Chem. Res. Toxicol. 2003, 16, 1581-1588
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Metabolism of the Polynuclear Sulfur Heterocycle Benzo[b]phenanthro[2,3-d]thiophene by Rodent Liver Microsomes: Evidence for Multiple Pathways in the Bioactivation of Benzo[b]phenanthro[2,3-d]thiophene Zhi-Xin Yuan, Harish C. Sikka, Sumaira Munir, Atul Kumar, A. V. Muruganandam, and Subodh Kumar* Environmental Toxicology and Chemistry Laboratory, Great Lakes Center, State University of New York College at Buffalo, 1300 Elmwood Avenue, Buffalo, New York 14222 Received June 27, 2003
Benzo[b]phenanthro[2,3-d]thiophene (BPT), a thia analogue of dibenz[a,h]anthracene (DBA), is a carcinogenic environmental pollutant. We have examined the metabolism of BPT by rodent liver microsomes to investigate the mechanism by which BPT produces mutagenic and carcinogenic effects. Both rat and mouse liver microsomes biotransformed [G-3H]BPT to various metabolites including BPT 3,4-diol and BPT sulfoxide, which are significantly more mutagenic than the parent compound. Liver microsomes from both control mice and rats metabolize BPT at similar rates. Treatment of mice with P450 inducers DBA, 3-methylcholanthrene (3-MC), Aroclor 1254, and phenobarbital enhanced the rate of metabolism of BPT by 74-, 28-, 77-, and 6-fold, respectively. In comparison, the treatment of rats with DBA and 3-MC increased the rate of metabolism of BPT by 22- and 34-fold, respectively, suggesting that P450 enzymes responsible for the metabolism of BPT are enhanced to different extents in rats and mice by a similar class of compounds. In general, the liver microsomes from mice treated with DBA or 3-MC were more active than those from similarly treated rats in metabolizing BPT to its 3,4diol, a precursor to the bay-region diol epoxide of BPT. BPT sulfone was a minor metabolite (if formed) in all cases. The liver microsomes from rats treated with DBA or 3-MC or from mice treated with PB produced a significant proportion of BPT sulfoxide (12-41%). In contrast, the liver microsomes from DBA- or 3-MC-treated mice formed BPT sulfoxide as a minor metabolite (98% purity) by multiple HPLC over a Zorbax C-18 silica column (4.6 mm × 250 mm) using 20% water-methanol isocratically. BPT 5,6-diol was synthesized by oxidation of cis-5,6-dihydroxy-5,6-dihydroBPT (22) with DDQ followed by the reduction of resulting BPT 5,6-quinone with an excess of sodium borohydride in ethanol following the general procedure reported earlier (23). The crude product was recrystallized from acetone to produce a colorless crystalline solid; mp 217-219 °C. 1H NMR (300 MHz, acetone-d6): δ 4.67 (dd, 1 H, J ) 4 and 10.4 Hz, 5-H or 6-H), 4.75 (dd, 1 H, J ) 4 and 10.4 Hz, 5-H or 6-H), 5.33 (d, 1 H, J ) 4 Hz, exchangeable with CD3OD, 5-OH or 6-OH), 5.45 (d, 1 H, J ) 4 Hz, exchangeable with CD3OD, 5-OH or 6-OH). HRMS for C20H14O2S: calcd M+, 318.0714; observed M+, 318.0693. 3-Hydroxybenzo[b]phenanthro[2,3-d]thiophene (3-hydroxyBPT) and BPT 3,4-diol were synthesized by unequivocal routes and characterized as previously described (24). 2-Hydroxybenzo[b]phenanthro[2,3-d]thiophene (2-hydroxyBPT) was synthesized according to the procedure analogous to that described for 3-hydroxyBPT (24). Thus, the Wittig reagent obtained from 2-bromo-4-methoxybenzyl chloride (25) and triphenylphosphine was stirred with dibenzothiophene-2-carboxaldehyde (21) in methylene chloride and 50% aqueous NaOH to produce a mixture of Z- and E-isomer of 1-(2-bromo-4methoxyphenyl)-2-(dibenzothiophene-3-yl)ethylene as an oil in a nearly quantitative yield. This mixture was refluxed in quinoline containing powdered KOH for 3 h. The usual workup of the reaction mixture produced a dark product, which was chromatographed over dry column grade silica gel. Elution of the column with hexane yielded nearly a 3:2 mixture of 2-methoxyBPT and 2-methoxybenzo[b]phenanthro[4,3-d]thiophene (1H NMR), which after demethylation (BBr3/CH2Cl2) followed by column chromatography over dry column grade silica gel using 10% EtOAc-hexane as a mobile solvent, produced 2-hydroxyBPT as a colorless crystalline compound; mp 241244 °C. 1H NMR (300 MHz, CDCl3 + DMSO-d6): δ 7.23 (dd, 1 H, J ) 8.5 and 2.3 Hz, 3-H), 7.48-7.91 (m, 7 H, ArH), 8.14 (d, 1 H, J ) 2.3 Hz, 1-H), 8.27-8.32 (m, 1 H, ArH), 8.36 (s, 1 H, 2-OH, exchangeable with MeOH-d4), 8.59 (s, 1 H, 7-H), 9.00 (s, 1 H, 13-H). HRMS for C20H12OS: calcd M+, 300.0603; observed M+, 300.0610. BPT sulfone was prepared by oxidation of BPT with an excess of 3-chloroperoxybenzoic acid in chloroform using the method described by Murphy et al. for analogous sulfone (14). BPT sulfone was obtained as a colorless solid; mp 349-351 °C. 1H NMR (500 MHz, CDCl3): δ 7.69-7.92 (m, 4 H, ArH), 7.99-8.14 (m, 4 H, ArH), 8.34 (d, 1 H, J ) 7.6 Hz, ArH), 8.82 (s, 1 H, 7-H), 9.13 (d, 1 H, J ) 7.9 Hz, 1-H), 9.56 (s, 1 H, 13-H). HRMS for C20H12O2S: calcd M+, 316.0558; observed M+, 316.0562. Oxidation of BPT with ceric ammonium nitrate (26) in 75% acetoni-
