Regioselective 2-hydroxylation of 17β-estradiol by rat cytochrome P4501B1

ology 216 (2006) 469–478www.elsevier.com/locate/ytaap

Toxicology and Applied Pharmac

Regioselective 2-hydroxylation of 17β-estradiol by rat cytochrome P4501B1

Mostafizur Rahman a,b, Carrie Hayes Sutter a, Gary L. Emmert b, Thomas R. Sutter a,b,⁎

a W. Harry Feinstone Center for Genomic Research, The University of Memphis, Memphis, TN 38152, USAb Department of Chemistry, The University of Memphis, Memphis, TN 38152, USA

Received 10 April 2006; revised 1 June 2006; accepted 5 June 2006Available online 15 June 2006

Abstract

Previous work demonstrated that human cytochrome P4501B1 (CYP1B1) forms predominantly 4-hydroxyestradiol (4-OHE2), a metabolitewhich is carcinogenic in animal models. Here, we present results from kinetic studies characterizing the formation of 4-OHE2 and 2-hydroxyestradiol (2-OHE2) by rat CYP1B1 using 17β-estradiol (E2) as a substrate. Km and Kcat values were estimated using the Michaelis–Menten equation. For rat CYP1B1, the apparent Km values for the formation of 4-OHE2 and 2-OHE2 were 0.61±0.23 and 1.84±0.73 μM; theturnover numbers (Kcat) were 0.23±0.02 and 0.46±0.05 pmol/min/pmol P450; and the catalytic efficiencies (Kcat/Km) were 0.37 and 0.25,respectively. For human CYP1B1, the apparent Km values for the formation of 4-OHE2 and 2-OHE2 were 1.22±0.25 and 1.10±0.26; theturnover numbers were 1.23±0.06 and 0.33±0.02; and the catalytic efficiencies were 1.0 and 0.30, respectively. The turnover number ratio of 4-to 2-hydroxylation was 3.7 for human CYP1B1 and 0.5 for rat CYP1B1. These results indicate that, although rat CYP1B1 is a low Km E2hydroxylase, its product ratio, unlike the human enzyme, favors 2-hydroxylation. The Ki values of the inhibitor 2,4,3′,5′-tetramethoxystilbene(TMS) for E2 4- and 2-hydroxylation by rat CYP1B1 were 0.69 and 0.78 μM, respectively. The Ki values of 7,8-benzoflavone (α-NF) for E2 4-and 2-hydroxylation by rat CYP1B1 were 0.01 and 0.02 μM, respectively. The knowledge gained from this study will support the rational designof CYP1B1 inhibitors and clarify results of CYP1B1 related carcinogenesis studies performed in rats.© 2006 Elsevier Inc. All rights reserved.

Keywords: Cytochrome P4501B1; Estrogen metabolism; 2,4,3′,5′-Tetramethoxystilbene; 7,8-Benzoflavone; 4-Hydroxyestradiol; 2-Hydroxyestradiol; Carcinogenesis

Introduction

Cytochrome P450 (CYP) enzymes catalyze various types ofreactions including the biotransformation of several endogen-ous substrates such as fatty acids, steroids, and cholesterol, aswell as xenobiotics, environmental carcinogens, and mutagens(Guengerich, 2001; He et al., 2005; Yano et al., 2000). CYP1B1

Abbreviations: CYP, cytochrome P450; E2, 17β-estradiol; 4-OHE2, 4-hydroxyestradiol; 2-OHE2, 2-hydroxyestradiol; EQ, equilin; B[a]P-7,8-diol,(−)-Benzo[a]pyrene-7,8-dihydrodiol; RTTC, (±)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol; RTCT, (±)-benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol;TMS, 2,4,3′,5′-tetramethoxystilbene; α-NF, 7,8-benzoflavone; HPLC-ECD,high performance liquid chromatography electrochemical detection; EROD, 7-ethoxyresorufin O-dethylation; DMBA, 7,12-dimethylbenz[a]anthracene.⁎ Corresponding author. W. Harry Feinstone Center for Genomic Research,

The University of Memphis, 201 Life Sciences Building, Memphis, TN 38152,USA. Fax: +1 901 678 2458.

E-mail address: [email protected] (T.R. Sutter).

0041-008X/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.taap.2006.06.004

is the third member of the CYP1 family and is the only knownmember of the CYP1B subfamily (Sutter et al., 1994). HumanCYP1B1 is an important isoform, capable of activating andmetabolizing a wide variety of chemically diverse environ-mental carcinogens and mutagens (Shimada et al., 1996).Orthologous forms of CYP1B1 have also been isolated frommouse and rat (Bhattacharyya et al., 1995; Savas et al., 1994;Walker et al., 1995). The amino acid sequences of human, rat,and mouse CYP1B1 are 80% similar (Bhattacharyya et al.,1995; Walker et al., 1995).

