Metabolic Activation of the (+)-S,S- and (−)-R,R ... - ACS Publications

Chem. Res. Toxicol. 1997, 10, 1161-1170

1161

Metabolic Activation of the (+)-S,S- and (-)-R,R-Enantiomers of trans-11,12-Dihydroxy-11,12-dihydrodibenzo[a,l]pyrene: Stereoselectivity, DNA Adduct Formation, and Mutagenicity in Chinese Hamster V79 Cells Andreas Luch,* Albrecht Seidel, Hansruedi Glatt,† and Karl L. Platt Institute of Toxicology, University of Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany Received January 16, 1997X

Polycyclic aromatic hydrocarbons require metabolic activation in order to exert their biological activity initiated by DNA binding. The metabolic pathway leading to bay or fjord region dihydrodiol epoxides as ultimate mutagenic and/or carcinogenic metabolites is thought to play a dominant role. For dibenzo[a,l]pyrene, considered as the most potent carcinogenic polycyclic aromatic hydrocarbon, the formation of the fjord region syn- and/or anti-11,12-dihydrodiol 13,14-epoxide (DB[a,l]PDE) diastereomers has been found to be the principal metabolic activation pathway in cell cultures leading to DNA adducts. In order to further elucidate the stereoselectivity involved in this activation pathway via the formation of the trans-11,12dihydrodiol, we have synthesized the enantiomerically pure 11,12-dihydrodiols of dibenzo[a,l]pyrene and investigated their biotransformation in rodents. Incubations with liver microsomes of Sprague-Dawley rats and CD-1 mice pretreated with Aroclor 1254 revealed that the enzymatic conversion to the fjord region DB[a,l]PDE strongly depends on the absolute configuration of the 11,12-dihydrodiol enantiomers. While oxidation at the 13,14-position of the (+)-(11S,12S)-dihydrodiol is limited to a small extent, the (-)-11R,12R-enantiomer is metabolized to its fjord region dihydrodiol epoxides in considerably higher amounts. Moreover, this substrate is transformed with high stereoselectivity to the corresponding (-)-antidihydrodiol epoxide by liver microsomes of Aroclor 1254-treated rodents. The metabolism results were in good accordance with the extent of stable adduct formation in calf thymus DNA as investigated by the 32P-postlabeling technique and with the mutagenicity in Chinese hamster V79 cells of the two enantiomeric 11,12-dihydrodiols mediated by hepatic postmitochondrial preparations of Aroclor 1254-treated rats. The results indicate that both genotoxic events occurred predominantly by the stereoselective activation of the (-)-(11R,12R)-dihydrodiol to the (-)-anti-DB[a,l]PDE with R,S,S,R-configuration.

Introduction The first synthesis and physicochemical characterization of the peri-condensed hexacyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P)1 (Scheme 1) had been unequivocally achieved in 1966 (1, 2). Indications about the extraordinary strong carcinogenic activity of this polycyclic aromatic hydrocarbon (PAH) were obtained after its topical administration on mice skin in an early carcinogenicity study (3). More recently, comparative investigations with other carcinogenic PAHs including benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene clearly showed that DB[a,l]P is a significantly * Address for correspondence: Technical University of Munich, Institute of Toxicology and Environmental Hygiene, Lazarettstrasse 62, D-80636 Munich, Germany. Tel: ++49-89-3187-2750. Fax: ++4989-3187-3449. E-mail: [email protected]. † Present address: German Institute of Human Nutrition, ArthurScheunert-Allee 114-116, D-14558 Potsdam-Rehbruecke, Germany. X Abstract published in Advance ACS Abstracts, September 15, 1997. 1 Abbreviations: B[a]A, benz[a]anthracene; B[a]P, benzo[a]pyrene; B[c]Ph, benzo[c]phenanthrene; P450, cytochrome P450; DB[a,l]P, dibenzo[a,l]pyrene; 11,12-dihydrodiol, DB[a,l]P-trans-11,12-dihydrodiol; syn- and anti-DB[a,l]PDE, syn- and anti-dibenzo[a,l]pyrene-11,12dihydrodiol 13,14-epoxide; FD-MS, field desorption mass spectrometry; MAA-Cl, menthoxyacetic acid chloride; (+)- and (-)-11,12-MAA-ester, (+)- and (-)-trans-11,12-bis[(-)-menthoxyacetoxy]-11,12-dihydrodibenzo[a,l]pyrene; MC, 3-methylcholanthrene; PAH, polycyclic aromatic hydrocarbon; S9, postmitochondrial fraction; tetrol, DB[a,l]P-11,12,13,14-tetrahydrotetrol.

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stronger tumor initiator than all other PAHs ever tested in mouse skin and rat mammary gland (4-6). The strong carcinogenicity of DB[a,l]P correlates well with a high level of DNA adducts formed in skin (7) or the mammary gland (8) as the target tissues in rats. On the basis of the carcinogenicity data in rodents, exposure to this PAH must be considered as a significant risk for human health. In fact DB[a,l]P has been detected in cigarette condensate (9) and has been identifed in low concentrations as a widespread environmentally occurring pollutant (10-13). Therefore, the assumption of an involuntary uptake of DB[a,l]P by individuals and its possible involvement in human cancer etiology appear to be evident (13, 14). Biotransformation of DB[a,l]P by liver microsomes of 3-methylcholanthrene (MC)-treated rats (15) and by several human recombinant cytochromes P450 (P450) (16) results in the formation of significant amounts of the DB[a,l]P-trans-11,12-dihydrodiol (11,12-dihydrodiol). This metabolite is the direct precursor of the fjord region DB[a,l]P-11,12-dihydrodiol 13,14-epoxides (DB[a,l]PDE) enzymatically generated by oxidation of the vicinal olefinic double bond (Scheme 1). Strong evidence for the involvement of this pathway in the bioactivation of the parent hydrocarbon stems from the analysis of corre© 1997 American Chemical Society

1162 Chem. Res. Toxicol., Vol. 10, No. 10, 1997

Luch et al.