Metabolism of Benzo[b]phenanthro[2,3-d]thiophene trile or iodobenzenediacetate (27) in acetic acid followed by purification of the resulting product by preparative TLC using 50% EtOAc-hexanes afforded BPT sulfoxide as a light yellow crystalline solid; mp 233-234 °C. 1H NMR (500 MHz, CDCl3): δ 7.50-8.00 (m, 8 H, ArH), 8.05 (d, 1 H, J ) 7.6 Hz, ArH), 8.24 (s, 1 H, 7-H), 8.72 (d, 1 H, J ) 8.2 Hz, 1-H), 9.29 (s, 1 H, 13-H). HRMS for C20H12OS: calcd M+, 300.0603; observed M+, 300.0614. Preparation of Liver Microsomes. Female CD-1 mice (25-34 g) and Sprague-Dawley rats (100-125 g) were obtained from Harlan (Almont, NY) and treated humanely in our animal care facility under the guidelines approved by Institutional Animal Care and Use Committee (IACUC). After an acclimation period of 7 days, the animals were treated with DBA (40 mg/kg in corn oil, single i.p. injection), 3-MC (20 mg/kg in corn oil, single i.p. injection), Aroclor 1254 (500 mg/kg in corn oil, single i.p. injection), PB (50 mg/kg/day in saline, i.p. for 4 days), or corn oil (1 mL/kg, single i.p. injection). Groups of 4-6 animals were killed by cervical dislocation 72 h after treatment except for PB in which case animals were killed 24 h after the last treatment. Liver microsomes from the pooled livers were prepared as described earlier (28), quickly frozen, and stored at - 80 °C. The protein concentration in each microsomal preparation was determined by the method of Lowry et al. (29) using crystalline bovine serum albumin as a standard. Metabolism of [G-3H]BPT by Mouse and Rat Liver Microsomes. Incubation mixtures containing 100 µmol of potassium phosphate buffer (pH 7.4), 4 µmol of MgCl2, 1 µmol of NADPH, and microsomal protein (0.05-1.5 mg) in a total volume of 1 mL were preincubated at 37 °C for 5 min. The reaction was started by the addition of [G-3H]BPT (2.5-60 nmol containing 1 µCi in 20 µL of DMSO). After incubation at 37 °C for 5-30 min with shaking, the total metabolism of BPT was determined according to Van Contfort et al. (30). For metabolite identification and quantification, [G-3H]BPT (1 µCi) and unlabeled BPT (in amounts to attain 10 µM for liver microsomes from 3-MC-induced mice and DBA-induced rats and 40 µM for all other liver microsomes) in 20 µL of DMSO was incubated with various microsomal preparations (0.5 mg protein/mL) for 10 min at 37 °C. The reaction was terminated by the addition of 3 mL of ice-cold ethyl acetate-acetone (2:1), and then, the unreacted substrate and metabolites were extracted. Control incubations were terminated at zero time. The organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated under a stream of nitrogen. The residue was dissolved in methanol (100 µL). Incubation in the presence of TCPO was carried out under similar conditions. In a typical experiment, nearly 90% of the radioactivity from the microsomal incubation was extracted into ethyl acetate. The values of metabolism for zero time blanks ranged from 1 to 5%. Analysis of Metabolites. Aliquots (20-30 µL) of the metabolite extracts were mixed with unlabeled synthetic standards and analyzed by HPLC on a Zorbax ODS column (0.46 cm × 25 cm) (Figure 2). Conditions for individual separations are given in Figure 2. The effluent was monitored for UV absorption on a UV detector (254 nm) and for radioactivity on a IN/US radiometric detector. The metabolites were also analyzed by HPLC on a Zorbax CN column (Figure 3), which can resolve BPT sulfone from BPT sulfoxide. UV spectra of the individual metabolites were obtained by a diode array detector interfaced with the HPLC by analyzing pooled metabolite extracts without unlabeled synthetic standards. Individual metabolites were quantitated by integration of appropriate radioactivity peaks. The metabolites were identified by comparing their UV spectra and retention times with those of authentic standards. The overall recovery of radioactivity from the HPLC column ranged from 89 to 93%.
