Document dQRVvxKwdgKZOj49oqL5Ybdp5

TISSUE MODIFICATIONS IN MONKEYS AS RELATED TO ABSORPTION, DISTRIBUTION, AND EXCRETION OF POLYCHLORINATED BIPHENYLS J. R. A.LLI.N, II. II. NOKHlVCK. ami I. ( . IISU Department of Pnthnhtgy VmverMty of Mst onsln Metliml School nd Regional Primate tieseareh Center Univertity of Wiicmtifn Stadium, WlTconsIn 5J 706 Adult rhesus monkeys were given a single dose of I.S or 3.0 ^ of a poly chlorinated biphenyl (PCB) (Aroctiior 1248) per kg body weight and t;ti.rifii.al four days later. In addition, one monkey from each group was placed in a meu* holism cage and the excreta collected during a two-wck period and ^milyced fut PCB content. Over 90 percent of the single PCB dosage was ahsor^d from the gastrointestinal tract and deposited in various tissues of the body, lamination <>f the PCBs, primarily through the biliary system, occurred at a slow rate. I he monkeys did not become obviously ill following ingestion of the PCBs. but de veloped moderate hepatic enlargement due primarily to an increase in lipid droplet* and proliferation of the endoplasmic reticulum of the hepatic cell*. C.astnhypertropliy and hyperplasia and focal ulceration of the stomach lining were prominent lesions in these animals. The polychlorinated biphenyls (PCBs) have been used extensively for various industrial purposes during tlte past 40 years. Because of their chemical inertness these compounds are highly resistant to degradation in the environment. Through industrial accidents and improper disposal and usage, they have become global environmental contaminants. Ingestion of minute amounts of the PCBs have produced widespread deleterious effects on the wildlife population throughout the world (Holmes et al. 1967; Jensen *r<af. 1969; Koeman et al. 1967, 1969). Industrial accidents leading to ingestion by man have led to serious illness and long-lasting morbidity (Kuratsune et al. 1972). The presently reported study was conducted to determine acute toxic effects on nonhuman primates of PCBs (Aroclor 12481) and to evaluate absorption, tissue levels, and routes of excretion of these compounds following exposure to a single dosage. TIris investigation was supported in part by U. S. Public Health Service grants CS-004^2 and RR-OU167, and the University ofWiscornin Sea Giant. Primate Center Publication No. 13-029. 1 Monsanto Company, St. Louis, Missouri. Archives of Environmental Contamination and Toxicology, Vol. 2, No. I, I 974 01974 by tapnrijtiT-Verla* New York Inc. 86 Tissue Modifications in Monkeys as Related to PCD 87 .Muleriuls and methods Seventeen male rhesus monkeys, ranging in age from two to 2.5 years and weighing between three and 3.5 kg, weie placed :n three groups. The two experimental groups con* fisting of six animals per group we:c given 1.5 or 3.0 g of a PCB (Aroclor 1248) per kg body weight by gastric intubation. The third group of five animals served as controls. All of the experimental animals weic deprived of food for one day prior to intubation. Following the administration o; the PCBs the animals were allowed to eat ad libitum for three days, after which five ai.m.ials ir. cadi experimental group received no food for one day and were subsequently sacrificed. Blood studies including evaluation of white blood cells (WBC), hemoglocm, hematocrit, differential white cell counts, serum glutamic oxalacctlc transaminase (SCOT), serum glutamic pyruvic transaminase (SGPT) (Reitman ind Ftankcl 1957), total protein (Wcrchselbaum 1946), scrum protein electrophoresis (Beckman, 1965), mid blood uica nitrogen (BUN) (Bohoun ef a/. 1968) were done both prior to intubation and before each animal was sacrificed. Prior to death the animals were anesthetized, the jugular vessels severed, and the animals allowed to exsanguinate. Tissues were obtained immediately for microscopic and biochemical studies. For histological evaluations, small sections of the tissues were placed in ten percent neutral buffered formalin for 24 hr. They were subsequently dehydrated, embedded in paraffin, sectioned at five microns, and stained with hematoxylin and eosin (Armed Forces.Institute of Pathology ' 960). Tire hepatic tissues employed in the electron microscopic studies were cut into small cubes