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Pathology of dimethylnitrosamine poisoning in pekin ducklings
TOXICOLOGYAND APPLIEDPtIAlZlVLACOLOG¥8, 224-234 (1966)
Pathology of Dimethylnitrosamine Poisoning in Pekin Ducklings1 WILLIAM W . CARLTON, 2 JULIET E. LORD, AND LEO FRIEDMAN
Department o] Nutrition and Food Science, Massachusetts Institute o] Technology, Cambridge, Massachusetts 02139 Received February 3, 1965
Pekin ducklings have been shown to be a sensitive test animal for detecting the presence of aflatoxins, toxic metabolites produced by some strains of the fungus Aspergillus flavus (Newberne et al., 1964b; Armbrecht and Fitzhugh, 1964). Ducklings respond to these compounds with hepatic necrosis and proliferation of bile ductule cells. Comparable lesions were observed in ducklings fed aflatoxin-containing peanut meals which were carcinogenic in rats (Newberne et al., 1964a; Lancaster et al., 1961). These results suggested that the duckling might be useful as a test animal to screen potential carcinogens, and preliminary studies were undertaken to determine the tissue response of ducklings to known carcinogenic agents. The purpose of this report is to present the marked histopathologic lesions that have been observed in several tissues of ducklings fed the carcinogen, dimethylnitrosamine (DMNA). METHODS
Male Pekin ducklings, received within 36 hours after hatching, were weighed and assigned to their respective diets. Ten ducks, allotted at random to each experimental group, were weighed at weekly intervals, and were fed ad libitum for a period of 30 days. The composition of the purified diet is given in Table 1. Minerals were supplied as the salt mixture of Wesson (1932). Feed and water were offered ad libitum. Ten ducks fed only the purified diet served as controls. It was proposed to feed the carcinogen at a level not to significantly reduce weight gains over the experimental period of 30 days. Groups which failed to grow as well as the control ducks were discontinued earlier and sacrificed by decapitation; tissues were taken for histopathology. At the end of the 30-day feeding period surviving ducks were killed by decapitation and necropsy was performed on all ducks. Organ weights were recorded for the liver, spleen, pancreas and for thyroid and parathyroids, which were weighed together. Organ weights were taken only for those groups which were fed for 30 days. The 0.02% DMNA group was discontinued earlier and only histopathologic examination of tissue sections was completed. Selected portions of the liver, spleen; pancreas, kidney, heart, aorta, thyroid, parathyroid, and proven1 Contribution No. 650 from the Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts. 2 Present address: Department of Veterinary Physiology and Pharmacology, School of Veterinary Medicine, Purdue University, Lafayette, Indiana. 224
DIIVfETHYLNITROSAIVfINE
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DUCKLINGS
triculus were fixed in 10% buffered formalin for histopathology. Portions of the tissues were prepared for paraffin sections and stained with hematoxylin and eosin. Additional sections were stained with Masson's trichrome stain, Van Gieson's stain, Gridley's reticulum stain, and Alcian Blue-PAS stain. The Congo Red stain for TABLE 1 COIV~POSITION
OF THE
PURIFIED'
DIET
FED
TO DUCKLINGS
Concentration Component
(%)
Casein Gelatin Cornstarch Sucrose Salt mixture Wa DL-Methionine Wesson oil Choline chloride Vitamin mixb
20.0 8.0 26.2 26.2 5.0 0.3 10.0 0.3 4.0
a Nutritional Biochemical Corporation, Cleveland, Ohio. b Vitamin mix supplied per kilogram of diet: biotin, 0.8 rag; menadione, 20 rag; riboflavine, 8 rag; thiamine, 16 rag; pyridoxine, 8 rng ; calcium pantothenate, 40 rag; folic acid, 20 rag; niacin, 100 mg; inositol, 500 rag; alphatocopberol acetate, 450 rag; vitamin D3, 500 ICU; vitamin A acetate, 1950 IU; and vitamin B12, 0.020 rag. amyloid and the Ziehl-Nielsen stain for acid-fast material were applied to certain kidney sections. Frozen sections of the spleen, kidneys, and liver were stained by the Oil Red O technique. RESULTS The level of 0.02% of dimethylnitrosamine was acutely toxic and average weights of test ducks were significantly lower than those of controls after 3 weeks of feeding. D M N A at a level of 0.0175% of the diet did not markedly lower weight gains in surviving ducks, but mortality was increased (Table 2). Three of the 4 ducks which failed to survive the experimental period died during the fourth week. The ducks remained clinically normal except for abdominal distention late in the experimental period. At necropsy striking alterations were observed in all ducklings fed the carcinogen. Marked hydroperitoneum was present with the peritoneal cavity filled with an opaque, milky fluid. 3 The ascites was not hemorrhagic as has been described in the rat (Jacobson et al., 1955). Twenty milliliters of ascitic fluid was present in the peritoneal cavity of some birds. A rough estimate of the amount of ascitic fluid was made by the withdrawal of fluid with a 10-ml syringe. N o attempt was made to withdraw all the fluid. More than 10 ml of ascitic fluid was present in all the test ducks. Hydropericardium, although not as severe as the ascites, was present in all test birds. The livers were obviously smaller than those of control ducks, gray in color, and cut with increased resistance as compared with livers from control birds. This atrophy is reflected in much reduced average liver weights (Table 2). The kidneys of carcinogen-fed ducks were gray in color, swollen with rounded edges, and protruded from their bony Birds do not have a diaphragm so there is no anatomical separation of the abdominal and thoracic cavities.
