CYCAD’ TGXICOSIS AND RELATED CARCINOGENESIS IN ANIMALS Thesis for III: Degree of DB. D. ' MICHIGAN STATE UNIVERSITY Gerald Munene Mugera 1965 THEFT: Fffinfl‘wulllfi't r - 'lll. .- " LIBRARY Michigan State University V“, This is to certify that the thesis entitled CYCAD TOXICOSIS AND TIELATED CARCINOGENESIS IN ANIMALS presented by Gerald Munene Mugera has been accepted towards fulfillment of the requirements for 0-169 ABSTRACT CYCAD TOXICOSIS AND RELATED CARCINOGENESIS IN ANIMALS by Gerald Munene Mugera Cycads are used as a source of food by man and animals in many parts of the world. They are of particular importance as a source of sustenance during hard times or in areas where the food supply is naturally limited. They are also used for medicinal purposes in geo- graphically widespread areas of the earth. The impetus for research on cycads has been the recognition of a possible relationship between ingestion of cycad products and the occurrence of a severe paralytic condition in man. .The possibility of other harmful effects from consumption of cycad products has also needed exploration. Horses, cattle. swine and rats were used to study the pathologic effects of cycad nut flour obtained from the plant Cycas circinalis, indigenous to Guam. The flour was incorporated into natural grain rations, making up from 1% to 2% of the ration. The toxicity in animals 'was evaluated by its effect on the growth rate, symptomatolOgy, hema- tology, changes in serum enzymes, gross pathology and histopathology. The transmission of the cycad toxic factor(s) through the mammary and placental tissues was evaluated by means of gross and histopatholOgical lesions of suckling animals and fetuses of dams fed cycad. Gerald Munene Mugera Although the experiments failed to demonstrate effects on the inerveus system. pathological changes were induced in certain organs in all species. In large animals, when trials were conducted for 3-4 months, the most significant gross and microscOpic lesions were ob- served in the liver and kidney; also present were serous effusions in the peritoneal and pleural cavities. The pathologic changes in the livers of cattle and horses were portal fibrosis and slight to extensive bile duct proliferation. Varied degrees of degeneration and regenera- tion of hepatic cells were noted in all animals fed cycad flour. In swine, a high daily intake of cycad produced both portal and centro- lobular fibrosis. The renal lesion was that of chronic interstitial nephritis of variable degree and extent. In rats, when trials were conducted for 6-10 months, benign and znalignant tumors developed, mainly in the liver and kidney. Most of the hepatic tumors were hepatocellular carcinomas or reticulum cell sarcomas. occurring either simultaneously or independently. In 2 rats, hemangiomas were seen in the liver occurring simultaneously with hepato- cellular carcinomas. The renal tumors were adenomas. embryonal ‘nephromas or fibrosarcomas. The histOpatholOgic lesions observed in suckling animals and fetuses of dams fed cycad were similar to those noted in the parent stock, and usually the lesions were more severe. This suggested that ‘the cycad toxic factor(s) are transmitted through the milk and placen- 'tal barriers and that suckling animals are mbre susceptible to the toxicosis than their mothers. Gerald Munene Mugera Rats which suckled mothers fed cycad,were then weaned and fed a control diet for 6 months, developed kidney tumors. In rats fed a cycad-containing ration for a short period of time and then returned to a control ration. the liver showed complete or' partial regeneration; but a few rats develOped kidney tumors. Pathologic changes were similar in rats fed (1) cycad flour. (2) cycasin, the glycoside isolated from cycads. (3) dimethylnitrosamine. and (h) diethylnitrosamine. The lesions were different from those induced by dimethylaminoazobenzene. This suggested that the cycad toxic factor yields in its metabolic breakdown a carcinogenic compound which may be similar to that produced by dimethyl- or diethylnitrosamine. The transmission of carcinogens via the milk and across the pla- centa appears to be a significant discovery. The likely interrelation- ship of cycad and high incidence of liver cancer in trans-Saharan .Africa and Southeast Asia, where cycads are used extensively as food and medicine. is discussed. This study suggests the need for additional research on tropical herbal medicine. trepical poisonous plants in relation to human and animal diseases. and the extent to which these plants are consumed by man. CICAD TOXICOSIS AND RELATED CARCINCGETESIS IN ANIMALS By \ Gerald Munene thera A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1965 AC KN GILEDGEMEIJ TS The author wishes to express sincere appreciation to Dr. C. K. 'a'hitehair and Dr. 0. Mickelsen for their inValuable guidance and assis- tmuuce throughout this study and their critical reading of this thesis. Gratitude is expressed to Dr. C. C. Morrill for his careful reading armi criticism of the manuscript. Sincere thanks are expressed to Miss M. 3. Campbell for her generous cooperation and suggestions during the transmission studies and to Miss Janice Fuller for typing the thesis. The author wishes to express his sincere appreciation to all members of the Department of Pathology for their generosity and con- sideration and for the technical and professional aid and guidance. Thanks are exoressed to the Kenva Government for Sponsoring the author to the Agency for International Development, during Which period this study was conducted. This research was supported in part by the National Institutes of Health, research grant CAO 7052. ii TABLE OF CONTENTS Page '1. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . 1 III. REVIEW OF THE LITERATURE . . . . . . . . . . . . . . . . 4 III. PART I. CYCAD TOXICOSIS IN SWINE AND HORSES . . . . . . 22 Experiment I. Swine . . . . . . . . . . . . . . . . . . 22 Experiment II. Horses . . . . . . . . . . . . . . . . . 26 GENERAI.DISCUSSION FOR PART I . . . . . . . . . . . . . 30 Tables of Part I . . . . . . . . . . . . . . . . . . . . 32 PiguresofPartI................... 36 IV. PART II. CYCAD TOXICOSIS AND RELATED CARCINOGENS IN RATS . . . . . . . . . . . . . . . . . . . . . . . . 45 Experiment I. Cycad Flour Toxicosis in Rats . . . . . . 45 Experiment II. Carcinogenesis in the Rats Induced by Specific Chemicals . . . . . . . . . . . . . . . . 67 Experiment III. The Effects of Feeding Cycad Flour for a Short Period of Time on Tumor Formation . . . . . . 79 GENERAL DISCUSSION FOR PART II . . . . . . . . . . . . . 81 Ta’blesofPartII................... 90 Figures of Part II . . . . . . . . . . . . . . . . . . . 97 V. PART III. TRANSMISSION OF CYCAD TOXIC FACTORS AND CHEMICAL CARCINOGENS THROUGH‘THE MAMMARY GLAND AND PLACENTA . . . 129 Experiment 1. Transmission Through the Mamary Gland and Placenta . . . . . . . . . . . . . . . . . . . 129 ° Trial 1. Transmission Through the Mammary Gland . 129 Trial 2. Transmission‘Through the Placenta . . . . 134 iii VI. VII. Page Experiment 11. Long Term Effects of Exposing Rats During Suckling to Carcinogens Transmitted Through Their mm's Milk 0 O O O O O O O O O O O O O O O O O O 0 13S Experiment III. Transmission of Toxic Factor(s) in the Milk of Swine and Cattle . . . . . . . . . . . . . 137 GENERAL.DISCUSSION OF TRANSMISSION EXPERIMENTS . . . . 144 Tables of Part III . . . . . . . . . . . . . . . . . . 147 Figures of Part III . . . . . . . . . . . . . . . . . . 157 GENERAI.DISCUSSION . . . . . . . . . . . . . . . . . . 172 REFERENCES CITED 0 O O O O O O O O O I O O O O O O O O 1 74 iv Table l O“ ‘0 10 ll LIST OF TABLES Page Body Weight Changes, Liver, Kidney, Spleen and Heart Weights of Pigs Fed 2% Cycad . . . . . . . . . . . . . . 32 Body Weight Changes, liver. Kidney, Spleen and Heart 'Jeights of Horses Fed Cycad . . . . . . . . . . . . . . 33 Transaminase, Eilirubin, HemOglobin and Packed Cell Volumes in Horses Fed cycad Flour . . . . . . . . . . . 34 Composition of Basal Ration (Ml) Fed to Rats . . . . . . 90 Average Body Weights (Gm.) of weanling Rats Fed a Basal Ration Plus Various levels of cycad Flour, Cycasin, Dimethylnitrosamine, Diethylnitrosamine or Dimethylamino- azobenzene. (Numbers in parentheses indicate number of rats.) . . . . . . . . . . . . . . . . . . . . . . . . . 91 Summary of the Major Pathological Findings, Serum SCOT and SGPT, and Packed Cell Volumes in Experiments I and II D O O O O O O O O C O O O O O O O O O O O O O O 92 Summary of the Incidence and Classification of NeOplasms Observed in Rats Fed Various Levels of Cycad and Specific Chemicals 0 e e s e e e s e s e e e o s e o e e e e e e 95 Histological Lesions in Rats Suckling Mothers Fed Cycad or CarcinOgenic Chemicals . . . . . . . . . . . . . . . 1“? Observations on Pups Born to Female Rats Fed Dimethylnitrosa- mine, Diethylnitrosamine, Dimethylaminoazobenzene or Cycasin During Gestation . . . . . . . . . . . . . . . . 154 Average Weights (Gm.) of Rats That Suckled Dams Fed the Rasal Pation and Various Levels of Dimethylnitrosamine. Diethylnitrosamine, Dimethylaminoazobenzene or Cycasin During Lactation and Then Placed on the Basal Ration for 26 Weeks . . . . . . . . . . . . . . . . . . . . . . . . 155 Serum SCOT. SGPT, Bilirubin, hemoglobin and Packed Cell Volume Values of the Cow and the Calf . . . . . . . . . 155 Figure 10 11 12 13 LIST OF FIGURES Fibrotic liver of pig 4, which had been fed a diet con- taining 2% cycad flour for 60 days. . . . . . . . . . . 36 Liver of a pig fed control diet for 60 days. H & E stain: x75. 0 O O I O O O O O O O O O O O O O O O O O O 37 Portal and centrolobular fibrosis of a liver of pig fed 2% cycad for 60 days. H & E stain: x75 . . . . . . 37 Centrolobular fibrosis. Higher power of Fig. 3. Arrows show globules in the nuclei. H & E stain: x187 . . . . 38 A small globule in the nuclei. Higher power of Fig. 4. H & E stain: x750 O C O D O O O O O O O O O O O I O O O 38 A large globule in the nucleus with nuclear content pushed into the periphery in a form of sickle-shaped structure. Higher power of Fig. 4. H & E stain: x750. . . . . . . . . . . . . . . . . . . . . . . . . . 39 Diffuse fibrosis in the liver of a pig fed 2% cycad for 90 days. Section around central vein. H & E atain: x187 O O O O O O O O O O O O O O O O O O O O O D 39 Portal fibrosis in a liver of a pig fed 2% cycad for 90 days. Same liver as in Fig. 7. H & E stain: x187. O C O O O O O O O O O O O O O O O O O O O D O O O 40 Fibrosis in renal cortex of a pig fed 22 cycad for 90 days. H & E stain: x75 . . . . . . . . . . . . . . . . 40 Fibrosis in renal cortex of a pig fed 2% cycad for 90 days. H & E stain:' x187. O C O O O O O O O O O O O O O 41 Edema of submucosa of stomach of a pig fed 2% cycad for 60 days. H & E stain: x187 . . . . . . . . . . . . 41 Occluded hepatic artery of a horse fed cycad for 147 days. H & E stain: x187. . . . . . . . . . . . . . . . 42 Hyalinized hepatic arterial wall in a liver of a horse fed cycad for 147 days. H & E stain: x75 . . . . . . . 42 vi Figure 14 15 16 17 18 19 20 21 22 24 25 26 27 28 Page Portal fibrosis with bile duct proliferation and leuko- cytic infiltration in a liver of a horse fed cycad for 147 days. H & E stain: x75 0 O O I O O O O O O O O O O 43 Bile duct proliferation with leukocytic infiltration. Higher power of Fig. 14. H & E stain: x187 . . . . . . 43 Fibrotic phlebitis of sublobular vein of a horse fed cycad for 147 days. H & E stain: x75.. . . . . . . . . 44 Fibrotic phlebitis of sublobular vein. Higher power of Fig. 16. Horse fed cycad for 147 days. H & E stain: x187. O D O O O O O O O O O O O O O O O O O O O O O O O M (1) Male rat fed 2% cycad for 8 months. (2) male rat fed control diet for 8 months. (3) Male rat fed 2% cycad flour for 2 weeks, then control diet for 7 months . . . . . . . . . . . . . . . . . . . . . . . . 97 IntracytOplasmic hyaline bodies in hepatic cells of a rat fed 2% cycad for 6 days. Periodic acid Schiff Sta in: x750 O O O O C O I O O O O O O O O O 0 O O O O O 98 Centrolobular hemorrhage in the liver of a rat fed 5% cycad for 4 days. H & E stain: x75 . . . . . . . . . . 98 Higher power of Fig. 20. Rat fed 52 cycad for 4 days. H & E 8:31“: x187 O O O O O O O O O O O O O O O O O I O 99 Modular hyperplasia in liver of a rat fed 21 cycad for 70 days. H & E stain: x75. . . . . . . . . . . . . . . 99 Reticuloendothelial cell proliferation adjacent to the sublobular vein, in the liver of a rat fed 1% cycad for 90 days. H & E stain: x187 . . . . . . . . . . . . 100 Proliferation of Kupffer cells in a dilated sinusoid of the liver of a rat fed 12 cycad for 90 days. H 6 E stain: x750 O 0' O O O O O O C O C O O O O O O O O O O O 100 Fibrotic phlebitis of sublobular vein in the liver of a rat fed 2% cycad for 180 days. H & E stain: x187 . . 101 Organized thrombus in sublobular vein in a liver of a rat fed 11 cycad for 180 days. H & E stain: x187. . . 101 Liver and kidneys of a rat that had been fed 2% cycad for 180 days. Arrows show nodular hyperplasia of the liver. (1) Tumorous kidney; (2) Normal kidney . . . . 102 Liver of a rat that had been fed 1000 p.p.m. of dimethyl- sminoazobenzene for 150 days. Arrows show cystade- “ms 0 O O O I O O I O O O O O O O O O O O 0 0 Q 0 O O 103 vii Figure Page 29 Cystadenoma. A section taken from the liver in Fig. 28, of a rat fed 1000 p.p.m. of dimethylaminoazoben- zene for 150 days. H & E stain: x75. . . . . . . . . . 104 30 Cystadenoma. A section taken from a liver of a rat fed 2% cycad for 180 days. H & E stain: x75. . . . . . 104 31 The liver of a rat fed 1% cycad flour for 8 months. Arrow shows liver hepatoma . . . . . . . . . . . . . . 105 32 Cross section of a hepatoma on the liver of a rat. - Same liver as Fig. 31 . . . . . . . . . . . . . . . . . 106 33 Liver of a rat fed 125 p.p.m. of diethylnitrosamine for 150 days. (1) Hepatoma; (2) Hemangioma . . . . . . 107 34 Hepatoma. Section taken from theliver in Fig. 32. H & E stain: x187 O O O O O O O O O O O O O O O O O O O 108 35 Hepatoma. Section taken from the liver in Fig. 33 at NO. 1. H & E stain: x187 O O O O O O O O O O O O O O O 108 36 Hemangioma. Section taken from a liver of the rat in Fig. 33 at No. 2. H & E stain: x75 . . . . . . . . . . 109 37 Reticulum cell sarcoma in a liver of a rat fed 12 cycad for 300 days. (1) Dilated liver sinusoids. H & E Stain: X75. 0 O O O C C O O O O O O O O O O O O O O O O 109 38 Reticulum cell sarcoma. Same section as Fig. 38. Gridley's reticulum stain: x187 . . . . . . . . . . . . 110 39 Reticulum cell sarcoma. Same section as Fig. 38. Gridley's reticulum stain: x187 . . . . . . . . . . . . 110 40 Reticuloadenoma on abdominal wall. Rat fed 12 cycad for 300 days. Same rat as in Fig. 38. H & E stain: x187 . 111 41 Solid renal adenoma in the kidney of a rat fed 2% cycad for 168 days. H 8 E stain: x75 . . . . . . . . . . . . 111 42 Solid adenoma in the kidneyof a rat fed 2% cycad for 168 days. Higher power of Fig. 41. H & E stain: x187. 112 43 Cross sections of kidneys with embryonal nephroma from rats fed 22 cycad for 240 days. Arrows show cysts on the tmorom mass 0 O O O O O O O O O O O O O O O O O O 1 13 44 Embryonal nephroma in the kidney of a rat fed 2% cycad for 240 days. Section taken at the junction of adenoma and sarcomatous region. H & E stain: x187. . . . . . . 112 viii F igure Page «45 Embryonal nephroma in a kidney of a rat fed 22 cycad for 240 days. Note epithelial sheets in a less dense sarcomatous region. H & E stain: x187. . . . . . . . . 114 46 Embryonal nephroma of a kidney of a rat fed 22 cycad for 240 days. Anaplastic sarcomatous area. H & E stain: x187. O O O O O O O O O I O O O O O O O O O O O O O O O 114 47 Large kidney tumor of a rat fed 2% cycad flour for 210 days. Arrow shows constriction on the tumor mass . . . 115 48 Fibrosarcoma in kidney of a rat fed 22 cycad for 210 days. Section taken from kidney shown in Fig. 47. H & E Stain: x187 O O O O O O O O O O O O O O O O O O O 116 49 Calculi removed from urinary bladder of a rat with a large fibrosarcoma . . . . . . . . . . . . . . . . . . 117 50 Edema of pancreas with vacuolation of cytoplasm of acinar cells in a rat fed 2% cycad for 8 days. H & E stain: 8187. O O O O O O O O O O O O O O O O O O O O O O O O O 118 51 Interlobular edema with leukocytic infiltration in the pancreas of a rat fed 2% cycad for 18 days. H & E Stain: x187 O O O O O O O O O O O O O O O O 0 O O O O O 118 52 Early fibrosis in acinar tissue of the pancreas of a rat fed 2% cycad for 26 days. H & E stain: x187. . . . 119 53 Replacement of acinar tissue by fibrous connective tissue in the pancreas of a rat fed 22 cycad for 70 days. H & E stain: x187. . . . . . . . . . . . . . . . 119 54 Patchy fibrotic myocarditis of a rat fed 22 cycad for 31 days. H & E stain: x187 . . . . . . . . . . . . . . 120 55 Hemangioma on diaphragm of a rat fed 2% cycad for 240 days. Section taken from the center of the lesion. H & E 8:31“: x187 O O O O 0 O O O O O O O O O O O o c o 120 56 Hemangioma on the diaphragm of a rat fed 2% cycad for 240 days. Section taken at the periphery of the lesion, where lesion appeared more fibrotic. Same rat as in Fig. 55. H & E stain: x187 . . . . . . . . . . . . . . 121 57 Hemangioma.in a diaphragm of a-rat fed 2% cycad for 240 days. H & E stain: x75 . . . . . . . . . . . . . . 121 58 Liver of a rat which had been fed 1000 p.p.m. of dimethylaminoazobenzene for 150 days. (1) Hepatoma; (2) Hemangio-endothelioma; (3) Adenocarcinoma. 122 ix Figure 59 6O 61 62 63 64 65 66 67 68 69 7O 71 Page Hemangio-endothelioma in the liver of a rat fed 1000 p.p.m. of dimethylaminoazobenzene for 150 days. Sec- tion taken from the liver in Fig. 58, at No. 2. H & E stain: x75. . . . . . . . . . . . . . . . . . . . . . . 123 Hemangio-endothelioma in the liver of a rat. Higher power of Fig. 59. H & E stain: x187. . . . . . . . . . 123 Liver of a rat which had been fed 1000 p.p.m. of dimethylaminoazobenzene for 150 days. (1) Adenocarci- noma; (2) Hepatoma .. . . . . . . . . . . . . . . . . . 124 Adenocarcinoma in the liver of a rat fed 1000 p.p.m. dimethylaminoazobenzene for 150 days. Section taken from the liver in Fig. 61. H & E stain: x187. . . . . 125 Fibrous metaplasia in the adenocarcinoma in liver of a rat fed 1000 p.p.ms dimethylaminoazobenzene. H & E Stain: X75. 0 o o e e e e e e e e e e o s e e e o e o e 125 Fibrous metaplasia in adenocarcinoma in the liver of a rat. Higher power of Fig. 63. H & E stain: x187 . . . 126 Cartilage metaplasia in adenocarcinoma in the liver of a rat fed 1000 p.p.m. of dimethylaminoazobenzene for 150 days. H & E stain: X75 0 O O O O O O O O O C O O O O O 126 Cartilage metaplasia in adenocarcinoma-in the liver of a rat fed 1000 p.p.m. dimethylaminoazobenzene for 150 days. H & E stain: x187. . . . . . . . . . . . . . . . 127 Bile duct adenoma adjacent to apparently normal hepatic cells in the liver of a rat fed 1000 p.p.m. dimethylamino- azobenzene for 150 days. H & E stain: x187 . . . . . . 127 World map illustrating the distribution of 2 most used species of cycad; world distribution of kwashiorkor; and the high incidence of the liver carcinoma in the human . . . . . . . . . . . . . . . . . . . . . . . . . 128 Liver of a fetus of a rat born from a dam on control diet during gestation period. H & E stain: x187. . . . 