and fixed in osmium tetroxide and buffered with veronal acetate (Caulfield 1957) for 15 hr. They were subsequently dehydrated through a graded series of ethanol and em .cddcd in an epoxy resin mixture (Mollenhauer 1964). Sections of the tissues were cut on an uhramicfotomc, placed on uncoated copper grids, stained with uranyl acetate, and examined with an electron microscope. Additional portions of the liver weie homogenized at 0C with two volumes of 0.25 M sucrose will) 0,010 M MgCIj and 0.015 M KC1 (SKM). Levels of protein (Lowry ft al. 1951) and DMA and RNA (Munro and Fleck 1966) were determined on portions of the homogenate. Mierosomcs were isolated by centrifugation at 100,000 G for 90 min of the poumilochondria! supernatant (which was obtained by centrifugation at 9,000 G for 20 mm), washed by homogenization with sucrose and recentrifuged, then resuspended in SKM equal io three volumes of liver and stored at --70C. The activities of aromatic hydroxylase, nitroreductase, A'-dcmcthylasc, esterase, and glucose-6-phosphatase and Concentrations of protein were determined by methods previously reported (Norback and Alien 1972). At the beginning of the experiment one animal from each of the two treatment levels was placed in a metabolism cage and the urine and feces collected prior to intubation and over a two*weck period tlieieaftcr. In addition to the urine and feces from these two animals, the brain, liver, and kidneys were also obtained from all animals and analyzed for content of BCDs. Liver a:iu kidney samples were homogenized in hexane (500 mg; 20 ml); urine was e.-. racial in hexane (10 ml: 15 ml); and feces were desiccated over CaCli for one week, pulverized in a mortar and pestle, and extracted in acetone (1 gm:25 ml). Bunn tissue was extracted in acetone (500 mg:20 ml) (Jennings 1968) and HONS "v;v\v-a.-; -*- #.vr- V* 'A 1 ''ri'J`7'ui`ft*"' -:'''-*'': - BS J. K. Allen et a). all tissue extracts were subsequently evaporated under dry nitrogen with anhydrous sodium sulfate in a 40C water bath. Clcan-up of the extracted material prior to analysis was carried out in a disposable pipe* vnicrocolumn (Curley et ol. 1971) containing silica gel 60 (0.05-0.2 mm) by elution with a 1:1 benzene-hexane mixture. Levels of PCBe were quantitated in a Hewlett-Packard Research (7000) Chromatograph employing a b3Ni electron capture detector. The Pyrex glass column packed with Gas Chrom Q (80-100 ntesli) coaled with two percent Sl>30 was operated at 170C with argon* methane (95%-5%) as carrier gas at a flow rate of 40 cc/min. PCB recovery studies were conducted on control samples of tissue and excreta. The PCBs were added directly to the urine, dried feces, and tissue homogenates and sub sequently handled in a manner similar to that previously described. Results Following the administration of l .5 or 3.0 g per kg of body weight of the PCB mixture, Aroclor 1248, to the experimental monkeys, there were no grosa manifestations offflnea during the subsequent four days and the general appearance of the animals remained unchanged. Body weight of the animals immediately before receiving the PCBs (.V14 kg 0.40 for the higher dosage group; 3.03 kg 0.33 for the lower dosage group) did not differ significantly from the body weight four days after PCB administration (3.02 kg 0.46 for the higher dosage group; 2.97 + 0.28 for (he lower dosage group). The animals that were allowed to survive for 14 days became increasingly anorectic and lethargic and at 14 days weighed 2.7 kg (after receiving 1.5 g PCBs/kg) and 1.8 kg (after receiving 3.0 g PCiis/kg). As shown in Table I the experimental animals had slight but significant decreases in hemoglobin and hematocrit levels. The moderate leukocytosis was attributed primarily to an increase in neutrophils. There was also a slight rise in the level of SCOT, while the levels of SGPT were moderately reduced. The total serum protein level was slightly decreased. Levels of BUN were not modified appreciably. Gross lesions of the external surface of the body or of the internal organs were absent at sacrifice on the fourth day following administration of the PCBs. Microscopic ex amination of the tissues revealed a moderate hypertrophy and hyperplasia of the gastric