Document 5k44KJk9VB6m9Yj6B1O3XNog4

that h1 l r Ccilrage zur Okologischen Chemic LXXXIV* Metabolism of Lower Polychlorinated Biphenyls-"C in the Kliesus Monkey by W. Gbeb, W. Klein, F. Coulston*, L. Colbebc*. and F. Kobte Inttilul fUr okologuctu' Chrmie tfpr Gmnclhchaft fiir Stnhlen- itnd Umvthforxchnng mill, Munchcn >5205 St. Augmtin /, It . Crmmv /ml/tut of Kxperimtntal Pathology and toxicology Albany Medical College, Albany, N.Y. 12208 * INTRODUCTION Metabolism studies on PCB are important for estimating | the toxicity of. metabolites after knowing their identiI ty and biological pathway. Some pure PCB-isomera were shown to be converted to hydroxylated products by rat I liver microsomes (GREB et al. 1974), rabbits (BLOCK and CORNISH 1959) , rats (HUTZINGER et al. 1972, I YOSHIMURA and YAMAMOTO 1973) and pigeons (YOSHIMURA ' and YAMAMOTO 1973). The purpose of this study was to I Identify all major metabolites after PCB-adminlstration ! to Rhesus monkeys and to measure the amounts of diffe' rent excreted metabolites. Correlation of conversions J and elimination pattern of PCBs with different chlorine content might lead to conclusions about the behavior of ! other PCBs. t The excretion rates of 2,4'-dichlorobiphenyl and .! 2,5,2'-trlchloroblphenyl have been described elsewhere (GREB et al. 1973); we now wish to report the identiflcation of their metabolites. EXPERIMENTAL ' ' i. ) i ] . I. The PCBs investigated were 2,4'-dichlorobiphenyl and 2,5,2,-trlchlorobiphenyl, both major components.of the lower AROCLOR series. They were labelled with c. Feme- le Rhesus monkeys were housed in metabolic cages and the PCB was injected once in the left forearm vein. Three animals received 2,4'-dlchloroblphenyl in doses of 16.8, 77.6 and 566/ag/kg, while 02.6>*g/kg of the 2,5,2*-trichlorobiphenyl were administered to one monkey. Urine and feces were collected dally. Conjugates were hydrolysed by refluxing urine with an equal volume of 8 N H~SO. for one hour and extracted with ether. Fe ces were mixed with anhydrous sodium~sulfate and extrac ted for two days in a Soxhlet apparatus with CH2C12 end then with methanol. The combined extracts were concen- ' !. .. |; ,| . . LXXXIII. Communication! GREB, W., W. Klein, F,COULSTON, L.GOLBERG,and F.KORTE: Bull .Environ.Contain. Toxicol., In press. BoUmIb tt VS.U.N*. C--hwImiIm 4 N,VktM. ' HONS 081305 lA '*' rY<<*`.V,yTi'!*+- . tV. , ; .:f<. ' ' "'"r*'--*f,- .. . . , , ... trated undar a nitrogen stream and purifiad by prepara tive layer chromatography on silica gal Merck Nr.5765, 5766(solvent* CH-Cl,). After mathylation by diasomethane <DE BOER and BACkER`1963) the metabolites ware characte * rized by GLC (Packard 873, EC-detector, It OV-lf 2a) and identified by GLC-MS (LK8 9000 A). ,, RESULTS AND DISCUSSION ' After 14 days about 771 of tha administered radioactivity ware recovered,and reaction of urine with Glusulase (Endo Laboratories) according to K.D. VOIGT (1965) showed that about 17% of metabolites were conjugated with sulfuric or glucuronic acid. Incubation of urine and feces in vi * tro with tha original PCBs revealed no formation of meta bolites due to reaction with bacteria from the intestines. , In vivo urinary and fecal metabolites were identical and the metabolic pattern in excreta was constant batwean tha first and fifth day after application. Only metaboli tes, no parant compound, were detected in the excreta. I Pig. 1 gives survey of the metabolites formed from both PCBs. ' i Pig.1. Metabolism of Polychlorinated Biphenyls by Rhesus Monkeys 473 i /, i i i? ---.U -JAUli'iWji' MONS 081306 t. t 1\ -9 ^S^TLC TABLE l GLC- and HS-Data of2,4'-Dlchlorobiphenyl Metabolites In Monkeys Data OH-Metabo- OH-Metabo- CH-Metabo- (CH) --Meta (OH),-Me- lite lite S2 lite S3 bolite S4 bol. S5 R> before nethylation x <ch2ci2) H- alter nethylation 1 (CH2C12) 0.66 o.ft2 0.50 0.70 . 