157 Coagulative necrosis of the liver of a rat fetus born from a dam fed 2% cycad during the gestation period. Note loss of hepatic cell cytoplasm and nuclei. Fetus killed immediately after birth. H & E stain: x187. . . 157 Coagulative necrosis of the liver of a rat fetus born from a dam fed 400 p.p.m. cycasin during gestation period. Killed immediately after birth. H 8 E stain: x187. . . . . . . . . . . . . . . . . . . . . . . . . . 158 X Figure Page 72 Coagulative necrosis of the liver of a rat fetus born from a dam fed 150 p.p.m. dimethylnitrosamine during gestation. H & E stain: x187 . . . . . . . . . . . . . 158 73 Coagulative necrosis of the liver of a rat fetus of a dam fed 150 p.p.m. diethylnitrosamine during gestation period. H 8 E stain: x187. . . . . . . . . . . . . . . 159 74 Coagulative necrosis of liver of a rat fetus from a dam fed 750 p.p.m. of dnmethylaminoazobenzene during gesta- tion period. H 8 E stain: x187 . . . . . . . . . . . . 159 75 Liver of a rat killed after 14 days of suckling mother fed on control diet during lactation. H & E stain: x187. O O O O O C O O O O O O O O O O O O O O O O O O O 160 76 Fatty infiltration in a liver of a rat killed after 7 days of suckling mother fed 2% cycad during lactation. H & E stain: x187 O O O O O C O O O C O C C O C O O C O 160 77 Fatty infiltration in the liver of a rat after suckling for 14 days a mother fed 1% cycad during lactation. H & E Stain: x187 O O O O O O O O O O O O O O O O O O O 161- 78 Coagulative necrosis of the liver of a rat killed 5 days after suckling a mother fed 600 p.p.m. cycasin during lactation. H & E stain: x187. . . . . . . . . . 162 79 Coagulative necrosis of proximal convoluted tubules of kidney of a rat which died 18 days after suckling a mother fed 2% cycad during lactation period. H & E stain: x187 . . . . . . . . . . . . . . . . . . . . . . 162 80 Necrosis of the liver of a young rat killed 8 days after suckling a mother fed 150 p.p.m. dimethylnitrosa- mine. (P) Portal area with proliferating fibroblasts. H & E stain: x75. . . . . . . . . . . . . . . . . . . . 163 81 Necrosis of the liver of a young rat killed 8 days after suckling a mother fed 150 p.p.m. of dimethylnitrosamine. Higher power of Fig. 80._ H S E stain: x187 . . . . . . 163 82 Liver of a cow that had been fed a diet containing cycad flour for 140 days. Arrows show focal necrotic lesims O C O O O O O O O O O O O O O O O O O O O C O O 164 83 Liver of a calf that had been suckling a cow fed 2% cycad for 140 days. Arrow shows focal necrotic lesions 0 O O O O O O O O O O O O O O O O O O O O O O O 165 84 Portal fibrosis in the liver of a cow fed cycad for 140 days. H & E stain: x75 . . . . . . . . . . . . . . . . 166 xi Figure Page 85 Portal fibrosis in the liver of a cow fed cycad for 140 days. Higher power of Fig. 84. H & E stain: x187. I I I I I I I I I I I I I I I I I I I I I I I I I 167 86 Coagulative necrosis in the liver of a cow. Section taken from focal necrotic lesion in the liver of Fig. 83. H & E Stain: X750. 0 I I I I I I I I I I I I I I I 167 87 Edema and portal fibrosis in the liver of a calf killed after 140 days of suckling a cow fed cycad during the lactation period. H & E stain: x75 . . . . . . . . . . 168 88 Caseous necrosis in the liver of the calf. Section taken from focal necrotic lesion on the liver of Fig. 83I H & E stain: x75 I I I I I I I I I I I I I I I I I 168 89 Edema and emphysema in the lung of a cow fed cycad for 140 days. H 8 E stain: x187. . . . . . . . . . . . . . 169 90 Edema with leukocytic infiltration in lung alveoli of the calf killed after 140 days of suckling a cow fed cycad during lactation. H & E stain: x187. . . . . . . 170 91 Fibroblastic proliferation in the myocardium of a calf killed after 140 days of suckling a cow fed cycad during lactation. H & E stain: x187. . . . . . . . . . 170 92 Portal and centrolobular fibrosis with occlusion of the central vein in the liver of a sow fed 2% cycad during lactation. Heidenhain's aniline blue stlin: x187 . . . 170 93 Portal and centrolobular fibrosis of the liver of a pig killed after 8 weeks suckling a sow fed 2% cycad during lactltion. Heidenhain's aniline blue stain: x187 . . . 171 94 Xnaplastic undifferentiated cell sarcoma with dilated tubules in a kidney of a rat that suckled mother fed 600 p.p.m. of cycasin until weaning then fed control diet for 26 weeks. Section taken at the junction of apparently normal kidney tissue and the tumorous mass. H & E stain: x75. . . . . . . . . . . . . . . . . . . . 171 xii INTRODUCTION Cycad seeds or stems are used mainly as a source of food starch in many parts of the world. They are of particular importance as a source of sustenance during hard times or in areas where the food supply is naturally limited. They are also used as medicine in geographically widespread areas of the earth. In some areas cycads are used as fodder for livestock (Thieret, 1958). The seeds or nuts have a coat differentiated into an outer, fleshy layer, usually some shade of red with a more or less orange cast, and an inner, stony layer. The bulk of the seed is a firm, starchy endo- sperm, containing the embryo. Seeds vary in length from 1.3 to 7.5 cm. The biggest are those of ngggl_p. (Chamberlin, 1919). Cycads are paLm-like plants with starch-rich stems which may be either aerial and cylindrical, as in Macrozamia reidlei, or subter- ranean and tuberous, as in Macrozamia spiralis. In either case the stem is surmounted by a crown of very large, coarse, stiff, featherlike leaves. Cycad leaves average perhaps 1 m. long. The extremes are those of 222;; 1a, only 5-6 cm. long, and those of some Species of Encephalartos, reported to reach a length of 5-6 m. Cycads are thought to be the surviving remnants of an ancient line of plant families which probably predominated over other vegetation during the mesozoic era. They are thought to represent an intermediate evolutionary step from fern to flowering plants and are now restricted to trapicsl and subtropical regions, where they are well adapted to adverse conditions and survive when other plants are destroyed by 2 hurricane or drought. They are found in all continents except Europe. Cycads belong to the Cycadacal family of the plant kingdom, which has 9 genera. These genera and their approximate distributions are as follows: Cycas: ranging from northern and northeastern Australia, New Caledonia, Samoa and Fiji north and west to the Ryukyu Islands, southern China, the Philippines, Indochina, Thailand, Malaya, Indonesia, Burma, India, Ceylon, south Pakistan, Madagascar, Zanzibar and the East African coast. Encephalartos: ranging from the Union of South Africa, Mozambique, Southern Rhodesia and northeast Angola north to Kenya, southwest Sudan, Chad and Ghana. Stangeria: eastern coastal region of South Africa. Bowenia: northeastern Queensland, Australia. Ceratozamia: southern Mexico and Guatemala. Dioon: southern Mexico and Honduras. Macrozamia: eastern, central and southwestern Australia. Microcycas: Pinar del Rio and Cuba. Zamia: Florida, West Indies, southern Mexico south to Brazil and Chile (Thieret, 1958). Scientific interest in the role of cycads in disease is not new. Search for the plants' toxic ingredients began in the 19th century, but little progress was made until COpper isolated a glycoside from 'Macrozamia spiralis (Copper, 1941). Several glycosides have since been isolated and found to be lethal to animals. 3 The new impetus for research on cycads has been the recognition of a possible relationship between ingestion of cycad material and occurrence of a severe paralytic condition in man. 0n the island of Guam, the incidence of amyotropic lateral sclerosis has been found to be more than 100 times greater among the natives than among the resi- dents of areas such as the Continental United States (Kurland and Mulder, 1963). In the search‘for causes of amyotropic lateral sclero- sis, investigations have been focused on the toxic factors of nggg circinalis, which Guamanians have utilized for a long time as a source of food starch. The present experiments were conducted to investigate the possible relationship of amyotropic lateral sclerosis to the ingestion of products of 91233 circinalis. The research is presented in 3 parts: (1) cycad toxicosis in swine and horses, (2) pathology of cycad toxi- cosis and related carcinogenesis in rats, and (3) transmission of cycad toxic factor(s) through the mammary and placental tissue. REVIEW OF THE LITERATURE chads as Medicine Whiting (1963) published a detailed literature review of cycad toxicity. According to her survey. cycad mixed with beverages and teas is prescribed as s purge, pain depressant and emmenagogue. The characteristics of the plant which may contribute to its therapeutic value for external as well as internal uses are the hardening of the exudate upon exposure to air and the astringent and mucilaginous prop- erties of the plant sap. Cycad starch likewise has a mucilaginous prOperty (Tryon, 1955). In the Dominican Republic the Zamia nut is fried in oil and mixed with mashed mamey and avocado seeds and the mixture is recommended for a therapeutic shampoo: and from Zamia stem gum is extracted which is used in treating ulcers. Whiting (1963) reported that Cycas seeds pounded to a paste in coconut oil are used for sores. swellings, wounds, boils and various skin complaints in parts of southern and southeastern Asia, Indonesia and the Philippines. The sap squeezed from the nut of gamia muriggta is described as a drastic purge in Venezuela. Drury (1958) in his description of useful plants in India stated that the gum extracted from the stem of gigag circinalis was used as an antidote for snake bite, the gum being applied to the wound, while the nut from the same plant was ground to fine powder, mixed with sugar and water and prescribed as a laxative. 5 Kirbikar (1933) reported that in Cambodia the mucilaginous termi- nal bud of chas circigalis crushed in rice water or in water holding in suspension fine particles of clay was used in the dressing of ulcera- ted wounds, swollen glands and boils. Whiting (1963) described the treatment of tropical ulcers in Guam with gigas circinalis nut as follows: “The mature nut is Opened and the meat is scraped out. The meat is then squeezed out and the juice is allowed to run on the ulcer. This is said to cause great pain. After the juice has been put on the sore, the grated flesh is then put on it and the sore covered. This is done once a day for three or four days." Okabe (l9h0) stated that gxcas revoluta nut was ground to fine powder and prescribed as an emmenagogue and astringent at the rate of 10-20 Gm. per day in the Ryukyu Islands. 0ndaatje (1862) stated that Qxcas cizcinalis seed flour, in Ceylon, was highly esteemed as a remedy in bowel complaints and hemor- rhoids, for which purpose the flour was boiled and eaten. Cycads as Food Cycad as food for man can be grouped into 4 categories: (1) uncooked, (2) boiled, (3) extracted dry starch, and (h) fermented starch. Whiting (1963), in her survey, mentioned that in Guam the green outer husks of the seeds of giggg cirginalis are chewed to relieve thirst and when dried are considered a tasty sweet. Harvey (1945) reported similar use of husks of gigg§_mggia in Australia by the Karawa tribe of Australian aborigines. Thieret (1958) reported that in several areas in Africa the raw gum or exudate from a species of Encephalartos is chewed by children. He also mentioned that succulent / 3 leaves of several species of Cycas are cooked and eaten as vegetables in the Malay Peninsula, the Philippinesand Indonesia; and the tender leaves of‘gzggg circinalis are said to be eaten by Ceylon natives as curries. On the Fiji Islands, Degener *1949) mentioned that the ripe nuts of gycas circinalis are boiled until they are soft and the white flesh is eaten. In Ceylon, among the Vaeddas, Thieret (1958) mentioned that the Cycas seed kernels are cut into slices, dried and ground into a meal. A dough made from this meal is then baked into cakes. Williams (19h9) reported that on the East African coast sun-dried Cycas kernels were fermented in a tin with layers of banana leaves for about a week. They were next cleared of mold, soaked a day in water to soften and finally powdered and used as a porridge. He also men- tioned that the spongy farinaceous center of the trunk gwede was used in Zanzibar as food in times of shortage. It was prepared by chopping small pieces, then heaping for about a week to allow fermentation to take place for neutralization of certain toxic substances. They were then washed, preferably in hot water, and dried in the sun, after which they were pounded and used in porridge; or the flour was placed in boiling water and cooked for some time, after which it was stirred into a thick paste known as "ugali". Schweinfurth (1874) described the use of Encephalartos stem for preparation of intoxicating beer in Central and East Africa. Juritz (1915) described the preparation of Kaffir bread from starch extracted from stems of Encephalartos sp, in South Africa and Mozambique. Maiden (1899) reported that one of the most important sources of food for the New South wales aborigines was seeds of Macrozamia. Before the kernels could be eaten they were made innocuous by a process of 7 roasting, pounding and soaking in water. He also described the methods of preparation of starch from nuts of Macrozamia in Australia. The methods were similar to those used in East Africa. Whiting (1963) reported that in Guam nuts of 9123; cirginalis were Split, soaked for varied lengths of time up to 18 days, with at least 4 changes of water, dried and ground, then used to thicken soup, mixed with coconut milk or fermented coconut sap and cooked or wrapped in banana leaves and steamed or boiled before eating. The dried powder or sliced seeds in Guam are kept from season to season. Whiting also mentioned that although cycad starch was no longer manufactured in the United States, it had been a pioneer industry in southern Florida as early as 1845. Most of this starch, which was marketed under the name "Florida arrowroot", was used for infant foods, biscuits, choco- lates and spaghetti. chad as Fodder for Animals Chamberlin (1919) reported that while he was in Africa, gibbons interrupted his botanical collections of Encephalartos by carrying away the cones of the plant just as the seeds reached maturity. White (1928) mentioned that roots and piths of MacrOZamia stems were ground and boiled to a thick jelly-like paste and used for feeding pigs, calves and poultry in western Australia. He also reported that cattle and sheep selectively grazed on young shoots of growing cycads. Cycads as fioisog to Humans Accompanying the uses of cycads as food and medicine, several ill effects have been reported and have been attributed to the eating of inadequately prepared cycad products. All genera of cycads have been 8 implicated except Ceratozamia and Stangeria. Banks (1770) reported that a number of Captain Cook's men during their exploration in the Pacific developed strong fits of vomiting after eating nuts of £3912 Egglé. Reitz (1930) in his book Commando related an incident wherein a number of men, including General Smuts of the Boer Commando, became violently ill from eating the nuts of Encephalgrtos altensteinii. Fitzgerald (1898) reported that in Madagascar it was well known that unless qycad starch were carefully prepared it would cause fatal diarrhea and vomiting. Safford (1905), of the United states NaTY. wrote that Guamanians were well aware that cycad starch was injurious to health if consumed for any length of time. Maiden (1819), in Australia, reported children developing fever lasting a few days followed by constipation and paralysis of the lumbar region after eating Zamia nuts. Standley (1931) reported that Zamia roots from 23mg; furfuraceas were used in Honduras as a poison for criminals and in Costa Rica for both criminals and enemies. Whiting (1963) reported that the Guamanian, even today, believes that "Iytiko" - amyotropic lateral sclerosis - is caused by the handling and consumption of cycad plant products. Cycads as Poison to Animals Outbreaks of cycad poisoning have been known in Australia since the beginning of the vast sheep and cattle industry in the 19th century. Bryant (1900) reported loss of 200 head of cattle in the Saltash Dis- trict in New South Wales between 1877 and 1884 after eating growing species of Macrozamia. Marks (1912) described the loss of #00 head of cattle from a herd of 900 after eating leaves of Macrozamia spiralis. 9 The animals developed paralysis of hind limbs and loosening of the horns before death. Cleland (1914) reported 2 effects in sheep and cattle after ingestion of Macrozamia spiralis material, one of which was an acute condition characterized by severe gastrointestinal dis- turbances and another of which was a partial paralysis involving the hind limbs. Seddon gt El. (1931) noted that death occurred in sheep ' within 18 hours after eating leaves of Maogggamia spirglis. This was followed by 80 deaths daily for a week in 2 flocks of sheep. They reported total mortality of 2,200 sheep out of 6,000. Chemical Research Chemical studies on cycads were focused on the isolation and identification of the toxic constituents of the plants. Lauterer (1896) and other early researchers in Australia directed their investi- gations towards such substances as resin, mucin and oxalic acid. Dongen (1903) isolated a toxic principle from nuts of gygég circinglis. The substance was an amorphous glycoside, which was soluble in water and insoluble in alcohol. He named the substance Pakoein. Nishida (1939) produced formaldehyde from Cycas reyolutg seeds by the action of emulsin which was present in the seeds. Cooper (1941) isolated a crystalline compound from the seeds of Macrozggia spiralis which she named Macrozamin. Macrozamin was shown to be toxic to guinea pigs when administered orally but not when injected subcutaneously. Langley inél- (1951) isolated Macrozamin from other Species of macrozamia and preposed a methyl azoxy-methanol CH3N=N-CH20H as the structure for the aglycone of Macrozamin. Riggs (1954) isolated Macrozamin from other genera of cycad, Cycas, Bowenia, and Encephalartos. All the compounds were similar except for the carbohydrate moiety. 10 Nishida g§_gl. (1955) isolated a glycoside, which they named cycasin, from seeds and pith of a Japanese species of Cy; g zevoluta. Matsumoto and Strong (1963) isolated cycasin from nggg gizcinalis nuts from Guam and confirmed the presence of methylazoxymethanol in cycasin. 