mucosa and the presence of isolated mucinous cysts within the epithelium of the stomach. In addition, there was an occasional penetration of the inuscularis mucosae and the invasion of the submucosa by isolated glandular elements of the mucosal epithelium. There was also moderate edema of the submucosa of the stomach. These gastric changes were markedly accentuated in the two animals that were housed in metabolism cages and allowed to survive for two weeks. In these animals, the major changes in the stomach were associated with hypertrophy and hyperplasia of the gastric mucosa. There were large collections of gastric mucosa! epithelium that had penetrated the muscularis mucosae forming large cysts and sheets of glandular epithelium Ihruughout the edematous sub* mucosa. In addition, segments of the hyperplastic gastric mucosa were eroded thereby funning distinct ulcers, HONS 071949 TtT i..vs,;. \luj;iicdUons in Monkeys as Relied to PCB fable i. Hematological Changes in Monkeys Four Days l-'olioi' mg PCtt jAroclor 1248* j Administration 89 A.'Ml v s emuiucted lit; (gin';,'.) Iici (';;) WUC ( 1 0J/uni:i) Neutrophils (L) lymphocytes (LA S(iOT (Reit inan-l-ranke! umis/ml) SCtl'T (H e; t ma:i -i : ai: k el units/nil) BUN (iiis',%) IT (gnp;;,) Control11 13.8 0.9 43.1 t 3.2 6.3 1.8 44.5 i 12.0 54.4 t 12.0 43.0 13.3 24.9 6.9 19.8 5.4 7.4 0.6 PCB dosage1' (3g/kg) (I S s/kg) 12.2 39.0 9.4 t 0.6C l.9d 2.4 63.0 16.0 37.0 IS.2 11.9 0.6 37.4 t 1.7* 9.8 3,0 63.0 14.8* 36.0 t 14.6* 69.6 1 1.3= 46.4 t 4.8 18.9 t 3.8 22.6 t 3.8 6.5 i. 0.4d 16.9 19.4 i 6.5 t 3.3* 2.7 0.34 'Monsanto Company, St. Louis, Missouri. ll Values expressed .is mc.m i 1 surnL.id devialion. cOiiferci'ict* with control.-; statistically significant: p < ,005, ^Difference with coniiois statistically significant: p < ,01. `'Difference with conliols statistically significant: p < ,05. The livers of the animals from the two experimental groups were slightly enlarged and comprised 2,8 percent of their body weight while (he control livers were 2.1 percent. UUraMruciurul examination of the hepatic tissue demonstrated that the increase in size of the liver was due primarily (o the proliferation of the smooth endoplasmic reticulum within the cytoplasm of :he hepatic cells. The other cytoplasmic organelles and nuclei were unaltered. The liver homogonalcs of the experimental animals showed an increase in total RNA (Table 11). However, due to the selected proliferation of the cytoplasmic endoplasmic ictteuinm, thaw.*, was a decrease in the DNA content per gram of liver. Small modifications were also apparent in the enzyme activity of the microsomal fraction (Tabic Hi). Thcic we;c significant decreases in glucose-6-phosphatase and esterase. The percent recovery of Aroclor 1 248 that was added to control tissues and samples of feecs and urine was: feces, 98.7 14.3; urine, 83.3 6.3; kidney, 81.1 12.3; brain, 32.2 3,5, and hvci, 94.8 t 4.2. . The ^.cutest pcreenluge of the PC 13s eliminated fiom the body occurred through biliary excretion into the gnMiuimcstina] tract. By the 14th day, 5.60 percent of the miginal dose o! the PC Iks had been eliminated in the urine (0.38 mg) and feces (373 mg) v'vV',` . ,t " i- .1 :v I'M-!-?' ) v? .'y^ "4 1' 4 '-i i '"1 ^V? ... \n -1 v. i y* .* a .V -iw vi W -?{ X.*n' . .v-n A*! /'-v. y y. .V. V :.-r '''C'.vy>y-V* , " .' ' V ; ^ Jy/y -. r y . : '.r- * '.lJ- . 90 J. R. Alien et al. from the animal that had received 3.0 g per kg body weight of PCBs, and S.75 percent (Q 88 mg in the urine and 237 mg in the Teces) of the dose had been eliminated by the monkey that received 1.5 g per kg body weight. The chromatogram of the standard PC* Table II. Biochemical Alterations in the Liver ofMonkeys Given PCBs (Aroclor 1248) Control" PCS dosage3 (J l/k|) (I.Sl/kf) mg protein/mg DNA mg DNA/I 00 mg liver mg RNA/mg DNA 107 28 .346 .032 1.49 .08 107 10 ,266.0I4*> 2.19 .24" 98.8 11.0 .293 1.031c 1.991.21* Values expressed as means 1 1 standard deviation. ^Difference with controls statistically significant: p<.01. <cDifference with controls statistically significant: p .05. Table 111. Hepatic Microsomal Alterations *in Monkeys Given PCBs (Aroclor 1248) Control PCB dosage 3 B/k 1.5 g/k, Aniline hydroxylation (mpimol p-aminophenol/30 