Document B48J11Yp7dmDQMD1enNnmdp8

]Wcl:iI)oJisni of : Polychlorinated Piplicnvl (Arodor5' ]2."> 1) Mixture in 1 in*. lull liy D. I.. Chant, \V. K. J. Phillips, :mhI 11. C. Villlni-.iivk Tirwirrll f.nlii,rtihtri-.i. I'n.-il f,inl I>rufi Dimtmntr Dvpniltiiiuit nf \ ufAum/ Ili-tilth mul H rlfurv Tummy'> J'tutiire.Oflimit .1. Ontnriu jroor.'1 Day 1 Polychlorinated biphenyls (PCB's) have been used in induatrv since at least 1930. In 1955, von Oettingen (1) reviewed th literature on the toxicity of these compounds. Industrial worker*', have developed lesions of chloracne as a result or leaking vapours of a chlorinated biphenyl (Aroclor'*0 *) used a a heat-oxchunge material (2) . Today the problem in not restricted to exposure of industrial workers as polychlorinated biphenyls are widely dispersed in fisn and wild life (3-11). I'estoo et al. (11) found low levels of re? (90X, <0.1 mg per kg fat) in a large number of foodstuffs. Th<y also reported that all 22 samples of human milk analysed contained PCB residues with 11 samples between 0-0.5 and tr.p other 11 between 0.6-1.0 mg per kg fat. Therefore, there is i need to study the metabolism, distribution, storage, effect on reproduction, etc., of PCB's in mammals. This paper will report on the metabolism and distribution of Aroclor 1254 in normal and carbon tetrachloride-treated rats. ty ? D.iy 3 Day 11 0 ly o Day 'p.irh oro "Corn oil *A or \\ vjo <"g orally a Corn oil *a ui <:c Experimental Experiment I The distribution of PCB's in various tissues of the rat and the effect of liver damage on their metabolism, was studied in twenty male wistar rats randomized into four groups and treated as described in TABLE I. Experiment II The effect of Aroclor 1254 on the organ weights, fat content of the liver and on the potentiation of the toxicity of carbon tetrachloride was investigated in thirty eight Wistar rntJ divided into four groups and treated as described in TABLE II. In Experiments I and II, the rnt 'were housed in air conditioned rooms and were supplied with food and water The rats were killed by decapitation, blood collected, and brain, livvr, heart, spleen, kidrey3, teoteu and omental removed, weighed and frozen pending analyses. Aroclor^, Monsanto Company (U.S.A.), registered trade name for polychlorinated biphenyls. Troupa 1 Group** 4 r.roup^ I Groupd .* jYivi1 in a. ii.r.cen f'M l.v/5 ' i m ,n-Uy . *A 1H C Jiv ' IIuIIh.o Knii*i**.nt*l (rM.min.iliirf* ( VL Is ,N<. 1?. 1071. putili.lml l.y 102 Nrw Vi.fk liir.. M0NS 08253<! TADLJS I Dosing regimen J.or experiment I Group* 1 23 4 industry i(;W`d tho vlustrial ICKUlt of used aa industrial J in fish 10 Of PCS fa. They analyzed and the rc lc a effect on report normal and Day 1 Day 2 Day 3 Day 4 Day 8 Day 24 Oilb PCBa Oil Killed -- -- Oilb PCBa Oil -- e Oil Killed CC4lf rcBa f CC14 Killed -- ccl 4 rcBd f CC14 -- f CCI4 Killed ?Each group contained five male rats, Wietar strain, av. b.w. 375 g. "Corn oil, 2 ml per kg, administered orally. JjA 1 + 1 cci4:com oil solution, 2 mi per kg, administered orally. "k 500 mg per ml com oil solution of Aroclor 1254, administered orally at 500 mg per kg. Jcorn oil, 1 ml per kg, administered orally. vv 1+1 CCl4:corn oil solution, 1 ml per kg, administered orally. rat nnd .vtudied in i treated content mI cibon Jt.r rats ;>LE II. i in .ilr 1 water .\g c ol J ret.-.'U, `ntal in:. ..o iu:;.o Dosing regimen for experiment It Group* 1 Groupb 2 Groupd 3 Groups 4 Nil PCBc on day 2 CClJ on days 1, 3, 8, 12, 16, 20, 24, 20, 32, 36, 40, 44, and 47 CCif4 on days 1, 3, and 8 and PCBc on day 2 .Vive male rat3, Wistar strain, av. b.w. 410 g, killed on day 1. ^Fifteen m.ile rats, Wist.nr strain, av. b.w. 440 g, 5 killed on diys 8, 12, and 47. 