1508
Chem. Res. Toxicol. 1998, 11, 1508-1515
Hydroxylation and Methylthiolation of Mono-Ortho-Substituted Polychlorinated Biphenyls in Rats: Identification of Metabolites with Tissue Affinity Koichi Haraguchi,*,† Yoshihisa Kato,‡ Ryohei Kimura,‡ and Yoshito Masuda† Daiichi College of Pharmaceutical Sciences, 22-1, Tamagawa-Cho, Minami-Ku, Fukuoka 815-8511, Japan, and School of Pharmaceutical Sciences, University of Shizuoka, 52-1, Yada, Shizuoka 422-8526, Japan Received August 5, 1998
The metabolism of three mono-ortho-substituted congeners, 2,3,3′,4,4′-pentachlorobiphenyl (CB105), 2,3′,4,4′,5-pentachlorobiphenyl (CB118), and 2,3,3′,4,4′,5-hexachlorobiphenyl (CB156), was investigated with regard to the identification of hydroxy- and sulfur-containing metabolites and their tissue retention in rats. Hydroxylation proceeded primarily at the meta or para position either via an arene oxide, involving NIH shift and dechlorination, or by direct insertion of a hydroxyl group. CB105 was hydroxylated preferably in the 2,3,4-trichlorinated ring to yield 4-OH-2,3,3′,4′,5-pentaCB, whereas CB118 was hydroxylated in the 2,4,5-trichlorinated ring to yield the same hydroxy metabolite to a similar extent. The concentration of 4-OH2,3,3′,4′,5-pentaCB in blood was >3 times higher than that in liver, lung, or kidney. The ratios of 4-OH-2,3,3′,4′,5-pentaCB to unchanged CB in blood were 11:1 for CB105 and 7:1 for CB118. The other two metabolites, 4′-OH-2,3′,4,5,5′-pentaCB from CB118 and 4′-OH-2,3,3′,4,5,5′hexaCB from CB156, also exhibited a high blood affinity. Another metabolism of mono-orthoPCBs PCBs involved methylthiolation in the vicinal ortho and meta unsubstituted positions to give methylthio metabolites, which were detected as methylsulfonyl metabolites in liver and adipose tissue. The tissue retention of these metabolites might contribute to the toxic and biologic effects of mono-ortho-substituted PCBs.
Introduction Polychlorinated biphenyls (PCBs)1 are environmental pollutants that accumulate in the food chain due to their high lipophilicity and low biotransformation rate (1, 2). The toxic responses as well as the potency of a PCB congener are highly dependent on its planarity and chlorine substitution. The non-ortho- and mono-orthosubstituted PCBs bind to the Ah receptor and exhibit a broad range of biochemical and toxic responses (3). Three mono-ortho-PCB congeners, 2,3,3′,4,4′-pentachlorobiphenyl (CB105),2 2,3′,4,4′,5-pentachlorobiphenyl (CB118), and 2,3,3′,4,4′,5-hexachlorobiphenyl (CB156), may contribute significantly to the 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD)-like activity of PCB mixtures because they are present in high concentrations in commercial mixtures (5) and environmental extracts (6). PCBs may be metabolically activated to arene oxide intermediates which are subsequently transformed to hydroxylated products, as well as sulfur-containing metabolites such as methylsulfonyl (MeSO2) PCBs, via the mercapturic acid pathway (7, 8). Some of hydroxylated * To whom correspondence should be addressed. Fax: (81) 92 553 5698. E-mail:
[email protected]. † Daiichi College of Pharmaceutical Sciences. ‡ University of Shizuoka. 1 Abbreviations: PCB, polychlorinated biphenyl; CB, chlorobiphenyl; MeSO2, methylsulfonyl; MeS, methylthio; CB105, 2,3,3′,4,4′-pentachlorobiphenyl; CB118, 2,3′,4,4′,5-pentachlorobiphenyl; CB156, 2,3,3′,4,4′,5hexachlorobiphenyl; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; CB77, 3,3′,4,4′-tetrachlorobiphenyl; AHH, aryl hydrocarbon hydroxylase; MeO, methoxy; ECD, electron capture detector. 2 Numbering according to Ballschmiter (4).
