Differential metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 variants
Dieter Schwarz,
Pyotr Kisselev1,
Ingolf Cascorbi,
Wolf-Hagen Schunck2 and
Ivar Roots
Institute of Clinical Pharmacology, University Medical Center Charité, Humboldt University of Berlin, D-10098 Berlin, Germany,
1 Institute of Bioorganic Chemistry, Academy of Sciences Belarus, 220141 Minsk, Belarus, and
2 Max Delbrueck Center for Molecular Medicine, D-13125 Berlin-Buch, Germany
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Abstract
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Cytochrome P450 1A1 (CYP1A1) plays a key role in the metabolism of carcinogens, such as benzo[a]pyrene (B[a]P) and metabolites to ultimate carcinogens. Three human allelic variants, namely wild-type (CYP1A1.1), CYP1A1.2 (I462V) and CYP1A1.4 (T461N), were coexpressed by coinfection of baculovirus-infected insect cells with human NADPH-P450 reductase. These recombinant enzymes (in microsomal membranes) were used to analyze whether CYP1A1 polymorphisms affect catalytic activities towards B[a]P and B[a]P-7,8-dihydrodiol. The complete spectrum of phase I metabolites, including the tetrahydrotetrols resulting from hydrolysis of the ultimate carcinogen, B[a]P-7,8-dihydrodiol-9,10-epoxide, was examined by HPLC. Wild-type enzyme showed the highest total metabolism of B[a]P, CYP1A1.2 was ~50%, and CYP1A1.4 ~70%. Km values for all metabolites with CYP1A1.2 were generally significantly lower than with wild-type enzyme (e.g. B[a]P-7,8-diol formation: 13.8 µM for wild-type, 3.5 µM for CYP1A1.2 and 7.7 µM for CYP1A1.4). Addition of epoxide hydrolase markedly increases the relative diol-to-phenol activities by all three variants. However, CYP1A1.4 exhibits the greatest efficiency to produce diol species. Each variant produced the diol epoxides from B[a]P-7,8-dihydrodiol. CYP1A1.1 exhibited with 10.4 pmol/min/pmol CYP1A1 the greatest total rate for 7,8-diol metabolites followed by CYP1A1.2 (7.2 pmol/min/pmol CYP1A1) and CYP1A1.4 (5.5 pmol/min/pmol CYP1A1). All enzyme variants produced about three times more diol epoxide 2-derived metabolites than diol epoxide 1-derived ones, whereby both rare allelic variants exhibited statistically significantly increased formation of diol epoxide 2. This study showed that the three CYP1A1 variants had different enzyme kinetics properties to produce both the diol metabolites from B[a]P and the ultimate mutagenic species diol epoxide 2 from B[a]P-7,8-dihydrodiol, which must be considered in the evaluation of individual susceptibility to cancer.
Abbreviations: B[a]P, benzo[a]pyrene; CYP, cytochrome P450; CYP1A1, cytochrome P450 1A1; DE1, (±)-B[a]P-r-7,t-8-dihydrodiol-c-9,10-epoxide; DE2, (±)-B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide; 4,5-diol, B[a]P-trans-4,5-dihydrodiol; 7,8-diol, B[a]P-trans-7,8-dihydrodiol; 9,10-diol, B[a]P-trans-9,10-dihydrodiol; DMSO, dimethylsulfoxide; EH, epoxide hydrolase; EROD, 7-ethoxyresorufin O-deethylation; 3-OH, 3-hydroxybenzo[a]pyrene; 9-OH, 9-hydroxybenzo[a]pyrene; OR, human NADPH-P450 reductase; RTCC, (±)-B[a]P-r-7,t-8,c-9,c-10-tetrahydrotetrol; RTCT, (±)-B[a]P-r-7,t-8,c-9,t-10-tetrahydrotetrol; RTTC, (±)-B[a]P-r-7,t-8,t-9,c-10-tetrahydrotetrol; RTTT, (±)-B[a]P-r-7,t-8,t-9,t-10-tetrahydrotetrol; Sf9, Spodoptera frugiperda insect cell line.