0 CH3N=N-CHZOH CH3-N=N-CH2006H1105 methylazoxymethanol cycasin Kabayashi and Matsumoto (1964) demonstrated that methylazoxymethanol ‘was the toxic factor in cycasin. This compound produced formaldehyde when heated with an acid, and cyanide when heated with a base. The compound decomposed about 12$ in 5 days' standing at room temperature. It decomposed 75% when heated for 10 minutes at 75 C. and 100% in boiling water for 10 minutes. The substance was soluble in water and ethyl ether. Campbell (1964) found that the toxic factor in nggg gircinalis nut flour was readily destroyed by both moist and dry heat. When cycad flour was cooked for 1 hour with enough water to make a thin gruel and then dried at 37 C., all its toxicity was destroyed. This was also true when the dry flour was heated to 90 C. for 1 hour. Animal Begegzch Farm Animals Banks (1770) reported the first animal feeding experiment with cycad. After members of his party had become violently ill from eating cycad nuts during Captain Cook's voyage to Australia, they decided to test the nuts on hogs. When the hogs ate the nuts, 2 died within a week and the rest recovered, but with difficulty. ‘ 11 Turner (1893) recorded an experiment conducted by Norton, a grazier of Ieppoon. in Australia. Norton fed a heifer in a small paddock with Zamia leaves and fruits. The animal developed paralysis of the hindquarters. Edwards (1894) reported results after feeding cattle with Mgcggzamia fruseri in western Australia. The animals developed the following symptoms: the skin covering the ears, particu- larly at the tips, became bare and had a tendency to curl, just as in cases of poisoning by ergot on rye. In the more severe cases Edwards stated that the hair loosened on the whole body, especially on the extremities. Between 3 and 9 days, animals developed paralysis of the hindquarters. At necropsy Edwards reported paleness, hemorrhage and atrophy of muscles of the hindquarters, patchy congestion of the liver and kidney, constriction and occlusion of the terminal branches of arteries, hemorrhagic exudate in the spinal column, congestion of the arachnoid membrane of the spinal cord, and softening of the spinal cord just posterior to the middle of the loins. Lauterer (1898) fed leaves of Macgozamia spigalis to 3 calves. The gait was not affected; however, all 3 died with severe gastro- enteritis. Hunt (1899) fed leaves, stem, bulbs and fruits of Macrozamia miggelii and £125; gizcinalig to cattle at the rate of 2-4 pounds per day. The animals develOped paralysis of the hindquarters after 14 days. Seddon gt_gl. (1931) reported some experiments after a heavy loss of sheep fed nuts of Macrozamia spiralis. They used the starchy kernels and membranes. In some of their trials they used boiled and dried nuts. They gave drenches of powdered kernels and membrane to 451 adult sheep. One pound of the material was finely minced and 12 suspended in 16 oz. of water for each animal. Death usually occurred “within 36 hours. When a smaller dose of the same material was given to h93 adult sheep. no ill effects were noted for 8 days. On the 9th day the feces were covered with blood-stained mucus and the appetite was depressed. On the 20th day the animals started eating well and gradually recovered. Seddon summarized the necrOpsy lesions as follows: A. Gross lesions. Ecchymotic hemorrhages in the subcutaneous and intramuscular connective tissue were present in sheep which died acutely. Those that died later (had lived for more than # days) exhi- bited icterus: hemorrhagic fluids in the abdominal cavity: venous congestion. friable liver; edema, congestion. and petechiation of the mucosa of the stomach; hemorrhages, congestion of small intestine: hydro- thorax and scattered subpleural hemorrhages on the lungs. B. Microscopic lesions. Cloudy swelling and coagulative necrosis of the liver. congestion. coagulative necrosis of the tubules, with interstitial hemorrhages in the kidney. and marked congestion of the spleen were present. The gastrointestinal tract showed congestion and necrosis of the epithelium. In experiments with cattle, Hall (1957) fed 3 groups different species of Macrozamia leaves. To the first group he gave fresh leaves of figgzozamia 252;; guilielmi, to the second group he gave a mixture of fresh and dry leaves of Macrozamia douglasii. and to the third group he gave dry leaves of throzamia spiralis. Feeding the fresh leaves of Maggozamig pgglipggilielmi produced an acute condition with ataxia terminating fathlly between the 28th and the 29th day in 2 heifers. No symptoms were noted in the animals fed leaves exposed to the air for 2-3 weeks. At necropsy of the heifers, the subcutaneous tissue 13 and visceral fat were edematous. with many petechiae. The liver was swollen. yellow. hard to cut and apparently fibrosed. MicroscOpic sections showed advanced portal cirrhosis and some centrolobular fibrosis and vacuolated hepatic cells. The abomasal mucosa was edema- tous and showed a loss of nuclei of epithelial cells and ragged cell walls. No lesions were demonstrated in the spinal cord or brain sec- tions when stained with hematoxylin and eosin and Marchi technique. even though ataxia was present. Laboratory Animals ‘Walt (194“) drenched 3 rabbits with 15. 30, and 70 Gm.. respectively. of finely mixed nuts of Encephalartog horridgs suspended in water. The rabbits died in 1-3 days. The post-mortem changes were general icterus. severe hydrothorax. slight emphysema and severe hyperemia and edema of the lungs. regressive changes in the myocardium and kidneys. very severe regressive changes in the liver and orange-colored urine. Histological sections revealed fatty degeneration of the myocardium, with initial focal myocarditis and a few intramyocardial hemorrhages. severe hyperemia and catarrh of the bronchi, perilobular interstitial hepatitis. as well as necrotic hepatitis of central and sublobular distribution. desqua- mating reticulo-endothelial reaction and severe hyperemia of the spleen, and fatty degeneration of the renal epithelium of the proximal convo- luted tubules and. to a lesser extent, of distal convoluted tubules. Similar lesions were observed in rabbits when fresh ripe nuts of figcephalartos lehmanii were fed. Steyn gt 2;. (19h8) drenched rabbits with minced fresh nuts of Encephalartos lehmanii and Encephalartos horridus at the rate of 6.3 Gm./kg. body weight for 3 days. Before death of the rabbits he 14 observed the following symptoms: anorexia. diarrhea and apathy. The rabbits died on the third day. and the following post-mortem lesions were noted: general icterus. hydrothorax. hydroperitoneum. hyperemia and edema of the lungs with petechiae throughout the lung tissue. con- gestion of the gastrointestinal mucosa. myocardial hemorrhages. pro- nounced degenerative and fatty changes in the heart and liver. and pronounced congestion of the spleen. They described the microscopic lesions as follows: ”There were myocardial petechiae. fatty degeneration of the myocardium and an incipient focal myocarditis. pronounced congestion of the lungs and desquamative bronchial catarrh in the early stage; central and sublobular hepatitis and a perilobular interstitial hepatitis associated with fatty degeneration; pro- nounced congestion of the spleen: and fatty degenera- tion of the epithelium of tubuli contorti." In guinea pigs. by Nishida gt 5;. (1959). guinea pigs were given cycasin isolated from gyg g revoluta. They observed the following symptoms: anorexia. atonic paralysis of hind legs in 20 hours. and respiratory distress. The guinea pigs died in 2 days. They summarized post—mortem lesions as stagnation and bleeding in the lungs. Micro- scopically they noted degenerative changes in the lungs. liver. kidney and adrenals. Rats were fed by Laqueur gt 5;. (1963). 1% and 2% cycad meal obtained from nggg girginalig nut indigenous to Guam. They reported the development of tumors in the rats after chronic ingestion of cycad nut meal as a part of their diet. They found hepatocellular carcinoma and reticulo-endothelial proliferation in the liver occurring simul- taneously but independently. adenomas and undifferentiated tumors of the kidney. 1 primary pulmonary adenoma of the lung with foci of squamous metaplasia. and 2 primary mucinous adenocarcinomas of the 15 large intestines. They also reported peculiar vascular changes charac- terized by ”subintimal hyalinosis" of capillaries, limited to the undifferentiated tumor of the kidney. With acute ingestion of cycad meal they noted edema of the pancreas. centrolobular necrosis of the liver lobules and marked fatty infiltration of the myocardium. We; The pathology induced by ingestion of cycad products simulated in some respects that induced by nitrosamines and butter yellow. There- fore. studies were carried out to determine whether the lesions pro- duced by these substances were similar to those produced by cycad. Dimethylnitrosamine (CH3)2N-NO is a mobile yellow liquid with a faint characteristic odor. It boils at 151 C., and its specific gravity is 1.815 at 20 C. It is soluble in all preportions with water. ethyl alcohol. methyl chloride or vegetable oil. Uhder ordinary conditions it is stable. but with zinc dust and acetic acid it can be reduced to dimethylhydrazine (CH3)2N-NH2. Diethylnitrosamine (C2H5)2N-NO is a mobile. slightly yellow liquid. soluble in water. ether and alcohol. The nitrosamines and their derivatives are used in industries. primarily in EurOpe. in the vulcanization of rubber and preparation of textile fibers. The appearance of 2 cases of cirrhosis of the liver among 3 men working in an industrial research laboratory using dimethyl- nitrosamine led to suspicion of dimethylnitrosamine as the causative agent (Barnes gt';l.. 1954). Barnes and Magee (1954) conducted an experiment with albino rats. mice. guinea pigs. rabbits and dOgs. They gave a single oral dose of dimethylnitrosamine in water to 1 group of animals in each species. The other group was administered the chemical 16 by injection. either undiluted or in an aqueous solution. The third group was given the chemical in food first mixed in arachis oil before being added to the diet at the rate of 10 ml./kg. They reported a single oral lethal dose of less than 50 mg./kg. in rats, 15 mg./kg. in rabbits. 20 mg./kg. in mice. and 50 mg./kg. in degs. They also reported extensive liver necrosis in all species which received a single lethal dose either by mouth or by injection. At necropsy they found various amounts of blood-stained fluid in the peritoneal cavity. a jelly-like edema of the pancreas and omentum. hemorrhages into the lumen of the upper part of the intestines, and swollen. dark and mottled livers. Histologically. they reported extensive necrosis of the liver. In a second report. Magee and Barnes (1956) described development of primary hepatic tumors in rats after chronic ingestion of dimethyl- nitrosamine in their diet at the rate of 50 p.p.m. Tumors developed between 26 and 40 weeks. They classified the tumors as cystadenoma. fibrosarcoma and hepatoma. with a few'metastases to the lung. They observed bile duct hyperplasia on several occasions. In another report. Magee and Barnes (1959) described incidences of tumors in rats living 40 weeks or longer after dimethylnitrosamine (DMN) was given at various dose levels. They observed liver tumors when DMN was fed at 50, 20 and 10 p.p.m. No kidney tumors were present. When 50 p.p.m. of DMN was given for 4 weeks and then animals given a nonmal diet until the end of the experiment, there were no tumors; but when this level was fed for 2-3 months and then the animals fed a normal diet. both kidney and liver tumors were observed. When a higher dose (100 p.p.m.) was given for 1 week. no tumors were seen; but when it was given for 2-4 weeks. out of 16 rats they observed 10 kidney tumors and 17 1 liver tumor. When 200 p.p.m. of DMN was given for 1 week and then rats fed a normal diet, out of 4 rats there were 4 kidney tumors and no liver tumors. In a recent study. Magee and Barnes (1962) described the condition in which high incidence of the kidney tumors could be produced by feeding dimethylnitrosamine in the diet. In this experiment a high dose for a short time produced tumors of the kidney. without liver tumors. Schmal gtflgl. (1959) reported development of liver tumors similar to those reported by Magee gt 5;. (1956) from chronic ingestion of dimethylnitrosamine and diethylnitrosamine. Zak gtflgl. (1960) reported renal and pulmonary tumors after chronic ingestion of dimethylnitrosamine. Argus (1961) reported tumors of the kidney. liver and lung after chronic ingestion of dimethylnitrosamine and diethylnitrosamine in the diet. From this work it appeared that diethylnitrosamine is a much more rapidly acting carcinogen than DMN for the liver. less active towards the kidney and inactive towards the lung. Magee and Hultin (1962) demonstrated methylation of rat liver protein and nucleic acid when rat liver slices were incubated with DMN in,zitgg. Magee and Farber (1962) described experiments in which they had injected Clu-labeled dimethylnitrosamine intraperitoneally to rats. Their experiments demonstrated methylation of protein and nucleic acids. The methylation of protein occurred in the histidine part of the protein molecule at positions 1 and 3 of the imidazole ring. Mbthylation in nucleic acids occurred in guanine. producing 7 methylguanine. P - Dimethylaminoazobenzene butter yellow CluH15N3 18 This compound has yellow crystals. is insoluble in water. soluble in alcohol, benzene. chloroform. ether petrol. ether. mineral acids and oils. Its uses are for determination of free HCl in gastric Juice. microscopic identification of fats (particularly margarine) and also as a pH indicator. Yoshida (1932) described the first induction of tumors of the liver as a result of administration of azo dyes. Sasaki and Yoshida (1935) described the histology and histogenesis of the tumors induced by o-aminoazotoluene. They described the sequence of the changes they saw in the liver. and appeared to regard the process as starting with hyperplasia of the periportal parenchyma. which is continuously pro- gressive until the stage of cancer is reached. Heep (1936-37) described the sequence of the changes induced by o-aminoazotoluene and suggested that degenerative and regenerative changes play an important role in the induction of cancer by this compound. Kinosita (1937) reported that liver tumors were much more rapidly induced by p-dimethylaminoazobenzene than by o-aminoazotoluene and reported the primary change of the liver as hyperplastic, although degenerative changes also occur. Orr (1940) described the sequences of histological changes in the liver of white rats induced by p-dimethylaminoazobenzene. or butter yellow. The butter yellow'was added to the rats' food and the rats were killed at intervals throughout the experiments. He described the usual sequence as proliferation of connective tissue cells in the portal system. extension of granulation tissue from the latter into the parenchyma, with degeneration of the contiguous liver cells. atypi- cal regenerative proliferation of bile duct and liver epithelium. 19 leading ultimately to nonarchitectural nodular hyperplasia and a macro- scopically hobnailed liver. in which. in a certain proportion of cases. tumors arose. He described 3 types of liver tumors resulting from chronic ingestion of butter yellow in the food. The first tumor to be noted was bile-duct adenocarcinoma. which was noted after 111 days of feeding a diet containing butter yellow. Other tumors seen were hepatic- cell carcinomas and bile-duct cystadenomas. In several livers he observed a combination or occurrence of all 3 types of the tumor. or 2 types in 1 liver. Edwards and White (1941) reported pathologic changes. with special reference to pigmentation and classification of hepatic tumors. in rats fed p-dimethylaminoazobenzene. They reported early changes as extensive fatty infiltration in hepatic cells within 2 weeks. Cirrhosis of the liver was noted in 100 days. and bile duct proliferation became apparent by the 60th day. Of 106 rats. 66 developed primary hepatic carcinomas. They divided the tumors into 2 major groups - hepatomas and adenocarcinomas. They divided hepatomas into 2 subtypes, desig- nating the more highly differentiated form Type I. In this type the cells resembled the hepatic tissue very closely. The less differentiated form was designated as Type II. This showed the general structural pattern of liver. but the tumor cells had no striking resemblance to hepatic cells. They described 3 types of visceral pigmentation. One was an iron- containing. granular. brown pigment found in the liver. spleen and lymph nodes. The second pigment was found in tubular cells and macro- phages of the renal cortex. This was brown and granular and gave nega- tive tests for iron. They suggested this pigment represented deironized 20 blood pigment. The third pigment. also iron-free. was deposited in the liver and lymph nodes as a canary-yellow material and stained with lipid stains. Richardson and Borso-Nachtrebal (1950) reported develOpment of liver tumors and histological changes in other organs in rats fed an azo dye. 3'-methy1-4-dimethyl-aminoazobenzene. They described 5 types of malignant neOplasms of the livers. Three types were parenchymal tumors. which they classified as hepatomas. both of small-cell and large-cell subtypes. adenocarcinomas and anaplastic carcinomas. The other 2 types were stromal types. classified as fibrosarcomas and angiosarcomas. respectively. In addition. they noted nonmalignant biliary adenomas on several occasions. Price gtlél. (1951) described progressive microscopic alterations in the livers of rats fed the hepatic carcinogens 3‘-methy1-4-dimethyl- aminoazobenzene (3-Me-DAB) and 4-fluoro-4-dimethyl-aminoazobenzene (4'-FHDAB). The carcinogens were fed in the diet at the rate of 0.064 mg./kg. of diet. Grossly. the first changes in the livers appeared at 18 days and consisted of pallor and a fine granularity of the surface. This was more conspicuous at 21 days, at which time ascites and pancreatic edema were also seen. At 48 days. the liver appeared very cirrhotic. At laparotomy on the 9th day. 3 of 17 rats fed 3'-Me-DAB and 6 of 19 fed 4'-F-DAB had small tumors. At 111 days. 9 of 14 animals fed 3'-MelDAB and 10 of 13 fed 4'-F-DAB had gross lesions at autOpsy. At 133 days. all survivors had tumors. Micro- scopically. the earliest changes in the livers were seen after 7 days of ingestion of the carcinogen. when small hyaline droplets appeared in the eytOplasm of some of the parenchymal cells. By 11 and 14 days 21 they were found in the cells of most liver lobules. As the size of the hyaline drOplets increased. they assumed the shape of a sausage or horseshoe. and eventually some of these hyaline inclusions encircled the nuclei. The second change in the livers in rats fed 3'-Me-DAB was an increased prominence and cytOplasmic basophilia of bile ducts. This occurred between the 11th and 14th days; with 4'-F-DAB. similar changes ‘were noted at the 20th day. At 18-24 days a slight increase of bile- duct cells had occurred. At 26-28 days these cells had penetrated close to the central vein. The malignancies that were produced were classified as hepatomas, cholangiomas and mixed tumors. In addition, bile-duct adenomas were noted on several occasions. The mixed tumors were often the larger ones and presented areas characteristic of hepatomas and cholangiomas. often with blending of one type into the other. Richardson gt_gl. (1959) reported that. for development of tumors of the liver. rats need only be fed 0.06% of dimethylaminoazobenzene for 7 weeks. They obtained their results by feeding the dye for several weeks. then discontinued the dye and placed the rats on a basal diet until the end of 16 weeks. PART I. CYCAD TOXICOSIS IN SWINE AND HORSES Experiment I. Swine INTRODUCTION Weanling pigs were used as the experimental animal in the first studies on cycad toxicity because (1) they were available. (2) they were of a size that would provide for collection of ample tissue material for pathologic studies. and (3) they facilitated clinical observations. The pigs were housed atbarn number 5 at the veterinary research farm and maintained in individual metabolism cages. The cycad flour used in this experiment and all later experiments 'was prepared as follows: the nuts of 919;; circinalis were harvested and collected at various times on the island of Guam and shipped to the National Institutes of Health. The cycad flour was prepared there in Dr. Jr C. Kerestesy's laboratory by first removing the shells, cut- ting the kernels into small pieces. drying in a vacuum oven at 40 C., and then grinding into a fine powder in a Wiley mill. The cycad flour was shipped to Michigan State University and stored at -20 C. until needed. EXPERIMENTAL PROCEDURE Four Yorkshire pigs weighing approximately 50 pounds and 10 weeks of age were used. .They were divided into 2 groups of 2 pigs each, as shown in TABLE 1. A balanced. growing type swine ration obtained from the swine unit of the Animal Husbandry Department was fed. The 2 con- trol pigs were fed the basal ration, and the 2 experimental pigs were 22 23 fed the basal ration plus 2% unwashed cycad flour. The rations were fed on an agilibitum basis and fresh water was supplied continuously. The experiment was started November 7, 1962, and terminated February 5. 