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Metabolism of 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by Human CYP1B1 Genetic Variants

School of Public Health/Environmental and Occupational Health Sciences Institute, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey (J.-F.H., X.-Y.H., J.S.H., J.-F.Z., J.-Y.H.); Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, New Jersey (L.A.W.)

(Received May 23, 2007; Accepted January 23, 2008)

	Abstract

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Human cytochrome P450 1B1 (CYP1B1) plays a critical role in the metabolic activation of a variety of procarcinogens, including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The existence of human CYP1B1 missense genetic variants has been demonstrated, but their activities in metabolizing PhIP are unknown. In this study, we expressed 15 naturally occurring CYP1B1 variants (with either single or multiple amino acid substitutions) and determined their activity changes in metabolizing PhIP to its two major metabolites, 2-hydroxyamino-PhIP and 4'-hydroxy-PhIP. Although the PhIP-metabolizing activities of four variants (Ala¹¹⁹Ser, Pro³⁷⁹Leu, Ala⁴⁴³Gly, Arg⁴⁸Gly/Leu⁴³²Val) were comparable with that of the expressed wild-type CYP1B1, five variants (Trp⁵⁷Cys, Gly⁶¹Glu, Arg⁴⁸Gly/Ala¹¹⁹Ser, Arg⁴⁸Gly/Ala¹¹⁹Ser/Leu⁴³²Val, Arg⁴⁸Gly/Ala¹¹⁹Ser/Leu⁴³²Val/Ala⁴⁴³Gly) exhibited more than 2-fold decrease in activity and a reduction in the catalytic efficiency (V_max/K_m) for both N- and 4-hydroxylation of PhIP. Six variants (Gly³⁶⁵Trp, Glu³⁸⁷Lys, Arg³⁹⁰His, Pro⁴³⁷Leu, Asn⁴⁵³Ser, Arg⁴⁶⁹Trp) showed little activity in PhIP metabolism, but the molecular mechanisms involved are apparently different. The microsomal CYP1B1 protein level was significantly decreased for the Trp³⁶⁵, Lys³⁸⁷, and His³⁹⁰ variants and was not detectable for the Ser⁴⁵³ variant. In contrast, there was no difference between the Trp⁴⁶⁹ variant and the wild-type in the microsomal CYP1B1 protein level and P450 content but the Trp⁴⁶⁹ variant totally lost its metabolic activity toward PhIP. The Leu⁴³⁷ variant also had a substantial amount of CYP1B1 protein in the microsomes, but there was a lack of detectable P450 peak and activity. Our results should be useful in selecting appropriate CYP1B1 variants as cancer susceptibility biomarkers for human population studies related to PhIP exposure.

Like most environmental carcinogens, PhIP requires metabolic activation to exert its carcinogenic effects. The metabolism of PhIP leads to the formation of two major metabolites: 2-hydroxyamino-PhIP (N²-OH-PhIP) and 4'-hydroxy-PhIP (4'-OH-PhIP) (Shirai et al., 1997; King et al., 1999) (Fig. 1). N²-OH-PhIP is highly mutagenic in both bacterial and mammalian cells, and its N-acetylated and -sulfated conjugates can form DNA adducts. In contrast, 4'-OH-PhIP is considered to be noncarcinogenic and is eventually excreted through urine after its conjugation with glucuronide or sulfate (Crofts et al., 1997). The metabolic activation of PhIP (i.e., N-hydroxylation) is primarily catalyzed by the enzymes belonging to the cytochrome P450 (P450) 1 family, including CYP1B1 (Crofts et al., 1997). Human CYP1B1 is constitutively expressed in many extrahepatic tissues and often overexpressed in a variety of human cancers (Murray et al., 1997). In addition to PhIP, it catalyzes the activation of some other environmental carcinogens including arylarenes, nitroarenes, arylamines, and polycyclic aromatic hydrocarbons (Shimada et al., 1997). Recently, the existence of the genetic variants of human CYP1B1 has been demonstrated (Stoilov et al., 1998; McLellan et al., 2000; Aklillu et al., 2002; Tanaka et al., 2002). Functional characterization of several CYP1B1 missense variants has been conducted with carcinogen substrates estradiol, benzo[]pyrene, and 7,12-dimethylbenz[]anthracene (Li et al., 2000; Shimada et al., 2001; Aklillu et al., 2002; Aklillu et al., 2005). However, the impact of these and other reported CYP1B1 missense variations on PhIP metabolism is yet unknown. It is important to emphasize that an alteration in catalytic function, induced by amino acid substitution, of a particular enzyme could be substrate-dependent. For example, while in comparison with the Val⁴³² form of CYP1B1, the Leu⁴³² form was reported to have a 3-fold increase in the K_m value for estradiol hydroxylation but little effect on benzo[]pyrene epoxidation (Li et al., 2000).

