Document 9JpkBeqyvNLpzqoOYqpRj5o7

Response of the nonhuman primate to polychlorinated biphenyl exposure1 J. R. ALLEN Regional Primate Research Center, University of Wisconsin Medical School, and Food Research Institute, University of Wisconsin, Madison, Wisconsin 53706 The polychlorinated biphenyls (PCBs) have been used extensively for industrial purposes during the past 40 years. These compounds are extremely stable, not hydrolyzed by water, acid or alkali, and are able to withstand temperatures up to 650 C without disintegrating. These prop erties make them ideal for use in adhesives, paints, varnishes, printing inks, and as general fillers. Since they do not conduct electricity, they have found widespread use in elec trical equipment such as transform ers. However, it was only during the past decade that the health signifi cance of these compounds in man and lower animals has been brought to public attention. Prior to. this time only workers exposed to the PCBs during the production process (15) or workers who were in direct con tact with manufactured products con taining PCBs (10) occasionally de veloped a skin rash on exposed poruons of the body. It was not un til 1966 that Jensen (14) reported the presence of PCBs in the tissues of wildlife suffering from what was thought to be DDT intoxication. As a result of these findings other investigators began to evaluate the magnitude of environmental con tamination of this newly discovered pollutant. The public health significance of the PCBs was brought to the fore front when over 1,000 Japanese con sumed rice oil that had been con taminated with a PCB mixture. In this outbreak the exposed persons developed chloracne and subcutane ous edema. Infants born to exposed mothers were small, exhibited dis colored skin and had an abnormal eye discharge. Many of the symp toms and lesions that developed in those persons exposed to the PCBs have persisted (17). The properties that make the PCBs ideal for commercial usage also en hance their resistance to degradation in the environment. The magnitude of PCB contamination is exemplified by their presence in coho salmon, milk fat, poultry, eggs and fish (16). Detectable levels of PCBs are also present in over 30% of randomly sampled inhabitants of the United States (22). In the following presentation a de tailed description of the biological responses of nonhuman primates to various levels of PCBs will be pre sented, thus establishing this animal species as an ideal model for eval uating PCB intoxication in man. Data are also presented which show that levels of PCBs presently permitted in some foods destined for human consumption will produce ill effects in nonhuman primates within a rela tively short period. Effects on nonhuman primates of high level exposure to PCBs When adult Macaca mulatto monkeys were given diets containing 100 and 300 ppm of a polychlorinated bi phenyl (Aroclor 1248, Monsanto, St. Louis, Mo.) for periods ranging from 2 to 3 months, they experienced ex treme morbidity within 1 month and mortality approaching 100% within 3 months. Although the signs and lesions that developed in these ani mals became apparent equally as soon in those receiving the lower dosage of PCBs, the total intake varied from 0.8 to 1.0 g for the 100 ppm group to 3.6 to 5.4 g for the 300 ppm group (5). There was a gradual weight loss experienced by all of the PCBfed animals, with the 300 ppm group having a 25% decrease in oody weight during the 3-month period of ex posure. Within 3 weeks to 1 month the animals had lost a majority oftheir hair from the face, head, and neck, and the mouth and eyelids were edematous (6). In addition, there was a loss of eyelashes, excessive lacrimation, and conjunctival congestion. Small comedones were particularly obvious around the mouth and on the cheeks and neck (Fig. 1). These animals also showed a gradual decrease in hemoglobin and hematocrit. Although the total white cell count was not altered appreciably, there was a decrease in