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Introduction
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Benzo[a]pyrene (B[a]P), a well-known environmental pollutant, is bioactivated by cytochrome P450 (CYP) enzymes to acquire its mutagenic and carcinogenic properties (1,2). The first step of activation is the formation of B[a]P-7,8-epoxide, followed by hydrolysis by epoxide hydrolase (EH) to the B[a]P-trans-7,8-dihydrodiol (7,8-diol), which is further metabolized by CYP enzymes to the ultimate genotoxic (±)-B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide (DE2) (3,4). Cytochrome P450 1A1 (CYP1A1) and CYP1B1 have been shown to be the principal catalysts of metabolism of B[a]P and other polycyclic aromatic hydrocarbons. In numerous studies (512), CYP1A1 was the enzyme that exhibited the greatest capacity for B[a]P metabolism and particularly for production of DE2. B[a]P was twice as cytotoxic in human than in rat CYP1A1-expressing V79 cells (13).
The human CYP1A1 gene is polymorphic. Beside wild-type CYP1A1*1A (protein: CYP1A1.1) (14), so far seven CYP1A1 alleles have been identified (see URL: http://www.imm.ki.se/CYPalleles), two of which show nucleotide exchange associated with an amino acid exchange near the active site of the enzyme: CYP1A1*2B (protein: CYP1A1.2, I462V variant) (15) and CYP1A1*4 (protein: CYP1A1.4, T461N variant) (16). The nucleotide exchange 2455A>G, which results in the amino acid substitution I462V, was found in Caucasians to be exclusively linked with 3801T>C in forming allele CYP1A1*2B (1518). The latter nucleotide mutation alone forms allele CYP1A1*2A. Allele frequencies of CYP1A1*2A are 6.618.1% in a Caucasian population (1618) and 33% in Japanese (19); respective frequencies of *2B are 2.28.9% (1618) and 19.8% (20). CYP1A1*4 occurs with 2.05.7% of alleles in Caucasians (1618). An increased risk for lung cancer was found in carriers of CYP1A1*2A and *2B alleles following epidemiological investigations (16,1924). A strong association between the CYP1A1*4 allele and the risk of endometrial cancer was reported (25), but not for lung cancer (16). Another study discussed breast cancer susceptibility associated with individual CYP1A1 genotypes (26). However, ethnic, regional or statistical issues complicate elucidation of an association between specific hereditary CYP variations and cancer incidence (17,18,27).
Thus, it is important to assess whether CYP1A1 polymorphism affects catalytic activities towards procarcinogens as substrate. A kinetic analysis of B[a]P and 7,8-diol-B[a]P metabolism by all known variants of CYP1A1 has not yet been reported, even for the wild-type enzyme. Previous in vitro studies characterized B[a]P metabolic rates by CYP1A1.2 in comparison with wild-type CYP1A1 (20,28,29). The CYP1A1.4 variant has been expressed by us recently as a functionally active enzyme with lowered catalytic efficiencies for specific steroid hydroxylations in cumene hydroperoxide-mediated reactions (30).
In this study, we undertake a systematic kinetic analysis of B[a]P metabolism, including B[a]P-7,8-diol as the precursor of DE2 (the ultimate genotoxic compound), by the three known variant CYP1A1 enzymes that differ in amino acid sequence: CYP1A1.1, CYP1A1.2 and CYP1A1.4. For CYP1A1.4 this is, to our knowledge, the first study on B[a]P metabolism. We utilized the baculovirus/insect cell system and the coexpression by coinfection approach (31,32). In this way, high levels of the different human CYP1A1 variants, stoichiometrically combined with coexpressed human NADPH-P450 reductase (OR), could be prepared. The complete spectrum of B[a]P and 7,8-diol-B[a]P phase I metabolites could be analyzed by application of HPLC.
