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PROC SOC 1909 mocudingj of thc locirrv rent ixrr mmental motoov and xitoiciNt 150. *47-853 (1973) Urinary Metabolites of 2, 5, 2', 5'-Tetrachlorobiphenyi in the Nonhuman Primate (38999) ; I. C. HSU, J. P. VAN MJLLER, J. L. SEYMOUR, and J. R. ALLEN Department of Pathology, University of Wisconsin Medical School; Food Research Institute; and Experimental Pathology Unit, Regional Primate Research Center; University of Wisconsin, Madison, Wisconsin 53706 2, 5, 2', 5'-tetrachlorobiphenyl (TCB) is a major component of commercial poly chlorinated biphenyls (PCBs) which are universally distributed pollutants and arc present in the food chain primarily as a result of improper disposal (1, 2). The complexity of the commercial PCB mixtures has made it necessary to use pure isomers (c.g., TCB) for evaluation of metabolism of the compounds. The metabolic behavior of several pure PCB isomers in the pigeon, rat, and trout has been investigated by Hutzinger el a!. (3). They found that pigeons and rats metabolized TCB to monohydroxy TCB, but they were unable to detect any metabo lites in trout. Gardner cl a!. (4) found 3hydroxy-TCB, 4-hydroxy TCB and Irons3,4-dihydro-3,4-dihydroxy TCB in the urine of rabbits fed TCB and postulated metabolism of TCB via an arene oxide inter mediate. Van Miller et al. (5) reported that rats receiving 'H TCB excreted about 66% of the 'H in the feces by 72 hr with an addi tional 10% being recovered in the urine. Vnonohydroxy TCB was found to be the only metabolite in the ether extract of the urine (5, 6). Due to the observed species difference in metabolism of TCB, it became important to study TCB metabolism in a species more closely related to man. In the present study, four metabolites of TCB (Fig. 1) were isolated from the urine of infant nonhuman primates. Two of these four metabolites have not previously been reported as metabolites of TCB in any species. Materials and methods. *H-iabcled TCB was prepared from 2, 5, 2', 5'-tctrachlorobenzidine by the method of Hutzinger and Safe (7) and was dissolved in corn oil before use. Four male infant rhesus monkeys weighing 600-800 g were given JH-TCB at a dose of 500 mg/kg body wt (6.1 rCi/mg) by fgnstric intubation. The animals had unlimited access to water and were incu bated at 24 and 48 hr with 20 ml of 5% glucose solution. The animals were sacrificed at 72 hr, at which time the urine was col lected for analysis of TCB and its metabo lites. The collected urine was extracted three times with equal amounts of ether. The ether extracts were chromatographed by thin layer chromatography (TLC) on flexible, silica gel plates (J. T. Baker Chemical Co.) with methylene chloride (Solvent A) as the solvent. The radioactive bands were extracted with methanol and cither further purified by TLC or converted to" trimethylsilyl (TMS) derivatives in preparation for gas liquid chromatography (GLC), mass spectrometery (MS) or GLC-MS. Further puri fication for infrared spectroscopy (IR) was done on a high pressure liquid chromato graph. Additional TLC purification was done as described above with hexane: ethyl acetate (7^^) (Solvent B) as the solvent. TMS derivatives were made by dissolving each compound in 0.1 ml of pyrridine and 0.06 ml of NjO-bis-(trimethylsilyl)-acetamide (BSA).` Dehydration of the hydroxylated derivative of fra/is-3,4-ditaydro-3,4-dihy- droxy TCB was done by dissolving the com pound in 2 ml of methylene chloride and adding three to four drops of concentrated sulfuric acid. GLC was done on a HewlettPackard Model 7620A gas chromatograph fitted with an electron capture detector (EC) of flame ionization detector (FID). The GLC was equipped with a glass column (1/8 in. X 6 ft) containing 3% SE-30 on Gaschrom Q (100-200 mesh) and was run at 200* with Nj (FID) or argon-methane (9STT5) (EC) as the carrier gas (40 ml/min). Samples for MS were collected from GLC in a capillary tube which Was then directly inserted into a MS-9 mass-spectrometer 1 2 tu <cu CAHopyrightfVKfvcil. PritnhteedSbkivtUcJt/Sf.Av.r r-Ajn.ntr>cnirtl 185 *mJ Mcdiclrx DSW 029237 STLCOPCB4013199 186 URINARY MKTAUOLITtS OF TCB IN PRIMATES TABLE I. Thin Layer Chromatography Data rOR THE HVOROXrLATED METABOLITES OF 2, 5, 2', 5'-Tetrachlorobiphenyl (TCB) Iso lated from the Urine of Infant Nonhuman Primates 3 Days rotcowiNG a Single Oral Dose of 'H-TCB. Thin.tayer chromatographic Kf values in solvents Fio. 1. Chemical structures of the metabolites of 2, 5, 2', 5'-tctrachlorobiphenyI (TCB) isolated in MetaliolUc Methylene chloride (Solvent A) Hexane : ethyl acetate (7:2) (Solvent B) (he urine of Intent nonhuman primates 3 days after a jingle oral dose of 'H-TCB. I - monohydroxy-TCB, II dihydroxy-TCB, HI * /ra;j-3,4ldih>dro-3,4dihydroxy TCB, IV * hydroxy-3,4-dihyclro-3,4dihydroxy TCB. TCB I` It* III' IV" 0.9 0.9 0.5 0 0 _ -- -- 0.46 0.18 (Associated Electrical Industries, Ltd.) or directly injected into a GLC-MS (HewlettPackard Model 3600). IR was done in a micro KBr disc on a Perkin-Elmer 247 IR Spectrometer fitted with a beam condenser. High pressure liquid chromatography was Dehydration product of IV 0.52 '-- * Monohydroxy-TCB. * Dihydroxy-TCB. . * 7>o/ij-3,4-dihydro-3,4-dihydroxy-TCB. * Hydroxy-3,4-dihydro-3,4-dihydroxy-TCB. done on a liquid chromatograph (Waters Associates Model M-6000) utilizing a C corasil column and eluting with methanol: water (3:2). . Results and discussion. The ether extracts were chromatographed into two major radioactive bands with Rj values (Solvent A) of 0.0 (a) and 0.45 (b) and a minor band TABLE II. Relative Retention Times'1 and Molecular Ion Data* for the Trimetkylsilyl Derivatives of the Hydroxylated. Meta bolites of 2, 5, 2', 5'-Tetrachlorobiphenyl CTCB^Isolateo from the Urine of Infant Non-human Primates 3 Days Following a Single Oral Dose of 3H-TCB. ` with an R/ value of 0.9 (c). The radioactive substance in (c) was identified by GLCMS as monohydroxy-TCB (I). The meta Trimelhylsilyl . derivatives of metabolites Relative retention time Molecular inn bolite in the extract of (b) did not show any I* peaks typical of chlorinated compounds in II" 2.55 ' 6.82 378 466 GLC (EC) (even at 340 for 30 min). How ever, the TMS derivative of this compound gave a symmetric peak with a relative retention time (RRT) of 6.82 on GLC. The III* IV' . Dehydration product oflV 2.76 7.85 7.35 468 556 466 metabolite in this peak was identified by MS as dihydroxy TCB (II). The radioactive substance in (a) was further chromato * Retention of tample/retenlion (ime1 of TCB (69 sec) (gas liquid chromatography). * Atomic mass units (a.m.u.) (mass spectrom graphed with Solvent B resulting in two etry). bands with R/ values of 0.05 (a) and 0.3 (a}). The TMS derivative of the metabolite in (as) had a RRT of 2.76 and was identified by MS as a dihydro TCB diol. Further * Monohydroxy-TCB. " Dihydroxy-TCB. * THF-3,4-dihydro-3,4-dihydroxy-TCB. > Hydroxy-3,4-dthydro-3,4-dihydroxy-TCB. purification and silylation produced a puri fied radioactive substance which showed a (Ill) which has been isolated and character single peak in GLC and had an infrared ized by Gardner c(al. (4) from (tT^bit urine. spectrum identical to /r//j-3,4-dihydroi3T' The radioactive substance in the extract of o4-dihydro-3,4-dihydroxy-2t 5, 2', 5'-TCB (a,) did not show a peak typical of chlori- *c--r>* I 5 ui I<D CL, SS LX DSW 029238 STLCOPCB4013200 URINARY METABOLITES OF TCP IN PRIMATES 187 noted compounds in GLC (EC). The TMS den'vative of this compound, "however, ex hibited a symmetric peak on GLC with a RRT of 7.85 and was identified by MS as dihydrotrihydioxy TCB (IV). The similarity of the fragmentation pattern of the TMS derivatives of III and IV indicates a probable ' structure for IV as a hydroxylatcd derivative Of //wj5-3,4-dihydro~3,4-dihydroxy TCB with the additional OH group on the aro matic ring. Further confirmation of the structure for IV was obtained by dehydra tion of the metabolite and subsequent silylation of the dehydration product. The result ing compound had R; 0.52 in Solvent A, RRT 7.35 on GLC (FID), and a molecular ion of 468 a.m.u. These values are consistent with dihydroxy TCB, which is the expected dehydration product if IV is a hydroxylated ICOI--I ' * derivative of 3,4-dihydro-3,4-dihydroxy TCB. The TLC Rj values of the four metabolites oh Solvents A and B are summari7.cd in Table I. The RRT and the molecular ion data for the TMS derivatives of the four metabolites and the dehydration product of IV are shown in Table JI. The mass spectra for IF, TMS derivative of I-IV, and TMS derivative of the dehydrated product of IV are shown in Fig. 2. Similar metabo lites have been found in the urine of adult monkeys (unpublished observations). Silylation of the metabolites of TCB made the identification of II and IV possible in addition to iniprovingthe resolution. Without silylation monohydroxy TCB usually shows a broad tailing peak on GLC and dihydro o\ iU i<(DK C co co --- ---- rx> - _ - - ~r~~j*r~ii *| - iii- ii'Y -i*. ill V ttw WO Hj^i _ XJO i--v400, i*. 