the number of circulating lymphocytes and a con comitant increase in neutrophils. De creases in the serum proteins were related to a reduction in the level of albumin. Reduced total serum lipids, cholesterol, and triglycerides accompanied the altered serum pro tein (I). The major microscopic changes in the skin of the affected animals were the development of large intrafollicular keratin cysts and epithelial hyperplasia of the hair follicles par ticularly of the face with the eye lids being most severely affected (Fig. 2). In addition, in many instances the tissue surrounding these affected hair follicles was edematous and con tained acute inflammatory cells (1,6). There were also major modifications in the morphological features of the stomach, particularly in the fundic and pyloric regions (Fig. 3). The markedly thickened gastric mucosa contained numerous large cysts filled with mucin and lined by epithelium. Penetration of the muscularis mucosa into the underlying submucosa by the glandular epithelium was a common feature of these affected stomachs. Cystic areas similar to those present in the mucosa were larger and more abundant in the submucosa. In addi tion, ulcerations of the gastric mucosa developed in areas of eroded epi thelium and where large mucinous cysts had ruptured (6). This hyper plastic gastritis has persisted in mon- 1 Supported in pan by Public Health grants RR-00167, ES-00472 and CA-13288 from the National Institutes of Health, and the Uni versity of Wisconsin Sea Grant Program. Primate Center Publication no. 14-026. Abbreviations: PCB, polychlorinated bi phenyl: TCB, 2,5,2',5'-tclrachlorobiphenyl. RESEARCH activities at regional primate centers 1675 DSW 0 2 5 8 6 7 STLCOPCB4009822 Figure 1. Facial edema is depicted in a monkey given 100 ppm Aroclor 1248 for 1 month. Swollen lips and eyelids and absence of eye lashes are common features of these animals. Smatl comedones are discernible on the upper lip, nose and forehead. keys for over 1 year following the discontinuation of PCB exposure (5). Ingestion of the PCBs by monkeys caused a decided hypertrophy of the liver which in many instances amounted to over a twofold increase Figure 2. Large keratin cysts (C) in the hair follicles are common on the face of monkeys exposed to PCBs. Tissue from animal given 100 ppm Aroclor 1248 for 3 months. Hema toxylin and eosin stain; x60. in size. Electron microscopically, the in the endoplasmic reticulum have hepatocytes of these livers showed a been designated by Huiterer et al. decided increase in smooth endo (13) as hyperplastic, hypoactive endo plasmic reticulum (Fig. 4). In addi plasmic reticulum. tion, there were segments of endo At necropsy, tissues from these plasmic reticulum that formed elabo rate concentric membrane arrays. Terminally, the livers of these animals continued to have enlarged hepato cytes. However, there was a rear rangement of the proliferated smooth endoplasmic reticulum in these cells. Instead of being distributed through out the cytoplasm it was arranged animals were analyzed by gas liquid chromatography for their PCB con tent. The major storage site for the PCBs was in the adipose tissue and to a lesser extent in the liver, adrenals, and pancreas. Regardless of the site of deposition the higher chlorine isomers predominated in the tissues (Fig. 6). in well circumscribed packets of closely associated membranes (Fig. Low level PCB exposure 5) (11Liver biopsies taken from these ani mals during the course of the experi ments showed a decided proliferation of the smooth endoplasmic reticulum of the hepatic cells. Biochemically, the liver homogenates contained a decreased level of DNA (mg/g liver) and RNA (mg/mg DNA) and in Adult female rhesus monkeys have been fed diets containing 2.5, 5.0 and 25.0 ppm of Aroclor 1248 over periods ranging from 2 months for the higher PCB level to 1 year for the two lower doses (4, 8). Animals on the 25 ppm dose developed facial edema, alopecia and acne within 1 month, and 1 of 6 animals