Results Rate of Metabolism of BPT by Liver Microsomes from Rodents Treated with Various P450 Inducers. Initial experiments were performed to establish linear
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Figure 2. Metabolites of BPT resolved on a Zorbax C-18 column eluted with 50% methanol-water for 0-10 min, 5070% methanol-water in 5 min, 70% methanol-water for 25 min, 70% methanol-water to 100% methanol in 10 min, and finally 100% methanol for 20 min with a flow rate of 1 mL/min. (A) Resolution of BPT and the following synthetic standards: BPT 3,4-diol, BPT sulfoxide, BPT sulfone, 2-hydroxyBPT, and 3-hydroxyBPT. (B) Metabolites of [G-3H]BPT produced by microsomes prepared from mice treated with DBA.
Figure 3. UV spectra of metabolite (- - -) eluted at 36 min (Figure 2B) and synthetic BPT sulfoxide (-).
conditions for the metabolism of BPT by mouse and rat liver microsomes with respect to substrate concentration, incubation time, and microsomal protein concentration. Total BPT metabolism by the liver microsomes was measured according to Van Canfort et al. (30) using 40 µM of BPT for 10 min for microsomal protein linearity, 0.5 mg/mL of microsomal protein for 10 min for substrate linearity, and 40 µM of BPT and 0.5 mg of microsomal protein/mL for time linearity. The metabolism of BPT showed linearity from 0.05 mg to at least 0.5 mg of protein/mL for all microsomal preparations from both species. The extent of BPT metabolism by all types of liver microsomes was linear from 2.5 to 30 µM substrate concentrations except for those from 3-MC-treated mice and DBA-treated rats. For these microsomal prepara-
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Table 1. Rate of Metabolism of BPT by Rat and Mouse Liver Microsomesa species mouse
rat
cytochrome P-450 inducer
rate of metabolism (nmol/mg protein/min)b
control DBA 3-MC araclor 1254 PB control DBA 3-MC
0.022 ( 0.00 1.469 ( 0.14 0.565 ( 0.05 1.538 ( 0.18 0.121 ( 0.01 0.017 ( 0.00 0.438 ( 0.03 0.673 ( 0.04
a The microsomal protein concentration was 0.5 mg/mL for all microsomal preparations. The substrate concentration was 10 µM for 3-MC-treated mice and DBA-treated rats and 40 µM for other microsomal preparations. The period of incubation was 10 min. Incubation conditions are given in the Materials and Methods. b Results are means ( SD of triplicate determinations.
tions, the metabolism of BPT was linear only up to 5 µM substrate concentration. The time course of metabolism of BPT appeared to be linear at least up to 10 min in all cases. The rate of metabolism of BPT by liver microsomes and the profile of metabolites formed were examined at a saturating substrate concentration (10 µM for liver microsomes from 3-MC-treated mice and DBA-treated rats, and 40 µM for all other liver micrsomes) under conditions that gave linearity with respect to protein concentration (0.5 mg/mL) and incubation time (10 min). Pretreatment of rodents with P450 inducers resulted in a 5.5-70-fold enhancement in BPT metabolism (Table 1). The liver microsomes from untreated mice and from the mice treated with DBA, 3-MC, Aroclor 1254, or PB metabolized BPT at a rate of 0.02 ( 0.00, 1.47 ( 0.14, 0.56 ( 0.05, 1.54 ( 0.19, and 0.12 ( 0.01 nmol/min/mg of microsomal protein, respectively. In the case of rat liver microsomes, the rate of metabolism of BPT was 0.44 ( 0.04, 0.67 ( 0.044, and 0.02 ( 0.00 nmol/min/mg of protein for liver microsomes from DBA- and 3-MC-treated and untreated rats, respectively. Profile of [G-3H]BPT Metabolites Formed by Mouse Liver Microsomes. A typical profile of metabolites of BPT generated by liver microsomes from DBApretreated mice is shown in Figure 2B. The following BPT metabolites were identified by cochromatography with authentic standards and by comparing the UV spectra of the isolated metabolites with those of authentic standards (BPT sulfoxide, BPT sulfone, and BPT 3,4diol). Under the HPLC conditions used for the separation of authentic standards on a Zorbax-C18 column, BPT sulfoxide and BPT sulfone were not completely resolved (Figure 2A). A poor separation of BPT sulfoxide and BPT sulfone and a similarity in the UV spectrum of these BPT derivatives prompted us to investigate an alternate HPLC condition for the separation of BPT sulfoxide and BPT sulfone. We found that a much better separation of BPT sulfoxide and sulfone can be achieved using a Zorbax CN column (Figure 3A). Analysis of BPT metabolite(s) using a Zorbax CN column indicated that the BPT metabolites coeluting with BPT sulfoxide on a Zorbax C-18 column and had UV spectrum (Figure 4) very similar to that of BPT sulfoxide contain 10-12% of BPT sulfone (Figure 3B). A poor separation of authentic BPT 3,4-diol and BPT 5,6-diol was also noted when a Zorbax ODS column was used (Figure 2A). However, a comparison of the UV spectra of these compounds with that of the metabolite coeluting with these dihydrodiols indi-
Figure 4. UV spectra of metabolite (- - -) eluted at 38 min (Figure 2B) and synthetic BPT 3,4-diol (-) and BPT 5,6-diol (- ‚ -).