Scheme 1. Stereoselective Bioactivation of Dibenzo[a,l]pyrene to the Fjord Region 11,12-Dihydrodiol 13,14-Epoxides, Catalyzed by Cytochromes P450 and Microsomal Epoxide Hydrolase (mEH)a

a

Arrows indicate the sterically hindered regions of the parent polycyclic aromatic hydrocarbon.

sponding DNA adducts in cell cultures (17, 18) and microsomal incubations (19). Stable DNA adducts of DB[a,l]PDE are most probably formed at the exocyclic amino groups of the purine bases (20). Also an alternate activation of DB[a,l]P by a one-electron oxidation has been reported based on studies with microsomes (19). Because the 11,12-dihydrodiol can be produced metabolically in two enantiomeric forms, further activation of these two enantiomers can lead, in principle, to the formation of four stereoisomeric fjord region DB[a,l]PDEs (enantiomeric pairs of two different syn- and antidiastereomers, Scheme 1). However, the action of P450 on PAH carbon bonds, which possess two stereoheterotopic sites, in general exhibits stereoselective character (21). Therefore the substrate specificity of P450 varying with the species involved in metabolism and depending on the stereochemical requirements of the active site of individual isoforms (22) determines the isomeric composition of the formed mixture of dihydrodiol epoxides. This is exemplified by the (-)-(3R,4R)-dihydrodiol of benzo[c]phenanthrene (B[c]Ph) and the (-)-(7R,8R)-dihydrodiol of B[a]P which are both preferentially converted to the corresponding anti-dihydrodiol epoxides with R,S,S,Rconfiguration by microsomes of MC-treated rats. In contrast, oxidation of the (+)-S,S-dihydrodiol enantiomers under the same conditions leads to an excess of the corresponding syn-dihydrodiol epoxide with S,R,S,Rconfiguration (23).

Recent studies have shown that both the syn- and antiDB[a,l]PDE possess high mutagenic and carcinogenic activity. Investigation of the intrinsic mutagenic activity of syn- and anti-DB[a,l]PDE in Chinese hamster V79 cells revealed that the anti-diastereomer exhibited a 3-fold higher specific mutagenicity than the syn-diastereomer (24). Furthermore, the anti-DB[a,l]PDE has been shown to be a very potent carcinogen in the newborn mouse assay (25) and a strong mammary carcinogen in rats (26). In a comparative study, the syn-DB[a,l]PDE has been reported to be a stronger tumor initiator in mouse skin than the anti-diastereomer at lower doses (27). In the present study, metabolic activation of the (+)and (-)-11,12-dihydrodiol enantiomers of DB[a,l]P to the corresponding fjord region dihydrodiol epoxides was examined using liver microsomes from either SpragueDawley rats or CD-1 mice both pretreated with Aroclor 1254. The syn- and anti-DB[a,l]PDE were determined indirectly by formation of their corresponding DB[a,l]P11,12,13,14-tetrahydrotetrols (tetrols), which are generated by acidic hydrolysis of the oxirane ring at the benzylic position (Scheme 2). Previous synthesis and characterization of the diastereomeric tetrols allowed not only detailed metabolism studies with racemic 11,12dihydrodiol but also those with the (+)- and (-)-11,12dihydrodiol enantiomers. To obtain further information about the genotoxicity of each individual set of metabolites produced from the enantiomeric 11,12-dihydrodiols,

Stereoselective Metabolism of DB[a,1]P-11,12-diol

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Scheme 2. Stereochemistry of the Hydrolysis of Enantiomeric Fjord Region Dihydrodiol Epoxides of Dibenzo[a,l]pyrene to Isomeric Tetrahydrotetrols

stable modification of calf thymus DNA (28) and forward mutations at the HPRT-locus of Chinese hamster V79 cells (29) after coincubation of the two enantiomers with hepatic preparations have been determined. Both genotoxic events have been shown to be causatively related to each other (30, 31), and furthermore, the quantitative effects are well correlated in most cases, as has already been demonstrated for various fjord and bay region dihydrodiol epoxides of other PAHs (32).

Experimental Procedures Caution: All synthetic DB[a,l]P derivatives and metabolites used in the present study should be considered potentially toxic, mutagenic, and/or carcinogenic and therefore should be handled in an appropriate manner. Instrumentation and Chemicals. Melting points were determined on a Buechi 510 apparatus using unsealed capillary tubes and are uncorrected. 1H NMR spectra were recorded on a Bruker AM 400 spectrometer (Bruker, Karlsruhe, Germany) operating at 400 MHz. Chemical shifts (δ) are reported as ppm downfield from tetramethylsilane. UV spectra were obtained with a Shimadzu MPS-2000 spectrophotometer (Shimadzu, Duesseldorf, Germany). The field desorption (FD) mass spectra were run on a MAT 90 mass spectrometer (Finnigan, Bremen, Germany). Specific optical rotations ([R]20D) were obtained with a model 241 polarimeter (Perkin-Elmer, Ueberlingen, Germany) at 20 °C. Preparative HPLC purifications were conducted on a DuPont 830 instrument using a silica gel (LiChrosorb Si 60, 5 µm) column (16 × 250 mm). Analytical separations of metabolites were performed by HPLC with a system consisting of two high-pressure pumps (model 740) and a gradient programmer (model 744) connected to a UV detector (model 230) (Spectra Physics, Darmstadt, Germany) operating at 280 nm. LiChrospher RP-18 (5 µm) columns (4 × 250 mm; Knauer, Berlin, Germany) were used as stationary phases in analytical HPLC.

(-)-Menthoxyacetic acid chloride [(-)-MAA-Cl] was synthesized by adding 40.0 g (0.187 mol) of (-)-MAA (Aldrich, Steinheim, Germany) in small portions to 62.6 mL (0.860 mol) of thionyl chloride (Fluka, Neu-Ulm, Germany) at room temperature and subsequent refluxing for 5 h. The excess of thionyl chloride was removed by distillation under a gentle vacuum before the (-)MAA-Cl (42.0 g, 97%) was obtained as a colorless oil: bp(0.02mbar) ) 67-69 °C [bp(3mbar) ) 117-120 °C (33)]. Biochemicals for metabolism studies were from Boehringer (Mannheim, Germany) and Sigma Chemie (Deisenhofen, Germany). Calf thymus DNA was purchased from Sigma Chemie. For enzymes the supplying firm is indicated individually in the context. [γ-32P]ATP with a specific activity of >185 TBq/mmol (>5000 Ci/mmol) was obtained from Amersham Buchler (Braunschweig, Germany). Poly(ethylenimine)-cellulose TLC plates (Polygram CEL 300 PEI) were from Macherey-Nagel (Dueren, Germany). The detection of radiolabeled compounds was accomplished by autoradiography using Kodak X-OMAT XAR-5 (8 × 10 in.) films (Sigma Chemie). The films were developed in Kodak LX24 and fixed with Kodak AL4 (Nordfoto, Hamburg, Germany). Radioactivity was quantified by the measurement of Cerenkov radiation in a liquid scintillation analyzer (Canberra Packard, Frankfurt/M., Germany). Phenol was purchased from Aldrich (Steinheim, Germany), melted at 60 °C, supplemented with 8-hydroxyquinoline (0.1%; Sigma Chemie), and extracted twice with 1 volume of 1 M Tris/HCl (pH 8.0). The obtained solution was stored at 4 °C and used as phenol (Tris saturated) in the extraction protocols. Synthesis of Racemic Metabolites of DB[a,l]P. The synthesis of the (()-11,12-dihydrodiol of DB[a,l]P and the two diastereomeric syn- and anti-DB[a,l]PDE was performed by a pathway published earlier (24). The tetrols required as reference compounds were obtained by hydrolysis of the diastereomeric syn- and anti-DB[a,l]PDE in an acidic solution of 1,4dioxane/water (containing 1-2 drops of concentrated hydrochloric