Estrogen is a known risk factor and plays an important role inthe etiology of breast and endometrial cancer (Liehr and Ricci,1996; Rylander-Rudqvist et al., 2003; Sasaki et al., 2003).Womenwith higher levels of exposure to estrogens have a higherrisk of developing breast cancer (Dunning et al., 2004), and areduction in estrogen production in women reduces theincidence of breast cancer (Jefcoate et al., 2000). CYP1B1 ishypothesized to play a role in mammary carcinogenesis becauseof its ability to form endogenous reactive catechol estrogens, as

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470 M. Rahman et al. / Toxicology and Applied Pharmacology 216 (2006) 469–478

well as its ability to activate exogenous procarcinogens(Shimada et al., 1996; Hayes et al., 1996; Liehr and Ricci,1996). Human CYP1B1, a predominantly extrahepatic enzyme(Shimada et al. 1996; Chang et al., 2003; Kim et al., 2004;Choudhary et al., 2005), was shown to be a low Km 17β-estradiol (E2) 4-hydroxylase (Hayes et al., 1996). This enzymefurther oxidizes the catechol estrogens to unstable chemicallyreactive estrogen semiquinones and quinones intermediates(Cavalieri et al., 1997; Dawling et al., 2004). The E2-3,4-quinone, which is formed from the oxidation of the catecholestrogen, 4-hydroxyestradiol (4-OHE2), produces more depur-inating adducts than E2-2,3-quinone, which is formed fromoxidation of 2-hydroxyestradiol (2-OHE2) (Cavalieri et al.,1997; Zahid et al., 2006). The apurinic sites may lead tomutations (Chakravarti et al., 2003; Zhao et al., 2006; Fernandezet al., 2006), cell transformation (Russo et al., 2003), and cancer.4-OHE2 has been implicated in animal models of estrogen-mediated tumor formation (Liehr et al., 1986). In addition,increased E2-4 hydroxylase activity has been measured inhuman breast and uterus tumors compared to normal tissue(Liehr and Ricci, 1996). Furthermore, CYP1B1 protein has beenshown to be increased in a wide range of human malignanttumors. This is of particular interest to the field of cancerresearch because it indicates that CYP1B1 may have a role intumor development and progression (Carnell et al., 2004;McFadyen et al., 1999;Murray et al., 1997; Spivack et al., 2001),providing a potential therapeutic target for the development ofnew anti-cancer drugs and immune-based therapies (Downie etal., 2004; McFadyen et al., 1999; Murray et al., 2001; Chun andKim, 2003). In addition, expression of CYP1B1 protein wasfound in normal human tissues, including breast (Jefcoate et al.,2000;Muskhelishvili et al., 2001), lung (Kim et al., 2004), brain,kidney, prostate, ovary, ectocervix, endocervix, endometrium,lymph node, and liver (Muskhelishvili et al., 2001). Suchexpression in normal tissue, particularly those susceptible to E2-mediated carcinogenesis, suggests that CYP1B1 may be a goodtarget for chemoprevention.

There is a high degree of sequence homology between thehuman, rat, and mouse forms of CYP1B1, yet there areconsiderable differences in their tissue-specific expression,regulation, and metabolic specificity (Bhattacharyya et al.,1995; Savas et al., 1994, 1997; Sutter et al., 1994). The rat isan important animal model for studies of estrogen-mediatedcarcinogenicity. Interpretation of such studies requires a firmunderstanding of estrogen metabolism in this species. Whilethe metabolism of E2 by human CYP1B1 has beenextensively studied (Hayes et al., 1996; Hanna et al., 2000),very little is known about the metabolism of E2 by its orthologin the rat. Because of the postulated significant role ofCYP1B1 in the carcinogenicity of E2, the present investiga-tion was aimed at determining whether rat CYP1B1 is an E2hydroxylase and to compare the kinetics of these reactionswith human CYP1B1.

Whether CYP1B1 is to become a target for chemotherapy orchemoprevention, or both, specific inhibition of CYP1B1 isessential. To this end, 2,4,3′,5′-tetramethoxystilbene (TMS) hasbeen shown to preferentially inhibit human CYP1B1, compared

to other CYP1 enzymes (Chun et al., 2001), while 7,8-benzoflavone [7,8-BF; also known as α-naphthoflavone (α-NF)] non-selectively inhibited CYP1 enzymes (Shimada et al.,1998). Because of the widespread use of the rat in preclinicalstudies of inhibitor effectiveness, we investigated the ability ofTMS, as well as α-NF, to inhibit the enzymatic activities of ratCYP1B1 and directly compared these values with those forhuman CYP1B1.

