Microsomal Biotransformation of Benzo[ghi]perylene, a Mutagenic

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Chem. Res. Toxicol. 2005, 18, 700-710

Microsomal Biotransformation of Benzo[ghi]perylene, a Mutagenic Polycyclic Aromatic Hydrocarbon without a “Classic” Bay Region Karl L. Platt* and Stefanie Grupe Institute of Toxicology, University of Mainz, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany Received November 3, 2004

Carcinogenic polycyclic aromatic hydrocarbons (PAH), e.g., benzo[a]pyrene (BaP), possess a bay region comprising an ortho-fused benzene ring. Benzo[ghi]perylene (BghiP) represents the group of PAHs lacking such a “classic” bay region and hence cannot be metabolically converted like BaP to bay region dihydrodiol epoxides considered as ultimate mutagenic and carcinogenic metabolites of PAH. BghiP exhibits bacterial mutagenicity in strains TA98 (1.3 his+-revertant colonies/nmol) and TA100 (4.3 his+-revertant colonies/nmol) of Salmonella typhimurium after metabolic activation by the postmitochondrial hepatic fraction of CD rats treated with 3-methylcholanthrene. Inhibition of microsomal epoxide hydrolase (mEH) with 1,1,1-trichloro2-propene oxide raised the bacterial mutagenicity of BghiP in TA98 almost 4-fold indicating arene oxides as ultimate mutagens. To confirm this assumption, the biotransformation of BghiP was elucidated. Incubation of BghiP with liver microsomes of CD rats treated with Aroclor 1254 yielded 17 ethyl acetate extractable metabolic products. Twelve metabolites were identified by a combination of chromatographic, spectroscopic, and biochemical methods. The microsomal biotransformation of BghiP proceeds by two pathways: Pathway I starts with the monooxygenase attack at the 7-position leading to the 7-phenol, which is transformed to the 7,8and 7,10-diphenols followed by oxidation to the 7,8- and 7,10-quinones. On pathway II, the K regions of BghiP are successively converted to arene oxides yielding the indirectly identified 3,4-oxide and the 3,4,11,12-bisoxides. Enzymatic hydrolysis of the 3,4-oxide leads to the trans3,4-dihydrodiol, which is oxidized to the 3,4-quinone. Similarly, the trans-3,4-trans-11,12bisdihydrodiols and the trans-3,4-dihydrodiol 11,12-quinone are generated from the 3,4,11,12bisoxides. The trans-3,4-dihydrodiol and the trans-3,4-trans-11,12-bisdihydrodiols are preferentially formed as R,R and R,R,R,R enantiomers, respectively. The intrinsic bacterial mutagenicity of the 3,4,11,12-bisoxides is rather low and hardly explains the strong increase in bacterial mutagenicity of BghiP after inhibition of mEH. Thus, we believe that the 3,4-oxide plays a more important role as the ultimate mutagenic metabolite of BghiP.

Introduction 1

Polycyclic aromatic hydrocarbons (PAH) are toxicologically important environmental pollutants since this group contains many strong carcinogens. A characteristic structural feature of carcinogenic PAH is a sterically hindered region comprising an ortho-fused benzene ring, e.g., the bay region in benzo[a]pyrene (BaP) flanked by C-10 and C-11 (cf. Scheme 1). Enzymatically formed vicinal dihydrodiol epoxides with the oxirane ring in the bay region are considered to be the ultimate mutagenic and carcinogenic metabolites of PAH (1). Several PAHs * To whom correspondence should be addressed. E-mail: platt@ mail.uni-mainz.de. 1 Abbreviations: BaP, benzo[a]pyrene; BghiP, benzo[ghi]perylene; 3,4,11,12-bisdihydrodiol, trans-3,4-trans-11,12-tetrahydroxy-3,4,11,12tetrahydro-BghiP; 3,4-dihydrodiol, trans-3,4-dihydroxy-3,4-dihydroBghiP; 11,12-dihydrodiol-3,4-quinone, trans-11,12-dihydroxy-3,4-dioxo3,4,11,12-tetrahydro-BghiP; 7,8-diphenol, 7,8-dihydroxy-BghiP (other diphenols are similarly designated); ee, enantiomeric excess; EI, electron ionization; FD, field desorption; 3MC, 3-methylcholanthrene; mEH, microsomal epoxide hydrolase; 3,4-oxide, 3,4-epoxy-3,4-dihydroBghiP (other arene oxides are similarly designated); PAH, polycyclic aromatic hydrocarbon(s); 3,4-quinone, BghiP 3,4-quinone (other quinones are similarly designated); 3-phenol, 3-hydroxy-BghiP (other phenols are similarly designated); TCPO, 1,1,1-trichloro-2-propene oxide.

