Document KEKYd4vkvyaKOv3G519RdMdK

CEC - EPA - WHO PAHIS JUKE 1974 IWTERN/iTIQjVAL SYMPOSIUM - EHVIKOSKEKT AI7I) HEALTH BIOLOGICAL EFFECTS OF THE POLYCHLORINATED BIPHENYLS IN NONMUMAN PRIMATES J.R. ALLKM, L.A. CAHSTKHS, and D.H. NORBACK Department of Pathology, and Regional Primate Research Center University of Wisconsin, Madison, Wisconsin $3706 U.S.A. Summary ' Daily ingestion of polychlorinated biphenyls (PCBs) at doses between 25 ppm and 300 ppm oyer a period of 2 to 3 months caused morbidity and mortality in nonhuman primates (Macaca mulatto). Changes included loss of body weight, alopecia, subcutaneous edema, acne, conjunctivitis, ascites, hydrothorax, gastric mucosal hyperplasia, liver hypertrophy, and bone marrow hypoplasia. Terminally, these animals had decreased hemoglobins, hematocrits, and serum protein, shifts in the serum, albu min to globulin ratios and a neutrophilia. A dose of 2.5 ppm PCB in the diet was sufficient to cause alopecia, focal edema and conjunctive- r tis in monkeys within 2 months. Animals that have survived`the inges tion of 250 to 300 mg of PCB over 2 months maintained levels of PCB in their adipose tissue for periods in excess of 1 year. In addition, offspring of these animals had detectable levels of PCBs in their tis sues. In contrast to the injurious effects experienced by adult nonhunan primates exposed to PCBs, one-month-old infant monkeys that were expo sed to 1 g of PCB for 50 days were essentially free of any of the clini cal, gross, and microscopic alterations of PCB intoxication. These obser vations as well as metabolic studies suggest that the metabolism of the PCBs is important in determining their toxicity. DSW 037677 STLCOPCB4021501 Z" 1. Introduction Although the polychlorinated biphenyls (PCUs) have been used exten .. sivcly for industrial purposes for over 40 years, it has only been during the last decade that they have been regarded as environmental contamin ants (Jensen [1)). These compounds arc extremely stable, not hydro- . lyzc-d by water, acids or alkali, and arc able to withstand temperatures up to 650!' without disintegrating. They have, been used in elastomers 1 adhesives, paints, varnishes, printing inks, putty, and as general . fillers. Since they do r.ot conduct electricity, they have also found' widespread use in electrical equipment such as transformers. Their stability and low vapor pressures make them well suited as lubricants, hydraulic fluids, liquid seals, cutting oils, vacuum'diffusion pump oils, and as vapor suppre*ssnnts and insecticide formulations. The same proper ties that make them ideal for commercial use also enhance their resis tance to degradation in the environment. The magnitude of PCB contamina tion is further exemplified by their presence in coho salmon, milk fat, poultry, eggs, and fish (Kolbyc [2]).. Primarily as a result of food contamination, detectable levels of PCBs were found to be present in over 30%. of randomly sampled inhabitants of the United States (Yobs [3]). Early outbreaks of PCD intoxication were limited to industrial acci dents' where workers reported incidents of chloracne (Jones and Aldcn, hi, Good and J'ensky [3]). * It was not until 1968 that the greater healt)i significance of PCB exposure became known. Following the consum ption of PCB contaminated rice oil by over 1,000 Japanese, diverse symp toms and lesions developed. These persons became nauseated and lethargic, and developed chloracne and subcutaneous edema. Infants born to exposed mothers were small and exhibited discolored skin and had an abnormal eye discharge. Many of the symptoms and lesions that developed during the tacutc phase of the illness have persisted for years (Kuratsune [6], Kuratsunc at aI- [7]). 2. Experimental Studies ... ,. .... - .. 