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rxoCEEDiNos or the society for experimental biology and medicine 148, 682-687 (1975) Distribution and Metabolism of 3H-2,5,2' ,5'-tetrachlorobiphenyl in Rats' (38610) ir ) cA u or J. P. VAN MILLER, I. C. HSU, and J. R. ALLEN 'i5 Department oj Pathology, University of Wisconsin Medical School, and Regional Primate Research Center, w cr University of Wisconsin, Madison, Wisconsin 53706 i 1C (trie Recently, the effects of isomeric mixtures of polychlorinated biphenyls (PCBs) have received considerable attention. It has been reported (I, 2) that humans develop chloracne and subcutaneous edema and exhibit lethargy and nausea as a result of PCB in toxication. Nonhuman primates exhibit similar effects as well as pathological changes in rabbits. Two of the metabolites were identified as monohydroxy-TCB with the hydroxyl group located in the three or four position, while the third metabolite was identified as trans-3,4-dihydro-3,4-dihydroxy-TCB. The presently reported study was under taken to establish the distribution of TCB i ci lit mcr :i tie nulf. .f r cih\ Ivfc proc which include hypertrophy of the hepatic smooth endoplasmic reticulum (ER) and mucosal lining of the stomach (3-5). Altera tions in protein levels and hepatic enzyme activity have also been observed in non human primates after PCB intoxication. No gross abnormalities have been observed in rats, although similar hepatic ER and en zyme alterations have been noted (6, 7). In addition, it has been shown that the ef fect on hepatic function is increased as the in the tissue and excreta of rats using *HTCB. Further information was sought as to the amount of TCB metabolized and confirmative identification of these me tabolites. Materials and Methods. *H-TCB was prepared from 2,5,2',5'-telrachlorobenzidine by the method of Hutzinger and Safe (14). The compound was shown to be greater than 99% pure by gas liquid chroma tography, with a specific activity of 1.13 DM V dS lone IT l s'r >. \! I fl I chlorine content of the PCB isomers in creases (8). The necessity for further understanding of the distribution and metabolism of these compounds is evident. Since the complexity /rCi/mg after dilution witli TCB. The com pound was dissolved in corn oil as final preparation for dosage. Four groups (three animals per group) of male Spruguc-Dawlcy ruts, weighing of the PCB mixtures makes such studies difficult, it has been necessary to use pure components. The compound 2,5,2', 5'letrachlorobiphenyl (TCB) has been shown to be one component of the PCB mixtures presently in use (9, 10). Hutzinger et al. (II) were first to report hydroxylatcd metabolites of TCB in the excreta of rats and pigeons. However, struc tural confirmation of these findings was lacking. Yoshimura and coworkers (12) identified monohydroxy-tetrachlorobiphenyl among several phenolic metabolites of 2,4,3',4'-(ctrachlorobiphenyl in rat feces. Recently, Gardner et al. (13) have isolated and characterized three metabolites of TCB 1 This investigation was supported in part by U.S. I'ublic Health Seivicc Grant Nos. E5-00472 and KR OOI67 from the National Institutes of Health. Primate Center publication number 14-015. 