Document DGg2Jn2dw88r4oYzwZ71ORYga

r ! i-l `'V\V. : ' -i .vS.V >. 7:* ..\Vf .,'',o9 ' ..'.. ' I1 . . V ^ ;$IKv- .. V\:'> ti: '. 1.' / - AVjJ wV* M-r VO ':'v ." - `y-^ASIb ' V* Jrjf. !-'V. ' \ fpB \>'>,;Vi.v'Vu''Av'b>1'- ' * *v> fj : j. Jft?**'** ^ PCI? Metabolism in Hats Following Prolonged Exposure to Aroclor 1242 and Aroclor 1016 by Vuilyn W. Buhsk Center for iJiseuse Control J100 Clifton ltd. Atlanta, Georgia 30333 Robest F. Moseman and G. Wayne Sovocooi, Pesticides and Toxic Substances Effects Laboratory National Eni iranniental Iteseurch Center Environmental Protection Agency Research Triangle Park, lOorih Carolina 27711 and Ellen C. Villanueva The Coca Cola Export Corporation 260 Peachtree, /V.IP. Atlanta, Georgia 30303 Determination of the level of metabolic products present in urine has been utilized as an indicator of type and degree of exposure to chemicals. Tvo of, the most widely studied urinary metabolites, namely DDA , the carboxylic acid metabolite of DDT and the paranitrophenol metabolite of parathion have been used to gain insight into the degree of exposure to these parent compounds (LAWS, et al. 1967; ELLIOTT, et al. i960). Polychlorinated biphenyls (PCB's) were first re ported as environmental contaminants in 1966 (JENSEN). The scientific literature now abounds with reports con cerning the analysis and toxicology of these highly stable and persistent materials. For many years it was generally believed that PCB's were not metabolized to any appreciable extent by manmalian systems (REYNOLDS, 1969). However, as early as 1959, BLOCK and CORNISH (1959) reported on the conver sion of biphenyl and b-chlorobiphenyl to monohydroxylated compounds in the rabbit. WEST, et al. (1956) isolated pure compounds resulting from the metabolism of biphenyl in the rat. Recently JENSEN, et al. (197*0 described the separation and identification of individ ual isomers of PCB's in the technical material and in human adipose tissue. Based on the percentages of various isomers stored in adipose tissue, he indicated that two adjacent unsubstituted carbon atoms were re quired for rapid metabolism. This supported the con tention of previous investigators who were working with chlorobenzenes (JONPORF, et al. 1955). KAISER and WONG (197*0 reported that microbial degradation of Aroclor 12 * 2 resulted in non-chlorinated aromatic and aliphatic products. GRANT and his group (197*0 demonstrated that Aroclor 125** was metabolized in rats. He noted signi ficant differences in GLC peak patterns for the PC3 standard and the PCB which was extracted from -the tis sues. Similar - finds' were reported for Aroclor 125** by CURLEY, et al. (1971) using Electron Capture-Gas Chromatography in their examinations of rat tissues and urine. No metabolites were isolated or identified. 122 Hullcun of KnvJronmental Coniaminailon A Tcmlcolo|:yp Veil. J}, No. I 1976 by Sprinyer-Vcrlag NVw York Inc. DSW 026050 V"''-1 ^ J* T, : '^V,*** t -'v ' ,/ ^ rV- '* . ', ' -* .. ........ * l-'h' V V 'V IT*** c ongcd J016 products icatcr of vo of the ely EGA, e paran used to these parent - 1S 6 0 ) . firs-- r e (JENSEN). eports ecnhighly d that PCB's nt by mans early as he ccnverohydroxyl- (1956 ) etabolism al. (19 7lj ) of i i! d 1 v i dal and in P. e s of indicated were rethe Con or k i r. g with E R and W0HG f Aroclor d aliphatic tratei that ted signithe PCB m the t i s or 12 5 ^ by -Gas tissues and tlfied. h , - > ; This paper reports on metabolic products observed in rat urine, through Coulson Confiuctivity-Gas Chroma tography (CC-GC) and combined Gas Chromatography-Mass Spectrometry (GC-MS) analyses, following a prolonged diet of Aroclor 1016 or Aroclor 1 2 !