1963. The pigs were observed daily and weighed at weekly intervals. The calculated daily intake of cycad flour was 0.86 Gm. /kg. body weight in each of the experimental pigs. At the end of 60 days, 1 pig on each treatment was killed. necropsied and selected tissues collected for histopathologic examination. At 90 days, the remaining 2 pigs were killed, necrOpsied and tissues collected for histopathologic studies. At necropsy the tissues were collected and prepared for histOpatho- logic examination in the following general manner: the sections were selected from all macroscOpic lesions and routinely from the following organs - brain. liver. heart, pancreas, kidney. lungs, stomach, duodenum. and jejunum. They were preserved in acetate-buffered, 10% formalin. Sections of liver and kidney were fixed in Carnoy's fixative. Routine sections were stained with hematoxylin and eosin. The following special stains were used for selected materials: Heidenhain's aniline blue stain for connective tissue; Gridley's reticulum stain; periodic acid-Schiff reaction: crystal violet stain; Best's carmine; and Sudan IV. The histological and staining procedures followed were according to the Armed Forces Institute of Pathology nanual of Histological and 'Special Staining Technics (1957). RESULTS The weight gains during the experimental period and the weights of selected organs at the time the pigs were necrOpsied are given in TABLE 1. While the total number of animals was limited, the average weight gains were much less in the eXperimental pigs than in the controls. \> 24 Gross and microscOpic lesions were very evident in the experimental pigs, while no significant lesions were noted in the control pigs. The lesions noted in the 2 experimental pigs are shown in Figures 1 to 11 and given in detail as follows: Lesions Liver. The livers of experimental animals killed at 60 and 90 days appeared enlarged. firm in consistency and had whitish patchy. apparently fibrotic areas. I Microscopically. the hepatic parenchymal cells showed a widely varied histological picture. Most of the cells were enlarged. Their cytoplasm was vacuolate. Many parenchymal cells had giant nuclei and some were multinucleated. with up to 5 nuclei. The cytoplasm of the enlarged cells showed many eosinophilic hyaline droplets. Nest of the lobules showed a generalized cirrhosis with bands of fibrous connective tissue connecting more solid masses of connective tissue in centrolobular and periportal areas. In the central areas of some lobules the central veins had been obliterated by the well formed fibrous tissue. In other cases the fibrous tissue was found in the form of a ring surrounding the central vein, where the wall of the central vein remained intact but collapsed due to fibrous tissue. In such cases the collapsed small slits of central veins were seen. The central fibrosis was equally extensive whether the central veins were occluded or not. The connectivd tissue connecting portal and central fibrous tissue varied in amount. In central areas within the proliferating connective tissue hemosiderin could be observed. In portal areas there were regenerating liver cells in the form of nodules. These consisted of small liver cells which in most cases were binucleate and very closely 25 packed without having the normal orientation pattern of hepatic cells. In addition. there was a varied proportion of mature and proliferating connective tissue and. in some areas, bile duct proliferation was very extensive. Kidney. There was no visible gross lesion in the kidneys. Microscopi- cally, there was a variable amount of fibrosis of the renal cortex. The fibrosis was mainly interstitial and was associated with mononucleated cell infiltration. In a few areas the glomerular tufts were hyalinized and only eosinophilic debris remained. The Bowman's capsule had pro- liferated at its parietal layer and was continuous with the interstitial connective tissue. Lungs, Grossly the lungs were congested and edematous. The pig killed after 90 days on the experimental diet had more extensive edema of the lungs and a few ecchymotic hemorrhages of the lung parenchyma. A catarrhal bronchitis with a foamy exudate was observed in both pigs. Peritoneum. Ascites with varied characteristics was seen in the 2 experimental animals. In the pig killed after 60 days on the experimen- tal diet, 500 ml. of clear yellowish fluid were removed from the peri- toneal cavity.. In the animal killed after 90 days, 800 ml. of clear serous fluid were removed from the peritoneal cavity and about 100 ml. from the pericardial sac. Only a very small amount of fat was present in the fat depots. Pancreas. MicroscOpically. slight interstitial edema and occasional vacuolation of acinar cells was seen. 26 Heart. Grossly. the heart appeared enlarged and flabby. MicroscOpi- cally. there appeared to be a slight increase of heart muscle nuclei which had an appearance of nuclear beading in some areas. Gastrointestinal tracts. The fundic and pyloric regions of the stomach revealed necrotic ulceration. Erosions were present in the duodenum. In the remainder of the small intestine a catarrhal enteritis was present. An exudate was seen in the submucosa and mucosa of the stomach and intestine. It was predominantly serocellular, including mainly eosinophils, macrOphages and a few neutrophils. Experiment IIa Hozses INTRODUCTION Horses were suggested as a desirable experimental animal to inves- tigate further the possibilities of the cycad flour producing a Specific neurologic lesion. The nervous system of the horse has been studied in some detail. and other researchers hays mentioned that the horse is more susceptible to neurotoxins than other experimental animals. EXPERIMENTAL PROCEDURE Four male horses. approximately 2 years of age. were used in this experiment. They were maintained at the veterinary research farm. The experiment started June 19, 1963, and terminated October 26. 196A. TWO horses were assigned to an experimental group to be fed 2% unwashed 27 cycad flour in an oat basal ration, and other 2 were the controls, fed only the basal oat ration. To improve the palatability of the ration containing the cycad flour and also to hold the cycad flour with the oats, h pounds of equal parts of water and molasses were added to 12 pounds of cats and cycad flour. The cycad flour was fed to the 2 horses at the rate of .l Gm./kg. body weight for the first 15 weeks and then at the rate of .19 Gm./kg. body weight for 9 weeks. The cats were fed at the rate of .5 pound per 100 pounds body weight. Each horse was individually fed its assigned ration. The horses were weighed and blood samples collected at weekly intervals for the first 3 months and thereafter at selected intervals. Blood samples were collected for analysis using ethylene diaminotetra- acetic acid as the anticoagulant. Two to five milliliters of blood were taken for hematologic examination. Five to ten milliliters of blood were taken without using an anticoagulant for serum enzume determinations. Hemoglobin was determined by the cyanmethemoglobin method. Packed cell volumes were determined by the micro method (capil- lary tube). white blood cell counts were done with Turk's diluting fluid and hemocytometer. Blood smears were made at the time of bleeding and then stained with Wright's stain and differential white blood cell, counts made. Serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) were determined according to the methods of Reitzman and Frankel (1957), as outlined in Sigma Chemical Company's 1963 Technical Bulletin No. 505. Blood bilirubins were determined according to Benjamin (1961). 28 RESULTS The data on weight changes for both experimental and control horses are presented in TABLE 2. TABLE 3 summariées the blood seer. so? . bilirubin, hemOglobin and packed cell volume. All animals gained weight at approximately the same rate. There was a rise in SGOT after 7 days of ingestion of the diet containing cycad flour. The rise reached its maximum after 28 days of ingestion of the diet containing cycad flour. After the maximum rise there was a steady fall of SGOT. but it remained higher in the experimental than in the control, even at the end of the experiment. Bilirubin began to rise after 35 days of ingestion of the cycad-containing diet and con- tinued to rise steadily until the end of the experiment. There was no significant difference in either SGPT or hemoglobin between the control and experimental groups, although there were individual varia- tions. Gross and microscopic lesions were noted in experimental horse number 3. which was fed cycad flour. No significant lesions were observed in the 2 control horses or in the horse fed cycad for 189 days and then fed a control diet for 301 days. Lesions Liver. Several light gray-colored depressed focal lesions were seen on the surface. The lesions were roughly spherical, with a diameter of ' approXimately 6 mm. The liver was firm in consistency. The microscopic lesions are shown in Figures 12 to 17. The grossly apparent light- colored depressed areas histologically appeared as necrotic areas where necrotic hepatic’cells had been replaced by macrophages. lymphocytes and a few eosinOphils. In portal areas there was extensive fibrosis with leukocytic infiltration and bile duct proliferation. Bile duct 29 proliferation varied widely from-one lobule to the other. In some lobules it was very extensive. In portal areas with extensive fibrosis and leukocytic infiltration. the hepatic arteries had hypertrophy of their media with some cases resulting in the obliteration of the lumen. A few vessels showed periarteritis with leukocytic infiltration in the vessel walls. Hepatic cells had different manifestations of degeneration, some having a Vacuolate cytoplasm. Coagulative necrosis characterized by pyknotic nuclei was present in some lobules. A few bile canaliculi were distended with bile pigments. Kidney. There was chronic interstitial nephritis With occasional exten- sive proliferation of fibroblasts and infiltration of mononucleate cells. Lungs. Several patchy consolidated areas were seen. In addition, areas of compensatory emphysema were seen adjacent to the consolidated areas. The bronchiolar epithelium in some areas washyperplastic. This was indicated by extensions of epithelial rugae into the lumens of the bronchioles, which appeared as small papillae. Many bronchioles and bronchi were filled with desquamated bronchial epithelial cells. There were peribronchiolar lymphoid hyperplasia, alveolar edema and emphysema in several areas. Gastrointestinal tracts. The submucosa of the stomach and intestine was edematous. There were leukocytic infiltration and fibroblastic proliferation in mucosal and submucosal layers of the gastrointestinal tracts. 30 Pancreas. Slight vacuolation of cytoplasm of acinar cells was seen in several lobules of the pancreas. GENERAL DISCUSSION FOR PART I Australian workers who had observed several field cases of cycad toxicosis in farm animals described paralysis of hind limbs in various animals. In the present studies, paralysis was not observed in swine or horses. and no lesion in the nervous system was demonstrated which could be associated with cycad toxicity. In these studies pigs fed cycad flour at daily intake of .86 Gm. per kg. body weight deveIOped centrolobular and portal cirrhosis of the liver. Hall (1957) had described an advanced portal fibrosis and less extensive centrolobular fibrosis in heifers fed leaves collected from cycad plants. His histological findings, both in the liver and gastrointestinal tract. were very similar to observations recorded in this group of pigs, but there was no paralysis of hindquarters in the pigs. while Hall observed paralysis in half of his experimental animals. In the horses, the most significant lesion was the portal fibrosis, observed in horse number 3. fed cycad for 147 days. The other experi- mental horse showed no evidence of portal fibrosis. In these studies there was an elevation of SGOT in the horses fed the cycad. However, there was no elevation of SGPT values. Elevation of plasma transaminases has been reported by Cornelius (1963) following ' carbon tetrachloride ingestion in dOgs. horses. sheep and calves. - u “A: ‘35 at. l L n 99' 1. en” :I‘P‘ C .ID“9 3‘59 e noub'vl 0A ‘y. o- " ”:16 ”Q ~ ' “ML .. V :‘ng‘l A‘ "“-:- 31 While paralysis. without demonstrable.lesions in the nervous system, has been associated with cycad feeding by previous workers, no signs of central nervous disturbances were noted in these studies with large animals. This might be partially accounted for by the lack of specific information as to the nature of compounds fed by earlier workers. Since edema of tissues was a consistent finding. it would be possible to have edema in areas of the central nervous system which would pro- duce paralysis and yet be very difficult to demonstrate. In the rat studies. the report of which follows, tumor formation was a constant finding in chronic studies. No tumors were observed in the larger species. However, the duration of cycad feeding was not as long in proportion to the life span of the large animals as in the rat studies. 32 TABLE 1. Body Weight Changes. Liver, Kidney, Spleen and Heart Weights of Figs Fed 2% Cycad Pig Identity 3M* 1F*‘ ' hF 2M Treatment none none cycad cycad Days on experiment 60 90 60 90 Initial wt. (1b.) 45 b6 52 6“ Final wt. (1b.) 130 133 6h 131 Average daily gain (1b.) 1.41 1.03 .20 .98 Liver wt. (Gm.) 1352 1378 #30 1649 Liver wt.. % body wt. 2.3 2.2 1.5 2.8 Kidney wt. (Gm.) 129 --- 112 141 Kidney wt., 3: body wt. .22 um .uo .25 Heart wt. (Gm.) 1&1 --- 160 223 Spleen wt. (Gm.) 10% 113 87 1&2 *M = male *‘F female TABLE 2. 33 Body weight Changes, Liver. Kidney, Spleen and Heart‘Weights of Horses Fed Cycad W Horse Numbers 1 2 3: he Days on treatment none none 147 189 Days on experiment 490 161 lb? 490 Initial weight (1b.) 51h 5&0 750 525 Final weight (1b.) 760 880 900 1120 Avg. daily gain (1b.) 0.5 2.11 1.02 1.21 Liver wt. (Gm.) 45% 4826 #800 6810 Liver wt., % body wt. 1.32 1.20 1.17 1.33 Kidney wt. (Gm.) 908 608 5&3 1362 Kidney wt., 2 body wt. 0.26 0.15 0.11 0.23 Heart wt. (G .) --- 2607 2300 --- Spleen wt. (Gm.) --— 885 830 --- *Fed cycad flour 38 TABLE 3. Transaminase, Bilirubin. Hemoglobin and Packed Cell Volumes in Horses Fed Cycad Flour W Horse SGOT* SGPT** Bilirubin Packed cell Days Number (Sigma-Frankel units) (mg. /100 m1. ) (Gm. /100 m1. ) Volumes % 1 1 160 8 0.0 10.48 37 2 136 8 0.0 7.2 25 3* 144 8 0.0 10.4 30 4* 144 8 0.0 11.1 30 7 1 160 8 0.50 11.2 32.5 2 134 8 0.50 8.0 --— 3 168 8 0.50 10.2 28.5 4 194 9 0.56 9.2 28.5 14 1 160 8 0.5 8.1 24 2 144 8 0.5 9.0 27 3 234 9 0.75 11.4 32 4 256 9 0.50 10.0 28 21 1 160 10 1.0 8.5 21 2 172 8 0.5 10.8 28 3 520 8 0.75 10.8 31 4 440 8 0.5 9.5 26 28 1 144 8 0.75 8.7 27 2 160 8 0.5 11.7 28 3 680 8 0.5 11.0 33 4 680 9 0.5 10.8 27 35 1. 144 8 0.5 12.0 29 2 144 8 0.75 11.7 31 3 238 8 1.5 11.7 31 4 264 8 1.0 10.0 27 42 1 144 8 0.5 12.2 34 2 144 8 0.5 13.7 35 3 240 8 1.5 14.7 37 4 220 8 1.0 9.2 28 49 1 160 8 0.5 10.8 32 2 166 8 0.5 11.2 33 3 360 8 1.0 11.7 34 4 320 8 0.75 9.2 28 56 1 160 8 0.5 10.0 33 2 172 7 0.5 10.0 31 3 400 8 1.25 9.7 31 4 440 6 1.75 9.2 28 TABLE 3--continued 'Horse SGOT‘l SGPT** 35 Bilirubin Days Number (Sigma-Frankel units) (mg./100 m1.) (Gm./100 m1.) Volumes % 63 7O 77 91 105 119 168 172 240 300 160 148 192 208 C—‘WNI" ¢WNH 172 160 380 320 168 140 420 340 F‘WNP CWNH 174 160 300 300 {7me 174 365 310 «(2"me *Fed cycad flour O\O\(I) CthhCD O ‘4 8 (1) \1000000 (DLDODCD 00000000 9- 0000! increase HHOO o o e 0 OK." 0 0mm 0 HNOO Q- I—‘NOO O O. O O O mmm HNOO 0.. U‘OU‘ U1 \l". O \OO\I \) (fit'mfl \Jwflkn \IN'flkn c+ \JXNVU‘ \nNUxUt U’vOIO \JJKJOOO O .00. U1 Hb Packed cell 12.5 38 11.5 32 11.2 32 10.5 29 13.7 36 11.3 29 13.7 33 9.9 27 11.3 32 10.2 28 12.3 34 11.0 30 12.3 32 11.6 29 11.6 30 10.5 28 14.0 35 13.0 33 14.8 38 12.0 30 13.3 33 13.7 35 13.2 38 12.6 34 .93. on .5 need e38 um 333:8 «on. e e.“ 83 e3 82: .e was we 8»: octane—:4 8E 37 Fig._2.--Liver of a pig fed control diet for 60 days. H & E stain; x 75. - Fig.fi3.--Portal and centrolobular fibrogis erg; liver of pig fed 2% cycad for 60 days. H & E stain: x 75. ~Vl " Fig. 4.--Centrolobular fibrosis. Higher power of Fig. 3. Arrows indicate globules in the nuclei. H & E stain: x 187. —Fig:m5::-A small globule in thi‘nuclei.”HI§Her power 61* Fig. l}. H & E 8t‘1n: X 7500 Fig. 6.--A large globule inithe nucleus with nuclear con- tent pushed into the periphery in a form of sickle-shaped structure. Higher power of Fig. 4. H & E stain: x 750. Fig. 7.--Diffuse fibrosis in file liver o’f’pig 136325 cycad for 90 days. Section .around central vein. H & E stain; x 187. 4 Fig. 8.--Porta1 fibrosis in the liver of pig fed 2%? cycad for 90 days. Same liver as in Fig. 7. H J: E stain; x 18?. Fig. 9.--Fi§rosis in¥rena1 icortex of, pig fed 21 cycad for _ 90 days. H 8: E stain; x 75. Fig. 10.--Fibrosis in the rehal cortex of pig @721 eycer for 90 days. Higher power of Fig. 9. H 8: E stain: x 187. Fig.111.--Edemin-the subnquFof t1; stomachVOf sigma 2% cycad for 60 days. H a a stain: x 187. 42 Fig.~12.--0cc1uded hepatic artery of a horse fed cycad for 147 days. H & E stain; x 187. L4 , . . _ ringL-rbelinized hepatic arterial wall Tni'ilver of 2 horse fed cycad for 147 days. H & E stain; x 75. J; . . ". t Fig. 14.-FPortal fibrosis with bile duct proliferation and leukocytic infiltration in a liver of a horse fed cycad for 147 days. H & 8 stain: x 75. Fig:415.4-Bile duct proliferation with leukocytic infiltra- tion. Higher power of Fig. 14. H & E stain; x 187. Fig. l6.--Fibrotic phlebitis of sublobular vein of a horse fed cycad for 147 days. ”:75. H 8: E stain 17.