226
WILLIAM W. CARLTON, JULIET E. LORD~ AND LEO FRIEDMAN
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DIMETHYLNITROSAMINE POISONING IN DUCKLINGS
227
fossae. Although organ weights were not recorded for the 0.02% D M N A group, the spleens were not obviously enlarged at necropsy. Spleens from ducks fed 0.0175% D M N A for 4 weeks were markedly enlarged as reflected by about a 9-fold increase in average organ weight (Table 2). Average organ weights of the pancreas and the thyroid and parathyroids did not vary significantly between control and test ducks. These organs were histologically normal. Microscopically, the liver presented hepatic parenchymal necrosis in either single cells or groups of cells (Fig. 1). Hepatocytes varied in nuclear size and in the size and number of nucleoli. Also present was bile duct proliferation (Fig. 2) as well as bile ductule cell hyperplasia (Fig. 3). Nodules of regenerative liver cells were absent from all sections. Fibrous tissue increase was present to a varying degree in all livers and cut across the lobular structure as irregular-shaped bands. Silver impregnation methods
FIO. 1. Section of liver from a duckling fed 0.02% DMNA for 3 weeks. Necrotic liver cells partially enclose a few viable cells. Hematoxylin and eosin stain. Magnification: X 1125. established that the fibrous tissue was composed predominantly of reticular fibers with only a minor collagen component. Glisson's capsule was markedly thickened in all livers; most of the increase in thickness was due to proliferation and condensation of reticular fibrils (Fig. 4). Extensions of the fibrous tissue into the hepatic parenchyma from the capsule was apparent in several sections. Isolated islands of vacuolated hepatic cells stained intensely with Oil Red O, confirming the presence of lipids in these cells. The lesions described above were present in livers from both carcinogen-fed groups but were of greater severity in the group fed for 4 weeks. In liver sections from animals fed 0.02% D M N A for 3 weeks, lymphoid hyperplasia was especially marked (Fig. 5). Lymphocytes were present in large nodules exhibiting germinal centers and also as a diffuse infiltration of cells beneath the thickened capsule of the liver. Lymphoid cells infiltrated the wall and formed perivascular cuffs around radicles of the portal vein (Fig. 6). Often, proliferated bile ductule cells were intermixed with the hyperplastic lymphatic nodules (Fig. 5).
228
WILLIAM W. CARLTON~ JULIET E. LORD~ AND LEO FRIEDMAN
A l t h o u g h l y m p h o i d h y p e r p l a s i a was p r e s e n t in birds fed for 4 weeks, the nodular a c c u m u l a t i o n s were not as extensive and fewer interlobular veins c o n t a i n e d intral a m i n a r cellular deposits. M a n y of the l y m p h a t i c nodules had u n d e r g o n e necrosis;
Fie. 2. Section of liver from a duckling fed 0.0175% DMNA for 4 weeks showing bi!e ductile cell proliferation with formation of definitive ducts. Hematoxylin and eosin stain. Magnification: X 1125.
FIG. 3. Section of liver from a duckling fed 0.02% DMNA for 3 weeks showing ductul~ cell proliferation in rows and clumps. Hematoxylin and eosin stain. Magnification: )K 1125.
DllV[ETHYLNITROSAMINE POISONING IN DUCKLINGS
229
nuclei of l y m p h o c y t e s p r e s e n t e d pycnosis a n d k a r y o r r h e x i s . T h e s e d e g e n e r a t i n g nodules were o f t e n infiltrated b y heterophils. P r o l i f e r a t i o n of the epithelial cells of the renal tubules resulted in c l u m p s of d a r k -
FIo. 4. Section of liver from a duckling fed 0.02% DMNA for 3 weeks. Glisson's capsule is thickened with an increase in reticular fibers. Penetration (condensation) of fibers into parenchyma is evident. Gridley's reticulum stain. Magnification: X 100.
Fla. 5. Section of liver from a duckling fed 0.0175% DMNA for 4 weeks showing lymphoid hyperplasia with intermixed bile ductile cells in rows. Hematoxylin and eosin stain. Magnification: X 100.
230
WILLIAB/f W. CARLTON~ JULIET E. LORD, AND LEO FRIEDMAN
FIo. 6. Section of liver from a duckling fed 0.02% DMNA for 3 weeks showing an accumulation of lymphoid tissue around portal veins. Hematoxylin and eosin stain. Magnification: X 100.
FIG. 7. Sections of kidney from a duckling fed 0.0175% DMNA for 4 weeks. On the right, the glomerulus presents a lack of cellularity and areas of hyaline change. Hematoxylin and eosin stain. On the left, much of the glomerulus gives a positive PAS reaction. Alcian Blue-PAS stain. Magnification: )< 1125.