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Materials and methods
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Materials
Spodoptera frugiperda (Sf9) insect cells, the initial baculovirus transfer vector pBlueBac4.5 and linear baculovirus DNA Bac-N-Blue DNA were purchased from Invitrogen (Groningen, The Netherlands). Insect cell medium Excell 401 was from JRH Biosciences (Andover, Hampshire, UK), fetal bovine serum and penicillin/streptomycin from Life Technologies (Karlsruhe, Germany) and the transfection kit from Pharmingen (San Diego, CA). Goat anti-rat CYP1A1 and anti-rat OR antibodies were purchased from Daiichi Pure Chemicals (Tokyo, Japan) and anti-goat secondary antibodies (horseradish peroxidase-labeled) from Boehringer Mannheim (Germany). In western blot analysis we used, as negative and positive control for the expression of CYP1A1 and OR, microsomes prepared from uninfected Sf9 cells, CYP1A1-supersomes (Gentest, Woburn, MA) and microsomes prepared from Sf9 cells expressing only OR, respectively. For supplementation of assays with EH, lymphoblastoid-cell microsomes containing microsomal EH were purchased from Gentest. [7,10-14C]B[a]P was bought from Amersham Pharmacia Biotech (Freiburg, Germany). 7-Ethoxyresorufin, resorufin, B[a]P and
-amino levulinic acid were purchased from Sigma (Deisenhofen, Germany) and 1,1'-bi-2-naphthol from Fluka (Buchs, Switzerland). The racemic 7,8-dihydrodiol and all metabolite standards were purchased from NCI Chemical Carcinogen Repository at Midwest Research Institute (Kansas City, MI) except 3-OH-B[a]P, which was a kind gift from Prof. F.Oesch and Dr A.Seidel (Institute of Toxicology, University of Mainz, Germany).
Construction and preparation of recombinant baculoviruses
Wild-type cDNA for human CYP1A1 was kindly provided by Dr F.J. Gonzalez (National Cancer Institute, NIH, Bethesda, MD). Construction of the CYP1A1.2 and CYP1A1.4 variants, cloning of cDNAs carrying the respective mutation into baculovirus transfer vectors pBlueBac4.5 under control of the polyhedrin promoter, cotransfection of Sf9 insect cells and preparation of large-scale high-titer stocks of recombinant baculovirus for the expression of the variants in insect cells were done as described (30). Recombinant baculovirus for coexpression of OR was kindly provided by Dr F.J.Gonzalez.
Coexpression by coinfection of human CYP1A1 variants and OR in Sf9 insect cells
Sf9 cells (400 ml) seeded at 1.8x106 cells/ml were simultaneously infected at a multiplicity of infection of 3.2 for baculovirus-CYP1A1 and 1.1 for baculovirus-OR. Twenty to twenty-four hours post-infection, media were fortified with
-amino levulinic acid (0.5 mM final). Cells were harvested after ~73 h. The optimal time to harvest was based on CYP content and enzymatic activity assessed by CO difference spectra and 7-ethoxyresorufin O-deethylation (EROD) activity. The harvested cell pellet was washed twice with PBS and resuspended in phosphate buffer. Catalytical studies were performed with microsomes prepared according to Buters et al. (33). Microsomal pellets were resuspended in 100 mM standard phosphate buffer [pH 7.4, 1 mM EDTA, 0.1 mM dithiothreitol, 20% glycerol (vol/vol)], aliquoted and stored at 80°C.
Immunoblot and analytical methods
Microsomal proteins were electrophoresed in 10% SDSPAGE and transferred to a nitrocellulose filter. The filter was incubated overnight with an antibody mixture of 1:1000 dilution of goat anti-rat CYP1A1/1A2 and 1:10 000 dilution of goat anti-rat OR. The blots were developed using 1:10 000 dilution of horseradish peroxidase-labeled anti-goat IgG as secondary antibody and detected using the BM chemiluminescence blotting system (Boehringer Mannheim).
CYP content was measured by reduced-CO minus reduced difference spectroscopy (34) and protein concentration was determined by Coomassie Plus protein assay (Pierce, Rockford, IL). OR activity was determined as NADPH-cytochrome c reductase activity. One unit is defined as the amount of OR reducing 1 µmol cytochrome c per min at 25°C.
Kinetic constants were determined by non-linear analysis using the computer program ENZFITTER (by J.R.Leatherbarrow, Elsevier-Biosoft, 1987). Error ranges represent standard errors based on analysis of at least three different preparations. Statistical significance of results between variants was analyzed using one-way ANOVA (GraphPad Software, San Diego, CA; URL: http://www.graphpad.com).