View larger version (12K): [in this window] [in a new window]   FIG. 1. Metabolic pathways of PhIP and its two major metabolites.

In the present study, we used a heterologous system to express a total of 15 naturally occurring CYP1B1 variant proteins (with either single or multiple amino acid substitutions) (Table 1) and compared them with the wild-type CYP1B1 protein in metabolizing PhIP to its major metabolites, N²-OH-PhIP and 4'-OH-PhIP. Our results are expected to provide direct evidence for the functional significance of these genetic variations in CYP1B1-mediated PhIP metabolism.

	Materials and Methods

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Materials. PhIP and N²-OH-PhIP were purchased from Toronto Research Chemicals (Toronto, ON, Canada). For standard validation, we also obtained N²-OH-PhIP as a gift from NCI Chemical Carcinogen Repository Midwest Research Institute (Kansas, MI). 4'-OH-PhIP was a generous gift from Dr. K. S. Kulp and Dr. M. G. Knize (Lawrence Livermore National Laboratory, Livermore, CA). Glucose 6-phosphate, glucose-6-phosphate dehydrogenase, NADP⁺, -aminolevulinic acid, ferric citrate, and HPLC grade methanol were purchased from Sigma-Aldrich (St. Louis, MO). Bac-to-Bac baculovirus expression system (including pFastbac1 expression vector) and Grace insect cell culture medium were obtained from Invitrogen (Carlsbad, CA). NADPH-P450 oxidoreductase was purified from rat liver microsomes as described previously (Yasukochi and Masters, 1976). A polyclonal rabbit anti-human CYP1B1 antibody was obtained from Gentest Corp. (Boston, MA). Goat anti-rabbit IgG horseradish peroxidase conjugate was obtained from Bio-Rad (Hercules, CA). The ECL-enhanced chemiluminescence reagents for immunoblotting detection were obtained from Amersham Biosciences Inc. (Piscataway, NJ). KOD Hot Start DNA Polymerase was obtained from Novagen (Madison, WI).

Construction of CYP1B1 Variant cDNAs. The wild-type human CYP1B1 cDNA (NM_000104 [GenBank] ) was used as a template to generate the variant cDNAs by site-directed mutagenesis as described previously (Han et al., 2006). The polymerase chain reaction primer sequences for the mutagenesis are shown in Table 2. Additional rounds of mutagenesis were conducted to generate the variant cDNAs containing more than one nucleotide variations. All the variant cDNAs were sequenced after mutagenesis to ensure there were no extra mutations produced during polymerase chain reaction amplification process.

Heterologous Expression and Microsome Preparation. A Bac-to-Bac baculovirus expression system was used for the expression of CYP1B1 proteins. Wild-type and variant cDNAs were individually subcloned into the expression vector pFASTbac1. The reconstructed bacmid DNAs obtained after DNA transpositions in DH10B_AC cells were transfected into Sf9 cells to obtain the recombinant baculovirus particles. Sf9 cells were subsequently infected in large scale with the virus particles for the production of the CYP1B1 proteins. -Aminolevulinic acid and ferric citrate stock solutions (100 mM each, dissolved in Grace medium) were added to the culture medium for a final concentration of 100 µM. The infected Sf9 cells were harvested 72 h after infection and resuspended in phosphate-buffered saline containing 5 mM imidazole, 20% glycerol, and 1 mM freshly added phenylmethylsulphonyl fluoride. Microsomes were prepared from the cells by sonication and differential centrifugation as previously described (Hong et al., 1999). The microsome preparations were stored at –70°C before use. Protein concentrations were determined by the protein assay reagents from Bio-Rad.

Immunoblot Analysis and P450 Content Determination. Microsomal proteins (1 µg) were loaded onto 10% SDS-polyacrylamide gels for electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked for 1 h using 5% (w/v) evaporated milk in Tris-buffered saline containing 0.0005% (v/v) Tween 20. The membrane was probed with a CYP1B1-specific polyclonal antibody (1:3300 dilution) as the primary antibody for 1 h and followed by goat anti-rabbit IgG, conjugated with horseradish peroxidase (1:5000 dilution), as the secondary antibody for 1 h. The immunoblot was visualized by ECL reagents according to the manufacture's protocol (Amersham Biosciences Inc.). Microsomal P450 content was determined by reduced CO-difference spectrum using a UV/visible spectrophotometer as previously described (He et al., 2004).