--Fibrotic phlebitis of sublobular vein. Higher H & E stain: Horse fed cycad for 147 days. Fig. power of Fig. 16. PART II. CYCAD TOXICOSIS AND RELATED CARCINOGENS IN RATS Egpegiment Ig chad Flour Toxicosis in Rats INTRODUCTION Rats were used as the experimental animal to eXplore the possible harmful effects from cycad. because large numbers of animals could be used and they could be maintained over a longer period of time more easily than larger species. The cycad preparation was from the same source as that described in PART I. EXPERIMENTAL PROCEDURE Animal; and tgeatments ' weanling male and female Sprague-Dawley rats. 146 in number and divided into 7 groups. were used in Experiment I. The initial weights varied from 39 to 54 Gm. They were housed in groups varying from 2 to 4 in all-metal cages which were provided with coarse-mesh wire bottoms. For identification they were marked with notches in their right ears. The groups were fed varying percentages of cycad flour in a natural grain ration. The ration, designated as Michigan State University Mi ration. is listed in TABLE 4. The M1 ration was miXed thoroughly in a Hobart food mixer with weighed cycad flour in the required percentage. The mixed diets were kept in tightly closed tin cans at approximately 4 C. until they were used. Each morning weighed amounts of fresh food were placed in food containers designed so as to minimize scattering. Fresh water was 45 46 available at all times. The different groups. treatments. numbers of rats and specific details for each group are shown below. Group Number of Animals‘ 1’of Cycad Flour No. per Cage 1 15 M. 5.0 3 2 30 M. & 30 F. 2.0 2 M. & 2 F. 3 30 M. . 1.0 3 ’4 5 Me OeS 3 5 6 M. 0.25 3 6 6 H. 0.125 3 7 15 M. & 15 F. -- 2 M. & 2 F. The rats in group 1 were weighed daily and post-mortem examination done when they died. Those in group 2 were weighed daily and, during the first 7 days, 1 animal was killed each day - the one which had lost the most weight. From the 8th to the let days. 1 animal was killed at each 2-day interval. then 1 each week up to 3 months. then 1 each month until the experiment was terminated after 9 months. These rats were allowed to reproduce. and the data thus obtained on the toxicity to the newborn is reported in PART III of this thesis. In group 3 the rats were weighed and 1 killed at each weekly interval for 4 months. then at monthly intervals until the experiment was terminated after 10 months. In groups 4-6. the animals were killed at monthly intervals. In group 7. no cycad was included in the diet. The animals were weighed weekly and 1 killed at each monthly interval. all remaining animals being killed at the termination of the experiment. These rats were allowed to reproduce and were also the controls for later experiments on transmission of the toxic factors through the placenta. Before the rats were killed at specified times. blood samples were taken by cardiac puncture under light chloroform anesthesia. Then the rats were killed by severing of the spinal cord. 1‘? mama Blood samples were collected. using ethylene-diaminetetraacetic acid (EDTA) as the anticoagulant. One to two milliliters of blood were used for hematologic procedures. Three to four milliliters of blood were taken without an anticoagulant for serum enzyme determination. Hemoglobin was determined by the cyanmethemoglobin method. Packed cell volumes were determined by the micro method (capillary tube). White blood cell counts were made using Turk's diluting fluid and the hema- cytameter. Blood smears were made at the time of bleeding. then stained with wright's stain and a differential white blood cell count determined. Serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) were determined according to the methods of Reitsman and Franket (1957). as outlined in Sigma Chemical Company’s 1963 Technical Bulletin No. 505. All the animals that were killed or died were examined by standard necropsy procedures. Thin sections from all macroscopic lesions. and routinely from the following organs. were preserved in acetate-buffered. 10% formalin for histological examination: brain. liver. heart. pan- creas. kidney. lungs. stomach. duodenum. and Jejunum. Sections of liver and kidney were also fixed in Carnoy's fixative. Routine sections were stained with hematoxylin and eosin. The following special stains were used for selected tissues: Heidenhain's aniline blue stain. Gridlqy's reticulum stain. periodic acid Schiff reaction. crystal violet stain. Best's carmine. and Sudan IV. The histological and staining procedures followed were according to the Armed Forces Institute of Pathology Manual of Histglogical and Special ‘ W (1957)- 48 Selected liver and kidney sections were stained for nucleic acids, alkaline phosphatase and acid phosphatase. using cold microtome (Cryo- stat) methods. Sections of tissue approximately 0.5 mm. in thickness were flattened on aluminum foil. The foil was plunged into acetone chilled with crushed ice. taking care not to allow the acetone to come into contact with the tissue. The tissue was then frozen on the foil for 10 seconds and transferred into tightly closed bottles and kept at -20 C. until needed. The sections were cut at 8 microns thickness and while being maintained constantly at -20 C. and stained immediately. For nucleic acids. the tissue was stained.using the methyl green-Pyronin method according to Brachet (l9h0a. l9h2. 19416). For alkaline phospha- tase. the Burstone method (Burstone. 1958) was used. The ester Naphthol AS-Mx was used as substrate. For acid phosphatase. the Naphthol AS-Bl phosphate method. as described by Pearse (1961) was used. Procedures for preparation of the stains and staining were those described by Pearse in his Textbook of gistgghgmistzy. Ihgoreticgl and Applied (1961). RESULTS Information on the growth rate of rats fed various levels of cycad is given in TIBLE 5. The rats fed levels of .125. .25 and .501 cycad were essentially the same as the controls (not fed cycad) and are not included in the table. Levels of 1% and 2% cycad resulted in a slower growth rate than occurred in the controls. The 5% level of cycad flour produced such an acute toximuns that growth rate information was diffi— cult to evaluate and is not included. The major pathologic findings and the serum SGOT. SGPT. hemoglobin and packed cell volume values are summarized in TABLE 6. “9 TABLE 7 summarizes the-types of neOplasms in rats fed the various levels of eye-d flour and other'chsmiaals. The control rats were maintained for 10 months on the basal ration and at no time were pathologic changes or any abnormalities noted in any of the animals. except a high incidence of murine pneumonia. The rats fed .125. .25 or .5% cycad flour for 6 months were essen- tially normal in all respects. and pathologic changes were not observed in any of these rats. . Significant changes were noted in the rats fed the 1%. 2% and 5% cycad flour. These changes varied from acute deaths and severe lesions in rats fed 5% cycad flour to a more chronic course and chronic tissue changes. including a significant incidence of neoplasms in those fed lesser amounts. The description of the gross and microscOpic lesions is given in the following pages in detail. The specific lesions are shown in Figures 18 to 56. o l Flou giggtoms On the third day after beginning feeding the experimental,diet. the rats developed a loss of appetite. They consumed 3/# as much food as they had consumed on the previous day. On the fourth day. Q rats were found dead in the cage. 0n the sixth day. 2 others died. By the eighth day. all the rats in the group except 2 were dead. The remaining rats were in a moribund state. with difficulty in breathing. The breathing was exaggerated and abdominal. They were destroyed by severing the spinal cord. \ 0.. N: u' H a] 5'3. 50 1:932:13 Liver. Scattered ecchymotic hemorrhages were noted over the surface of the liver parenchyma. The liver was also slightly yellowish in color and friable. Microscopically. there was centrolobular hemorrhage with pronounced leukocytic infiltration. No changes were noted in the vicinity of the portal triads. lungs. The lungs of the rats which died on the fourth day appeared congested. The congestion was primarily in the apical lobes. The lungs of the rats dying between the fourth and eighth days had scattered hemorrhages varying in size from small petechial to ecchymotic in character. Microscopically. the pulmonary alveoli of the lungs of the rats dying on the fourth day were filled with serous exudates. and the lung capillaries were very congested. The pulmonary alveoli of the rats which died later were filled with either blood or serous exudate with some leukocytes. mainly eosinophils and monocytes. Several atelec- tatic areas were noted. Gastrointestinal tracts. Grossly. a catarrhal to hemorrhagic gastro- enteritis was noted with severe congestion of the duodenum. MicroscOpi- cally. there was a hemorrhagic duodenitis. W was Liver. In the first h months there were no noteworthy changes in the liver. except for occasional congestion. exaggeration of the lobular Pattern and pale depressed areas located centrolobularly. The first definite changes were seen in a rat killed at 121 days. in which the . 51 liver appeared to be small in size. firm. with several scattered whitish nodular lesions. The nodules varied from 1 to 2 mm. in diameter and were firm in consistency. .Thereafter. the incidence of the nodules increased. sometimes evenly distributed over the entire surface and at times limited to certain lobes. On no occasion did the median lobe of the liver not contain approximately a to 6 whitish nodules. The second time gross lesions were observed on the liver was in a rat killed at 205 days. The liver had a distorted surface. There were many small. hard nodules l to 2 mm. in diameter and. in addition. several translucent. cystlike nodules were noted at the periphery of the lobules. They varied from 2 to h mm. in diameter. 0n section. colorless fluid oozed from these nodules. On the ventral surface of the median lobe there was a large nodule with a depressed surface. The nodule was approximately 7 mm. in diameter and whitish in color. 0n section it was softer than the rest of the liver tissue and greasy. The animals killed after 205 days showed an increased incidence of cystlike nodules. In some livers they were very numerous and. on section. the liver had a honeycomb appearance. The incidence of grossly visible tumors also increased. Several rats had multiple tumors which varied in size. In 3 rats rupture of the tumors resulted in intra- abdominal hemorrhage. On cross section of a large tumor. necrotic and hemorrhagic patches. irregularly distributed. were noted. In 2 rats killed at 2&0 days. in addition to the whitish nodules. reddish-pink nodules about 4 to 6 mm. in diameter were noted. On sectioning. blood-red fluid oozed out. MicroscOpically. the earliest change noted in the liver was seen 3 days after ingestion of the cycad flour-containing diet. There was a 52 loss of cytoplasmic basophilia and demonstrable glycogen in the cells around the central veins. Some cells had eosinophilic condensed cyto- plasm encircling the nuclei: others had well formed. round hyaline drap- lets. The droplets appeared as intracytoplasmic inclusions with a colorless rim between them and apparently normal cytoplasm. The drop- lets were noted in cells close to the central veins. Many hepatic cells showed fatty infiltration. as demonstrated with the Sudan IV stain. on the fourth day pyknosis and karyorrhexis were noted in the nuclei of hepatic cells. An increase of alkaline phosphatase activity was recorded on the fifth day. and there was a decrease of acid phosphatase activity. On the seventh day the number of hyaline droplets had increased and there was a change in their distribution. At this time they were noted mostly around the central veins and about the portal areas. A few were seen in the midzonal area of the liver lobules. The drOplets stained magenta with the periodic acid Schiff reaction and purple with crystal violet. There was uniform centrolobular necrosis with hemorrhagic com- ponents in all lobules of the liver. In some lobules there was leuko- cytic infiltration. both in the center of the lobule and in the portal area. The leukocytes were mainly mononuclear cells. with a few neutro- phils and eosinophils. At lh days the centrolobular areas consisted of necrotic debris mixed with erythrocytes a. dying leukocytes. The rest of the liver lobules showed fatty infiltration and occasional necrotic cells in all areas. including cells around the portal triads. The fat vacuoles varied from small fine vacuoles to large ones twice the diameter of the hepatic cell nuclei. 53 Within 26 days most of the necrotic debris in the centrolobular area had disappeared. The central veins were obliterated in a few cases by collapsed reticulum framework and proliferating fibroblasts. Many macrophages were noted with ingested hemosiderin. The hepatic cells around portal triads were vacuolated. and there was slight pro- liferation of fibroblasts. with leukocytic infiltration. There were several isolated necrotic areas with mononuclear and neutrophil infil- tration. Several individual necrotic cells characterized by pyknosis and karyorrhexis and eosinophilic cytOplasms were seen among large regenerating cells. ‘lhe regenerating cells had hyperchromatic nuclei. enlarged nucleoli and vacuolate cytoplasm. In rats fed the 2% cycad for #2 days there was marked destruction of liver cells. It was more evident in the central areas than in the periphery of the lobule. Most of the central veins were obliterated by proliferating fibroblasts. In the portal areas there were both mature and poliferating fibroblasts. Bile duct proliferation was seen at this time and there were macrophages with ingested hemosiderin and eosinophils surrounding the proliferating bile ducts. In rats killed between 60 and 112 days there was diffuse liver cirrhosis of varying degrees. In addition. the Kupffer cells of may dilated sinusoids were seen prolifera- ting. In some cases a Iver of dark staining cells l+-5 cells thick. was noted continuous with the wall of the sinusoid. In other instances Similar cells were seen forming isolated islands. In these islands most 01' the cells had an elongated. spindle-shaped appearance with little Wplasm. but there were some resembling those seen in the sinusoids. Similar cells were seen around the central veins and occasionally ”‘tensively infiltrating the portal areas. Nodular hyperplasia was 51.; noted in animals killed at 121 days. Thereafter. it was seen in all rats. MicroscOpically. the nodular hyperplasia was in sharply defined rounded areas. They were composed of closely packed parenchymal cells. which lacked the normal trabecular arrangement. The cells were large. with enlarged nucleoli. The cytoplasm was acidophilic and very much vacuolated. In most cases the vacuoles contained no fat. Also charac- teristic of the lesions was the presence of many hyaline droplets in the cytoplasm of these enlarged cells. These droplets were sharply defined and often surrounded by a halo. These nodules appeared to be compressing the surrounding structures. with no capsule around them; but their acidOphilic cytoplasm contrasted sharply with the smaller. slightly basophilic hepatic cells. Bile duct proliferation was seen in all rats after #2 days on the experiment. Extent of this change was variable within each liver. In the earlier cases. clusters of bile ducts were lined with cuboidal or columnar epithelium and were associated with extensive fibrosis. In later cases of bile duct proliferation it corresponded with the gross translucent cystlike nodules. MicroscOpically. these were relatively large cavernous spaces lined by cuboidal epithelium in some cases. In others the spaces were lined by flattened endothelioid cells. The stroma between the cysts consisted of well formed. slender collagen fibers and. occasionally. normal columns of hepatic cells at different stages of degeneration. Intermediate types of cells between cuboidal and endothelioid cells were noted. lining adjacent cysts. This led to a conclusion that all were probably of the same origin. In some areas these cystic lesions were very extensive and resembled hemangiomas; knit instead of blood. they contained serous fluid. Such cases were d14agnosed as bile duct cystadenomas. 55 Nuclear changes were seen in the livers of some animals killed 60 days after ingestion of the cycad flour diet. At this time most of the liver cells were enlarged. They had large nuclei. irregular in shape. with one or more nucleoli and prominent chromatin granules. There were also l-h globules in a single nucleus. The globules were unstained with hematoxylin and eosin and appeared as a vacuole with a narrow baso- philic rim. With periodic acid Schiff reaction. their staining proper- ties varied. The nucleoli stained a green of varying shade. Some of the globules had colorless centers with red peripheries; others had small reddish granules which appeared as a precipitate. The number of the red-colored granules varied greatly. Changes in blood vessels were rare. The central veins were seen occasionally obliterated by proliferating fibroblasts and later by forma- tioh of mature connective tissue. This lesion was very variable. as in many occasions the mature tissue formed around the central vein with- out a break in the intimal layer of the vein; occlusion resulted from compression. In some cases fibrous tissue proliferation was seen partially occluding the lumen of the vessel resulting from a break in the intimal layer. In a few cases sublobular veins were seen with organized thrombi. Proliferation of the intimal layer of the vein was also noted in vessels of the lungs in a few cases. Hepatic arteries were occasionally seen. with proliferated endothelial cells forming several layers and partially occluding the lumen. The tumors of the liver were mainly of 3 types: hepatoma. hemangioma. and fibrosarcoma. 56 Hepatoma. HistolOgically, the cellular pattern varied greatly. Some tumors had large cells and other tumors were composed of compressed cells smaller than the normal liver cells but with an abnormal orienta- tion into trabeculae. ‘Where the trabeculae-like livers were seen. they appeared broader than normal. In others. the tumor had a tubular appearance with a blood sinus at the center surrounded by syncytial structures made up of hepatic-like cells. The width of the cellular column varied greatly. In all cases the hepatomas were associated with cirrhosis. Several rats had multiple tumors. In 2 cases metastasis had occurred in the lungs. Tumor emboli were seen locally in veins of other lobes of the liver. The tumor cells in some areas were very vacuolated and contained fat. Hyaline drOplets similar to those seen in adjacent normal liver cells were very numerous in tumor cells. Two cases of hemangioma. one associated with hepatoma. were seen. The fibrosarcoma type of liver tumor will be described with tumors seen in animals fed 1% cycad diet for 7 months. Kidney. The first gross lesion seen on the kidney was a tumor found in a rat killed after 168 days. Twelve of 22 rats killed between 168 and 270 days had kidney tumors. Grossly. the tumors can be divided into 3 groups: (1) small whitish solid nodules. 