PCBs have been shown to inhibit mitochondrial oxidative phosphorylation in mice (9), and to inhibit thyroid hormone sulfation in rats in vitro (10), and also to exhibit estrogenic or antiestrogenic activity (11-13). On the other hand, some MeSO2 metabolites are known to exhibit strong induction of several cytochrome P450 isoenzymes in rats (14, 15), may act as tumor promoters in rat liver (16), and also may reduce the thyroid hormone levels in minks (17) and rats (18). Previous studies have shown that 3,3′,4,4′-tetraCB (CB77) is relatively readily metabolized to 4-OH-3,3′,4′,5tetraCB in mice or rats (19, 20). The metabolite is known to be selectively retained in blood of wildlife due to their strong binding affinity for transthyretin, a plasma thyroxine-transporting protein, and to cause alteration in the thyroid hormone metabolism (21-23). In addition, we found that CB77 was biotransformed in rats to methylthio (MeS) and MeSO2 metabolites, of which 5-MeSO2-CB77 was localized in the liver (24). This type of MeSO2 metabolite has been shown to effectively inhibit aryl hydrocarbon hydroxylase (AHH) activity induced by TCDD in cultured human lymphoblastoid cells (25, 26) and to affect AHH activity in the liver microsomes taken from mice with different levels of genetic responsiveness (27, 28). Similar metabolic features and activities may be, therefore, expected from the other PCB congeners with 3,4-chlorine substitution. In this study, we chose to investigate the metabolism of CB105, CB118, and CB156, since they have accumulated to a significant extent in human milk (29) and adipose tissue (30). This paper describes the structural
10.1021/tx980183r CCC: $15.00 © 1998 American Chemical Society Published on Web 11/14/1998
Metabolism of Mono-Ortho Chlorinated Biphenyl
identification of oxygen- and sulfur-containing metabolites, their fecal excretion, and tissue distribution.
Materials and Methods Chemicals. CB105, CB118, and CB156 were prepared by the method of Cadogan (31). The chemical purities of the compounds were >99% as determined by GC. 4′-Methyl-3′MeSO2-2,3,4,5,5′-pentaCB and 2,3,3′,4,4′,5,5′-heptaCB were used as internal standards. Four reference compounds, 5-MeSO22,3,3′,4,4′-pentaCB, 5′-MeSO2-2,3,3′,4,4′-pentaCB, 5′-MeSO22,3′,4,4′,5-pentaCB, and 5′-MeSO2-2,3,3′,4,4′,5-hexaCB, were synthesized as described previously (32). Methoxy derivatives used for identification were synthesized by the method of Bergman et al. (33). Other chemicals and solvents used in the extraction procedure were analytical grade. Caution: Synthetic PCBs and their metabolites should be considered potentially toxic and hazardous and therefore should be handled in an appropriate manner. 5-MeS-2,3,3′,4,4′-pentaCB: 1H NMR (500 MHz, chloroformd) δ 2.48 (s, 3H, SCH3), 6.92 (s, 1H, 6-H), 7.24 (dd, J ) 2.2, 8.5 Hz, 1H, 6′-H), 7.48 (d, J ) 2.2 Hz, 1H, 2′-H), 7.53 (d, J ) 8.5 Hz, 1H, 5′-H); MS (EI) m/z (relative intensity) 370 (65) [M+], 372 (100) [M+ + 2], 374 (70) [M+ + 4], 337 (8), 320 (20) [M+ CH3Cl]. 6-MeS-2,3,3′,4,4′-pentaCB: 1H NMR (500 MHz, chloroformd) δ 2.38 (s, 3H, SCH3), 7.05 (dd, J ) 2.2, 8.5 Hz, 1H, 6′-H), 7.18 (s, 1H, 5-H), 7.31 (d, J ) 2.2 Hz, 1H, 2′-H), 7.56 (d, J ) 8.5 Hz, 1H, 5′-H); MS (EI) m/z (relative intensity) 370 (52) [M+], 372 (100) [M+ + 2], 374 (60) [M+ + 4], 320 (58) [M+ - CH3Cl]. 