DIMETHYLNITROSAMINE
POISONING IN
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231
staining basophilic cells which formed incomplete and complete tubules. An occasional tubular cell was observed in mitosis. These focal areas of cellular proliferation, present in both treatment groups, were especially prominent and numerous at the periphery of the kidney. Glomerular alterations consisted of hyalinization and loss of cellularity in the vascular tufts (Fig. 7). Some of the glomeruli were markedly atrophic and apparently nonfunctional. Erythrocytes were less frequently observed in the capillary tufts of the test ducks. The hyaline material of the glomeruli was not distinctly stained by Masson's trichrome, Van Gieson's stain or the Congo Red stain for amyloid as the color of the material was not different from that of the cytoplasm of the renal tubular cells. It was not acid fast. However, the material was strongly PAS positive (Fig. 7) and nearly replaced the epithelial and endothelial cells in many glomeruli. Protein material was not observed in the dilated Bowman's space, nor did Gridley's reticulum stain establish structural differences between renal glomeruli of control and treated ducks. The hyaline areas in the glomerular tufts did not contain lipid. Lipid droplets were observed, however, in the epithelial cells of some renal tubules. These changes, although present in the kidneys of ducks fed 3 weeks, were more severe in the ducks fed for the additional week. In sections of spleen from test ducks fed 4 weeks, much of the splenic pulp had been replaced by large pale-staining cells (Fig. 8) with vesicular nuclei containing clear vacuoles and cell debris in their granular cytoplasm (Fig. 9). The cytoplasmic vacuoles of these cells, interpreted as reticuloendothelial components, did not stain distinctly with Alcian Blue-PAS stain or Congo Red stain. They were negative for lipids and did not react to Oil Red O stain. Proliferation of reticuloendothelial and lymphoid cells was restricted to the spleen and liver, and lymphoid deposits were absent from the proventriculus, heart, kidney, and thyroid. DISCUSSION Very early hepatic damage induced in rats by DMNA has been described (Bailie and Christie, 1959). Confluent hepatoparenchymal necrosis was present by 16 hours posttreatment. More chronic lesions include fibrosis, condensation of reticulum, pleomorphism of hepatic cell nuclei, and bile duct pro]iferation (Magee and Barnes, 1956). Similar microscopic lesions were present in liver sections from the test ducks of this study. These liver lesions in ducklings probably represent subacute toxic effects of DMNA. At 4 weeks there was continuing necrosis of hepatocytes. These data suggest that ducklings are more susceptible to the toxic effects of the drug than rats. No significant increase in collagen was demonstrable by special stains, and hyperplastic nodules of liver cells were absent. Thus, the liver lesions do not resemble histologically postnecrotic cirrhosis. The pathologic pattern of biliary hyperplasia and pleomorphism of hepatocytes may represent changes which could progress to neoplasia. These alterations have been observed in the rat after DMNA administration and after the feeding of aflatoxin. Both of these substances are known to produce hepatic tumors in rats (Magee and Barnes, 1956; Carnaghan and Crawford, 1964). Additional studies in which DMNA is fed over many months to ducks could establish the carcinogenicity of the drug for this species. Until such studies are completed, the liver lesions induced by DMNA can only serve as presumptive evidence of its carcinogenicity for the duck. An additional lesion observed in this study was lymphoid hyperplasia. The latter
232
WILLIAM W. CARLTON, JULIET E. LORD~ AND LEO FRIEDMAN
change may well be a species difference due to the diffuse nature of lymphoid tissue in the duck. The degeneration and necrosis of the lymphoid elements at 4 weeks suggest exhaustion of these components, but a direct toxic effect cannot be excluded. Micro-
FIG. 8. Section of spleen from a duckling fed 0.0175:% DMNA for 4 weeks. Much of the pulp has become displaced by large pale cells. Hematoxylin and eosin stain. Magnification: X 100.
FIG. 9. High power view of section shown in Fig. 8. Large pale-staining cells with vacuolated cytoplasm occupy the pulp area. Hematoxylin and eosin stain. Magnification: X 600.
