1 relative to several other P450 enzymes.

Reagents.

E2, 4-hydroxy E2, 2-hydroxy E2, ethoxyresorufin, resorufin, DMSO, thiamine, IPTG, and δ-aminolevulinic acid were purchased from Sigma Chemical Co. (St. Louis, MO). TB broth, bactopeptone, and bacto-agar were obtained from Difco (Detroit, MI). Other chemicals were of the highest grade commercially available.

Chemical Synthesis.

The synthesis of TMS (Fig. 1) was carried out by using a modified Wittig reaction and isomerization reaction (11, 12, 13, 14). To a stirred solution of triphenyl(3,5-dimethoxybenzyl)phosphonium bromide (1.0 mmol), 2,4-dimethoxybenzaldehyde (1.0 mmol), and 18-crown-6 (0.1 mmol) in 2 ml of CH2Cl2 was added freshly powdered KOH (2.0 mmol) at room temperature (11). After 1 h, the mixture was diluted with 10 ml of CH2Cl2 and filtered, and the filtrate was washed with H2O. The organic layer was dried over MgSO4 and concentrated under reduced pressure, and the residue was purified by silica gel chromatography with CH2Cl2 to afford the desired stilbene (0.91 mmol) as a mixture of E- and Z-isomers (∼1:1 ratio). To the solution of this mixture in heptane (5 ml), a catalytic amount of I2 (1 crystal) was added. This was heated to reflux for 12 h (12). The reaction mixture was diluted with 20 ml of diethyl ether and washed with 10 ml of saturated aqueous sodium bisulfite and H2O. The organic layer was dried over MgSO4 and concentrated in a vacuum, and the residue was purified by silica gel chromatography to afford the desired E-stilbene as white needles (0.9 mmol, 90% yield): melting point 75.2–75.6°C (13, 14); 1H NMR (300 MHz, CDCl3) δ 7.42 (d, J = 8.4 Hz, 1H), 7.28 (d, J = 16.2 Hz, 1H), 6.87 (d, J = 16.2 Hz, 1H), 6.59 (d, J = 2.4 Hz, 2H), 6.44 (dd, J = 8.4, 2.4 Hz, 1H), 6.39 (d, J = 2.1 Hz, 1H), 6.29 (t, J = 2.1 Hz, 1H), 3.80 (s, 3H, -OCH3), 3.76 (s, 3H, -OCH3), 3.75 (s, 6H, -OCH3); 13C NMR (75 MHz, CDCl3) 160.88, 160.61, 158.10, 140.37, 127.35, 126.98, 123.85, 119.33, 107.08, 104.99, 104.38, 99.41, 98.50, 97.50, 55.51, 55.40, 55.36, 55.22; C18O4H20 mass spectrum (electron impact) calculated average 300.34, observed 300 (Mt).

Enzymes.

Bacterial coexpression (bicistronic) plasmids for human P450s (1A1, 1A2, 1B1, or 3A4) and NADPH-P450 reductase were transformed into Escherichia coli DH5αF′Iq (15). A single ampicillin-resistant colony of transformed cells was selected and grown in overnight culture to saturation at 37°C in Luria Broth medium containing 100 μg ampicillin ml−1. A 10-ml aliquot was used to inoculate each liter of TB medium containing 0.2% bactopeptone (w/v), 100 μg ampicillin ml−1, 1.0 mm thiamine, trace elements, 0.5 mm δ-aminolevulinic acid, and 1.0 mm IPTG. The cultures were grown at 30°C with shaking at 200 rpm for 24 or 48 h. After incubation, cells were harvested by centrifugation at 6500 × g for 20 min. Spheroplasts were prepared using lysozyme and disrupted by sonication. The cellular sonicates were centrifuged at 104 × g for 20 min, and the membranes were pelleted by centrifugation at 1.1 × 105 × g for 90 min and were resuspended in 10 mm Tris-HCl buffer (pH 7.4) containing 1.0 mm EDTA and 20% glycerol (v/v; Ref. 16).

Human Liver Microsomes.