min)1* II I t 3.9 10.0 2.0 +13.6 4 0 Af-demcthylation (m/imol formaldehyde/30 min)b 218 36 257 52 289 1 89 Nitroreduction (m^mol p-aminobenzoate/hr)b 16.0 3.3 21.5 8.5 24.3 98 Clucose-6-phosphatase Oimol P04/I5 min)b +1.71 .37 1.13 .18c 1.47 1.18 Esterase (/imol p*nitrophenol/min)b 3.44 .33 1.47 .184 3.37 1.62 4 Values expressed as means I standard deviation. b Per mg microsomal protein. cDifference with controls statistically significant: p < .001. d Difference with controls statistically significant: p < .02. HONS 07195i Tidin' ModiJiCdliujis m Monkeys <n Related to PCB 91 (Aroulor 1248) contatr.inc uumecaus peaks having retention times between 40 and 325 see, differed markedly from the chromatograms of the urinary lesidue obtained on the fourth day winch contained an isolated peak at 120 see, and from that obtained from the urinary residue of the Mth day which contained a major peak at 120 sec (representing 93% of the material) and minor peaks at 280 and 400 see. The chromatogram of the biliary residue contained five major peaks at 45, 80, 160, 300, and 355 sec (Figure 1, D). At the time the animals wc-c sacrificed-on the fourth day, levels of PCBs in the brain and kidney were about half those found in the liver (Table IV), However, in tissues taken fiom the two animals ::.m survived for two weeks there was a marked increase in liver PCBs and a deemed decrease m levels of PCBs in the brain and kidney. Tracings of chromatograms of the liver residues taken at four days and 14 daysaftcr PCB administra tion are depicted in Figure : B and C. At four days there is a greater percentage of isomers and perhaps polar me abolitcs having longer retention times than those found In the 0 0 240 Seconds 0 240 480 Seconds Fig. 1. GLC-liC (racings of A) Aroclor 1 248; (8) the residues found in the liver of a monkey 4 days, and (C) 14 days jftcr PCB administration; and (D) the residue found in (he gallbladder 14 days after PCB administration. ' v'- u y: v.v::.., . ,{-, 92 J. R. Alien ei *1 Table IV. Levels of PCB (Aroclor 1248) in the Tissues of Monkeys* No. of animals 5 5 1 Days after exposure 4 4 14 Dose per kg body wr 1.5 3.0 J .5 Liver m/m 24.9 8.2 52.9 27.7 92.1 Kidney Wl/lm 12.) 4.4b 27.2 18.2 3.3 l 14 3,0 72.5 Values expressed as means i I standard deviation. b Difference with liver value statistically significant: p < .02. 1.8 Hg/|m 16.7 7 : 28.1 7.j No* detectable Not detectabV standard (Figure 1, A). At 14 days, the chromatograms of the hepatic residue contain four major peaks with a greater proportion of (he material having longer retention times than those found in the standard. Discussion These data indicate that a large percentage of the PCBs administered in this experiment is readily absorbed from the gastrointestinal tract and is subsequently slowly eliminated from the body. These findings are in agreement with the percentage absorption of PCBs by rodents recently reported by AJbro and Fishbein (1972). Probably as a result of the PCB metabolism and selective excretions, the chromatograms of the tissue residues and of the body excreta are modified from (he chromatograms of the PCB standard. The residues of the liver samples which contained greater precentages of material with longer retention times suggest selective excretion of compounds having few chlorine atoms with the resultant hepatic residue consisting of a higher percentage of highly chlorinated and more polar compounds. Of the tissues examined for PCB content at four days after administration of the material, the liver contained the highest levels. In the two animals that were allowed to survive for 14 days, the hepatic levels of PCBs were higher thin the hepatic levels at four days, while the kidney and brain levels decreased to very low levels. High levels of PCBs were reported by Grant ct ai (1971) to be present in the rat adipose tissue and it is assumed that the fat depots of the monkeys also contained high concentrations of the PCBs. Ait explanation for the PCB location within the liver at 14 days is that PCBs from other tissues, including that fiom brain, kidney, and adipose tissue, have been mobilized and subsequently selectively taken up by the liver. During the period of reduced food intake following PCB administration, mobilization of lipid tissue containing the hydrophobic PCBs presumably occurred and resulted in the HONS 071953 jm " ifi.e 1 'ie .1", `V>v* .v-- ' A.' Jittu Modifications m Monkeys as Related to PCB 93 release of fatty acuH and PCBs into the circulatory system. The continued exposure of lire liver to the (.