Materials and methods

Materials. Acetone, nicotinamide adenine dinucleotide phosphate(NADPH), dimethylsulfoxide (DMSO), 7-ethoxyresorufin, resorufin, α-NF,and analytical standards of E2, 2-OHE2, 4-OHE2, and equilin (EQ) werepurchased from Sigma-Aldrich Co. (St. Louis, MO). TMS was purchased fromCayman Chemical Company (Ann Arbor, MI). Analytical standards of (−)-benzo[a]pyrene-trans-7,8-dihydrodiol (B[a]P-7,8-diol) and its metabolites (±)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol (RTTC) and (±)-benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol (RTCT) were purchased from the NationalCancer Institute Chemical Carcinogen Repository (Kansas City, MO).Acetonitrile, methanol (both HPLC grade), and ammonium acetate used inmobile phase were obtained from Fisher Scientific (Pittsburgh, PA). The purewater (18.2 MΩ cm) used to make aqueous solutions and used in mobile phasewas obtained from the Millipore Milli-Q System (Bedford, MA). The nylonmembrane filter (0.2 μm, 47 mm) was purchased from Waters Corporation(Milford, MA). The extinction coefficient (ε) for E2 and 4-OHE2 at 280 nmwas 2 mM−1 cm−1 and the ε for 2-OHE2 was 3.6 mM−1 cm−1 at 289 nm. Theconcentration of each standard solution was cross-checked and corrected by theBeckman Coulter DU® 640 Spectrophotometer (Fullerton, CA). The stock andstandard solutions were prepared in methanol and kept under argon and storedin the dark at −20 °C.

B[a]P-7,8-diol metabolism assay. The recombinant human and rat CYP1B1protein (cDNA from Sprague–Dawley rat) was expressed in Saccharomycescerevisiae as described previously (Hayes et al., 1996). Specific CYP1B1protein content was estimated as described (Mammen et al., 2003; Kim et al.,1998). The average P450 levels (pmol/mg) of human and rat were 32.79 and11.74, respectively. In order to evaluate and to compare these microsomepreparations with our previous studies (Kim et al., 1998), we measured theenzymatic activity of rat and human CYP1B1 towards B[a]P-7,8-diol.Incubation mixtures contained microsomes corresponding to 10 pmol ofCYP1B1 enzyme and a final concentration of 10 μM B[a]P-7,8-diol assubstrate, dissolved in DMSO. Each reaction contained 0.1 M sodiumphosphate (NaPO4) buffer at pH 7.4 with BSA (2 mg/mL), 1.4 mM NADPH,2 mM ascorbic acid, 5 mM MgCl2, and a fixed concentration of B[a]P-7,8-diol (4 μL in DMSO) as substrate. The total reaction volume was 1 mL,yielding a final DMSO concentration of 0.4% in the reaction mixture. Thereaction mixtures were pre-incubated for 3 min at 37 °C followed by theaddition of 10 pmol of CYP1B1 microsomal protein to initiate the enzymereaction. The reactions were incubated for 15 min (reactions were linear tothis time, data not shown) at 37 °C and were stopped immediately by theaddition of 1 mL of ice-cold acetone followed by vortexing for 2 min. Analiquot (10 μL) of internal standard ‘equilin’ (20 μM) was added, and thereactions were again vortexed for 2 min. The reaction mixtures were extractedtwice with 2 mL ethyl acetate and then spun for 5 min at 4302×g. The organicextracts were separated and pooled together. The extracts were evaporated anddried down under nitrogen (N2). The residue was dissolved in MeOH (50 μL)and analyzed by high pressure liquid chromatography (HPLC) withelectrochemical detection (ECD). The metabolites were identified bycomparison to authentic standards. The extraction recovery was between 85and 95%.

HPLC-ECD analysis of B[a]P-7,8-diol and metabolites. Chromatographicmeasurements were performed with Waters Alliance 2695 HPLC systemconsisting of a pump, an autosampler, and an in-line degasser, coupled with fourchannel ESA CoulArray Detector (Model 5600A) using C18 reversed-phase

471M. Rahman et al. / Toxicology and Applied Pharmacology 216 (2006) 469–478

analytical column, Waters Symmetry, 4.6×75 mm, 3.5 μm particle size (WAT066224) and guard column, Waters Symmetry Sentry, 3.9×20 mm, 5 μmparticle size (WAT 054225).

Eluent compositions. The mobile phases used for the analysis of B[a]P-7,8-diol were binary linear gradient elution and composed of two solvent systems.Solvent A contained a mixture of CH3CN:MeOH:H2O:CH3COONH4 (1 M)(15:5:70:10, v/v/v/v) and solvent B contained CH3CN:MeOH:H2O:CH3-

COONH4 (1 M) (50:20:20:10, v/v/v/v). Both solutions were at pH 6.0. Eluentflow rate was 1.0 mL/min using initially 90% A and 10% B. After 5 min,70% A and 30% B, after 10 min, 50% A and 50% B, and finally after20 min, 30% A and 70% B were used. The injection volume was 20 μL. Thecell potentials were set at 600, 675, 750, and 850 mV, respectively. ESAsoftware CoulArray for Windows version 1.04 was used for data acquisitionand processing. Analysis time for B[a]P-7,8-diol and its metabolites was lessthan 20 min. With this method, RTTC and RTCT were well separated.