7-Ethoxyresorufin O-deethylation (EROD) assay
EROD activity was determined fluorometrically (35) (spectrofluorometer RF5000-PC; Shimadzu, Japan). In standard assays, 2.5 pmol of microsomal CYP1A1 were diluted with EROD buffer (50 mM TrisHCl, pH 7.5, 3 mM MgCl2). 7-Ethoxyresorufin dissolved in 10 µl dimethylsulfoxide (DMSO) was added and the solution was preincubated for 3 min at 37°C (final substrate concentration: 4.3 µM). The reaction was started by addition of 10 µl NADPH to give a final concentration of 0.5 mM. After 5 min at 37°C, the reaction was stopped by addition of 2 ml ice-cold acetone. After centrifugation, the fluorescence at 585 nm was recorded. Product was calibrated using authentic resorufin.
B[a]P and 7,8-diol metabolism
B[a]P oxidation assays were based on previous methods (6,36,37). In brief, microsomes containing 20 pmol CYP1A1 and 10 µl DMSO solution containing a mixture of B[a]P and [14C] B[a]P were diluted in EROD buffer (total volume 990 µl) and preincubated for 3 min at 37°C. For certain assays, EH was included in the preincubation mixture because of its effect on the metabolism of B[a]P (37). Typical EH addition was 250 µg per 20 pmol CYP1A1. The reaction was started by addition of 10 µl of NADPH and continued for 30 min at 37°C (final volume: 1 ml, final concentration: 10 µM B[a]P substrate, 0.5 mM NADPH). The reaction was stopped by addition of 1 ml ice-cold acetone. All assays were performed in parallel with the three enzyme variants and with equal CYP concentration, to minimize variation.
Substrate stock solutions were prepared as follows. A certain amount of B[a]P stock solution (8 mM in acetone) was mixed with 14C-B[a]P in toluene (corresponding to 200 000 c.p.m.; sp. act.: 2.0 GBq/mmol). The solvent was evaporated and the B[a]P dissolved in a certain volume of DMSO giving a concentration whereby 10 µl contained the amount of substrate necessary for the assay. After the incubation substrate and products were extracted twice with ethyl acetate. Ethyl acetate (2.5 ml) was added to the reaction suspension, vortexed for 30 s and centrifuged for 20 min. The upper phases were combined, followed by evaporation of the solvent. Residual substrate and products were redissolved with 50 µl of methanol, sonicated in an ultrasonication bath and injected into the HPLC apparatus.
Metabolism of 7,8-diol was assayed analogously with only minor modifications. Substrate stock solutions were prepared in methanol. The reaction was stopped on ice by adding 100 µl of 1 M potassium phosphate buffer (pH 3.5) and held on ice for 1 h. Following this, 10 µl methanol containing 10 µg 1,1'-bi-2-naphthol as internal standard was added, followed by extraction of the substrate and products as described above for the B[a]P assay.
HPLC analysis
B[a]P metabolism was analyzed using an HPLC system (Shimadzu) consisting of two solvent delivery systems LC-10 AD VP, a UV detector SPD-10 A VP and run with CLASS VP software. Separation was performed with a reversed-phase column (Nucleosil 100-5 C18, 5 µm, 250x4 mm; Macherey-Nagel, Dueren, Germany) at 25°C with a flow rate of 0.8 ml/min using a linear methanolwater gradient of 60% methanol rising to 100% in 80 min. 7,8-Diol metabolism was analyzed analogously, at 40°C with a flow rate of 1 ml/min. Initial solvent conditions were 40% methanol with a linear gradient to 47.3% in 35 min followed by isocratic elution with 60% methanol for another 35 min. Absorbance was monitored at 254 nm (B[a]P) and 344 nm (7,8-diol), respectively. 14C radioactivity was evaluated using a radioactivity monitor (LB 507 A, Berthold, Wildbad, Germany). Metabolites were identified by comparison with retention times of authentic standards. The level of each tetrol was determined by comparison with a standard curve generated from the absorption areas of authentic tetroles analyzed in parallel under identical assay and extraction conditions using the internal standard.