1164 Chem. Res. Toxicol., Vol. 10, No. 10, 1997 acid) and subsequent separation by HPLC as previously described.2 Synthesis of the Enantiomeric 11,12-Dihydrodiols. The synthesis of the enantiomerically pure 11,12-dihydrodiols of DB[a,l]P was accomplished via chromatographic separation of their diastereomeric bis-(-)-MAA-esters. For this purpose 50 mg (0.15 mmol) of (()-11,12-dihydrodiol was reacted with 0.21 g (0.90 mmol) of (-)-MAA-Cl in 10 mL of pyridine under argon. When the reaction was completed after 24 h at 4 °C (monitored by TLC), the solvent was removed by distillation under reduced pressure. The residue was purified by flash chromatography on silica gel using CH2Cl2/n-hexane (4:1, v/v) as mobile phase giving a mixture of the diastereomeric bis-(-)-MAA esters (94 mg, 86%) as a colorless solid (mp ) 125 °C). HPLC separation of the two diastereomeric esters was achieved under conditions used previously for the separation of similar compounds (34). Thus, the mixture was loaded onto a LiChrosorb Si 60 (5 µm) column (16 × 250 mm) which was subsequently eluted with cyclohexane/diethyl ether (97:3, v/v, 8 mL/min). Early eluting diastereomeric ester (-)-trans-11,12-bis[(-)menthoxyacetoxy]-11,12-dihydrodibenzo[a,l]pyrene [(-)-11,12MAA-ester] (38.8 mg, 35.5%): retention time 9.3 min; mp ) 110-112 °C; [R]20D ) -299° (0.481 mg/mL THF); UV λmax (nm), (, cm2 mmol-1) 208 (49 600), 242 (25 800), 249 (27 300), 270 (31 100), 281 (28 400), 292 (32 800), 304 (38 300), 315 (20 500), 344 (19 000), 361 (23 000); 1H NMR (CDCl3) δ (ppm) 8.82 (d, 1, H-5, J5,6 ) 7.5 Hz), 8.78 (d, 1, H-4, J3,4 ) 7.5 Hz), 8.44 (d, 1, H-1, J1,2 ) 7.3 Hz), 8.13 (d, 1, H-7, J6,7 ) 7.3 Hz), 8.10 (s, 1H, H-10), 7.98 (pseudo-t, 1, H-6), 7.97 (AB-system, 2, H-8, H-9, J8,9 ) 8.9 Hz), 7.75-7.71 (m, 1, H-3), 7.66-7.61 (m, 2, H-2, H-14), 6.57 (d, 1, H-11, J11,12 ) 5.6 Hz), 6.31 (dd, 1, H-13, J12,13 ) 4.4 Hz, J13,14 ) 10.0 Hz), 5.79 (pseudo-t, 1, H-12), 4.16 (AB-system, 2, Hmethylene, J ) 16.5 Hz), 4.13 (AB-system, 2, Hmethylene, J ) 16.6 Hz), 3.19-0.71 (m, 38, Hmenthoxy); FD-MS m/z (relative intensity) 730 ([M + H]+, 7), 729 ([M]+, 100), 516 (730 - MAA, 7). Late eluting diastereomeric ester (+)-trans-11,12-bis[(-)menthoxyacetoxy]-11,12-dihydrodibenzo[a,l]pyrene [(+)-11,12MAA-ester] (42.0 mg, 38.4%): retention time 12.1 min; mp ) 155-157 °C; [R]20D ) +129° (0.565 mg/mL THF); UV λmax (nm) (, cm2 mmol-1) 209 (47 000), 240 (25 300), 250 (27 500), 270 (30 600), 281 (28 200), 292 (31 800), 304 (37 300), 315 (21 900), 344 (17 200), 361 (20 900); 1H NMR (CDCl3), δ (ppm) 8.81 (d, 1, H-5, J5,6 ) 7.6 Hz), 8.77 (d, 1, H-4, J3,4 ) 7.6 Hz), 8.44 (d, 1, H-1, J1,2 ) 7.6 Hz), 8.13 (d, 1, H-7, J6,7 ) 7.4 Hz), 8.09 (s, 1, H-10), 7.98 (pseudo-t, 1, H-6), 7.97 (AB-system, 2, H-8, H-9, J8,9 ) 8.9 Hz), 7.74-7.70 (m, 1, H-3), 7.65-7.60 (m, 2, H-2, H-14), 6.57 (d, 1, H-11, J11,12 ) 6.1 Hz), 6.30 (dd, 1, H-13, J12,13 ) 4.3 Hz, J13,14 ) 10.0 Hz), 5.84 (pseudo-t, 1, H-12), 4.18 (AB-system, 2, Hmethylene, J ) 16.6 Hz), 4.14 (AB-system, 2, Hmethylene, J ) 16.5 Hz), 3.18-0.73 (m, 38, Hmenthoxy); FD-MS m/z (relative intensity) 730 ([M + H]+, 51), 729 ([M]+, 100), 516 (730 - MAA, 8). The (-)- and (+)-11,12-dihydrodiols were obtained by methanolysis of the (-)- and (+)-MAA-ester, respectively. Thereby 73 mg (0.10 mmol) of the ester was dissolved in 40 mL of THF, and a solution of 13 mg (0.24 mmol) of sodium methanolate in 20 mL of methanol was added. The mixture was stirred at room temperature until the reaction had been shown by TLC to be complete (about 4 h). Then 10 mL of ice-cold water was added, and the organic solvent was removed under vacuum. The crude mixture was poured into 50 mL of water, and the separated product was filtered, washed with water, and dried over silica gel. The enantiomerically pure (-)- or (+)-11,12-dihydrodiol was obtained as colorless crystals by sonication of a suspension in methanol. Determination of the absolute configuration of (+)and (-)-11,12-dihydrodiol was unequivocally achieved using the exciton chirality method by recording the CD spectra of the 4-[4(dimethylamino)phenylazo]benzoic acid ester derivatives (35). Specific optical rotation of (-)-(11R,12R)-dihydrodiol (25 mg obtained, 75% yield): [R]20D ) -121° (0.414 mg/mL THF). 2

Luch et al., manuscript submitted for publication.