3-4 mm. in diameter. localized in the renal cortex or at the junction of the cortex and medulla; (2) large tumors. whitish-gray in color and firm in consistency. sometimes occupying most of the abdominal cavity (The largest tumor of this group is shown in Figure #7. It had a constriction which divided a large mass from a smaller mass - both were continuous and weighed 70 Gm. The mass was was 8.6 cm. in longest diameter.); and (3) tumors varying from 6 to 57 30 mm. in diameter. The largest of these weighed 10 Gm. These tumors were reddishawhite in color and of a fleshy. gelatinous or friable con- sistency. On cross section the tumors were cystic. and the cysts con- tained either serosanguineous fluid or blood. In 3 group-3 rats with large tumors. whitish elongated calculi. shown in Figure #9. were found in the urinary bladder. The calculi were resistant to cutting with a knife and had a few cavities. MicroscOpically. the earliest lesions in the kidney occurred 3 days after ingestion of the cycad diet. These consisted of fatty infiltra- tion in the ascending lOOps of Henle. coagulative necrosis of the proximal convoluted tubules. interstitial hemorrhages and congestion of the glomeruli. Thereafter. the lesions in the kidney progressed into chronic interstitial nephritis. characterized by proliferating fibroblasts. The first sign of a neoplastic process in the kidneys was observed in rats fed the diet for 128 days. This consisted of a nodule which microscopically was composed of cords of large cells without lumen forma- tion. The size of these solid cords of cells did not exceed that of a single tubule. The cells forming the cord were large and basophilic with rarefied cytOplasm. which was granular without vacuoles. The cell boundaries were very clear. They had vesicular or oval nuclei with rather scanty chromatin and basophilic nucleoli located eccentrically. Histologically. the kidney tumor followed the same pattern as seen grossly. The small whitish nodules were solid renal tubular adenomas (Figures #1 and #2). Microscopically. these were well organized neo- plastic masses. The tumors were sharply demarcated from the renal parenchyma by their baSOphilic cells. but they had no capsule. The adjacent tubules appeared compressed and narrow. The neOplastic struc- ture was composed of very closely packed cells with round or oval 58 vesicular nuclei. hardly any chromatin and a basophilic nucleolus. which in most cells was centrally located and in others was just next to the nuclear membrane. The boundary of the cell was seen very clearly. The cytoplasm of these cells was more deeply basophilic than that of the adjacent tubular cells. There were fine intersecting fibrous strands with capillaries. giving a clear lobular structure to the mass. The large tumors were typical fibrosarcomas (Figure 48). The cellular components were very anaplastic and pleomorphic. including cells fusiform and polyhedral in shape. The tumors showed many whorls and interwoven bundles of immature fibroblasts. The tumor cell nuclei were round and hyperchromatic. Mitotic figures were very common. In some areas there were undifferentiated cells which were light-colored and grouped together. In other areas secondary infection and necrosis were present. In addition. in a few areas tumorous cells were distributed in vacuolated basophilic mucinous stroma that resembled myxoma. .Tumors noted in the kidneys of the third group were diagnosed as embryonal nephromas. Histologically. these tumors varied from each other. and they also varied in different areas of the same tumor. One variation seemed to be a coexistence of 2 separately well formed tumors adjoining each other in the tumorous mass (Figure nu). One type was the well formed. typical solid adenoma. hist010gically almost identical to the tumors described previously. The modifications included occasional excessive necrosis. hemorrhages and a few areas within the adenoma of parts well differentiated into cystadenoma with eosinOphilic staining material within the cyst. The other tumor was very anaplastic. The main cellular components were elongated spindle-shaped cells with hyper- chromatic nuclei. In some areas these anaplastic cells were sheets and 59 cords of clearly differentiated epithelial cells surrounded by the spindle-shaped cells (Figure #5). A few of these epithelial cells were undergoing necrosis. and a few seemed to be forming glandular structures. The boundary between the adenoma and this anaplastic tumor was not marked. The anaplastic cells were invading the well differentiated adenoma. but in a few areas adenomas were clearly separated from the anaplastic tumor by bundles of collagenous fibers. There was no indi- cation that the anaplastic tumor was derived from the well differentiated adenoma. ‘Within these anaplastic tumors were dilated cavernous spaces lined with a single layer of endothelial cells. These spaces were either filled with blood or eosinophilic protein-like material with fibrin strands. In 2 cases a cluster of tumor cells was seen inside a cystic blood vessel. No metastasis was evident in any of the kidney tumors. A variation of the above tumor was one that was composed mainly of closely packed anaplastic cells with no definite arrangement. The cells were spindle-shaped with elongated or oval nuclei. The boundary of the cell cytoplasm was not clear. but nuclei were clearly demarcated by sharply hyperchromatic nuclear membranes. The tumor mass was very cystic. These cysts were of two types - one filled with blood and the other filled with a granular eosinophilic deposit with fibrinous strands. In areas where the stroma was scanty. scattered tubular structures with a single layer of cuboidal epithelium were seen. Occasionally. partially formed glomeruli were present. These consisted of well formed Bowman's capsule with poorly organized glomerular tufts. associated with elongated fibrous cells. In some areas degeneratingttubules were seen surrounded by concentrically arranged layers of elongated cells. giving an appearance 60 of whorls like those of hemangioperiqytoma of the dog. In the centers of some of these tumors there appeared to be excess capillaries with hyalinized walls. The vessels appeared as hyalinized tubules with narrow lumens which contained blood. This resembled the lesion described by Laquer _e_t 5;. (1963). Peritoneum. Ascites with fluid excess ranging between 5 and 20 ml. in amount was found occasionally. The first ascitic case occurred in a male rat_killed after 18 days in which 10 ml. of strawacolored fluid were removed from the abdominal cavity. Of the #7 rats killed between 18 and 270 days. 2“. including both males and females. had ascites. Nine of the 2h ascitic cases had no neOplasm. Ascites was serous in character except in 3 cases. in which there was intraperitoneal hemorrhage as a result of rupture of the liver tumors. Pancreas. In all cases of ascites. the pancreas had a jelly-like inter~ stitial edema. In a few rats killed after 54 days a few patchy areas. whitish-gray in color. firm in consistency and raised above the surface of the pancreas. were noted. These usually involved one or two adjacent lobes and were irregularly scattered in the organ. These lesions were not noted in the rats which were killed after 3 months on the experiment. Microscopic lesions are shown in Figures 50-53. The earliest lesion noted in the pancreas was interstitial edema seen in the rat after 6 days of ingesting the cycad-containing diet. Rats killed after 8 days had vacuolation of the cytOplasm of the acinar cells with eosino- philic staining of the nonvacuolated cytOplasm (Figure 50). No nuclear changes were seen until the 17th day. and at this time a few nuclei were pyknotic. A characteristic lesion in the pancreas was seen in rats 61 which died after 18 days of ingestion of the cycad-containing diet. This was extensive interstitial edema with infiltration of leukocytes. mainly lymphocytes. polymorphonuclear leukocytes and eosinOphils (Figure 51). A rat killed at 26 days had proliferating fibroblasts in the interstitial areas with some infiltrating into the degenerating acinar tissue (Figure 52). A rat killed at 5h days. in addition to intralobular fibrosis. had hyper- plastic pancreatic ducts and periductal fibrosis. At this time the inflammatory cells were essentially eosinOphils. One rat killed at 70 days had most of the acinar cells completely destroyed and replaced by fibrous tissue in many of the lobules (Figure 53); this area corresponded with the gray areas noted grossly. In the rats killed between 70 days and 8 months. the incidence of pancreatic lesions was low and when present consisted of microscOpic focal fibrotic areas. Heart muscle. The earliest lesion in heart muscle was noted in a rat killed after 3 days. This consisted of fatty infiltration and progressed into patchy hyalinization. After 22 days of ingestion of cycad-containing diet. a few Anitschkow cells were noted in these patchy necrotic areas. Fibrosis of these areas was noted after 31 days. These lesions were very patchy and could be easily missed. They had no uniform distribution. Lungs. The earliest lung lesion appeared in a rat killed on the 6th day. The lung was dark red and firm in consistency. with patchy areas of atelectasis and emphysema. Thereafter. the lungs had either hreas of consolidation without signs of acute inflammation or typical lesions of chronic murine pneumonia. Microscopically. the earliest lesion seen in the lung was noted h days after ingestion of the cycad-containing diet. The lung capillaries 62 were very congested. A rat killed on the 6th day had serous to hemorrhagic exudate in the pulmonary alveoli with several emphysematous alveoli. Rats killed after 18 days on the cycad diet had various lesions. Some had thickening of interalveolar septa due to increases of fibroblasts and septal cells. Several lung alveoli were atelectatic with mononuclear and eosinophilic infiltration and edema of varying degree. Several animals had chronic murine pneumonia with marked foamy cell infiltration in the alveoli. Gastrointestinal tract. In most rats no lesions were demonstrable in the digestive tract. Occasionally. catarrhal to ulcerative enteritis was noted. In one killed at 270 days. a slightly rough elevation. whitish-gray in color. was noted at the ileocecal junction. Histologically, this nodule was of glandular components. It was lined by columnar epithelium.and some had a mucous exudate. Between the glands were infiltrations with neutrophils and a few monocytes. Other lesions.' Two rats killed at 240 days had small nodules on the diaphragm. In one case the nodule was on the middle of the abdominal surface of’the diaphragm and clearly demarcated. In the other case the nodule was on the diaphragm and was adherent to the liver capsule. The nodules were whitishered in color. On sectioning. a few cavities with blood were noted. separated by distinct trabeculae of white-colored tissue. The consistency of the nodule was firm in the white part and rubbery in the reddish parts. The part adjacent to the muscle appeared to be continuous with the muscle without a definite capsule. Histologically. these were tumors made up of vascular elements in a connective tissue stroma. The vascular elements were cavernous spaces 63 filled with blood. and in some portions the components of the blood in these spaces resembled those of normal blood. The spaces were lined with a single layer of endothelium and a wall which in some areas was very thin; but in others the central areas. especially. were very thick and made up of fibrous connective tissue. This varied greatly in its cellular elements. In some cases fibroblastic proliferation was very extensive. but there was neither mitosis nor anaplasia present. The skeletal muscle was found in all parts of the tumor at different stages of degeneration. The degenerative changes seen in the muscle were atrophy. loss of striation. and hyaline degeneration. Between the muscle bundles were lines of fatty vacuoles and histiocytes with ingested pigments. Several areas had localized homepoietic centers with undifferentiated. darkly staining nucleated round cells. Occa- sionally. these dark staining cells were seen forming a ring around the necrotic muscle fibers and appeared as though they were the origin of the endothelium lining the vascular element. These lesions were diag- nosed as hemangioma of the striated muscles (Figures 55-57). G ou l cad Flo r Lesions In this group of rats. with the exception of lesions of chronic murine pneumonia. no gross lesions were demonstrable until the 8th month of ingestion of the cycad-containing diet. Liver. Six of the 10 rats killed between the 8th and 10th month had neOplasms of the liver. In one rat there was metastasis to the lungs and peritoneum. Grossly. the tumors could be divided into 2 groups: (1) large. firm and whitish in color. and (2) small. slimy and pinkish- white in color. which on cross section had small cavities. The tumor of 05$ the latter type was 1.8 cm. in diameter and was found in 3 livers. These tumors were similar in appearance to the 2 tumors seen in 2 rats fed 2% cycad and killed after 270 days. Occurring with the large tumors were smaller. discrete. whitish and shiny nodules noted when the liver was cut on cross section. MicroscOpically. the earliest changes were noted 14 days after ingestion of the cycad flour-containing diet. These consisted of slight fatty infiltration in the cells around the central veins and absence of glycogen. After 28 days of feeding the cycad ration. hyaline eosinophilic droplets with similar staining characteristics as those described in Group II rats were noted. At 56 days. ballooned hepatic cells with gigantic nuclei and occasional mitotic figures were noted. Among the enlarged cells were individual necrotic cells characterized by a con- tracted cytOplasm that surrounded the nuclei. The contracted cytOplasm left a halo between it and the cell membrane. At 84 days. proliferation of Kupffer cells lining the hepatic sinusoids were seen. In addition. isolated accumulations of cells resembling reticuloendothelial cells were seen in different locations. forming scattered islands of cells. In 2 of the 4 rats fed the diet for 8 to 10 months. reddish-white nodules were noted in the liver. These nodules were 5 mm. in diameter. Hist010gically. they were localized in the portal triads and consisted of proliferating reticuloendothelial cells. with dark staining nuclei and scanty cytoplasm. Among these cells were dilated vessels filled with blood. These vessels were lined by endothelial cells with similar nuclei. The nuclei were dark staining and elongated. Similar lesions were seen in the livers with large tumors. In some areas these lesions 65 were associated with bile duct proliferation. Occasionally. islands of reticuloendothelial cells were seen in the dilated sinusoids. Four of the 6 grossly visible liver tumors were hepatomas with a similar histological pattern as those described in Group II. The small. pinkishdwhite. slimy tumors had a sarcomatous cellular component. Histo- logical appearance of this tumor was varied (Figures 37-39). The cellular components showed great pleomorphism. In some areas the cells had scanty cytoplasm and round hyperchromatic nuclei with prominent nucleoli. These cells were closely packed. In less dense areas the cells had spindle- shaped nuclei with slightly eosinophilic cytoplasm and prolonged fibers. Among these spindle-shaped cells were cells with hyperchromatic nuclei and no cytoplasm. resembling the cells seen in the dilated sinusoids. A few contained mitotic figures. There were many dilated blood vessels in the less dense areas with cells lining the vessel resembling the dark staining areas in the sinusoids. There were other vessels lined with endothelial cells and eosinophilic staining exudate in the lumen. In less dense areas. remains of portal tracts surrounded in a few areas by 2 or 3 cords of strephic liver cells were noted. Reticulum stain demon- strated reticulum fibers continuous with the cytoplasm of the cells. These tumors were classified as reticulum cell sarcomas originating from cells lining the sinusoids or from the reticuloendothelial cells with the liver stroma. Kidney. No gross lesions were seen in this group of rats. MicroscOpi- cally. most of the rats killed after the 3rd month of ingestion of the cycad diet had slight chronic interstitial nephritis. 66 Pancreas. No gross lesions were noted. MicroscOpically. a few patchy microscOpic fibrotic areas were noted near the interlobular ducts. lungs. More than 50% of the rats had chronic murine pneumonia. One rat had.metastatic tumors in the lung. These were scattered in all the lobes. They were nodular and about 2 mm. in diameter. The nodules had a translucent. reddish center surrounded by a hemorrhagic periphery. The hemorrhagic line clearly demarcated the lesion from the rest of the lung tissue. Histologically. the metastatic tumors were hepatomas with similar cellular patterns as those found on the liver of the same rat. Other lesions. The rat with the largest whitish slimy tumor on the liver had a whitish mass 1 cm. in diameter adhered to the inner wall of the abdominal cavity. The mass was firm in consistency. Histologically. it was composed of thick fibrous sheets oriented in different directions. Among these fibers were glandular structures lined with cuboidal epi- thelium with a prominent basement membrane. The lumens of these glands contained a mucous exudate. Reticulum stain demonstrated reticulum fibers. The tumor was diagnosed as a fibroadenoma. Two rats had small oval whitish growths at the ileocecal junction. These were glandu- lar structures with a cuboidal desquamated epithelium lining. The fibrous stroma had leukocytic infiltration. mainly monocytes. The lesion appeared more or less like a granulomatous reaction. 67 Egperiment gz. Carcinogenesis in the Rats Induced by Specific Chemicals INTRODUCTION A number of chemical compounds have been known to induce carcino- genesis in animals. Miller gt 3;. (1953) demonstrated that at least 1 n-methyl group is required for carcinogenic activity of dyes related to dimethylaminoazobenzene. Both dimethylaminoazobenzene and dimethyl- nitrosamine have n-dimethyl groups, as shown. (CH3)2NNO (CHBIZWN-eflj dimethylnitrosamine P-dimethylaminoazobenzene Schoental (1960) suggested that dimethylnitrosamine owes its carcino- genic activity to its metabolic breakdown in the form of diazomethane. Argus gt él~ (1961) suggested that a similar mechanism may exist by ana10gy with diethylnitrosamine (CZHSDZNNO. A toxic constituent in the cycad nuts has been identified as cycasin (methylazoxymethyl-B-glucoside) (Matsumoto and Strong, 1963). The compound is nontoxic when administered orally. Matsumoto gt 2;. (unpublished) have demonstrated that the toxic component of cycasin is the aglycone methylazoxymethanol, or its metabolic product. Laqueur Elwéi- (1963) suggested that a similar metabolic breakdown to diazomethane could result from cycasin or its aglycone (C6H1105—O-CH2NNO=CH3). If all these compounds produce their toxic reactions only after being converted to diazomethane. it would suggest that they should produce similar lesions. This experiment was designed to test this theory. 