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Biological Responses of the Nonhuman Primate, Chicken, and Rat to Chlorinated Dibenzo-^dioxin Ingestion* by D.K. Norbackt and J.R. Alien1 In 1908 Schmittle et al, (/) reported the development of hydropericardium and as cites in poultry following ingestion of feeds containing industrially contaminated fats. The toxic component was demonstrated to have the chlorinated dibenzo-p-dioxin (CDD) structure by Cantrell et a), in 1969 (2). Experimental animal studies in our laboratory have shown that CDD adminis tration causes varying responses in different animal species. Gastric hyperplasia and ul ceration, hydropericardium, ascites, reduced spermatogenesis, focal liver necrosis, de creased hematopoiesis, skin lesions, and eventual mortality have been demonstrated in nonhuman primates ($). Chickens suc cumbed very rapidly to the same dietary concentration with hydropericardium, hy drothorax, and ascites. They also developed liver necrosis, hypoplastic testes, and al tered capillary permeahility and decreased hematopoiesis (4~6). The rat was more re sistant to the morbid efTecta of CDD but de veloped a hypertrophied liver composed of enlarged hepatocytes with a proliferated in- Tht* investigation vu supported in part by U.S. Public Health Service grants ES-00472 and Rlt00137 from the National Institutes of Health. Pri mate Center Publication No. 13-010. tDeiiartment of Pathology, University of Wiscon sin Morlical School, and Rrgionnl Primate Research Center. University of Wisconsin. Madison. Wisconsin 5370G. tracellular membrane system (7). The re sults of these investigations are reviewed and the variable responses and pathogenesis of lesions are discussed. The material used in the investigations was crude industrial fat capable of produc ing hydropericardium, ascites, and death in the chicken. Gas-liquid chromatographic and nuclear magnetic resonance analysis of the materials demonstrated bi-, tritetra-, penta-, hexa-, and heptachlorodibenzo-pdioxin present in the material, with the tetrachtorinated compound comprising 64% (mass) of the total dioxins present, in ad dition, rats were given radioactive octachloro- or radioactive tetrachlorodibenzo-pdioxin in separate experiments (8, 9). Maeoca mulatta monkeys were given a diet that contained varying quantities of the crude industrial fat. The percentage of fat that allowed survival of the nonhuman pri mate for 100 days produced 50% mortality in chickens within 15 days. The survival time of the monkeys was inversely related to the percentage of the CDD-containing fat given to the animals. Clinical and patho logic changes occurring at their demise were similar regardless of concentration of ma teria) in their diet. At death alt had devel oped ascites, hydropericardium, and anasar ca. Prior to their demise the monkeys de veloped a decrease in total serum protein September 1973 HONS 207260 233 from 7.5 to 5 4 g/100 ml and a decrease in the percentage albumin from 61% to 35%. There was a decrease in hematocrit from 41% found in the control animals to 16% in the experimental group, a decrease in the white blood count from 6.8 x 103 per mm3 to 3.0 X 10J and a decrease in the red cell count from 6-5 x 10* per mm3 to 2,5 x 10*. The hemoglobin values were correspondingly reduced. Analysis of the sternal bone mar row showed a hypoplastic bone marrow with diminished myeloid and erythroid cells being replaced by fatty tissue. The lymphoid tissue of the spleen and lymph nodes was hypoplastic. The skin changes of the monkeys included alopecia and subcutaneous edema which pro gressed from the eyelids to the remainder of the face, eventually involving the sub cutaneous tissue of the trunk, extremities, and scrotum. Microscopically, there was edema of the derma) layer with disarray of the collagen fibers. Hair follicles, particularly of the face, contained numerous keratin cysts with hyperplasia of the epithelium (Fig. 1). cells. However, there was a decreased num ber of primary and secondary spermato cytes, and spermatids were inapparent in most instances (Fig. 2). The interstitial cells of Leydig appeared normal, Figure 2. In a monkey fed CDD, seminiferous tub ules of testes contained decreased number* of pri mary and secondary apermatocytea without (permatids. Spermatogonia, Sertoli cell*, and Inter stitial cell* were normal in appearance. Light micrograph of teaticular tissue fixed with formalin and stained with hematoxylin and eoain. X115. Figure 1, Hair follicle*, particularly of the face and eyelids, of monkey* fed CDD contained numerous keratin cyst*. Light micrograph of akin fixed with formalin and stained with hematoxylin and eosin. x 15. The seminiferous tubules of the testes con tained abundant spermatogonia and Sertoli 234 Figure 3. Cardiac