Figure 5. Metabolites of BPT resolved on a Zorbax CN column eluted with 30% methanol-water for 0-5 min, 30-78% methanol-water in 60 min, and 78% methanol-water to 100% methanol in 5 min with a flow rate of 1 mL/min. (A) Resolution of BPT, BPT sulfoxide, and BPT sulfone. (B) Metabolites of [G-3H]BPT produced by microsomes prepared from control rats.
cated that the metabolite is BPT 3,4-diol (Figure 5). The radioactive peak coeluting with BPT 3,4-diol and some of the metabolites eluting before 30 min (Figure 2 B) was significantly reduced when [G-3H]BPT was incubated with liver microsomes in the presence of epoxide hydrolase inhibitor TCPO (data not shown). The relative proportion of the ethyl acetate extractable BPT metabolites formed by various types of liver mi-
Metabolism of Benzo[b]phenanthro[2,3-d]thiophene
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Table 2. Profile of [G-3H]BPT Metabolites Formed by Mouse and Rat Liver Microsomes species mouse
cytochrome P-450 inducer none DBA 3-MC araclor PB
rat
none DBA 3-MC
sulfoxide
sulfone
63.41 ( 0.88 (13.98 ( 1.94) 1.62 ( 0.72 (23.79 ( 10.57) 4.32 ( 2.12 (24.41 ( 11.97) 2.81 ( 1.12 (43.21 ( 17.22) 25.67 ( 5.21 (31.04 ( 6.40) 58.14 ( 0.83 (9.89 ( 0.14) 12.18 ( 0.50 (53.35 ( 2.24) 40.77 ( 18.12 (93.35 ( 41.50)
11.19 ( 0.156 (2.42 ( 0.34) ND ND ND 4.52 ( 0.93 (5.48 ( 1.13) 10.26 ( 0.15 (1.74 ( 0.02) 2.15 ( 0.09 (9.41 ( 0.39) 7.19 ( 3.20 (16.47 ( 7.32)
% of total metabolitesa 3,4-dihydrodiol ND 15.32 ( 6.07 (225.05 ( 89.16) 26.06 ( 0.84 (147.24 ( 4.74) 26.61 ( 0.80 (409.26 ( 12.30) 7.63 ( 4.27 (9.23 ( 5.16) 3.97 ( 1.18 (0.67 ( 0.20) 3.51 ( 0.19 (15.37 ( 0.83) 15.21 ( 5.48 (34.83 ( 12.55)
phenols
25.28 ( 0.52 (5.56 ( 0.11) 26.74 ( 2.71 (392.81 ( 39.80) 25.15 ( 4.68 (142.09 ( 26.44) 26.15 ( 1.73 (402.18 ( 26.60) 56.52 ( 9.95 (68.38 ( 12.04) 5.20 ( 3.38 (0.88 ( 0.57) 28.20 ( 1.84 (123.51 ( 8.06) 11.53 ( 10.05 (26.40 ( 23.01)
unknown ND 56.30 ( 9.65 (827.04 ( 141.75) 44.42 ( 5.42 (250.97 ( 30.62) 44.40 ( 3.13 (682.87 ( 48.13) 5.65 ( 0.15 (6.83 ( 0.18) 22.43 ( 2.02 (3.81 ( 0.34) 53.91 ( 0.20 (236.12 ( 0.87) 25.29 ( 5.03 (57.91 ( 11.51)
a Values are means ( SD of triplicate determinations and represent the percentage of total radioactivity, which emerges from the column prior to [G-3H]BPT. The figures in parentheses represent the rate of BPT metabolites formed (pmole/min/mg of microsomal protein).
Figure 6. Pathways for the formation of the major metabolites of BPT by rodent liver microsomes. Heavy arrows represent the pathways by which BPT is activated to mutagenic metabolites.
crosomes is shown in Table 2. Among the identifiable metabolites, BPT sulfoxide and BPT 3,4-diol were formed by all types of microsomal preparations except those from control mice. In addition, metabolites having retention times similar to that of 2-hydroxy- and 3-hydroxyBPT (summed up as phenolic metabolites) and polar metabolites having retention time lower than that of BPT sulfoxide were also produced in significant amounts in most of the cases. As compared to BPT sulfoxide, insignificant formation of BPT sulfone was noted in all cases. The formation of some of the polar metabolites was significantly inhibited by TCPO, suggesting the involvement of epoxide hydrolase in their formation (data not shown). Among the BPT metabolites produced by liver microsomes from DBA-, 3-MC-, or Aroclor 1254-treated mice, the most prominent metabolite was BPT 3,4-diol (precursor to the bay-region diol epoxide of BPT), constituting 15-27% of the total ethyl acetate extractable metabolites. In all of these cases, BPT sulfoxide accounted for only 1.6-4.3% of the total metabolites. A significant formation of phenolic metabolites (25-27% of the total metabolites) and unknown metabolites (44-56% of the total metabolites) was also observed. In contrast,
among the ethyl acetate extractable metabolites of BPT generated by liver microsomes from PB-treated mice, BPT sulfoxide accounted for a major portion of the metabolites (∼26% of the total metabolites) whereas BPT 3,4-diol accounted for only 7.6% of the total metabolites. While phenolic metabolites accounted for 57% of the total metabolites, polar metabolites accounted for a minor fraction (5.6% of the total metabolites) of the metabolites formed by liver microsomes from PB-treated mice. The profile of BPT metabolites generated by liver microsomes from DBA- or 3-MC-induced rats was significantly different from the profile produced by liver microsomes from DBA- or 3-MC-induced mice. In both cases, rat liver microsomes produced a higher proportion of BPT sulfoxide as compared to BPT 3,4-diol. However, the liver microsomes from 3-MC-treated rats were more efficient than those from DBA-treated rats in metabolizing BPT to its sulfoxide (41 vs 12% of total metabolites) but less efficient in metabolizing BPT to BPT 3,4-diol (3.5 vs 15.2% of the total metabolites). No significant difference was noted in the relative formation of phenolic and polar metabolites by liver microsomes from rats and mice treated with DBA. However, this was not true in the case of liver microsomes from rats and mice treated with 3-MC. In general, the proportion of phenolic and polar metabolites produced by the liver microsomes from 3-MCtreated rats was nearly half of that produced by the liver microsomes from DBA-treated mice (Table 1).