E2 hydroxylation assay. The aforesaid B[a]P-7,8-diol metabolism assaymethod was followed for E2 hydroxylation assay. Incubation mixtures contained

Fig. 1. HPLC-ECD analysis of hydroxylation of E2 catalyzed by CYP1B1 expressedfrom yeast transformed with vector alone; (B) analysis of equimolar concentrationmixtures contained 30 pmol of microsomal human protein plus 10 μM E2; (D) incub250 mV; 2, 325 mV; 3, 450 mV; 4, 600 mV.

10 pmol of microsomal CYP1B1 enzyme and varying concentrations of E2 (0.0,0.1, 0.3, 1.0, 3.0, 10, and 20 μM) as substrate. The remainder of the assayprotocol was as described for B[a]P-7,8-diol analysis.

HPLC-ECD analysis of E2 and metabolites. Methods to analyze E2 and itsmetabolites by HPLC-ECD were developed and modified slightly from thepreviously described B[a]P-7,8-diol analysis method. The same mobile phasecompositions were used with eluent flow rate of 1.2 mL/min using initially90% A and 10% B. After 5 min, 60% A and 40% B, and finally after 12 min,10% A and 90% B were used. The injection volume was 20 μL. The final setof potentials in the detector was optimized in order to separate mixed standardsof E2, 4-OHE2, 2-OHE2, and EQ (used as internal standard) at cell potentialsof 250, 325, 450, and 600 mV, respectively. The run time for analysis ofestrogens, its metabolites, and equilin was less than 12 min.

Method detection limit (MDL), limit of quantitation (LOQ), and limit of

linearity (LOL). For determining the reliability and applicability of theanalytical method, MDL, accuracy, and precision studies were conducted. TheMDL signifies and characterizes the sensitivity of a particular method for a

in S. cerevisiae. (A) Incubation mixtures containing 213 μg microsomal proteinof mixed standards containing E2, 4-OHE2, 2-OHE2, and EQ; (C) incubationsations mixtures contained 30 pmol of microsomal rat protein plus 10 μM E2. 1,

Fig. 2. Michaelis–Menten curve of human and rat CYP1B1. Incubationmixtures contained 10 pmol of microsomal protein expressed in yeast and 0.0,0.1, 0.3, 1.0, 3.0, 10, and 20 μM E2 as substrate. The curves were obtained byfitting the data to the Michaelis–Menten equation; values for the derivedconstants, Km and Kcat are presented in Table 1. Each point represents anaverage of three determinations, and the error bars indicate standard deviation.(A) human CYP1B1; (B) rat CYP1B1.▪, 4-OHE2; ▴, 2-OHE2.

Table 2IC50 and Ki values of the CYP1B1 inhibitors TMS and α-NF

Inhibitor Rat CYP1B1 Human CYP1B1

Kia (μM) IC50

b (μM) Kia (μM) IC50

c (μM)

4-OHE2

2-OHE2

4-OHE2

2-OHE2

4-OHE2

2-OHE2

4-OHE2

2-OHE2

TMS 0.69 0.78 0.13 0.15 0.43 0.70 0.15 0.71α-NF 0.01 0.02 0.01 0.02 0.13 NED 0.01 NED

NED=not enough data points due to inhibition.a Each chemical was tested with 10 pmol of CYP1B1 in a single

determination at 1, 3, and 5 μM of E2 in the presence of five concentrationsof TMS or α-NF, as described in Materials and methods.b Reaction contained 10 pmol of CYP1B1 in a single determination at 3 μM

of E2 with TMS (0.0, 0.1, 0.3, and 1.0 μM) or α-NF (0.0, 0.03, 0.05, and0.1 μM) as inhibitors.c Reaction contained 10 pmol of CYP1B1 in a single determination at 3 μM

of E2 with TMS or α-NF (0.0, 0.1, 1.0, and 10.0 μM) as inhibitors.


particular analyte. The MDL establishes the lowest concentration of analyte thatcan be statistically distinguished from noise at the 98% confidence level with atwo-tailed test. Accuracy is measured as mean percent recovery, and precision isdetermined as percent relative standard deviation. The MDL, accuracy, andprecision for E2 and its metabolites were calculated using EPA procedures andrecommendations (DBP/ICR, 1996; Federal Register, 2003; Glaser et al., 1981).The LOQ is the lowest concentration at which a quantitative measurement canbe made. The LOL is the highest concentration at which a calibration curvedeparts from linearity.