Frozen human liver samples were thawed in 0.1 m Tris-acetate buffer (pH 7.4) containing 0.1 m KCl, 1.0 mm EDTA, and 20 μm butylated hydroxytoluene and homogenized in a Teflon-glass homogenizer. The homogenate was centrifuged at 104 × g for 20 min at 4°C, and the resulting supernatant was centrifuged for 60 min at 105 × g at 4°C. The microsomal pellets were resuspended in 10 mm Tris-acetate buffer (pH 7.4) containing 1.0 mm EDTA and 20% glycerol (v/v; Ref. 17). Protein concentrations were estimated using the bichinchoninic acid method according to the supplier’s recommendations (Pierce Chemical Co., Rockford, IL) using bovine serum albumin as a standard. The isolated microsomes were stored at −80°C.

Expression and Purification of Recombinant His-tagged P450 1B1.

To facilitate expression and purification of P450 1B1, six histidine residues were introduced at the position before the termination codon. The PCR primers were designed to contain EcoRI and XbaI restriction sites at the 5′ and 3′-ends of the amplified fragment, respectively (forward: 5′-GTTTTCCGCGAATTCGAGCAG-3′; reverse: 5′-AATTATTTCTAGATCTTAGTGATGGTGATGGTGATGTTGGCAAGTTTCCTTGGCTTGTAAATTTTGGAC-3′). The PCR reaction was carried out in a 50-μl volume consisting of 10 mm Tris-HCl (pH 8.3), 50 mm KCl, 1.5 mm MgCl2, 200 μm each deoxynucleotide triphosphate, 1 μg of wild-type P450 1B1 cDNA, native Pfu DNA polymerase (2.5 units; Stratagene, La Jolla, CA), and each primer at 50 ng·ml−1. The PCR cycles consisted of denaturation at 95°C, annealing at 62°C, and extension at 72°C for a total of 30 cycles. The amplified PCR fragment was digested with EcoRI and XbaI, purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA), and ligated into the EcoRI/XbaI-digested pCW′/1B1/hNPR plasmid. The ligated plasmids were transformed into E. coli DH5αF′Iq, and the correct DNA sequence was judged by restriction enzyme digestion and nucleotide sequence analysis. Picked colonies were grown, and the membranes were prepared as described above. The purification of His-tagged P450 1B1 was performed as described previously (18) with slight modifications. The membranes were solubilized in 0.1 m potassium phosphate (pH 7.7) buffer containing 20% glycerol (v/v), 0.5 M NaCl, 10 mm β-mercaptoethanol, 0.5% sodium cholate (w/v), 1% Triton N-101 (w/v), and 30 μm α-naphthoflavone at 4°C overnight with stirring. The solubilized membranes were centrifuged at 105 × g for 90 min, and the supernatant was applied to a nickel-nitrilotriacetic acid column (Qiagen) that had been pre-equilibrated with 0.1 m potassium phosphate (pH 7.7) buffer containing 20% glycerol (v/v), 0.5 m NaCl, 10 mm β-mercaptoethanol, 0.25% sodium cholate (w/v), and 10 mm imidazole. The column was washed with ≥50 column volumes of the same buffer followed by a second wash with the same buffer containing 40 mm imidazole. The His-tagged P450 1B1 protein was eluted with two column volumes of the same buffer containing 400 mm of imidazole. The eluate was dialyzed against 0.1 m potassium phosphate (pH 7.4) buffer containing 20% glycerol (v/v), 0.25 M NaCl (w/v), 1.0 mm EDTA, and 0.1 mm DTT.

The purified preparation contained 18.1 nmol P450 (mg protein)−1 (protein estimated by quantitative amino acid analysis in the Vanderbilt Protein Chemistry Facility; theoretical value 17.6). The purity of the protein was assessed by SDS-PAGE and Coomassie Brilliant Blue R-250 staining (Fig. 2 A).

EROD Assay.

EROD activity was used as a measure of P450 1A1, 1A2, and 1B1 activities (17, 19). Bicistronic membranes containing 5 nm of P450 1A1, 1A2, or 1B1 were added to 0.10 m potassium phosphate buffer (pH 7.4) containing 2 μm ethoxyresorufin and varying concentrations of TMS. For the reconstitution studies, purified P450 1B1 enzymes (20 nm) were mixed with a 2-fold molar excess of rabbit liver NADPH-P450 reductase, 0.01% sodium cholate (w/v), and 5 μg of l-α-dilauroyl-sn-glycero-3-phosphocholine·ml−1. The reaction mixtures were preincubated at 37°C for 3 min. The reactions were initiated by addition of an NADPH-generating system consisting of 5 mm glucose 6-phosphate, 0.5 mm NADP+, and 0.5 unit glucose 6-phosphate dehydrogenase ml−1. Incubations were performed in a shaking water bath at 37°C for 10 min and terminated by addition of 1 ml of CH3OH. The formation of resorufin was determined fluorometrically with a Perkin-Elmer LS 5 spectrofluorometer (with excitation and emission wavelengths of 550 nm and 585 nm, respectively).