PhIP Metabolism and Kinetic Analysis. The incubation mixture for PhIP metabolism consisted of 100 mM sodium phosphate buffer (pH 7.4), 5 mM MgCl₂, an NADPH generating system (15 mM glucose 6-phosphate, 1 unit of glucose-6-phosphate dehydrogenase, 1 mM NADP⁺), NADPH-P450 oxidoreductase (reductase/P450 = 1:1 in molar ratio), 100 pmol of CYP1B1, and PhIP in a total volume of 1 ml. After a 5-min preincubation, the reaction was initiated with the NADPH generating system and incubated at 37°C for 30 min. The reaction was terminated by adding one volume of ice-cold methanol. The mixture was then centrifuged at 4°C and filtered. Samples were stored at –70°C prior to analysis by HPLC within 48 h after the preparation. For the determination of kinetic parameters, eight different PhIP concentrations, ranging from 0.3 to 50 µM, were used for incubation. The selection of substrate concentrations for activity assay and the determination of kinetic parameters were based on literature (Crofts et al., 1997) and our pilot experiments. Commercially available human CYP1B1, CYP1A1, and CYP1A2 (Gentest) were used as positive controls to confirm the formation of PhIP metabolites under our assay conditions. Incubation without P450 enzymes or with preinactivated CYP1B1 proteins (adding methanol before initiation of the reaction) was used as a negative control.

HPLC Analysis. PhIP metabolites were analyzed using an HPLC-fluorescence system (Thermo Separation Products Inc., Fremont, CA) consisting of Spectra Physics P4000 Mobile Phase Pump, Spectra Physics AS3000 Autosampler, Spectra Physics FL2000 Fluorescence Detector, a Waters (Milford, MA) µBondapak C18 column (3.9 x 300 mm, 125 Å, 10 µm), a guard cartridge (µBondapak, 125 Å, 10 µm), and software PC1000 for data collection and analysis. The metabolites were eluted with 55% methanol in water containing 0.1% diethylamine (pH 4.0 with acetic acid). The isocratic solvent was held at a flow rate of 1.25 ml/min for 20 min. The sample injection volume was 100 µl. The excitation wavelength was 316 nm, and the emission wavelength was 370 nm. The analytical protocol was able to clearly resolve N²-OH-PhIP and 4'-OH-PhIP in less than 20 min. Concentrations of the PhIP metabolites were determined by calibration curves prepared using a standard solution of N²-OH-PhIP and 4'-OH-PhIP. Under our analytical conditions, the limits of detection were 0.02 pg for N²-OH-PhIP and 3.2 pg for 4'-OH-PhIP. For the validation of HPLC analysis, we used PhIP metabolite standards as well as the positive control and negative control samples from the in vitro metabolism.

	Results

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Heterologous Expression of the Wild-Type and Variant CYP1B1 Proteins. All the variant CYP1B1 cDNAs were successfully constructed and used for protein expression in the Bac-to-Bac baculovirus/Sf9 insect cell system. For most of the variant proteins, immunoblot analysis of the microsomal proteins detected a single protein band with the same expected molecular weight as the wild-type CYP1B1 (Fig. 2A). There were no detectable CYP1B1 proteins in the microsomes prepared from the Sf9 cells infected with the vector only (containing no CYP1B1 cDNA) or the bacmid containing the Asn⁴⁵³Ser variant cDNA. The microsomal CYP1B1 protein levels for the Gly³⁶⁵Trp, Glu³⁸⁷Lys, and Arg³⁹⁰His variants were reproducibly lower than the wild-type and most of the variants.

View larger version (24K): [in this window] [in a new window]   FIG. 2. Heterologous expression of human CYP1B1 cDNAs in Sf9 cells. Microsomal proteins were prepared from the Sf9 cells infected for 72 h with recombinant bacmids containing either wild-type or variant CYP1B1 cDNAs. A, immunoblot analysis. Microsomal proteins (1 µg) of each sample were used for electrophoresis. The membrane was probed with a CYP1B1-specific polyclonal antibody as the primary antibody, followed by goat anti-rabbit IgG conjugated with horseradish peroxidase as the secondary antibody for 1 h. Microsomes from the Sf9 cells transfected with the pFASTBAC1 vector alone (without CYP1B1 cDNA) were used as a negative control (–). B, representative CO-difference spectra. The assay solution containing microsomal proteins (2 µg/µl) was bubbled with CO for 60 s followed by addition of dithionite into the test cuvette and gentle mixing. The absorption spectrum was recorded from 500 to 400 nm. Microsomes prepared from the Sf9 cells expressing wild-type CYP1B1 and the Ser119 and Trp469 variants showed the characteristic P450 absorption peak, whereas the Leu437 only displayed a major absorption peak at 420 nm. wt, wild-type.