500 ing per ml corn oil solution of Aroclor 1254 administered j.oraily nt 500 mg per kg. Nine male rats, wiotar strain, av. b.w. 442 g. CA 1 + 1 ccl.pcom oil solution, 2 ml per kg administered orally on day 1 and 1 ml per kg on other days. i 101 }\ 1 MQNS 082533 I Polychlorinated hinhynyl anM.vr.nn. Tissues wore blended with 100 ml hexrtJH* and fiO y anhydrous sodium sulfate for 10 minutes. The extracts were filtered, concentrated to 10 ml, 3 ml of cone. - fuming H2 SO.) (1:1) added and shaken. The hexane solutions were dried and neutralized with 4 g of anhydrous sodium sulfate-BO.Uum carbonate (10:1). The solutions were subjected to GLC-JGC analyses on a V.irian Aerograph Model 600D ga3 chromatograph, fitted with a coiled 4` x 1/4" o.D. glass column containing Chromonorb W, 00-100 mesh, coated with 43 S-30 and 63 QK-1. The nitrogen flow rate was 120 ml per minute with column and injection temperatures of 193 and 225C, respectively. Aroclor 1254 standards and test solutions wore chromatographed and the concentration of PCB's in the test solutions determined by comparing the heights of the six major peaks of the standards with corresponding peak heights of the test, solutions. I Apioen, hi'.'Od, (killed on <J respectively, i t values :;how ' various tissue Resit Problems were not encountered with sensitivity during analyses and the lower limit of detection would depend on the tissue being analyzed, hexane and acetone gave equal recoveries of Aroclor residues. Quadruplicate analyses of a rabbit liver gave values of 431, 430, 464, and 474 ppm. Recoveries of spiked samples were greater than 955. Lipid was determined by the method of Bligh and Dyer (12). Results and Discussion Experiment I Prom day 1 to day 4 the rats dosed with corn oil and Aroclor 1254 (groups 1 and 2) or carbon tetrachloride and Aroclor 1254 (groups 3 and 4) lost an average of 19 and 55 g (b. w.), respectively. On day 8, group 2 rats had regained their lost weight, while group 4 rats continued to lose weight. Four of the five rats in group 4 v/hich received the oral doso of carbon tetrachloride on day 8 died within the next 24 hours. However, by day 24 the 1 remaining rat in group u and the five rats in group 2 had gained 20 and an average of 30 g, respectively. The livers of the group 2 rats were enlarged and averaged 5.3 2*0.003 of their body weight. Residues of Aroclor 125 (expressed as ppm wet tissue, TADL III) ware found in all tissues analysed, with fat and UlooJ having the greatest and least concentration, respectively. The residues in the blood, testeo, liver, kidney and heart wore siqnificavvt.ly greater in the group 3 rats than in the group 1 rots. This shows that the liver is the main site of Aroclor 1254 metabolism and rats with carbon tetrachloride damaged livers are not able to metabolize this mixture nu rapidly as rate with normal liver;*. The residues in the spleen, brain and fat were similar tor both groups. The residues in the brain, liver, 101 Group Blood Testes Heart Spleen Kidney | Brain | Liver | Liverd ' Tat 1.' 19.; 24. 29. 31. 39.' 1 15.' 1560, 09 6. Mran of five. v. I"Standard error \ ^Single vain/*. "l`pm on a 1 jV fc GLC-l uv, residue > i,.md 1. The ai.iount. the total zr'i< MONS 08.1534 Hided with minute;*. , of cone. ie hexane up nodi, un >j#ctc:l to 600D cj.in is column 30 and 6**, ,th column lactivoXy. itographod letermined standards during nd on the !covaries bit liver f spiked (12). id Aroclor slor 1254 > (b. w.)r oir lost .ur of the of carbon However, re rats in i*ly. The >.32*0.30* .'O, TA3LB and blood .nd heart the group * Aroclor ;ed 1 Ivors rats with fat were in, liver. blood, testes, heart, kidney and fat of group 2 rats (killed on day 2) were 10, 16, 20, 21, 22, 24, 36 and 67*, respectively, of those of group 1 (killed on day 4). These valnnji show that the TOO residues were being cleared from the various tissues at different rates. TABLE III Residues in tissues of rats orally dosed with Aroclor 1254 (500 mg/kg) Residue found (ppm, wet tissue) Group 1 2 34 Blood Testes Heart 8pleen Kidney Brain Liver Liver** Fat 1. 