2,2',4,5,5'-Pentachlorobiphenyl: Comparative Metabolism in Mink

1340

Chem. Res. Toxicol. 1996, 9, 1340-1349

2,2′,4,5,5′-Pentachlorobiphenyl: Comparative Metabolism in Mink (Mustela vison) and Mouse Eva Klasson Wehler,*,† Lotta Hovander,† and Bert-Ove Lund‡ Department of Environmental Chemistry, Wallenberg Laboratory, Stockholm University, S-106 91 Stockholm, Sweden, and Department of Pharmacology and Toxicology, Swedish University of Agricultural Sciences, S-751 23 Uppsala, Sweden Received July 2, 1996X

The metabolism of 14C-labeled 2,2′,4,5,5′-pentachlorobiphenyl was studied in mink (Mustela vison) and, for comparison, in the mouse (C57Bl). Both species were dosed orally and kept in metabolism cages for 5 days. Distribution in tissues and excretion rate were determined radiometrically, and metabolites were analyzed by GC/MS. Within 5 days, the mink excreted 17% of the dose, and the mouse excreted 74%, mainly via the feces. For both species, the excreted radioactivity consisted primarily of metabolites, a large proportion of which were covalently bound to macromolecules and to lipids. A smaller proportion consisted of watersoluble metabolites. Phenolic and, in trace amounts, methylsulfonyl metabolites were also excreted. In the mink, a salivary gland in the neck region demonstrated the highest concentration of radiolabeled material of all tissues examined (3 times higher than in adipose tissue and liver on a lipid weight basis). The radioactive material in the salivary gland consisted of the parent compound and phenolic and methylsulfonyl metabolites. In the mouse, the highest concentration of 14C was found in the lung and consisted of 4-methylsulfonyl-2,2′,4′,5,5′pentachlorobiphenyl. Hydroxylated and methylsulfonyl metabolites were retained to various degrees in tissues of both species. The mink, but not the mouse, also formed metabolites that were hydroxylated in the trichlorinated phenyl ring.

Introduction Polychlorinated biphenyls (PCBs)1 are widely distributed throughout the ecosystem. PCBs are both lipophilic and persistent and, therefore, accumulate readily in food chains. Approximately 130 of the 209 possible chlorobiphenyls (CBs) may be found in technical products (1), and over 50 of these have been identified in biological samples. A small group of 10 CBs constitutes more than 80% of the total PCB content in biota, e.g., blubber from common porpoise and in fish-eating birds (2, 3). The differences in the CB composition in the technical mixtures and biota are primarily due to different biotransformation rates. CBs with unsubstituted adjacent meta-/para-positions are readily metabolized whereas CBs lacking adjacent unsubstituted positions are more resistant to metabolism (4, 5). Thus, CBs such as 2,2′,4,4′,5,5′-hexaCB (CB153),2 2,2′,3,4,4′,5′-hexaCB (CB138), 2,2′,3,4,4′,5,5′-heptaCB (CB180), and 2,2′,3,4′,5,5′,6-heptaCB (CB187), all lacking adjacent unsubstituted meta-/para-positions, predominate in biological samples from the top of the food webs (2, 7-9). The more readily metabolized CBs with unsubstituted meta-/para-positions, such as 2,4′,5-triCB (CB31) and 2,2′,5,5′-tetraCB (CB52), are metabolized * Corresponding author. † Stockholm University. ‡ Swedish University of Agricultural Sciences. X Abstract published in Advance ACS Abstracts, November 1, 1996. 1 Abbreviations: PCBs ) polychlorinated biphenyls; OH-CB ) hydroxychlorobiphenyl; MSF-CB ) methylsulfonyl chlorobiphenyl; CB101 ) 2,2′,4,5,5′-pentachlorobiphenyl; CB105 ) 2,3,3′,4,4′-pentachlorobiphenyl; MeO ) methoxy; OH ) hydroxy; GPC ) gel permeation chromatography; GPC-MF ) metabolite fraction from GPC; GPC-IF ) intermediate fraction from GPC; GPC-LF ) lipid fraction from GPC; MTBE ) methyl tert-butyl ether; DDT ) di(chlorophenyl)trichloroethane; DDE ) di(chlorophenyl)dichloroethene; MAP ) mercapturic acid pathway. 2 Numbering according to Ballschmiter (6).