Scheme 1. Structural Formulas and Numbering of BaP and BghiP

exhibit genotoxicity although they lack the “classic” bay region of BaP, e.g., benzo[b]fluorene (2), naphthacene (3), benzo[ghi]fluoranthene (4), benzo[ghi]perylene (BghiP) (4-8), and anthanthrene (5, 9). We are interested in the metabolic activation of these PAHs. Recently, we reported our results concerning metabolism and mutagenicity of anthanthrene (9). In the present study, we investigated the microsomal biotransformation of BghiP in order to identify the metabolite(s) responsible for its genotoxicity. BghiP (Scheme 1) occurs ubiquitously in products of incomplete combustion and represents a major compo-

10.1021/tx049698a CCC: $30.25 © 2005 American Chemical Society Published on Web 03/09/2005

Metabolism and Mutagenicity of Benzo[ghi]perylene

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Table 1. 1H NMR (400 MHz) Spectral Data of Hydroxylated Metabolites of BghiPa metabolite no. 8

1/2 16 9 (R2)b

NMR data 4.99 (d, 1H, -OH, J ) 5.1), 5.04 (d, 1H, -OH, J ) 5.1), 5.14-5.19 (m, 1H, H3 or H4), 5.23-5.28 (m, 1H, H3 or H4), 7.80 (pseudo-t, 1H, H6, J6,5 ) 7.4, J6,7 ) 8.2), 8.02 (d, 1H, H5, J5,6 ) 7.4), 8.06-8.13 (m, 3H, H9,11,12), 8.26-8.30 (m, 2H, H1,2), 8.34 (d, 1H, H10, J10,9 ) 7.8), 8.86 (d, 1H, H7, J7,6 ) 8.2), 9.04 (d, 1H, H8, J8,9 ) 8.2) after 1H-2H exchange with D2O: 5.14 (d, 1H, H3 or H4, J3,4 ) 9.8), 5.23 (d, 1H, H3 or H4, J3,4 ) 9.8) 4.77-4.87 (m, 4H, -OH), 4.98-5.08 (m, 4H, H3,4,11,12), 7.70 (pseudo-t, 2H, H6,9, J6,5 ) J9,10 ) 7.4, J6,7 ) J9,8 ) 7.8), 7.90 (s, 2H, H1,2), 7.91 (d, 2H, H5,10, J5,6 ) J10,9 ) 7.4), 8.66 (d, 2H, H7,8, J7,6 ) J8,9 ) 7.8) 7.65 (d, 1H, H6, J6,5 ) 8.0), 8.01 (pseudo-t, 1H, H9, J9,8 ) 8.0, J9,10 ) 8.4), 8.13-8.22 (m, 4H, H3,4,11,12), 8.42-8.45 (m, 2H, H1,2), 8.51 (d, 1H, H10, J10,9 ) 8.4), 9.00 (d, 1H, H5, J5,6 ) 8.0), 9.03 (d, 1H, H8, J8,9 ) 8.0), 9.67 (s, 1H, -OH) 7.56 (d, 2H, H6,9, J6,5 ) J9,10 ) 8.5), 8.12 (d, 2H, H4,11, J4,3 ) J11,12 ) 9.0), 8.37 (s, 2H, H1,2), 8.45 (d, 2H, H3,12, J3,4 ) J12,11 ) 9.0), 8.82 (d, 2H, H5,10, J5,6 ) J10,9 ) 8.5), 9.36 (s, 2H, -OH)

a NMR spectra were recorded in acetone-d ; shown are the chemical shifts in ppm, multiplicity, number of protons, their position, and 6 coupling constants in Hz. b R2, 7,8-dihydroxybenzo[ghi]perylene (see Experimental Procedures).

nent of the total content of PAHs in the environment (10). Among other sources, BghiP has been identified in the exhaust emissions from gasoline engines, in mainstream cigarette smoke, and in charcoal-broiled steaks (10). BghiP was found inactive in several carcinogenicity studies in experimental animals (10, 11). However, after intrapulmonary injection of BghiP in rats, lung tumors were formed (12). In addition, BghiP possesses moderate cocarcinogenic activity when applied to mouse skin in combination with BaP (11). Despite these in vivo results concerning the genotoxicity of BghiP and the fact that this PAH was found to be mutagenic in many studies (4-8), no attempts were made till now to elucidate the biotransformation of BghiP apart from theoretical (13) or speculative (14) considerations.

