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Chem. Res. Toxicol. 1996, 9, 418-425
Simulation of Human Benzo[a]pyrene Metabolism Deduced from the Analysis of Individual Kinetic Steps in Recombinant Yeast Jean-Charles Gautier,† Philippe Urban,‡ Philippe Beaune,*,† and Denis Pompon‡ INSERM U75, CHU Necker-Enfants-Malades, Universite´ Rene´ Descartes, 75015 Paris, France, and Centre de Ge´ ne´ tique Mole´ culaire du CNRS, Laboratoire Propre associe´ a` l’Universite´ Pierre et Marie Curie, 91198 Gif-sur-Yvette, Cedex France Received May 31, 1995X
Human cytochrome P450 1A1 (1A1) and microsomal epoxide hydrolase (mEH)-dependent metabolic activation of benzo[a]pyrene (BP) have been reconstituted with microsomes from yeast cells expressing the two enzymes. The formation of the postulated ultimate mutagen 7β,8R-dihydroxy-9R,10R-epoxy-7,8,9,10-tetrahydro-BP, the so-called diol epoxide-2 (DE2) derived from the reoxidation of BP-7,8-dihydrodiol by 1A1, was estimated by HPLC measurement of its hydrolysis product 7β,8R,9R,10β-tetrahydrotetrol-BP (T2-tetrol). The 1A1/mEH coupled system was analyzed by varying the incubation time, initial substrate concentration, and molar ratio of the two enzymes. A minimum kinetic model of BP metabolism by 1A1 and mEH was constructed on the basis of the overall kinetic parameters (Vmax, Km) for a number of individual steps determined with human 1A1 and mEH expressed in yeast. The model was converted into a set of differential equations including 30 independent kinetic constants, 15 chemical species, and 8 enzymes and enzyme/substrate complexes. Numerical simulation of the model enabled us to satisfactorily reproduce the experimental kinetics of formation of BP-phenols, -dihydrodiols, and -tetrols for all tested conditions. Such a validated model was used to investigate the kinetics of unstable genotoxic species such as BP-epoxides and diol epoxides, which were not directly measurable. Based on numerical simulation, BP-7,8-oxide and -9,10-oxide appear to accumulate rapidly to reach a plateau after 2 min, while maximal accumulation of DE2 occurs after about a half-hour and declines during the following 2 h. A contribution of BP-9,10-dihydrodiol metabolism to T2-tetrol formation via the formation of a BP-7,8-oxide-9,10-dihydrodiol is predicted to be detectable after 2 hours due to the preferential accumulation of BP-9,10-dihydrodiol following 1A1-dependent consumption of initially accumulated BP-7,8-dihydrodiol. Benzo[a]pyrene (BP),1 a polycyclic aromatic hydrocarbon, is a widespread environmental pollutant found in incomplete combustion products. This compound was described as a possible causative agent of lung cancer in smokers (1, 2). It has been shown that BP can be oxidized by human cytochrome P450 1A1 (1A1) into BP7,8-epoxide (7,8-ox), which is hydrolyzed by microsomal epoxide hydrolase (mEH) into BP-7,8-dihydrodiol (7,8diol). The latter compound can then be reoxidized by 1A1 to give the postulated ultimate mutagen 7β,8R-dihydroxy9R,10R-epoxy-7,8,9,10-tetrahydro-BP, the so-called diol epoxide-2 (DE2) (3). Either this diol epoxide can be spontaneously hydrolyzed into tetrols, or it reacts with DNA to form adducts. Alternatively, BP can be oxidized by 1A1 on other positions of the molecule to produce epoxides which can be either spontaneously rearranged into phenols such as 3-hydroxy-BP (3-OH) and 9-OH, or enzymatically hydrolyzed by mEH into dihydrodiols such as 4,5-diol and 9,10-diol. Although the metabolism of BP has been investigated with human 1A1 and mEH ex* Author to whom correspondence should be addressed. † INSERM U75. FAX: 33 1 40 61 55 82; e-mail:
[email protected]. ‡ CNRS. FAX: 33 1 69 07 55 39; e-mail:
[email protected]. X Abstract published in Advance ACS Abstracts, February 1, 1996. 1 Abbreviations: BP, benzo[a]pyrene; 7,8-ox, BP-7,8-oxide; 3-OH, 3-hydroxy-BP; 7,8-diol, BP-7,8-dihydrodiol (other BP-oxides, BP-phenols, and BP-dihydrodiols are similarly abbreviated); DE2, diol epoxide2; DE3, diol epoxide-3; T2-tetrol, 7β,8R,9R,10β-tetrahydrotetrol; C2tetrol, 7β,8R,9R,10R-tetrahydrotetrol; P450, cytochrome P450; mEH; microsomal epoxide hydrolase; 1A1, cytochrome P450 1A1.