92-103 g initially, were housed in metabo lism cages and allowed to acclimate for 3 days prior to dosage. The rats were subse quently given a single dose of 'H-TCB by gastric intubation. Each animal received 50.0 mg (56.5 >rCi) of the compound in 0.5 ml corn oil. The animals were given access to unlimited food and water through out the experiment. Feces and urine were collected at 24-hr intervals. The animals were sacrificed al 1,3, 7, and 14 days and tissue samples collected for radioactivity analysis and histologic evaluation. All tissue and fecal samples (200-500 mg) were oxi dized in a Packard Model 306 sample oxi dizer, collected in Monophase 40 (Packard) scintillation cocktail, and the radioactivity measured in a Packard Tri-Carb liquid scintillation counter. Samples of blood and 682 I' K 1' :\y. I !. (r ,i on w. . c.r i S -oo' 1 m, in in \A [ !' CAollpnyrgiRhhiiire0se1rv9e7d5. by the Society for Experimental Biology and Medicitx DSW 025969 TLTRACHLOKODirilliNYL METABOLISM IN RATS 68J urine (0.5 nil) were treated similarly. In addition, subccllular fractions of the liver were prepared by the method of Hogeboom (15) and analyzed us described above. Isolation und identification of metuboliles were done on fecal and urine samples from the first 48 hr after dosage. Urine was freeze- dried und extracted with hexane and ethyl ether by two different methods. The first method involved hydrolyzing the sample after hexane extraction with glucuroniduse- sulfatasc (G-S) by the method of Reid ct at. (16), followed by extraction with ethyl ether and a subsequent acid hydrolysis before a second ether extraction. The second procedure was similar to the above except no G-S hydrolysis was performed. Feces DM5 Fig. I. Excretion of`H by rats for the first 8 days following a single oral dose of 'H-i.S.Z'.S'-tetrachlorobiphenyl. Each point represents mean I was extracted continuously first with ace SD for three rats. Daily excretion values for feces and tone und then with methanol. TCB and urine after 8 days were less than 0.2% of dose. metabolites were purified from the hexane extract of urine und acetone and methanol on a Hitachi Model 247 Grating Infrared extracts of feces by thin layer chroma Spectrometer fitted with a beam condenser tography und the samples analyzed by gas (Perkin-Elmer Corp.). liquid chromatography, mass spectrometry, Results. A. Distribution. The experimental and infrared spectrometry. Ether extracts animals appeared normal, exhibited normal of urine could not be purified by TLC. weight gain, and in general showed no del These samples will be analyzed in future eterious effects from (he TCB dosage. experiments upon development of a system Microscopically, the tissues showed no for purification. abnormalities. The greatest percentage of TLC samples were applied to silica gel the recovered tritium was found in the ex IB, flexible plates (J. T. Baker Chem. Co.) creta (Fig. I). Approximately 66 7, of the and developed with hexane, hexanctethyl tritium was excreted in the feces by 72 hr acetate (8:2), or hcxane:acetone (1:1). with an additional 10'If recovered in the The plates were scanned on a Model 7201 urine. At 14 days the total tritium in the Rudiochromaiogram Scanner (Packard) and feces and urine was 72.5 5.7and 12.9 the peaks eluted with methanol for further 4.0M, respectively. Through the first 7 analysis. GLC was done on a Model 7620A days the largest internal concentrations of Hewlett-Packard gas liquid chromatograph tritium were located in the adipose tissue, fitted with an F.C detector. Glass columns hlood, liver, and skin (Table 1). Signifi Cs" id X 6') containing 2C Apiczone L cantly high concentrations of tritium were on Gaschrom Q (100-120 mesh) at 215 found in several other tissues at 24 hr (in were used. Argon-methane was used as cluding the thymus, brain, lung, spleen, carrier gas at approximately 40 cc/min. heart, small intestine, and muscle), but Samples for mass spectral analysis were these values decreased rapidly and at 3 days collected Tront the GLC in capillary tubes were less than 2'"< of that present in the which can be inserted directly into a MS-0 tissue conUut ing the highest conccntriilson mass spectrometer (Associated Electrical i.e., adipose tissue (0.243 0.18'..' of Industries, Ltd.) equipped with a direct dosc/g). Although the