Document 4a5GBr9KwDzkQL23yGQ8B2D2e

it rod- ihat tel lor. U. tty on. 974) 12 '"'*"-`J ...:.. .-i*.i:,.1.^.^.j....f _ ,L :..^i ... ,.'. ... --- -uU J>, .^. a^-xrvV Beilriigc zur Okolo^ischcn Chcmie LXXX1V+ Metabolism of Lower Polychlorinated Biplienyls,4C in the Ulicstis Monkey by W. Gum, W. Ki.r.tN, F. Com.ston*,I,. Coi.di.iu;*, and K. Konte Inxlitut fur oliolngitehr Chcmie tier ('.r<rll*ch<\lt fur Strahlen-11ltd Uinn-rltforscituitK iitbll, flliniclicii D S20S Si. /fiigti.Vin /. If'. Germany *lnHi<u<e of Experimental I'athology ami toxicology Albany Medical College, Albany, N.Y. 12200 INTRODUCTION Metabolism studies on PCB are important for estimating | the toxicity of metabolites after knowing their identi: ty and biological pathway. Some pure PCB-isomers were shown to be converted to hydroxylated products by rat 1 liver microsomes {GREB et al. 1974), rabbits {BLOCK I and CORNISH 1959), rats (HUTZINGER et al. 1972, I Y0SH1MURA and YAMAMOTO 1973) and pigeons (YOSHIMURA ' and YAMAMOTO 1973). The purpose of this study was to i identify all major metabolites after PCB-administration | to Rhesus monkeys and to measure the amounts of diffe rent excreted metabolites. Correlation of conversions and elimination pattern of PCBs with different chlorine ' content might lead to conclusions about the behavior of : other PCBs. i The excretion rates of 2,4-dichlorobiphenyl and .1 2,5,2'-trichlorobiphcnyl have been described elsewhere (GREB et al. 1973)? we now wish to report the identifi; cation of their metabolites. I EXPERIMENTAL t ----------------------- The PCBs investigated were 2,4 ' -dichlorobiphenyl and I 2,5,2'-trichlorobiphenyl, both major components.of the t lower AROCLOR series. They were labelled with *C. FemaI le Rhesus monkeys were housed in metabolic cages and ' the PCB was Injected once in the left forearm vein. * Three animals received 2,4'-dichlorobiphenyl in doses i of 16.8, 77.6 and 566/*g/k9# while 82.6/H.g/kg of the | 2,5,2'-trichlorobiphenyl were administered to one mon key. Urine and feces were collected daily. Conjugates were hydrolyzed by refluxing urine with an equal volume . of 8 N H2S04 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 CH-Cl- and then with methanol. The combined extracts were concen- +LXXXIII. Communication: GREB, W., W. Klein, F.COULSTON, L.GOLBERG,and F.KORTE: Bull.Environ.Contam. Toxicol., in press. ' . lull,lift ol En.ltonmti.ta1 Conlominollon A Toiltolo,,. , ( Vol. II, No. 4 lt b, York lor. i MOMS 08226? I trated under a nitrogen stream and purified by prepara tive layer chromatography on silica gel Merck Nr.5765, 5766 (solvent: CH^Cl-)* After methylatlon by dia2omethane (DE BOER and BACKER 1963) the metabolites were characte rized by GLC (Packard 873, EC-detector, 1% OV-1, 2m) and identified by GLC-MS (LKB 9000 A). . RESULTS AND DISCUSSION After 14 days about 77% of the administered radioactivity were 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 the 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 between the first and fifth day after application. Only metaboli tes, no parent compound, were detected in the excreta. Fig. 1 gives a survey of the metabolites formed from both PCBs. ` i" ICl ! i! Fig.1. Metabolism of Polychlorinated Biphenyls by Rhesus Monkeys 472 HUNS 0d226d ) I i I s' s f r * i I 5 C 2 (/) O 03 K t-u a %> ! $ 9 \'__- r s: fht>3 < --a t- -- 3ftH * ? * TABLE 1 TLC -, GLC- and MS-Oata of 2,4'-Dlchlorobiphenyl Metabolites In Monkeys Data OH-Metabo- OK-Metabo- CK-Metabo- (CH),-Meta- (OH),-Ke- lite S1 lite S2 lite S3 bolite bol. Ss :0K)2-Me:abof. Sg R> before inethylation L (CH2C12) R* after methylation * (c:i2ci2) 0.66 0.82 0.50 0.70 . 0.25 0.44 0.13 0.59 < 0.1 0.64 i 0.1 0.52 . GLC after methylation Rt (min.) MS-fragment a|ter methylation M 10.2/179C 12. 1/185C 11. 4/185C 14.8/200C 13.1/200C 11.6/20CC for 9 min. for min. for 8 min., for 8 min. for 8 min., then temp, then tenp. then temp, then temp, then ter.o. raised 5C raised 5C raised 50/ raised 5Q raised 5C/ mfn. min. min. min. min. 252 s 252 S 252 S 282 s 282 s 282 s " M+-CH3 " metastabie " M+-C1 " M+-HC1 " m+-ch3-co 217 1 _ 209 ra 237 1 - 217 m _ 209 S 237 m 223* in - 209 m 267 1 - 247 m 24 6 m 239 m 267 m - 247 m 246 m 239 1 267 m - 239 1 " m+-ch3-ci -- m+-ch3-co-hci 173 1 - ' M+-CH3-C0-C12 - - Peak-Intens!ties: s=strong, m=medium, lIow 202 1 173 m 139 m 204 1 - - - n qp i y i T g lT^. 