fiber* in the hearts of monkeys fed CDD were hypertrophied, and the myofila, ments were widely separated by the increased intracellular fluid. Light micrograph of heart fixed with formalin and stained with hematoxylin nd eosin. XUS, Environmental Health Perspectives HONS 2072S1 The heart was dilated, particularly in the right chamber, and an increase in the circumference of both the tricuspid and mi tral valves was noted. Microscopically, hy pertrophic muscle fibers were separated by fluid (Fig. 3). Electron microscopic exam ination demonstrated separation of the myo fibrils and swelling of the mitochondria with widely separated cristae (Fig. 4). In over BO 9b of the experimental monkeys, marked hypertrophy of the gastric mucosa occurred in the fundic and pyloric regions. The hypertrophied mucosal layer penetrated the muscularis mucosae to form crypts and mucin.containing cysts in the submucosa (Fig. !)). In the same areas, gastric ulcera tions of the mucosa) layer were present (Fig. 6). The livers were moderately yellow. On microscopic examination, enlarged multiuucleated hepatocytes and fat vacuoles were apparent. Terminally the animals developed centrilobular necrosis and bile duct hyper plasia. Changes in the biliary tree were ob served as proliferation and stratification of the bile duct epithelial cells of the small ducts within the portal area and in the larg er ducts, including the common bile duct running through the head of the pancrea(Fig. 7). Electron microscopic examination of the hepatocytes demonstrr.ed hypertro phied cells with numerous autophagosomes and fat droplets, an increase in the smooth endoplasmic reticulum with a decrease in the rough endoplasmic reticulum, and swol len mitochondria (Fig. 8). In the morbid animals the parenchymal cells showed num erous degenerative changes. Many of the celts Figure 4. Myofibril* of dilated cardiac fiber* within the heart of a monkey fed CDD were eeparatcd, and the mitochondria were moderately swollen. Electron micrograph of heart fixed with Veronal acetatebufTerod osmium tatroxid* solution and stained with uranyl acetate. X9.700. September 1973 HONS 207282 233 Figure 5. (o) Normal gland* of the gastric mucoea are separated by the muaeularts mucosae from the ubmucoaa in thia section of a stomach taken from a control monkey, (t) Following CDD ingestion, marked hypertrophy of the gastric mucosa occurred in the fundic and pyloric regions. Crypts and mucin-containing cysts within the submucosa are extensions of the hypertrophied mucosa which pene trated the muscularis mucosae. Light micrograph of stomach Axed with formalin and stained with hema toxylin and eosin. (a) x30; (6) x29. were shrunken and electron-dense, while t)tiiers were swollen and very lucent. Of the experimental animals evaluated, the chicken waa the most sensitive to the toxic effects of CDD. Low levels of the com pounds resulted in a decrease in growth rate and the development of hydropericardium, hydrothorax, and ascites, resulting in death of the animals. Following doses sufficient to cause 50% mortality within 15 days, hemoglobin was reduced from 10 g/100 nil to 6 g/100 ml, hematocrit was reduced from 31% to 18%, and the total serum protein decreased from 3.4 g/100 ml to 2.3 g/100 ml. The percentage albumin also decreased in these animals. The animals developed 236 large hearts, and on microscopic examina tion the cardiac fibers were separated by edematous fluid. A lymphocytic infiltrate was observed between the myocardial fibers and in the perivascular areas. The capsule of the liver was thickened with adherent fibrinous material. Lymphoid hyperplasia waa apparent in the portal areas, and the hepatic cells were infiltrated with fat (Fig. 9). Electron microscopic examination dem onstrated viable cells dispersed among de generative cells. Degenerative changes of hepatocytes and Kupffer cells included elec tron dense cytoplasm, irregular mitochon dria. and indistinct nuclear envelopes. The Sertoli cells and spermatogonia of the semi- Envlronmental Health Perspectives MOHS 2072S3 isif' Figure C. Gastric ulcerstions of the hypertrophied mucosal layer were present in the stomachs of monkey* receiving CDD. Ulcers were particularly prevalent over areas of epithelial cell-lined sub mucosal cysts. Edema of the subinucosa, portion of s submucosal cyst, fibrotic granulation tissue, a fibrinous exudate and overhanging edges of the. mucosa are present. Light micrograph of stom ach fixed with formalin and stained with hema toxylin and eoain. X35. Ficurb 8. Liver* of monkeys fed CDD contained hypertrophied cells with swollen mitochondria, In creased numbers of lipid droplets, and a prolifera tion of smooth endoplasmic reticulum. Electron micrograph of liver tissue fixed in Veronal acetatebuffered osmium tetroxide