specific activity of insertion probe. Samples for IR analysis the blood (0.009 0.002' i- of dose, g) were collected from the GLC on KBr in was slightly higher, the levels throughout capillary tubes. A 1.5 mm KBr pellet was the body were generally constant (average: made with a KBr Ultra Micro Die (Perkin- 0.004 0.002of dose g) at 14 days. Elmer Corp) and the IR spectrum measured In addition to the tritium present in the DSW 025970 684 TETRACHLOROBIPHLNYL MUTAUOLISM fN RATS TAULE I. Tissue Concentrations* of *H in Rats at I, 3 and 7 Days Following a Single Oral Dose OF *H -2,5,2',5'-TetrachloroBIPHENYL. I day1* J days* 7 d>V Adipose tissue BIoodJ Liver Stomach Kidney Testes Large intestine Skin 1.345 0.680 0.593 0.193 0.281 0.055 0.792 0.613 0.540 0.082 0.143 0.037 0.445 0.147 0.344 0.144 0.243 0.180 0.126 0.017 0.035 0.004 0.013 0.009 0.010 0.002 0.012 0.006 0.011 0.004 0.026 0.007' 0.058 0.044 0.044 0.019 0.008 0.002 0.004 0.001 0.005 0.00 0.005 0.003 0.003 0.001 0.005 0.002 " J'crccnt of dose per g tissue standard devi ation. > Moan for tlirce rats. ' Mean for two rats. * Percent dose per ml. excreta and specific tissues evaluated, 2.1 0.57'if of the original radioactivity was de tected in the remaining tissues of the animals on the 14th day of the experiment. Total recoveries for 1, 3, 7 and 14 days were 36.R 6.4%, 79.9 11.2%, 82.6 4.0%, and 87.6 7.8'if, respectively. The low recovery obtained for the 24-hr animals was probably due to (lie large quantity of tritiated compounds in the intestinal tract which was not measured. This conclusion is substantiated by the fecal excretion value for the 24- to 48-hr period. Subccllular fractions of the liver showed (he largest specific activity of tritiated com pounds in the microsomal fraction (Fig. 2). Significant specific activities were also found in the mitochondrial fraction with relatively low specific activity in the nuclei. B. Metabolites. The procedures employed in metabolite extraction and TLC separation produced six purified compounds of me tabolized TCB. Since complete identifica tion of all these compounds was not possible, it is not known if any are identical and hence each will be termed a metabolite. Fig. 2. Distribution of *H in liver homogenates and subccllular fractions of liver I, 3, 7, and t4 days (L lo R) following a single oral dose of >1-1-2,5.2', 5'tetrachlorobiphenyl. I signifies mean 1 SD. TABLE II. Distribution* of Tritium in Extracts of 24- and 48-Hour Urine from Rats Given a Single Oral Dose of 'H-2,5,2',5'-Tetrachlorobi phenyl. Extract 24 hr G-S* 4* hr 0S" 24 hr 4R hr Hexane Eilier-I' Ether-21' Aqueous 49.05 33.71 7.00 4.93 47.14 20.67 17.10 9.38 37.22 11.11 29.03 11.99 34. 14 7.37 21.53 17.67 * Percent of freeze-dried urine. 1 Olucuronidasc-sulfatase-trcatcd. * Before acid hydrolysis. * After acid hydrolysis. Freeze-drying of the urine sumples caused a loss of radioactivity between 50 and 65%. Table 11 shows the distribution of radio activity remaining in the freeze-dried urine for the 24- and 48-hr urine samples. The percentage of radioactivity remaining after extraction is reported as percent in the aqueous phase. Glucuronidase-sulfatase treatment yielded a slightly improved re covery over the samples that were only acid hydrolyzed. No significant difference was noted between the 24- and 48-hr samples as to the amounts extracted or the metabo lites found. A single metabolite (Metuboiite 1) was isolated from the hexane extracts of the urine samples. No unmetabolized TCB was found in these samples. The tritium recoveries in the acetone and methanol extracts of feces were 77.4% I TA BLF i AMnl I *<ILA Umrsii Oral mi put? M.J-aitbX o* I It III IV v VI TLC ft-- hexar and 18 8.7 ; o tabolizc' TCB w; Of the fraction (Mvlabi coiiiprist in I be fr; of lecus (Met abo data for Tabic 11 Metuboi ion at droxv -T( III and these sa nuix-. spi identified and 1R spectrum that for el rl t ocem 1 insti a. No SUtlica OSH 025971 1 ** .' 