Luch et al. Specific optical rotation of (+)-(11R,12R)-dihydrodiol (30 mg obtained, 89% yield): [R]20D ) +118° (0.491 mg/mL THF). Metabolism of the Enantiomeric 11,12-Dihydrodiols. The (()-, (+)- and (-)-11,12-dihydrodiols were each incubated with liver microsomes of adult male Sprague-Dawley rats (200-240 g; Interfauna Sueddeutsche Versuchstierfarm, Tuttlingen, Germany) or CD-1 mice (16-24 g; Charles River Wiga, Sulzfeld, Germany). The microsomes were prepared 6 days after a single intraperitoneal injection of 500 (rats) and 300 (mice) mg/kg of body weight Aroclor 1254 (diluted in tricaprylin; 2.5 mL/kg of body weight), respectively. Each dihydrodiol was incubated at 37 °C for 10, 30, or 60 min in a phosphate-buffered solution (pH 7.4; total volume: 2.0 mL) containing the following reagents: 950 µL of KCl (150 mM, pH 7.4), 200 µL of sodium phosphate buffer (0.5 M, pH 7.4), 100 µL of MgCl2 (100 mM), 500 µL of an NADPH-regenerating system [32 mM glucose 6-phosphate, 2.4 mM NADP+, 17.2 µg (2.4 units)/mL glucose 6-phosphate dehydrogenase (Boehringer) in an isotonic solution of KCl, pH 7.4], and 200 µL of a suspension of microsomes being equivalent to a total amount of 5 nmol of P450 as determined spectrophotometrically (36). After the incubation mixture was held for 2 min at 37 °C in an open vessel, the incubation was started by adding 80 µM dihydrodiol dissolved in 50 µL of Me2SO. After shaking (80 min-1) at 37 °C the incubation was stopped by adding 150 µL of 1.5 M perchloric acid which lowered the pH of the mixture to 2-3. Three hours later at room temperature the reaction mixture was neutralized with 100 µL of water-saturated Tris. The substrate and metabolites were then extracted by adding 3 mL of ice-cold ethyl acetate followed by vortexing for 1 min and separation of the organic layer by centrifugation at 3500 rpm. The extraction was repeated twice with 2 mL of ethyl acetate. The combined organic phases were dried over anhydrous MgSO4 and evaporated to dryness with a gentle stream of N2 at 35 °C. The residue was stored at -20 °C until HPLC analysis, for which the sample was redissolved in 80 µL of Me2SO. Aliquots of 20 µL were analyzed by HPLC using a LiChrospher-100 RP-18 (5 µm) column (4 × 250 mm). Elution conditions were as follows: linear gradient of acetonitrile/methanol/water (10:30:60, v/v/v) in 100 min to 100% methanol at a flow rate of 0.8 mL/min. The fjord region synand anti-DB[a,l]PDE were indirectly quantified by the amounts of the corresponding tetrols formed by their acidic hydrolysis. The tetrols were detected and identified by cochromatography and comparison of their UV spectra with the synthetic reference compounds. Their amount was photometrically determined at 280 nm from a calibration curve obtained by plotting the amounts of synthetic tetrols versus their peak areas (external standardization). DNA Adduct Analysis by 32P-Postlabeling. Calf thymus DNA was covalently modified by coincubation with either the racemic or enantiomerically pure 11,12-dihydrodiol in a Trisbuffered solution (pH 7.4) similar to the method described earlier (37). Biotransformation of the substrate was achieved by the added hepatic microsomal protein of Aroclor 1254-treated rodents, prepared as described above. Consequently, the total incubation mixture of 1 mL consisted of 500 µg of DNA, 1.0 mg of microsomal protein (equivalent to a total amount of 1.52.2 nmol of P450), 10 mM MgCl2, 0.5 mM NADP+, 5.0 mM glucose 6-phosphate, 0.35 U of glucose 6-phosphate dehydrogenase, 0.1 M Tris/HCl (pH 7.4), and 0.1 µmol (33.6 µg) of 11,12dihydrodiol (dissolved in 10 µL of Me2SO). When the incubation at 37 °C in an open vessel was terminated after 1 h, the mixture was extracted with 1 volume of phenol (Tris saturated)/ chloroform/isoamyl alcohol (25:24:1, v/v/v) followed by vortexing for 1 min and separation of the phases by centrifugation at 13 000 rpm. This step was repeated twice before the extraction protocol was continued as follows: two times with 1 volume of chloroform/isoamyl alcohol (24:1, v/v) and further two times with 1 volume of ethyl acetate. Then the DNA was precipitated by addition of 2 volumes of ethanol and 0.1 volume of 5 M NaCl, washed with 70% ethanol, dried under vacuum, and dissolved in water. The concentration of the nucleic acid in the preparation was photometrically determined at 260 nm.

Stereoselective Metabolism of DB[a,1]P-11,12-diol Quantification and analysis of the covalent DNA adducts was achieved by the 32P-postlabeling technique (38) modified by the nuclease P1 enhancement as described elsewhere (7, 28). Briefly, PAH-modified DNA (5.0 µg) was digested in a total volume of 4.8 µL overnight at 37 °C by addition of 2.5 mU of phosphodiesterase from calf thymus (EC 3.1.16.1; Boehringer), 145 mU of micrococcal endonuclease (EC 3.1.31.1; Sigma Chemie), 16.5 mM of sodium succinate (pH 6.0), and 8.5 mM CaCl2. Afterwards 1.2 U of nuclease P1 (EC 3.1.30.1; Sigma Chemie), 45 µM ZnCl2, and 63 mM buffered sodium acetate (pH 5.0) in a total volume of 4.8 µL were added and incubated at 37 °C for 90 min. Finally the enzymatic digest was stopped by addition of 1.92 µL of 0.5 M Tris. DNA samples (5.0 µg), enzymatically digested as described above, were 32P-labeled at 37 °C during an incubation time of 30 min after addition of 6 U of T4-polynucleotide kinase (3 U/µL diluted in kinase buffer, EC 2.7.1.78; Amersham Buchler, Braunschweig, Germany), 1.0 µL of kinase buffer (0.2 M bicine, 0.1 M MgCl2, 0.1 M DTT, 10 mM spermidine, pH 9.0), and 0.925 MBq (25 µCi) of [γ-32P]ATP (370 MBq/mL). The reaction was terminated by addition of 40 mU of apyrase (20 U/mL diluted with water, EC 3.6.1.5; Sigma Chemie) and subsequent incubation at 37 °C for 30 min. Separation of 32P-labeled 3',5'-bisphosphates was performed in the 4-directional mode by TLC on 20 × 20 cm poly(ethylenimine)-cellulose plates according to Randerath et al. (38) with the following elution buffers: direction D1, 1 M sodium phosphate (pH 6.0); wash solution D2, 2.5 M ammonium formate (pH 3.5); direction D3, 5.3 M lithium formate, 8.5 M urea (pH 3.5); direction D4, 1.2 M lithium chloride, 0.5 M Tris-HCl, 8.5 M urea (pH 8.0); D5 (same direction as D4), 1.7 M sodium phosphate (pH 6.0). Levels of DNA modification were calculated as described (28). Mutagenicity in V79 Cells. The racemic or enantiomerically pure 11,12-dihydrodiols were tested for mutagenicity in Chinese hamster V79 cells in the presence of the postmitochondrial fraction (S9) from livers of Aroclor 1254-treated male Sprague-Dawley rats as activating system (39) essentially as described for the fjord region DB[a,l]PDE (24). Dulbecco’s PBS (15.6 mL) supplemented with 10 mM 4-(2-hydroxyethyl)-1piperazine ethanesulfonic acid (Hepes; pH 7.4), 1.8 mL of freshly prepared S9, and 600 µL of a solution of cofactors (200 mM glucose 6-phosphate, 30 mM NADP+, 30 mM NADH, and 10 mM NADPH in Dulbecco’s PBS containing 10 mM Hepes, pH 7.4) were added together with the solution of the test compound in Me2SO (60 µL).