Most of the microsomes containing the expressed recombinant CYP1B1 proteins displayed the characteristic P450 absorption peak in CO-difference spectrum analysis with the average of 219 pmol/mg (range: 118–324 pmol/mg). A significant decrease in P450 content was observed in the microsomes from the cells expressing Gly³⁶⁵Trp, Pro³⁷⁹Leu, and Glu³⁸⁷Lys variant proteins with an average of 39 pmol/mg (range: 24–55 pmol/mg) (Table 3). Microsomes from the cells infected with the Arg³⁹⁰His and Asn⁴⁵³Ser variant cDNAs showed no detectable P450 (data not shown) and little or no detectable CYP1B1 proteins (Fig. 2A). The Pro⁴³⁷Leu variant showed a substantial amount of expressed CYP1B1 protein but no detectable P450 peak (Fig. 2B). All these results were confirmed by 3 to 5 independent expression experiments in the Sf9 cells. For the CYP1B1 variants with significantly lower levels of P450 content, the repeated expressions started from the expression vector reconstruction.

Activity of CYP1B1 Variants and Enzyme Kinetics. The formation of N²-OH-PhIP and 4'-OH-PhIP during CYP1B1-mediated PhIP metabolism was determined by a reported HPLC-fluorescence method (Crofts et al., 1997). We modified the original protocol by changing gradient elution (from 33 to 55%) to isocratic elution with 55% methanol. This change resulted in a satisfactory resolution of PhIP, N²-OH-PhIP, and 4'-OH-PhIP in less than 20 min instead of 40 min in the original method, which reduced the possibility of metabolite degradation when a large number of samples were analyzed. As shown in Fig. 3, the retention times of N²-OH-PhIP, PhIP, and 4'-OH-PhIP are 4, 8, and 13 min, respectively.

View larger version (13K): [in this window] [in a new window]   FIG. 3. HPLC chromatograms of PhIP and its metabolites. The metabolites were eluted with 55% methanol in water containing 0.1% diethylamine (pH 4.0 with acetic acid). The isocratic solvent was held at a flow rate of 1.25 ml/min for 20 min. The sample injection volume was 100 µl. The excitation wavelength was 316 nm and the emission wavelength was 370 nm. A, mixture of the PhIP metabolite standards: 4'-OH-PhIP (5 pg) and N2-OH-PhIP (5 pg). B, metabolites produced during the incubation of PhIP (0.63 µM) with microsomes containing expressed wild-type human CYP1B1 enzyme.

When PhIP was incubated with microsomes containing wild-type CYP1B1, the production of N²-hydroxy PhIP and 4'-hydroxy PhIP was linear up to a 60-min incubation time and up to 120 pmol CYP1B1/ml reaction volume (data not shown). We therefore conducted the PhIP-metabolizing activity assay using a 30-min incubation time and 100 pmol CYP1B1/ml reaction volume. For the CYP1B1 variants with no detectable activity, the assay was repeated with increased amount of microsomal proteins (20-fold higher than the wild-type CYP1B1). The activity was determined at both high (10 µM) and low (1 µM) substrate concentrations. Figure 4 shows that, at 10 µM PhIP, the variants Ala¹¹⁹Ser, Pro³⁷⁹Leu, Ala⁴⁴³Gly, and Arg⁴⁸Gly/Leu⁴³²Val displayed almost the same activity as the wild-type CYP1B1 in both N-hydroxylation and 4-hydroxylation of PhIP. The activities of the variants Trp⁵⁷Cys, Gly⁶¹Glu, Arg⁴⁸Gly/Ala¹¹⁹Ser, Arg⁴⁸Gly/Ala¹¹⁹Ser/Leu⁴³²Val, and Arg⁴⁸Gly/Ala¹¹⁹Ser/Leu⁴³²Val/Ala⁴⁴³ were lower than the wild-type CYP1B1. There was no detectable PhIP-metabolizing activity in the variants Gly³⁶⁵Trp, Glu³⁸⁷Lys, Arg³⁹⁰His, Pro⁴³⁷Leu, and Asn⁴⁵³Ser. It is of interest to note that, although the Arg⁴⁶⁹Trp variant showed the same level of CYP1B1 protein and P450 content (190 pmol/mg) as the wild-type, it had a total loss of PhIP-metabolizing activity. This activity profile at 10 µM substrate concentration was consistent with the result with 1 µM substrate concentration (data not shown).