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Results
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Characterization of CYP1A1 variants coexpressed with OR in Sf9 insect cells
The three CYP1A1 variants could be successfully coexpressed with OR in Sf9 insect cells as native, spectrally and catalytically active enzymes. Expression levels for CYP1A1 and OR, as determined by CO difference spectroscopy and cytochrome c reduction assay, respectively, were similar for all three CYP1A1 variants. In whole cell extracts using 1.5x107 cells/ml, they were ~1020 nmol CYP/l culture and 160220 U OR/l culture. Formation of cytochrome P420 was negligible. Microsomes with each enzyme variant had a similar stoichiometry to CYP and OR (Table I
).
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Table I. Characteristics of microsomes prepared from Sf9 insect cells coexpressing the human CYP1A1 variants and OR
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The three expressed CYP1A1 variants were assayed for EROD. Table I
illustrates that they were characterized by almost equal Km values; Vmax values for the rare variants were slightly higher, indicating only minor differences in kinetic behavior.
Immunoblot analysis proved expression of all CYP1A1 variants and OR, as revealed by simultaneous probing of the blot with polyclonal goat anti-rat CYP1A1/1A2 and goat anti-rat OR antibodies (Figure 1
, lanes 13). The same antibodies did not detect any immunoreactive proteins in microsomes from uninfected Sf9 cells as negative control (lane 5). As expected, they showed strong immunoreactivity with baculovirus-expressed wild-type CYP1A1 in CYP1A1-supersomes (lane 6, positive control) as well as with OR in microsomes expressing OR exclusively (lane 4).

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Fig. 1. Immunoblots of microsomal preparations of human CYP1A1 variants coexpressed with OR in Sf9 insect cells. Microsomal protein (~7 µg per lane, except for lane 6 which contains 1.7 µg) was subjected to SDSPAGE and transferred to nitrocellulose membranes. Membranes were probed simultaneously with goat anti-rat CYP1A1/1A2 and goat anti-rat OR and detected by the chemiluminescent system using horseradish peroxidase-labeled secondary antibodies. Lanes 13, variants CYP1A1.1, CYP1A1.2 and CYP1A1.4, respectively, each coexpressed with OR. Lane 4, microsomes prepared from cells expressing only OR (positive control for OR). Lane 5, uninfected Sf9 cells as negative control. Lane 6, CYP1A1-supersomes as positive control for CYP1A1. Molecular weight standards are indicated at the left (in kDa).
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B[a]P metabolism by human CYP1A1 variants
CYP1A1 variants were assayed using B[a]P as substrate with and without addition of EH. The B[a]P-trans-9,10-dihydrodiol (9,10-diol), B[a]P-trans-4,5-dihydrodiol (4,5-diol), 7,8-diol, 9-hydroxybenzo[a]pyrene (9-OH), 3-hydroxybenzo[a]pyrene (3-OH) and quinone metabolites were well separated. We could not detect the 7-hydroxybenzo[a]pyrene metabolite eluting shortly before 3-OH under our conditions. The quinones were identified by comparison with published chromatograms recorded under comparable conditions (11). Incubations with control microsomes not expressing CYP1A1 and OR were negative for B[a]P metabolism, as were assays performed without NADPH.
Rates of B[a]P metabolite formation by CYP1A1 variantswith and without EH supplementationare shown in Table II
. In the absence of EH, total phenol product formation was 1.87, 0.85 and 1.11 pmol/min/pmol CYP for CYP1A1.1, CYP1A1.2 and CYP1A1.4, respectively. Diol metabolite formation was determined to be 0.51 (CYP1A1.1), 0.28 (CYP1A1.2) and 0.50 pmol/min/pmol CYP (CYP1A1.4). The 4,5-diol metabolite formation was almost negligible in the absence of EH. Total quinone production was 0.92, 0.42 and 0.72 pmol/min/pmol CYP for CYP1A1.1, CYP1A1.2 and CYP1A1.4, respectively. Thus, the summed rates of phase I metabolites were 2.79, 1.27 and 1.83 pmol/min/pmol CYP for CYP1A1.1, CYP1A1.2 and CYP1A1.4, respectively. Taken together, total metabolism of B[a]P by wild-type enzyme always exhibited the highest activity (3.3 pmol/min/pmol; 100%), CYP1A1.2 was 1.55 (~50%), and CYP1A1.4 2.33 (~70%).