Results Incubation of Racemic or Enantiomerically Pure 11,12-Dihydrodiols with Liver Microsomes from Aroclor 1254-Treated Rodents. The stereochemistry and the amount of fjord region DB[a,l]PDE enzymatically formed from the (+)-(11S,12S)- and (-)-(11R,12R)-dihydrodiol were determined by measuring the tetrols as hydrolysis products of the dihydrodiol epoxides. Racemic tetrols, synthesized as described before,2 were suitable for an unequivocal elucidation of the stereochemistry involved in the formation of fjord region DB[a,l]PDE from the enantiomeric dihydrodiols (Scheme 2) since they are chromatographically indistinguishable from their enantiomers when separated by HPLC on achiral stationary phases. According to the findings for the fjord region B[c]PhDE (40), the syn-DB[a,l]PDE was hydrolyzed upon acidic workup (pH 2-3) of control incubations without microsomes by trans- and cis-attack of water to a 1:4 mixture of the (r,t,c,t)- and (r,t,c,c)-tetrols (Scheme 2), while acidic hydrolysis of the anti-DB[a,l]PDE yielded exclusively the trans-opened product, the (r,t,t,c)-tetrol. Both trans-opened tetrols, i.e., (r,t,c,t)- and (r,t,t,c)-tetrol, coeluted in a single peak (peak A in Figure 1) well separated from the cis-opened (r,t,c,c)-tetrol (peak B in

Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1165

Figure 1. Chromatographic separation of metabolites of racemic or enantiomerically pure 11,12-dihydrodiols of dibenzo[a,l]pyrene formed by rat liver microsomes from Aroclor 1254treated rats after an incubation time of 30 min. Experimental conditions are described in Experimental Procedures. Arrows depict retention times of tetrols: A, (()-(r,t,t,c)- and (()-(r,t,c,t)tetrols; B, (()-(r,t,c,c)-tetrol (cf. Scheme 2).

Figure 1), indicative of the syn-DB[a,l]PDE. From this information calculation of the relative amounts of the enantiomeric syn- and anti-DB[a,l]PDE-derived tetrols was possible (Table 1) by employing a calibration curve obtained with the synthetic tetrol. The results indicate that P450-catalyzed monooxygenation of the (-)-(11R,12R)-dihydrodiol enantiomer by liver microsomes of Aroclor 1254-treated rodents (Figure 1) leads to the formation of fjord region (-)-anti-DB[a,l]PDE with high stereoselectivity in both Sprague-Dawley rats and CD-1 mice (see Scheme 1, Table 1). In contrast, conversion of the (+)-(11S,12S)-dihydrodiol by rat liver microsomes yields nearly equivalent amounts of (+)-synand (+)-anti-DB[a,l]PDE, while corresponding subcellular fractions from mice again form a considerable excess of the anti-diastereomer (Table 1). It should be noted that the amounts of the measured tetrols could somewhat differ from the quantity of the corresponding DB[a,l]PDE (see Scheme 2) formed during incubation. Further enzymatic oxidation of dihydrodiol epoxide intermediates (41) or covalent interaction with proteins (42) or other nucleophilic constituents like phosphate groups (43) (e.g., from the phosphate buffer) has been observed with B[a]PDE and, therefore, could also not be excluded for the DB[a,l]PDE. Experiments incubating the authentic fjord region syn- and anti-DB[a,l]PDE with microsomes under the same conditions used for the dihydrodiol precursors (cf. Experimental Procedures) indicate that both diastereomers were meta-

1166 Chem. Res. Toxicol., Vol. 10, No. 10, 1997

Luch et al.

Table 1. Photometrically Quantified Amounts of 11,12,13,14-Tetrols Derived from Fjord Region Dihydrodiol Epoxides by Hydrolysis after Incubation of Either Racemic or Enantiomerically Pure Dibenzo[a,l]pyrene-trans-11,12-dihydrodiols with Liver Microsomes from Aroclor 1254-Treated Rodentsa metabolic conversion (pmol of tetrol/nmol of cytochrome P450) of DB[a,l]P-trans-11,12-dihydrodiol microsomes

tetrol

incubation time (min)

racemic

(+)-S,S

(-)-R,R

rat (Sprague-Dawley)

anti-dihydrodiol epoxide-derived tetrol

10 30 60 10 30 60 10 30 60 10 30 60

920 ( 120 1800 ( 200 2500 ( 200 110 ( 20 44 ( 4 39 ( 1 590 ( 170 660 ( 20 600 ( 80 260 ( 20 210 ( 50 180 ( 10

72 ( 14 31 ( 5 74 ( 14 91 ( 4 45 ( 1 62 ( 4 480 ( 40 360 ( 50 390 ( 90 210 ( 7 200 ( 20 160 ( 10

2300 ( 300 3500 ( 700 4900 ( 700 110 ( 10 94 ( 25 97 ( 25 1200 ( 100 1500 ( 200 1800 ( 200 340 ( 10 240 ( 20 160 ( 10

syn-dihydrodiol epoxide-derived tetrols mouse (CD-1)

anti-dihydrodiol epoxide-derived tetrol syn-dihydrodiol epoxide-derived tetrols

a Values represent means ( SD (n ) 3). Conditions of identification and quantitation are described in Experimental Procedures and Results.