3 IK'S \ ii.ijs %.' . y- vl IV \ 1 ilM -.1' OF . I . I -1 7. : i." 17 .(' atiscd 65 ' radiourinc .. Iht alter n i lie Tut.isc .'(.! re s' acid ,, \s ;is m-ples . i i ho- l-.llllC .1: icts olizcd and .j % TETRACHLOROBIPHENYL METABOLISM IN RATS 685 TABLE III. Chromatographic Data for Me identical mass spectra, however, led to the tabolites of 2,5,2',5'-Tetrachlorobiphenyl Isolated from 24 and 48 Hour Feces and Urine Samples from Rats Given a Single Oral Dose of 'H-2,5,2', J'-TetracjiloroDIPHENYL. conclusion that metabolites I, II, IV and V are all monohydroxy-TCB, although the positions of the hydroxyl groups were indeterminable. The mass spectrum and IR spectrum for Metabolite 11 are given in Metabo lite Origin Friction TLC olvent" / GLC reten tion time (MC) Fig. ?' . Discussion. The data indicate that over 95% of the recovered tritium was in the . ' excreta of which over 90% was metabolized. 1 Urine Hexane A 0.8 120 The unmetabolized TCB found in the feces u Feces in Feces IV Feces Acetone MeOH MeOH B 0.4 125 B 0.0 C 0.4 124 B 0.25 C 0.34 122 was probably the portion of the initial dose which was not absorbed. The loss of radio activity in the urine from freeze-drying is probably due either to loss of tritium during V Feces MeOH B 0.0 hydroxylation of TCB or to a greater ability C 0.0 of the hydroxylated metabolites to exchange A 0.6 134 tritium with water. At least 60% and prob VI Feces MeOH B 00 ably a considerably larger fraction of the C 0.0 A 0.45 137 metabolites are in the form of monohydroxy-TCB. However, the possibility of TLC solvents were: A--hexanetacetone (1:1); B--hexane; C-- hexanc:ethyl acetate (8:2). dihydroxy-TCB as a major metabolite as reported by Gardner et al. (13) cannot be ruled out from these data. und 18.4% respectively. It was found that 8.7% of the acetone fraction was unme tabolized TCB, while no unmetabolized TCB was found in the methanol extract. Of the remaining activity in the acetone fraction only one metabolite was isolated (Metabolite II) which after purification comprised 47.3'% of the total radioactivity in the fraction. TLC of the methanol extract of feces yielded four additional metabolites (Metabolites 111--VI). The TLC and GLC data for the six metabolites are given in Table III. Mass spectra were identical for Metabolites I, 11, IV and V with a molecular ion ut 306 a.m.u. indicative of monohydroxy-TCB. Low quantities of Metabolites III and VI made it impossible to purify these samples in sufficient quantities for mass spectral analysis. Metabolite II was identified on the basis of the mass spectrum and IR spectrum as 3-OH-TCB. The IR spectrum compared extremely well with that for 3-OH-TCB reported by Gardner et al. (13), ulthough minor dilTerences occurred due to the use of KBr pellets instead of CCL solutions of the metabolites. Allen et at. (17) have reported significantly higher concentrations of Aroclor 1248, a mixture of polychlorinated biphenyls, in the lipid of chronically treated rats than was observed for similarly treated rats given 2,5,2',5'-tetrachlorobiphenyl. Fur thermore, the proliferation of the hepatic endoplasmic reticulum und alterations of other hepatic functions are more severe for Aroclor 1248 than for the single isomer. The present study shows that TCB is rapidly metabolized to one or more hy droxylated metabolites and excreted. There is apparently ho appreciable storage depot for the compound within the animal, us the specific activities in the tissues are all re duced to very low values after 14 days. The relatively high specific activity in the blood of the animals indicates that