EH was added because it might have a large effect particularly on diol metabolites (37). Our results with insect cell microsomes show that diol and phenol metabolite production was differently affected by EH. Whereas 9,10-diol and 4,5-diol formation was greatly increased, the influence on 3-OH and 9-OH was only slight, and formation of 7,8-diol was almost unaffected. Thus, the overall effect of increased EH activity resulted in an increased formation of diols at the expense of phase I metabolites for all CYP1A1 variants.
Km and Vmax values for major phenol and diol metabolites were determined (Table III
); 4,5-diol formation was too small for a kinetic analysis. Clearly, CYP1A1.2 exhibited the lowest Km values for all reactions towards B[a]P as substrate. For example, in the presence of EH the Km for 3-OHB[a]P formation by CYP1A1.2 is nearly four times lower compared with that of wild-type enzyme, whereas that of CYP1A1.4 is comparable with that of wild-type enzyme. A similar relationship holds for 7,8-diol formation. On the other hand, the Km for 9,10-diol production by CYP1A1.2 is comparable with that for CYP1A1.4, but both are less than half that of the wild-type. Figure 2
depicts the efficiency in the formation of products leading finally to mutagenic species, i.e. the combined efficiencies for (7,8-diol + 9,10-diol) formation related to those for (3-OH + 9-OH) formation. CYP1A1.4 appears to have the greatest capacity in terms of this ratio of diolphenol production. Relative efficiencies of diol formation by wild-type enzyme and CYP1A1.2 are similar in the absence of EH; however, wild-type enzyme exhibited a significantly greater efficiency in the presence of EH (P < 0.05; ANOVA). EH supplementation led to a pronounced overall increase (23-fold) in relative diolphenol activities of all CYP1A1 variants.

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Fig. 2. Relative catalytic efficiencies of diol formation from B[a]P by variants of CYP1A1. Diolphenol formation efficiencies were calculated as the ratio of the sum of catalytic efficiencies (Vmax/Km) of diol formation (7,8-diol + 9,10-diol) and the sum of catalytic efficiencies of phenol formation (3-OH + 9-OH). The ratios represent mean and standard deviation of individual values (n = 4) obtained for each variant by ANOVA.
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7,8-diol metabolism by human CYP1A1 variants
Separation of all 7,8-diol oxidation products could be well achieved according to the HPLC method described in the Material and methods. Each variant of CYP1A1 produced the diol epoxides 1 (syn) and 2 (anti) which finally hydrolyze non-enzymatically to the four tetrol metabolites (±)-B[a]P-r-7,t-8,c-9,c-10-tetrahydrotetrol (RTCC), (±)-B[a]P-r-7,t-8,c-9,t-10-tetrahydrotetrol (RTCT), (±)-B[a]P-r-7,t-8,t-9,c-10-tetrahydrotetrol (RTTC) and (±)-B[a]P-r-7,t-8,t-9,t-10-tetrahydrotetrol (RTTT) (8). Additional minor, unknown oxidation products were present, some of which may represent triols. Assays of incubations with uninfected microsomes as negative control not expressing CYP1A1 showed no production of 7,8-diol metabolites, and neither did assays performed without NADPH (not shown).
The summed rates RTCC+RTCT, representative for (±)-B[a]P-r-7,t-8-dihydrodiol-c-9,10-epoxide (DE1) formation, and RTTC+RTTT, representative for DE2 formation, were measured at three different 7,8-diol concentrations (Table IV
). The rates only slightly increased with substrate concentration indicating approximation of saturation conditions. CYP1A1.1 had the greatest total rate for 7,8-diol metabolism at all substrate concentrations (averaged for the three concentrations investigated: 10.4 pmol/min/pmol CYP), followed by CYP1A1.2 (7.2 pmol/min/pmol CYP) and CYP1A1.4 (5.5 pmol/min/pmol CYP). The DE2-derived metabolites were formed at a clearly higher rate by each CYP1A1 compared with DE1-derived metabolites. DE2:DE1 ratios in Table IV
indicate a slightly higher value for both rare variants as compared with wild-type enzyme.