--'pounds and a suggested preferential hepatic uptake resulted in the higher hepatic levels observed at 14 days. The hematological changes that occurred in the animals during the four-day examina tion period were inimnul. However, there were indications of slight modifications in the hemoglobin and hematocrit values of the experimental animals. The moderate leuko cytosis and neutrophilia which occurred in this acute study are patterns observed in monkeys exposed to PCBs for longer periods. Monkeys fed lower levels of PCBs for a period of three months were anemic and developed a decided leukocytosis (Allen et aL 1973). The increase in circulating white blood cells may be a response to the gastric irritation that develops in animals following exposure to the PCBs. Although not partlculady severe in the acute animals, ulcerations of the gastric mucosa and subsequent acute inflammatory responses may be sufficient to induce a leukocytosis. In addition, focal hemorrhage in the gastric mucosa of the more chronically affected animals w&s apparent. Hemorrhagic gastric ulceration would also account fora portion of the decrease in hemo globin and hematocrit that occurred in the PCB-intoxicated animals. There may also be an inhibiting effect of the PCBs on the hematopuietic tissue of the hone marrow and l\ mph tissue. The exact mechanism by which the PCBs produce these hypoplastic changes is not dear. Vos and Dc Roij (1972) have shown a decided decrease in antibody forming cells m the lymph nodes of guinea pigs that had been exposed to the PCBs. In addition, there was a decrease in circulating lymphocytes in the PCB-fed animals (Vos 1972). Tire PCBs appear to have minimal immediate deleterious effects on liver function in the nonhuman primate, even when large doses arc given. It has been shown in a number of animal species, including the monkey, that the PCBs are activators of microsomal drug metabolizing enzymes (Norhnck and Allen 1970; Allen and Abrahamson 1972). Degen erative changes in the hepatic cells s.id focal liver necrosis are observed in monkeys that luve been fed high levels of PCBs for extended periods (Allen et al 1973). The lesions that developed in the gastric mucosa are possibly unique to the primate species. In the work that lias been done on rodents (Allen and Abrahamson 1972), rabbits (Vos 1972), dogs (Kephngcr a al. 1971), and other species (Vos and fCoeman 1970), gastric hypertrophy. In pcrplasu, or dysplasia of the mucosa! epithelium have not been recorded. However, in human outbreaks of PCB intoxication, nausea and vomiting, both of which aic consistent with gastric irritation, were a constant observation (Kuratsune 1969). The presently reported research indicates that a single large dose of the PCBs to non human primates is capable of producing injurious effects particularly to the gastric niuensa after short periods of time. Because of the high degree of absorption and sub sequent tissue deposition, as well as the slow elimination from the body, large amounts of the PCBs remain m !.c body for long periods. Evidence suggests they are gradually *' ! 1 ! f j { ! ( yswpwwi ey >w r?9w- -I.'''' HONS 071954 '< yr., - .iv':' ' \ i fgSfil i ;. ' 1 A Si V' ``V : ' \* ' * 94 }. R. Alien et al. mobilized from the tissue stores, sequestered in the liver, and eliminated by biliary ex cretion. Mobilization of the PCBs ensures a continuing exposure of tissues to low iev) PCBs for an extended period. Acknowledgments The authors thank L. J. Abrahamson, L. A. Carstens, and J. M Schefflet for their technical assistance. References Aibro, F. W., and L. Fishbein; Intestinal absorption of poiychloiinated biphenyls in rats. Bull. Environ. Contamin. Toxicol. 