96a 0.23b 0. 42 * 0.07 3.85 * 0.46 0.25 19.22* 0.59 4. 30* 0.44 33.18* 1.35 5.62 24.16* 2. 84 5.83* 0.53 62.40* 4.37 6.17 29.17* 3.44 31.14* 2.09 5.82* 1.17 11.20* 1.76 36.60* 4.39 57.38* 3.91 -- 11.08 39.9B* 5.91 4.01* 0.31 41.91* 3.30 5.96 115.66* 10. 55 18.85* 1.65 796.47* 64.96 18.79 1869.14*166.63 996.16* 98.58 672.66*155.12 6137.64*556.06 900.46*106.16 -- 1149.05 *f!can of five values. "Standard error of the mean. ^Single value. "Ppm on a fat basis. GI.C-EC tracings from 24 ng of Aroclor 1254 and from the residue found in the liver of a group 2 rat, are nhovn in Figure 1. The amount (%) that each of the six major peaks contribute to the total residue in the tissues is presented in table IV. 10$ i! * i: l! I I! I MGNS 082535 9 MOWS 0 S 2 5 3 6 Figure 1 GLOEC tracings fron 24 ng of Arocler 12S4 (left) and found in the liver of a group 2 rat. the residue \ i / (b aw c H i rr oa Ibxt 3 Ce3* U i K) (ft (J1 s a rbr 3a0c3 3 ft ii i TABLE IV The omount (*) that each of the six major peaks o the Aroclor 1254 standard contribute* to the total residue in the tissues . i- peak number Percent 1 2 3 4 56 Aroclor 1254 12 25 18 17 15 13 spleen Testes Liver Kidney ; Fat 1 Brain Blood 1> Heart 1 - Spleen ;* Testes i . Liver ** Kidney 9 1 Fat | J Brain Blood Heart Group 1 6 21 9 22 21 21 3 15 2 11 6 21 27 28 3 30 28 27 8 21 11 21 19 19 7 25 11 25 17 16 1 13 4 13 5 23 30 29 6 24 26 26 4 17 6 21 26 26 Group 3 7 25 14 19 18 17 6 23 14 19 19 19 4 21 11 21 22 21 8 22 15 IB 10 17 10 29 16 21 13 12 6 25 13 20 18 18 5 22 14 22 18 18 5 27 15 18 16 17 Percent i' I \ 2 3 4 5 -6 j 12 25 18 17 15 13 Group 2 3 17 2 11 34 32 1 ' : ! i ; !' ! 1 19 0 25 0 18 2 11 35 32 2 18 26 2B 2 14 34 32 ii ' i; : i!! 1 19 2 19 1 20 4 20 28 28 4 12 34 29 3 12 33 31 :'i| t; I! 1 17 2 8 31 29 Group 4 ii: i 1 20 0 19 1 21 1 20 1 22 2 22 1 20 4 12 31 32 4 20 26 31 4 a 32 34 5 20 26 28 4 ii 30 31 3 ii 29 33 3 10 32 34 i ,i \i '! l:. ; i i 1! 107 -* - fr ... i; i i! 1h MQNS 062537 Figure 1 and TABLE IV show that the components of the Aroclor 1254 mixture with the shorter retention times, peaks 1, 2 and 3, and presumably with the lowest chlorine content# (10) wv.c metabolized to a greater degree than those with the longer retention times. This observation agrens with that reported in studies with Phonochlor Dl'6* fed to Japanese Quail (7). in group 2, pmk 2 accounted for 25, 20, 19 and 1954 of the residue in the liver, blood, brain and testes, respectively, whereas in group i the percents were 11, 13, 13 and Vj*C, respectively. Thus, the metabolism of this component (peak 2) was very slow between day 4 and 24 or one or more of the other components of the mixture were metabolized to the peak 2 component. The residue pattern in the fat of animals treated with carbon tetrachloride (Group 3) was similar to that of the standard Aroclor 1254 indicating little metabolism. Expression of the Aroclor 1254 residues in the tissues relative to those in blood (TABLE V) shows that the ratios depend on the length of time following the oral dose (kidney, brain, liver and fat, group 1 vs 2) and the metabolic activity of the liver (liver and fat, group 1 vs 3). TABLE V Concentrations of the Aroclor 1254 residues in tissues relative to those in blood Group Blood Testes llenrt Spleen Kidney Brain Liver Fat 1 1.00 9.81 12.33 14.68 15.09 20.40 59.17 508.24 2 1.00 10.24 13.88 13.86 26.67 9. 