E2 Hydroxylation Assay.

E2 hydroxylation activities were determined as described (20, 21) with slight modifications. The incubation mixture (final volume of 0.25 ml) consisted of 0.10 m potassium phosphate buffer (pH 7.4) containing 1.0 mm ascorbic acid; bicistronic membranes containing 100 nm P450 1A1, 1A2, 1B1, or 3A4; an NADPH-generating system (see above); and 100 μm E2. For the reconstitution studies, purified P450 1B1 enzymes (200 nm) were mixed with a 2-fold molar excess of rabbit liver NADPH-P450 reductase, 0.01% sodium cholate (w/v), and 5 μg l-α-dilauroyl-sn-glycero-3-phosphocholine ml−1. Incubations were carried out at 37°C for 30 min and terminated by adding 1 ml of CH2Cl2. The organic phase was taken to dryness under a nitrogen stream. The residue was dissolved in CH3CN. The reaction products were separated by HPLC on a 5-μm Ultrasphere C18 column (4.6 × 250 mm; Beckman, Fullerton, CA) with a linear gradient increasing from 20% CH3CN (v/v) in 1% CH3CO2H (v/v) to 30% CH3CN(v/v) in 1% CH3CO2H (v/v) over 20 min then for an additional 10 min with 30% CH3CN (v/v) in 1% CH3CO2H (v/v) with UV detection at 280 nm. The flow rate was 2.5 ml/min−1.

NADPH Dependence of Inhibition.

Bacterial membranes containing human P450 1B1 (5 nm) and NADPH-P450 reductase were preincubated in 0.10 m potassium phosphate (pH 7.4) buffer containing various concentration of TMS at 37°C for 10 min in the presence or absence of 1.0 mm NADPH (9). At various times during the preincubation, an aliquot of the preincubation mixture was diluted 10-fold into a second reaction mixture containing 0.10 m potassium phosphate (pH 7.4), 2 μm ethoxyresorufin, and 120 μm NADPH. The mixtures were additionally incubated at 37°C for 10 min. The formation of resorufin was measured by fluorescence assay as described above.

Genotoxicity Assays.

Genotoxicity assays were performed using a reversion strategy for β-galactosidase activity (22, 23, 24). Briefly, pCW′/1B1/hNPR plasmid was transformed into E. coli DJ3109 pNM12 tester strain cells overexpressing bacterial N-acetyltransferase. The cells were plated on Luria Broth plates containing ampicillin (50 μg·ml−1) and chloramphenicol (10 μg ml−1) and incubated at 37°C overnight. A single colony was selected for growth at 30°C in TB medium. P450 1B1 expression was induced by treatment of the cultures with 1 mm IPTG. After a 24-h induction of P450 1B1 expression, 500 μl aliquots of the culture were mixed with 5 ml of 0.7% top agar (w/v) containing 200 nm MeIQ and varying concentrations of TMS (0–100 μm). The mixture was immediately plated on lactose minimal agar. Colonies were counted after a 40-h incubation at 37°C.

Spectral Binding Titrations.

Purified P450 1B1 enzyme was diluted to 1 μm in 0.10 m potassium phosphate (pH 7.4) and divided between two glass cuvettes. A baseline (against the reference cuvette) was recorded (350–500 nm) on an Aminco DW-2a/OLIS spectrophotometer (OLIS, Bogart, GA). TMS (0–300 μm) was subsequently added, and spectra were recorded (350–500 nm) after each addition. The difference in absorbance between the wavelength maximum and minimum was plotted versus the ligand concentration (25).

Data Analysis.

Kinetic parameters were analyzed by nonlinear regression methods with Graph-Pad Prism software (San Diego, CA). A quadratic expression was used when Ki and Ks values were similar to the enzyme concentration, with the assumption that a single molecule of ligand interacted with P450.

Expression and Purification of COOH-terminal His6-tagged P450 1B1.