0893-228x/96/2709-0418$12.00/0
pressed in several expression systems (4-7), the kinetics of DE2 formation from BP has not been studied. Knowledge of the time course for unstable species is of critical importance when genotoxic events are associated. In particular, this opens up the possibility of analysis of the potential role of the dynamics of genotoxic damage in modulating the mutagenesis/repair balance. In the case of BP, accumulation of reactive species could be critically determined by the ratio between 1A1 and mEH, and by the rate of nonenzymatic processes such as spontaneous rearrangement, chemical hydrolysis, and adduct formation. In addition, competition between BP and derivatives at the 1A1 and mEH active sites could result in rather unpredictable behavior in the absence of detailed knowledge of individual reaction rates and substrate affinities. The qualitative description of the BP metabolite pattern is thus not sufficient for predicting the consequences of polymorphisms which could affect both enzymatic kinetic parameters and relative expression levels. The first two steps of BP activation leading to the formation of 7,8-diol have been previously reconstituted by coexpression of human 1A1 and mEH in the yeast Saccharomyces cerevisiae (7). Efficient metabolic coupling was observed even when the two enzymes were included in separate microsomal membranes. We now report a complementary investigation using an optimized HPLC detection system permitting simultaneous quan© 1996 American Chemical Society
Simulation of Human Benzo[a]pyrene Metabolism
titation of a large number of metabolites (phenols, dihydrodiols, and also tetrols derived from spontaneous hydrolysis of diol epoxides). The results obtained with the yeast-expressed human enzymes were used to establish a minimal kinetic model for BP multistep metabolism. The present study confirms that numerical simulation of the presented model can satisfactorily reproduce experimental kinetics, and thereafter can be used to predict the concentration courses for highly unstable epoxides and diol epoxides which cannot be quantified directly.
Materials and Methods Chemicals and Reagents. Caution: BP and its derivatives are probable human carcinogens. They should be handled in accordance with NIH guidelines (8). BP, benzo[ghi]perylene, NADP(H), and trifluoroacetic acid were purchased from Sigma (St. Louis, MO, USA). [3H]BP (specific activity 80 Ci/mmol) was from Amersham (Les Ulis, France). Racemic 3-OH, 9-OH, and trans-7,8-diol were obtained from the National Cancer Institute Chemical Repository (Midwest Research Institute, Kansas City, MO, USA). Racemic trans-4,5-diol and trans-9,10-diol were kindly donated by Dr. P. Dansette. Racemic 7β,8R,9R,10β-tetrahydrotetrol (T2-tetrol) and 7β,8R,9R,10R-tetrahydrotetrol (C2-tetrol) were a kind gift from Dr. F. Oesch and Dr. A. Seidel. Octyldecylsilyl Newguards (7 µm particle size, 15 mm × 3.2 mm) and Spheri-5 columns (5 µm particle size, 100 mm × 2.1 mm) were from Brownlee (San Jose, CA, USA). Yeast Strains, Culture, and Microsome Preparation. Yeast strains W[R]-pV60, W[R]-p1A1, W[R]-pEH, and W[E,R]p1A1 have been described previously (7). Briefly, all these strains were genetically engineered to overexpress yeast NADP(H) cytochrome P450 reductase when grown in the presence of galactose. W[R]-pV60 corresponds to the control strain transformed with cDNA-free pV60. W[R]-p1A1 and W[R]-pEH express respectively human 1A1 and mEH. W[E,R]-p1A1 coexpresses human 1A1 and mEH. All strains were grown in minimum galactose medium SW5 until cell density reached A600 of 3 (9) and diluted 3-fold with complete medium YPL containing galactose, and cell growth was continued up to A600 of 10. Microsomes were prepared using an enzymatic lysis method (10). Total P450 content was calculated from the reduced carbon monoxide difference spectra (11) using a Perkin-Elmer Lambda 2 spectrophotometer. The concentration of mEH was evaluated by measuring the styrene oxide hydrolase activity as described (7). Protein concentration was determined using the Pierce bicinchoninic acid procedure, according to the manufacturer’s instructions. Reverse Phase HPLC of BP Metabolites. BP (0-15 µM) was incubated for 15 min at 37 °C in 50 mM Tris-HCl buffer, pH 7.4, 1 mM EDTA, 0.1 mg/mL NADP(H), and 150 µg of microsomal proteins in a final volume of 200 µL. All experiments were performed using the same membrane to buffer volume ratio to limit possible artifacts resulting from partition effects of BP between membrane and aqueous phases. Adjustment of membrane to buffer ratio was achieved when required by addition of control yeast microsomes (from strain W[R]-pV60) devoid of 1A1 and mEH. Reactions were generally stopped with 16 µL of trifluoroacetic acid 25% in water in order to secure hydrolysis of oxides. A drawback of this procedure is the almost stoichiometric conversion of the 9,10-diol to 9-OH. Trifluoroacetic acid was thus omitted when measuring the formation of 9,10-diol. We checked that the latter procedure did not affect the formation of T2-tetrol in our experimental conditions. Extractions were performed using 200 µL of ethyl acetate and 200 µL of NaCl-saturated water. Benzo[ghi]perylene (1.2 nmol) was added as an internal standard. Organic extracts were evaporated under a nitrogen stream, and residues were solubilized in 80 µL of methanol 50% in water. A 10-µL aliquot was