View larger version (18K): [in this window] [in a new window]   FIG. 4. PhIP N-hydroxylation and 4-hydroxylation activity of heterologously expressed wild-type and variant CYP1B1. The activity was determined at 10 µM PhIP. The incubation mixture for PhIP metabolism consisted of 100 mM sodium phosphate buffer (pH 7.4), 5 mM MgCl2, and an NADPH generating system. After a 5-min preincubation, the reaction was initiated with the NADPH generating system and NADPH-P450 oxidoreductase incubated at 37°C for 30 min. The reaction was terminated by adding 1 volume of ice-cold methanol. The results are the average of duplicated experiments with less than 10% variations. A, PhIP N-hydroxylation activity. B, PhIP 4-hydroxylation activity. There were no detectable activities in both N-hydroxylation and 4-hydroxylation of PhIP for the CYP1B1 variants Trp365, Lys387, His390, Leu437, Ser453, and Trp469. wt, wild-type.

	Discussion

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Although previous studies demonstrated that human CYP1A2 is the major enzyme in the metabolic activation of PhIP, it is mainly expressed in the liver (Shimada et al., 1996). In contrast, CYP1B1 is predominantly expressed in extrahepatic tissues, including prostate, mammary gland, and colon (Aklillu et al., 2005). Therefore, metabolic activation of PhIP in situ by CYP1B1 and other extrahepatic enzymes such as CYP1A1 is believed to play a critical role in PhIP-related carcinogenesis in these tissues, such as prostate cancer (Holme et al., 1989; Patterson and Murray, 2002). Because the amino acid substitutions in the CYP1B1 protein induced by genetic variations may alter the enzyme activity and/or protein stability, the present study characterized the functional significance of 15 CYP1B1 genetic variants in PhIP metabolism. As a result, we identified several CYP1B1 missense variants with changes in protein expression level and/or catalytic activity.

The microsomal CYP1B1 protein level was not detectable for the Asn⁴⁵³Ser variant and was significantly reduced for the Gly³⁶⁵Trp, Glu³⁸⁷Lys, and Arg³⁹⁰His variants. These CYP1B1 variants also lost their activity in both N-hydroxylation and 4-hydroxylation of PhIP. Based on a published computer modeling study (Stoilov et al., 1998), Gly³⁶⁵ belongs to the conserved core structures of CYP1B1. Substitution of Gly³⁶⁵ with Trp may interfere with the proper folding and heme-binding of CYP1B1 protein. Both Glu³⁸⁷ and Arg³⁹⁰ residues of CYP1B1 are localized in the conserved helix K, which are one helical turn apart and are predicted to form a salt bridge. Substitutions with Lys and His at these locations probably alter the secondary structure of CYP1B1 protein, which may affect the enzyme in its coupling with NADPH-cytochrome P450 reductase and catalytic function. The lack of detectable Asn⁴⁵³Ser variant protein in our observation is consistent with a recent study in which there was a significant decrease in the level of Asn⁴⁵³Ser variant protein transiently expressed in the COS-1 cells (Bandiera et al., 2005). The same authors also demonstrated that the degradation of the Asn⁴⁵³Ser variant protein by the proteasomal pathway is much faster than the wild-type CYP1B1 protein (Bandiera et al., 2005), suggesting that the substitution of Asn⁴⁵³ with Ser leads to an unstable protein structure. In contrast to a reported finding that the Gly⁶¹Glu variant has a greatly diminished protein stability when it is expressed in Escherichia coli (Jansson et al., 2001), we did not observe a significant change in protein content of this CYP1B1 variant in our expression system.

It is of interest to observe that both the Pro⁴³⁷Leu and Arg⁴⁶⁹Trp variants showed the same protein expression levels in the microsomes as the wild-type CYP1B1 but lost the PhIP-metabolizing activity. However, the involved inactivation mechanisms for these two variants are apparently different. The Pro⁴³⁷Leu variant protein lacks the characteristic P450 absorption peak in CO-difference spectrum analysis, suggesting the amino acid substitution affects the heme binding. This may be explained by the modeling result that Pro⁴³⁷ is localized in the meander region and precedes the heme binding region (Stoilov et al., 1998). On the other hand, the P450 absorption peak was clearly present for the Arg⁴⁶⁹Trp variant, which is consistent with the observation of Jansson et al. (2001) that the stability of this variant protein was normal. As predicted by the same model (Stoilov et al., 1998), Cys⁴⁷⁰ in CYP1B1 is the cysteine that is completely conserved among all the microsomal cytochrome P450 enzymes and provides the axial heme ligand in the heme-binding region. Arg⁴⁶⁹ immediately precedes Cys⁴⁷⁰ and a replacement with Trp, a larger amino acid residue with a rigid side group, may interfere with the normal conformation of the highly conserved cysteine pocket and catalytic function.

According to the CYP1B1 model (Stoilov et al., 1998), Trp⁵⁷ is located in a hinge region and precedes the last proline residue of the hinge. As one of the most conserved amino acid residues in the same region, Gly⁶¹ is also very close to the proline. Previous studies revealed that substitution of the corresponding proline residue in CYP2C11 and CYP2C2 could influence heme incorporation and reduce enzymatic activity (Yamazaki et al., 1993; Chen and Kemper, 1996). Substitution of nonpolar and hydrophobic tryptophan with polar cysteine or replacement of noncharged glycine with charged glutamic acid in this hinge region may interfere with the proper folding of the CYP1B1 protein and result in decreased enzymatic activity as observed in PhIP metabolism.

