Document 2yokzNG4vbYqR6LaMNaMaEVp

Metabolism of a Polychlorinated Biphenyl (AfocIoi-^ 1254) Mixture in the Kat by D. I.. On ant, \V. E. J. J'im.ui's, ;md D. C. Yii.i.kneuvk Hrsrnrrh l.nlittrntfrif*. lt>"tl tintI Drilf: Dirvrtorate l)r/*arltncni nj ^n/intinl I Itiil'h anti Welfare , TunnrY t I 'tt<1 it r<\ ()l Itntn .V, Ontario Polychlorinated biphenyls (PCR's) have been used in industry since at least 1930. In 1955, von oettingen (1) reviewed the literature on the toxicity of these compounds. Industrial workers have developed lesions of chloracne as a result of leaking vapours of a chlorinated biphenyl (Aroclor*) used as a heat-exchange material (2). Today the problem is not restricted to exposure of industrial workers as polychlorinated biphenyls are widely dispersed in fish and wild life (3-11). Kcstoo et al. (11) found low levels of PC3 (90S, <0.1 mg per kg fat) in a largo number of foodstuffs. They also reported that all 22 samples of human milk analyzed contained PCB residues with 11 samples between 0-0.5 and the other 11 between 0.6-1.0 mg per kg fat. Therefore, there is a 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. 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 TftBLK 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 rats divided into four groups and treated as described in TABLE II. In. Experiments I and II, the rats were housed in air conditioned rooms and were supplied with food and water ad libitum. The rats were killed by decapitation, blood collected, and brain, liver, heart, spleen, kidneys, testes and omental fat removed, weighed and frozen pending analyses. AroclorlR),'Monsanto Company (U.S.A.), registered trade name for polychlorinated biphenyls. ,Rultrlin of Lnvir..nmcnl.l Contaniinjli<-n & Toxic..In,'r. VuL 6, No. 2 lV7],,pubii--lu-J by ^prin^cr-Vrilag Nt-w YorL Inc. DSW 332474 STLCOPCB4079050 TABLE I Dosing regimen for experiment I Group4 1 234 Day 1 Day 2 Day 3 Day 4 Day 8 Day 24 Oil b PCBd Oile Killed -- Oilb PCBd Oil e Oil Killed CClf 4 PCBd f CC14 Killed -- CC1 PCBd f CC14 f CCI4 Killed dbcaCAAEao5c1rnh0*10ogCmrioglCu, lpp42e:ccrmoommnl tlapoicenorierldnksqo,ofivliauledtismomoninlau,liestit2oernrametldso,pf oeWArrarikoslglcty,al.orards1mtr2ai5ni4ins, ,tearadevmd. inboi.srwtae.lrleyd. orally at 500 mg per kg. e f Corn A 1+1 oil, 1 ml CCl4:com per oil kg, administered solution, 1 ml orally. per kg, administered orally. g- TABLE II Dosing regimen for experiment II Group4 1 Group*5 2 Groupd 3 Groupd 4 Nil PCB c CClf 4 ccf 4 3, 8, 12, 16, 20, 24, 28, 32, 36, 40 44, and 47 3, and 8 and PCBC on day 2 Five male rats, wistar strain, av. b.w. 410 g, killed on day 1. "Fifteen male rats, Wistar strain, av. b.w. 440 g, 5 killed on days 8, 12, and 47. CA 500 ng per ml corn oil solution of Aroclor 1254 administered .orally at 500 mg per kg. Tline male rats, Wistar strain, av. b.w. 442 g. eA 1*1 CCl4:com oil solution, 2 ml per kg administered orally on day 1 and 1,ml per kg on other days. 10J DSW 332475 STLCOPCB4079051 Polychlorinated biphenyl analyses. Tissues were blended with 100 ml hexane and SO g anhydrous sodium sulfate for 10 minutes. The extracts were filtered, concentrated to 10 ml, 3 ml of cone. H2SO4 - fuming H2 SO4 (1:1) added and shaken. The hexane solutions wore dried and neutralized with 4 q of anhydrous sodiun sulfate-sodium carbonate (10:1). The solutions were subjected to GLC-EC analyses on a Varian Aerograph Model 600D gas chromatograph, fitted with a coiled 4' x 1/0" O.D. glass column containing Chromosorb W, 80-100 mesh, coated with 4X SS-30 and 6% QF-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 were 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. 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, 438, 464, and 474 ppm. Recoveries of spiked samples were greater than 9575- Lipid was determined by the method of Bligh and Dyer (12). Results and Discussion Experiment I From day 1 to day 4 the rat3 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 which received the oral dose of carbon tetrachloride on day 8 died within the next 24 hours. However, by day 24 the 1 remaining rat in group 4 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 5.320.301 of their body weight. Residues of Aroclor 1254 (expressed as ppm wet tissue, TABLE III) were found in all tissues analyzed, with fat and blood having the greatest and least concentration, respectively. The residues in the blood, testes, liver, kidney and heart were significantly greater in the group 3 rats than in the group 1 rats. 