Discussion A substantial body of evidence has suggested that bayregion diol epoxides are the ultimate carcinogenic metabolites of PAHs (7-10). Because of structural similarities between PAHs and thia-PAHs, we postulate that bay-region diol epoxides are also involved in the metabolic activation of thia-PAHs. In addition to bay-region diol epoxides, the formation of metabolites produced via sulfoxidation of thia-PAHs has been suggested in the metabolic activation of these chemicals (3, 16). It has been reported that BPT induced more tumors than its carbon analogue DBA in XVII nc/Z strain mice that received subcutaneous injection of 0.6 mg of BPT at 4 week intervals (4). This finding is consistent with the bayregion theory (7), which predicts that the bay-region diol epoxides of BPT should be chemically more reactive than
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those of DBA and would, presumably, have higher biological activity. This prediction is based on the reasoning that the thiophene moiety is more effective than the benzene ring at stabilizing the carbocation generated from the diol epoxide (31). The purpose of the present investigation is to investigate the pathway(s) by which BPT may be bioactivated by examining its metabolism. As observed with a number of PAHs, the rate of metabolism of BPT by liver microsomes from mice pretreated with either a PAH or Aroclor 1254 was 2570-fold higher than the rate of BPT metabolism by liver microsomes from untreated mice. However, this rate was only 13-26- fold higher for liver microsomes from DBAor 3-MC-treated rats. These observations suggest that P450 enzymes that are involved in the metabolism of PAHs are also capable of metabolizing BPT, but these enzymes are induced to a greater extent in mouse liver than in rat liver. A considerable enhancement (5-fold) in the rate of metabolism by PB treatment shows that the P450 enzymes induced by PB exhibit a considerable specificity for BPT. Generally, the hepatic microsomes of PB-treated animals show similar or less activity toward the metabolism of PAHs as compared to liver microsomes of untreated animals (32). These data indicate that in addition to PAH inducible P450 enzymes (e.g., P450 1A1 and P450 1B1), P450 enzymes induced by PB (e.g., P450 2B1, 3A1, and/or 3A2) are involved in the metabolism of sulfur heterocyclic PAHs. PAHs, on the other hand, are metabolized primarily by P450 1A1 and P450 1B1 (33). Liver microsomes from control mice or rats metabolized BPT predominantly to BPT sulfoxide. However, a significant shift in the metabolism from sulfur to angular benzo-ring was observed when BPT was incubated with liver microsomes from mice treated with P450 inducers, especially DBA, 3-MC, and Aroclor 1254. The BPT metabolites formed by liver microsomes of mice treated with different inducers included BPT 3,4-diol (8-27%), BPT sulfoxide (2.8-26%), BPT phenols (25-56%), and several unidentifiable polar metabolites (6-56%) (Table 1). No significant amount of BPT sulfone or BPT 5,6-diol (a K-region metabolite) was detected. BPT 3,4-diol was a major BPT metabolite formed by liver microsomes from Aroclor 1254- or 3-MC-treated mice, accounting for nearly 27% of the total metabolites. Only a minor amount of BPT sulfoxide (2.8-4.3% of total metabolites) was formed by these microsomes. The metabolism of BPT is similar to that of the weakly carcinogenic benzo[b]naphtho[2,1d]thiophene (BNT), the only other thia-PAH studied for characterization of its metabolites (14, 15). Both of these hydrocarbons are converted to a considerable extent to putative procarcinogenic dihydrodiol with a bay-region double bond, accounting for as much as 27% of the total metabolites in the case of BPT. It is interesting to note that a significant proportion of the corresponding procarcinogenic dihydrodiol was also produced from BPT isosters, such as DBA (32) and dibenz[a,h]acridine (34). A similarity in the regioselective metabolism of isosteric hydrocarbons to their corresponding dihydrodiols has also been noted for dibenz[a,j]anthracene and its azasters dibenz[a,j]acridine and dibenz[c,h]acridine, which are metabolized predominantly to their K-region diols (3537). Thus, the growing evidence that the metabolism of a PAH and its isosters to the corresponding dihydrodiols occurs with a similar regioselectivity suggests that the same cytochrome P450 enzymes are involved in the
Yuan et al.