the compound remains mobile until metabolized and ex creted. Gardner et al. (13) has reported that the probable mechanism for formation of hy droxylated metabolites of TCB is through an epoxide intermediate. It is likely that the components of Aroclor 1248 with higher chlorine content which cannot form epoxide No other metabolites could be purified in intermediates are the cause of these major sufficient quantity for IR analysis. The differences between Aroclor 1248 and TCB. OS M 025972 STLCOPCB4009934 686 TLTR ACHLOROBII'HLNYL MliTADOLISM IN RATS I 8. A:h, 7T Ii c rr 10 \'lI o.lii n. iim, i\ s /ilk. 12. Yosl C her 13. Gate 2,5,2', 5'-lclrachlorobiphenyl. Acute lethiil doses of TCB and Aroclor 1248 Itavc been reported to be approxi mately 1.2-1.5 g/kg body wt. Further evidence of the importance of metabolism to toxicity is that both Aroclor 1248 and TCB were shown to be lethal to rats at significantly lower doses than 1.2 g/kg when metabolism was inhibited by admin istration of SKF-525A. Furthermore, no mortality was observed in rats at doses of 1,25 g/kg when metabolism was enhanced by treatment with phenobarbital (17). It thus appears that metabolism of poly chlorinated biphenyls in rats is a method of detoxification of these compounds. Summary. Distribution and metabolism of an isomeric polychlorinated biphenyl were determined in rats. Over 70% of a single dose of 3H-2,5,2',5'-tetrnchlorobiphcnyl was excreted in the feces and another 13% in the urine during a 14-day period. Adipose tissue, blood, skin, and liver were found to contain low levels of the compound. Over 90 % of the excreted tritiated compound was found to be metabolized, the major metabolite being identified as 3-OH2,5,2',5'-letrachlorobiphenyl. All of the unmetabolizcd 2,5,2', 5'-letrachlorobiphenyl was eliminated by the alimentary route. 1. Kuralsune, M., Fukuoka Acta Medica, 60. 513 (1969). 2. Kuratsune, M., Yoshimura, T., Matsuzaka, 3., and Yamaguchi, A., Environ. Health Persp. 1, 119 (1972). 3. Allen, J. R., and Norback, D. H., Science 179, 498 (1973). 4. Allen, J. R., Abrahamson, L. J , and Norback D. H., Environ. Res. 6, 344 (1973). 5. Allen, J. R , Carstcns, L. A., and Barsolli, D. A., Toxicol. Appl. Pharmacol. 30, 440 (1974), 6. Allen, J. R. and Abrahamson, L. J., Arch. Environ. Conlam. Toxicol. I, 265 (1973). 7. Litters!, C. L., Farbcr, T. M., Baker, A. M., and Van Loon, E. J., Toxicol. Appl. Pharmacol. 23, 112 (1972). DSW 025973 tetrachlorobiphenyl METABOLISM in rats 687 8. Johnstone, G. J., Ecobichon, D. J., and Huizinger, O., Toxicol. Appl. Pharmacol. 28, 66 (1974). 9. Sissons, D., and Welti, D., J. Chromatography, 60, 15 (1971). 10. Webb, R. G., and McCall, A. C., J. Assoc. Oflic. Anal. Chem. 55. 746 (1972). 11. Hutzinger, O., Nash, D. M., Safe, S., de Freitas, A. S. W., Norsirom, R. J., Wildish, D. J., and Zitko, V., Science 178, 312 (1972). 12. Yoshimura, H., Yamamoio, H., and Saeki, S., Chem. Pharm. Bull. 21, 223! (1973). 13. Gardner, A. M., Chen, J. T., Roach, J. A. G., and Ragelis, E. P., Biochem. Biophys. Res. Commun. 55, 1377 (1974). 14. Hutzinger, O. and Safe, S., Bull. Environ. Contam. Toxicol. 7, 374 (1972). 15. Hogeboom, G. H., Methods Enzymol. 1, 16 1955. 16. Reid, W. D., Christie, B., Krishna, G , Mitchell, J. R., Moskowilz, J., and Brodic, B. B , Pharma cology 6, 41 (1971). 17. Allen, J. R., Carstens, L. A., Abrahamson, L. J., and Marlar, R. J., Environ. Res., in press. Received September 30, 1974. P.S.E.B.M. 1975, Vol. 148. u 4led as 3-OH- !(.) compound d. lbe mujor l .is 3-OHA!i of the unhtorobiphenyl .try route. M> .hca, 60, 513 , M.ttsuraka, J., 11- .CiIt Pcrsp, I, 11 , Science 179, 1., and Norback !3) .1 U.nsotli, D. A., 10 1974). it. I.. J., Arch. .5 1973). .ik.., A. M., and I I'harmacol. 23, OSW 025974 STLCOPCB4009936