Experimental Procedures Chemicals. BghiP (99% purity), benzo[e]pyrene, and benzo[c]phenanthrene were obtained from Promochem (Wesel, Germany). 3-Methylcholanthrene (3MC), perylene, triphenylene, and (-)-menthoxyacetic acid chloride were supplied by SigmaAldrich (Taufkirchen, Germany). Aroclor 1254 was obtained from Bayer (Leverkusen, Germany), trioctanoin was obtained from Sigma, and 1,1,1-trichloro-2-propene oxide (TCPO) was obtained from EGA (Steinheim, Germany). Benzo[e]pyrene 4,5-quinone was prepared as described (15). NADP+ was purchased from AppliChem (Darmstadt, Germany). Other biochemicals were supplied by Roche Diagnostics (Mannheim, Germany). Solvents for HPLC were obtained from Baker (GrossGerau, Germany) or from Roth (Karlsruhe, Germany); all other chemicals of analytical grade were purchased from VWR International (Darmstadt, Germany). Spectral Methods. UV/vis spectra of metabolites and of synthetic reference compounds were recorded on-line during HPLC with the diode array detector (1100 series; Agilent Technologies, Karlsruhe, Germany; cell volume, 13 µL; path length, 10 mm; peak width, >0.1 min; response time, 2 s; and slit, 2 nm) operated with LC 3D ChemStation software (Rev. A.08.03 [847]; Agilent Technologies). Field desorption (FD) mass spectra were obtained with a Finnigan MAT 95 (emitter heating current rate, 10 mA/min; extraction voltage, -5 kV; and acceleration voltage, 5 kV). Electron ionization (EI) mass spectra were recorded with a Varian MAT CH 7A spectrometer at 70 eV. 1H NMR spectra were measured on a Bruker AM 400 spectrometer at 400 MHz. Chemical shifts (in ppm) are relative to tetramethylsilane. Deuterated solvents (Deutero, Kastellaun, Germany) were used as indicated for each compound. Preparation of Tritium Labeled BghiP. [G-3H]BghiP was obtained from the parent hydrocarbon by tritium exchange with 3H2 in the presence of a soluble catalyst (Hartmann Analytic, Braunschweig, Germany). After removal of labile

tritium, the raw product (chemical purity, 98%; radiochemical purity, 17%) was dissolved in toluene and kept at -20 °C. Prior to use as substrate in microsomal incubations, an aliquot of the stock solution containing 27 µg of the crude product was evaporated, and the residue was dissolved in acetone (20 µL) and purified by HPLC [stationary phase: LiChrospher 100 RP18e (5 µm; Merck, Darmstadt, Germany); mobile phase: 100% MeOH; flow rate, 0.8 mL/min]. The fraction of the eluate containing BghiP was collected and brought to dryness with a stream of N2 resulting in a chemical purity of 99% and a radiochemical purity of 94%. Repeated fractionation did not considerably improve the radiochemical purity. The rather low calculated radiochemical purity was caused by radioactivity eluting after BghiP without exhibiting UV absorption typical for aromatic compounds. Because the radioactive impurity was not influenced by microsomal metabolism, it did not interfere with the biotransformation of BghiP and the quantification of its metabolites. Preparation of Quinones of BghiP and Their Reduction. BghiP (27.6 mg; 0.1 mmol) was oxidized with sodium dichromate as described for other PAHs (16). HPLC of the raw product (26.4 mg) employing the conditions mentioned below for the separation of the metabolites of BghiP revealed a mixture of three products (retention time, percentage of overall peak area, and molecular ion determined by FD-MS): Q1 (33.8 min; 84.3%; m/z ) 306), Q2 (37.9 min; 4.5%; m/z ) 306), and Q3 (38.6 min; 11.2%; m/z ) 306). The UV/vis spectra of Q1, Q2, and Q3 are displayed in Figures 4 and 7 and Figure S1 of the Supporting Information, respectively. The mixture of Q1, Q2, and Q3 was reduced with sodium borohydride according to ref 16 omitting acetylation and yielded solely three products characterized as described above: R1 (29.1 min; 3.8%; m/z ) 310), R2 (31.5 min; 78.3%; m/z ) 308), and R3 (34.5 min; 17.9%; m/z ) 308). The main product R2 was chromatographically isolated and identified by 1H NMR (see Table 1) as 7,8-dihydroxy-BghiP (7,8-diphenol); its UV/vis spectrum is shown in Figure 7. The UV/vis spectra of R1 and R3 are displayed in Figure S1 of the Supporting Information. From the relative amounts of the quinones Q1, Q2, and Q3 and their reduction products R1, R2, and R3, it can be deduced that Q1 is transformed to R2, Q2 to R1, and Q3 to R3. This assumption was verified by reoxidation of chromatographically isolated R2 and R3. Preparation of Subcellular Fractions. Adult male CD rats (180-220 g) obtained from the animal care facility of the University of Mainz were treated ip either with Aroclor 1254 (500 mg/kg body wt at day 6 before sacrifice) or with 3MC (40 mg/kg body wt at days 3, 2, and 1 before sacrifice). Aroclor 1254 and 3MC were dissolved in trioctanoin (2.5 mL/kg body wt). The postmitochondrial fraction, i.e., the 9000g supernatant, as well as the microsomal fraction of the liver were prepared as previously described (17) under sterile conditions and kept at -70 °C until use. Protein concentrations were determined