* 2 . EXPERIMENTAL Aroclor 1016 and 12 li 2 were of electrical grade, lot Ho. K3-06-756 and KB-05-^15, respectively. They were supplied by Monsanto Chemical Company, St Louis, Missouri. The Aroclors were fed in parallel experi ments to rale Sherman strain rats, 6l to 73 days old at a dietary level of 100 ppm each. Twenty-four hour urine was collected from four experimental rats fed Aroclor 12^*2 or Aroclor 1016 and one control under the following schedule: 2 weeks after onset of the exper iment; one and 2 months after onset of experiment; U , 6, 8 ar.d 10 months after onset of experiment. After 6 months on the diet, some rats were allowed to recover by removal of PCB diet, for time periods of 2, t and 6 months. Urine was collected at these intervals. A more detailed accounting of the experimental protocol can be found in another publication (3UP.SE, et al . 1971*). The amount of Aroclor 12t2 consumed ranged from 6.6 mg/kg bodyweig.ht / day to 3.89 mg/kg body we i ght / day while Aroclor 10l6 ranged from 6.9 - 3.5 mg/kg bodyweight/day. The twenty-four hour urine Earnpies were combined from four rats in each experimental group. Total urine volume ranged from 1*0 to 80 ml. The urine was refluxed in an equal volume of concentrated HC1 for three hours and extracted 3 times with 50 ml of benzene. The benzene extract was washed with 20 ml of 3% NaOHu/v followed by 20 ml water. The aqueous phases were com bined and acidified with 20 ml of 1.2 H HC1. The acidic aqueous phase was extracted 3 tines with 10 ml of benzene and dried over sodium sulfate. To each sample was added diazomethane (STANLEY, 1966). Each sample in a volume of 0.5 ml was eluted from a micro column containing 3% u/w deactivated silica-gel using 10 ml of a 1:1 benzene: hexane mixture. A .'-'i crotek --2000 gas chromatograph, equipped with a Coulson Conductivity Detector was used for prelimi nary screening for halogen in the sample extracts. Pyrex glass columns (1.83m X tnn i.d.), packed with 5)5 0V-210 on 80/100 mesh 'Supel coport , and 3% 0V-1 on 70/80 nest Ohroraosorb G were operated at 1650 and 170 respectively. . Composited urine sample extracts were adjusted to 0.5 ml with, pesticide grade hexane. Eight micro], iters 123 OSW 026051 STLCOPCB4010013 f ,.iv. ., >-. . kf.r-.V4 '* , -f\, s, '* Jffl 4:. v;v -X;' ]Cy>n. t 1 m' 'j 1 r;-l <> vere injected into a Hewlett Packard 5700 A gas chromatogruph containing a 1.22r. x 2mm i.d. stainless steel column packed wit!) 3? OV-i on 80/100 mesh Can Chrom Q. The initial column temperature was 150, and the oven was programmed at 20 / m i r. . to 210. The injection, port, transfer line and silicone membrane separator vere kept at 200, 220, and 200 respectively. Helium fiow rate past the membrane was 32 ml/min. Compounds transmitted through the membrane separator entered the ion source of a Hewlett Packard 5930A dodecapole mass spectrometer. Parameters of the mass spectrometer were as follows: ion source, 200; mass filter, 110; electron impact source at 70 eV; filament emission current, 250y amps; target current, 220y amps, scan rate 100 amu/sec from 15 - ^50 amu. Ions were detected with a Bendix Continuous Dynode Electron Multiplier. Data were acquired, stored and plotted using the Hewlett Packard 5932A Data System. RESULTS AND DISCUSSION Recently HUTZINCER and co-workers (1972) demon strated the metabolism of pure chlorobiphenyl isomers in pigeons and rats. The l-chloro- , , 1) '-d i chi oro-, and 2,21,5,5'-tetrachlorobiphenyl isomers were metabo lized to monohydroxy compounds. A dihydroxy monochloro metabolite was also reported. GARDNER, et al. (1973) fed 2,5,21 ,51-tetrachlorobipheny1 to rabbits and iden tified 3-hydroxy-2,5 ,2 1 , 51-tetrachlorobipheny1 , hydroxy-2,5,2 ' , 5 1-tetrachlorobiphenyl and trans-3,1!d1 hydro-3 ,^-dihydroxy-2,5,2',5-tetrachlorobipheny1 in urine samples. In this study we have identified at least six hy droxylated biphenyl metabolites (Table I). Five of these compounds were found in both treatment groups. Of the three dihydroxy1 ated metabolites, two were pre viously unreported. These compounds, a diehlorodihydroxy and a trichlorodihydroxy biphenyl, were found in both groups of animals. The only significant difference noted between the two treatment groups was the absence of a t e t r achl oro di hy dr oxy biphenyl in Aroclor 12l)2. Significant quantities of hydroxylated chloroben zenes were found in the urine extracts of both treat ment groups (Table II). The source of these metabolites has not been determined but there is reason to suspect that they may have arisen from hexachlorobenzene which was found in the adipose tissue samples of the experi mental animals. 