DIMETHYLNITROSAMINE
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233
scopic lesions in lymphatic tissue of rats fed D M N A appear not to have been reported. Hyperplasia of splenic reticuloendothelial cells appears to be a species-mediated response to D M N A . No reports of such changes in other species are known to the authors. D M N A administration over a short period results in an impairment of kidney function in rabbits without demonstrable histologic changes (O'Leary et al., 1957). Chronic administration of the compound to rats resulted in the formation of kidney tumors (Magee and Barnes, 1962). D M N A fed to ducks over the experimental period of 30 days induced proliferation of the epithelial cells of the renal tubules. Histologically, the hyperplasia resulted in the formation of clumps of basophilic cells with and without lumens. These hyperplastic nodules may represent precursors of kidney tumors, but in the absence of more chronic studies with resultant tumor formation a more definite statement cannot be made at this time. Marked alterations were present in the glomeruli of the kidneys of the ducklings of this study. The histologic evidence suggests that impairment of kidney function must have been severe. This damage coupled with the progressive loss of liver tissue may well account for the marked ascites and deaths during the fourth week of the study. Ascites present in our test ducks was not hemorrhagic as described in rats, dogs, and guinea pigs (Barnes and Magee, 1954). Intestinal hemorrhage and the bleeding tendency des.cribed in dogs (Jacobson et al., 1955) was not a feature of D M N A poisoning in ducks. Additional studies have been completed in which other known carcinogens have been fed to Pekin ducklings (Carlton, Lord and Friedman, unpublished data). The drugs studied included: 1,2,3,4-diepoxybutane, 4-nitroquinoline-N-oxide, {~-propiolactone, and p-dimethylaminoazobenzene. Liver lesions were minimal to absent in the test ducks of these groups, and no consistent pattern of hepatic damage has been observed. These preliminary studies suggest that the duckling has no great advantage over other species as an animal for the screening of potential carcinogens. Butler (1964) noted that aflatoxin (15 9g) induced within 3 days an extensive biliary proliferation in the livers of ducklings. A comparable lesion was observed after a single dose of dimethylnitrosamine or cycasin. Hepatotoxic agents such as carbon tetrachloride, ethionine, and thioacetamide did not induce comparable lesions. Our data and the findings of Butler (1964), indicate that the liver of ducklings does not respond with a consistent morphologic pattern of injury to hepatotoxins or carcinogens either in acute or subacute toxicity studies. SUMMARY Pekin ducklings were fed a purified diet containing dimethylnitrosamine at levels of 0.02% and 0.0175%. The level of 0.02% was acutely toxic, weight gains were significantly reduced, and feeding was discontinued after 3 weeks. Abdominal distention was observed in some ducks fed 0.0175% dimethylnitrosamine for 4 weeks. A nonhemorrhagic ascites and hydropericardium were observed at necropsy in both test groups. The livers were atrophic and gray in color. The spleens of ducks fed for 4 weeks were markedly enlarged. tlistopathologic lesions in the liver included necrosis of hepatocytes with condensation of the reticulum, bile duct proliferation, bile ductule cell hyperplasia, and thickening of the liver capsule with an increase in reticular fibers. Lymphoid hyperplasia was especially prominent in the ducks fed dimethylnitrosamine for 3 weeks. The lymphocytes were present as nodules and as a diffuse infiltration of cells around radicles of the portal vein. Degeneration and necrosis was observed in the lymphoid tissue of ducks fed 4 weeks. Hyalinization of renal glomeruli was prominent in all
234
WILLIAM W. CARLTON~ JULIET E. LORD~ AND LEO FRIED1VfAN
ducks, and the hyaline material was strongly PAS-positive but was negative to Congo Red, Oil Red O, and the Ziehl-Nielsen stain. En]argement of the spleen was restricted to ducks fed 4 weeks. Much of the red pulp of the spleen was replaced by large pale cells with vacuolated granular cytoplasm. This change was considered to be hyperplasia of the reficuloendothelial components of the spleen. ACKNOWLEDGMENT This investigation was supported in part by Public Health Service Research Grant No. CA-0698801 from the National Cancer Institute. REFERENCES AI~BREC~IY, B. H., and FITZI-IIYGI-I,O. G. (1964). Mycotoxins. II. The biological assay of ariatoxin in Peking White ducklings. Toxicol. Appl. Pharmacol. 6, 421-426. BAILIE, M. J., and CI-IRISTIE, G. S. (1959). The acute toxic action of dimethylnitrosamine on liver cells. Biochem. J. 72, 473-479. BARNES, J. M., and MAGEE, P . N . (1954).. Some toxic properties of dimethylnitrosamine. Brit. J. Ind. Med. 11, 167-174. BIJTL~R, W. H. (1964). Acute liver injury in ducklings as a result of aflatoxin poisoning. J. Pathol. Bacteriol. 88, 189-196. CAmCACHA~¢,R. B. A., and CI~AWFO~D,M. (1964). Relationship between ingestion of aflatoxin and primary liver cancer. Brit. Vet. J. 19.0, 201-204. JACOBSON, K. H., WHEELWRIGI:LT,H. J., JR., CLEIVI, J. H., and St~A~cxTo~,R . N . (1955). Studies on the toxicology of N-nitrosodimethylamine vapor. A.M.A. Arch. Ind. Health 12, 617-622. LANCASTER, M. C., JENxI~,Ts, F. P., and PHII~I~, j'. M. (1961). Toxicity associated with certain samples of groundnuts. Nature 192, 1095-1096. MAOE~, P. N., and BARNES,J . M . (1956). The production of malignant primary hepatic tumours in the rat by feeding dimethylnitrosamine. Brit. J. Cancer 10, 114-122. MAOEE, P. N., and BARNES, J . M . (1962). Induction of kidney tumors in rats with dimethylnitrosamine (N-nitrosodimethylamine). J. Pathol. Bacteriol. 8~, 19-31. NEwnE~TE, P. M., CARL¢O~,W. W., and WOCA~¢, G.N. (1964a). Hepatomas in rats and hepatorenal injury in ducklings fed peanut meal or AspergiUus flavus extract. Pathol. Vet. 1, 105-132. lkrEW~R~CE, P. M., WOCA~¢, G. N., CARLTO~¢,W. W., and A~D~L KADER, M . M . (1964b). Histopathologic lesions in ducklings caused by Aspergillus flavus cultures, culture extracts, and crystalline aflatoxins. Toxicol. Appl. Pharmacol. 6, 542-556. O'LEARY, J. F., WILLS, ~. H., HARRISON,B., and OIKE.MlYS,A. (1957). Some effects of N-nitrosodimethylamine on rabbit kidney function. Proc. Soc. Exptl. Biol. Med. 94, 775-776. WESSON, L. G. (1932). A modification of the Osborne-Men~del salt mixture containing only inorganic constituents. Science 75. 339-340.