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by the method of Bradford et al. (18) employing Roti-Quant (Roth) as dye and bovine serum albumin for calibration. Metabolism Studies. Incubations contained 2 mg of microsomal protein of rat liver, 0.6 mM NADP+, 8 mM glucose-6phosphate, 1.5 units of glucose-6-phosphate dehydrogenase, and 5 mM MgCl2 in a final volume of 2 mL of 50 mM isotonic (150 mM KCl) sodium phosphate buffer (pH 7.4). In some cases, the incubation mixture contained 1 mM TCPO in 10 µL of Me2SO. This mixture was preincubated for 5 min at 37 °C. The incubation was started by the addition of 80 µM unlabeled or tritium-labeled BghiP (95-115 MBq [2.6-3.1 mCi]/mmol) in 50 µL of Me2SO and continued with shaking (120 oscillations/ min) at 37 °C. The incubation was stopped with 2 mL of icecold ethyl acetate. After the sample was vortexed for 1 min, the organic and aqueous phases were separated by centrifugation at 900g and 5 °C for 5 min. The extraction was repeated twice more with 2 mL of ethyl acetate. Incubations in the presence of TCPO were extracted three times with 2 mL of n-hexane/ethyl acetate/triethylamine [97.1/2.5/0.4, v/v (19)]. The organic phases were combined, dried over anhydrous magnesium sulfate, and brought to dryness at 37 °C with a stream of nitrogen; then, the residue was stored at -20 °C until HPLC separation was performed, for which the sample was dissolved in 30 µL of Me2SO. All steps were performed under subdued light. The recovered radioactivity in the organic and aqueous phase was 91 ( 6% of that applied. HPLC Analysis of Metabolites of BghiP. Chromatographic separations were performed with a system consisting of a ternary high-pressure pump (SP 8700; Spectra-Physics, Darmstadt, Germany), a sample injection valve (C6W; Valco, Schenkon, Switzerland) with a 25 µL sample loop, and a diode array detector (see Spectral Methods). For the chromatographic separation, 20 µL of the solution of the metabolites dissolved in Me2SO was injected onto a Vertex column (4 mm × 250 mm; Knauer, Berlin) filled with LiChrospher 100 RP-18e as the stationary phase. The mobile phases kept at 1.5 bar under an atmosphere of heliumsconsisted of a mixture of MeOH and bidistilled water with a linear increase in MeOH content from 50 to 100% (v/v) in 40 min followed by 100% MeOH for 25 min at a flow rate of 0.8 mL/min. When metabolites formed in the presence of TCPO had to be separated, the water content in the mobile phase was replaced by 10 mM aqueous NH4HCO3 (pH 8.0). Metabolites containing a transdihydrodiol moiety were chromatographically separated into the stereoisomers either directly by employing a stationary phase derived from (R)-(-)-2-(2,4,5,7-tetranitrofluoren-9-ylideneaminooxy)butyric acid covalently bound to silica gel (20) or after transformation into diastereomeric R-menthoxyacetates on silica gel (LiChrospher Si 60, 5 µm, 4 mm × 250 mm; Merck) as a stationary phase (21). For the detection and quantification of radioactivity, the eluate was collected (2112 RediRac, LKB, Bromma, Sweden) in 18 s (240 µL) fractions, mixed with liquid scintillator (3.5 mL; Aquasafe 300 Plus, Zinsser Analytik, Frankfurt, Germany), and counted (Tri-Carb 2100 TR, Canberra Packard, Germany). Quantification of Metabolic Conversion. The amount, m, of a distinct metabolite was calculated from the radiochromatogram with the formula: m ) (p‚R)/(100‚S), where p is the percentage of radioactivity in the combined fractions of the metabolite in relation to the total radioactivity collected during radiochromatography, R is the radioactivity in the organic phase after extraction, and S is the specific activity of the substrate, respectively. The total metabolic conversion, M, was determined by the formula: M ) (A + [P‚R]/100)/S, where A is the radioactivity remaining in the aqueous phase after ethyl acetate extraction and P is the percentage of the radioactivity eluting prior to BghiP in relation to the total radioactivity collected during radiochromatography. The amount of metabolites could also be determined from incubations of unlabeled BghiP by using calibration curves of the peak area at 254 nm vs the amount of the metabolite derived from incubations with radioactively labeled substrate.