2.1 High Level PCB Exposure. In order to further clarify the bio logical effects of various levels of PCB exposure in primates, a series vof experiments have been conducted. In these experiments, adult Mneaca mulotta monkeys were given diets containing 28, 100, and 300 ppm of a "T f 1 PCB (Aroclor 1248, Monsanto Co., Inc., St. Louis, Missouri, U.S.A.) for periods of 2 to 3 months (Allen and Norbnek [&)> Allen ct ah (9], DSW 037678 STLCOPCB4021502 % [10]). Although each of these dosages produced signs and lesions com patible .with PCB intoxication within one month, the total PCB intake varied from 250 to 400 mg for the 25 ppm group, 0.8 to 1.0 g for the 100 ppm group, and 3.6. to 5.4 g for the 300 ppm group. Mortality occurred in the groups receiving the two larger doses within 2 months and within 4 months on the lower regime. Hematological changes developed gradually during the period of experimentation; however, they were most obvious in the 300 ppm group. These animals showed a decrease in hemoglobin (grams per 100 ml) from 13.0 1.0 to 10.6 0.3 within 12 weeks, and a corres ponding decrease in hematocrit from 39.0 2.0% to 32.0f .1.0%. Although the total white cell count was not appreciably altered, there was a decrease in the number of lymphocytes and a concomitant increase in Hie neutrophil population. Decreases in the level of.serum protein were accompanied by a reduction in the percentage of albumin and increases in the globulin fraction. Reduced levels of serum lipids, cholesterol, and triglycerides accompanied the alterations in scrum protein (Allen et al. [10]). . : A gradual weight loss was recorded throughout the experimental period for all PCB-fed animals.- The 25% decrease in body weight of the 300 ppm group was particularly striking. Following one month of expo sure, there was moderate to marked loss of hair from the head, neck, and back, and edema of the mouth and eyelids. Loss of eyelashes, exces sive lacrjmation, and conjunctivial congestion were also apparent. Small pustules involving hair follicles were particularly obvious around . the mouth and on the checks and neck. - .. , Liver biopsies taken from the animals after a short period of expo sure to PCBs showed a decided increase in the smooth endoplasmic reticu lum of the hepatic cells (Figure 1). Biochemically, the liver homogen ates contained decreased levels of DMA (mg/mg liver) and RNA (mg/mg DXA) and increased levels of protein (mg/mg DXA) and increased activity of microsomal mixed function oxidases. In those animals that were .sacri- . ficcd, the enlarged livers continued to show proliferation of the endo plasmic reticulum. However, instead of the endoplasmic reticulum being distributed throughout the cytoplasm it was arranged in'distinct packets of closely associated membranes (Figure 2), Microsomes prepared from 0 those livers showed a decided decrease in activity of mixed function oxi dases (Allen t^ aK [9]). . ' DSW 037679 STLCOPCB4021503 The most severe pathological changes'recorded at necropsy were loca ted in the.'Stomach where marked hypertrophy of the mucosal lining and edema.of the stomach wall, particularly of the fundic and pyloric regions, were apparent. Microscopically, the hypertrophic, gastric mucosa was several times thicker than the control mucosa and was comprised of . greatly elongated hyperplastic glands containing mucous secreting cells (figures 3 and 4). Prevention of discharges of the secretions owing to- the-depth and apposition of the glands predisposed to the development of large mucous cysts. There was also widespread penetration of the muscu- laris niucosaewith invasion of the submucosa by the mucosal epithelium. There were microscopic lcsi.ons of the skin, particularly of the face, of PCB-cxposed animals. Numerous intrafoilicular cysts surrounded by edema tous leukocyte-filled connective tissue were apparent. ' , At necropsy a portion of the tissues from the animals was. obtained for'the determination of PCB levels by the previously reported gas chroma tographic procedures (Allen et fd., [9]). Sites of major storage, in addition