Pathology of Dimethylnitrosamine Poisoning in Pekin Ducklings1 WILLIAM W . CARLTON, 2 JULIET E. LORD, AND LEO FRIEDMAN
Department o] Nutrition and Food Science, Massachusetts Institute o] Technology, Cambridge, Massachusetts 02139 Received February 3, 1965
Pekin ducklings have been shown to be a sensitive test animal for detecting the presence of aflatoxins, toxic metabolites produced by some strains of the fungus Aspergillus flavus (Newberne et al., 1964b; Armbrecht and Fitzhugh, 1964). Ducklings respond to these compounds with hepatic necrosis and proliferation of bile ductule cells. Comparable lesions were observed in ducklings fed aflatoxin-containing peanut meals which were carcinogenic in rats (Newberne et al., 1964a; Lancaster et al., 1961). These results suggested that the duckling might be useful as a test animal to screen potential carcinogens, and preliminary studies were undertaken to determine the tissue response of ducklings to known carcinogenic agents. The purpose of this report is to present the marked histopathologic lesions that have been observed in several tissues of ducklings fed the carcinogen, dimethylnitrosamine (DMNA). METHODS
Male Pekin ducklings, received within 36 hours after hatching, were weighed and assigned to their respective diets. Ten ducks, allotted at random to each experimental group, were weighed at weekly intervals, and were fed ad libitum for a period of 30 days. The composition of the purified diet is given in Table 1. Minerals were supplied as the salt mixture of Wesson (1932). Feed and water were offered ad libitum. Ten ducks fed only the purified diet served as controls. It was proposed to feed the carcinogen at a level not to significantly reduce weight gains over the experimental period of 30 days. Groups which failed to grow as well as the control ducks were discontinued earlier and sacrificed by decapitation; tissues were taken for histopathology. At the end of the 30-day feeding period surviving ducks were killed by decapitation and necropsy was performed on all ducks. Organ weights were recorded for the liver, spleen, pancreas and for thyroid and parathyroids, which were weighed together. Organ weights were taken only for those groups which were fed for 30 days. The 0.02% DMNA group was discontinued earlier and only histopathologic examination of tissue sections was completed. Selected portions of the liver, spleen; pancreas, kidney, heart, aorta, thyroid, parathyroid, and proven1 Contribution No. 650 from the Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts. 2 Present address: Department of Veterinary Physiology and Pharmacology, School of Veterinary Medicine, Purdue University, Lafayette, Indiana. 224
DIIVfETHYLNITROSAIVfINE
POISONING
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DUCKLINGS
triculus were fixed in 10% buffered formalin for histopathology. Portions of the tissues were prepared for paraffin sections and stained with hematoxylin and eosin. Additional sections were stained with Masson's trichrome stain, Van Gieson's stain, Gridley's reticulum stain, and Alcian Blue-PAS stain. The Congo Red stain for TABLE 1 COIV~POSITION
OF THE
PURIFIED'
DIET
FED
TO DUCKLINGS
Concentration Component
(%)
Casein Gelatin Cornstarch Sucrose Salt mixture Wa DL-Methionine Wesson oil Choline chloride Vitamin mixb
20.0 8.0 26.2 26.2 5.0 0.3 10.0 0.3 4.0
a Nutritional Biochemical Corporation, Cleveland, Ohio. b Vitamin mix supplied per kilogram of diet: biotin, 0.8 rag; menadione, 20 rag; riboflavine, 8 rag; thiamine, 16 rag; pyridoxine, 8 rng ; calcium pantothenate, 40 rag; folic acid, 20 rag; niacin, 100 mg; inositol, 500 rag; alphatocopberol acetate, 450 rag; vitamin D3, 500 ICU; vitamin A acetate, 1950 IU; and vitamin B12, 0.020 rag. amyloid and the Ziehl-Nielsen stain for acid-fast material were applied to certain kidney sections. Frozen sections of the spleen, kidneys, and liver were stained by the Oil Red O technique. RESULTS The level of 0.02% of dimethylnitrosamine was acutely toxic and average weights of test ducks were significantly lower than those of controls after 3 weeks of feeding. D M N A at a level of 0.0175% of the diet did not markedly lower weight gains in surviving ducks, but mortality was increased (Table 2). Three of the 4 ducks which failed to survive the experimental period died during the fourth week. The ducks remained clinically normal except for abdominal distention late in the experimental period. At necropsy striking alterations were observed in all ducklings fed the carcinogen. Marked hydroperitoneum was present with the peritoneal cavity filled with an opaque, milky fluid. 3 The ascites was not hemorrhagic as has been described in the rat (Jacobson et al., 1955). Twenty milliliters of ascitic fluid was present in the peritoneal cavity of some birds. A rough estimate of the amount of ascitic fluid was made by the withdrawal of fluid with a 10-ml syringe. N o attempt was made to withdraw all the fluid. More than 10 ml of ascitic fluid was present in all the test ducks. Hydropericardium, although not as severe as the ascites, was present in all test birds. The livers were obviously smaller than those of control ducks, gray in color, and cut with increased resistance as compared with livers from control birds. This atrophy is reflected in much reduced average liver weights (Table 2). The kidneys of carcinogen-fed ducks were gray in color, swollen with rounded edges, and protruded from their bony Birds do not have a diaphragm so there is no anatomical separation of the abdominal and thoracic cavities.