had ex creased activity of microsomal mixed pired as a result of PCB intoxica function oxidases. Terminally, as the tion 2 months after having been re morphological features of the pro moved from the experiment diet. The liferated endoplasmic reticulum were total intake of PCBs during the ex modified there was a decided de periment ranged from 250 to 400 mg crease in the activity of the mixed per animal. As was the case with function oxidases (1). Such changes the higher doses, these animals de- Figure 3. a) The morphological features of the normal gastric mucosa (M) of the monkey are depicted. The muscularis mucosa is intact and glandular elements in the submucosa (S) are absent. Hematoxylin and eosin stain; X28. b) Hypertrophy and hyperplasia involving primarily the mucous secreting cells of the gastric mucosa (M) developed in monkeys fed diets containing Aroclor 1248 ranging from 25 to 300 ppm for 2 to 3 months. Penetration of the muscularis mucosa by the glandular epithelial elements and disruption of the underlying submucosa is a common feature of these affected stomachs. 1676 FEDERATION PROCEEDINGS VOL. 34, NO. 8 JULY 1975 STLCOPCB4009823 Figure 4. Hepatocyieofa monkey that had received 100 ppm Aroclor 1248 inlhe diet for 1 month. Abundant smooth endoplasmic reticulum (ER) and concentric membrane arrays (CMAs) develop in the cytoplasm of these cells. Large lysosomes (L) (acid phosphatase positive) containing numerous large lucent vacuoles arc dispersed throughout the cytoplasm. Uranyl acetate stain; X2970. veloped anemia, hypoproleinemia, bone marrow hypoplasia and severe hypertrophic hyperplastic gastritis. PCB concentrations in samples of sub cutaneous adipose tissue obtained from the animals averaged 127 ^tg/g fat at the time the experimental diet was discontinued. Eight months later the PCB content of the adipose tis sue had decreased to 34 /zg/g fat. However, the surviving animals con tinued to show clinical signs and lesions of PCB intoxication 2 years following PCB exposure. Infants born to these PCB-fed females were small and contained detectable levels of PCB in their tissues (Table 1). When adult rhesus monkeys were fed diets containing PCB equal to and one-half that permitted in some foods destined for human consumption (2.5 and 5.0 ppm) for I year, the females of the group developed periorbital edema, alopecia, erythema and acneform lesions that involved the face and neck within 1 to 2 months. Even though the males consumed niore PCB, they exhibited only moderate periorbital edema and erythema. During the course of the experiment the females on the higher PCB dose consumed 364 mg while those on the tower dose ingested 182 mg. The males which received only the higher dosage consumed 383 mg of PCB. After having established a relatively steady tissue level of PCB at 6 months, the females were bred to control males. Following 3 matings 12.5% of the 5 ppm group and 37.5% of the 2.5 ppm group was pregnant as compared to 90% in the control group. However, the conception rate of females bred to PCB-fed males was equally as great as that in females bred to control males. Throughout the course of the experiment the fe males that consumed the PCBs had higher levels of urinary ketosteroids and a decided increase in the length of menses and amount of menstrual bleeding (8). Absorption, metabolism, tissue deposition and excretion of PCB When adult rhesus monkeys were given a single oral dose of Aroclor 1248 and the urine and feces eval uated chromaiographically for 14 days, over 90% of the compound was absorbed from the gastrointes tinal tract. Ten percent of the original dose was detected in the excreta within 14 days with the greatest per centage occurring between the second and eighth day (7). When a tritiated PCB isomer (2,5,2',5'-tetrachlorobiphenyl) (TCB) was given to monkeys, less than 5% of the tritium had been eliminated in the excreta within 72 hours (12). Analysis of the ex creta indicated that direct hydroxylation was not a major