to fat, were the adrenals, liver, and pancreas. . . The persistence of the PCBs in the tissues of exposed animals was demonstrated in the group receiving 25 ppm PCBs in the diet for 2 months (total intake approximately 300 mg). Immediately after the PCB-supple mented diet was discontinued, the subcutaneous adipose tissue contained an-average of 127 yg per g fat (Allen et_ aK (11]). Even after one year, concentrations of PCBs in the fatty tissue averaged 30 vg per g fat. Additional distribution data were obtained from a PCB-fed female that delivered an infant eight months after being taken off the PCB diet. Levels of PCB (yg/g tissue) in the adipose tissue of-this infant were approximately one-half that present in the tissues of the mother, thus indicating transplacental movement of these compounds (Allen et aj_., . 2.2 Low Level PCB Exposure. Recently,'experiments have been initi ated to determine if levels of PCBs permitted in fopds destined .for human consumption would be toxic when consumed on'a continuous basis. Adult rhesus monkeys are'being given diets containing 2..5 and 5.0 ppm PCBs (Aroclor 1248). These levels are one-half and equal to the levels per mitted in certain foods. Interestingly enough, these animals developed loss'of hair and eyelashes within 2 to 3 months. They also have swollen upper eyelids and congested conjunctiva. Dsw 037680 STLCOPCB4021504 2.3 Infant Studies. In order to determine if adult and infant pri mates wolild respond similarly to JO exposure, onc-nonth-old rlicsus non hoys were given 1 g (35 mg/kg body weight) of PCB over a 30 day period. TJic total consumption of PCDs by these infants was essentially the same as that received by the adults given 100 ppm in tlie diet as mentioned previously. These animals were able to survive without any obvious ill effects at doses tliat had been fatal to adults (Abrahamson and Allen, (12)). When the tissues from the infant and adult monkeys were evaluated chromatograpliically it was establislicd that tlic concentration of PCB was lower and fewer isomers were present in tlic adult tissues. Those isomers tlint were present in infants and absent in the adult tissues consisted primarily of those having lower chlorine levels (Table 1). ... 2.4 .Metabolic Studies. Metabolic studies were conducted in adult rhesus monkeys that had been given a single intragastric dose of a PCB containing multiple isomers (Aroclor 1248) and a single isomer (2,5,2', S'-tetrachlorobiphcnyl). It was demonstrated that approximately 90% o.f the compound was absorbed from the gastrointestinal tract. Ten percent of the original dose was'delected chromatographically in tlic urine and feces within 14 days with the greatest percentage being excreted between the second and eighth day. Mien similar studies were conducted witli tritiated 2,5,2*,5'-tetrachlorobiplicnyl, approximately 85% of the radio activity was recovered, primarily in the feces within 2 weeks, and major ity of it was eliminated by 72 liours. An additional 10% was detected in the animal tissues. Of the PCB that remained in the pnimal, the . largest percentage was present in the adipose tissue and secondly in organs having high fat content. , --r; , ; -. .. . .. 3. Discussion .. . _ .. Mien nonhuman primates were exposed to levels of PCBs similar to tliat experienced by tlic Japanese in the 1968 PCB outbreak, they deve loped signs and lesions similar to those recorded in human cases. Sub cutaneous edema, particularly around tlic eyes and lips, and hypercr.iic conjunctivitis witJi excessive secretion of the Meibomian glands were "consistently present. There were also follicular accentuations of the skin characterized by dilatation of tlic sebaceous ducts and development of keratin cysts. The severely affected patients and primates liad a decrease in cry.tlirocytcs, reduced hemoglobin, and a noutropliilia. Although not documented in the exposed humans, tlic nausea and anorexia