Platt and Grupe

Figure 1. Bacterial mutagenicity of BghiP in strains TA100 and TA98 of S. typhimurium without (empty symbols) and with (filled symbols) external metabolic activation (postmitochondrial hepatic fraction of CD rats pretreated with 3MC); values are means ( SD (n ) 3). Mutagenicity Studies. The mutagenicity experiments were performed in the plate incorporation mode as described by Ames et al. (22). The minimal glucose agar plates were supplied by VWR International. The strains TA98 and TA100 of Salmonella typhimurium auxotrophic for histidine were generously provided by Dr. B. N. Ames (Berkeley, CA). The preparation of the postmitochondrial liver fraction of rats treated with 3MC is described above in the Metabolism Studies section; an amount containing 3.0 mg of protein was employed per plate. The test compounds were dissolved in 30 µL of Me2SO. The specific mutagenicity (his+-revertant colonies/nmol) was calculated as the slope of the linear part of the dose-response curve. Mutagenicity experiments in the presence of TCPO could only be performed with strain TA98 since this alkene oxide is a potent mutagen for strain TA100 (23).

Results Bacterial Mutagenicity of BghiP. Without external metabolic activation, BghiP was devoid of mutagenic activity in histidine-dependent S. typhimurium strains TA100 and TA98 (Figure 1) as well as in strain TA104 (data not shown). Metabolic activation with the postmitochondrial hepatic fraction of rats pretreated with 3MC yielded a mutagenic effect with a specific mutagenicity of 4.3 his+-revertant colonies of TA100 per nmol (Figure 1). Under identical activation conditions, the specific mutagenicity in strain TA98 reached only 1.3 his+revertant colonies per nmol (Figure 1). When, however, the mutagenicity assay with TA98 was performed in the presence of 1 mM TCPO, a potent inhibitor of microsomal epoxide hydrolase (mEH) (24), the mutagenic effect rose almost 4-fold to 5.0 his+-revertant colonies per nmol (Figure 1). Identification of Microsomal Metabolites of BghiP. Incubation of BghiP (80 µM) for 40 min with liver microsomes of adult male CD rats pretreated with Aroclor 1254, extraction with ethyl acetate, and separation by reverse phase HPLC consistently yielded 13 metabolites (Figure 2A; 1-4, 6, 8, 9, and 11-16) distinguishable by their retention time as well as their UV spectra taken during chromatography. Peaks that appeared erratically and displayed UV spectra untypical for derivatives of PAH were not considered metabolites of BghiP. Characterization and identification of the metabolites were performed with a combination of chromatographic, spectroscopic, and biochemical methods. Metabolite 8 obviously plays a central role in the biotrans-


Figure 2. Reverse phase HPLC chromatograms of the ethyl acetate extractable metabolites obtained from incubations of BghiP (80 µM, 40 min) with liver microsomes (1 mg of protein/ mL) of CD rats pretreated with Aroclor 1254: (A) without TCPO and (B) with TCPO (1 mM). For the meaning of the numbers, see Table 2; for experimental conditions, see Experimental Procedures.

formation of BghiP since it is the metabolic precursor of metabolites 1-4, 6, and 13 (Table 2). Metabolite 8 exhibits the UV chromophore of benzo[e]pyrene (Figure 3) indicating a 3,4-dihydro derivative of BghiP. The fact that TCPO abolished the enzymatic formation of metabolite 8 (cf. Figure 2B) suggested the presence of at least one dihydrodiol moiety in 8. The FD mass spectrum supported this assumption by the molecular ion at m/z ) 310 and fragmentation by the loss of one molecule of water (Table 2). Finally, the 1H NMR spectrum of the chromatographically isolated metabolite 8 unequivocally proved its structure as the trans-3,4-dihydroxy-3,4-dihydro-BghiP (3,4-dihydrodiol) of BghiP (Table 1). The coupling constant J3,4 ) 9.8 Hz observed after 1H-2H exchange indicates the quasi-diequatorial conformation of the hydroxyl groups at C-3 and C-4 (25). Reduction of quinone Q2 (see Experimental Procedures) with sodium borohydride yielded R1, which was identical with the 3,4-dihydrodiol based on chromatographic and UV spectroscopic evidence (cf. Figure 3 and Figure S1 of the Supporting Information). Consequently, Q2 is the BghiP 3,4-quinone (3,4-quinone). Metabolite 13 exhibited a molecular ion of m/z ) 306 indicative of a quinone of BghiP, it was rapidly formed from the 3,4-dihydrodiol 8 upon exposure to air, and it showed the same UV spectrum as Q2 (Figure 4), which demonstrated its identity with the 3,4-quinone. The most polar metabolites 1 and 2 (cf. Figure 2A) displayed identical UV spectra, which resembled that of triphenylene (Figure 5). The FD mass spectrum of metabolites 1 and 2 exhibits a parent ion at m/z ) 344