68 EXPERIMENTAL PROCEDURE Dimethylnitrosamine. diethylnitrosamine and Pedimethylaminoazobenzene were purchased from Eastman-Kodak Company. Cycasin was obtained from the National Institutes of Health. weanling male Sprague-Dawley rats. 50 in number. were used. The rats were divided into 5 groups of 10 each and housed in individual metal cages. The dimethylnitrosamine. diethylnitrosamine and P-dimethyl- aminoazobenzene chemicals were first dissolved in olive oil before mixing with the basal M1 ration. Cycasin was dissolved in water. Small portions of Mi ration were placed in trays. The dissolved chemical was sprayed over the ration and premixed by hand. The weighed M1 ration was then added to make the required percentage and was mixed in a Hebart mixer. The diets were kept in tightly closed tin cans. as described in Experiment I. The different groups and treatment for each are as follows: Group A. 125 p.p.m. dimethylaminoazobenzene Group B. 125 p.p.m. diethylnitrosamine Group C. 1000 p.p.m. dimethylaminoazobenzene Group D. #00 p.p.m. cycasin Group E. controls. basal M1 ration The feeding routine. as described in Experiment I. was followed. In this experiment. the rats were weighed weekly and killed (1 from each group) at 3. 7. 1h, and 21 days. The remainder were kept until found dead. in a moribund condition. or were killed after 6 months. when the experiment was terminated. 69 RESULTS Information on the growth rate of rats fed various levels of carcino- genic chemicals is given in TABLE 5. All of these chemicals caused some reduction in the normal growth rate of the rats. and it was most noticeable in the rats fed dimethylnitrosamine. The major pathologic findings and serum SGOT and SGPT. hemoglobin and packed cell volume values are given in TABHE 6. No significant changes are noted in hemoglobin or packed cell volumes that could be attributed directly to the feeding of a specific chemical. However. significant increases are noted in SGOT and SGPT values. which varied somewhat with the chemicals. Most noticeable are the increased values associated with dimethylnitrosamine. The incidence and types of neoplasms associated with the various chemicals are summarized in TABLE 7. The results on the group of rats fed cycasin are not included. as most of the rats in this group were killed and sent to Dr. Laqueur at the National Institutes of Health for his examination. The results in this group will be given in another report. The dimethylnitrosamine caused deaths in the rats before neoplasm formation would have occurred. Diethylnitrosamine and dimethylaminoazo- benzene appear to be stronger hepatic carcinogens than cycad flour. The cycad flour appeared to be a specific liver carcinogen. and the ability to induce kidney neOplasms appeared to be related to the concentration Of’that fed. A higher concentration of cycad appeared to induce a liigher incidence of kidney tumors. No kidney tumors were found in the lasts fed lfi cycad. while liver tumors were noted in 60% of the rats fed ‘the same level. Two per cent cycad flour induced an incidence of 60% 7O kidney tumors. The description of the gross and microscopic lesions in the rats fed the different chemicals is described in detail in the following pages. The specific lesions are shown in Figures 57 to 68. Dimethylnitzosgmige Legions All the rats in this group except one died or were killed, when moribund. after 96 days on the diet. Deaths started after feeding the diet for 70 days. The one which survived longer than 96 days lived for 150 days. The only pathological lesion seen grossly in animals killed before 70 days was edema of the pancreas. After 70 days of experimenta- tion. the rats had various lesions in the viscera. Liver. In all animals killed or dead after 70 days. the liver appeared small and firm in consistency. There were hemorrhages on the surface varying from ecchymotic to large blood splashes. In one animal killed ‘/ at 96 days, the liver had several grayish-white nodules similar in size and appearance to those described in rats fed 2% cycad flour. In the rat killed at 150 days. the liver had both firm and cystlike nodules. Microscopically. the earliest lesions seen were noted in the hepatic cells around the central veins. These consisted of congestion of sinusoids. loss of cytoplasmic basOphilia and accumulation of fine fatty vacuoles. This occurred on the 3rdfday of the experiment. At the 7th day. there was a contraction of the cytoplasm of these cells and a characteristic eosinophilic staining. In a few lobules there was centrolobular necrosis with hemorrhagic components. A few cells had eosinophilic cytOplasmic droplets. The distribution of these droplets was very varied. At 1“ days there was an accumulation of leukocytes, mainly in the necrotic 71 central areas of the lobules and occasionally in the portal areas. At 21 days. fibroblast proliferation was noted in the necrotic debris in central areas of the lobules. At the portal areas there was a slight proliferation of fibroblasts and large oval nucleated cells resembling those of the bile duct. At 28 days many hepatic cells were enlarged and had large nuclei. A few were binucleated. The cytoplasm of these cells was eosinophilic and vacuolated. In the necrotic debris there were many macrophages and a few neutrophils. In those rats killed or that died between 70 and 96 days. the main histological lesion was extensive centrolobular hemorrhage. Many lobules appeared homageneous. containing only necrotic debris. In the midzonal and portal areas of the lobules were apparently normal cells with very vacuolated cytOplasm. Some had 1 to 4 intracytoplasmic eosinOphilic hyaline draplets. In the portal areas there was proliferation of both fibroblasts and bile ducts. Mature fibrous connective tissue was seen ‘winding around regenerating hepatic cells. Accumulation of reticulo- endothelial cells was noted in a few scattered areas. together with macrOphages containing ingested pigment. The rat killed at 96 days had nodular hyperplasia similar in histo- logical pattern to those described in rats fed 2% cycad flour. The rat killed at 150 days had dilated bile ducts. corresponding to gross cyst- like nodules. The histological picture was similar to that seen in animals fed 2% cycad. Kidney. No significant gross lesion. except paleness. was seen in this organ. The earliest histological lesion was seen after the rats had been fed the diet for 3 days. This consisted of fatty infiltration of 72 the cells of the ascending loop of Henle. At 14 days coagulative necrosis of the proximal convoluted tubules was noted. In animals killed after 1h days. different lesions were noted. In some, the kidney appeared normal. and in others there was slight interstitial nephritis with brown pigments in the cells of the proximal convoluted tubules. Peritoneum. Ascites in amounts between 20 and #5 ml. occurred in all animals killed or dying after 21 days. The ascites was hemorrhagic in character. Pancreas. In ‘11 rats killed after 21 days. the pancreas was edematous. with a jelly-like appearance. MicroscOpically. the edema was inter- ' stitial. All rats killed after 21 days had edema in varying degrees. There was no inflammatory cell infiltration in the edematous exudate. but several macrophages with ingested pigment were seen in the inter- stitial areas. There was very little precipitated material in the edematous areas. The acinar tissue had vacuolation of cytoplasm. and in 2 rats coagulative necrosis of the acinar cells. but no fibrosis. was 800?! e Lungs. Lesions in the lung varied from congestion to extensive hemorrhage. where every lung alveolus was filled with erythrocytes. Gastrointestinal tract. Hemorrhagic gastroenteritis Was observed in the anterior portion. 73 Diethylnitrosamine Lesions The chronic patholOgical lesions in this group. in general. were similar to those reported by Argus 23,31. (l96l).with the exception of the development of tumors of the vascular system. which are reported in this thesis for the first time (in rats) as far as known. Liver. The first gross liver change was seen in a rat killed after 139 days on the diet. This consisted of a great distortion in the shape of the liver. One large whitish tumor on the median lobe. measuring 2.5 cm. in diameter. was present. In addition. there were many other smaller tumors varying between 0.6 and 1 cm. in diameter. The large tumor had metastasized into the peritoneum and lung. On the peritoneum the lesions were mainly granule-like nodules. whitish in color and firm in con- sistency. The whole peritoneum was edematous. Forty milliliters of serous fluid were removed from the abdominal cavity. . Four more rats were killed between 139 and 180 days. and they all had tumors on the liver. The tumors Varied greatly in size, location and number in any one particular lobe of the liver. All the lobes con- tained tumors. Most of the tumors were whitish in color and of firm consistency. In 2 rats. tumonsof different types were seen. In one rat a reddish mass was noted on the right lateral lobe of the liver. 0n cross section. blood oozed out. The other rat had a large tumor. 2.6 cm. in diameter. which was whitish-red in color and firm in con- sistency. In addition, there were 2 small nodules. reddish in color and of soft consistency. On cross section they had cavities filled with blood. 7b MicroscOpically. in the rats killed during the first 2 weeks on the diet, the liver cells appeared essentially normal. with the exception of a very slight rarefication of the cytoplasm. At 14 days, a few hyaline droplets were seen around the central veins. A few liver cells had fatty vacuoles. At 21 days a few cells had a contracted eosinOphilic cytoplasm and intracytOplasmic droplets. Many enlarged hepatic cells were seen with enlarged nuclei. Three types of neOplasms appeared in the liver. The large tumors measuring Z-h cm. in diameter. together with many smaller ones. histo- logically were hepatomas. In 2 rats. an adenocarcinoma occurred adjacent to a hepatoma. The reddish tumors which appeared in 2 rats were hemangiomas. Hepatoma. These tumors varied considerably in histological pattern. Some were composed of small cells resembling hepatic cells with hyper- chromatic nuclei. Others had a cell type which appeared larger than normal liver cells. The cells were arranged in cords alternating with endothelial-lined blood sinuses. The sinuses were filled with neutro- phils and macrOphages. The hepatomas were associated with cirrhosis of the adjacent liver tissue. Extramedullary hematopoietic centers were noted in most of the tumors. The cytOplasm of the tumor cells had many hyaline draplets. with staining characteristics similar to those described in rats fed 2% cycad flour. The adjacent liver tissue occasionally had fatty infiltration in the hepatic cells. Adenocarcinoma. The 2 cases of adenocarcinoma were similar in histological pattern to those described for dimethylaminoazobenzene fed rats. 75 Hemangioma. These tumors were composed of large blood spaces. lined by a single layer of epithelial cells. Between the blood spaces were atrOphied hepatic cells. Meet of the stroma was made up of mature collaginous fibers. among which were proliferating fibroblasts. Within the stroma there was a.yellowish pigment. Histologically. the metastasized nodules in the peritoneum and lungs noted in one animal were similar to the adenocarcinoma in the liver of the same animal. lungs. Chronic murine pneumonia and consolidation of lung parenchyma were seen in all rats. In one rat the bronchial epithelium showed meta- plasia of the epithelium into a squamous type of epithelium with keratinization. Peritoneum. In 2 rats. ascites amounting to 40 and 50 ml. was present. The fluid was hemorrhagic in both cases. Kidney. The epithelium of the proximal convoluted tubules contained a yellowish pigment. Efdimethylgminoazobgnzene The pathological changes in the livers of the rats fed dimethyl- aminoazobenzene and liver tumors induced by this chemical have been studied in detail by Edwards gt 3;, (l9hl-t2), Kinosita (1937), Opie (l9hk). Orr (l9h0) and Price (l9h9). Changes seen in this group of animals were similar to those described by the above authors. Lesions Liver. The first gross changes were seen in a rat killed after 120 days on the experiment. These were tumors on the liver. All 5 rats killed 76 between this time and 180 days had tumors. In addition to the large solid tumors. the livers had multiple cysts, 2 to 3 mm. in diameter. In some animals the cysts were very numerous. forming localized clusters. The cysts contained clear fluid on cross section. All rats had multiple tumors. In some, all lobes of the liver were involved. The tumors varied in consistency. size and color. Their colors were whitish-gray and pinkish-red. The whitish-gray tumors had a firm and fibrous con- sistency. The largest of these tumors had a diameter of “.9 cm. Others varied from 2 to h.6 cm. in diameter. In addition. there were smaller firm nodules.about 0.6 to 1 cm. in diameter. which gave the external surface of the liver a rough and nodular appearance. Histologically. these were nodular hyperplasias. The large tumors contained necrotic and hemorrhagic centers.' The pinkish-red tumors had a soft to rubbery consistency. and on cross section they were hemorrhagic. The largest of these was seen in the rat killed after 120 days. In most cases the tumors were adherent to the adjacent diaphragm. stomach. pancreas. spleen and omentum. Grossly. metastatic tumors were seen in the lung. peritoneum. spleen. pancreas. and intestinal lymph nodes. The earliest microscOpic change in the liver was present in rats after 3 days of ingestion of the diet containing dimethylaminoazobenzene. This consisted of a characteristic eosinOphilic staining of the cytOplasm 0f cells at the periphery of the lobules. A few cells had fine fat vacuoles. as demonstrated with Sudan IV stain. At 7 days. small hyaline droplets similar in staining characteristics to those described in rats fed 2% cycad flour were present. 77 The third change in the liver was seen at the let day of the experiment. This consisted of proliferating basOphilic oval-shaped cells around the portal areas. Next to the bile ducts many of the pro- liferating cells were seen arranged in a glandular pattern with narrow lumens. Extending away from the portal areas towards the central veins the proliferating cells were arranged in a form of cell cords or sheets. Among these cell sheets neutrOphilic leukocytic infiltration was noted in a few instances. The surviving hepatic cells were very enlarged. with rarefied and vacuolated cytoplasm and an enlarged nuclei. Five types of neOplasms appeared in the livers in these rats. They were classified as bile duct adenomas. cystadenomas. adenocarcinomas. hepatomas and hemangioendotheliomas. Cystadenomas and hepatomas were generally similar in histological pattern to those described in previous animals. Bile duct adenomas. These tumors were grossly small - about 1 cm. in diameter. Histologically. they were composed of glandular structures with their lumens filled with mucous coagulated exudate. The epithelium lining these glands was atrOphied and in some was completely desquamated. Associated with these glands were wide bands of connective tissue which separated the glands. These tumors in some areas were adjacent to a hepatoma or associated with liver parenchyma with excess fatty infil- tration and many hyaline droplets within their cytoplasm. Adenocarcinoma. The large tumors were generally adenocarcinomas or adenocarcinomas associated with hepatomas. They were composed of columnar epithelium several layers thick forming glandular structures. 78 On some occasions no clear lumen was formed. The cells of the gland were hyperchromatic and had lost the normal gland cell polarity. Numerous atypical mitotic figures were seen. Some tumors had papillary projections in a cystic glandular structure. There were marked inflam- matory reactions. necrosis. and hemorrhages within and around some of the large tumors. Within the glandular structure was some connective tissue metaplasia (Figures 63 and 64). This was seen as Spindle-shaped cells proliferating in the lumen of the glandular structures and in some cases connected to the stroma of it. In 2 rats. hyaline cartilage meta- plasia was seen in the adenocarcinomas (Figures 65 and 66). In these rats similar cartilages were seen in the metastatic tumors on the peri- toneum. All grossly visible metastatic tumors in different organs histologically were adenocarcinomas. Hemangioendothelioma. Histologically. these tumors were composed of spindle-shaped cells with hyperchromatic. oval. or elongated nuclei. These cells in some areas had anastomosing processes and tended to form variable cell sheets. There were many blood spaces lined with a single layer of endothelial cells. The blood spaces varied greatly in size and were filled with blood. Mitotic figures were quite common in most of the cells. In areas with the cell sheets there was infiltration of a few macrOphages with ingested pigment. Kidney. Microscopically. the epithelium of the proximal convoluted tubules of the kidney was filled with a yellowish pigment. Peritoneum. Serous to hemorrhagic ascites, measuring from 20 to 50 m1.. occurred in all rats with tumors. 79 Pancreas. Interstitial edema of the pancreas was seen in allrats with liver tumors. In 2 rats. metastatic adenocarcinoma was present. Spleen. The spleen appeared enlarged and reddish-purple in color in most rats. MicroscOpically. the splenic sinusoids were engorged with erythrocytes. Within the splenic stroma there was a lot of hemosiderin and macrOphages with ingested pigments. In one case the splenic lymphoid tissue had been replaced with fibrous tissue. chasin The acute lesions in animals fed #00 p.p.m. of cycasin were similar to those described for rats on 2% cycad flour. The incidence and type of tumor on rats fed cycasin for a long period are still under study by Dr. Laqueur. . erimen II e Effects of edin ad Flo fo a Sho t eriod of Time on or o ation INTRODUCTION This experiment was conducted to determine if cycad flour fed to rats for a short period of time would result in a progressive lesion which might result in formation of liver tumors. EXPERIMENTAL PROCEDURE TWenty-five weanling Sprague-Dawley rats. 38 to 62 Gm. in weight. were used. The rats were put into 2 groups. 10 females and 15 males. The rats were housed in groups of h. The housing and feeding techniques were the same as those described in Experiment I. The females were fed Mi ration containing 1% cycad flour for 3 weeks. They were then fed the basal Mi ration for 6 months and killed. 80 RESULTS TABLE 7 summarizes the incidence and types of tumors observed in this experiment. Lesions Liver. The first gross lesions noted in this experiment were seen in 2 of the 10 rats. The lesions were translucent. cystlike nodules at the periphery of the liver lobules. One had 3 large nodules, and the other one had 2 small nodules. On cross section, serous fluid was noted. MicroscOpically, thé nodules were dilated bile ducts similar in llistological structure to those described in EXperiment I as cystadenomas. Piistologically, in the male rats fed the diet containing 2% cycad for 22 weeks and then normal diet for 6 to 10 months. regeneration of hepatic cells was noted occasionally. The regenerated areas were patchy in distribution and consisted of large hepatic cells very closely packed with no colmvmar pattern. The cytoplasm was vacuolated. but no fibrosis was seen. - Kiciney. In the male group of rats. the gross lesions were noted in 3 rats after consuming the 2% cycad flour for 2 weeks and then the basal I‘81:.ion for 10 months. 