226
WILLIAM W. CARLTON, JULIET E. LORD~ AND LEO FRIEDMAN
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DIMETHYLNITROSAMINE POISONING IN DUCKLINGS
227
fossae. Although organ weights were not recorded for the 0.02% D M N A group, the spleens were not obviously enlarged at necropsy. Spleens from ducks fed 0.0175% D M N A for 4 weeks were markedly enlarged as reflected by about a 9-fold increase in average organ weight (Table 2). Average organ weights of the pancreas and the thyroid and parathyroids did not vary significantly between control and test ducks. These organs were histologically normal. Microscopically, the liver presented hepatic parenchymal necrosis in either single cells or groups of cells (Fig. 1). Hepatocytes varied in nuclear size and in the size and number of nucleoli. Also present was bile duct proliferation (Fig. 2) as well as bile ductule cell hyperplasia (Fig. 3). Nodules of regenerative liver cells were absent from all sections. Fibrous tissue increase was present to a varying degree in all livers and cut across the lobular structure as irregular-shaped bands. Silver impregnation methods
FIO. 1. Section of liver from a duckling fed 0.02% DMNA for 3 weeks. Necrotic liver cells partially enclose a few viable cells. Hematoxylin and eosin stain. Magnification: X 1125. established that the fibrous tissue was composed predominantly of reticular fibers with only a minor collagen component. Glisson's capsule was markedly thickened in all livers; most of the increase in thickness was due to proliferation and condensation of reticular fibrils (Fig. 4). Extensions of the fibrous tissue into the hepatic parenchyma from the capsule was apparent in several sections. Isolated islands of vacuolated hepatic cells stained intensely with Oil Red O, confirming the presence of lipids in these cells. The lesions described above were present in livers from both carcinogen-fed groups but were of greater severity in the group fed for 4 weeks. In liver sections from animals fed 0.02% D M N A for 3 weeks, lymphoid hyperplasia was especially marked (Fig. 5). Lymphocytes were present in large nodules exhibiting germinal centers and also as a diffuse infiltration of cells beneath the thickened capsule of the liver. Lymphoid cells infiltrated the wall and formed perivascular cuffs around radicles of the portal vein (Fig. 6). Often, proliferated bile ductule cells were intermixed with the hyperplastic lymphatic nodules (Fig. 5).
228
WILLIAM W. CARLTON~ JULIET E. LORD~ AND LEO FRIEDMAN
A l t h o u g h l y m p h o i d h y p e r p l a s i a was p r e s e n t in birds fed for 4 weeks, the nodular a c c u m u l a t i o n s were not as extensive and fewer interlobular veins c o n t a i n e d intral a m i n a r cellular deposits. M a n y of the l y m p h a t i c nodules had u n d e r g o n e necrosis;
Fie. 2. Section of liver from a duckling fed 0.0175% DMNA for 4 weeks showing bi!e ductile cell proliferation with formation of definitive ducts. Hematoxylin and eosin stain. Magnification: X 1125.
FIG. 3. Section of liver from a duckling fed 0.02% DMNA for 3 weeks showing ductul~ cell proliferation in rows and clumps. Hematoxylin and eosin stain. Magnification: )K 1125.
DllV[ETHYLNITROSAMINE POISONING IN DUCKLINGS
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nuclei of l y m p h o c y t e s p r e s e n t e d pycnosis a n d k a r y o r r h e x i s . T h e s e d e g e n e r a t i n g nodules were o f t e n infiltrated b y heterophils. P r o l i f e r a t i o n of the epithelial cells of the renal tubules resulted in c l u m p s of d a r k -
FIo. 4. Section of liver from a duckling fed 0.02% DMNA for 3 weeks. Glisson's capsule is thickened with an increase in reticular fibers. Penetration (condensation) of fibers into parenchyma is evident. Gridley's reticulum stain. Magnification: X 100.
Fla. 5. Section of liver from a duckling fed 0.0175% DMNA for 4 weeks showing lymphoid hyperplasia with intermixed bile ductile cells in rows. Hematoxylin and eosin stain. Magnification: X 100.
230
WILLIAB/f W. CARLTON~ JULIET E. LORD, AND LEO FRIEDMAN
FIo. 6. Section of liver from a duckling fed 0.02% DMNA for 3 weeks showing an accumulation of lymphoid tissue around portal veins. Hematoxylin and eosin stain. Magnification: X 100.
FIG. 7. Sections of kidney from a duckling fed 0.0175% DMNA for 4 weeks. On the right, the glomerulus presents a lack of cellularity and areas of hyaline change. Hematoxylin and eosin stain. On the left, much of the glomerulus gives a positive PAS reaction. Alcian Blue-PAS stain. Magnification: )< 1125.