11>w'" Ff ^ y w '.-F' wi f u 'ipy-wy n y TABLE 2 TLC-, GLC - and MS-Data of 2,5,2*-Trich.lorobiphenyl Metabolites in Monkeys . Data OH-Metabo- OH-Metabo- OH-Metabo- (OH)--Meta .(OH)--Meta (OH)3-Me- lite Gj lite G2 lite Cj bolite g4 bolite g5 tabol. Gg l'A R-- before methylation (CH2C12) 0.49 0.25 0,25 0.11 <0.1 < 0.1 -1 Rf after methylation' (ch2ci2) 0.76 0.72 0.72 0.64 0.53 0.32 - GLC after methylation 13.8/180C 15.4/185C 16.2/185C 17.0/200C 13.7/200C 17.8/200C -1 R. (min.) 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 after ' methylation 286 s 286 s 266 s 316 s 316 s 34 6 s : " M+-CH3 271 1 271 1 - 301 1 301 1 - ; -- "--- ' ............. ~~ * M -Cl 251 m - 251 1 281 1 - - .i .c iA > M+-CH3-CO i M -CHj-Cl + * -C12 243 m - 186 1 243 1 - 243 1 - 273 m 266 1 246 1 273 1 - 246 s 276 1 a " M+-CH3-C0-C1j 173 1 173 - 203 1 - fu -- -' - Peak-Intensities: s= strong, m * medium, l=low 1 v-j 5 U4 l--`-^'-^"- i ' 1 | j . i \ 1 I j I ,, ' . 1 8 H N -,;-.,.77ii:..-..il u.-i.-. ji. .,V, I.,1 mii1....,...^ 'lii, Hi Iii^tfii I .fi.fcl. n'.,. * J|,^` 1 -I&), M ---- >- ' "', After methylation all purified metabolites showed in the mass spectra -OCH3-groups indicating that the original metabolites were phenolic derivatives. In both cases a number of isomers had been formed. Isomers only differed 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 *-dichlorobiphenyl there was no conversion exceeding the introduction of 2 OH-groups per molecule. For the 2,5,2*-trlchlorobiphenyl we observed a metabolite with three OH-groups. Corresponding to its formation the monohydroxy-metabolite with the lowest concentration should be the precursor of the highest concentrated dihydroxy-compound. Only three monohydroxy-derivatives were formed although six (resp. seven for the trichlorobiphenyl) structures are possible. For each PCB a highly polar zone 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 dichlorobiphenyl was mainly excreted as monohydroxy-derlvatives# while the trlchlorobiphenyl was mainly eliminated as dihydroxy-compounds (Table 3). TABLE 3 Comparison of Metabolites formed of PCBs by Rhesus Monkeys Metabolites Dichloro biphenyl Trichlorobiphenyl Monohydroxy-metabolites Dihydroxy-metabolites Trihydroxy-metabolites Polar metabolites, unidentified 66.6% 32.4% 1% 7.8% 62.2% 6% 4% P e a k - In te n s itie s : s-- s tro n g , n * medium, I i i i .1IIIFI.*.* 'V,.'-1.. H" - 475 HONS 082271 II"'. in m u, |. ' t." ' 11^^.----- - -v^ ^^ f -- - - - The following might explain this different behavior. Due to the additional chlorine atom, the trichlorobiphenyl is more lipophilic than the dichlorobiphenyl. The monobydroxy-metabolites react in the same way: the monohydroxy-dichloroblphenyl is excreted rapidly, while the water solubility of the monohydroxy-trichlorobiphenyl 1b still low. After further hydroxylation the trichloroblphenyl-metabolites reach a polarity to be easier eli minated. A dihydroxy-trichlorobiphenyl thus should correspond to a monohydroxy-dichlorobiphcnyl as regards speed 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. I CONCLUSION I 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 tions! "the PCBs investigated in primates indicate that accumulation of lower chlorinated biphenyls at low doses nor'occur,' .................... * " REFERENCES : BLOCK, W.D., and H.H. CORNISH: J. Biol. Chem. 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. KORTE: Chemosphere 2, 143 (1973). GREB, W., W. KLEIN, F. COULSTON, L. GOLBERG, and F. KORTE: Bull. Environ. Contam. Toxicol., in press (1974). HUTZ1NGER, 0. et al.i Science U8, 312 (1972), VOIGT, K.D. in H.D. BERGMEIER; Methods of Enzymatic Analysis. Acad. Press 1965, p. 462. Y0SH2MURA, H., and H. YAMAMOTO: Chem. Pharm. Bull.21, 1168 (1973). nnM^.iMur* 476 IllffyP" HONS QZ2.Z11 inflow--ipi, ii'