Document DM5pe3eDQEE9Z8bMgEgY46Z0n

PCB Metabolism in Kats Following Prolonged Exposure to Aroclor 1242 and Aroclor 1016 by Viulyn W. Buhse Center for iJiteuse Control 3190 Clifton ltd. Atlanta, Georpiu 30333 RoBKnr F. Moseman and G. Wayne Sovocool Pesticides and Toxic Substances Effects Laboratory /VuiionuJ Environmental Heseurch Center Environmental Protection Agency ' Hesearch Triangle Park, North Carolina 27711 and Ellen C. Villanueva The Coca Cola Export Corporation 260 Peachtree, NAP. 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. Two of the most widely studied urinary metabolites, namely DDA, the carboxylic acid metabolite of DDT and the parenitrophenol 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 (JE*!SE:i). 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 -ere not metabolized to any appreciable extent by n&n"alian systems (REYNOLDS, 1969)- However, as early as 1959. BLOCK and CORNISH (1959) reported on the conver sion of biphenyl and U-chlorobiphenyl to raonohydrc/.ylftted 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 (JONDORF, 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. Simi lar f i ndc 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 Bulletin o! r.nvitoumeMal ContammaHwi A TcMlcoiupy. \ ol. i>. No. I 1976 hy Sprlniter-VctUp SY York Inc. MU No 082104 ongcri I 1016 products icator of vo of the ly TDA, t paran used to these parent . 19*3). first reUtssxn). eports coiihighly Id that PCB's nt by man ia early as he ccnverohydroxyl(me) etabolism al. {19TU ) !of individ ual and In ges of indicated were rei the conorkirg with Eft and WONG f Aroclor d aliphatic trated that 'ted signithe PCB m the tisor 1251* by -Gas tissues and ,tlfled. This paper reports on metabolic products observed in rat urine, through Coulson Conductivity-Gas Chroma tography (CC-GC) and combined Gas Chromatography-Mass Spectrometry (GC-MS) analyses, following a prolonged diet of Aroclor 1016 or Aroclor 12h2. EXPERIMENTAL Aroclor 1016 and 121*2 were of electrical grade, lot No. 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 male 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 12U2 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; l, 6, 8 and 10 months after onset of experiment. After 6 months on the diet, some rats were allowed to recover by removal of PCD diet, for tine periods of 2, U and 6 months. Urine was collected at these intervals. A more detailed accounting of the experimental protocol can be found in another publication (3URSE, et al. 197**). The amount of Aroclor 12^2 consumed ranged from 6.6 mg/kg bodyweig.ht/day to 3.89 ng/kg body we i ght/day while Aroclor 10l6 ranged from 6.9 - 3.5 mg/kg bodyveight/day. The twenty-four hour urine samples 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 HCl for three hours and extracted 3 times with 50 ml of benzene. The benzene extract was washed with 20 ml of 5% Na0Hw/v followed by 20 ml water. The aqueous phases were com bined and acidified with 20 ml of 1.2 II HCl. The acidic aqueous phase was extracted 3 tines with 10 ml of benzene and dried over sodium sulfate. To each sample vis added diazomethane (STANLEY, 1966). Each sample in a volume of 0.5 ml was eluted from n micro column containing 3% w/v deactivated silica gel using 10 ml of a 1:1 benzene: hexane mixture. A Microtek-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 Lnm i.d.), packed with 5% 0V-210 on 80/100 mesh Supelcoport, and 3J 0V-1 on 70/80 mesh Chromosorb G were operated at l65 and 170 respectiv ely. Conposited urine sample extracts wore adjusted to 0.5 ml with pesticide grade hexane. Eight microliters 123 HONS 082109 f' 4 S' i! 1(1. ' M- 'SUil 1^; vere injected into a Hewlett Packard 5700 A gao chronato^raph containing a 1.22r. x 2mm i.d. stainless steel column packed with 3lC 0V-1 on 80/100 mesh Cae Chrom Q. The initial column temperature was 150, and the oven was programmed at 2/rain. 