8, 26 (1972). .Allen, J. R , and L. J. Abrahamson: Enzymatic changes in the liver of rats fed poly chlorinated biphenyls, triphenyls, and DDT. Proc. Amer. Chem. Soc, Div. Water, Air, and Waste Chem. 12,97 (1972). Allen, J. R., L. J. Abrahamson, and D. H. Norback: Biological effects of chlorinated biphenyls and triphenyls on the subhuman primate. Environ. Rea. 6,344 (1973). Armed Forces Institute of Pathology. Manual of Histologic and Special Staining Techniques. New York, McGraw-Hill (I960). Beckman Model R-l01 Microzone Electrophoresis Cel) Instruction Manual RM-IM-3, (1965). Bohoun, C..J.C. Delarue, and E. Comoy: Direct method for the determination of urea in blood with diacetyl monoxime. Clin. Chim. Acta 18,417 (1968). Caulfield, 3. B.. Effects of varying the vehicle for OtO, in tissue fixation. J. Biophys. Biochem. Cytol. 3, 827(1957). Cu.-ley, A., V. W. Burse, M. E. Grim, R. W. Jennings, and R. E. Linder: Polychlorinated biphenyls: Distribution and storage in body fluids and tissues of Sherman rats. Environ. Res, 4,481 (1971). Grant. D. L.. W, E. J. Phillips, and D. C. Villeneuve: Metabolism of a polychlorinated biphenyl (Aroclor 1254) mixture in the rat. Bull. Environ. Contamin. Toxicol. 6, 102 (1971). Holmes, D. C, J. H Simmons, and J. 0*G. Tatton: Chlorinated hydrocarbons in British wildlife. Nature 216,227 (1967). Jennings. R. W.: Biological sample clean-up for chlorinated hydrocarbon and organo phosphorus analysis using eiectron-capture gas-liquid chromatography. Paper pre sented at Am. Chem. Soc. Meeting, Atlantic City, New Jersey (1968). Jensen, S., A. C. Johnels, M. Olsson, and G. Otterlind: DDT and PCB in marine animals from Swedish waters. Nature 224, 247 (1969). Rcplinger, M. L., 0. E. Fancher, and J. C.Calandia: Toxicological studies with chlorinaied mons 071S55 S i i iii- i i'it ir 'irMrfrm ijfnV'iOT Tissue Modifications in Monkeys as Related to PCB 95 biphenyls. Proceedings NIEHS Polychlorinated Biphenyl Conference, Research Triangle Park, N. C., Dec. 20-21 ()97l). Koeni2n. j. H.. A A. G. Oskrunp, J. Veen, E. Brouwer, J. Rooth, P. Zwart, E. V. D, Broek, and H. Van Genderen: Insecticides as a factor in the mortality of the sandwich tern (Sterna sandv:censis). Moded. Rjjksfac. Landbouwwetenschappen Gent. 32, 841 (1967). Koeman. J. H.. M. C. Ten Nocver De Brauw.and R. H. De Vos: Chlorinated biphenyls in fish, mussels, and birds from the River Rhine and the Netherlands coastal area. Nature 221. J1 26 (1969). Kuratsune, M.: An epidemiologic study on "Yusho'' or chlorobiphenyls poisoning. Fukuoka Acta Medica 60, 513 (1969). Kuratsune, M.. T. Yoshhriura, J. Matsuzaka, and A. Yamaguchi: Epidemiologic study on Yuslio, a poisoning caused by ingestion of rice oil contaminated with a commercial brand of polychlorinated biphenyls. Environ. Health Persp. 1.119 (1972). Lowry, 0. H., N J Rosebrough, A. L. Farr, and R. J. Randall: Protein measurement with Folln phenol reagent. J. BioJ. Chcm. 193, 265 (1951). Mollcnhauer, H. H,: Plastic embedding mixture for use in electron microscopy. Stain Ted). 39. 1J I (1964). Munro, H. M . and A. J Fleck. The determination of nucleic acids. Methods of Biochem. Anal. 14, 115 (1966). Norback, D. H.. and J. R. Allen: Enzymatic and morphologic alterations of hepatic endoplasmic reticulum induced by chlorinated aromatic hydrocarbons. Fed. Proc. 29, 816 (1970). ' Norback, D. H., and J. R. .Alien: Chlorinated tripheny! induced extension of the hepatic endoplasmic reticulum. Proc. Soc. Exp. Biol. Med. 139, 1127 (1972). Redman, S., and S. Ftankcl: A colorimetric method for the determination of serum glutamic uxalacetic and glutamic pyruvic transaminases. Am. J. Clin, Path. 28, 56 (1957). Vos, J. G.; Toxicology of the PCBs for mammals and birds, Environ. Health Persp. 1, IQS (1972). Vos, J. G., ar.d i. M. Kweman: Comparative toxicologic study with polychlorinated bh phenyl m chickens with special reference to porphyria, edema formation, liver necrosis, and tissue residues. Toxicol. Appl. Pharmacol. 17. 656(1970). Vos, J. G.. <tna T. De Roij: Immunosuppressive activity of a polychlorinated biphenyl preparation cn the humoral immune response in guinea pigs. Toxicol. Appl. Pharmacol. 21, 549 (1972). Hcrchselbaum, T. E.: Air accurate and rapid method for the determination of proteins in small amounts uf blood serum and plasma. Am. J. Clin. Path. 28. 56 (1946). Manuscript received July 12, 1973; accepted October 28, 1973 1 i \ .......... .r*;: V: HONS 071956