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both to hydroxylated products and products of the mercapturic acid pathway, such as methylsulfonyl metabolites (10-13). Generally, the metabolites are excreted, but they may occasionally be retained in tissues due to binding to proteins. For example, several hydroxylated CB metabolites (OH-CBs) are selectively retained in the blood plasma of both humans and wildlife due to their affinity for transthyretin, a thyroxine-transporting protein (14-16). These OH-CBs have a hydroxy group in a para- or metaposition and chlorine atoms on the adjacent carbon atoms, making them structurally similar to thyroxine, and they exhibit an affinity for transthyretin that is even higher than that of thyroxine itself (16). Another example are the PCB methylsulfones (MSF-CBs), some of which, depending on structure, bind to a uteroglobin-like protein in the lung (17, 18). This protein, called the PCBbinding protein, binds progesterone and calcium (19). Competition between PCB metabolites and endogenous compounds e.g., thyroxine and progesterone, for binding to proteins may cause endocrine disturbances. Furthermore, certain 3-MSF-CBs are potent inducers of cytochrome P450 isozymes at levels 100-fold lower than required for equivalent induction by the parent compounds (20, 21). Induction of enzymes by xenobiotics may influence the catabolism of endogenous compounds such as steroids (22). These toxicological and ecotoxicological aspects of PCB metabolism motivated further study of the metabolism of individual congeners in different species. Mink is a particularly interesting species, since it is a representative fish-eating marine mammal resembling otter and seal, which have both shown correlations between poor reproductive ability and high PCB content (23, 24). Several studies on the reproductive effects of PCB in © 1996 American Chemical Society

Metabolism of Pentachlorobiphenyl

Figure 1. Structures of CB101 and identified metabolites. The abbreviated designation is presented under each structure.

mink using both technical PCB (25-27) and PCBcontaminated fish (28) have revealed that PCB affects both the number of kits born and their survival rate. Individual PCB congeners have also been found to cause toxic effects in mink, including disrupted molting patterns, deformed nails, and decreased plasma levels of thyroid hormones (26, 29). Although many such toxicity studies have been performed, there is much less knowledge on the metabolism of CBs by the mink. We have previously studied the metabolism of a mono-ortho-substituted CB, 2,3,3′,4,4′pentaCB (CB105), in mink and mouse. This compound is slowly metabolized to hydroxylated metabolites as well as to metabolites that bind covalently bound to lipids and macromolecules (30). However, no MSF metabolites of CB105 were detected in that study. In the present investigation, we extend our comparisons of PCB metabolism in mink and a laboratory animal by studying the metabolism of a CB known to form MSF metabolites in rodents, i.e., 2,2′,4,5,5′-pentachlorobiphenyl (CB101), which is also a major congener present in technical PCB products (31, 1). Our goal has been to describe the distribution, metabolite formation, and excretion rates of CB101 in these two species, with special emphasis on the determination of possible selective tissue localization of CB101 and/or its metabolites.

Materials and Methods Chemicals. 14C-Labeled 2,2′,4,5,5′-pentachlorobiphenyl (CB101) was synthesized from 2,4,5-trichloro[14C]aniline (1 mCi, specific activity 24.5 Ci/mol, Sigma Chemicals) and 1,4-dichlorobenzene (Aldrich) by the Cadogan diaryl coupling reaction as described by Bergman et al. (32). Unlabeled CB101 was synthesized as described by Sundstro¨m (33) and used to dilute the specific activity. In order to aid the reader, the numbering of substituents on CB101 has been based on the numbering of CB101 rather than using correct IUPAC nomenclature. The demethylated structures of reference compounds used in the analysis are shown in Figure 1. The reference compounds 4′-MeO-2,2′,4,5,5′pentachlorobiphenyl (4′-MeO-101) and 3′-MeO-2,2′,4,5,5′-pentaCB (3′-MeO-101) were synthesized as described earlier (34). 3-Methoxy-2,2′,4,5,5′-pentaCB (3-MeO-101) and 4-methoxy2,2′,3,5,5′-pentaCB (4-MeO-pentaCB) were synthesized by cou-

Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1341 pling 2,5-dichloroanisole and 2,4,5-trichloroaniline according to Bergman et al. (35). The two isomers 3-MeO-101 and 4-MeOpentaCB could not be separated from each other and were therefore purified and used as a mixture. These two products were characterized by GC/MS as follows: 3-MeO-101: m/z (relative abundance %): 354 (48, (M)+), 356 (100, (M + 2)+), 358 (62, (M + 4)+); 339 (4, (M - 15)+), 341 (7.8), 343 (5.7); 311 (27, (M - COCH3)+), 313 (45), 315 (34). 4-MeO-pentaCB: 354 (49, (M)+), 356 (100, (M + 2)+), 358 (60, (M + 4)+); 339 (20, (M - 15)+), 341 (36), 343 (25); 311 (30, (M - COCH3)+), 313 (52), 315 (39). 3′- and 4′-Methylsulfonyl-2,2′,4,5,5′-pentaCB (3′- and 4′-MSF101) and 4′-methylthio-2,2′,4,5,5′-pentaCB (4-MeS-101) were synthesized as described previously (36). 2,3,3′,4,4′,5,5′-Heptachlorobiphenyl (CB189, synthesized according to Sundstro¨m (33)), 4-OH-2,3,3′,4′,5,5′,6-heptachlorobiphenyl, and 4-methyl3-methylsulfonyl-2′,3′,4′,5,5′-pentachlorobiphenyl were used as internal standards (35, 37). All solvents, i.e., acetone (Riedel de Haen), methyl tert-butyl ether (MTBE, from Rathburn), dimethyl sulfoxide, methanol, 2-propanol, and chloroform (Merck), used in the extraction and cleanup procedures were of p.a. quality. Dichloromethane and hexane were of pesticide grade and obtained from Riedel de Haen. Sulfuric acid (98%) was purchased from Merck, and potassium hydroxide was purchased from EKA (Bohus, Sweden). Diazomethane was synthesized as described by Fieser and Fieser (38). Caution: Diazomethane is a hazardous chemical that is carcinogenic to humans and should therefore be handled carefully in a well-ventilated fume hood. Instruments. Homogenization of tissues was performed using an Ultra-Turrax IKA homogenizer. Gel permeation chromatography (GPC) was performed as described elsewhere (39) with the column connected on line to a UV detector (Gilson) and a A500 Flow-one radiodetector (Packard). The radiodetector was equipped with a flow cell (0.5 mL) and a splitter, sampling 5 or 10% of the sample, depending on its total amount. Fractions were collected on the basis of the UV and radioactivity recordings: a lipid fraction (GPC-LF eluted at 50-110 mL), an intermediate fraction (GPC-IF, 110-125 mL), and a metabolite fraction (GPC-MF, 125-200 mL) containing unconjugated metabolites and the parent compound. For tissue extracts of less than 30 mg, a smaller GPC column, Pharmacia SR10 (10 mm i.d. × 400 mm, 10 g Bio Beads SX-3), was used with the same parameters as above but at a flow rate of 1 mL/min. The fractions collected were in this case GPC-LF, 9-25 mL; GPC-IF, 25-29 mL; and GPC-MF, 29-40 mL. GC analysis (4) and GC/MS (35) were performed as described by Bergman et al., using the temperature program described for GC in both cases. Radioactivity Measurements. Radioactivity measurements were performed on a Wallac 1409 scintillator (Wallac Oy, Finland). For lipophilic extracts and purified samples, scintillator 299 (Packard) was used, while Optiphase II (Wallac Oy) was used as the scintillation cocktail for aqueous samples. Solid biological samples (tissue and fecal samples, 50-100 mg wet weight or 20 mg dry weight) were digested with Soluene 350 (1 mL, Packard) for 2 h or until a clear solution was obtained. When necessary, the samples were bleached with hydrogen peroxide (30%, Kebo) in 2-propanol (Merck) (2:1, 1 mL) for 2 h before counting. Hionic fluor (Packard) was used as the scintillation cocktail for the solid biological samples. Animals. (A) Female C57Bl mice (19 ( 1 g) (ALAB, Solna, Sweden) were divided into four groups of four and kept in metabolism cages with a 12-h light/dark cycle and water and food ad libitum. These animals were treated po with 14C-labeled CB101 (10 mg/kg, 1 Ci/mol) dissolved in corn oil (200 µL/mouse). Feces and urine were collected for 5 days after which the animals were sacrificed, and the liver, lung, adipose tissue, adrenal, and blood (separated into erythrocytes and plasma) were removed and stored at -20 °C. (B) Female minks (Mustela vison) (1 year old, 1.2-1.3 kg) were kept individually in four separate metabolism cages. These animals received 14C-labeled CB101 (10 mg/kg, 1.7 Ci/

1342 Chem. Res. Toxicol., Vol. 9, No. 8, 1996

Klasson Wehler et al.