55 44.68 1601.57 3 1.00 8.62 16.21 9.51 14.90 10.89 206.08 233.89 4 1.00 22.48 24.68 -- 44.32 23.84 75.16 4596.20 Phenochlor DPG, Frodelec's (France) registered trade name for polychlorinated biphenyls. 1M low^ver, sine could to used providing tlv Estimates. Experiment II In Expecii therefore exj ^oclor 1254 and (M alter: Eight of ` receiving th rats in group it is cnnel carbon trtrac phenobarbital Treatment changes in or increase In 1 Signific in orge Day killed Organ Spleen Liver Heart Kidney Testes JlExprecrod as expresr d as MONS 088538 n > Tloi .1 3, 7r in roup the n1 the day :u re i in 3) :ing :uet end in, the liow^ver, since Mood namplou are relatively easy to obtain they could to used tor estimatin'! the residue in other tiumirs, providing the investigator is aware of the limitationu ot the estimates. . gxrcr^msnt 11 In Experiment I no rats received carbon tetrachloride alone, therefore experiment II was conducted to determine whether Arcelor 1254 (a) potentiates the toxicity of carbon tetrachloride and (b) alters the weights of other organs besides the liver. Eight of the 9 rats in group 4 died within 24 hours of receiving the carbon tetrachloride on day 8, while none of the rats in group 3 died up to day 47, when they were killed. Thus, it in concluded that Aroclor 1254 potentiated the toxicity of carbon tetrachloride in a manner similar to that reported for phenobarbital (13-15) and for DDT (13, 15). Treatment with Aroclor 1254 evoked a number of significant changes in organ weights (TABLE VI), the mout consistent being an increase in liver weight. TABLE VI Significant differences (Student Fisher's t test) found in organs weights from rats treated with Aroclor 1254 and untreated rats Day killed Organ 8 12 47 Px*obabil ity Spleen Liver Heart Kidney Testes nil <0.005a'b nil - ^Expressed as % body weight, Lxprcoued as actual organ weight. A O o w XT nil <0.005a'b <0.05 <0.05 <0.01 <0.005'b <0.05b - !i \S ! r !!: i I '! w> MQNS 081539 Thin agrcen with rcnult.n from experiments in this laboratory on rabbitu treated with rcn'n, which will be reported clncwhare. More differences were observed on day 12 than on days 0 and 47. The only organ to significantly decrease in weight was the spleen on day 47. The results of the lipid analyser of some livers from experiments I and II are presented in TABLE VII. TABLE VII Percent lipid of rat livers from experiments I and II Treatment Nil, killed on day 1* Aroclor 1294 on day 2, killed on day 4a Carbon tetrachloride on day 1, killed on day 4 Aroclor 1254 on dayf2, killed on day 6 Aroclor 1254 on day 2, killed on day 121 Lipid (ft) 4.4Sbi0.008c 6.19 0.23 13.22 1.17 8.35 0.99 7.78 0.38 ^Experiment II, group 1. Msan of five values. cStandar<3 error of the mean. Experiment I, group 1. ^Experiment I, group 3. Experiment II, group 2 In experiment II, the lipid content of livers from rats killed 6 and 10 days after receiving the Aroclor were significantly higher than the controls (P<0.005, student Fisher's t tct). Tanaka et al. (1f>) reported that chlorobiphenyli; given orally (0.1 g per kg per day) to rats for four weeks caused loss of body weight, hepatomegaly and marked increase in serum lipid components. As expected, livers from rats which received the carbon tetrachloride and Aroclor 1254 had a very high content of lipid. A number of the tissues from the rats killed on day 47 were analysed for PCD residues (TADLE VIZI). no F<?r.ilu. u admin i-J.fr Residue Group" ppm. Blood Heart Kidney Brain Liver Fat 0. 2. 3. 