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 as rapidly as rats with normal livers. The residues in the spleen, brain and fat were similar for both groups. The residues in the brain, liver, in 0S\N 332476 STLCOPCB4079052 spleen, blood, tonter,, heart, kidney and fat of group 2 rats (killed on day 24) were 10, 16, 20, 21, 22, 24, 36 and 67S, respectively, of thone of group 1 (killed on day 4). These values show that the PCB residues wore 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 Spleen Kidney Brain Liver Liverd Fat 1.9 6 a 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 5.82* 1. 17 36.60* 4.39 -- 31.14* 2.09 11.20* 1.76 57.3S* 3.91 11.08 39.98* 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 1868.14166.63 -- 6137.64*556.06 -- 996.16 98.58 672.66*155.12 900.46*106.16 1149.05 ?Mean of five values. "Standard error of the mean. ^Single valjue. dPpm on a fat basis. GLC-EC tracings from 24 ng of Aroclor 1254 and from the residuo found in the liver of a group 2 rat, are shown 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. ias DSW 332477 STLCOPCB4079053 ( I 106 DSW 332478 STLCOPCB4079054 F ig u re 1 GLC-EC found i t n r ac th in e gl isv efrro m o f 2" a ng o f group A ro clo r 2 ra t. 1254 (le ft) and from the re sid u t TABLE IV The amount (%) that each of the six major peaks of the Aroclor 1254 standard contribute to the total residue in the tissues Peak number Percent 1 23 4 56 .Percent 1 234 56 Aroclor 1254 12 25 18 17 15 13 12 25 18 17 15 13 Spleen Testes Liver Kidney Fat Brain Blood Heart Spleen Testes Liver Kidney Fat Brain* Blood Heart Group 1 6 21 9 22 21 21 3 15 6 21 27 28 2 11 3 30 28 27 8 21 11 21 19 19 7 25 11 25 17 16 1 13 5 23 30 29 4 13 6 24 26 26 4 17 6 21 26 26 Group 3 7 25 14 19 10 17 6 23 14 19 19 19 4 21 11 21 22 21 8 22 15 18 18 17 10 29 16 21 13 12 6 25 13 20 18 18 5 22 14 22 18 18 5 27 15 18 18 17 Group 2 3 17 2 11 34 32 1 19 2 11 35 32 0 25 2 18 26 28 0 18 2 14 34 32 1 19 4 20 28 28 2 19 4 12 34 29 1 20 3 12 33 31 1 17 2 8 31 29 Group 4 1 20 0 19 1 21 1 20 1 22 2 22 1 20 4 12 31 32 4 20 26 31 4 0 32 34 5 20 26 20 4 11 30 31 3 11 29 33 3 10 32 34 107 DSW 332479 STLCOPCB4079055 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 contents (10) were metabolized to a greater degree than those with the longer retention times. This observation agrees with that reported in studies with Phenochlor DP6* fed to Japanese Quail (7). in group 2, peak 2 accounted for 25, 20, 19 and 19% of the residue in the liver, blood, brain and testes, respectively, whereas in group 1 the percents were 11, 13, 13 and 15%, 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. I 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 Heart Spleen Kidney Brain Liver Fat - 1 1.00 9.81 12.33 14.88 15.89 20.40 59.17 508.24 2 1.00 10.24 13.88 13.86 26.67 9.55 44.88 1601.57 3 1.00 8.62 16.21 9.51 14.90 10.89 206.88 233.89 4 1.00 22.48 24.68 -- 4 4.32 23.84 75.16 4596.20 ; Phenochlor DP6, Prodolec's (France) registered trade name for polychlorinated biphenyls. 10* OS\N 332480 STLCOPCB4079056 However, since blood samples are relatively easy to obtain they could fcc used for estimating the residue in other tissues, providing the investigator is aware of the limitations of the estimates. Experiment T1 In Experiment I no rats received carbon tetrachloride alone, therefore experiment II was conducted to determine whether kroclor 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 is concluded that Aroclor 1254 potentiated the toxicity of carbon tetrachloride in a manner similar to that reported for phenobarbital (13-15) and for DOT (13, 15). Treatment with Aroclor 1254 evoked a number of significant changes in organ weights (TABLE VI), the most 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 4 7 Probability Spleen Liver Heart Kidney Testes nil <0.005a'b nil - nil <0.005*'b <0.05b <0.05* <0.05a AO o & <0.01a <0.005a,b -' Expressed as % body weight, `Expressed as actual organ weight. 