To facilitate expression and purification of P450 1B1, six histidine residues were introduced just before the termination codon. Yields as high as 200 nmol spectral P450 (liter culture)−1 were routinely detected in the cells. The one-step nickel-nitrilotriacetic acid agarose column chromatography of solubilized membranes purified the protein to near homogeneity (Fig. 2,A). The purified P450 1B1 had a typical Fe2+-CO versus Fe2+ difference spectrum, with little evidence of cytochrome P420 (Fig. 2,B). The absorption maximum was at 447 nm. Examination of the oxidized spectrum indicated that purified P450 1B1 has both high- and low-spin states (Fig. 2,C). Analysis of the Soret band of the oxidized protein by second-derivative spectra indicated the presence of both high- and low-spin Fe3+, as shown by the negative peaks at 390 nm and 418 nm (Fig. 2 D).

Selective Inhibition of P450 1B1 Activity by TMS.

The effect of TMS on EROD activity was determined in human liver microsome samples (HL29, HL97, and HL111). TMS inhibited EROD activities (catalyzed mainly by P450 1A2 in liver) with IC50 values of ∼4 μm (Fig. 3). To determine the selectivity of the effect of TMS on P450 1A1, P450 1A2, and P450 1B1, a bicistronic expression system was used to produce bacterial membranes containing human NADPH-P450 reductase and each of several P450 enzymes. TMS showed a potent inhibitory effect on P450 1B1 (IC50 = 6 nm) and, to a lesser extent, on P450 1A1 (IC50 = 300 nm) and P450 1A2 (IC50 = 3 μm; Fig. 4,A). TMS exhibited 50-fold selectivity for P450 1B1 over P450 1A1 and 500-fold selectivity for P450 1B1 over P450 1A2. These results indicate that TMS is one of the most potent P450 1B1-selective inhibitors discovered to date. TMS also blocked the EROD activity of purified P450 1B1 enzyme reconstituted with purified rabbit NADPH-P450 reductase (Fig. 4 B).

TMS was examined for its ability to inhibit P450 1B1-dependent E2 4-hydroxylation activity (Fig. 5). Significant inhibition by TMS of E2 4-hydroxylation in bicistronic membranes and reconstituted purified enzyme was shown with IC50 values of 90 nm and 390 nm, respectively. Inhibition of E2 2-hydroxylation by TMS was compared with P450 1A1, P450 1A2, P450 1B1, or P450 3A4. TMS selectively inhibited P450 1B1-dependent E2 2-hydroxylation (Fig. 6). The difference in Ki compared with the EROD assays (see above) is attributed to the different substrates and reactions used (IC50 is more sensitive to differences than is Ki). Although the IC50 was higher for E2 4-hydroxylation than EROD activity, it is still submicromolar and indicates strong affinity for P450 1B1.

Mechanism of Inhibition by TMS.

Kinetic studies were performed with human P450 1A1 and 1B1 enzymes to examine the mechanism of inhibition by TMS. Analysis of the mode of inhibition indicated mixed-type inhibition of P450 1A1 with a Ki of 130 nm and competitive inhibition of P450 1B1 with Ki of 3 nm (Fig. 7). To examine the possibility that TMS is a mechanism-based inactivator, preincubation experiments were done (Fig. 8,A). The inhibition of P450 1B1 was not enhanced by preincubation for 10 min. The protective effects of various trapping agents on the inhibition were determined. Trapping agents such as glutathione, N-acetylcysteine, or DTT failed to protect P450 1B1 from inhibition (Fig. 8 B).

Binding Properties of TMS.

Titration of purified P450 1B1 with TMS gave reverse type I spectral changes on binding, suggesting possible coordination of the oxygen to the P450 heme (Fig. 9). The spectra were characterized by an increase in absorbance at 420 nm and a decrease in absorbance at 390 nm. The binding of TMS yielded a Kd of 3 μm.

Metabolism of TMS by P450 1B1.

Incubation of TMS with bicistronic P450 1B1 membranes and NADPH yielded one apparent product at a slow rate (<0.1 min−1 at a TMS concentration of 20 μm, as judged by comparison of peak areas on HPLC). This product, which has not yet been identified, was more polar and had absorbance maxima at 295 and 325 nm (compare with 325 nm for TMS), suggesting that it might be an O-demethylated product. These results indicate that TMS is relatively stable in the presence of P450 1B1. The metabolic stability of TMS has not yet been evaluated in other enzyme systems.