For RTTC, the MDL was found to be 4.0 pmol per 20 μL injection volume.The average percent recovery, percent standard deviation, and percent relativestandard deviation were 95.53%, 0.58%, and 0.60%, respectively. The LOQ forRTTC was found to be 6 pmol. For E2, the MDL was found to be 2.0 pmol andfor both 4-OHE2 and 2-OHE2 it was 1.0 pmol for per 20 μL injection volumes.The average percent recovery, percent standard deviation, and percent relative

Table 1Estradiol hydroxylation by rat and human CYP1B1

CYP1B1 4-OHE2 2-O

Km (μM) Kcat (pmol/min/pmol P450)

Kcat/Km Km

Rat 0.61±0.23 0.23±0.02 0.37 1.8Human 1.22±0.25 1.23±0.06 1.01 1.1

Data represent mean±SD from triplicate assays at six concentrations of E2 as descsubstrate binding (affinity constant) was determined by the formation of product 4-O

standard deviation varied from 97% to 87%, 0.06% to 0.22%, and 0.06% to0.26%, respectively. The correlation coefficients for the calibration curves usedin the MDL, accuracy, and precision studies were found to be in good linearity(0.99). The LOQ for E2 was found to be 6 pmol, and for 2-OHE2 and 4-OHE2,the LOQs were approximately 5 pmol per 20 μL injection volume. Limits oflinearity were above 300 pmol per 20 μL injection volume.

Data analysis. Kinetic parameters (Km and Kcat) were determined by non-linear regression analysis using GraphPad Prism software version 3.0 (SanDiego, CA).

Inhibition kinetics assay using the microsomal fraction containing recombinant

CYP1B1. Inhibition kinetics of rat and human CYP1B1 by TMS and α-NF were evaluated using concentrations expected to produce 30 to 90%inhibition. A fixed substrate concentration and varying inhibitor concentra-tions were used. Percent inhibition was calculated by comparing theamount of product to that formed without the inhibitor. An IC50 value wasdetermined at the point where 50% inhibition of the enzyme's catalyticactivity occurred. The assay method was similar to those described in previoussections with the addition of inhibitors as follows. The inhibitor (4 μL) wasadded simultaneously with 4 μL of substrate to get the desired concentration ofinhibitor and substrate. Ten picomoles of CYP1B1 microsomal protein wasadded to each reaction. A fixed substrate concentration of 3 μM E2 was chosenbecause we observed that the Vmax of product formation is achieved atapproximately 10 μM substrate concentration (Fig. 2). In addition, the averageKm values for 4- and 2-OHE2 from previous experiments were approximately1.23 μM. We know that Km is the substrate concentration at half Vmax.Therefore, 2×Km (2×1.23=2.46 μM), i.e. 3 μM, was selected for determiningIC50 values (Marangoni, 2003). Inhibition was calculated as percent of productformation compared to the corresponding control (enzyme–substrate reaction)without inhibitors.

To determine the Ki values from Dixon plots (1/rate versus inhibitorconcentration) using human and rat CYP1B1, the inhibitors (TMS and α-NF)were tested at five concentrations in the presence of three substrate

HE2 Kcat ratio(4-OHE2/2-OHE2)

(μM) Kcat (pmol/min/pmol P450)

Kcat/Km

4±0.73 0.46±0.05 0.25 0.50±0.26 0.33±0.02 0.30 3.73

ribed using three separate microsomal preparations. The apparent Km value forHE2 and 2-OHE2.


concentrations. The substrate concentrations of 1, 3, and 5 μM of E2 wereapproximately equal to 0.5×Km, 2×Km, and 4×Km, as determined previously.The accurate values were 0.612 (0.5×Km), 2.45 (2×Km), and 4.89 (4×Km) μM.The concentrations of inhibitors were chosen and varied based on our previouslydetermined IC50 for these two inhibitors. For human CYP1B1, TMS and α-NFconcentrations were 0.0, 0.05, 0.1, 0.3, and 1 μM. For rat CYP1B1, TMSconcentrations were 0.0, 0.05, 0.1, 0.3, and 1 μM and α-NF concentrations were0.0, 0.01, 0.03, 0.05, and 0.1 μM. Linear regression analyses for each of thethree substrate concentrations were plotted on a single graph of 1/rate versusinhibitor concentration for each inhibitor. The rate of product formation wasdetermined as pmol of product per min per pmol of P450. The coordinates of theintersection of regression lines in Dixon plots are −Ki and 1/Kcat. The Ki value isestimated by averaging the results from individual intersections when the threeregression lines did not intersect at a single point.

Results

B[a]P-7,8-diol metabolism by rat and human CYP1B1

To evaluate our microsome preparations, we analyzed themetabolism of B[a]P-7,8-diol for both rat and humanCYP1B1. We had previously shown that rat CYP1B1 hadactivity similar to that of human CYP1B1 for this substrate(Kim et al., 1998). We also observed here that rat CYP1B1showed similar stereoselectivity in the metabolism of B[a]P-7,8-diol to that of human CYP1B1. The turnover numbers for

Fig. 3. Dixon plots for the inhibition of human CYP1B1 by TMS and α-NF. Each inhi1, 3, and 5 μM of E2. Reactions contained 10 pmol of CYP1B1 and were run for 15 mhuman CYP1B1 (4-OHE2). 1 μM α-NF was not plotted due to its complete inhibit

the formation of major tetrol metabolite RTTC produced bythe oxidation of B[a]P-7,8-diol by both human and ratCYP1B1 were 1.13±0.22 and 1.93±0.10 pmol/min/pmolP450 (n=3) for human and rat CYP1B1, respectively. The ratCYP1B1 and human CYP1B1 rates are similar and consistentwith our previously reported results (Kim et al., 1998;Mammen et al., 2003).

