Document BoQKKQ15rBmRkRZ9zdQ6LLqJ

I 74). Itcilriig'CKiir OUoloniscIuii Qicmiii LXXXIV* Metabolism of I.ower I'olycbloriniitcil l!i|>henyls-'`C in the IUicsiis Monkey by W. Giii.m, W. Ki.r.iN, F. Coiii.ston*.L. Coi.ui;hc*, aim! F. KortTC InttiUil /iir iihnlofliiclir Clicnti* tier Cetrllsehaft fiir Strahlfib utitl l/miiWi/oM<7int> mbll, Mtiucbrn t)-520$ Si. Jnf-it.ilin I, HGermany ' /mfiliifr vf Kxitrrinttnlal l*atklnfiy ami Toxicology Albany Medical Callege, Albany, A'.V. I220B INTRODUCTION Metabolism studies on PCD are Important for estimating the! toxicity of. metabolites after knowing their identi ty land biological pathway. Some pure PCB-isomers were shown to be converted to hydroxylated products by rat liver microsomcs (GREB et al. 1974), rabbits (BLOCK and CORNISH 1959), rats (HUTZINGER et al. 1972, YO^HIMURA and YAMAMOTO 1973) and pigeons (YOSHIMURA YAMAMOTO 1973) . The purpose of this study was to identify all major metabolites after PCB-administration to Rhesus monkeys and to measure the amounts of differeit excreted metabolites. Correlation of conversions an< elimination pattern of PCBs with different chlorine content might lead to conclusions about the behavior of other PCBs. ,Thf excretion rates of 2,4'-dlchlorobiphenyl and 2 ,2*-trichloroblphenyl have been described elsewhere (CRlEB et al. 1973)} we now wish to report the identifi- ion of their metabolites. EXPERIMENTAL Thk PCBs investigated were 2,4'-dlchlorobiphenyl and 2,5,2'-trlcMorobiphenyl, both major components.of the lower AROCLOR series. They were labelled with *C. Fema le Rhesus monkeys were housed in metabolic cages and this PCB was Injected once in the left forearm vein. Three animals rocolved 2,4'-dlchlorobiphenyl in doses off 16.8, 77.6 and 566>|ag/kg, while 82.6/sg/kg of the 2,p,2'-trlchlorobiphenyl were administered to one mon key. Urine and feces were collected daily. Conjugates were hydrolysed by refluxing urine with an equal volume of 8 N h2SO. for one hour and extracted with ether. Fe ces were mixed with anhydrous sodium-sulfate and extrac ted for two days in a Soxhlct apparatus with CHjCl, end then with methanol. The combined extracts were concen- +ljxxxin. Communication: GREB, W., W. Klein, F.COULSTON, L.GOLDERG,and F.KORTE* Bull.Environ.Contam. Toxicol., in press. >0.1 .( 4, II. N. 4 I#7J 4, Siln,rr>Vrti| New Vth Ut. 471 0a*lis i !i VVv trited under a nitrogen stream and purified by prepara tive layer chromatography on silica gel Merck Nr.5765# 5766(solvent: CfUCl-). After methyJation by diazomethane (DR DOER and BACKEk 1963) the metabolites were characte rised by GLC (Packard 873, EC-detector, 1% ov-l, 2m) and identified by GLC-MS (LKB 9000 A). . RESULTS AND DISCUSSION Afiter 14 days about 771 of the administered radioactivity were rocovered,and reaction of urine with Glusulase (Endo Laboratories) according to K.D. VOIGT (1965) showed that out 17% of metabolites were conjugated with sulfuric ojj: glucuronic acid. Incubation of urine and feces in vi tro with the original PCBs revealed no formation of meta bolites due to reaction with bacteria from the intestines. Ii vivo urinary and fecal metabolites were identical and tie metabolic pattern in excreta was constant between e first and fifth day after application. Only metaboli te, no parent compound, were detected in the excreta. lg. 1 gives a survey of the metabolites formed from both l|CD8. Ipig.l*Metabolism of Polychlorinated Biphenyls by Rhesus Monkeys 472 MONS 084130 !1 ii TABLE 1 TLC - GLC- and MS-Data of 2.4,-Dichlorobiohenvl Metabolites in Monkeys Data OK-Metabo- CH-Metabo- CK-Metabo- (CH)--Meta (OK),-Me- lite sx lite S2 lite S3 bolite s4 bol. S$ R- before methylation 1 (ch2ci2) 0.66 0.50 0.25 0.13 < 0.1 fe- after^metfiylation 1 (ch2ci2) 0.82 0.70"! oTT6 0753 0.64 --------------- -- [OHTj-Me- :abbl. Sg < 0.1 0.52 , GLC after methylation Rt (min.) MS-fracnent ater methylation M 10. 