Inhibition of P450 1B1-dependent MeIQ Genotoxicity.

To characterize the effects of TMS on the activation of promutagens by P450 1B1, a β-galactosidase-based E. coli lacZ reversion mutagenicity test was used (23, 26). P450 1B1, NADPH-P450 reductase, and N-acetyltransferase were expressed using a bicistronic expression plasmid (pCW′/1B1/hNPR), and pNM12 plasmid cotransformed into E. coli DJ3109. In this system, metabolic activation of MeIQ by P450 1B1 evokes −2 frameshift mutations in the E. coli lacZ strain and resulted in growth of colonies on lactose minimal medium. The number of MeIQ revertants was significantly reduced in the presence of TMS with an IC50 of <1 μm (Fig. 10). TMS (100 μm) itself did not increase mutations.

Estrogen is a known risk factor in human breast cancer. Human P450 1B1 participates in the metabolism of E2, catalyzing the hydroxylation of E2 primarily at the C-4 position and to a lesser extent at C-2 (5). 4-Hydroxy E2 can be oxidized to E2–3,4-semiquinone and E2–3,4-quinone. E2–3,4-quinone binds exclusively to the N-7 position of guanine and forms 4-hydroxy-E2–1(α, β)-N7-deoxyguanosine, resulting in destabilization of the glycosidic bond and subsequent depurination. Apurinic sites in DNA can yield mutations in critical genes. Ratios of 4-hydroxy:2-hydroxy E2 formation in neoplastic tissues have been reported to be elevated over values in normal breast (27), and the carcinogenic effect of 4-hydroxy E2 in male hamster kidney strongly suggests that 4-hydroxy E2 causes estrogen-induced carcinogenesis (28). P450 1B1 has been detected in 73% of the human breast tumor samples analyzed (29).

Because P450 1B1 is responsible for carcinogenic metabolism of E2 and is regarded as the target enzyme for blocking mammary tumor initiation, its inhibition remains a logical target for breast cancer protection. Availability of potent and selective inhibitors of P450 1B1 should facilitate the development of P450 1B1-targeted cancer chemotherapy and prevention. Recently we found that rhapontigenin (Fig. 1), a hydroxystilbene compound isolated from the oriental medicinal plant Rheum undulatum, showed selective inhibition of human P450 1A1 activity (10). To obtain a potentially selective inhibitor of P450 1B1, the moieties of stilbene structure were chemically modified, and inhibitory effects were determined using recombinant human P450 family 1 enzymes. In this study we demonstrated that TMS is a potent and selective competitive inhibitor of P450 1B1. TMS significantly prevented P450 1B1-dependent P450 activities such as EROD and E2 hydroxylation in P450 1B1-expressing E. coli membranes and the purified enzyme. The result that TMS blocked P450 1B1-dependent MeIQ genotoxicity suggests that inhibition of P450 1B1 activity by TMS can also occur in cells. TMS itself is not mutagenic in this system, up to 100 μm.

Binding studies with purified P450 1B1 suggested TMS is a ligand that directly binds near the heme region of P450 1B1 with high affinity. Binding of TMS produced a reverse type I binding spectrum, i.e., an increase in low-spin Fe3+ and a decrease in high-spin Fe3+. This change involves the movement of a water molecule to the distal ligand position (of Fe3+), presumably triggered by a conformational shift in the protein. However, this change may not be directly correlated to the inhibition. Moreover, the effect Ki values (3 nm for EROD and 90 nm for E2 4-hydroxylation) are less than for TMS binding, arguing that this change does not directly reflect the (competitive) inhibition. Many P450 1B1 inhibitors are rapidly metabolized (30), compromising their effective use in vivo. TMS was not a good substrate for P450 1B1 as judged by the slow rate of disappearance (<0.1 min−1). The metabolic product might be an O-demethylated TMS. The inhibitory effect of the product will need to be determined to understand the mechanism of TMS inhibition. The desmethyl derivative of TMS is known to be a mitochondrial inhibitor (31, 32) but does not appear to be formed rapidly.