124 xr:i V-. A'.- **1 -i V . 7* .r-.; - -TO1 11* - I| | Tli i f I II J v- -w '., w., ..v '..4;.,- ..'-v. ' , -i ' " i-"' -.'iv':-. -s !;- 'V, /V- .< <; ;.v 0SW 026052 J. V .1 -*'** ^12` ^12^12' ' c12," c12b Ci2H -- c6> C6C C of pos preclu Hoveve id en t i phenyl f r a g it. e 1 some sent chroma obs e r obtains ?-00 A gas chroi. stainless steel -esh Gas Chroro Q. and the oven r. jrijectibn port, carator were kept Helium flow rate rounds transmitted i the ion source pass spectrometer. ;re as follovB: lectron impact rent, 250y amps; 10 acu/sec from a Be.ndix Con- ata were acquired, sckard 5932A Data . j 972 ) demon ic henyl Isomers , - 1-aichloro-, srs were met abo. proxy monochloro , et al. (1973) iVbit s and iden- iphenyl, 1*:r. d t r an s - 3 , ^ . zrobiphenyl in tc least six hy1 ) . Five of [tocnt groups. , two were pre- di chlorodihy, were found in ficant difference -as the absence roclor 121)2. seed chlorobencf both treat.hese metabolites i.scn to suspect -cbenzene which cf the experi- ' - V- n'Aiuiiwiifa iiati tin r r, it' -- -/ TABLE I PCB Metabolites Found in Urine of Rats Fed Arocldr 10l6 or Aroc lor i2 U 2 (as Methyl Ether Derivatives) Composition Aroclor Aroclcr Molecular Ion (M+) lqi6 igtg C12H7C12CH3 252 yes ye s c12h6ci2(och3)2 282 ' yes yes c12k6c13och3 286 yes yes C12H5C13(CH3)2 316 yes yes C12H5G1U0CH3 c12h14C1i4(CH3)2 320 350 yea yes yes no TABLE II Phenolic Metabolites Found in Urine of Rats Fed Aroclor 1016 or Aroclor 12l2 (as Methyl Ether Derivatives) Composition CgCljOCH-j CgHClljOCH-j C6Cli,(0CH3)2 Molecular Ion (M + 278 2Uh 271) Quantitative estimation and exact identification of positional isomers of the observed metabolites was precluded because of the lack of authentic standards. However, GARDNER, et al. (1973) established positional identity for a specific tetrachi orohydroxylated bi phenyl. By analogy, based on GLC retention and MS fragmentation data, it appears that more than one isomer of some of the hydroxylated biphenyls were pre sent in these samples. Total ion current reconstructed chromatograms are presented in Figures 1 and 2. Assignment of the molecular formulae of the observed metabolites was based on numerous mass spectra obtained during GLC separation for each component. In 125 DSW 026053 STLCOPCB4010015 --------------------------------------- ----. - v ' \ ' i. r : " - V . i *'. Vi O (/> sc o t tv O- o U1 4> i Figure 1. Reconstructed chromato grams of urine extracts from rats fed a diet of Aroclcr 12l*2. 3% OV-1 column programed from 150 to 210 at 2 per minute. Figure 2. Reconstructed chromatograms of urine extracts from rats fed a diet of Aroclor 1016. 3% OV-1 column pro gramed from 150? to 210 at 2C per minute . t- !T>*-iOUJr+Orraa.(Dt-'-<-t-3 ifOpJO:r>-^p>Ol-- -fOCTP: T -) (I M- c+ 3 O O O M p> cr oorrn) w i- 3 o e >-- ~ -' ~ - - 1 O O W c+ O ~i rtMOCO-iOt-^- C'^cT'O t-- < tn ui n r* *-. <* *<0> m. rp ^ - r * I STLCOPCB4010016 \ O u Ah 4 Ga D 30 r-t OJ 0 V P0) 1 1o >o OH CM oo o o \0 o U"N O H -< a f* o o^ o T3 41 G 0) -P < ecd s o ma X0 O0 ITS H -J B O VJ u <u -< 3 ., 'O G H J sJ a AJ u u n O Q< U P.O CM ) 1 ue r--3 +> s) O u 00 H OJ \ 1 O 4O-> a. : / : 'i 1 ' ' <v** * ' . S.'`' -- ,v , ^ ' 'l- .' ' 1. --*. ,*vi ' *1, *' - ' , . M.l mm .. . II ifi llWi.h*, r^fc-i.W MfcgdUtfisay.'i. . tfr. ,.