bolically converted to a minor extent detectable only after prolonged periods of incubation (30 and 60 min). However, those metabolites were only detectable in significant amounts after incubation of the (-)-dihydrodiol and have been found to elute very early between 15 and 25 min under the conditions used, as a consequence of their stronger hydrophilic character (see Figure 1). In contrast, addition of denaturated (boiled) microsomes to the incubation mixture did not provide evidence that relevant amounts of the DB[a,l]PDE were lost by covalent interaction with proteins. No difference between the amounts of tetrols formed after incubation with boiled microsomes or buffer alone was observed (data not shown). As calculated from the earlier experiments employing the 3H-labeled (()-11,12-dihydrodiol, the metabolic conversion of this substrate increases even after long incubation times due to an extraordinarily low but constant total turnover rate of 0.34 ( 0.16 nmol/(nmol of P450‚ min).2 Interestingly, already after 10 min of incubation of both 11,12-dihydrodiol enantiomers the amounts of tetrols derived from the syn-DB[a,l]PDE seem to remain nearly constant or slightly decreases. In contrast, the amount of the corresponding (-)-anti-DB[a,l]PDE formed from the (-)-dihydrodiol increases to high excesses dependening on the incubation time (Table 1). Taken together, the extent of fjord region biotransformation of the 11,12-dihydrodiol strongly depends on its absolute configuration (Table 1). Calculations based on tetrol formation indicate that the (-)-(11R,12R)-dihydrodiol was enzymatically transformed to the corresponding (-)anti-DB[a,l]PDE with 11R,12S,13S,14R-configuration in much higher amounts (factors of ∼60-80 and ∼4-11 after 60 min incubation with rat and mouse liver microsomes, respectively) than the (+)-(11S,12S)-dihydrodiol to the corresponding (+)-(11S,12R,13R,14S)-anti- or (+)-(11S,12R,13S,14R)-syn-DB[a,l]PDE (see Scheme 1). Calf Thymus DNA Adducts Formed by Metabolically Activated Racemic or Enantiomeric 11,12Dihydrodiols. As demonstrated by the results of the 32P-postlabeling assay (Figure 2), various stable adducts were formed upon coincubation of the racemic or enantiomeric 11,12-dihydrodiols with microsomes of Aroclor 1254-treated rodents in the presence of calf thymus DNA. As expected, after chromatography the DNA adduct pattern of enzymatically activated (()-11,12-dihydrodiol originated from a superposition of those of the enantiomerically pure dihydrodiols (Figure 2). Identical adduct

Figure 2. Autoradiograms of thin layer chromatograms on cellulose sheets after 32P-postlabeling of mixtures obtained by coincubation of racemic or enantiomerically pure 11,12-dihydrodiols of dibenzo[a,l]pyrene with calf thymus DNA in the presence of microsomes from Aroclor 1254-treated rodents. The numbers given indicate the same adduct spot in one set of experiments (rat or mouse liver microsomes) on the basis of their identical chromatographic behavior. Experimental conditions are described in Experimental Procedures. Inset: Results after reduced exposure time.

spots in each set of experiments (left row in Figure 2 obtained with rat, right row in Figure 2 obtained with mouse microsomes) are marked with the same number. Consequently, adducts with the same number in the two different sets of experiments are not necessarily identical. Depending on the species origin of the microsomes, the patterns of adducted nucleotides obtained were somewhat different. As indicated by different numbers the activation with rat liver microsomes has produced only two major adduct spots (1 and 2) with two further spots not well separated from spot 2 (2a and 2b). In experiments with mouse liver microsomes, four major adduct spots (2-5) were obtained which are nicely separated, and

Stereoselective Metabolism of DB[a,1]P-11,12-diol Table 2. Quantities of DNA Adducts after Coincubation of Either Racemic or Enantiomerically Pure Dibenzo[a,l]pyrene-trans-11,12-dihydrodiols with Calf Thymus DNA in the Presence of Microsomes from Aroclor 1254-Treated Rodentsa

Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1167 Table 3. Mutagenicity in Chinese Hamster V79 Cells of Either Racemic or Enantiomerically Pure Dibenzo[a,l]pyrene-trans-11,12-dihydrodiols Metabolically Activated by Postmitochondrial Preparations from Livers of Aroclor 1254-Treated Ratsa

specific DNA binding of enzymatically activated DB[a,l]P-trans-11,12-dihydrodiols

substrate/type of microsomes (()-dihydrodiol/rat liver (+)-dihydrodiol/rat liver (-)-dihydrodiol/rat liver (()-dihydrodiol/mouse liver (+)-dihydrodiol/mouse liver (-)-dihydrodiol/mouse liver

fmol of adducts/µg of nucleic acid

adducted sites/1010 nucleotides

9.7 1.2 36.0 1.4 1.3 5.3

32000 3800 120000 4600 4100 17000

a

32P-postlabeling

Individual adducts were separated by the technique (cf. Figure 2) and subsequently quantified by measuring the Cerenkov emission in a scintillation counter after spots had been cut out of the cellulose sheets. Detailed descriptions are given in Experimental Procedures. Numbers represent mean values of two independent experiments.

adducts in the area near the starting point were detectable which consist of two not well-separated spots (1a and 1b). Furthermore, also quantitative differences in the formation of stable adducts were observed by subsequent measurement of the radioactivity via Cerenkov counting. As compiled in Table 2, biotransformation of the dihydrodiols to DNA-binding metabolites was quite more efficient during incubation with hepatic microsomes from rats as compared to those of mice. Moreover, the DNAbinding capacity of individual enantiomers of the 11,12dihydrodiol of DB[a,l]P after enzymatic activation by liver microsomes from rodents was remarkably different. Irrespective of the source of microsomes, activation of the (-)-(11R,12R)-dihydrodiol results in a significantly larger extent of total DNA binding compared to the (+)-(11S,12S)-dihydrodiol (Table 2). For example, the adduct level from the (-)-11,12-dihydrodiol activated by rat liver microsomes was about 31-fold higher than that of its enantiomer (Table 2). Similar differences were observed after activation by mouse liver microsomes although the ratio was considerably smaller (factor of ∼4). S9-Mediated Mutagenicity of Racemic or Enantiomeric 11,12-Dihydrodiols in Chinese Hamster V79 Cells. On the basis of the number of mutants yielded after coincubation of V79 cells with racemic or enantiomeric 11,12-dihydrodiols in the presence of S9 from livers of Aroclor 1254-treated rats (Table 3), the specific mutagenicity, calculated according to the method of Glatt et al. (44), strongly depended on the stereochemistry of the 11,12-dihydrodiol. Thus, enzymatically activated (-)-(11R,12R)-dihydrodiol caused 6-fold more mutations in V79 cells [130 × 106 mutants/(nmol‚mL)] than its (+)-11S,12S-enantiomer [22 × 106 mutants/ (nmol‚mL)].