The CYP1B1 variants Arg⁴⁸Gly/Ala¹¹⁹Ser (CYP1B1.2), Arg⁴⁸Gly/Ala¹¹⁹Ser/Leu⁴³²Val (CYP1B1*6), and Arg⁴⁸Gly/Ala¹¹⁹Ser/Leu⁴³²Val/Ala⁴⁴³Gly (CYP1B1*7) exist as common haplotypes in several different populations (Aklillu et al., 2002; Aklillu et al., 2005). During the enzyme kinetic study of the CYP1B1 variants with reduced PhIP-metabolizing activity, the variants Arg⁴⁸Gly/Ala¹¹⁹Ser/Leu⁴³²Val (CYP1B1.6) and Arg⁴⁸Gly/Ala¹¹⁹Ser/Leu⁴³²Val/Ala⁴⁴³Gly (CYP1B1.7) were found to have the most significant change in the K_m values for N²-OH-PhIP formation (14.8 and 30.1 µM, respectively, versus 3.6 µM in wild-type CYP1B1) but not in the 4'-OH-PhIP formation. Because the Arg⁴⁸Gly/ Ala¹¹⁹Ser variant (CYP1B1.2) showed no K_m value change for PhIP N-hydroxylation and in the metabolism of other substrates (Aklillu et al., 2002; Aklillu et al., 2005), it is reasonable to speculate that the K_m value changes in the variants Arg⁴⁸Gly/Ala¹¹⁹Ser/Leu⁴³²Val (CYP1B1*6) and Arg⁴⁸Gly/Ala¹¹⁹Ser/Leu⁴³²Val/Ala⁴⁴³Gly (CYTP1B1*7) are mainly due to the inclusion of a common Leu⁴³²Val substitution. Ala⁴⁴³ is conserved in the mouse, rat, and human CYP1B1. Inclusion of an additional Ala⁴⁴³Gly substitution in the CYP1B1.7 variant resulted in a further increase in the K_m value for PhIP N-hydroxylation, suggesting that the substitution also affects substrate binding. In addition, the alteration of enzymatic function in CYP1B1 variants is clearly substrate-dependent. Although the capacity for N-hydroxylation of PhIP was significantly decreased in both CYP1B1.6 and CYP1B1.7 variants, a significantly decreased capacity for oxidation of benzo[]pyrene was only observed in the CYP1B1.7 variant, not in the CYP1B1.6 variant (Aklillu et al., 2005).

In summary, the present study demonstrates that several genetic variants of human CYP1B1 have significant changes in the protein expression level and PhIP-metabolizing activity. These results appear to be consistent with the prediction of a published CYP1B1 protein model (Stoilov et al., 1998). Our study provides new information on the role of involved amino acid residues in protein stability and catalysis of CYP1B1. In addition, identification of these functional CYP1B1 variants should help understand the interindividual variations in PhIP metabolism and their potential impact on the risk to human cancers related to PhIP exposure.

	Acknowledgments

 
We thank Yi-Hua Jan for help in the enzyme kinetics study and Emerson Liu for editing the manuscript.

	Footnotes

 
This work was supported by the U.S. Army Medical Research Prostate Cancer Program DAMD 17-02-0250 (J.-Y.H.).

J.-F.H. and X.-Y.H. contributed equally to this work.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.107.016824.

ABBREVIATIONS: HCAs, heterocyclic amines; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; HPLC, high-performance liquid chromatography; P450, cytochrome P450; N²-OH-PhIP, 2-hydroxyamino-PhIP; 4'-OH-PhIP, 4-hydroxy-PhIP.

Address correspondence to: Dr. Jun-Yan Hong, School of Public Health, University of Medicine and Dentistry of New Jersey, Room 385, 683 Hoes Lane West, Piscataway, NJ 08854. E-mail: jyhong{at}eohsi.rutgers.edu

	References

Top Abstract Materials and Methods Results Discussion References

 


Aklillu E, Oscarson M, Hidestrand M, Leidvik B, Otter C, and Ingelman-Sundberg M (2002) Functional analysis of six different polymorphic CYP1B1 enzyme variants found in an Ethiopian population. Mol Pharmacol 61: 586–594.[Abstract/Free Full Text]