solution and stained with uranyl acetate. X8500. Fictnm 7. Following CDD Ingeatinn by the monkeys, bile duct hyperplasia occurred within the liver. Tall columnar epithelial cell*, many of which ap peared stratified, replaced cuboidal cells found in the normal bile duct and epithelial folds extended into the lumen. Light micrographs of liver tissue fixed in formalin and stained with hematoxylin and eosin. x 115. September 1973 Figure 0. Light and dark staining cells were present in the livers of chickens fed CDD. Numerous fst droplet* infiltrated the hepatocytes. Light micro graph of liver tissue fixed in Veronal acetatebuffered osmium tetroxide solution and stained with toluidine blue. xfilO- 237 MONS 207284 niferous tubules within the testes were nor mal ; however, there was a reduction in the number of primary and secondary sperma tocytes, and no spermatozoa were present. Gastrointestinal changes were not observed. The perfusion of the mesenteric vessels with ferritin, thorium dioxide, iron oxide, and car bon black demonstrated a decided alteration in capillary permeability of these experi mental animals. Following administration of approximate ly five times the concentration of the CDD- containing fat in the diet sufficient to cause hydropericardium, ascites, and focal necrosis of the liver in chickens and in nonhuman primates, the rat developed liver alterations with a 50% mortality at 80 days. At 6 weeks, enlarged livers contained hypertrophied hepatocytes with an increase in droplets and a higher quantity of extractable lipid. The histologic pattern of the livers consisting of sinusoids separated by single sheets of hepatocytes radiating from the portal areas to the central veins was maintained. Within the large hepatocytes a prolifera tion of the smooth endoplasmic reticulum and reorganization of the parallel cisternae of the rough endoplasmic reticulum to form large agranular concentric membrane arrays was demonstrated electron microscopically (Fig. 10). Figure 30. Hepatocytes of rate which ingested CDD developed a proliferated smooth tndoplasmic retic ulum consisting of numerous vesicles and concentric arrays of agranular membranes. The number of lipid droplets was increased. Electron micrograph of liver tissue fixed in Veronal acetate-buffered os mium tetroxide solution and stained with uranyl acetate. X 20,400. 238 Environmental Health Perspectives HONS 207285 Administration of tetrachlorodibenzop-dioxin (1 pg/day) resulted in a 50% mor tality of the rats at 21 days. The morpho logic appearance of the livers was similar to that produced by ingestion of the crude CDD-containing lipid material by rats. The smooth endoplasmic reticulum was prolifer ated, and large concentric membrane arrays were present. Over a 21-day period of administration of labeled octachlorodibenzo-p-dioxin to rats (100 ^ig/rat/day, approximately 12.4 mg/kg administered over 21 days), 93% of the com pound passed unabsorbed through the gas trointestinal tract. An additional 5% was excreted in a lipid-soluble form in the urine. The administration of the octachloro compound produced few morbid alterations in the rats. The animals continued to gain weight and maintained normal activities and gross appearance. Approximately 50% of the material present within the body tis sues was located in the liver. Other reser voirs containing radioactivity at lesser lev els were the adipose tissue, skeletal muscula ture, and skin. Over 95% of the CDD pres ent within the liver was located in the mi crosomal fraction with equal distribution within the rough and smooth fractions. Discussion The data from these experimental animal studies have shown that CDD administra tion causes varying responses in the chicken, monkey, and rat. The chicken develops ex treme morbidity and mortality at dietary concentrations that are only mildly toxic to rats while the monkey is intermediate in its response to the CDD, The chicken and monkey developed ascites, hydrothorax, hydropericardium, and anas arca; however, the rat failed to develop in creased extracellular fluid. These modifica tions in the fluid content of the tissues and body cavities were attributed in part to hep atic degenerative changes and altered cap illary permeability of the chicken and monkey. The low serum albumin, a direct result of hepatic dysfunction, was associ ated with decreased osmolarity of the blood and subsequent extravasation of the fluid. In addition, the capillaries were dem onstrated to be more permeable to colloidal particles before the decline in serum protein was sufficiently severe to produce an accu mulation of fluid in the tissues. Gastric hyperplasia and ulceration were limited to the nonhuman primate. Hyper plastic changes are thought to be related to the chronic irritation following ingestion