metabolic path way for TCB in the monkey as is the case in the rat (20). Instead, hydroxylation occurred through the arene oxide intermediate (12). The TCB which remained in the body of these animals was primarily associated with the tissue protein and nucleic acids and serum albumin ofthe blood. In addition, the analysis of hexane extracts of liver and skin homog enates revealed that over 95% of the TCB present was in an unmetab olized form (12). When phenobarbital and SKF-525A were given to adult monkeys to increase and decrease their hepatic microsomal enzyme ac tivities respectively, the response of these animals to TCB was decidedly different. The animals pretreated with SKF 525A died within 48 hours on a dose of TCB that produced no obvious ill effects in animals that had been pretreated with pheno barbetal (8). COMMENTS In experiments that have been con ducted to determine the effects of polychlorinated biphenyls on non human primates it has been estab lished that the signs and lesions that appear in these animals are similar to those that occur in man exposed to similar levels of PCBs. Acne, sub cutaneous edema, particularly about the face, and conjunctivitis along with excessive secretion of the mei bomian glands were constantly pres ent in PCB exposed man and lower primates (6, 17). The more severely affected animals have a decrease in erythrocytes, reduced hemoglobin and a leukocytosis. The most de bilitating lesions in the monkeys were the severe gastric mucosal hyper plasia and ulceration. Whether simi- RESEARCH activities at regional primate centers 1677 OSH 0 2 5 8 6 9 STLCOPCB4009824 Figure 6. GLC-EC tracing of Arodor 1248 (lower); the residue in the Fat of a monkey given a diet containing 25 ppm Aroclor 1248 for 2 months (upper). posure to PCB, they succumbed rapidly. Such data suggest that the PCBs and not their metabolites are responsible for the acute toxic effects of the PCBs. Figure 5. A very fine network of smooth surfaced membranes (SM) is present in the It has recently been shown that cytoplasm of hepatocytes from monkeys fed Arodor 1248 in the diet for 3 months. Note rats were able to survive for a period the vesicular appearance of the remaining portions of the endoplasmic reticulum (ER). Con of one year on doses of PCB (100 trast these membranes with those of Fig. 4. Uranyl acetate stain; X5930. lar changes occur in the stomach of man exposed to PCBs has not been established. However, the nausea and anorexia they experienced was sug gestive of gastritis. Liver hyper trophy, proliferation of the hepatic endoplasmic reticulum and increased microsomal enzyme activity were con sistent hepatic changes in both species (1,11). Altered ketosteroid levels, dys menorrhea, reduced birth weights of infants born to exposed mothers and transplacental movement of PCBs have also been recorded. Of major significance is the persistence of lesions in both species for years fol lowing exposure to PCBs which may be related in part to the continued presence of detectable levels of PCB in the tissues of those exposed (4, 19). The previously presented data indi cate that the PCBs are toxic to pri mates over a wide dose range. It is particularly significant that monkeys develop signs of PCB intoxication within 1 to 2 months at doses as low as 2.5 and 5.0 ppm in the diet. Although the possibility of man consuming a diet containing these concentrations of PCB on a continuous basis is remote, these data do point out the fact that only small amounts of these compounds are required to produce toxicity and that a safe level has not yet been established. Since PCBs are known to accumulate in the tissues of exposed animals, continuous exosure to even minute quantities may e sufficient to eventually cause toxic effects. Indications are that the toxicity of PCB is directly related to the ability of the animal to metabolize and ex crete it (3, and Allen and Van Miller, ms in preparation). This has been demonstrated in rats and monkeys by the use of drugs