DIMETHYLNITROSAMINE
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DUCKLINGS
231
staining basophilic cells which formed incomplete and complete tubules. An occasional tubular cell was observed in mitosis. These focal areas of cellular proliferation, present in both treatment groups, were especially prominent and numerous at the periphery of the kidney. Glomerular alterations consisted of hyalinization and loss of cellularity in the vascular tufts (Fig. 7). Some of the glomeruli were markedly atrophic and apparently nonfunctional. Erythrocytes were less frequently observed in the capillary tufts of the test ducks. The hyaline material of the glomeruli was not distinctly stained by Masson's trichrome, Van Gieson's stain or the Congo Red stain for amyloid as the color of the material was not different from that of the cytoplasm of the renal tubular cells. It was not acid fast. However, the material was strongly PAS positive (Fig. 7) and nearly replaced the epithelial and endothelial cells in many glomeruli. Protein material was not observed in the dilated Bowman's space, nor did Gridley's reticulum stain establish structural differences between renal glomeruli of control and treated ducks. The hyaline areas in the glomerular tufts did not contain lipid. Lipid droplets were observed, however, in the epithelial cells of some renal tubules. These changes, although present in the kidneys of ducks fed 3 weeks, were more severe in the ducks fed for the additional week. In sections of spleen from test ducks fed 4 weeks, much of the splenic pulp had been replaced by large pale-staining cells (Fig. 8) with vesicular nuclei containing clear vacuoles and cell debris in their granular cytoplasm (Fig. 9). The cytoplasmic vacuoles of these cells, interpreted as reticuloendothelial components, did not stain distinctly with Alcian Blue-PAS stain or Congo Red stain. They were negative for lipids and did not react to Oil Red O stain. Proliferation of reticuloendothelial and lymphoid cells was restricted to the spleen and liver, and lymphoid deposits were absent from the proventriculus, heart, kidney, and thyroid. DISCUSSION Very early hepatic damage induced in rats by DMNA has been described (Bailie and Christie, 1959). Confluent hepatoparenchymal necrosis was present by 16 hours posttreatment. More chronic lesions include fibrosis, condensation of reticulum, pleomorphism of hepatic cell nuclei, and bile duct pro]iferation (Magee and Barnes, 1956). Similar microscopic lesions were present in liver sections from the test ducks of this study. These liver lesions in ducklings probably represent subacute toxic effects of DMNA. At 4 weeks there was continuing necrosis of hepatocytes. These data suggest that ducklings are more susceptible to the toxic effects of the drug than rats. No significant increase in collagen was demonstrable by special stains, and hyperplastic nodules of liver cells were absent. Thus, the liver lesions do not resemble histologically postnecrotic cirrhosis. The pathologic pattern of biliary hyperplasia and pleomorphism of hepatocytes may represent changes which could progress to neoplasia. These alterations have been observed in the rat after DMNA administration and after the feeding of aflatoxin. Both of these substances are known to produce hepatic tumors in rats (Magee and Barnes, 1956; Carnaghan and Crawford, 1964). Additional studies in which DMNA is fed over many months to ducks could establish the carcinogenicity of the drug for this species. Until such studies are completed, the liver lesions induced by DMNA can only serve as presumptive evidence of its carcinogenicity for the duck. An additional lesion observed in this study was lymphoid hyperplasia. The latter
232
WILLIAM W. CARLTON, JULIET E. LORD~ AND LEO FRIEDMAN
change may well be a species difference due to the diffuse nature of lymphoid tissue in the duck. The degeneration and necrosis of the lymphoid elements at 4 weeks suggest exhaustion of these components, but a direct toxic effect cannot be excluded. Micro-
FIG. 8. Section of spleen from a duckling fed 0.0175:% DMNA for 4 weeks. Much of the pulp has become displaced by large pale cells. Hematoxylin and eosin stain. Magnification: X 100.
FIG. 9. High power view of section shown in Fig. 8. Large pale-staining cells with vacuolated cytoplasm occupy the pulp area. Hematoxylin and eosin stain. Magnification: X 600.
DIMETHYLNITROSAMINE
POISONING IN DUCKLINGS
233
scopic lesions in lymphatic tissue of rats fed D M N A appear not to have been reported. Hyperplasia of splenic reticuloendothelial cells appears to be a species-mediated response to D M N A . No reports of such changes in other species are known to the authors. D M N A administration over a short period results in an impairment of kidney function in rabbits without demonstrable histologic changes (O'Leary et al., 1957). Chronic administration of the compound to rats resulted in the formation of kidney tumors (Magee and Barnes, 1962). D M N A fed to ducks over the experimental period of 30 days induced proliferation of the epithelial cells of the renal tubules. Histologically, the hyperplasia resulted in the formation of clumps of basophilic cells with and without lumens. These hyperplastic nodules may represent precursors of kidney tumors, but in the absence of more chronic studies with resultant tumor formation a more definite statement cannot be made at this time. Marked alterations were present in the