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, 250u amps; target current, 220y amps, scan rate 100 amu/sec from k5 - ^50 amu, Ions were detected with a Bendix Con tinuous Dynode Electron Multiplier. Data were acquired, stored and plotted using the Hewlett Packard 5932A Data Gy st en. RESULTS AND DISCUSSION Hecently HUTZINCER and co-workers (1972) demon strated the metabolism of pure chlorobiphenyl isomers in pigeons and rats. The b-chloro-, b , b '-dichloro-, and 2 ,2,5,5'-tctrachlorobiphenyl isomers were metabo lized to monohydroxy compounds. A dihydroxy monochloro metabolite was also reported. GARDNER, et al. (1973) fed 2,5,25'-tetrachlorobiphenyl to rabbits and iden tified 3-hydroxy-2,5 ,2' ,5'-tetrachlorobiphenyl, bhydroxy-2,5,21,51-tetrachlorobiphenyl and trana-3,fr ailly dro-3b-dihydr ox y-25,2' ,5-tetrachlorobiphenyl in urine samples. In this study we have identified at least six hydroxylated biphenyl metabolites (Table I). Five of these compounds were found in both treatment groups. Of the three dihydroxylated metabolites, two were pre viously unreported. These compounds, a diehlorodihydrexy 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 tetrarhlorodihydroxy biphenyl in Aroclor 12U2. 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 hexachlorobenaene which was found in the adipose tissue samples of the experi mental animals. C' C12l C12i C1?K C1SH c12h Ci C6( C6> C6C Q of pos preclu Howeve identi phenyl fragme lsomer sent i chroma At observ* obtain? ;*r fV: Nyy.1|Ml nil^imfjlljyill'l/ PI "Iiyii -1.PIK9 .. ;: . HUNS OttillO Gu*. f"00 A gas chro1, stainless steel :.eh Gaa Chrom Q. :c, and the oven *.* injection port, rparator were kept helium flow rate ::unda transmitted l the Ion source uit spectrometer. re as follows: lectron Impact rent, 250u amps; 10 acu/aec from a Bendix Contta were acquired, arkard 5932A Data :i9T2) demonLphenyl isomors 1-dichloro-, ir* vtri metaboiroxy monochloro , t tl. (19T3) ibbits and lden- iphenyl, h,r.d trans-3,* crobiphenyl in it least six hyI). Five of tsent groups. , two were pre- dichlorodihy, were found In ficent difference vas the absence roclor 12l2. ated chloroben-* of both treat:hesc metabolites tt?n to suspect cbentene which cf the experl- TABLE T PCB Metabolites Found in Urine of Rats Fed AroclOr 10l6 or Aroclor 121.2 (a. Methyl Ether Derivatives) Composition Aroclor Arocicr Molecular Ion (M+ 1 1016 1212 C^2^7C120CH ^ 252 yes yes C12H6C12(CH3)s 282 yes yes c12h6c13och3 c12h5ci3(och3)2 286 316 yes yes y yes C12H5C1i4OCH3 320 yes yes c12hIc1 1* (chT > 2 350 yes no TABLE II Phenolic Metabolites Found in Urine of Rats Fed Aroclor 10l6 or Aroclor l?U2 (as Methyl Ether Derivatives) Composition C^CljOCH^ CgHCl^OCHj CgCl|,(0CH3)2 Molecular Ion cv; 278 2U4 27I* 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 tetrachlorohydroxylated bi phenyl. By analogy, based on CLC 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 nan spectra obtained during GLC separation for each component. In 