E2 metabolism by rat and human CYP1B1

To determine the activity of rat CYP1B1 towards E2, weanalyzed extracts of microsomal incubations by HPLC/ECD.To directly and accurately compare the activities of ratCYP1B1 with those of human CYP1B1, human CYP1B1was analyzed at the same time. Representative chromatogramsare shown in Fig. 1. These analyses showed that, like humanCYP1B1, the rat enzyme produced 4-OHE2 and 2-OHE2when the concentration of E2 was 10 μM (Fig. 1D). Incontrast to human CYP1B1, where the product ratio of 4- to 2-OHE2 was 4:1 (Fig. 1C), the product ratio for rat CYP1B1was close to 1:2 (Fig. 1D). Plasmid control microsomes didnot produce any metabolic products (Fig. 1A). The reactionkinetics were determined for human and rat CYP1B1 usingseven different concentrations of E2, in triplicate. The

bitor was tested in a single determination at five concentrations in the presence ofin at 37 °C. (A) human CYP1B1 (4-OHE2); (B) human CYP1B1 (2-OHE2); (C)ion of 4-OHE2. ▾, 1 μM; ▴, 3 μM; ■, 5 μM E2.


Michaelis–Menten curves of human and rat CYP1B1 areshown in Fig. 2. The resulting Km and Kcat values arepresented in Table 1. Human CYP1B1 forms predominantly 4-OHE2, and the results are fully consistent with earlier reportedwork (Hayes et al., 1996). The apparent Km value for 4-OHE2was 1.22±0.25 μM; the turnover number was 1.23±0.06 pmol/min/pmol P450; and the catalytic efficiency(Kcat/Km) was 1.01. The apparent Km value for 2-OHE2 was1.10±0.26 μM; the turnover number was 0.33± 0.02 pmol/min/pmol; and the catalytic efficiency was 0.30. The turnovernumber ratio of 4- to 2-hydroxylation was found to be 3.7 forhuman CYP1B1. For rat CYP1B1, 2-OHE2 was the predomi-nant product. The apparent Km value for 4-OHE2 was 0.61±0.23 μM; the turnover number was 0.23±0.02 pmol/min/pmolP450; and the catalytic efficiency was 0.37. The apparent Km

value for 2-OHE2 was 1.84±0.73 μM; the turnover number was0.46±0.05 pmol/min/pmol P450; and the catalytic efficiencywas 0.25. The turnover number ratio for rat CYP1B1 4- to 2-hydroxylation was 0.5.

Inhibition of CYP1B1 activity by TMS and α-NF

Inhibition properties of rat and human CYP1B1 by TMS andα-NF were determined using microsomal preparations ofrecombinant CYP1B1 expressed in yeast. Effects of TMS andα-NF on rat and human CYP1B1 dependent E2 hydroxylation

Fig. 4. Dixon plots for the inhibition of rat CYP1B1 by TMS and α-NF. Each inhibof 1, 3, and 5 μM of E2. Reactions contained 10 pmol of CYP1B1 and were run f(C) rat CYP1B1 (4-OHE2); (D) rat CYP1B1 (2-OHE2). 0.1 μM α-NF was not p1 μM; ▴, 3 μM; ▪, 5 μM E2.

were examined. The IC50 values of TMS and α-NF arepresented in Table 2. α-NF was a more potent inhibitor thanTMS. The IC50 values for TMS for rat CYP1B1 E2 4- and 2-hydroxylation were found to be 0.13 and 0.15 μM, respectively.The IC50 values for rat CYP1B1 E2 4- and 2-hydroxylation byα-NF were found to be 0.01 and 0.02 μM, respectively. TheIC50 values for TMS for human CYP1B1 E2 4- and 2-hydroxylation were found to be 0.15 and 0.71 μM, respectively.Inhibition of human CYP1B1 E2 4-hydroxylation by α-NF wasobserved with an IC50 value of 0.01 μM.We could not calculatethe IC50 value of α-NF for human 2-hydroxylation due to stronginhibition and lack of enough data points.