5′-MeS-2,3,3′,4,4′-pentaCB: 1H NMR (500 MHz, chloroformd) δ 2.49 (s, 3H, SCH3), 7.01 (d, J ) 2.2 Hz, 1H, 6′-H), 7.17 (d, J ) 8.5 Hz, 1H, 6-H), 7.24 (d, J ) 2.2 Hz, 1H, 2′-H), 7.46 (d, J ) 8.5 Hz, 1H, 5-H); MS (EI) m/z (relative intensity) 370 (60) [M+], 372 (100) [M+ + 2], 374 (72) [M+ + 4], 337 (12), 320 (15), 285 (11), 267 (15), 250 (17). 6′-MeS-2,3,3′,4,4′-pentaCB: 1H NMR (500 MHz, chloroformd) δ 2.40 (s, 3H, SCH3), 7.10 (d, J ) 8.5 Hz, 1H, 6-H), 7.20 (s, 1H, 5′-H), 7.31 (s, 1H, 2′-H), 7.45 (d, J ) 8.5 Hz, 1H, 5-H); MS (EI) m/z (relative intensity) 370 (0.8) [M+], 372 (1.2) [M+ + 2], 374 (0.7) [M+ + 4], 335 (80) [M+ - Cl], 337 (100) [M+ - Cl + 2], 320 (74) [M+ - CH3Cl], 300 (27), 250 (28). 5′-MeS-2,3′,4,4′,5-pentaCB: 1H NMR (500 MHz, chloroformd) δ 2.50 (s, 3H, SCH3), 7.03 (d, J ) 2.2 Hz, 1H, 6′-H), 7.27 (d, J ) 2.2 Hz, 1H, 2′-H), 7.43 (s, 1H, 6-H), 7.61 (s, 1H, 3-H); MS (EI) m/z (relative intensity) 370 (60) [M+], 372 (100) [M+ + 2], 374 (68) [M+ + 4], 355 (4), 337 (8), 320 (15), 303 (12), 285 (10). 6′-MeS-2,3′,4,4′,5-pentaCB: 1H NMR (500 MHz, chloroformd) δ 2.41 (s, 3H, SCH3), 7.20 (s, 1H, 5′-H), 7.31 (s, 1H, 2′-H), 7.35 (s, 1H, 6-H), 7.60 (s, 1H, 3-H); MS (EI) m/z (relative intensity) 370 (2) [M+], 372 (4) [M+ + 2], 374 (3) [M+ + 4], 335 (74) [M+ - Cl], 320 (80) [M+ - CH3Cl], 300 (20), 250 (25). 5′-MeS-2,3,3′,4,4′,5-hexaCB: 1H NMR (500 MHz, chloroformd) δ 2.49 (s, 3H, SCH3), 7.00 (d, J ) 2.2 Hz, 1H, 6′-H), 7.24 (d, J ) 2.2 Hz, 1H, 2′-H), 7.38 (s, 1H, 6-H); MS (EI) m/z (relative intensity) 404 (50) [M+], 406 (100) [M+ + 2], 408 (82) [M+ + 4], 371 (7), 354 (4), 301 (7), 284 (7). 6′-MeS-2,3,3′,4,4′,5-hexaCB: 1H NMR (500 MHz, chloroformd) δ 2.42 (s, 3H, SCH3), 7.19 (s, 1H, 5′-H), 7.31 (s, 1H, 2′-H), 7.32 (s, 1H, 6-H); MS (EI) m/z (relative intensity) 404 (2) [M+], 406 (4) [M+ + 2], 408 (2) [M+ + 4], 369 (52) [M+ - Cl], 354 (55) [M+ - CH3Cl], 356 (100) [M+ - CH3Cl + 2], 334 (8), 284 (20). 6-MeSO2-2,3,3′,4,4′-pentaCB: 1H NMR (500 MHz, chloroformd) δ 2.77 (s, 3H, SO2CH3), 7.16 (d, J ) 2.2, 8.5 Hz, 1H, 6′-H), 7.39 (d, J ) 8.5 Hz, 1H, 5′-H), 7.60 (d, J ) 2.2 Hz, 1H, 2′-H), 8.30 (s, 1H, 5-H); MS (EI) m/z (relative intensity) 402 (56) [M+], 404 (100) [M+ + 2], 406 (62) [M+ + 4], 338 (8), 304 (20) [M+ SOCH3Cl], 288 (32) [M+ - SO2CH3Cl]. 6′-MeSO2-2,3,3′,4,4′-pentaCB: 1H NMR (500 MHz, chloroformd) δ 2.87 (s, 3H, SO2CH3), 7.25 (d, J ) 8.5 Hz, 1H, 6-H), 7.42 (s, 1H, 2′-H), 7.49 (d, J ) 8.5 Hz, 5-H), 8.30 (s, 1H, 5′-H); MS (EI) m/z (relative intensity) 402 (5) [M+], 404 (8) [M+ + 2], 406 (7)
Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1509 [M+ + 4], 367 (77) [M+ - Cl], 369 (100) [M+ - Cl + 2], 304 (45) [M+ - SOCH3Cl], 288 (16) [M+ - SO2CH3Cl]. 6′-MeSO2-2,3′,4,4′,5-pentaCB: 1H NMR (500 MHz, chloroformd) δ 2.89 (s, 3H, SO2CH3), 7.41 (s, 1H, 2′-H), 7.48 (s, 1H, 6-H), 7.63 (s, 1H, 3-H), 8.28 (s, 1H, 5′-H); MS (EI) m/z (relative intensity) 402 (5) [M+], 404 (8) [M+ + 2], 406 (7) [M+ + 4], 367 (78) [M+ - Cl], 369 (100) [M+ - Cl + 2], 304 (48) [M+ - SOCH3Cl], 288 (15) [M+ - SO2CH3Cl]. 