125 MQNS 0821U it' . S Pi U 6: o 1 o fH wn i o -t fe o ou ob 0 *> <83 P V. u * o *fi -I V. 4* a *o a > V -H :r r-4 tW I >O O *4 . wv; $ :,v W> , Figure 3. Mess spectrum of a methylated dihydroxytetrachlorobiphenyl (M+*350) metabo- lite found in urine of rats fed a diet of Aroclor 1016. a fev instances, overlap of two cr more component* yielded spectra with more than cne molecular ion. The structural assignment of the methoxy derivetives of polychlorinated biphenyls rests upon the following: (l) The presence of intense molecular ions of the correct masses, (2) The correct chlorine ''isotope 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 typical features of ths mass spectra of the methylated ether derivatives of the phenolic PCB metabolites. The intense molecular ion at m/e 350 and four chlorine "isotope cluster" are consistent with the assigned composition of a dinethoxytetrachlorobiphenyl, ^OgCl ^ . Loss of methyl radical leads to the fragment at 335 amu con- taining four chlorines. Loss of methyl radical and of carbon monoxide from the aromatic ring, produces the largest fragment of M+-^3 with four chlorines at 307 erau. The loss of methyl radical and extrusion of carbon nonoxide from aromatic rings is well documented for methoxy and dimethoxy aromatic eihera (BUUZIKIEWTCZ, et al. iy67). 127 HUNS 082113 If R Ev KU- ^ f : ' ^ fc !" , 1 , : ; ; .i. SUMMARY Several mono- and dihydroxy metabolites of di-, tri, and tetrach.Lorobiphenyl have been identified in the urine of rata fed prolonged diet# of Aroclor 10l6 or Aroclor 1242 . Combined gas chromatography-mass .spectrometry was used for characterization of the metabolic products. REFERENCES BLOCK, V/.D. and H.H. CORNISH: J. of Biol. Chem., 234 . 3301, (1959). BUDZIKIEWICZ, H., C. DJERASSI, and D.H. WILLIAMS: Mass Spectrcr.etry of Organic Compounds, San Francisco, Holden-lay, 1967* BURSE, V.W., R.D. KIMBROUGH, E.C. VILLANUEVA, R.W. JENNINGS, R.E. LINDER, and C.W. SQVOCOOL: Arch, of Environ. Health, 29., 301, (1974 ). CURLEY, A., V.W. BURSE, M.E. GRIM, R.W. JENNINGS, and R.E. LINDER: Environ. Res., 4_, 48l, (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. RAOELIE: Biochem. and Biophys. Res. Cor.m., 55., 1377, (1973). GRANT, D.L., W.E.J. PHILLIPS, and D.C. VXLLEUEUVA: Bull, Environ. Contam. Toxicol., 102, (1974). HUTZINUER, 0., D.M. NASH, A.S.W. DEFREITES, R.J. N0R8TR0M, D.J. WILDISH, and V. ZITKO: Science 178, 312, (1972). JENSEN, S.: New Scientist, p. 612, Dec., (1966). JEN3BN, B.: Ambio, 70, (1974). JONDORF, W.R., D.V. PARKE, .and F.T. WILLIAMS: Biochem. Jour. , 6l_, 512, (1955) KAISER, K.L.E., and P.T.S. WONG: Bull. Environ. Contam Toxicol., II,, 291, (1974 )i LAWS, E.R., JR., A. CURLEY, and F.J. BIROS: Arch, of Environ. Health, 1, T66, (1967). REYNOLDS, L.M.: Bull. Environ. Contam. Toxicol., 4^, 128, (1969). ~ STANLEY, C.W.: J. Agr. Food Chem., lU., 321, (1966 ). WEST, H.D. , J.R. LAWSON, I.H. MILLER, and O.R. MATHURA: Arch, of Biochem. and Biophys., 60. 14 , (1956 ). IKS* The original typescript and subject category; if there is should be sent to the Editor Manuscripts should he in I french provided a compielu Papers in this journal am re; essential that manuscripts I 1. Manuscripts should be I paper using an electric should be numbered ligi 2. The typed area on the f space at the* top. In tin aHiliation(s) will be typi affifiation(J should be 1 not excci dinjj x 9 11. 3. 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