4>0 ' 600 so 0 P4O.. !,l 4.00 *so xo MO 4CO )0 oo FSOC SOC 1909 (00 CO too tv> WO ' MO Vt t _ Mg. 2. Mass spectra of the hydroxytated metabolites or lhctruimeihylsil>1 (IMS) derivatives of 2, 5, 2\ 5b IcliaeMorobipiirnyl (TCB) isolated in tlic urine of infant nonhumxn primates 3 days following n single ora! dos of (1) TMS derivative of the dchjrirated product orh>dfo.\y-3f4-dihydro-3,4*di!ydroxy TCB; (>) TMS derivative of hydrox> -3t4-diliydrO'3(4-t!ih>dro\y TCD; (3) TMS derivative of fra'/J-3,4-dihydro-3,d-di-. X hjdio.ty TCB; (4) dih>droxy-TCD; (5) TMS derivatives ofdih>dto>y-TCF);and (6) TMS derivative of monohy- . droxy-TCD. * . QSW 029239 \ STLCOPCB4013201 188 URINARY METABOLITES OF TCB IN PRIMATES TCB dio! decomposes inside the column at the temperatures employed. Identification of metabolites in this experi ment, particularly II and IV, suggests that the mechanism of metabolism is through arene oxide intermediates which are then ^converted to the hydroxylated metabolites, ^"Aromatic hydrocarbons that" have been reported to be metabolized through arene oxide intermediates often exhibit carcino genic, teratogenic, mutagenic, and necrogenic potential in experimental animals (8). These arene oxide intermediates are potent alkylat ing agents of nucleic acids.and proteins; hence the alkylation of the macromolecules Is suspected to be the cause of these wide ranging effects. The strong evidence reported here that TCB is metabolized through arene oxide intermediates suggests the possibility that similar macromoiecutar alterations may occur in the nonhuman primate subjected to PCB intoxication. Summary. The metabolism of 2, 5, 2', 5'- tctrachlorobiphenyl (TCB) in nonhuman primates was found to be different from that previously reported in lower species. Mono hydroxy TCB (I), the only metabolite in the ether extracts of rat urine, is a minor metabolite in the urine of nonhuman pri mates. The two major metabolites identified in the urine were dihydroxy TCB (II) and trans-3,4-dihydro-3,4-dihydroxy TCB (III). A second minor metabolite was identified as hydroxy-3,4-dihydro-3,4-dihydroxy TCB (IV). None of the above mentioned metabo lites have been reported in primates and only I and III have been identified in lower animals. It is concluded that a likely mech anism for metabolism of TCB in primates is through arene oxide intermediates. This observation is of particular importance in that these types of intermediates arc known to alkylate cellular components causing carcinogenic, mutagenic, necrogenic and teratogenic effects. We thank Dr. Bruce M. Johnson, Department of Clinical Oncology, University of Wisconsin Medical School for the use of the mass spectrometer. This Investigation was supported in part b> U.S. Public Health Service Grant Nos. ES00472, HL-10941, CA-13288 and RR-00167 from the National Institutes of Health. Primate Center publication no. 00-000. 1. Riscbrough, R. W., Reiche, P., Peakail, D. B., Herman, S. G., and Kirven, M. N., Nature (London) 220, 1098 (1968). 2. Kolbye, A. C., Environ. Health Persp. 1,85 (1972). 3. Hutzinger, O., Nash, D. M., Safe, S., Defrcitas, A. S. W. Norstrom, R. J., Wildish, D. J.f and Zitko, V., Science 178, 312 (1972). ' 4. Gardner, A. M., Chen, J. T., Roach, J. A. G., and Ragelis, E. P., Biochem. Biophys. Res. Commun. SS, 1377 (1974). 5. Van Miller, J. P., Hsu, I. C., and Allen, J. R., Proc. Soc. Exp. Biol. Med. 148, 682 (1975). 6. Hsu, I. C., Van Miller, J. P., and Allen, J. it; Bull. Environ. Contam. Toxicol, in press (1975)., . ' 7. Hutzingcr, O,, and Safe, Sr S.,-Bull. Environ. Contam. Toxicol. 7, 314 (1972). 8. Jerina, D. M., and Daly, J. W., Science 185, 573 (1974). Received March 3, 1975. P.S.E.B.M. 1975, Vol. 15^ -o > G3 m NJ o Q^ PROC SOC 1909 DSW 029240 STLCOPCB4013202