0.25 6.66 0.13 5759 < 0.1 oTT? !0H) 2-Me:abof. Sg < 0.1 o.52 . GLC after nethylation * Rt (min.) MS-fragment after * nethylation M 10.2/179C 12.1/185C 11.4/185C 14.8/2OO0C 13.1/200C 11.6/200C for 9 nin. for 9 nin. for 8 min., for 8 nin. for 8 min., then temp, then temp, then temp, then temp, then temp, * raised 5C raised 5C raised 5C/ raised 5Q raised 5C/ rofn. min. nin. min. min. 252 a 252 s 252 s 282 s 282 s 282 s M+-CH3 " netastable N+-C1 " H+-HC1 " M+-CH3-CO - - 217 1 209 m 237 1 - 217 n _ 209 s 237 m _ _ 209 m 267 1 - 247 m 24 6 m 239 m 267 m - 24 7 m 246 m 239 1 267 m - 239 1 ' M+-CH3-C1 M+-CH3-C0-HC1 173 1 - 202 1 173 m - - - ' M+-CH3-C0-C12 - - 139 n 204 1 - - Peek-Intensities: s*strong, m*nediun, 1-Xow it, ' * * m A 1 ; !1 ^ a J :$ * k -< - - *'4i %; 'j g .. TABLE 2 TLC-# GLC - and MS-Data of 2,5,2,-Trlchlorobiphenyl Metabolites in Monkeys bats . - OB-Metabo- 0H-Met*bo- OH-Ketabo- (OH)--Meta . (OH) --Meta-i (OH)--Me- lite Gx lite G2 lite G3 bolite G4 bolite G$ tabol. Gg R* before methylation 1 *ch2ci2) 0.49 0.25 0.25 o.u <0.1 < 0.1 R. after methylation1 (CH2C12) 0.76 0.72 0.72 0.64 0.53 0.32 GLC after methylation Rt (min.) 13.8/180C 15.n85C 16.2/185C 17,0/200C 13.7/20OC 17.8/2O0C for 8 min., for 8 min., for 8'min. then temp, then temp, then temp, raised 5C/ raised 5C/ raised 5C/ min. min. min. * MS-fragment a|ter methylation M* 286 s 288 a 286 s 316 s 316 s 346 s ' M+-CHJ, M -Cl + M -CHj-CO 271 1 251 m 243 m 271 1 . 243 1 - 251 1 243 1 301 1 281 1 273 m 301 1 - 273 1 - - M+-CHj-Cl - - - 266 1 - '- . * n+-cij 186 1 - - 246 1 246 S 276 1 M+-CH3-C0-C12 173 1 173 - 203 1 - - Peak-Intensities; a* strong, m median, l*low I r 8 0 CT 9 0 SNOW `-----------'-- "*- ---- k: i-.u*. .ti*---ii-tl& ' - " -A 1 _ ;?:> :.`y" * i i After methylation all purified metabolites showed In the ; mass spectra -OCH3-groups indicating that the original I metabolites were phenolic derivatives. In both cases | a number of Isomers had been formed. Isomers only dif- JI fered in peak-intensities. All Isolated compounds showed the typical Isotopic distribution pattern corresponding . to the number of chlorine atoms in the parent PCB. For ' 2,4'-dlchloroblphenyl there was no conversion exceeding v the introduction of 2 OH-groups per molecule. For the 2,5,2'-trichlorobiphenyl we observed a metabolite with | three OH-groups. Corresponding to its formation the t monohydroxy-metabolite with the lowest concentration should be the precursor of the highest concentrated di| hydroxy-compound. Only three monohydroxy-derivatives | were formed although six (resp. seven forthetrichloro; biphenyl) structures are possible.For each PCB a highly I polar zone of 1 and 41 resp. of radioactivity could not ( be identified. Deta 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, 1 while the trichloroblphenyl was mainly eliminated as <31- < hydroxy-compounds (Table 3) s p4 TABLE 3 Comparison of Metabolites formed of PCBs by Rheaua Monkeys I4 1 M.t.bolit., Dichlorobiphenyl Trlchlorobiphenyl a Monohydroxy-metabol1tes 66.61 7.8* , Dlhydroxy-mataboUt.a 32.4* 82.2* Trihydroxy-netabolites - 6t Polar metabolites, 1t 4t l unidentified I .* 475 'yw .'imimwpv!1*1" . ijwpffeswfssymir i MONS 0Sii09 1 <*tfflui& The following might explain this different behavior. Due to the additional chlorine atom, the trichloroblphenyl is more lipophilic than the dichloroblphenyl. The mono hydroxy-metabolites react in the same way: the monohydroxy-dichlorobiphenyl is excreted rapidly, while the water solubility of the monohydroxy-trichlorobiphenyl la still low. After further hydroxylation the trichlorobiphenyl-metabolltes reach a polarity to be easier eli minated. A dihydroxy-trlchloroblpheriyl thus should correspond to a monohydroxy-dichlorobiphenyl as regards apeed of elimination. 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 PC&s Investigated in primates indicate that accumulation of lower chlorinated biphenyls at low doses will not occur. REFERENCES BLOCK, W.D., and H.H. CORNISH: J. Biol. Chcm. 234, 3301 (1959). DE BOER, Th. J., and H.J.BACKER: Org. Syn. Coll. 4, 943 (1963). GREB, W., W. KLEIN, F. COULSTON, L. GOLBERG, and F. FORTE: Chemosphere 2, 143 (1973). GREB, W., H. KLEIN, F. COOLETON, L. GOLBERG, and F. FORTE: Bull. Environ. Contain. Toxicol., in press (1974). HUT22NGER, 0. et al.: Science 178, 312 (1972) VOIGT, K.D. in H.D. BERGMEIER: Methods of Enzymatic Analysis. Acad. Press 1965, p. 462. YOSHZMURA, H., and H. YAMAMOTO: Chem. Phorm. Bull.21, 1166 C1973) MONS OdlilO