fragmented by the successive loss of two molecules of water (Table 2). Together with their UV spectral properties, this led to the assumption that metabolites 1 and 2 are trans-3,4-trans-11,12-tetrahydroxy-3,4,11,12-tetrahydro-BghiP (3,4,11,12-bisdihydrodiols). Additional proof of this structural assignment was furnished by the suppression of the metabolic formation of 1 and 2 by TCPO (Figure 2B). Finally, the 1H NMR spectra of the chromatographically isolated metabolites 1 and 2 (Table 1) unequivocally proved their structures as the 3,4,11,12bisdihydrodiols of BghiP. Metabolite 4 exhibited a UV spectrum similar to that of benzo[e]pyrene 4,5-quinone (Figure 6). This indicated a quinone moiety at the 3,4-position and a saturated 11,12-position in the BghiP molecule. The parent ion at m/z ) 340 and the loss of one molecule of water (Table 2) together with its polarity (Figure 2A) suggest that metabolite 4 has the structure of the trans-11,12-dihydroxy-3,4-dioxo-3,4,11,12-tetrahydro-BghiP (11,12-dihydrodiol-3,4-quinone). Metabolite 9 was identical with the synthetically available 7,8-diphenol (R2; see Experimental Procedures) on the basis of their UV spectra (Figure 7A), retention times, and FD mass spectra (Table 2). The same analytical parameters (Figure 7B and Table 2) demonstrated the identity of metabolite 11 with the synthetic precursor of the 7,8-diphenol, the 7,8-quinone (Q1). While this metabolite was always detected in microsomal incubations of BghiP, the 7,8-diphenol 9 sometimes appeared only in traces probably due to its nonenzymatic oxidation to the more stable 7,8-quinone 11. Microsomal incubations of the isolated metabolite 16 led to the formation of metabolites 9, 11, 12, and 14 (Table 2). Because metabolites 9 and 11 are oxygenated at the 7- and 8-positions of BghiP and metabolite 16 displayed the parent ion at m/z ) 292, it very likely bears the structure of the 7-phenol. Finally, the 1H NMR spectrum of the chromatographically isolated metabolite 16 (Table 1) proved its structure as 7-hydroxy-BghiP. Upon microsomal incubation of BghiP in the presence of TCPO, the polar metabolites 1-6 and 8 disappeared while two prominent metabolites, 7a,b, were formed (Figure 2B). Metabolites 7a,b exhibited identical UV spectra resembling those of metabolites 1 and 2 as well as of triphenylene (Figure 5). Metabolites 7a,b were isolated and incubated with microsomes generating the 3,4,11,12-bisdihydrodiols 1 and 2 (data not shown). This information taken together indicated that 7a,b are the 3,4,11,12-bisoxides. Further support of this assumption was provided by the FD mass spectra of 7a,b displaying the expected molecular ion at m/z ) 308 (Table 2). On searching for the 3,4-epoxy-3,4-dihydro-BghiP (3,4-oxide) as a metabolite of BghiP, 14a was detected as a small peak in the chromatogram of microsomal incubations in the presence of TCPO (Figure 2B) exhibiting the UV spectrum of the 3,4-dihydrodiol 8 (Figure 3A). Thus, metabolite 14a probably represents the 3,4-oxide. Its isomerization product, the 3- and/or 4-phenol, was found hidden under the peak of the 7-phenol 16. When metabolite 16 was isolated from a microsomal incubation of BghiP in the presence of TCPO and rechromatographed by isocratic elution with methanol/acetonitrile/ water (48.0/30.8/21.2%; v/v), thereby optimizing the selectivity of the mobile phase (26), partial separation into two peaks, 15a and 16, was achieved (Figure 2C).