Dixon plots were used to demonstrate the mechanism ofinhibition of TMS and α-NF and also to estimate thedissociation constant, Ki, for binding of inhibitor to enzyme.The intersection of the regression lines in the upper leftquadrant of a Dixon plot indicates competitive inhibition(Dixon, 1953). Figs. 3 and 4 show Dixon plots for TMS andα-NF by human and rat CYP1B1. The Ki values of TMS andα-NF for both E2 4- and 2-hydroxylation by human and ratCYP1B1 were determined except for that of α-NF for E2 2-hydroxylation by human CYP1B1 because there were notenough data points due to the strong inhibition and low rate ofproduct formation. The Ki values of E2 4- and 2-hydroxylationby TMS and α-NF of both human and rat CYP1B1 are alsoshown in Table 2. The Ki values of TMS for E2 4- and 2-

itor was tested in a single determination at five concentrations in the presenceor 15 min at 37 °C. (A) Rat CYP1B1 (4-OHE2); (B) rat CYP1B1 (2-OHE2);lotted due to its complete inhibition of 4-OHE2; rat CYP1B1 (2-OHE2). ▾,

Fig. 5. E2 metabolism by rat adrenal microsomes and inhibition by α-NF. Ratadrenal microsomes were incubated with 10 μM of E2 plus 1 μM α-NF andwithout α-NF. Reactions were carried out for 15 min at 37 °C.


hydroxylation by human CYP1B1 were determined to be 0.43and 0.70 μM, and the Ki values of TMS for E2 4- and 2-hydroxylation by rat CYP1B1 were found to be 0.69 and0.78 μM, respectively. For human CYP1B1, the Ki value of α-NF for E2 4-hydroxylation was 0.13 μM. The Ki values of α-NF for E2 4- and 2-hydroxylation by rat CYP1B1 were 0.01and 0.02 μM, respectively.

E2 metabolism by rat adrenal microsomes and inhibition byα-NF

To determine the activity of rat CYP1B1 protein inextrahepatic tissues, E2 metabolism and its inhibition by α-NFwere studied in rat adrenal microsomes, a tissue where CYP1B1is highly expressed (Bhattacharyya et al., 1995; Walker et al.,1995). Rat adrenal microsomes formed 4-OHE2 as thepredominant product. The rates of formation of 4-OHE2 and2-OHE2 were 8.6 and 6.1 pmol/min/mg protein, respectively.However, α-NF completely inhibited the E2 2-hydroxylationactivity of rat adrenal microsomes, but only 19% of 4-hydroxylation (Fig. 5), supporting our observations thatCYP1B1 favors 2-hydroxylation and is strongly inhibited byα-NF.

Discussion

The kinetic parameters of E2 hydroxylation catalyzed byheterologously expressed rat CYP1B1 protein demonstratethat, although rat CYP1B1 is a low Km estrogen hydroxylase,its product ratio favors 2-hydroxylation over 4-hydroxylation.This is in contrast to the predominantly E2 4-hydroxylaseactivity of human CYP1B1, which we previously determined(Hayes et al., 1996) and repeated here for a directcomparison to rat CYP1B1. Moreover, the relative rates ofestrogen 4-hydroxylation by rat CYP1B1 were approximately5 times lower than that of the human enzyme, while therelative rates of 2-hydroxylation were approximately 1.5times greater than human CYP1B1. This is the first studyinvestigating the metabolism of E2 specifically by ratCYP1B1. Previously, mouse CYP1B1 enzyme expressed in

E. coli was reported to show no activity towards estrogens(Savas et al., 1997).

Such a difference between rat and human CYP1B1 activityis an important factor in using rat models to study the roles ofspecific E2 metabolites in human carcinogenesis anddetermining the effectiveness of CYP1B1 inhibitors aschemotherapeutic or chemopreventive agents. CYP1B1 hasbeen implicated in E2-mediated carcinogenesis because inhumans it predominantly catalyzes the formation of 4-OHE2,a metabolite that not only binds and activates the estrogenreceptor (Van Aswegen et al., 1989), but also generatesreactive species capable of binding DNA and formingdepurinating adducts (Zahid et al., 2006; Cavalieri et al.,1997). The apurinic sites that result from these adducts maycause mutations (Zhao et al., 2006; Fernandez et al., 2006)that could ultimately lead to cell transformation (Russo et al.,2003) and cancer. In an animal model of carcinogenesis, 4-OHE2 was carcinogenic in the hamster kidney, where 2-OHE2 was not (Liehr et al., 1986). Furthermore, elevated 4-hydroxylase, but not 2-hydroxylase, was measured in breast(Liehr and Ricci, 1996) and uterine tumor samples (Liehr etal., 1995), when compared to normal tissues. Detection ofCYP1B1 in tumor cells of multiple organs and not in normaltissues indicates that CYP1B1 may be an effective target forchemotherapy (Murray et al., 1997). However, additionalstudies have demonstrated that human CYP1B1 is expressedin normal tissues (Jefcoate et al., 2000; Kim et al., 2004;Muskhelishvili et al., 2001), suggesting that CYP1B1 mayalso be a target for chemoprevention.