of the compounds and other closely related chlorinated aromatic hydrocarbons (f 0). The dysplastic histologic and cytologic pat tern as demonstrated by the invasion of the mucosal cells'through the muscularis muc osae and the atratlAcation of the epithelial cells within the cysts are changes suggestive of an eventual neoplastic transformation. Monkeys developed widespread alopecia, moderate hyperkeratosis, follicular keratin cysts, and hyperplasia of the epithelium of the hair follicles, particularly of the face. However, the skin of rats or chickens was not altered appreciably. Hypoplasia of the lymph tissue and bone marrow was present in all three animal spe cies. However, the blood-forming tissues of the chicken and the monkey were affected earlier and more severely than were those of the rat. As a result of these changes in the lymph tissue and bone marrow, the ani mals became anemic and displayed a pro gressive leukopenia. Due to the reduced re sistance of these animals they became prime hosts for opportune pathogens which in many instances were responsible for their death. Hypoactlvity of the seminiferous tubules of the testes was associated with chronic intoxication of the monkeys and chickens. Young chickens exposed to low levels of the dioxins experienced retardation in the de velopment of the testes and at maturity were of normal size with hypoplastic gon ads. There were no other alterations in growth, blood elements, or histologic ap pearance of the tissues. Enlargement of the liver occurred in all animats used in these investigations. In creased size was related to the cell hyper- September 1973 NONS 2072*6 trophy resulting from proliferation of the smooth endoplasmic reticulum and accumu lation of lipid, within the cytoplasm of the hepatocytes. The chicken rapidly developed widespread liver necrosis; similar degenera tive changes occurred at a less rapid rate in the liver of the monkey, and the rat was very resistant to hepatic necrosis. Radioactive studies which determined tis sue and cell fraction levels of the CDD in the rat and possibly in other animal species demonstrated the proliferated hepatic en doplasmic reticulum present in animals fol lowing exposure to the CDD may serve not only as a source of enzymes to enhance the metabolism of foreign substances but may also function as an area of localization for these toxic compounds. The presence of a large portion of the ingested CDD within the microsomal fraction of the hepatic tis sue may be one explanation as to why the rat is able to tolerate larger doses of the dioxins. The localization of the dioxins in these membranes may prevent their move ment to other tissues of the body that are more susceptible to the toxic effects of these compounds. Although the specific reason for the difference in response of various animal species to the dioxins has not been estab lished, further studies on absorption, meta bolism, body distribution, excretion, or sen sitivity of the tissues to the toxic effects of the dioxins are avenues of research that will likely clarify these questions. REFERENCES 1. Schmjttle, S. C-, Edward*, H. M., *nd Morris, D. A disorder of ehiekana probably due to a toxic feed--preliminary report. 1. Amer. Vet. Med. Aisn. 132; 218 (1068). 2. Cantrell, J. S,, Webb, N. C., and M*bii, A. J. The identification and cryatai structure of a hydro pericardium producing factor; 1,2,3,7,8,9-hexachlorodibenxo-p-dioxin. Acta Cryit. B23: ISO (1909). 3. Allen, J. R, and Caratena, L. A. Light and elec tron microscopic observations in Macaea mu/a I la monkeys fed toxic fat. Amer. J. Vet. Kes. 28; 1613 (1967). 4. Allen, J. R. The role of toxic fat In the produc tion of hydropericardfum and ascites in chickens. Amer. J. Vet, Re*. 25; 1210 (1984). 5. Allan, J. R., and Canton*. L. A. Electron microacoptc alterations In the liver of chickens fed toxic fet. Lab. Invest. 15; 970 (1966). 6. Allen, J. R., and Lalich. J. J. The effects of "toxic fat" on apermatoganesis. Proc. Soc. Exp. Biol. Med. 109; 46 (1962). 7. Norback, D. K., and Allen, J. R. Morphogenesis of the toxic fat-induced concentric membrane array* in rat hepatocytes. Lab. Inveat. 20; 336 (1969). 8- Norback, D. H. Morphological end biochemical responses of the rat hepatic endoplasmic re ticulum to polychlorinated triphenyl* and to chlorinated bibenzo-p-dioxins. Ph.D. disserta tion, University of Wisconsin, Madison, Wise., August 1973; Dissertation Abatr., in press. 9. Norback, D. K., snd Engbiom, J. F. Chlorinated dibenzo-p-dioxin distribution within rat tissue and subtraction* of the liver. Fed. Proc. 32; 236 (1973). 10. Allen, J. R., and Norback, D. H- Polychlorinated biphenyl- and triphenyl-induced gastric mucosal hyperplasia in primates. Science 179; 498 (1973). 240 Environmental Health Perspectives MONS 207287