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Table 2. Chromatographic, Spectroscopic and Biochemical Characterization of the Microsomal Metabolites of BghiP metabolites formed from BghiPa b

no.

tR (min)

FD spectral data (m/z)

1 2 3 4 6 7ad 7bd 8 9 11 12 13 14 14ad 15 15ad 16

5.9 6.9 14.2 15.7 26.5 27.5 29.0 29.1 31.5 33.8 34.5 37.9 38.6 41.1 43.0 43.4h 43.4h

344, 326, 308 344, 326, 308 326, 308 340, 322 326, 308 308 308 310, 292 308, [392]f 306 308, [308, 279, 250]g 306 306

a

biotransformation from 8 8 8 1, 2, 8 8

16 9, 16 16 8 16

UV spectral characterization

into

chromophore of

4 4

spectrum identical with that ofc

triphenylene triphenylene benzo[e]pyrene-4,5-quinone

[1 + 2]e [1 + 2]e 1, 2, 3, 4, 6, 13 11

triphenylene triphenylene benzo[e]pyrene

14

R1 7,8-diphenol (R2) BghiP-7,8-quinone (Q1) R3 BghiP-3,4-quinone (Q2) Q3

benzo[e]pyrene 292 292

9, 11, 12, 14 b

Numbers as in Figure 2. Retention time upon reversed-phase HPLC; for conditions, see the Experimental Procedures. c Numerals indicate synthetic derivatives of BghiP; see the Experimental Procedures. d Formation from BghiP exclusively in the presence of TCPO. e Formation from mixture of 7a,b. f Acetylation product of 9. g EI-MS. h Chromatographic separation achieved by isocratic elution with acetonitrile/methanol/water; see the Experimental Procedures.

Figure 3. UV spectra of (A) metabolites 8 and 14a and of (B) benzo[e]pyrene. Spectra were taken during HPLC separation.

Figure 4. UV/vis spectra of metabolite 13 (broken line) and of 3,4-quinone (Q2; solid line). Spectra were taken during HPLC separation.

Metabolite 15a was also formed as a product of acidic dehydration (27) of the 3,4-dihydrodiol 8 (data not shown). It should be emphasized that arene oxides 14a and 7a,b could only be detected by reverse phase chromatography when using a slightly alkaline eluent (9, 28, 29). Furthermore, microsomal incubations in the absence of TCPO provided no indication for the formation of the 3,4-oxide or its isomerization product(s). Because no metabolites were detected exhibiting the UV chromophore of perylene or of benzo[c]phenanthrene, the formation of considerable amounts of 1,2-dihydro or 5,6,7,7atetrahydro derivatives of BghiP is unlikely.

Figure 5. UV spectra of (A) metabolites 1 and 2, (B) triphenylene, and (C) metabolites 7a,b. Spectra were taken during HPLC separation.

Figure 6. UV/vis spectra of (A) metabolite 4 and of (B) benzo[e]pyrene 4,5-quinone. Spectra were taken during HPLC separation.

Chiral Separation of Metabolites of BghiP. The chromatographic separation of the enantiomers of 3,4-dihydrodiol 8 was attempted either without derivatization on a chiral stationary phase based on (R)-(-)-2(2,4,5,7-tetranitrofluoren-9-ylideneaminooxy)butyric acid covalently bound to silica gel or as diastereomeric bis(R-menthoxyacetates) on silica gel. Both methods were successful and revealed an enantiomeric purity of the metabolically formed 3,4-dihydrodiol 8 of 50% ee (Figure 8A,B). While the separation of underivatized 3,4,11,12bisdihydrodiols 1 and 2 on different chiral phases failed, the separation of 1 as tetrakis(R-menthoxyacetate) succeeded and revealed an enantiomeric purity of 96% ee


Figure 7. UV/vis spectra of (A) 7,8-dihydroxybenzo[ghi]perylene (R2; solid line) and metabolite 9 (broken line) and of (B) BghiP 7,8-quinone (Q1; solid line) and metabolite 11 (broken line). Spectra were taken during HPLC separation.


incubations employing unlabeled substrate (see Experimental Procedures). While the amount of the metabolites 1/2, 11, and 14 rose time-dependently up to an incubation time of 60 min, the increase of metabolites 8 and 16 ceased after ca. 20 min (Figure 9). When liver microsomes of rats after treatment with 3MC were employed, the quantitative relations after 40 min were similar; however, when the incubation was performed in the presence of TCPO (1 mM), the total metabolic conversion dropped to 22.0 nmol while the amount of metabolites remaining in the aqueous phase rose to 38.2%. About one-fifth of the total metabolic conversion of BghiP at 40 min consisted of the 3,4,11,12-bisdihydrodiols 1/2 followed by the 3,4-dihydrodiol 8 and the 7,8-quinone 11. When TCPO was present in the microsomal incubation of BghiP, metabolites 1, 2, and 8 could not be detected; under these conditions, the 3,4,11,12-bisoxides 7a,b (11.4%) and the 7,8-quinone 11 (10.9%) were the principal metabolites. Bacterial Mutagenicity of Metabolites of BghiP. To determine their genotoxic potential, the most prominent microsomal metabolites of BghiP were isolated and tested for bacterial mutagenicity. As Figure 10 shows, the 3,4,11,12-bisdihydrodiols 1 and 2 were not further activated by the postmitochondrial hepatic fraction of rats treated with 3MC whereas under the same conditions the 3,4-dihydrodiol 8 and the 7-phenol 16 were activated to mutagens for strain TA100. Only the 3,4,11,12-oxides 7a,b are metabolites of BghiP exhibiting mutagenicity in strain TA98 without enzymatic activation (Figure 10).