Several compounds to act as inhibitors of P450 1B1 have been reported. A potent inhibition of P450 1B1 by α-naphthoflavone, the well-known P450 1 inhibitor was reported (30, 33, 34). However, α-naphthoflavone did not show the selectivity for inhibiting P450 1B1 compared with P450 1A2. Pyrene showed strong inhibition of P450 1B1 with an IC50 of 2 nm but not much selectivity relative to P450 1A2 (30). Homoeriodictyol selectively inhibited P450 1B1 with a relatively high IC50 of 0.24 μm(35).

Thus far, TMS is the most selective and potent compound inhibiting P450 1B1. Inhibition of P450 1B1-dependent E2 metabolism by TMS may be beneficial in breast cancer prevention. We propose that TMS may also be a useful compound for characterizing the enzymatic properties of P450 1B1 because of its strong selectivity among P450 family 1 enzymes.

Fig. 1.

Structures of TMS and rhapontigenin.

Fig. 1.

Structures of TMS and rhapontigenin.

Close modal
Fig. 2.

Electrophoresis and absorbance spectra of purified P450 1B1. A, SDS-PAGE. The Mr was estimated to be 57,000 using protein standards (results not shown). Spectra were recorded with 1 μm protein in 0.10 m potassium phosphate (pH 7.4) buffer containing 20% glycerol (v/v), 0.25 m NaCl (w/v), 1.0 mm EDTA, and 0.10 mm DTT. B, Fe2+-CO versus Fe2+ difference spectrum. C, absolute spectra: Fe3+, Fe2+, and Fe2+-CO forms, as indicated (reduction with excess Na2S2O4). D, second-derivative spectrum of Fe3+ form.

Fig. 2.

Electrophoresis and absorbance spectra of purified P450 1B1. A, SDS-PAGE. The Mr was estimated to be 57,000 using protein standards (results not shown). Spectra were recorded with 1 μm protein in 0.10 m potassium phosphate (pH 7.4) buffer containing 20% glycerol (v/v), 0.25 m NaCl (w/v), 1.0 mm EDTA, and 0.10 mm DTT. B, Fe2+-CO versus Fe2+ difference spectrum. C, absolute spectra: Fe3+, Fe2+, and Fe2+-CO forms, as indicated (reduction with excess Na2S2O4). D, second-derivative spectrum of Fe3+ form.

Close modal
Fig. 3.

Effects of TMS on EROD activity catalyzed by human liver microsomes. Human liver microsome sample HL-29 (•), HL-97 (□), or HL-111(▵) was incubated with TMS for 10 min at 37°C in the presence of NADPH. Assays included the substrate 7-ethoxyresorufin in the presence of the indicated concentrations of TMS. Each point represents the mean of duplicate assays; bars, ± SD.

Fig. 3.

Effects of TMS on EROD activity catalyzed by human liver microsomes. Human liver microsome sample HL-29 (•), HL-97 (□), or HL-111(▵) was incubated with TMS for 10 min at 37°C in the presence of NADPH. Assays included the substrate 7-ethoxyresorufin in the presence of the indicated concentrations of TMS. Each point represents the mean of duplicate assays; bars, ± SD.

Close modal
Fig. 4.

Effects of TMS on P450 family 1-dependent EROD activities. A, bicistronic membranes. EROD activities catalyzed by P450 1A1, 1A2, and 1B1 in bicistronic E. coli membranes were determined in the presence of TMS. P450 concentrations for determination of catalytic activities were 5 nm, and the substrate concentration was 2 μm in all cases. Control activities in the absence of chemical were 20, 5, and 2 nmol of resorufin formed min−1(nmol of P450)−1 for P450 1A1, 1A2, and 1B1, respectively. Assays included EROD by P450 1A1 (•), P450 1A2 (▪), or 1B1 (▵) in the presence of the indicated concentrations of TMS. Each point represents the means of duplicate assays. B, purified P450 1B1. EROD activity of purified P450 1B1 (20 nm) reconstituted with rabbit NADPH-P450 reductase was determined in the presence of various concentrations of TMS.

Fig. 4.

Effects of TMS on P450 family 1-dependent EROD activities. A, bicistronic membranes. EROD activities catalyzed by P450 1A1, 1A2, and 1B1 in bicistronic E. coli membranes were determined in the presence of TMS. P450 concentrations for determination of catalytic activities were 5 nm, and the substrate concentration was 2 μm in all cases. Control activities in the absence of chemical were 20, 5, and 2 nmol of resorufin formed min−1(nmol of P450)−1 for P450 1A1, 1A2, and 1B1, respectively. Assays included EROD by P450 1A1 (•), P450 1A2 (▪), or 1B1 (▵) in the presence of the indicated concentrations of TMS. Each point represents the means of duplicate assays. B, purified P450 1B1. EROD activity of purified P450 1B1 (20 nm) reconstituted with rabbit NADPH-P450 reductase was determined in the presence of various concentrations of TMS.