Table 1. Concentration (nmol/g) of 14C-Labeled Compounds in Fresh Tissues (Wet Weight) and Tissue Extracts (Lipid Weight), Lipid Content (%), and Relative Amount of Non-Extractable and Lipid-Bound 14C in Tissues (% of Total in Tissue) tissue

lipid content (%)

14C in fresh tissue (nmol/g wet weight)

adipose tissue liver lung adrenala salivary gland plasma

92 ( 0.5 9(2 7.8 ( 1 23 4.8 ( 1.5 n.a.

52 ( 11 8.6 ( 2.9 2.8 ( 1.25b n.a.d n.a. 1.3 ( 0.3

adipose tissue liver lung adrenala plasma

66 ( 14 4.7 ( 0.2 4.5 ( 0.7 28 n.a.

37 4.7 13b n.a. 2.2

14C

in extracts (nmol/g lipid)

non-extracted 14C (% of total)

60 ( 14 65 ( 9.4 47 ( 8.4 31.6 157 ( 52 n.a.

90% of the 14C in the GPC-MF, but only for 55% of the total 14C, the remainder being covalently bound and/or other metabolites (cf. Tables 1 and 2). In plasma, CB101 represented only 0.2% of the total 14C, and thus >99% of the radioactivity represented metabolites. The concentrations of metabolites and CB101 in tissues are expressed on a lipid weight basis (i.e., nmol/g of lipid weight), in order to reveal possible mechanisms of accumulation other than passive partitioning in lipids, e.g., reversible binding to proteins. Methylsulfonyl metabolites were present in all tissues. The highest concentrations were found in the salivary gland and the liver, with the concentration in the former organ being 5 times higher than that in the latter (Table 2). Generally, 3′MSF-101 was present at higher concentrations than 4′MSF-101. Phenolic metabolites were generally present in approximately the same concentrations as the methylsulfonyl metabolites, except in adipose tissue (where the concentration of the lipophilic methylsulfones was much higher) and in plasma (where phenolic metabolites dominated) (Table 2). Several hydroxylated metabolites were identified (cf. Figure 1). 3′-OH-101 and 4′-OH-101 were present in all tissues, with the highest concentration in the liver. 3-OH-101 and 4-OH-pentaCB (Figure 1) were present in liver, lung, and plasma, but generally at slightly lower concentrations than 3′-and 4′-OH-101. In addition, all tissues contained a metabolite that after methylation yielded a mass spectrum with a molecular ion of m/z ) 384 and an isotopic cluster corresponding to five chlorine atoms, which corresponds to a methyl derivative of a dihydroxylated pentaCB (di-OH-pentaCB) (Figure 5A). The presence of a major fragment (M - 15)+ (corresponding to the loss of a methyl group) and another fragment (M - 43)+ (corresponding to the loss of (COCH3)) supports a structure with at least one hydroxy group in a para-position (34). Feces was the major route of excretion in the mink; 17% of the total dose was excreted by this route within 5 days. Only 0.6% was excreted with the urine, with the highest level of excretion on day 2. The total amount of

0.095 1.9 0.01 0.02 6 0.041 ( 0.02 1.1 ( 0.3 0.0009 ( 0.0004 0.00003 0.044 ( 0.006 a

The hydroxylated metabolites have all been quantified as their methyl derivates, using 4-MeO-101 as standard. b n.d., not detected.

n.d. n.d. n.d. n.d. n.d. 0.056 ( 0.03 2.7 ( 1.1 0.0047 ( 0.005 0.0001 0.095 ( 0.047 22 ( 6 6.2 ( 2 19 ( 4 11 0.024 ( 0.01 adipose tissue liver lung adrenal plasma

0.67 ( 0.3 1.1 ( 0.19 0.81 ( 0.32 0.71 0.0059 ( 0.002

0.48 ( 0.19 1.1 23 ( 4 0.66 0.024

1.4 1.0 0.035 1.1 0.24

Mouse 0.012 ( 0.004 0.37 ( 0.089 0.24 ( 0.076 0.027 0.040 ( 0.017

n.d. n.d n.d. n.d. n.d.