glomeruli of the kidneys of the ducklings of this study. The histologic evidence suggests that impairment of kidney function must have been severe. This damage coupled with the progressive loss of liver tissue may well account for the marked ascites and deaths during the fourth week of the study. Ascites present in our test ducks was not hemorrhagic as described in rats, dogs, and guinea pigs (Barnes and Magee, 1954). Intestinal hemorrhage and the bleeding tendency des.cribed in dogs (Jacobson et al., 1955) was not a feature of D M N A poisoning in ducks. Additional studies have been completed in which other known carcinogens have been fed to Pekin ducklings (Carlton, Lord and Friedman, unpublished data). The drugs studied included: 1,2,3,4-diepoxybutane, 4-nitroquinoline-N-oxide, {~-propiolactone, and p-dimethylaminoazobenzene. Liver lesions were minimal to absent in the test ducks of these groups, and no consistent pattern of hepatic damage has been observed. These preliminary studies suggest that the duckling has no great advantage over other species as an animal for the screening of potential carcinogens. Butler (1964) noted that aflatoxin (15 9g) induced within 3 days an extensive biliary proliferation in the livers of ducklings. A comparable lesion was observed after a single dose of dimethylnitrosamine or cycasin. Hepatotoxic agents such as carbon tetrachloride, ethionine, and thioacetamide did not induce comparable lesions. Our data and the findings of Butler (1964), indicate that the liver of ducklings does not respond with a consistent morphologic pattern of injury to hepatotoxins or carcinogens either in acute or subacute toxicity studies. SUMMARY Pekin ducklings were fed a purified diet containing dimethylnitrosamine at levels of 0.02% and 0.0175%. The level of 0.02% was acutely toxic, weight gains were significantly reduced, and feeding was discontinued after 3 weeks. Abdominal distention was observed in some ducks fed 0.0175% dimethylnitrosamine for 4 weeks. A nonhemorrhagic ascites and hydropericardium were observed at necropsy in both test groups. The livers were atrophic and gray in color. The spleens of ducks fed for 4 weeks were markedly enlarged. tlistopathologic lesions in the liver included necrosis of hepatocytes with condensation of the reticulum, bile duct proliferation, bile ductule cell hyperplasia, and thickening of the liver capsule with an increase in reticular fibers. Lymphoid hyperplasia was especially prominent in the ducks fed dimethylnitrosamine for 3 weeks. The lymphocytes were present as nodules and as a diffuse infiltration of cells around radicles of the portal vein. Degeneration and necrosis was observed in the lymphoid tissue of ducks fed 4 weeks. Hyalinization of renal glomeruli was prominent in all
234
WILLIAM W. CARLTON~ JULIET E. LORD~ AND LEO FRIED1VfAN
ducks, and the hyaline material was strongly PAS-positive but was negative to Congo Red, Oil Red O, and the Ziehl-Nielsen stain. En]argement of the spleen was restricted to ducks fed 4 weeks. Much of the red pulp of the spleen was replaced by large pale cells with vacuolated granular cytoplasm. This change was considered to be hyperplasia of the reficuloendothelial components of the spleen. ACKNOWLEDGMENT This investigation was supported in part by Public Health Service Research Grant No. CA-0698801 from the National Cancer Institute. REFERENCES AI~BREC~IY, B. H., and FITZI-IIYGI-I,O. G. (1964). Mycotoxins. II. The biological assay of ariatoxin in Peking White ducklings. Toxicol. Appl. Pharmacol. 6, 421-426. BAILIE, M. J., and CI-IRISTIE, G. S. (1959). The acute toxic action of dimethylnitrosamine on liver cells. Biochem. J. 72, 473-479. BARNES, J. M., and MAGEE, P . N . (1954).. Some toxic properties of dimethylnitrosamine. Brit. J. Ind. Med. 11, 167-174. BIJTL~R, W. H. (1964). Acute liver injury in ducklings as a result of aflatoxin poisoning. J. Pathol. Bacteriol. 88, 189-196. CAmCACHA~¢,R. B. A., and CI~AWFO~D,M. (1964). Relationship between ingestion of aflatoxin and primary liver cancer. Brit. Vet. J. 19.0, 201-204. JACOBSON, K. H., WHEELWRIGI:LT,H. J., JR., CLEIVI, J. H., and St~A~cxTo~,R . N . (1955). Studies on the toxicology of N-nitrosodimethylamine vapor. A.M.A. Arch. Ind. Health 12, 617-622. LANCASTER, M. C., JENxI~,Ts, F. P., and PHII~I~, j'. M. (1961). Toxicity associated with certain samples of groundnuts. Nature 192, 1095-1096. MAOE~, P. N., and BARNES,J . M . (1956). The production of malignant primary hepatic tumours in the rat by feeding dimethylnitrosamine. Brit. J. Cancer 10, 114-122. MAOEE, P. N., and BARNES, J . M . (1962). Induction of kidney tumors in rats with dimethylnitrosamine (N-nitrosodimethylamine). J. Pathol. Bacteriol. 8~, 19-31. NEwnE~TE, P. M., CARL¢O~,W. W., and WOCA~¢, G.N. (1964a). Hepatomas in rats and hepatorenal injury in ducklings fed peanut meal or AspergiUus flavus extract. Pathol. Vet. 1, 105-132. lkrEW~R~CE, P. M., WOCA~¢, G. N., CARLTO~¢,W. W., and A~D~L KADER, M . M . (1964b). Histopathologic lesions in ducklings caused by Aspergillus flavus cultures, culture extracts, and crystalline aflatoxins. Toxicol. Appl. Pharmacol. 6, 542-556. O'LEARY, J. F., WILLS, ~. H., HARRISON,B., and OIKE.MlYS,A. (1957). Some effects of N-nitrosodimethylamine on rabbit kidney function. Proc. Soc. Exptl. Biol. Med. 94, 775-776. WESSON, L. G. (1932). A modification of the Osborne-Men~del salt mixture containing only inorganic constituents. Science 75. 339-340.