Close modal
Fig. 5.

Effects of TMS on P450 1B1-dependent E2 4-hydroxylation. A, bicistronic membranes. E2 4-hydroxylation catalyzed by 1B1 in bicistronic E. coli membranes was determined in the presence of TMS. The P450 concentration used for determination of catalytic activities was 100 nm, and the E2 concentration was 100 μm in all cases. Control activities in the absence of chemical were 20 nmol of 4-hydroxy E2 formed min−1(nmol P450)−1 for 1B1. Each point represents the means of duplicate assays. B, purified P450 1B1. Activity of purified P450 1B1 (200 nm) reconstituted with rabbit NADPH-P450 reductase was determined in the presence of TMS.

Fig. 5.

Effects of TMS on P450 1B1-dependent E2 4-hydroxylation. A, bicistronic membranes. E2 4-hydroxylation catalyzed by 1B1 in bicistronic E. coli membranes was determined in the presence of TMS. The P450 concentration used for determination of catalytic activities was 100 nm, and the E2 concentration was 100 μm in all cases. Control activities in the absence of chemical were 20 nmol of 4-hydroxy E2 formed min−1(nmol P450)−1 for 1B1. Each point represents the means of duplicate assays. B, purified P450 1B1. Activity of purified P450 1B1 (200 nm) reconstituted with rabbit NADPH-P450 reductase was determined in the presence of TMS.

Close modal
Fig. 6.

Inhibition by TMS of E2 2-hydroxylation activities catalyzed by P450s 1A1, 1A2, 1B1, and 3A4 in bicistronic E. coli membranes. P450 concentrations for determination of catalytic activities were 100 nm. Formation of 2-hydroxy E2 was determined using HPLC. Control activities in the absence of chemical were 0.4, 1.0, 0.7, and 1.4 nmol of 2-hydroxy E2 formed min−1(nmol of P450)−1 for P450 1A1, 1A2, 1B1, and 3A4, respectively. Assays included E2 2-hydroxylation by P450 1A1 (⋄), P450 1A2 (▪), 1B1 (•), or 3A4 (▴) in the presence of the indicated concentrations of TMS. Each point represents the means of duplicate assays; bars, ± SD.

Fig. 6.

Inhibition by TMS of E2 2-hydroxylation activities catalyzed by P450s 1A1, 1A2, 1B1, and 3A4 in bicistronic E. coli membranes. P450 concentrations for determination of catalytic activities were 100 nm. Formation of 2-hydroxy E2 was determined using HPLC. Control activities in the absence of chemical were 0.4, 1.0, 0.7, and 1.4 nmol of 2-hydroxy E2 formed min−1(nmol of P450)−1 for P450 1A1, 1A2, 1B1, and 3A4, respectively. Assays included E2 2-hydroxylation by P450 1A1 (⋄), P450 1A2 (▪), 1B1 (•), or 3A4 (▴) in the presence of the indicated concentrations of TMS. Each point represents the means of duplicate assays; bars, ± SD.

Close modal
Fig. 7.

Kinetic analysis of P450 1A1 or 1B1 inhibition by TMS. EROD activity was determined with P450 1A1 or 1B1 in E. coli membranes. A, Lineweaver-Burk plot of P450 1A1 inhibition in the presence of TMS. The estimated Ki for TMS (using quadratic analysis) was 110 nm. B, Lineweaver-Burk plot of P450 1B1 inhibition in the presence of TMS. The estimated Ki for TMS (using quadratic analysis) was 3 nm.

Fig. 7.

Kinetic analysis of P450 1A1 or 1B1 inhibition by TMS. EROD activity was determined with P450 1A1 or 1B1 in E. coli membranes. A, Lineweaver-Burk plot of P450 1A1 inhibition in the presence of TMS. The estimated Ki for TMS (using quadratic analysis) was 110 nm. B, Lineweaver-Burk plot of P450 1B1 inhibition in the presence of TMS. The estimated Ki for TMS (using quadratic analysis) was 3 nm.