Discussion To better understand the underlying mechanisms of the strong carcinogenicity of DB[a,l]P, the present study investigated differences between the (+)-(11S,12S)- and (-)-(11R,12R)-dihydrodiols of DB[a,l]P regarding the extent of P450-catalyzed fjord region syn- and anti-DB[a,l]PDE formation, DNA binding, and DNA mutagenicity in Chinese hamster V79 cells. Since the liver repre-

S9 mix-mediated mutagenicity of DB[a,l]P-trans-11,12-dihydrodiols

substrate (()-11,12-dihydrodiol

(+)-11,12-dihydrodiol

(-)-11,12-dihydrodiol

mutant specific dose frequency mutagenicity (nmol) (×106) [×106 mutants/(nmol‚mL)] 0.61 1.3 2.5 5.0 1.0 2.0 4.0 6.0 0.5 1.0 2.0 4.0 6.0

19 40 170 720 16 16 45 130 47 79 200 510 870

66

22

130

a Conditions are described in Experimental Procedures. Values represent means with n ) 3. Mutant frequencies in control experiments were in the same range (3-4 × 106) as previously described (24). Specific mutagenicity was calculated from mutant frequencies at low concentrations of the test compound according to the method of Glatt et al. (44).

sents the major site for metabolism of PAHs in mammals, rat and mouse liver microsomes from Aroclor 1254treated animals have been used as a metabolizing system. Quantifying the proportions of the syn- and antidiastereomers and determination of the stereochemistry involved in the formation of the fjord region DB[a,l]PDEs are of particular importance because it has been shown for many other PAHs including the strong carcinogens B[a]P and 7,12-dimethylbenz[a]anthracene (5, 45) or the weak tumorigen B[c]Ph (46), that the metabolic formation of vicinal dihydrodiol epoxides in a bay or fjord region (see Scheme 1) is one of the most important toxification pathways (21, 47). Moreover, comparative investigations of the four possible stereoisomers of sterically crowded dihydrodiol epoxides derived from a series of PAHs including B[a]P, benz[a]anthracene (B[a]A), chrysene, and B[c]Ph demonstrated that the anti-diastereomers with R,S,S,R-configuration displayed the strongest mutagenic and/or carcinogenic activity (23). Monitoring the formation of the different DB[a,l]PDEderived tetrols, the present work demonstrates that the enzymatic conversion of the (-)-(11R,12R)-dihydrodiol enantiomer occurs to the corresponding (-)-anti-DB[a,l]PDE with high stereoselectivity using rat and mouse liver microsomes from Aroclor 1254-treated animals (Table 1 and Scheme 1). In contrast, the observed levels of (-)syn-DB[a,l]PDE formation were only small (mouse liver microsomes) or marginal (rat liver microsomes). Correspondingly, the incubation experiments with the (+)(11S,12S)-dihydrodiol have revealed in general a significantly smaller extent and a lack of stereoselectivity in the oxidation of the 13,14-position by rat liver microsomes (Table 1). The elevated level of DB[a,l]PDE-derived tetrols after incubation of the (+)-(11R,12R)-dihydrodiol with mouse liver microsomes indicated a predominant formation of the corresponding anti-DB[a,l]PDE, the (+)isomer with S,R,R,S-configuration [ratio between (+)anti- and (+)-syn-diastereomer ∼2:1] (Table 1). In addition to these findings, the experiment employing rat liver preparations revealed a strong time-depend-

1168 Chem. Res. Toxicol., Vol. 10, No. 10, 1997

ent increase in the amount of the fjord region (-)-antiDB[a,l]PDE. This latter result can be partly understood taking into account the extraordinarily small conversion rate observed for the 11,12-dihydrodiol2 (cf. Results). As indicated by the relative amounts of the (r,t,t,c)tetrol formed (Scheme 2), the conversion of the (-)-(11R,12R)-dihydrodiol to the (-)-anti-DB[a,l]PDE with 11R,12S,13S,14R-configuration was by far more efficient than that of the (+)-11S,12S-enantiomer to both the (+)-antiand (+)-syn-DB[a,l]PDE (cf. Scheme 1). These results were independent of the animal species employed for the preparation of liver microsomes. Thus, the (-)-(11R,12R)-dihydrodiol appears to be a much better substrate for the P450 isoforms present in microsomes of Aroclor 1254-treated rats and mice than the (+)-(11S,12S)dihydrodiol. This difference in the metabolism of the 11,12-dihydrodiol enantiomers is also reflected by the measurements of two genotoxic end points which are both a direct consequence of the activation of the 11,12dihydrodiols to fjord region dihydrodiol epoxides. Both the total amount of stable DNA adducts as well as the number of mutations found in Chinese hamster V79 cells after coincubation with racemic or enantiomerically pure 11,12-dihydrodiols in the presence of different types of microsomal preparations correlated well with the total amount of metabolically formed fjord region DB[a,l]PDE (cf. Tables 1-3). The differences between the (-)-(11R,12R)- and (+)-(11S,12S)-dihydrodiol regarding their DNAmodifying capacity after metabolic activation are in the range of nearly 31-fold (rat liver microsomes) and 4.2fold (mouse liver microsomes), respectively (Table 2). On the other hand, a 6-fold difference between the specific mutagenicity of the (-)- and (+)-enantiomer can be calculated from the results in V79 cells incubating the 11,12-dihydrodiols with liver S9 preparations of Aroclor 1254-treated rats (see Results and Table 3). Taken together with the quantitatively different metabolic conversion of the (-)-(11R,12R)- and (+)-(11S,12S)dihydrodiol to the corresponding fjord region DB[a,l]PDEs, which reaches the factor of 37 (rat liver microsomes) and 3.6 (mouse liver microsomes), respectively, these findings represent an excellent correlation between the stereoselective metabolism of the 11,12-dihydrodiols, their DNA binding after activation with liver microsomes, and their S9-mediated mutagenicity in V79 cells. The preferential formation of the (-)-anti-DB[a,l]PDE with R,S,S,R-configuration has possibly strong implications not only for the DNA binding and mutagenicity but possibly also for the carcinogenicity in mammals. Many studies have shown that the carcinogenic potency can vary strongly depending on the absolute configuration of the dihydrodiol epoxide involved. It is known, for example, that the fjord region anti-dihydrodiol epoxide of B[c]Ph (48) and the bay region anti-dihydrodiol epoxide of B[a]P (23), both with R,S,S,R-configuration, exhibit the highest carcinogenic potency compared to the other three stereoisomers. Preliminary results of our tumorinitiation studies using the enantiomerically pure 11,12-dihydrodiols of DB[a,l]P point in the same direction. The individual enantiomers were topically applied to the back skin of NMRI mice following promotion with 4-Otetradecanoylphorbol acetate for another 10 weeks. The tumor-initiating activity of the (-)-(11R,12R)-dihydrodiol, which is the metabolic precursor of the (-)-anti-DB[a,l]PDE with R,S,S,R-configuration (Scheme 1), was by far higher than that of the (+)-(11S,12S)-dihydrodiol [tumor rate of 94% vs 0% and 88% vs 12% after administration