0.09 2.6 1.1 0.52 0.56 23 0.002 ( 0.0006 0.100 ( 0.012 0.032 ( 0.01 0.010 0.111 0.008 ( 0.002 n.d. 0.063 ( 0.028 0.033 ( 0.003 n.d. 0.11 0.020 ( 0.003 n.d.b 0.017 ( 0.0009 0.043 ( 0.006 n.d. 0.11 0.024 ( 0.003 0.006 ( 0.004 0.35 ( 0.15 0.053 ( 0.013 0.022 0.21 0.009 ( 0.004 14 ( 1.9 15 ( 4.0 11 ( 2.9 8.8 43 0.11 ( 0.04 adipose tissue liver lung adrenal salivary gland plasma

0.14 ( 0.08 0.29 ( 0.06 0.16 ( 0.05 0.073 1.5 0.003 ( 0.001

0.034 ( 0.019 0.052 ( 0.013 0.040 ( 0.013 0.022 0.24 0.0008 ( 0.0004

4.1 5.6 4.1 3.3 6.2 4.0

Mink 0.006 ( 0.001 0.36 ( 0.1 0.054 ( 0.024 0.018 0.44 0.027 ( 0.006

4-MeO-pentaCB 3′-MeO-101 4′-MeO-101 ratio 3-/4-MSF-101 4-MSF-101 CB101

3-MSF-101

Klasson Wehler et al. 14

tissue

Table 2. Concentrations of Metabolites and Parent CB101 in Tissues (µg/g Lipid)a

3-MeO-101

Di-MeO-pentaCB

∑MeO/∑MSF


C in the urine was considered to be too low for further analysis. The cumulative excretion of 14C in feces and urine is depicted in Figure 6. After extraction of the fecal samples, 14C-labeled material in the combined aqueous solution and methanol extract contained 22% of the excreted radioactivity (4% of the dose) and was assumed to be water-soluble metabolites, such as glucuronides, sulfates, and products of the mercapturic acid pathway (MAP). After hydrolysis of an aliquot of the methanol extract, 77% of the radioactivity could be extracted into an organic solvent, indicating that 3% of the dose or 16% of the excreted material corresponded to glucuronic and/or sulfate conjugates. The remaining water-soluble compounds may have been MAP metabolites since these are not deconjugated by the hydrolytic conditions used. The presence of methylsulfones is also indicative of glutathione conjugation. The lipophilic extract was fractionated by GPC, and the GPC-LFs contained a slowly increasing portion of the total 14Cs25% on day 1, 30% on days 2-3, and 32% on days 4-5sthe remaining radioactivity eluting in the GPC-MF. These values represent about 5% of the dose administered. The non-extractable 14C in the feces corresponded to 15% of the total excreted 14C (2.6% of the dose). The relative distribution of 14C in the different fractions of the fecal samples is shown in Figure 7A. GC/MS analysis revealed that the fecal GPC-MFs consisted primarily (>70%) of 3′- and 4′-OH-101 and that all metabolites detected in tissues, except for 4-OHpentaCB, were also present in feces, although in different relative amounts (Figure 8). 3′- and 4′-MSF-101 were also excreted in the feces but only in trace amounts (0.11% and 0.03%, respectively; see the mass spectrum in Figure 5C). The relative amounts of CB101 and of its phenolic metabolites in each mink fecal sample are presented in Figure 8. The di-OH-pentaCB detected in tissues was also recovered in feces (Figure 5B). Two additional metabolites had longer retention times than the dihydroxy metabolites, molecular ions of 380, isotopic clusters corresponding to four chlorine atoms, and spectra with a major fragment of (M - 30)+, which may correspond to two methyl groups (see Figure 5D). The data indicate that these metabolites are isomers of tri(MeO)-pentaCBs. No ortho-substituted hydroxylated metabolites were found in feces or in tissues. Mouse. The distribution of 14C in mouse tissues confirmed the findings from previous autoradiographic studies (44, 45), with a high concentration of 14C in adipose tissue and lung bronchial mucosa. Five days after exposure, the concentration of 14C in the lung was three times higher (on a wet weight basis) than in the liver (Table 1). In the extracts, the concentration of 14C in the lung (161 ( 9 nmol/g lipid) was twice as high as that in the adipose tissue (80 ( 24 nmol/g of lipid). The concentration of 14C in plasma was low on wet weight basis (lipid weight was not determined for these samples). In the liver and lung samples, 45 and 17%, respectively, of the total 14C could not be extracted. Plasma and adrenals also contained non-extractable 14C (28% and 5%, respectively), whereas the 14C in the adipose tissue was quantitatively extracted (Table 1). Only the liver contained water-soluble 14C, this fraction being about 4% of the total 14C. In the case of the liver extract, 32 ( 3% of the 14C present eluted in the GPC-lipid fraction, which indicates


Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1345

Figure 5. Mass spectra of metabolites in mink and mouse (after methylation of the samples). (A) Di(OH)-pentaCB found in the tissue and feces of both mink and mouse. (B) Di(OH)-pentaCB isomer detected in mouse feces. (C) Isomer of MeS-101 found in mouse feces. (D) An unknown metabolite found in mink feces.