Chem. Res. Toxicol., Vol. 9, No. 2, 1996 419 analyzed by HPLC. Metabolite separations were accomplished on a 100-mm octyldecylsilyl column (Brownlee). Elution was carried out at a flow rate of 1 mL/min, using a rapid gradient just after the injection start from 0% to 15% acetonitrile in water (containing 0.02% trifluoroacetic acid), followed successively by a linear gradient of 15-40% acetonitrile for 16 min, a 6-min gradient to 50% acetonitrile, a 9-min gradient to 75% acetonitrile, and finally a rapid gradient to pure acetonitrile. We checked that 9,10-diol was not converted into 9-OH in our HPLC conditions. The column effluent was monitored by using a Jasco 821 FP fluorescence detector, set with the following program: λexc ) 340 nm, λem ) 402 nm from 0 to 12 min (for tetrols); λexc ) 278 nm, λem ) 407 nm from 12 to 14 min (for 9,10-diol); λexc ) 263 nm, λem ) 388 nm from 14 to 16 min (for 4,5-diol); λexc ) 348 nm, λem ) 402 nm from 16 to 19 min (for 7,8-diol); λexc ) 375 nm, λem ) 435 nm from 19 to 40 min (for 9-OH, 3-OH, BP, and benzo[ghi]perylene). The BP metabolite concentrations were deduced from the peak areas on the chromatograms using authentic compounds for calibration. Incubations with labeled BP were performed as described above, except that 25 µCi of [3H]BP was added. BP-quinones were quantified by scintillation counting after HPLC fraction collection from 18 to 21 min using a Gilson 201 collector. Enzymatically synthesized 7,8- and 9,10-diols were purified by preparative HPLC using a Gilson 201 collector and were subsequently used as substrate for 1A1, as described for BP. Numerical Simulation. The kinetic model of BP metabolism by 1A1 and mEH presented in Figure 3 was converted into a set of differential equations listed in Table 2. Numerical integration was used to solve the set of differential equations for the given initial concentrations of reagents (see legends of Figures 4-7 for BP, 1A1, and mEH concentrations) and the given rate constants (Table 3). An interactive home made simulation program for a Macintosh microcomputer was used to calculate all the presented data. This integration program was written and compiled using Symantech Think Pascal objectoriented language and Water’s Edge Software ToolsPlus interface libraries. Integration was based on a fourth order RungeKuta method (12) with an automatic step control, interactive graphic interface, and batch mode capability. The program can be made available as a standard self-sufficient Macintosh application associated with predefined rate constants and initial concentration files. From a practical point of view, 4 cases of increasing complexity can be considered: Case 1: The model is used as such with the given rate constants and the given initial concentrations of chemicals and enzymes. The program, once launched, allows the reader to recalculate the time course of all species in the model. Case 2: The model is used as such, but with different rate constants and/or different enzyme or chemical concentrations. This case is similar to Case 1, except that rate constant files and concentration files must be rebuilt. The self-sufficient application stated in Case 1 enables rebuilding through a standard Macintosh dialogue in various windows. This gives readers the possibility of testing the influence of relative rate constants and concentrations on species kinetics. Case 3: The model also has to be changed (a different model, species, new reactions, and new constants), but the total numbers of constants and species are not significantly increased. The differential equation set has to be rebuilt accordingly and introduced using the “Pascal” language syntax into the “model” module of the source code (written Pascal). The new module must be compiled and linked to the main simulation program, which requires a commercial program, i.e., “Think Pascal” from Symantech (and the corresponding license) or, for the forthcoming version of the simulation program, the CW6 version of the CodeWarrior C++ and Pascal Macintosh development package. Case 4: Extensive changes in the model are required, including much more complex models. Full knowledge of the program and total source code (several thousand lines) is required. Please contact the author, Denis Pompon.
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Table 1. Metabolism of BP by Human 1A1 and mEH Expressed in Yeasta BP metabolite formation rate (pmol‚(pmol of P450)-1‚min-1) microsomes
T2-tetrol
9,10-diol
4,5-diol
7,8-diol
9-OH
3-OH
1A1b 1A1+ mEHc (mixtures) 1A1+ mEHd (coexpression)