metabolism of isosteric hydrocarbons to their corresponding dihydrodiols. BPT sulfoxide was the major BPT metabolite formed by liver microsomes from PB-treated mice, accounting for nearly 26% of the ethyl acetate extractable metabolites. In contrast, BPT sulfoxide was formed only as a minor metabolite (2.8-4.3%) by liver microsomes from mice treated with DBA, 3-MC, or Aroclor 1254. These data suggest that PB inducible P450 (such as P450 2B1, 3A1, and/or 3A2) is predominantly responsible for metabolizing BPT to BPT sulfoxide in mice. Consequently, liver microsomes from mice treated with DBA or 3-MC (P450 1A1 inducers) produce BPT sulfoxide only in minor amounts. On the other hand, the low metabolism of BPT to BPT sulfoxide by liver microsomes from mice treated with Aroclor 1254, which induces both PB and 3-MC inducible P450 enzymes, may be due to the possibility that PB inducible P450 enzymes induced by Aroclor 1254, because of its lower degree of substrate specificity for BPT as compared to that of 3-MC inducible P450 enzymes, is not able to compete for BPT in the presence of 3-MC inducible P450 enzymes. An extensive metabolism of BPT to its phenolic metabolites by liver microsomes from PBtreated mice (Table 2) indicates that PB inducible P450 enzymes are also efficient in catalyzing ring epoxidation of BPT to produce BPT epoxides that rearrange to form the corresponding phenols via NIH shift, presumably due to low epoxide hydrolase activity for such BPT epoxides. In contrast to liver microsomes from DBA- or 3-MCtreated mice, liver microsomes from DBA- or 3-MCtreated rats metabolize BPT to BPT sulfoxide to a greater extent. Sulfoxidation of thia-PAHs by liver microsomes from rats treated with a P450 1A1 inducer (5,6-benzoflavone) has been previously reported (15), but no such data are reported for mice. In addition to a significant formation of BPT sulfoxide, the proportion of BPT sulfoxide produced by liver microsomes from 3-MC-treated rats was 3.4-fold higher than that produced by liver microsomes from DBA-treated rats. These results suggest that the degree of biotransformation of BPT to its sulfoxide varies with the species as well as with the P450 inducers of a similar type and may be related to the differential induction of sulfoxidation specific P450 enzymes by different PAHs in these species (38). However, a greater formation of BPT 3,4-diol by liver microsomes from 3-MC-induced rats as compared to DBA-induced rats appears to be due to a differential induction of epoxide hydrolase activity in rats by chemicals of the same class of compounds (PAHs). Thus, DBA, unlike 3-MC, may not be a good inducer of epoxide hydrolase in rat liver, which catalyzes the conversion of BPT 3,4-oxide to BPT 3,4-diol, thereby producing a higher proportion of phenols (Table 2). Recent studies of Kumar et al. (20) have shown that among the various potential metabolites of BPT tested for mutagenicity in Salmonella typhimurium TA100 in the presence of hepatic S9 fraction from the rats treated with a mixture of PB and β-naphthoflavone (Aroclor 1254 type inducer), BPT 3,4-diol and BPT sulfoxide are the most active mutagenic metabolites. In fact, BPT sulfoxide is not only inherently mutagenic, its bioactivated product(s) is up to 3.3-fold more mutagenic than the bioactivated product(s) of BPT 3,4-diol. Therefore, the metabolism studies in conjunction with our mutagenicity data have provided evidence for the first time that BPT and possibly other thia-PAHs are metabolically activated via the
Metabolism of Benzo[b]phenanthro[2,3-d]thiophene
formation of both the dihydrodiol (subsequently to diol epoxide) and the sulfoxide. In conclusion, these studies demonstrate that P450 enzymes induced by PAHs (e.g., P450 1A1 and P450 1B1) and by PB (e.g., P450 2B1, 3A1, and/or 3A2) are involved in the metabolism of BPT to mutagenic products BPT 3,4-diol and BPT sulfoxide. However, the relative contribution of these pathways in inducing mutagenicity/ carcinogenicity may depend not only upon the species but also upon the exposure of a species to different P450 inducers. Further studies are planned to assess the relative contribution of individual recombinant human P450s in the formation of these mutagenic metabolites.
Acknowledgment. This work was supported by the U.S. Environmental Protection Agency Grant No. R826192-01 (S.K.).