Figure 6. Cumulative excretion of

14C-labeled

CB101 in (A) the feces and (B) the urine of mink (() and mouse (0).

that 18% of the total radioactivity in this organ was covalently bound to lipids. In the lung, the corresponding figure was 6.8% whereas no lipid-bound 14C could be detected in adrenals and adipose tissue (Figure 4). The same metabolites as detected in mink tissues were also present in mouse tissues, with the exceptions that 3-OH-101 and 4-OH-pentaCB could not be detected in any of the mouse tissues (Table 2). The parent CB101 was the major radioactive compound present in adipose tissue (99% of the total 14C) and in the adrenals (85%), whereas metabolites dominated in the liver, lung, and plasma. In liver, the major metabolite 3′-OH-101 was present in an almost 10-fold higher concentration than

the isomer 4′-OH-101, whereas 3′- and 4′-MSF-101 were present in equal amounts. In mouse lung, the major metabolite 4′-MSF-101 was present at a 28-fold higher concentration than its isomer 3′-MSF-101 and at almost at the same concentration as CB101 (Table 2). The amount of 4′-MSF-101 was greater than that of 3′-MSF101 in plasma as well. In other tissues, the ratio between these two methylsulfonyl metabolites was approximately one. Except for liver and plasma, the methylsulfonyl metabolites were more abundant than the phenolic metabolites. The 4′-OH-101 to 3′-OH-101 ratio in the mouse tissues varied from 0.14 in the liver to 51 in the lung to 270 in the adrenals.


Klasson Wehler et al.

in its mass spectrum (36). As in mink feces, only trace amounts of 3′- and 4′-MSF-pentaCB were excreted (0.0005% and 0.0014%, respectively, of the GPC-MF on day 1). The relative amounts of these metabolites increased with time (days 2-3, 0.049% and 0.17%, days 4-5, 0.13% and 0.45% for 3′- and 4′-MSF-101, respectively). The di(OH)-pentaCB detected in tissues was also present in feces. In addition, another compound with the same molecular weight (m/z ) 384) but a longer retention time was observed. This metabolite had an abundant fragment at (M - 43)+ but no (M - 15)+ fragment (characteristic for para-substitution) or (M - 50)+ fragment (characteristic for ortho-substitution). It is therefore suggested that this metabolite is a meta-substituted dihydroxy-pentaCB (Figure 5B) (35). No ortho-substituted hydroxylated metabolites were detected in mouse tissues or feces.

Discussion Figure 7. Relative distribution of 14C in different fractions of the fecal samples from (A) mink and (B) mouse. The percentage of the total dose is given above each circle diagram.

Figure 8. Relative amounts of CB101 and phenolic metabolites in mink (left) and mouse (right) fecal samples.

The mice excreted 74 ( 4% of the total dose in their feces within 5 days, with approximately 45% being excreted the first day. Only 1% of the dose was excreted in urine, with the largest amount again being excreted on day 1. The urine was not further analyzed. The cumulative excretion of 14C in feces and urine is shown in Figure 6. A total of 90% of the fecal radioactivity excreted on day 1 was lipophilic and only 3% was water-soluble. The remaining 14C (7%) was considered non-extractable. On the following days, the proportions of non-extractable 14C and water-soluble metabolites increased (Figure 7B). Most of the 14C in the fecal lipophilic extracts represented non-conjugated metabolites and/or parent compound at all time points. The remaining 14C represented lipidbound metabolites and constituted 7% of the excreted 14C, i.e., 5% of the dose administered. GC/MS analysis of the GPC-MF verified the presence of CB101, at high concentration on day 1 (51% of the excreted 14C or 23% of the dose), but this level decreased rapidly to 99% and 65% of the total dose, respectively (calculated from data reported in ref 31). It can be assumed that the more efficient absorption by the mink is due its larger size and intestinal system. Excretion by the mouse (74% of the dose) was more rapid than by the mink (17%), but for both species the excreted radioactivity consisted primarily of metabolites (73% and 94%, respectively). CB101 is thus metabolized to a large extent prior to excretion in these animals. These findings are consistent with those of Berlin and co-workers (44), who reported that 75% of an oral dose of CB101 given to mice was excreted in the feces within 5 days and that no unmetabolized CB101 was recovered in the fecal extract. In contrast, CB105 is less efficiently metabolized and excreted: 8% of an oral dose of this compound to mink was excreted within 5 days and approximately 60% of the material excreted consisted of metabolites, assuming that feces day 3 is representative (30). The mouse excreted 60% of the administered CB105, with 23% of the excreted material consisting of metabolites. Thus, CB101, with an unsubstituted meta-/ para-position, is more easily and efficiently metabolized and excreted than is CB105 with chlorine atoms in both para-positions. There were also qualitative differences in the CB101derived 14C excreted. The mink excretes larger relative amounts of water-soluble metabolites (25% of the excreted material) than does the mouse (4%). This may reflect different capacities for conjugation (e.g., formation of cysteine conjugates, since these are not hydrolyzed by the method used in the present study) and/or different efficiencies of the gut flora in deconjugating, e.g., glucu-