2/179C 12.1/185C 11.4/185C 14.8/200C 13,1/200C 11.6/200C for 9 min. for 9 min. for 8 min., for 8 min. for 8 min. then temp, then temp, then temp, then temo. then ter.p. raised 5C raised 5C raised 5C/ raised 5C raised 5C nrn. rain. min. min. 252 s 252 s 252 s 232 s 282 s 282 s " M+-CHj " metastable " M+-C1 " M+-HC1 " M*-CHj-CO - 217 1 _ 209 m 237 1 - 217 m _ 209 s 237 m 2 23* m _ 209 m 267 1 - 247 ra 246 m 239 m 267 m - 247 m 246 m 239 1 267 m - - 239 1 * M+-CH3-C1 - - 202 I - -- ' +-CH3-CO-HCl 173 1 - 173 m - - - +-CH3-CO-Cl2 - - 139 m 204 1 - - Peak-Intensities; sstrong, m=niedium, llow MQNS 0 8 4 1 3 1 -- TABLE 2 TLC-, GW - and MS-Data of 2,5,2*-Trichlorobiphenyl Metabolites in Monkeys Data OH-Metabo- OH-Metabo- OH-Metabo- (OH)--Meta . (OK)--Meta (0H)3-e- lite G1 lite G2 lite G3 bolite g4 bolite g5 taboi. Gg Rf before methylation 1 (ch2ci2) 0.49 0.25 0.25 0.11 <0.1 < 0.1 k- after methylation' r (CH2C12) 0.76 0.72 0.72 0.64 0.53 0.32 GLC after methylation (min.) MS-fragment a|ter methylation M " -M*-CH3 13.8/1B0C 286 s 271 1 1S.4/185C 16.2/185C 17.0/200C for 8 min., then temp, raised 5C/ min. 13.7/200C for 8 min., then temp, raised 5C/ min. 17.8/200C for 8 min., then temp, raised 5C/ min. 236 s 271 1 286 s - 316 s 301 1 316 s 301 I 346 s - " M+-C1 M+-CH3-CO 251 m 243 s> - 243 1 251 1 243 1 281 1 273 m - 273 1 - - " M*-CH3-C1 - - - 266 1 - - * +-CI2 186 1 - - 246 1 246 s 276 1 " M+-CH3-CO-CX2 173 1 173 - 203 1 - - ? fT V B O SNOW Beak-Intensities: s* strong, m * medium, l*lov mLidii'Utim. Aftpr methylation all purified metabolites showed in the mass spectra -OCIK-groups indicating that the original metabolites were phenolic derivatives, in both cases a number of isomers had been formed. Isomers only difforud in peak-intensities. All Isolated compounds showed the typical isotopic distribution pattern corresponding to :he number of chlorine atoms in the parent PCB. For 2,4'-dichlorobiphenyl there was no conversion exceeding the introduction of 2 OH-groups per molecule. For the 2,5f2'-trichloroblphenyl we observed a metabolite with thrse OH-groups. Corresponding to its formation the monphydroxy-metabolite with the lowest concentration should be the precursor of the highest concentrated dlhydproxy^compound. Only three monohydroxy-derivatives wore formed although six <rcsp. seven for the trichloroblphenyl) structures are possible. For each PCB a highly poljar tone of 1 and 4! resp. of radioactivity could not be identified. Data of the metabolites are shown in Table 1-2. Comparing the excreted amounts (in % of totally excreted radioactivity) of metabolites formed, the dichlorobi phenyl was mainly excreted as monohydroxy-derivatives, while the trlchlorobiphenyl was mainly eliminated as dihyproxy-compounds (Table 3). TABLE 3 Comparison of Metabolites formed of PCBs by Rhesus Monkeys Me :abolites Dichlorobiphenyl Trichlorobiphenyl Mohohydroxy-metabo11tes Dlnydroxy-metabolltes Trihydroxy-metabolites Polar metabolites, identified 66.6% 32.4% 1t 7.6% B2.2% 6% 4% ft MONS 084133 hAumi >ii.4miinn, -- S lin This following might explain this different behavior. Due to, the additional chlorine atom, the trichlorobiphenyl ix' more lipophilic than the dichlorobiphenyl. The monohyldroxy-motaboliteB react in the same way: the mono* hydroxy-dichloroblphenyl is excreted rapidly* while the witor solubility of the monohydroxy-trichlorobiphenyl is still low. After further hydroxylation the trichlorobiphenyl-mctabolites reach a polarity to be easier eli minated. A dihydroxy-trichloroblphenyl thus should correspond to a monohydroxy-dlchlorobiphenyl as regards speed of elimlnatlon. for higher chlorinated BPS, a higher degree of hydroxylation is necessary and will cause a longer retention time of the compound in the bo* dy, that is, a slow excretion rate. This might explain their accumulation. CONCLUSION Metabolism of PCB with high chlorine content will be dif ficult and proceed slowly. Concerning lower chlorinated biphenyls, however, this study reveals that total degra dation to hydroxylated metabolites and the rapid excre tion of the PCBs investigated in primates indicate that accumulation of lower chlorinated biphenyls at low doses will not occur. REFERENCES BLOCK, W.D., and II.H. CORNISH: J. Biol. Chem. 234, 3301 (1959). DEI BOER, Th. J., and H.J.BACKER: Org. Syn. Coll. 4, 943 (1963). GREB, W., VI. KLEIN, F. COULSTON, L. GOLBERG, and F. KORTEt Chemosphere 2, 143 (1973). CREB, W., W, KLEIN, F. COULSTON, L. GOLBERG, and F. KORTE: Bull. Environ. Contam. Toxicol., In press (1974), HUTZ1NGER, O. et al.t Science 178, 312 (1972). VOIGT, K.D. in H.D. BERGMEIER: Methods of Enzymatic Analysis. Acad. Press 19G5, p. 462. YOSHIMURA, H., and N. YAMAMOTO: Chem. Pharm. Bull.21, 1168 (1973). ' i } i l 476 -n:11 .. ..................... I MOMS 084134