6′-MeSO2-2,3,3′,4,4′,5-hexaCB: 1H NMR (500 MHz, chloroform-d) δ 2.92 (s, 3H, SO2CH3), 7.41 (s, 1H, 2′-H), 7.43 (s, 1H, 6-H), 8.28 (s, 1H, 5′-H); MS (EI) m/z (relative intensity) 436 (2) [M+], 438 (3.8) [M+ + 2], 440 (3.5) [M+ + 4], 401 (64) [M+ - Cl], 403 (100) [M+ - Cl + 2], 338 (45) [M+ - SOCH3Cl], 324 (18) [M+ - SO2CH3Cl]. Animal Treatments. Male Wistar rats (body weight of 200 g) were housed in an air-conditioned room with free access to a commercial chow and tap water. Rats were given ip each of CB105, CB118, and CB156 (80 µmol/kg) dissolved in Panacete 810, a mixture of glycerides of medium chain fatty acids (Nippon Oils and Fats Co., Tokyo, Japan). Feces were collected daily for 4 days. Rats were killed 96 h after administration, and blood, lung, liver, kidney, and adipose tissue were removed and analyzed for metabolites. Isolation of Metabolites. Sample cleanup and quantification were carried out according to our previous methods (24). Briefly, dry powdered feces were extracted with acetone/nhexane (2:1, v/v) in a Soxhlet apparatus for 24 h. Two internal standards were added to each extract and the mixtures subjected to a gel permeation column packed with Bio-Beads S-X3 (50 g, Bio-Rad Laboratories, Hercules, CA). Dichloromethane/ n-hexane (1:1) was used as a mobile phase at a flow rate of 4 mL/min. The metabolite fraction (120-200 mL) was collected and partitioned between n-hexane and a 2 M KOH/ethanol (5: 2) solution. The aqueous solution was acidified with HCl and then extracted with n-hexane/tert-butyl methyl ether (9:1) for acidic metabolites. The acidic fraction was methylated by diazomethane, whereas the neutral fraction was analyzed without any further purification. Blood and tissue samples were homogenized with acetone/n-hexane (2:1, v/v) and purified in a same way as described above. Analytical Methods. 1H NMR was obtained on a JEOL GSX-500 spectrometer (500 MHz). The samples were dissolved in chloroform-d with TMS as an internal standard. GC/MS was carried out on a JMS-AX505W (JEOL) apparatus connected to a JMA-DA5000 data system in the EI mode. The GC instrument was fitted with a DB-5 fused silica capillary column (60 m × 0.25 mm i.d., J&W Scientific Inc., Folsom, CA) or an SP2330 column (30 m × 0.25 mm i.d., Supelco, Inc., Bellefonte, PA) with helium as a carrier gas. Injection was carried out in the splitless mode. The oven temperature was programmed from 70 (2 min) to 220 °C at a rate of 20 °C/min and then to 290 °C at a rate of 4 °C/min. The GC for quantification was performed on a GC-14A (Shimadzu Co., Kyoto, Japan) instrument equipped with a 63Ni electron-capture detector (ECD) with column conditions analogous to those described for GC/MS. The metabolites in all samples were identified on GC/MS by comparison with authentic synthesized metabolites. The hydroxy metabolites were assessed as their methoxy derivatives after methylation by diazomethane. Recoveries of 4′-hydroxy2,3,3′,4,5,5′-hexaCB added to blood and liver samples prior to extraction were 84-89%. The quantification of metabolites was performed on a GC/ECD apparatus using two internal standards, 2,3,3′,4,4′,5,5′-heptaCB and 4′-methyl-3′-MeSO2-2,3,4,5,5′pentaCB.
Results Identification of Fecal Metabolites. (1) CB105. GC/ECD profiles of fecal extracts from CB105-treated rats are shown in Figure 1. The acidic fraction contained five phenolic metabolites, and their methylated derivatives (M1, M3, and M4) were identified as 4-MeO-
1510 Chem. Res. Toxicol., Vol. 11, No. 12, 1998
Haraguchi et al.
Table 1. GC Retention Times and Mass Spectral Data of MeS Metabolites Isolated from Feces of Rats Treated with Three Mono-Ortho-PCBs mass spectral data relative abundance (%) PCB congener
GC peak
GC tRa
m/z (M+)
M+
M+ - 33
M+ - 35
M+ - 50
structure
CB105
S1 S2 S3a S3b S4 S5 S6 S7
0.6613 0.6813 0.7541 (0.7023)b 0.7541 (0.7094) 0.6338 0.7143 0.7341 0.8598
370 370 370 370 370 370 404 404