In addition to metabolizing E2, human CYP1B1 is knownto activate numerous chemical procarcinogens (Shimada etal., 1996). Here, and in our previous study (Kim et al., 1998),we demonstrated that the catalytic activity of rat CYP1B1was similar to that of human CYP1B1 in the metabolism ofthe carcinogen benzo[a]pyrene, as well as its metabolite,benzo[a]pyrene-7,8-diol, indicating that the rat is a goodmodel for studying chemical carcinogenesis. The importanceof CYP1B1 to chemical carcinogenesis was clearly demon-strated by the resistance of CYP1B1-null mice to 7,12-dimethylbenz[a]anthracene-induced lymphomas (Buters et al.,1999). Therefore, inhibition of CYP1B1 may be important notonly to the field of estrogen-mediated carcinogenesis but tochemical carcinogenesis as well.

In the present study, we demonstrated that α-NF was amore potent inhibitor of both rat and human CYP1B1 thanTMS. Furthermore, it was observed that α-NF more stronglyinhibited rat CYP1B1 than human CYP1B1. In contrast, TMSwas found to be a stronger inhibitor of human CYP1B1 thanof rat CYP1B1. A number of chemicals including TMS andα-NF have been tested to inhibit the activity of hetero-logously expressed human CYP1B1, mostly by determiningIC50 or Ki values for EROD activity (Chan et al., 2003;Chang et al., 2001; Chun et al., 2001; Don et al., 2003;Doostdar et al., 2000; Green et al., 2004; Henderson et al.,2000, Mammen et al., 2005; Pang et al., 1999; Piver et al.,2003; Rochat et al., 2001; Shimada et al., 1998; Shimada etal., 1997; Sparfel et al., 2004; Takahashi et al., 2002). Chun


et al. (2001) examined the inhibition of human CYP1B1expressed in E. coli by TMS using the EROD assay andconcluded that TMS is a potent and selective competitiveinhibitor of CYP1B1. They also evaluated by HPLC-UV theability of TMS to inhibit human CYP1B1 dependent 4-hydroxylation using membranes of CYP1B1 expressing E.coli and CYP1B1 purified from E. coli. They estimated theIC50 values for the inhibition of 4-OHE2 formation to be90 nM and 390 nM, respectively. Inhibition of E2 2-hydroxylation was only investigated using the membranesfrom CYP1B1 expressing E. coli. The IC50 value for theinhibition of 2-OHE2 formation was approximately 200 nM.These authors did not determine Ki values for 4- and 2-OHE2. Our results for the IC50 values of 4- and 2-OHE2 onthe inhibition of human CYP1B1 by TMS were 150 and710 nM, respectively, which were slightly higher than thosereported by Chun et al. (2001). Shimada et al. (1998) reportedan IC50 value of 5 nM for α-NF by EROD assay andsuggested that it was a competitive inhibitor of humanCYP1B1 expressed in E. coli. Our IC50 value using α-NF toinhibit the formation 4-OHE2 was 10 nM, which is similar tothese reported results.

In addition to studies on recombinant CYP1B1, we studiedmetabolism in microsomes prepared from female Sprague–Dawley (SD) rat adrenal gland, where CYP1B1 is constitu-tively expressed (Bhattacharyya et al., 1995; Walker et al.,1995). Our results indicate that rat adrenal microsomes form4-OHE2 as the predominant product. We found that α-NFcompletely inhibited the E2 2-hydroxylase activity of ratadrenal microsomes, but only 19% of 4-hydroxylase activity.Thus, in rat adrenal microsomes, 81% of the 4-OHE2 isformed by enzymes other than CYP1B1 (Fig. 5). Thecomplete inhibition of 2-OHE2 activity by α-NF supportsour observation that 2-hydroxylation is the predominant formof catechol estrogen produced by rat CYP1B1. It isnoteworthy that the total level of E2 hydroxylation found inthe adrenal is low compared to liver microsomes from femaleSD rats, where the rates of formation of E2 2- and 4-hydroxylation by liver microsomes have been reported to be343.5 and 14.5 pmol/min/mg microsomal protein, respectively(Mesia-Vela et al., 2002).

In summary, the results presented here demonstrate that thetotal E2 4- and 2-hydroxylase activity of rat CYP1B1 isapproximately 40% of human CYP1B1 and that rat CYP1B1predominantly forms 2-OHE2, with its rate of formation closeto 1.5 times that of human CYP1B1. As with human CYP1B1,TMS and α-NF are potent inhibitors of rat CYP1B1 activity andanimal studies to determine inhibitor effects on estrogen-mediated or chemical carcinogenesis will be informed by theresults presented here.

Acknowledgments

We thank Ms. Kimberly Reid, Ms. Sufang Zhang, and Ms.Monali Master for assistance with preparation of microsomes.This work was supported by the W. Harry Feinstone Center for

Genomic Research and grants from the NIH, ES 08148, and theDOD, DAMD 17-03-1-0229.

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