DSW 0 3 7 6 8 1 STLCOPCB4021505 -6- % they experienced was suggestive of a gastritis ns was the case in the experimental animals. Liver hypertrophy, proliferation of the hepatic endoplasmic reticulum, and increased microsomal enzyme activity were apparent in-both species. In addition, the persistence of these lesions for extended periods following the discontinuation of PCB exposure was recorded in the Japanese outbreak and has also been observed in experi mentally exposed nonhuman primates. . Tho data and observations cited in this report clearly indicate that PCBs are toxic to primates over a wide dose range. Particularly signi ficant is the appearance of lesions within less than 2 months in monkeys that were fed diets containing 2.5 and 5.0 ppm. Although the intake of PCBs by these animals was on a continuous basis and the likelihood of the general population consuming a diet containing this quantity of PCBs is small, it does point out that only small'amounts of these compounds are required to produce toxicity in primates. In addition, since PCBs accu mulate in tissues of exposed mammals, continuous exposure to even very minute amounts may eventually reach a level sufficient to cause toxic manifestations. .* It is of interest that the toxicity of PCBs in infant and adult monkeys is markedly different. Levels of exposure that were fatal to adults produced no gross effects in one-month-old infants. It lias been shown that infant monkeys have a poorly developed mixed function oxidase system (Allen and Chesnc.y [13]) .These, observations are further substantiated by the chromatographic data obtained on the tissues cf the two age groups of animals. Adult tissues contained lower levels and fewer isomers of PCBs than did the infant tissues, thus indicating more rapid metabolism or excretion of these compounds by older animals. : These data suggest that PCB metabolites are responsible for the dele terious effects produced by PCBs in monkeys. ' ' Unlike primates, infants of lower animals suffered much more severely from PCB exposure than did adults (McLaughlin ext aK [Id], Keplinger ct al. [15], Dahlstrom [16], Hays and Risebrough [17]; Ringer ct_ al_. [18], PeLong et al. [19]). Clarification of these findings was' recently o' bt.ained in rats by the use of liver enzyme inhibi*tors1 (S*KP S25-A -- p-dimethyl .uninoethyldiphcnylpropylactatc and chloramphenicol) and indu cers (phenobarbita1). In these experiments, rats pretreated with enzyme .inhibitors, which may parallel the enzymatic status of the infant liver, DSW 0 3 7 6 8 2 STLCOPCB4021506 ~7 ~ experienced heavy mortality while animals pretreated with enzyme inducers suffered no deaths. Thus it would `appear that the metabolism of PCBs may be necessary for toxicity in monkeys while the reverse may be true in rats. Many of the lesions that develop in PCB intoxicated primates may be directly related to tissue exposure to the PCBs while other lesions may be secondary. The loss of weight, decrease in scrum protein and modcrato anemia could be related to the reduced food intake that occurs in the animals with severe hyperplastic gastritis .which develops as a result .' t of the irritating effects of the PCBs on the gastric mucosa. The hyper trophic hyperactive livers that develop in these animals arc a result of the stimulatory effects of these compounds on the hepatic endoplasmic reticulum. However, when the liver exposure to the PCBs is sufficiently great, degenerative changes develop particularly in the organelle con taining the majority of PCBs, the'endoplasmic reticulum. The skin lesions may also be directly related to localization of high levels of PCBs in their tissue. Keratinization of the sebaceous gland ducts and hair follicles following the irritating exposure to PCBs leads to foliation of keratin cysts. In addition, edema and inflammation that develops around the effected glands and hair follie!cs'may be a direct result