References (1) IARC (1973) IARC Monographs on the Evaluation of Carcinogenic Risk of the Chemicals to Man. Pt. 1. Certain Polycyclic Aromatic Hydrocarbons and Heterocyclic Compounds, Vol. 3, pp 1-271, IARC, Lyon, France. (2) IARC (1983) IARC Monographs on the Evaluation of Carcinogenic Risk of the Chemicals to the Humans, Polycyclic Aromatic Compounds, Pt. 1, Vol. 32, p 477; (1984) IARC Monographs on the Evaluation of Carcinogenic Risk of the Chemicals to the Humans, Polycyclic Aromatic Compounds, Pt. 2, Vol. 33, p 245; (1984) IARC Monographs on the Evaluation of Carcinogenic Risk of the Chemicals to the Humans, Polycyclic Aromatic Compounds, Pt. 3, Vol. 34, p 219; (1984) IARC Monographs on the Evaluation of Carcinogenic Risk of the Chemicals to the Humans, Polycyclic Aromatic Compounds, Pt. 4, Vol. 35, p 219, IARC, Lyon, France. (3) Jacob, J. (1990) Sulfur Analogues of Polycyclic Aromatic Hydrocarbons (Thia-Arenes), pp 1-281, Cambridge University Press, Cambridge. (4) Croisy, A., Mispelter, J., Lhoste, J. M., Zajdela, F., and Jacqignon, P. (1984) Thiophene analogues of carcinogenic polycyclic hydrocarbons. Elbs pyrolysis of various aroylmethyl-benzo[b]thiophenes. J. Heterocycl. Chem. 21, 353-359. (5) Grimmer, G., Jacob, J., and Naujack, K.-W. (1983) Profile of the polycyclic aromatic compounds from crude oils. Part 3. Inventory by GCGC/MSsPAH in environmental materials. Fresenius Z. Anal. Chem. 314, 29-36. (6) Grimmer, G., Jacob, J., Dettbarn, G., and Naujack, K.-W. (1985) Determination of polycyclic aromatic hydrocarbons, azaarenes, and thiaarenes emitted from coal-fired residential furnaces by gas chromatography/mass spectrometry. Fresenius Z. Anal. Chem. 322, 596-602. (7) Lehr, R. E., Kumar, S., Levin, W., Wood, A. W., Chang, R. L., Conney, A. H., Yagi, H., Sayer, J. M., and Jerina, D. M. (1985) The bay-region theory of polycyclic aromatic hydrocarbon carcinogenesis. In Polycyclic Aromatic Hydrocarbons and Carcinogenesis (Harvey, R. G., Ed.) ACS Symposium Series 283, pp 6384, American Chemical Society, Washington, DC. (8) Wislocki, P. G., and Lu, A. Y. H. (1988) Carcinogenicity and mutagenicity of proximate and ultimate carcinogens of polynuclear aromatic hydrocarbons. In Polycyclic Aromatic Hydrocarbons: Structure-Activity Relationships (Yang, S. K., and Silverman, B. D., Eds.) Vol. 1, pp 1-30, CRC Press, Boca Raton, FL. (9) Harvey, R. G. (1991) Polycyclic Aromatic Hydrocarbons: Chemistry and Carcinogenicity, pp 1-359, Cambridge University Press, Cambridge, U.K. (10) Jerina, D. M., Yagi, H., Thakker, D. R., Sayer, J. M., van Bladeren, P. J., Lehr, R. E., Whalen, D. L., Levin, W., Chang, R. L., Wood, A. W., and Conney, A. H. (1984) Identification of the ultimate carcinogenic metabolites of polycyclic aromatic hydrocarbons: Bay-region (R,S)-diol-(S,R)-epoxides. In Foreign Compound Metabolism (Caldwell, J., and Paulson, G. D., Eds.) pp 257-266, Taylor and Francis Ltd., London. (11) Karcher, W., Nalen, A., Depaus, R., Van Eijk, J., Glaude, P., and Jacob, J. (1981) New Results in the detection, identification and mutagenic testing of heterocyclic polycyclic aromatic hydrocarbons. In Polynuclear Aromatic Hydrocarbons: Chemical Analysis and Biological Fate (Cooke, M., and Dennis, A. J., Eds.) pp 317321, Battelle Press, Columbus, Ohio.
Chem. Res. Toxicol., Vol. 16, No. 12, 2003 1587 (12) Pelroy, R. A., Steward, D. L., Tominaga, M., Iwao, M., Castle, R. N., and Lee, M. L. (1983) Microbial mutagenicity of 3- and 4-ring polycyclic aromatic sulfur heterocycles. Mutat. Res. 117, 31-40. (13) McFall, T., Booth, G. M., and Lee, M. L. (1984) Mutagenic activity of methyl substituted tri and tetracyclic aromatic sulfur heterocycles. Mutat. Res. 135, 97-103. (14) Murphy, S. E., Amin, S., Coletta, K., and Hoffman, D. (1992) Rat liver metabolism of benzo[b]naphtha[2,1-d]thiophene. Chem. Res. Toxicol. 5, 491-495. (15) Jacob, J., Schmoldt, A., Augustin, C., Raab, G., and Grimmer, G. (1991) Rat liver microsomal ring- and S-oxidation of thiaarenes with central and peripheral thiophene rings. Toxicology 67, 181194. (16) Jacob, J., Schmoldt, A., and Grimmer, G. (1986) The predominant role of S-oxidation in rat liver metabolism of thiaarenes. Cancer Lett. 32, 107-116. (17) Thakker, D. R., Levin, W., Yagi, H., Wood, A. W., Conney, A. H., and Jerina, D. M. (1988) Stereoselective biotransformation of polycyclic aromatic hydrocarbons to ultimate carcinogens. In Sterochemical Aspects of Pharmacological Active Compounds (Wainer, I. W., and Drayer, D., Eds.) pp 271-296, Marcel Dekker, NY. (18) Lau, P. P., Pickett, C. B., Lu, A. Y. H., and Strobel, H. W. (1982) Comparison of cytochrome P-450 with high activity toward benzo[a]pyrene purified from liver microsomes of β-naphthoflavone and 3-methylcholanthrene-pretreated rats. Arch. Biochem. Biophy. 