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Discussion
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Controlled coexpression of CYP1A1 and OR
The baculovirus/insect cell expression system gives relatively high levels of expression of native and functionally active CYP1A1 (33). Using this approach, we had previously expressed the three CYP1A1 variants and characterized their catalytic properties in steroid hydroxylations employing a reconstituted system with cumene hydroperoxide as the oxygen donor (30). In the present study, we took advantage of the coexpression by coinfection approach to prepare CYP1A1 variants using two different recombinant baculoviruses for coexpression of CYP1A1 and OR. To compare activities of the CYP1A1 variantsour major objectiveit was of particular importance that coinfection allowed a fine adjustment of the OR:CYP ratio and that we used simple microsomal preparations. By changing the relative multiplicities of infection by the two viruses we achieved molar OR:CYP ratios of around 20 in microsomes prepared for all variants, thus establishing standardized conditions to elucidate possible differences in catalytic properties of the variants. Microsomes prepared from this system exhibited enzyme-kinetic properties in standard assays (EROD) comparable with those with purified and reconstituted CYP1A1, as expressed in the baculovirus (33) and Escherichia coli system (28), and also comparable with yeast-expressed microsomal CYP1A1 (29).
Differential formation of precarcinogenic B[a]P metabolites by CYP1A1 variants
B[a]P metabolism leads to the formation of phenols, quinones and epoxide-derived diol products. Phenols and quinones have been found to be non- or less mutagenic compared with diols (13). In contrast, diol epoxides, particularly DE2 (the major ultimate carcinogenic species), have been associated with an increased risk of cancer (4,38). We concentrate our discussion on which allelic variant of human CYP1A1 is the most effective catalyst of B[a]P to diols and of 7,8-diol to diol epoxide DE2, as discussed below.
Most active in B[a]P metabolism was wild-type CYP1A1.1, followed by enzyme variants CYP1A1.4. and CYP1A1.2 (Table II
). Here, differential substrate specificity becomes evident, since, conversely, EROD activity tended to be highest in CYP1A1.4 and lowest in wild-type enzyme (Table I
). There is another observation concerning substrate specificity. Table III
shows that Km values of B[a]P for production of all measured metabolites were lowest with CYP1A1.2 (up to a quarter of wild-type enzyme). This enzyme species is distinct due to an I462V amino-acid exchange just close to the region of substrate binding. Interestingly, the neighboring change of T461N, occurring in CYP1A1.4, is of lesser consequence on Km. Such differential Km values were not observed with 7-ethoxyresorufin (Table I
), but were also seen with steroid hormones as substrates (30).
The total B[a]P metabolism rate determined in our study is roughly comparable with data reported by Kim et al. (11) for insect-cell expressed human wild-type CYP1A1. Prior data on B[a]P metabolism by other variants of CYP1A1 are from Kawajiri et al. (20) who found 1.5-fold higher aryl hydrocarbon hydroxylase activity for CYP1A1.2 compared with wild-type enzyme. Likewise, Persson et al. (29) investigated the formation of 3-OH-B[a]P, but observed no difference in activities of both variants. On the other hand, Zhang et al. (28) measured rates of CYP1A1.1- and CYP1A1.2-mediated diol formation for higher B[a]P concentrations. While the authors could not detect any metabolites at 10 µM B[a]P, their data for diol formation at 40 and 100 µM are similar to our results for 10 µM B[a]P. As in our study, CYP1A1.2 was clearly the enzyme variant exhibiting a significantly smaller activity (about half) compared with wild-type. Why our data and those of Zhang et al. disagree with those of Kawajiri et al., who used a yeast-expression system with relative low level of CYP expression, is not obvious.
There are interesting differences between the CYP1A1 variants (Table II
). Total diol formation by CYP1A1.1 and CYP1A1.4 was approximately equal, whereas that by CYP1A1.2 was only half. However, the markedly lower rates of phenol formation by CYP1A1.4 compared with CYP1A1.1 are contrasted by preferred formation of premutagenic diol products by the CYP1A1.4 variant. These differences become evident by comparing the diolphenol formation efficiency of CYP1A1 variants (Figure 2
). CYP1A1.4 is the variant exhibiting the highest relative diol product-formation potency.