of injurious effects on these tissues by the PCBs. . Many of the lesions that develop following PCB exposure may result .secondarily from the increased metabolism of endogenous substances by thet hyperactive hepatic endoplasmic reticulum. Fo* llowing , PCB exposure there is enhanced metabolism of steroid and steroid-like compounds. Vitamin A which is steroid-like compositionally is deficient in quail, rats (Bitman et al. [20]) and monkeys (Allen, unpublished observations) following PCB exposure. Since vitamin A deficiencies resulting from an increased rate of metabolic decomposition h^ve been reported to produce gastric hyperplasia (Cramer [21]), gastric carcinoma (Fujimaki [22]), an follicular dermatitis (Mason [23]), it is not unlikely that a deficiency of this vitamin may be responsible at least in part for many of .the lesions resulting from P.CB intoxication. . DSW 037683 Acknov/lcd penents ' _. Thic research was supported by the University of Wisconsin Sea Grant and United States Public Health Service grants ES-00'72 and KR-00167 from .the national Institutes of Health. Primate Center Publication Ho. 1J-057. STLCOPCB4021507 3M M t References . .;. .;> 1. JENSrEN, S., "Report of a new chemical hazard", New Scicnt., 32, 612 ^,:-./(l%6). ' ' . . , ... .. . . t, , , 2. 'KOLRYE, A.C., "Food exposures to polychlorinated biphenyls", F.nviron. Heal tli Persp. , 1, 85 (1972). . . .. ; . * ... , .. . . 3. '* YOBS, A.R., "Levels of polychlorinated biphenyls in adipose tissue ; . of -the general population of the nation", Environ. Health Persp., 1, 79 (1972). ... . ....... ' ' `: "' t'' 4. - JONES, J.W., ALDEN, H.S., "An acneform dermatergosis", Arclv. Dermatol. Syphilol., 33, 1022 (1956). 5. -GOOD, C.K., PENSKY, N., "Ilalownx acne (cable rash); cutaneous erup tions in marine electricians due to certain chlorinated naphthalenes v'and diphenyls"* Arch. Dermatol. Syphi. lol., 48, 251 (1943). ' 6. KURATSUNE, M., "An epidemiologic study on "Yusho" or chlorobiphenyl '' "poisoning"', Fukuoka Acta Medico, 60, 513 (1969). . 7. KURATSUNE, M., YOSI11MURA, T.# MATSUZAKA, J., YUMAGUCI1I, A., "Epidemiologic study of Yusho, a poisoning caused by rice oil con ' laminated with a commercial brand of polychlorinated biphenyls", Environ. Health Persp., 1, 119 (1972). . 8. ALLEN, J.R., NORBACK, D.H. , '.'Polychlorinated biphenyl and triphenyl- induced gastric mucosal hyperplasia in primates", Science, 179, 4SS (1973). . ... 9. ALLEN, J.R., A15 RAM.AMS ON, L. J., NORBACK, D.H., "Biological effects of polychlorinated biphenyls and triphenyls on the subhuman primate, . Environ. Res., 6, *344 (1973). . .. . 10. ALLEN, J.R., CARSTENS, L.A., BARS0TT1, D.A., "Pathological changes in nonhuman primates exposed to low levels of polychlorinated biphenyls", ' Toxicol, Appl. Pharmacol., accepted (1974). ;_ 11, ALLEN; J.R., NORBACK, D.H., HSU, I.C., "Tissue modifications in mon keys as related to absorption, distribution and excretion of poly- 'chlorinated biphenyls", Arch.' Environ.' Contain. Toxicol., in press' ' (1974). ...... ; . ; .. . . 12. ABRAI1AMS0N, L.J,, ALLEN, J.'R,, "The biological response of infant nonhuman primates to a polychlorinated biphenyl1', Environ. Health Persp., 2, 81 (1973). *. '. 13. ALLEN, J.R., CHESNEY, C.F., "Effect of age on the development of cor pulmonale in nonhumnn primates following pyrrolizidine alkaloid intoxication", Exp. Molcc, Path., 17, 220 (1972). DSW 037684 STLCOPCB4021508 -9- 14. MCLAUGHLIN, J., MAULIAC, J.P., VERRDTT, M.J., MUTCHLEU, M.K., " pITZHUpll, 0.11. , "The inject ion'of chemicals into the yolk sac of fertile eggs prior to incubations as a toxicity test", Toxicol. Appl. Pharmacol., 5, 760 (1963). 15. KEPUNGER, M.L., FANCHER, O.E., CA1.ANDRA, J.C., K1IEELER, E.P., "Toxi cological studies with polychlorinated biphenyls", Proceedings of . 11 (IS Polychlorinated biphenyl Meeting,, December 20-21 (1971). 