|r| ^ . > i,migi^ yp im"w y u m ;v^ Figure 3. Mass spectrum of a methylated di hydroxy tet rachl orobi ph enyl (M+=350) metabo lite found in urine of rats fed a diet of Aroclor 1016 . a few instances, overlap of two cr more components yielded spectra with more than one molecular ion. The structural assignment of the methoxy deriva tives of polychlorinated biphenyls rests upon the following: (l) The presence of intense molecular ions of the correct masses, (2) The correct chlorine "iso tope clusters", to establish the number of chlorine substituents and (3) rational fragments, yielding the correct mass ions and appropriate chlorine "isotope clusters" for the fragments. Figure 3 illustrates the typ ical features of the mass spectra of the methylated et her derivatives of the phenolic PCB metabolites. Th e intense molecular ion at n/e 350 and four chlorine "isotope cluster" are consistent with the assigned composition of a dinethoxytetrachlorobiphenyl , C-^i Hi gC^Cl^. Loss of methyl radical lead3 to the fragr. er.t at 335 amu containing four chlorines. Loss of r.ethyl radical and of carbon monoxide from the aroma tic ring, produces the largest fragment of M + - h 3 wi h four chlorines at 307 acu. The loss of methyl rail cal and extrusion of carton monoxide from aromatic rin cs is well documented for r.ethoxy and dimethoxy aromati c ethers (BUDZIK1EWICZ , et al. 1967 ) . 127 DSW 026055 STLCOPCB40 SUMMARY Several mono- and dihydroxy metabolites of di-, tri, and tetrachlorobi phenyl have been identified in the urine of rats fed prolonged diets of Aroclor 10l6 or Aroclor 12142. Combined gas c hroma t og r ephy-mas s spectrometry vas used for characterization of the. metabolic products. REFERENCES ' . BLOCK, VI. D. and H.H. CORNISH: J. of Biol. Chem., 23h , 3 3 C1 , ( 1959 ). BUDZIKIEWICZ, H., C. DJERASSI, and D.H. WILLIAMS: Mass Spectrometry of Organic Compounds, San Francisco, Holden-Cay, 1967. BURSE, V. v;., R.D. KIMBROUGH, E.C. VILLANUEVA, R.W. JENNINGS , R.E. LINDER, and G.W. SOVOCOOL: Arch, of Environ. Health, 29, 301 , (197*4). CURLEY, A., V.W. BURSE, M.E. CRIM, R.W. JENNINGS, and R.E. LINDER: Environ. Res., , L S1 , (1971). ELLIOTT, J.W., K.C. WALKER, A.E. PEHICK, and W.F. DURHAM: J. Agr. Food Chem, 8, 111, (i960). GARDNER, A.M., J.T. CHEN, J.A.G. ROACH, and E.P. BAGELIS: Biochem. and Biophy6. P.es. Comm., 5_5_, 1377, (1973). : GRANT, D.L., W.E.J. PHILLIPS, and D.C. VII1EIJEUVA: Bull, Environ. Contain. Toxicol., 6_, 102, (197*0HUTZINGER, 0., D.M. NASH, A.S.W. DEFREITES, R.J. NORSTROM, D.J. WILDISH, and V. ZITKO: Science 178, 312, (1S 72 ) . JENSEN, S.: New Scientist, p. 6l2, Dec., (1966). JENSEN, S.: Ambio, J3> TO, (1970. JONDORF, W.R., D.V. PARKE, .and R.T. WILLIAMS: Biochem. Jour. , 6^, 512, (1955 ). KAISER, K.L.E., and P.T.S. WONG: Bull, environ. Contam Toxicol., 1_1, 291, (1970LAWS, E.R., JR., A. CURLEY, and F.J. BIROS: Arch, of Environ. Health, 1_5., 7 66, (1967). REYNOLDS, L.M.: Bull. Environ. Contam. Toxicol., 129, (1969). STANLEY, C.W.: J. Agr. Food Chem., l*j_, 321, (1966). WEST, H.D., J.R. LAWSON, I.H. MILLER, and G.R. MATHURA: Arch, of Biochem. and Biophys., 60, lO U956 ). 140 IKE The original typescript anc subject cato;.cry; it there 1 should be sent to 'he Editor Manuscripts should bG in French provided a cumpteh Papers in this journal are rt essential that manuscripts 1. Manuscripts should be paper using an electrr should be numbered lip 2. The typed area on the space at Hie top. In tlr affiliation(s) v/rll be typ affiliation!:) should be not excei.ding 5!,> x h r 3. Tables should be typer text. The word TARLC 4. Figures may he hue dr appropriate places, C. dirc-cliy he I i-v the rev.;' figures should not exo 5. Footnote:, should b.> from the text by ,1 siior' 6. References used in It authors' last namefsl v. authors, nrly the first theses should follow th is cited, the letters "a "This im.-lht d is nrn sp In the Reference Si etc name nf the first autii of publication, follw i the COMi'l.r I FLY CAPi the title c' the rt-rir,:: cals Abstracted, Vo1. ' page, and the ypar 111 0 Examples: MTI.7FP.H Mac.!'' d EDWARF'S. C A . and I' H Books should be chcd will title, edition, place of pub1 Fxample: METCALF, It L.: Organ, London: I nlurscituice l' REPRINTS - -A reprint pi Please return this piompt charges in then rcpioducl Printed in U.S.A j A'r V" OSM 026056 STLCOPCB4010018