4 16 397 jVlroup 2 of E ^4ean of five cStandard err The residues were 9, 11, 1 group 1, Ex ratios again following individual Ax amount 1%) total residue 12, 25, 10, 1 Male rats were found concentration difforent froi all component; residua* wort Aroclor 1254 ? kidney and f.v respectively the sire of Aroclor 1254 i tctrachlcrida MGNS 082540 |.ir.itory vh'*i o, |nl <17. eplivn In* from -11 oil 6 higher ^aka et )>i r kg | weight, ta. As carbon lipid. TABLE VIII Residues in tissues of rats 4f> days after being orally administered a single doso of Aroclor 1264 (500 mg/krj) Residue Group-3 Blood Heart Kidney Brain liver Fat ppm, wet tissue 2 bc 0. 18 tO.05 2.71 0.61 3.39 0.29 4.19 0.34 16.0 3.79 397.30 41.86 Relative to Blood 2 1.00 15.06 IB. 83 23.26 89.11 2207.22 ' "Group 2 of Experiment II killed on day 47. *Wan of five values. cStandard error of the mean. The residues in the blood, heart* kidney* brain* liver and fat were 9* 11* 11* 10* 19 and <10% respectively* of those found in group 1* Experiment I rats (TABLE I). The tissue-blood residue ratios again show these values depend on the length of time following dosing. The uneven rate of metabolism of the individual Aroclor components was again noted. For example* the amount (X) that each of the six major peaks contributed to the total residue in the fat was 0* 15* 1* 12, 90 and 39* compared to 12* 25* 18, 17, 15, and 13 for Aroclor 1254 standard. Summary Male rata were orally dosed with Aroclor 1259 and residues were found in all tissues analyzed* with the greatest concentration in the fat. The GLOEC pattern of the residues was different from the standard mixture administered. Indicating that all components ware not metabolized at the same rate. Higher residues were found in the carbon tetrachloride-treated rats. Aroclor 1254 residues in tho brain* spleen* blood, testes, heart, kidney and fat were reduced by 90, 04, 80, 79, 78, 76, 64 and 33X respectively in 20 days. Aroclor 1254 significantly increased tho size of the liver and .also the percent lipid in the liver. Aroclor 1254 wax found to potentiate the toxicity of carbon tetrachloride in the rat. m MONS 082541 Acknowledgment We acknowledge C.R. Uenterby lor technical flHaistnncc ar.c* D.U. Colcwell and i.A. McCully for review of thin manuscript. References 1. von Oettingon, W.F., Public Health Service Publication Ho 414, 306 (1955). * 2. Meigs, J.V\, 7klbom, J.J., and Martin, B.L., J. An. Med Assoc. 254, 1417 (1954). ' 3. Risebrough, R.W., .Rcicho, !., Ponkall, D.R., and Kirven, M.N., Mature 220, 1090 (1968). 4. Jensen, New Scientist 32, 612 (1966). Herman, S.G. 5. Holmes, D.C., Simmons, J.H., Tatton, J.O'G., Nature 216, 227 C. Holden A.V., and Marsdcn, K., Mature 216, 1274 (1967). 7. Koemon, J.ll., Ten Noevcr Dc Urauw, M.C., and DeVos, R.H Mature 221, 1126 (1969). 8. Jensen, S., Johnels, A.G., Olsson, M., and Otterlind, G., Nature 24, 247 (1969). 9. Duke, T.U., Lowe, J.I., and Wilson, A.J., Jr., Bulletin Environmental contamination and Toxicology 5, 171 (1970). 10. Dagley, G. E., Reichel, W. L., and Cromartie, E., J. Assoc. Offic. Anal. Chem. 53, 251 (1970). 11. Westoo, G,, Noron, X., and Andersson, M., Varfoda 22, 9 (1970). 12. Uligh, E.G., and Dyer, W.J., Can. J. Biochem, and Physiol. 37, 911 (1959). 13. McLean A.E.M. and McLean, E.K., Diochcm. J. 100. 564 (1966). 14. Purber, T.M., lleider. A., Peters, E.l.., Ritter, D.L., Di3raeiy, M., and Van Loon, Ninth Annual Meeting, Society of Toxicology, Atlanta, Georgia, March 15-19 (1970). 15. Cr.wthorra, M.A., Dunyan, J., Scnnitt, M.V., and Green, J., Or. J. Nutr. 24, 357 (1970). 16. Tanaka, K, Fujita, S., Komatsu, F., and Tamura, N., FukuokaIgaku-Zaoshi 6, 544 (1969). m CJiror Aro liy D.. Eviduncc of t (PCfl'a) cont these petals have been fo. (2), in seal bitda, thr.lr fish,`and ft at thiu liibc edit.icnt. an toxicity to of this yCB was not ecut laboratory t effects of 1c (Lclostomus we matured spot. Registers 1 Gulf Bret lVli.r<i.lrlinA...l /K,n*J<vr7>l mqns 08254*