5A1H.STROM, N., as quoted-by TINKER, J., "The PC!) study: seagulls aren't funny anymore", New Scicnt.Science J., 16 (1971). * 17. HAYS, H., RISEBUOUGH, R.W., In Natural History, as Reported in ("Controversy continues over PCBs", Chem. ling. News 49, 32 (1971). 18. RINGER, R.K.,.AULERICI1, R.J., ZABIK, M.,"Effect of dietary poly- chlorinated biphenyls on growth and reproduction in mink", Proc. Amer. Chem. Soc., 12, 149 (1972). . 19, DELONG, R.L., GI1.MARTIN, W.G., SIMPSON, J.G., "Premature births in California sea lions: association with high organochlorine pollutant levels", Science, 181, 1168 (1973). ' 20. PITMAN, J., CECIL, H.S., HARRIS, S.J., "Biological effects of poly , chlorinated biphenyls in rats and quail", Environ. Health Perse., 1, 145 (1972). -. 21. CRAMER, W., "Papillomatosis in the forestomach of the rat and its bearing on the work of fibiger," Amer. J. Cancer, 31, 537 (1957). 22, PUJIMAKI, Y.f "formation of gastric carcinoma in albino rats fed on deficient diets", J. Cancer Res, 10, 469 (1926), 23. MASON, K.E., "Effects of vitamin A-deficiency in human beings", In The Vitamins, W.H. Sobrel, Jr., R.H. Harris (eds.), Academic Press, New York, 1, 137 (1954). ' ' , - t DSW 037685 , STLCOPCB4021509 - 10 - TABLE 1. CHROMATOGRAPHIC DATA ON ADI POST. TISSUE OBTAINED PROM RHESUS MONKEYS EXPOSED TO ABOCLOR 124 8a Retention Time ` Standard (Sec) Mother^ Newborn Infant0 Adultd ' Infantc 57/ 73 82 102 130 146 156 ' 201 236 306 . 392 472 ` 507 0.12 2.85 4.07 18.45 6.91 1.52* ' 1.30 40.12 2.86 10.61 7.65 . . .3.46 0.03 7.70 ' 1.56 0.17 ' . . .' * ' . * *' * ' . *' ' " 14.04 '; ' . ; / . < . " . . .. 2.80 ` .... ' * 14.27 N, 10.49 7.58 0.30 50.96 20.14 . * ,f ' - 19.33 47.56 '. 't 7.93 11.34 . 63.52 . '11.63 4.14 15.57 3.92. . 76.79 . 0.47 7.17 .i i a Monsanto Co. Inc., St, Louis, Missouri U.S.A. ' k Tissue taken from female monkey at time of cesarian section, 8 months .after discontinuation of PCB diet (total PCB intake - 300 g). c Infant tissue taken immediately following cesarian delivery, d Tissues of adult monkey taken after ! month on PCB diet (total PCB intake - 1 g). ' 0 Tissue from infant monkey taken after 1 month on PCB diet (total PCD intake - 1 g). * `' . . DSW 037686 STLCOPCB4021510 -n - FIGURE LEGENDS 4 FIGURE 1. Jlcpatocyt.es obtained from a liver biopsy of a monkey that had received 300 ppm PCD in the diet for 2 weeks. Note the abundant smooth endoplasmic reticulum (SER) and short segments of rough endoplasmic reticulum (RER). Normal appearing mitochondria (M) are randomly dispersed among the HR, Small lipid droplets (L) appear as round lucent areas in the cytoplasm. X 8,420. . : : ' - t FIGURE 2. Large dense packets of smooth endoplasmic reticulum (SER) pre vail in the cytoplasm of hepotocyt.es obtained from monkey exposed to 300 ppm PCD in the diet for.2 months. Microsomcs prepared from liver cells having similar morphological features were hypoactive enzymatically. Abundant lipid droplets prevail in the cytoplasm not occupied by ER. The mitochondria (M) appear normal. X 6,920. FIGURE 5. The gastric mucosa of a normal adult rhesus monkey is depicted. Note the narrow band of muscularis. muco.sac(M) and underlying submucosa (SM) and smooth muscle (S) components o'f the stomach wall; hematoxylin and cosin stain. X 25-. ' . . FIGURE 4. Stomach of a monkey that had received 300 ppm PCD'in the diet for 2 months. Note the hyperplastic gastric mucosa and presence of dilated glands. The majority of the submucosa lias been replaced by glandular elements of the mucosal epithelium. Note the large cystic spac.es that have resulted from the stasis of mucous in Chese glands (C); hematoxylin and cosin stain. X 25. .. n il i > .i- --------- - t n f - n i i t l - I . :- OSW 037687 1 STLCOPCB4021511 1 i i ! 4 'I 1 1 DSW 037688 STLCOPCB4021512 I V '.* * ' r ' . V.x*.^ fcV.V. </. ` V.`V V ' v V>?t, -sr' VC/( !.: ' ?,( i^'sV V.v, ' f i ,ya - .,'V'r " vV' * ( r~* > v. .'; .v. j v\ppp- km:-pp 'A / DSW 037689 V \ `VT tyr S' l STLCOPCB4021513