It has been known for a long time that EH greatly affects the pattern of B[a]P metabolites (37,8). From our studies it is evident that the formation of 9,10-diol is greatly increased, as shown in Tables II and III
for all three enzyme variants. Formation of phenols is little affected by EH. Therefore all CYP1A1 variants showed a marked increase in the diolphenol formation efficiency (Figure 2
). Thus, EH activity shifts B[a]P metabolism towards more promutagenic products. The finding that considerable amounts of diols were present even in the absence of added EH suggests that our microsomal preparations contained some constitutively expressed EH activity. Whereas we have not determined such activity directly, the data are in accordance with observations of Kim et al. (11) who found for the wild-type CYP1A1 expressed in insect Sf9 cells remarkable amounts of dihydrodiols in the absence of EH as well. Another finding supports this assumption. The absence of 7-OH-B[a]P observed in assays without EH can be best understood assuming preferable transformation by endogenous EH of the 7,8-epoxide to the 7,8-dihydrodiol. This interpretation is supported by a report by Shou et al. (8). In this report, a striking effect was shown after coexpression of EH with CYP1A1 in lymphoblastoid cells, particularly on 7-OH-B[a]P formation, which decreased to one eighteenth.
Differences in ultimate carcinogen DE2 formation among CYP1A1 variants
In previous studies, wild-type CYP1A1 was reported to produce ~2- to 2.6-fold more DE2 than DE1 from 7,8-diol (8,11). Our data are in agreement with this, although our absolute velocities are slightly higher. Since DE2 is the ultimate carcinogenic species that binds to DNA, it is important to note that wild-type enzyme exhibits the highest production rates of DE2 (Table IV
). However, comparing the relative formation of diol epoxides in terms of the ratio DE2:DE1, both rare allelic variants show a significantly increased potency in the production of the ultimate carcinogenic DE2 species.
Impact on cancer susceptibility
We conclude that CYP1A1 polymorphisms markedly affect the catalytic properties of the enzyme in both the formation of diols from B[a]P and in the production of the ultimate carcinogenic DE2 from 7,8-diol as precursor substrate. Considering only formation rates, wild-type CYP1A1 may be associated with higher carcinogenic risk than the mutated enzyme variants. Under physiological conditions, however, only low intracellular concentrations of B[a]P might be assumed. Here, the low Km of CYP1A1.2 (3.5 versus 13.8 µM of wild-type) might result in relatively higher 7,8-diol formation rates in vivo. Additionally, the significantly increased potency of both rare allelic variants, CYP1A1.2 and CYP1A1.4, to produce more of the ultimate carcinogenic species DE2 should be considered. However, a more general conclusion must take into account data on inducibility of the CYP1A1 variants, e.g. by cigarette smoke. A more readily inducible variant can produce greater carcinogenic activation. In this connection it is interesting to note that it had been hypothesized that CYP1A1 mutations introduce a higher potential of inducibility (39,40), thereby explaining why individuals carrying alleles CYP1A1*2A and *2B possess a higher risk of lung cancer (20,41). On the other hand, a recent study showed that interindividual variation in levels of induced CYP1A1 EROD activity appears to be associated more with regulatory factors (Ah receptor polymorphism) than polymorphism in the CYP1A1 gene (42). Further, the impact of CYP1A1 on cancer risk may be modified by possible coregulation with detoxifying enzymes such as glutathione S-transferase M1 (43,44), or by interference from other polymorphic enzymes, such as myeloperoxidase (45) or microsomal epoxidehydrolase (46), that also metabolize B[a]P or its metabolites.
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Notes
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3 To whom correspondence should be addressed Email: schwarz{at}mdc-berlin.de 
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Acknowledgments
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We are grateful to Dr F.J.Gonzalez (National Cancer Institute, NIH, Bethesda, MD) for providing CYP1A1 cDNA and virus for OR expression and Prof. F.Oesch and Dr A.Seidel (Institute of Toxicology, University of Mainz, Germany) for metabolites standards. We thank C.Andrée for western blotting and Dr H.Honeck and R.Zummach (Max Delbrueck Center for Molecular Medicine, Berlin-Buch) for their help in HPLC and A.Sternke for skilfully culturing insect cells. This study was supported by grants of the German Research Foundation (DFG) to I.R. and D.S. (RO 1287/2-1 and 436 WER 17/4/99) and the Volkswagen-Stiftung to D.S. (I/75 468).
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Received September 13, 2000;
revised October 31, 2000;
accepted November 2, 2000.