109 DSW 332481 STLCOPCB4079057 I .This agrees with results from experiments in this laboratory on rabbits treated witii PCU's, which will be reported elsewhere. More differences were observed on day 12 than on days 8 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 arc presented in TABLE VII. . TABLE VII Percent lipid of rat livers from experiments I and II Treatment Lipid (X) Nil, killed on day 1a Aroclor 1234 on day 2, killed on day 4 Carbon tetrachloride on day 1, killed on day 4 Aroclor 1254 on dayf2, killed on day 8r Aroclor 1254 on day 2, killed on day 121 4.45b0.008c 6.19 0.23 13.22 1.17 8.35 0.99 7.78 0.38 ^Experiment II, group 1. Mean of five values. M cStandard 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 test). Tanaka et al. (16) reported that chlorobiphenyls 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 analyzed for PCB residues (TABLE VIII). no DSW 332*82 STLCOPCB4079058 TABLE VIII Residues in tissues of rats 45 days after being orally administered a single dose of Aroclor 1254 (500 mg/kg) Residue Group Blood Heart Kidney Brain Liver Fat ppm, wet tissue 2 bc 0. 18 0.05 2.71 0.61 3,39 0.29 4.19 0.34 16,04 3.79 397.30 41.86 Relative to Blood 2 1.00 15.06 18.83 23.28 89.11 2207.22 j^Group 2 of Experiment II killed on day 47. Tlean of five values. cStandard error of the mean. The residues in the blood, heart, kidney, brain, liver and fat were 9, 11, 11, 10, 14 and 4051 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 coinponeiyi^was again noted. For example, the amount (X) that each oi^^^hsix major peaks contributed to the total residue in the fat 15, 1, 12, 40 and 34, compared to 12, 25, 18, 17, 15, and 13^^w Aroclor 1254 standard. Male rats were orally dosed with Aroclor 1254 and residues were found in all tissues analyzed, with the greatest concentration in the fat. The GLC-EC pattern of the residues was different from the standard mixture administered, indicating that all components were not metabolized at the same rate. Higher residues were found in the carbon tetrachloride-treated rats. Aroclor 1254 residues in the brain, spleen, blood, testes, heart, kidney and fat were reduced by 90, 84, 80, 79, 78, 76, 64 and 33S respectively in 20 days. Aroclor 1254 significantly increased the size of the liver and also the percent lipid in the liver, Afoqlor 1254 was found to potentiate the toxicity of carbon tetrachloride J.n the rat. m DSW 332483 STLCOPCB4079059 Acknowledgment We acknowledge C\R. Westerby for technical assistance and B.B. Coldwell and K.A. McCully for review of this manuscript. References 1. von Octtinyen, W.F., Tublic Health Service Publication Ho. 41<l, 306 (1955) . 2. Meigs, J.W., Albom, J.J., and Kartin, B.L., J. An. Med. Assoc. 1_54, 1417 (1954) . 3. Risebrough, R.W., Reiche, P., Peakall, D.B., Herman, S.G., and Kirven, M.N., Nature 220, 1090 (1968). 4. Jensen, New Scientist 32^, 612 (1966). 5. Holmes, D.C., Simmons, J.H., Tatton, J.O'G., Nature 216, 227 (1967). ------ 6. Holden A.V., and Marsden, K., Mature 216, 1274 (1967). 7. Koeman, J.H., Ten Hoever De Brauw, M.C., and DeVos, R.H., Nature 221, 1126 (1969). 8. Jensen, S., Johnels, A.G., Olsson, M., and Otterlind, G., Nature 22^, 247 (1969) . 9. Duke, T.W,, Lowe, J.I., and Wilson, A.J., Jr., Bulletin Environmental Contamination and Toxicology !>, 171 (1970). 10. Bagley, G. E., Reichel, W. L., and Cromartio, E., J. Assoc. Offic. Anal. Chem. 53^, 251 (1970). 11. Wcstoo, G., Noren, K., and Andersson, M., (1970). Varfoda 2J2,9 12. Bligh, E.G., and Dyer, W.J., Can. J. Biochcm. and Physiol. 37, 91 1 (1959) . 13. McLean A.E.M. and McLean, E.K., Biochcm. J. 100, 564 (1966). 14. Farbdr, T.M., Heider, A., Peters, E.L., Ritter, D.L., Disraoly, M., and Van Loon, E.J., Ninth Annual Meeting, Society of Toxicology, Atlanta, Georgia, March 15-19 (1970). 15. Cawthorne, M.A., Bunyan, J., Sennitt, M.V., and Green, J., Br. J. Nutr. 24., 357 (1970). 16. Tanaka, X., Fujita, S., Komatsu, F., and Tamura, N., FukuokaZgaku-Zasshi 60., 544 (1969). m DSW 332484 STLCOPCB4079060