ronides. Deconjugation of glucuronic acid, sulfate, and cysteine conjugates by the gut flora in several mammals has been reported (46). Quantitatively important types of metabolites are those irreversibly bound to lipids and those which are nonextractable and presumably bound to macromolecules (Figure 7A,B). Thus, 27% of the radioactivity in mink feces is bound to lipids, and approximately 15% is bound to macromolecules. In mouse feces, 7% is bound to lipids, and 13% is bound to macromolecules. Lipid-bound metabolites were also detected in tissues: 8% and 18% of the total radioactivity in the mink and mouse livers, respectively, were covalently bound to lipids (Table 1). Lipid-bound metabolites have previously been observed for other PCB congeners, e.g., in the feces of mink exposed to CB105 (20% of the excreted 14C) (30) and in feces from mouse exposed to 3,3′,4,4′-tetraCB (CB77) (30% of the excreted 14C) (43). Lipid-bound metabolites have also been reported in the case of other xenobiotics, such as pentachlorophenol, which was found as a palmitic acid ester at a concentration of 240 ppb in human adipose tissue (47). Fatty acyl xenobiotic esters have also been detected in rat feces after exposure to tetrahydrocannabinol (48) and DDT (49). Furthermore, other sorts of lipids, including acyl glycerols, cholesterol, and phospholipids, have also been shown to bind covalently to xenobiotic metabolites (reviewed by Dodds (50)). The formation of lipid-bound metabolites thus seems to be quantitatively important for xenobiotics and deserves further study. The presence of lipid-bound metabolites in tissues and feces indicates the formation of reactive intermediates during metabolism. Such reactive intermediates, e.g., arene oxides, are produced in metabolically active tissues such as the liver. Accordingly, we found non-extractable radiolabeled material in the liver especially but in other tissues as well. As much as 32% and 45% of the total 14 C in the mink and mouse livers, respectively, was nonextractable. Considered together, this covalent binding of radioactivity to lipids and macromolecules indicates extensive metabolism of CB101 via reactive intermediates. Generally, PCBs are considered to be metabolized via arene oxides, and in the case of 2,2′,5,5′-tetraCB, an arene oxide has been isolated and identified (51). The presence of isomeric methylsulfonyl metabolites of CB101 constitutes evidence for arene oxides as the reactive intermediates (10, 11). Other evidence for the existence of such intermediates is the formation of 1,2-shifted metabolites (52), such as the 4′-OH-2,2′,3′,5,5′-pentaCB observed in mink tissues in the present study. An alternative pathway demonstrated recently for chlorobiphenyl is the formation of catechols that can be further metabolized to highly reactive quinones, which in turn may react with endogenous nucleophiles (53). This is of particular interest in the light of the dihydroxylated metabolites found in tissues in the present study. The distribution of xenobiotics and their metabolites in the body is governed by the chemical and physical properties of these compounds, particularly their lipophilicity, but structural similarities to endogenous compounds may also be present and allow interaction for binding sites on specific proteins. The latter process may give rise to selective retention, or accumulation, of xenobiotics in certain tissues. Selective retention may be observed using whole-body autoradiography (40) and/