Aklillu E, Ovrebo S, Botnen IV, Otter C, and Ingelman-Sundberg M (2005) Characterization of common CYP1B1 variants with different capacity for benzo[a]pyrene-7,8-dihydrodiol epoxide formation from benzo[a]pyrene. Cancer Res 65: 5105–5111.[Abstract/Free Full Text]

Augustsson K, Skog K, Jagerstad M, Dickman PW, and Steineck G (1999) Dietary heterocyclic amines and cancer of the colon, rectum, bladder, and kidney: a population-based study. Lancet 353: 703–707.[CrossRef][Medline]

Bandiera S, Weidlich S, Harth V, Broede P, Ko Y, and Friedberg T (2005) Proteasomal degradation of human CYP1B1: effect of the Asn453Ser polymorphism on the post-translational regulation of CYP1B1 expression. Mol Pharmacol 67: 435–443.[Abstract/Free Full Text]

Chen CD and Kemper B (1996) Different structural requirements at specific proline residue positions in the conserved proline-rich region of cytochrome P450 2C2. J Biol Chem 271: 28607–28611.[Abstract/Free Full Text]

Crofts FG, Strickland PT, Hayes CL, and Sutter TR (1997) Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by human cytochrome P4501B1. Carcinogenesis 18: 1793–1798.[Abstract/Free Full Text]

Edwards RJ, Murray BP, Murray S, Schulz T, Neubert D, Gant TW, Thorgeirsson SS, Boobis AR, and Davies DS (1994) Contribution of CYP1A1 and CYP1A2 to the activation of heterocyclic amines in monkeys and human. Carcinogenesis 15: 829–836.[Abstract/Free Full Text]

Guengerich FP (2000) Metabolism of chemical carcinogens. Carcinogenesis 21: 345–351.[Abstract/Free Full Text]

Gunter MJ, Probst-Hensch NM, Cortessis VK, Kulldorff M, Haile RW, and Sinha R (2005) Meat intake, cooking-related mutagens and risk of colorectal adenoma in a sigmoidoscopy-based case-control study. Carcinogenesis 26: 637–642.[Abstract/Free Full Text]

Hammons GJ, Milton D, Stepps K, Guengerich FP, Tukey RH, and Kadlubar FF (1997) Metabolism of carcinogenic heterocyclic and aromatic amines by recombinant human cytochrome P450 enzymes. Carcinogenesis 18: 851–854.[Abstract/Free Full Text]

Han JF, Wang SL, He XY, Liu CY, and Hong JY (2006) Effect of genetic variation on human cytochrome p450 reductase-mediated paraquat cytotoxicity. Toxicol Sci 91: 42–48.[Abstract/Free Full Text]

He XY, Shen J, Ding X, Lu AY, and Hong JY (2004) Identification of critical amino acid residues of human CYP2A13 for the metabolic activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco-specific carcinogen. Drug Metab Dispos 32: 1516–1521.[Abstract/Free Full Text]

Hecht SS (2002) Tobacco smoke carcinogens and breast cancer. Environ Mol Mutagen 39: 119–126.[CrossRef][Medline]

Holme JA, Wallin H, Brunborg G, Soderlund EJ, Hongslo JK, and Alexander J (1989) Genotoxicity of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP): formation of 2-hydroxyamino-PhIP, a direct acting genotoxic metabolite. Carcinogeneis 10: 1389–1396.[CrossRef]

Hong JY, Wang YY, Bondoc FY, Lee M, Yang CS, Hu WY, and Pan JM (1999) Metabolism of methyl tert-butyl ether and other gasoline ethers by human liver microsomes and heterogously expressed human cytochrome P450: identification of CYP2A6 as a major catalyst. Toxicol Appl Pharmacol 160: 43–48.[CrossRef][Medline]

Jansson I, Stoilov I, Sarfarazi M, and Schenkman JB (2001) Effect of two mutations of human CYP1B1, G61E and R469W, on stability and endogenous steroid substrate metabolism. Pharmacogenetics 11: 793–801.[CrossRef][Medline]

King RS, Teitel CH, Shaddock JG, Casciano DA, and Kadlubar FF (1999) Detoxification of carcinogenic aromatic and heterocyclic amines by enzymatic reduction of the N-hydroxy derivative. Cancer Lett 143: 167–171.[CrossRef][Medline]

Li DN, Seidel A, Pritchard MP, Wolf CR, and Friedberg T (2000) Polymorphisms in P450 CYP1B1 affect the conversion of estradiol to the potentially carcinogenic metabolite 4-hydroxyestradiol. Pharmacogenetics 10: 343–353.[CrossRef][Medline]

Malfatti MA, Kulp KS, Knize MG, Davis C, Massengill JP, Williams S, Nowell S, MacLeod S, Dingley KH, Turteltaub KW, et al. (1999) The identification of [2-(14)C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine metabolites in humans. Carcinogenesis 20: 705–713.[Abstract/Free Full Text]