Close modal
Fig. 8.

A, effect of preincubation with NADPH on inhibition of P450 1B1 activity by TMS. The P450 1B1 bicistronic membranes were preincubated for the indicated times with NADPH (1 mm) and TMS. The concentrations of TMS were 0 (♦), 0.01 (▪), 0.1 (□), and 1 μm (▴). B, effects of the trapping agents on the inhibition of P450 1B1 by TMS. The P450 1B1 bicistronic membranes were preincubated for 10 min with NADPH (1 mm), TMS (10 nm or 50 nm), and the indicated trapping agents (2 mm glutathione, N-acetylcysteine, or DTT) or H2O. At the end of the incubation, aliquots were assayed for EROD activity. Each point represents the mean of duplicate determinations; bars, ± SD. The loss of P450 1B1 activity in the absence of inhibitors has also been observed earlier (30).

Fig. 8.

A, effect of preincubation with NADPH on inhibition of P450 1B1 activity by TMS. The P450 1B1 bicistronic membranes were preincubated for the indicated times with NADPH (1 mm) and TMS. The concentrations of TMS were 0 (♦), 0.01 (▪), 0.1 (□), and 1 μm (▴). B, effects of the trapping agents on the inhibition of P450 1B1 by TMS. The P450 1B1 bicistronic membranes were preincubated for 10 min with NADPH (1 mm), TMS (10 nm or 50 nm), and the indicated trapping agents (2 mm glutathione, N-acetylcysteine, or DTT) or H2O. At the end of the incubation, aliquots were assayed for EROD activity. Each point represents the mean of duplicate determinations; bars, ± SD. The loss of P450 1B1 activity in the absence of inhibitors has also been observed earlier (30).

Close modal
Fig. 9.

Titration of ferric P450 1B1 with TMS. A, spectral changes for P450 1B1 with varying TMS concentration. Purified P450 1B1 (1 μm) was divided into each of two 1-ml glass cuvettes, and a baseline was set. Aliquots of TMS dissolved in DMSO were added to the sample cuvette, and equal volumes of DMSO were added to the reference cuvette. B, plot of ΔA420–390versus concentration of TMS.

Fig. 9.

Titration of ferric P450 1B1 with TMS. A, spectral changes for P450 1B1 with varying TMS concentration. Purified P450 1B1 (1 μm) was divided into each of two 1-ml glass cuvettes, and a baseline was set. Aliquots of TMS dissolved in DMSO were added to the sample cuvette, and equal volumes of DMSO were added to the reference cuvette. B, plot of ΔA420–390versus concentration of TMS.

Close modal
Fig. 10.

Effects of TMS on MeIQ mutagenicity in the E. coli lac system using P450 1B1 activation. DJ3109 pNM12 tester strain cells overexpressing P450 1B1 were treated with 200 nm MeIQ and various concentrations of TMS (0–100 μm), 100 μm TMS, or DMSO alone, as indicated, and plated on minimal lactose agar. The number of revertants was counted after a 40-h incubation at 37°C.

Fig. 10.

Effects of TMS on MeIQ mutagenicity in the E. coli lac system using P450 1B1 activation. DJ3109 pNM12 tester strain cells overexpressing P450 1B1 were treated with 200 nm MeIQ and various concentrations of TMS (0–100 μm), 100 μm TMS, or DMSO alone, as indicated, and plated on minimal lactose agar. The number of revertants was counted after a 40-h incubation at 37°C.

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported in part by NIH Grants R35 CA44353, R01 CA90426, and P30 ES00267 (to Y-J. C., D. K., and F. P. G.) and a Korea Health 21 R&D Project Grant 01-PJ2-PG3-21605-0001 from the Ministry of Health and Welfare, Republic of Korea (to Y-J. C., S. K., and S-K. L.).

3

The abbreviation used are: P450, cytochrome P450; hNPR, human NADPH-P450 reductase; E2, 17β-estradiol; TMS, 2,4,3′,5′-tetramethoxystilbene; HPLC, high-performance liquid chromatography; EROD, ethoxyresorufin O-deethylation; resveratrol, trans-3,4′,5-trihydroxystilbene; TB, Terrific Broth; IPTG, isopropyl-1-thio-β-d-galactopyranoside; MeIQ, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline; DTT, dithiothreitol.

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