Luch et al.

of 10 and 20 nmol of dihydrodiol, respectively (A. Luch, Ph.D Thesis, University of Mainz)]. The stereochemical course of the biotransformation of PAHs to bay or fjord region dihydrodiol epoxides via their precursor trans-dihydrodiols can strongly depend on the content and the specificity of individual P450 isoforms present in the metabolizing system (49-53). Previous studies demonstrate that the fjord region activation of B[c]Ph with liver microsomes of MC-treated rats occurs via a preferential formation of the (-)-(3R,4R)-dihydrodiol which is subsequently metabolized to the corresponding (-)-anti-dihydrodiol epoxide with R,S,S,Rconfiguration (23). In contrast, incubation of the enantiomerically pure (+)-(3S,4S)-dihydrodiol exclusively produced the corresponding (+)-syn-dihydrodiol epoxide with S,R,S,R-configuration (23). Despite major differences in the extent of metabolism occurring in cell culture and mouse skin, a similar stereoselectivity was observed for the dihydrodiol epoxide pathway of B[c]Ph in rodent or human cell lines (54, 55) and mouse skin (55). The preferential formation of the bay region (+)-anti- and (+)syn-dihydrodiol epoxides of B[a]P follows, in principle, the same stereoselectivity in different metabolizing systems including liver preparations (49) and cell lines (56). More recent findings for the activation of DB[a,l]P and its racemic 11,12-dihydrodiol by analyzing the DNA adducts (18, 19) indicate that they were produced by both the (-)-anti- and (+)-syn-DB[a,l]PDE with 11R,12S,13S,14R- and 11S,12R,13S,14R-configuration, respectively, after incubation of the parent hydrocarbon with liver microsomes of MC-treated rats (19) or with the human mammary carcinoma MCF-7 cells (18). However, a different picture ermerged after the employment of the enantiomerically pure 11,12-dihydrodiols using the MCF-7 cell-mediated V79 cell mutation assay as an indicator system (57). The (-)-(11R,12R)- but not the (+)-(11S,12S)-dihydrodiol was mutagenic under these assay conditions indicating that the conversion of the (-)-(11R,12R)-dihydrodiol to the (-)-anti-DB[a,l]PDE, but not the corresponding activation of the (+)-(11S,12S)-dihydrodiol to the (+)-syn-DB[a,l]PDE, proceeds in MCF-7 cells (57) (cf. Scheme 1). Similarly to the findings with MCF-7 cells, the present study shows a very low level of fjord region DB[a,l]PDE formation and DNA binding using the (+)-(11S,12S)-dihydrodiol as the substrate for P450 isoforms present in liver microsomes of Aroclor 1254-treated rodents. Accordingly, a high mutagenicity was found in V79 cells only for the (-)-(11R,12R)-dihydrodiol mediated by S9 liver preparations of rats. Thus, the metabolic competence of MCF-7 cells to activate the 11,12-dihydrodiol enantiomers appears to be similar to that of liver microsomes from Aroclor 1254-treated rats. Different stereoselectivity observed for the activation of the 11,12-dihydrodiol enantiomers in the present study as compared to those mentioned above might indicate a different composition of various P450 isoforms in the different metabolizing systems and liver preparations of rodents after induction by Aroclor 1254. This may be due to a so-called “mixed-type induction” of various liver P450 isoenzymes after ip administration of the mixture of different polychlorinated biphenyl congeners present in Aroclor 1254 (58), while MC causes solely an increase of P450 members of the 1A subfamily (59). In this context it is of interest to note that measurement of mutants after coincubation of the (-)-(11R,12R) and (+)-(11S,12S)dihydrodiol of DB[a,l]P with V79 cells stably expressing the rat P4501A1 isoform (A. Luch, Ph.D Thesis, Univer-

Stereoselective Metabolism of DB[a,1]P-11,12-diol

sity of Mainz) revealed that under these conditions the differences between both enantiomers found after S9 activation (∼6-fold; see above) were increased to a factor of nearly 81-fold [1200 × 106 mutants/(nmol of (+)dihydrodiol‚mL) vs 97 300 × 106 mutants/(nmol of (-)dihydrodiol‚mL)]. These observations clearly indicate a much less efficient binding of the (+)-(11S,12S)-dihydrodiol to the active site of rat P4501A1 than that of the (-)-(11R,12R)-dihydrodiol. On the basis of the available binding site model of rat P4501A1 (21) and in accordance with our findings of the metabolic rates with rat liver microsomes as mentioned above, one might expect that the 11,12-dihydrodiols of DB[a,l]P are poor substrates for this P450 isoform. Interestingly, a similar substrate selectivity of rat P4501A1 toward the 3,4-dihydrodiols of B[a]A has been reported. The (-)-(3R,4R)-dihydrodiol of B[a]A is a poor substrate, but activated to the corresponding (-)-anti-dihydrodiol epoxide, the (+)-(3S,4S)dihydrodiol of B[a]A is not converted to a dihydrodiol epoxide at all (60). However, the role of individual P450 isoforms in the stereoselective biotransformation of DB[a,l]P and its 11,12-dihydrodiol enantiomers is not yet completely understood, and further investigations using cell lines expressing individual P450 isoforms are urgently needed to clarify this point. In conclusion, the present study demonstrates a clear relationship between the stereoselectivity of the biotransformation of the (-)-(11R,12R)- and the (+)-(11S,12S)dihydrodiol of DB[a,l]P to the corresponding fjord region DB[a,l]PDEs by liver preparations of Aroclor 1254treated rodents and the extent of stable DNA adduct formation as well as the detected mutations in Chinese hamster V79 cells. As supported by the preliminary results of our tumor-initiation studies (vide supra), differences between the individual 11,12-dihydrodiol enantiomers of DB[a,l]P during the initial events of biotransformation, DNA binding, and DNA mutagenicity can be also considered as an indicator for similar differences in their ability to cause cancer.

Acknowledgment. We thank M. Tommasone, K. Pauly, and A. Schwierzok for excellent technical assistance. This work was financially supported by the Deutsche Forschungsgemeinschaft (SFB 302). A.L. received a fellowship from the Astrid-Haugstrup-Soerensen Gedaechtnis Stiftung (Stifterverband fuer die Deutsche Wissenschaft) during his Ph.D. Thesis work.

Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1169

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