that inhibit and in crease the activity of hepatic micro somal enzymes. Animals that had been pretreated with phenobarbital prior to PCB administration suffered no ill effects from doses of PCB that under control conditions would have been very toxic. However, when the animals were given SKF 525A during the initial 24 hours of ex- TABLE 1. PCB content of tissues obtained from mother and infant monkey following consumption of Aroclor 1248" by mother prior to pregnancy* Organ Mother Liver Fat Placenta DSW Weight, gm PCB content, fig/gm tissue 025870 56.3 50.0 0.9 Infant Fat Adrenals Muscle Stomach Small intestine Skin Brain Lung Kidney Large intestine Liver 0.2 52.9 6.1 2.8 11.9 27.70 24.40 0.98 0.55 0.52 0.31 0.29 0.21 0.10 0.08 0.01 * Monsanto Co., Inc., St. Louis. Mo. * Adult on diet containing 25 ppm PCB for 2 months and sub* sequenlly placed on a control diet for 2.5 months before breeding. 1678 FEDERATION PROCEEDINGS VOL. 34, NO. 6 JULY 1975 STLCOPCB4009825 ppm in'tlie dki) lhal were letha! t.o particularly in the organelle con monkeys- within 2 to 3 months (2, 6). taining the highest level of PCB, An explanation for the variation in the endoplasmic reticulum (1). The species response is associated with the skin lesions may also be directly re rate at which they metabolize PCB. lated to the presence of high levels of In the rat the half-life of the PCB PCB. It has been shown that following isomer 2,5,2',5'-tetrachlorobiphenyl exposure a considerable amount of was less than 24 hours (21) due to PCB accumulates in the skin and its rapid metabolism and excretion. underlying tissue and persists in this However, the half-life of 2,5,2',5'- area for an indefinite period (4). letrachlorobiphenyl in the monkey Many of the injurious effects of is much longer (Allen and Van Miller, PCB intoxication may result sec ms in preparation), primarily due to ondarily from the rapid metabolism its slow metabolism. This difference of endogenous substances by the in the rate of metabolism of 2,5,2',5'- hyperactive endoplasmic reticulum. letrachlorobiphenyl in the rat and Following PCB exposure there is an monkey may be related to the dif increased metabolism of steroids and ference in metabolic pathways. There steroid-like compounds. These al are data which indicate that most of tered steroid hormone levels may be the PCBs undergo direct hydroxyla- responsible for the inability of PCB- tion in the rat (21) while an arene fed primates to conceive (8). De oxide intermediate is formed prior to creased levels of vitamin A as have hydroxylation in the monkey (12). been reported to occur in quail, The presence of arene oxide as rats (9) and monkeys (D. A. Barsotti, an intermediate metabolite of PCB M. H. Zilc and Allen, unpublished in the primate broadens the scope of observations) could also be at least in possible injurious effects that may be part responsible for the follicular experienced following PCB exposure. dermatitis that occurs in these The arene oxides have been shown to animals. be strong alkylating agents capable of producing sufficient changes in the A majority of the data presented in this proteins and nucleic acids to cause death or neoplastic transformations in affected cells (18). These cell modi fications may be rather subtle and paper were obtained through the efforts of my colleagues in the Experimental Pathology Laboratory: L. J. Abrahamson, D. A. Barsoui, L. A. Carstens, I. C. Hsu, R. J. Marlar, D. H. Norback, j. P. Van Miller. require years to be manifested. Therefore, continuous low level ex REFERENCES posure to PCBs may be insufficient to produce obvious signs of PCB in 1. Allen, J. R., L. J. Abrahamson and toxication yet the dose may be ade quate to cause macromolecular alterations. Many of the lesions that develop in PCB intoxicated primates are D. H. NORBACK. Biological effects of polychlorinated biphenyls and triphenyls on the subhuman primate. Environ. Res. 6: 344, 1973. 