McLellan RA, Oscarson M, Hidestrand M, Leidvik B, Jonsson E, Otter C, and Ingelman-Sundberg M (2000) Characterization and functional analysis of two common human cytochrome P450 1B1 variants. Arch Biochem Biophys 378: 175–181.[CrossRef][Medline]

Murray GI, Taylor MC, McFadyen MC, McKay JA, Greenlee WF, Burke MD, and Melvin WT (1997) Tumor-specific expression of cytochrome P450 CYP1B1. Cancer Res 57: 3026–3031.[Abstract/Free Full Text]

Nakagama H, Nakanishi M, and Ochiai M (2005) Modeling human colon cancer in rodents using a food-borne carcinogen, PhIP. Cancer Sci 96: 627–631.[CrossRef][Medline]

Ochiai M, Imai H, Sugimura T, Nagao M, and Nakagama H (2002) Induction of intestinal tumors and lymphomas in C57BL/6N mice by a food-borne carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Jpn J Cancer Res 93: 478–483.[CrossRef]

Patterson LH and Murray GI (2002) Tumour cytochrome P450 and drug activation. Curr Pharm Des 8: 1335–1347.[CrossRef][Medline]

Peluso M, Castegnaro M, Malaveille C, Friesen M, Garren L, Hautefeuille A, Vineis P, Kadlubar F, and Bartsch H (1991) 32P Postlabelling analysis of urinary mutagens from smokers of black tobacco implicates 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) as a major DNA-damaging agent. Carcinogenesis 12: 713–717.[Abstract/Free Full Text]

Shimada T, Gillam EM, Sutter TR, Strickland PT, Guengerich FP, and Yamazaki H (1997) Oxidation of xenobiotics by recombinant human cytochrome P450 1B1. Drug Metab Dispos 25: 617–622.[Abstract/Free Full Text]

Shimada T, Watanable J, Guengerich FP, Inoue K, and Gillam EMJ (2001) Specificity of 17β-oestradiol and benzo[]pyrene oxidation by polymorphic human cytochrome P450 1B1 variants substituted at residue 48, 119, and 432. Xenobiotica 31: 163–176.[CrossRef][Medline]

Shimada T, Yamazaki H, Mimura M, Wakamiya N, Ueng YF, Guengerich FP, and Inui Y (1996) Characterization of microsomal cytochrome P450 enzymes involved in the oxidation of xenobiotic chemicals in human fetal liver and adult lungs. Drug Metab Dispos 24: 515–522.[Abstract]

Shirai T, Sano M, Tamano S, Takahashi S, Hirose M, Futakuchi M, Hasegawa R, Imaida K, Matsumoto K, Wakabayashi K, et al. (1997) The prostate: a target for carcinogenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) derived from cooked foods. Cancer Res 57: 195–198.[Abstract/Free Full Text]

Stoilov I, Akarsu AN, Alozie I, Child A, Barsoum-Homsy M, Turacli ME, Or M, Lewis RA, Ozdemir N, Brice G, et al. (1998) Sequence analysis and homology modeling suggest that primary congenital glaucoma on 2p21 results from mutations disrupting either the hinge region or the conserved core structures of cytochrome P4501B1. Am J Hum Genet 62: 573–584.[CrossRef][Medline]

Sugimura T and Sato S (1983) Mutagens-carcinogens in foods. Cancer Res 43: 2415s–2421s3.[Medline]

Tanaka Y, Sasaki M, Kaneuchi M, Shiina H, Igawa M, and Dahiya R (2002) Polymorphisms of the CYP1B1 gene have higher risk for prostate cancer. Biochem Biophys Res Commun 296: 820–826.[CrossRef][Medline]

Ushiyama H, Wakabayashi K, Hirose M, Itoh H, Sugimura T, and Nagao M (1991) Presence of carcinogenic heterocyclic amines in urine of healthy volunteers eating normal diet, but not of inpatients receiving parenteral alimentation. Carcinogenesis 12: 1417–1422.[Abstract/Free Full Text]

Williams JA, Martin FL, Muir GH, Hewer A, Grover PL, and Phillips DH (2000) Metabolic activation of carcinogens and expression of various cytochromes P450 in human prostate tissue. Carcinogenesis 21: 1683–1689.[Abstract/Free Full Text]

Yamazaki S, Sato K, Suhara K, Sakaguchi M, Mihara K, and Omura T (1993) Importance of the proline-rich region following signal-anchor sequence in the formation of correct conformation of microsomal cytochrome P-450s. J Biochem 114: 652–657.[Abstract/Free Full Text]

Yasukochi Y and Masters BS (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.[Abstract/Free Full Text]

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