218, 472-477. (19) Nakamura, A., Koshu, O.-f., and Hokoku, E.-K. K. (1983) Studies on technology for environmental assessment.2. Analysis and indexing on capillary columns. Shokuhin Eisei Hew 14, 69-73. (20) Kumar, S., Lin J.-M., Whysner, J., Sikka, H. C., and Amin, S. (2003) Mutagenicity of benzo[b]phenanthro(2,3-d)thiophene (BPT) and its metabolites in TA100 and base-specific tester strains (TA7001-7006) of Salmonella typhimurium: evidence of multiple pathways for the bioactivation of BPT. Mutat. Res. In press. (21) Tedjamulia, M. L., Tominaga, Y. R., Castle, N., and Lee, M. L. (1983) The synthesis of benzo[b]phenanthro[d]thiophenes and anthra[b]benzo[d]thiophenes. J. Heterocycl. Chem. 20, 861-866. (22) Blum, J., Shalom, Y., and Harvey, R. G. (1993) K-region oxides and imines of benzo[b]phenanthro[2,3-d]thiophene and benzo[b]phenanthro[3,2-d]thiophene. J. Heterocycl. Chem. 30, 1521-1523. (23) Platt, K., and Oesch, F. (1982) K-region trans-dihydrodiols of polycyclic arenes; and efficient and convenient preparation from o-quinones or o-diphenols by reduction with sodium borohydride in the presence of oxygen. Synthesis, 459-461. (24) Kumar, S. (2001) Synthesis of trans-3,4-dihydroxy-3,4-dihydro phenanthro[3,2-b][1]benzothiophene, a potentially carcinogenic metabolite of sulfur heterocycle phenanthro[3,2-b][1]benzothiophene. J. Chem. Soc., Perkin Trans. 1, 1018-1023. (25) Lythgoe, B., Trippett, S., and Watkins, J. C. (1956) Califerol and its relatives. Part II. An alternative synthesis of trans-1,2′cyclohexyliene-ethylidene-2-methylenecyclohexane. J. Chem. Soc. 4060-4065. (26) Ho, T.-L., and Wong, C. M. (1972) Ceric ammonium nitrate oxidation via diaryl sulfides. Synthesis, 561-562. (27) Lucas, H. J., and Kennedy, E. R. (1955) Iodobenzene dichloride. In Organic Syntheses Collection Volume III (Horning, E. C., Ed.) pp 482-484, John Wiley and Sons, New York. (28) Nordqvist, M., Thakker, D. R., Vyas, K. P., Yagi, H., Levin, W., Ryan, D. E., Thomas, P. E., Conney, A. H., and Jerina, D. M. (1981) Metabolism of chrysene and phenanthrene to bay-region diol epoxides by rat liver enzymes. Mol. Pharmacol. 19, 168-178. (29) Lowry, O. H., Rossebrough, Farr, A. L., and Randall, A. J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275. (30) Van Cantfort, J., DeGraeve, and Gielen, J. (1992) Radioactive assay for aryl hydrocarbon hydroxylase. Improved method and biological importance. Biochem. Biophys. Res. 79, 505-512. (31) Sinsheimer, J. E., Hooberman, B. H., Das, S. K., Savla, P. M., and Ashe, A. J., III (1992) Genotoxicity of chryseno[4,5-bcd]thiophene and its sulfone derivative. Environ. Mol. Mutat. 19, 259-264. (32) Thakker, D. R., Yagi, H., Levin, W., Wood, A. W., Conney, A. H., and Jerina, D. M. (1985) Polycyclic aromatic hydrocarbons: metabolic activation to ultimate carcinogens. In Bioactivation of Foreign Compounds (Anders, A. W., Ed.) pp 177-241, Academic Press, NY. (33) Shimada, A., Hays, C. L., Yamazaki, H., Amin, S., Hecht, S. S., Guengerich, F. P., and Sutter, T. (1996) Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1. Cancer Res. 56, 2979-2984.
1588 Chem. Res. Toxicol., Vol. 16, No. 12, 2003 (34) Steward, A. R., Kumar, S., and Sikka, H. C. (1987) Metabolism of dibenz[a,h]acridine by rat liver microsomes. Carcinogenesis 8, 1043-1050. (35) Nair, R. V., Nettikumara, A. N., Cortez, C., Harvey, R. G., and DiGiovanni, J. (1992) Comparative metabolism of dibenz[a,j]anthracene and 7-methyldibenz[a,j]anthracene in primary cultures mouse kiratinocytes. Chem. Res. Toxicol. 5, 532-540. (36) Gill, J. H., Duke, C. C., Rosario, C. A., Ryan, A. J., and Holder, G. M. (1986) Dibenz[a,j]acridine metabolism: identification of in vitro products formed by liver microsomes from 3-methylcholanthrene-treated rats. Carcinogenesis 7, 1371-1378.
Yuan et al. (37) Lehr, R. E., Wood, A. W., Levin, W., Conney, A. H., and Jerina, D. M. (1988) Benzacridines and dibenzacridines: metabolism, mutagenicity and carcinogenicity. In Polycyclic Aromatic Hydrocarbon Carcinogenesis: Structure-Activity Relationships (Yang, S. K., and Silverman, B. D., Eds.) Vol. 1, pp 31-58, CRC Press, Boca Raton, FL. (38) Guengerich, F. P. (1997) Comparison of catalytic selectivity of cytochrome P450 subfamily enzymes from different species. Chem.-Biol. Interact. 106, 161-182.
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