or by determining the ratio of parent compound and/or isomeric metabolites in various tissues and/or excreta. Thus, in the mouse lung a selective retention of 4′-MSF-101 is pronounced, with a ratio of this compound to 3′-MSF-101 of 28; whereas in adipose tissue the corresponding ratio is only 1.4. This retention is a result of the affinity of certain 4-substituted MSF-CBs, including 4′-MSF-101, for a uteroglobin-like, progesterone- and calcium-binding protein present in the lung and known as “PCB-binding protein” (17-19). Accumulation of CB101-derived radioactivity in the mouse lung has previously been observed using autoradiography (44, 45), and it was later shown by Bergman et al. (31) that this phenomena reflected accumulation of the 4′-MSF metabolite in the bronchi of the lung. The autoradiograms of mink exposed to CB101 demonstrated the presence of MSF metabolites in the lung of this animal as well, although no selectivity in the accumulation of 3- or 4-MSF -101 was observed. This lack of selectivity may be due to the short exposure time used here since selective accumulation of 4′-MSF-CBs in the lung of wild mink has been reported (54). The toxicological implications of the retention of MSFPCBs in tissues are not clear, but the interactions of these metabolites with the PCB-binding protein in lung may be related to the respiratory disturbances occurring in PCB-poisoned victims in Japan in 1968 (19, 55). 3′-MSFCBs but not 4′-MSF-CBs are also potent inducers of drugmetabolizing enzymes (cytochromes P450 (2B1, 2B2, 3A2, and 2C6) and cytochrome b5, aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase, and benzo[a]pyrene hydroxylase). Indeed, 3′-MSF-101 is 700 times more potent than the parent CB101 in inducing drug-metabolizing enzymes in the rat (20, 21). MSF-CBs may thus affect the metabolism of both endogenous compounds and xenobiotics. Drug-metabolizing enzymes in mink liver are also induced by MSF-CBs.3 In addition, phenolic metabolites may be selectively retained in tissues, as may be exemplified by the difference in 3′-OH-101 to 4′-OH-101 ratio in the mouse lung and adrenal (0.02 and 0.004, respectively) as compared to the ratio of 10 in feces. This difference indicates either selective synthesis or selective retention of 4′-OH-101 in mouse lung and adrenal. Both isomers can be formed via an arene oxide in the 3′-/4′-position, but the 3′-OH101 can also be formed by direct hydroxylation, as demonstrated for 2,2′,5,5′-tetraCB (56). In the mink tissues, the 4-OH-pentaCB is present in approximately equal amounts as 3-OH-101, but is not detectable in feces. This metabolite pair, formed by metabolism in the 2,4,5-trichlorinated phenyl ring, is present in mink tissues at approximately the same concentration as the 3′-/4′-OH-101 pair, although the latter metabolites are obviously produced to a much larger extent (40 times more in the feces; Figure 8). This may indicate selective retention of, at least, 4-OHpentaCB but perhaps also of 3-OH-101, although the possible underlying mechanism is presently unknown. Selective retention of hydroxylated PCB metabolites has previously been reported to occur in the blood of humans and seals exposed environmentally (14). This retention was explained by the affinity of the OH-PCBs for a thyroxine-transporting protein TTR (16). The OHPCBs retained had a hydroxyl group in the para-position 3

Unpublished, manuscript in preparation by Lund, B.-O.


and chlorine atoms on the adjacent carbon atoms, which is similar to the structure of 4-OH-pentaCB (Figure 1) and that of thyroxine. The high concentration of 14C in a salivary gland of the mink was shown to correspond primarily to CB101. The concentration of CB101 here was 3 times higher than in any other tissue, and the concentrations of metabolites were also highest in this gland (e.g., a 5-fold higher concentration of 3′-MSF-101 than in the liver). One explanation for these high concentrations in this gland may be that it has an excretory function. It has previously been reported that CB101-related 14C was present at high concentrations in a salivary gland in mice (44). The mouse salivary gland was not analyzed in the present study. MSF metabolites of DDE have previously been reported to be adrenotoxic in the mouse via a mechanism involving bioactivation (57). Furthermore, one MSF-PCB has been shown to be bioactivated by the mink adrenal in vitro.3 The adrenal was therefore included in the present study. Both CB101 and metabolites there of were found in mink and mouse adrenal, indicating the possibilities for interference with adrenal function. In conclusion, mink and mouse tissues produce several metabolites of CB101. Lipid-bound metabolites constitute a quantitatively large group, both in feces and tissues and particularly in the liver, where there is also a high degree of covalent binding to macromolecules. The mink produces metabolites hydroxylated in the 2,4,5trichlorinated phenyl ring, metabolites that were not observed in the mouse. The mink also excretes relatively more water-soluble metabolites, presumably conjugates, than does the mouse. The mouse selectively accumulates 4-MSF-101 in its lungs. Specific tissue localization of metabolites due to binding to proteins may be involved in toxic processes.

Acknowledgment. We gratefully acknowledge Prof. A° ke Bergman for valuable discussions and for providing us with the synthetic standards. Financial support was granted by the Swedish Environmental Protection Agency.

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TX960112X

2,2',4,5,5'-Pentachlorobiphenyl: Comparative Metabolism in Mink

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