Discussion

Figure 8. Chromatographic separations of the stereoisomers of trans-3,4-dihydroxy-3,4-dihydrobenzo[ghi]perylene (A,B) and of trans-3,4-trans-11,12-tetrahydroxy-3,4,11,12-tetrahydrobenzo[ghi]perylene (C, early-eluting; D, late-eluting diastereomer on reverse phase) underivatized {A: stationary phase, (R)-(-)-2(2,4,5,7-tetranitrofluoren-9-ylideneaminooxy)butyric acid covalently bound to silica gel; mobile phase, dichloromethane/ methanol [1/1, v/v]; and flow, 2 mL/min} or as R-menthoxyacetates (B-D: stationary phase, silica gel; mobile phase, n-hexane/ diethyl ether [95/5, v/v]; and flow, 1 mL/min); numbers denote metabolites of BghiP (see Table 2); dotted line, chromatogram of synthetic compound.

(Figure 8C). Under identical conditions, the tetrakis(Rmenthoxyacetate) of 2 exhibited only one peak upon chromatography on silica gel (Figure 8D). Quantification of the Microsomal Conversion of BghiP and the Time-Dependent Formation of Its Metabolites. The metabolic conversion of BghiP (Table 3) was determined from microsomal incubations of the tritiated substrate. Liver microsomes of rats after treatment with Aroclor 1254 converted in 40 min 33.8 of 160 nmol of BghiP (80 µM) to metabolites that could be extracted with ethyl acetate (71.3%) or remained in the aqueous phase (28.7%) probably bound to proteins. The amount of the metabolites could also be obtained from

Like other PAHs, BghiP failed to exert bacterial mutagenicity without external enzymatic activation (6). Because monooxygenases of the P450 1A subfamily were found to be most efficient in activating BghiP to mutagenic metabolites (5), the postmitochondrial hepatic fraction of rats after treatment with 3MC was employed as metabolizing system. Under these conditions, BghiP exhibited mutagenicity in strain TA100 that is reverted to histidine prototrophy by base pair substitutions. The specific mutagenicity (4.3 his+-revertant colonies/nmol) was about twice as high as that reported in the literature (1.5-2.2 his+-revertant colonies/nmol) (4-6) probably due to the fact that we employed 3MC as an inducer instead of Aroclor 1254. The mutagenicity of BghiP in strain TA98 reverted by frame shift mutagens was much weaker and amounted to 1.3 his+-revertant colonies/nmol as compared to 1.9 his+-revertant colonies/nmol calculated from the results of Sakai et al. (6). Because the inhibition of mEH by TCPO raised the mutagenicity of BghiP in strain TA98 dramatically by 385%, an important role of arene oxides in the mutagenicity of this PAH could be anticipated (30). Although there are reports dealing with the metabolic conversion of BghiP (14, 31), nothing is known on the structure of its metabolites. To determine the metabolites responsible for the genotoxicity of BghiP, the biotransformation of this PAH was elucidated. BghiP is transformed to 17 microsomal metabolites, 12 of which could be identified by a combination of chromatographic, spectroscopic, and biochemical methods (Table 2). On the basis of this structural elucidation, it is concluded that the microsomal biotransformation of BghiP proceeds via two distinct metabolic pathways (Scheme 2). Pathway I

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Table 3. Metabolic Conversion of BghiP by Rat Liver Microsomesa nmolc (% of total metabolic conversion) inducer/inhibitor

Aroclor 1254

3MC

Aroclor 1254/TCPOb

total metabolic conversion EtOAc extractable metabolites metabolites remaining in the aqueous phased metabolites 3,4,11,12-bisdihydrodiols (1 + 2) 3,4,11,12-bisoxides (7a + 7b) 3,4-dihydrodiol (8) 7,8-quinone (11) 7,10-quinone (14) 3,4-oxide (14a) 7-phenol (16) other metabolites

33.8 ( 8.1 (100) 24.1 ( 6.2 (71.3) 9.7 ( 2.0 (28.7)

32.0 ( 10.2 (100) 21.4 ( 4.7 (66.9) 10.6 ( 5.4 (33.1)

22.0 ( 0.8 (100) 13.6 ( 0.5 (61.8) 8.4 ( 1.3 (38.2)

6.9 ( 1.5 (20.4)

Microsomal Biotransformation of Benzo[ghi]perylene, a Mutagenic

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