2. Allen, J. R., L. A. Carstens and L. J. Abrahamson. Responses of rats directly related to the deposition of the compound in the affected tissue while other changes may be sec ondary. The loss of weight, decrease exposed to polychlorinated biphenyls for fifty-two weeks. I. Comparison of tissue levels of PCB and biological changes. Arch. Environ. Conlam. Toxicol. In press. in scrum protein and moderate anemia were likely related to the re duced food intake that occurred in the animals with hyperptastic gastritis, the latter having developed due to the 3. Allen, J. R., L. A. Carstens, L. J. Abrahamson and R. J. Marlar. Response of rats and nonhuman pri mates to 2,5,2',5'-letrachlorobiphenyI. Environ. Res. In press. 4. Allen, J. R.. L. A. Carstens and D. a. irritating effect that the PCB had on the gastric mucosa. The hypertrophic hyperactive livers that developed in PCB exposed animals were a result of the stimulatory effect they had on the hepatic endoplasmic reticulum and their enzymes. However, when the exposure to PCB is sufficiently great, degenerative changes develop Barsotti. Residual effects of short term, low level exposure of nonhuman primates to polychlorinated bi phenyls. Toxicol. Appl. Pharmacol. 30: 440, 1974. 5. Allen, J. R,, L. A. Carstens and D. H. Norback. Biological effects of the polychlorinated biphenyls in non human primates. Proceedings of the International Symposium on Recent Advances in the Assessment of (he Health Effects of Environmental Pollution, World Health Organization. In press. 6. Allen, J. R,, and D, A. Norback. Poly chlorinated biphenyl anti rriphenjl induced mucosal hyperplasia in pri mates. Science 179: 498, 1973. 7. ALLEN, J. K,, D. H. NORBACK AND I. C. HSU. Tissue modifications in monkeys as related to absorption, distribution and excretion of polychlorinated biphenyls. Arch. Environ. Conlam. Toxicol. 2: 86. 1974. 8. Barsotti, D. a., and J. R. Allen. Ef fects of polychlorinated biphenyls on reproduction in the primate. Federation Proc. 34: 338, 1975. 9. Bitman, J.. h. S. Cecil and s. j. Harris. Biological effects of poly chlorinated biphenyls in rats and quail. Environ. Health Persp. I: 145, 1972. 10. Good, C. K.t and N. Pensky. Halowax acne; cutaneous eruptions in marine electricians due to certain chlorinated naphthalenes and diphenyls. Arch. Dermatol. Syphilol. 48: 215. 1943. 11. Hirayama, C., T. Irisa and t, Yama moto. Fine structural changes of the liver in patients with chlorobiphenyl intoxication. Fukuoka IgaJtu Zasshi 60: 455, 1969. 12. Hsu, I. C,, J. P. Van Miller and J. R. ALLEN. Metabolic fate of 'H 2.5.2'.5'tetrachlorobiphenyl in nonhuman pri mates. Bull. Environ. Contam. Toxicol., submitted. 13. HUTTERER, F,, F. Schaffner, F. M. Klion and H, Popper. Hypertrophic, hypoaettve smooth endoplasmic re ticulum: A sensitive indicator of hepatotoxicity exemplified by dieldrin. Science 161: 1017, 1968. 14. Jensen, S. Report on a new chemical hazard. Hew Sci. 32: 612, 1966. 15. Jones, j. W,, and H. S. Alden. An acneform dermatergosis. Arch. Dermatol. Syphilol. S3: 1022. 1936. 16. Kolbye, A. C. Food exposure to poly chlorinated biphenyls. Environ. Heallh Persp. 1: 85, 1972. 17. Kuratsune, M. An epidemiologic study of "Yusho" or chlorobiphenyl poisoning. Fukuoka Igaku Zasshi 60: 513, 1969. 18. Kuratsune, M., t. Yoshimura, j. Matsuzaka and A. Yamacuchi. Epidemiologic study on Yusho. a poisoning caused by ingestion of rice oil contaminated with a commercial brand of polychlorinated biphenyls. Environ. Heallh. Persp. 1: 119, 1972. 19. Miller, J. A. Carcinogenesis by chemi cals. Cancer Res. 30: 559, 1970. 20. Okimura, M,, C. Hirayama and M. UzaWa, Study of Yusho (chlorobi phenyl poisoning) in clinical examina tion. Fukuoka Igaku Zasshi 62: 123, 1971. 21. Van Miller, J. P.. 1. C. Hsu and J. R, ALLEN. Distribution and metabolism of *H 2,5,2',5'-tetrachlorobiphenyl in rats. Proc. Soc. Exptl. Biol. Med. 148: 682, 1975. 22. Yobs, A, R. Food exposure to poly chlorinated biphenyls. Environ. Health Persp. 1: 79, 1972. RESEARCH ACTIVITIES AT REGIONAL PRIMATE CENTERS DSW 025871 STLCOPCB4009826