'One had a large oval whitish mass on the left kiciney. The tumor was firm in consistency and on cross section extended from cortex to the pelvis. The other 2 had small round nodules localized in the renal cortex. Histologically. all rats in this group had chronic interstitial nephritis which varied. greatly from one rat to the other. In some. maPk‘ed f ibroblastic proliferation was demonstrated. The tumors seen 81 O grossly in the 3 rats were diagnosed as embryonal nephromas. The tumors had in general cellular components similar to those described in Experi- ment I. in which rats were fed 2% cycad for a long period‘of time. GENERAL DISCUSSION FOR PART II The earliest histological change induced by all the carcinogens was similar.. This consisted of develOpment of hyaline draplets. or vvhat has been referred to in the literature as hyaline inclusion bodies. in the cytOplasm of the parenchymal cells. This histological altera- tion occurred between 3 and 7 days after the first ingestion of 3 of the carcinogens. In the case of diethylnitrosamine. it occurred by the 2nd week. All the hyaline droplets had similar histochemical staining characteristics. Initially, they differed in distribution. With cycad flour and the nitrosamines. the draplets were of centrolobular distri- bution and later they occurred around the portal areas. With dimethyl- aminoazobenzene they were of peripheral distribution in the first week but later were found centrolobularly. These draplets probably repre- sented a degenerative change. as they were followed by coagulative necrosis in the regions of their earliest appearance. either centro- lObularly or peripherally. The appearance of hyaline droplets was a><=o::ompanied by fatty infiltration of varying degrees. The distribution or fatty'infiltration followed that of the hyaline droplets and was r"101-e marked with dimethylaminoazobenzene than with the other carcinogens. this alteration was followed by necrosis. with or without hemorrhagic °°mponents. and infiltration of leukocytes. The differences in histological alteration were noted in animals killed on the let day after ingestion of the carcinogens. In rats fed 82 cycad flour, cycasin or the nitrosamines. the centrolobular areas were composed of collapsed central veins with leukocytes and proliferating fibroblasts within the necrotic debris. In animals fed the diet con- taining dimethylaminoazobenzene. the zone of necrosis was being replaced by extensive hyperplasia of oval basophilic cells and leukocytic infil- tration. All livers of the animals fed any one of the carcinogens had hepatic cell regeneration 'in the necrotic areas. The regenerating hepatic cells were characterized by their large size. with enlarged nuclei. and were occasionally binucleated. The nuclei had enlarged nucleoli. The caytOplasm was vacuolated. The sequence of histological changes up to this point in rats fed diets containing cycad flour is similar to that described by Laqueur (1963). who described the sequence of changes for 2 weeks. He also described in general the chronic lesions. The sequence of histolOgical changes in rats fed the dimethylaminoazobenzene diet is similar to that described by Kinosita (1937). Orr (l9lf0), Opie (1944) and Price 21; gl_. ( 19 52). In rats fed the diet containing nitrosamine. the lesions are simi- 1 lar to those described by Barnes gt 2.1: (195“). Fibrosis of the liver of the rats fed the 2% cycad flour started at, .‘the central veins. This was seen as a proliferation of fibroblasts Almost at the same time. fibrosis was develOping in the necrotic debris. wound the portal areas. In rats. diffuse fibrosis was not observed as r98111arly as in pigs. where tissues occluding the central veins were connected by thick fibrous strands with portal fibrosis. In some rats. hyperplasia of the arterial intima and lesions simu- 19ting a chronic thrombus were seen in the sublobular veins and in the 83 venules. They were in areas with extensive chronic inflammation. These are probably processes of chronic irritation. as they could not be demonstrated elsewhere. Magee _e_t_ a}. (1956) described intravascular ante-mortem thrombosis in the vessels of the liver of rats following chronic ingestion of dimethylnitrosamine. They were unable to identify the vessel due to the distortion of the liver. Laqueur 9; g_l_. (1963) described a lesion which they called reticulo- endothelial proliferation. With use of reticulum stains they could not ciecide the derivation of these cells. They considered the possibility of it being an angiomatous tumor due to the large cystic blood spaces. This lesion was followed in these animals from early develOpment of dark staining cells to formation of large tumors of similar cells. The cells were of reticuloendothelial origin. Several were seen pro- liferating from the Kupff er cells of the hepatic sinusoids. Others were seen developing around the portal areas. where they formed localized lesions. In more chronic lesions composed of similar cells. reticulum fibers were easily demonstrated with reticulum stain as projections from these spindle-shaped cells. Gillman gt 2;. (l9h9) have described pro- liferation of reticuloendothelial cells lining the sinusoids of the rat liver and also reticuloendothelial cell proliferation around central Venus and portal areas after repeated injection of trypan blue. They 1‘ Ollowed the deveIOpment of this lesion to the formation of a high incidence of what they diagnosed as reticulum cell sarcoma. The reticulum cell sarcoma tumors produced morphoIOgically resemble hUman tumors described as Kupffer cell sarcoma or hemangioendothelioma of the liver (Gray. 1929. Puhr. 1931. White. 1933. Videback. 19h6. and Baker :2 31., 1956). as summarized by Baker §_t_ gal. (1955). 8h Nodular hyperplasia of hepatic cells was common in rats after chronic ingestion of all the carcinogens. It was characterized by changes in staining and arrangement of cells. This lesion was more frequent with other carcinogens than dimethylaminoazobenzene. This lesion appears to be the one which develOps into a hepatoma, although no gradual differentiation from one to the other was noted. Its fre- quency in the rats fed the carcinogens producing a high incidence of hepatomas supports this theory. The proliferating oval cells around portal areas are the possible origin of the adenocarcinomatous tumors. which occurred in high incidence in the rats fed dimethylaminoazobenzene and not in those fed low levels of cycad flour. and in only 2 cases after the chronic ingestion of diethylnitrosamine. The adenocarcinomas and hepatomas observed were morphologically similar to the tumors produced by different carcinOgens which have been described .in the literature. Cartilage and bone formation had been noted previously in hepatic adenocarcinoma after chronic ingestion of dimethylaminoazobenzene by Edwrd and White (191d). Firminger _e_t_ 3;. (1952) have described islands of cartilage in adenocarcinoma induced by chronic ingestion of dimethylaminoazobenzene. In these studies 2 rats had cartilage islands in the adenocarcinoma. In one. sarcomatous metaplasia occurred inside the glandular part of the adenocarcinoma. Cartilage islands were found in the metastatic lesion in the peritoneum in both rats. with cartilage in the adenocar- cinoma of the liver. Richardson 3; 9;. (1951) reported angiosarcoma in rats fed . 3‘l'flethyl-Jk-dimethylaminoazobenzene. In these studies. hemangioendo- theliomas and hemangiomas were seen only in rats fed p-dimethylamino- az obenzene. 4i. i. ‘.’l Iskafll D r 85 Hemangiomas have not been reported after chronic ingestion of nitrosamine in rats. In this study. hemangiomas of the liver were seen in rats fed 2% cycad flour and in those fed diets containing diethyl- Hemangiomas of diaphragm muscle were seen in 2 animals fed Morphologically. these hemangiomas nitrosamine . a diet containing 2% cycad flour. were not different from those seen in rats injected at birth with 9 .lo-dimethyl-l-Z-benzanthrene. (Howell. 1961+) . Kirsten g_t_ $2... (1962) produced similar hemangiomas in rats by injecting polyoma virus into rats at birth. mm (1933. 193a) induced hemangioma and hemanglo-endothelioma in mice following treatment with carcinogenic hydrocarbons. Andervont (1950) had a high incidence of hemangb-endothelioma and sarcoma after treating mice with o-aminoazotoluene. Cystic ducts and cystadenomas consisted of dilated tubules lined by endothelial-like or cuboidal epithelium. Those termed cystadenoma had lumens filled with homogeneous material which grossly had a serous This type of lesion was very extensive in animals which appearance. The number of had been fed a diet containing dimethylaminoazobenzene. c33’sts and their size assembled in one spot varied widely. Histologically. all lesions were similar in all groups of animals fed diets containing the different carcinogens. The periphery of the cysts was lined by a single layer of cuboidal epithelium. while there was progressive flat- tening of the epithelial lining towards the center. resembling the end othe 1i a1 cells . Renal adenomas. with the exception of the lack of cystic appearance. in this study were in general similar to those described by Boyland 9st: 49.1. (1962) and Van Esch 93;. 5‘22 (1962) after chronic lead intoxication. They Were also similar. in general. to those described by Morris g3 _a_l_. 86 (1957) in rats after treatment with N-h-(u-fluoro)-bipheny1acetamide. Mathews and Walpole (1958) described similar adenomas in rats fed ll-fluoro-br-aminodiphenyl. They had a similar histological appearance to what Magee g; 31. (1962) described as well differentiated tumors in rats after chronic ingestion of diethylnitrosamine. Laqueur gt g_l,. (1963) fed rats cycad flour and observed adenomas morphologically similar. except for the cystic part. which was not seen in this study. However. cysts were observed in adenomatous parts of the embryonal nephromas in all animals with this tumor. The embryonal nephromas observed in rats after chronic ingestion Of 2% cycad and in rats after ingestion of 2% cycad for 2 weeks then control diet for 9 months were in general similar in histological morphology to what Magee g. g. (1962) described as anaplastic tumors after chronic ingestion of dimethylnitrosamine. However. no cartilage was seen in the embryonal nephromas in this study, while in their study they observed cartilage metaplasia in a few cases. In this study. no kidney tumors were seen in any group other than thalt fed a diet containing 2% cycad flour. Laqueur g; g. (1963) described 2 cases of sarcomas of the kidney. with one case having metastasized into the lung. No metastases were 8°01) in this study in animals fed diets containing less than 2% cycad fl<-"‘l.1r up to 9 months. but the 3 fibrosarcomas seen were very large.and thO invasive character and high number of mitotic figures leaves no doubt of their malignancy. The failure of metastasis may be related to thO lack of renal vessel involvement and early killing of the animals. Most of the rats fed diets containing one of the carcinogens de- v°l°P°d ascites of varying amounts and edema of the pancreas. Only 8 rats 87 fed the diet containing 2% cycad flour showed generalized degeneration of acinar tissue of the pancreas with extensive inflammatory reaction or fibrotic pancreatitis. Generalized pancreatic lesions were normally associated with a large amount of edema and more damage of hepatic parenchymal tissue than have been seen in the other animals. The relationship betwun fatty changes and cirrhosis of the liver. on the one hand. and pancreatic disease. on the other. have been reported in man by several investigators. Davis (19%) stated that pancreatic lesions in hashiorkor occur first and are responsible for hepatic changes. Hartz (191+9) expressed a similar opinion when describing pancreatic atrophy in infants with fatty liver. Gillman _e_t_ g. (19‘4'9). however. stated that fatty liver was the first lesion. followed by pancreatic lesion. In our work (unpublished). when weanling rats were fed milk from a cow ingesting cycad. fatty liver appeared first. followed by pancreatic lesions. In man. Steinhaus (1902) described interstitial fibrosis of the Pancreas in all of 12 cases of cirrhosis of the liver. Kirshbaum gt_. 2;. (1918) studied 356 cases of alcoholic cirrhosis and found a 363$ incidence of pancreatic fibrosis. Stinson gt gl_. (1952) compared 75 cases of cirrhosis of the liver with 75 control cases without any liver In 72 out of the 75 subjects with cirrhosis of the liver. The reaction was J~<3aions. there was some inflammatory reaction of the pancreas. characterized by interstitial infiltration of leukocytes. In the con- tI’Ols they observed 3? cases of interstitial pancreatitis. and most of the cases were mild. 88 Netik (1957) described a high frequency of pancreatic fibrosis in liver cirrhosis among Africans. No definite conclusion can be drawn as to the pathogenesis of the pancreatic lesions. but there are 3 possible contributory factors: (1) the same factors that produced the hepatic injury could have produced the pancreatic injury: (2) edema and accumulation of metabolic products around the interstitial areas of the pancreas could have produced the injury: and (3) damage of the liver could have reduced the ability of the liver to perform its normal synthesis of particular amino acids which indirectly would produce a deficiency of the particular amino acid important for maintenance of the integrity of pancreatic acinar tissue. Veghelyi ggigl. (1950) ‘produced degenerative changes and fibrosis of the pancreas in rats by :feeding yeast or gelatin as the only source of protein. The pancreatic ILesions preceded by many weeks the changes in the liver. The complete regeneration of liver cells after short-term feeding <>f rats with 2% cycad flour and development of kidney tumor was similar ‘to that described by Magee ggbgl. (1959) with dimethylnitrosamine. The incidence of the kidney tumors was very low in our series in ccmrarison ‘Vith their report. The finding of a naturally occurring carcinogen in a plant so widely used by man and having such wide distribution raises a theoretical consideration of the possible association of this plant and cancer in Knuth. Available information suggests a high liver carcinoma in all areas ‘Wllere cycads are indigenous. Higginson (1963) summarized epidemiOIOgical (iitta on primary liver carcinoma. This summary indicates a high inci- dence of primary liver cancer in Africa south of the Sahara and in southeast Asia. Higginson (1963) states: 89 "The highest incidence of primary liver cancer is reported in Bantu males from Lourenco Marques. Mozambique. where in the 25 to 3“ year age group. the rate is nearly 500 times that in the United States and 15 times that in South African Bantu males . " Kwashiorkor has been suggested. but it has been demonstrated that Ictwashiorkor itself does not lead to cirrhosis of the liver; and the liver of the young African adult is histologically normal (Broek. 1957. Higginson g; 5;” 1957. and Trowell. 1960). Higginson (1953) indicated tzfiat.liver cancer is rare in India and Latin America. where kwashiorkor its: common. There is no other single agent other than the cycad which has been sakmown to be common in these 2 communities of widely differing cultures fier.Asia and Africa. Cycad is widely used in these areas both uncooked aund cooked. Campbell (1964) demonstrated that both dry and moist heat destroy cycad toxic factors. A map of the world (Figure 68) shows the aureas of known or suspected high incidence of primary liver cancer. Clistribution of kwashiorkor and distribution of 2 cycads which are used <>ften uncooked. This distribution suggests a possible connection IJetween the ingestion of uncooked cycad products and a high incidence Of liver cancer. 90 TABLE 4. Composition of Basal Ration (M1) Fed to Rats e Ingredient Pounds per 100 1b."I Yellow corn. ground 60.70 Soybean oil meal. 50% protein 27.98 Dehydrated alfalfa meal. 17% protein 2.00 Fish solubles. condensed (50% solids) 2.50 Dried whey product, 50% lactose 2.50 DL’methionine 0.50 Calcium Carbonate (38% Ca) 1.60 Dicalcium Phosphate. 20% P. 24% Ca 1.75 Salt _ 0.50 Vitamin A. stabilized (4000 IU/Gm.) 0.20 Vitamin D3 (1500 IU/Gm.) 0.05 Choline Chloride 0.07 Total 100.00 fTo each 100 1b. of ration the following vitamins or additives 'were added: Riboflavin. 150 mg.; calcium pantothenate. 250 mg.: Niacin. 1.5 Gm.: vitamin 812. .3 mg.; Alpha Tocopherol acetate. 200 mg.: ‘vitamin K (menadione), 100 mg.; and Arsanilic acid. 4.5 Gm. TABLE 5 . 91 Average Body Weights (Gm.) of weanling Rats Fed a Basal Ration Plus Various Levels of Cycad Flour. Cycasin. Dimethylnitrosamine. (Numbers in Diethylnitrosamine or Dimethylaminoazobenzene. parentheses indicate number of rats.) W Weeks of Study, 12345791115192328 (Zontrol Male 90 141 188 216 251 320 367 397 438 457 472 478 M1 (10) (9) Female 81 123 152 161 180 210 226 236 255 264 272 276 Cycgd Flour M (88)].10 137 143 168 225 262 311 339 361 386 391 2 * '10 F (70) 95 119 126 131+ 153 177 208 217 239 258 250 10 (2ycad Flour M 90 139 181 205 238 291 340 372 410 415 446 478 15’ (10) ' (chasin M 90 108 126 194 176 240 275 306 352 374 392 396 £400 P-Pom- (18) (17) (16) (15) (13) F 81 92 100 118 133 168 183 196 218 235 236 244 (Eycasin M 90 121 194 173 206 273 316 316 403 “21 442 446 200 p.p.m. (20) (19) F 81 101 121 134 157 185 200 210 229 243 259 257 (20) (18) (17) Dimethyl- M 120 149 173 209 211 235 24-4 248 279 278 280 nitrosamine (8) (7) (5) (5) ‘ (4) 125 p.p.m. Dimethylamino- M 120 1214 153 197 157 167 166 183 210 237 244 237 azobenzene (8) (7) (6) (5) l.Gm./kg. 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N new .30: ueeho RN can 0e.— eHe: AC 35:3. 0 he.“ veg Hone—Bo den .«eu ode: A3 «nausea w you nacho KN new ueh edex A5106." .Rh ‘ Fig. l9.--Intracytop1asmic hyaline bodies in hepatic cells of a rat fed 2% cycad for 6 days. Periodic acid Schiff: x750. . i . I l - ’ v . ' ' . r U ‘ ‘ i l - ll. ‘\ o t , 1 ' ‘ ' ‘ . “ (‘2 ‘ J .; /.".$‘}(‘ .‘3 F12. 20.-;Centfil3§ulariheifrhage in the liver "ofpairaf Ted 5% cycad for 4 days. H & E. stain: x75 99 Fig. 21.--Higher power of Fig. 20. Rat fed 5% cycad for 4 days. H 8: E stain: x 187. ‘ Fig. 22.--Nodular hyperplasia in liver of a rat fed72% cycad fer 70 days. H 8: E stain: x75. I ' c .. > ' Fig. 23.--Reticuloendothelia1 cell proliferation adjacent to the sublobular vein. in the liver of a rat fed 1% cycad for 90 days. H 8: E stain: x187. D Hg. 24.--Prolfl‘fitio_n of Kuprer cellsflin a dilated’svifisoid 0f the liver of a rat fed 1% cycad for 90 days. H 8: E stain: x750. 101 Fig. 25.--Fibrotic phlebitis of sublobular vein in the ’ liver of a rat fed 2% cycad for 180 days. H & E stain: x187. Fig. '267—10rganized’thrombus' in sublobular vein in a livEr of I a rat fed 1% cycad for 180 days. H 8: E stain: x187. ‘ 102 $2.63 Henna: ANV ”heaven nachos—a. CU do»: ea» «c edeeanuedhn 5.260: :2: 25.5 6.3. 8” see .263 cacao uN coo seen can use... a: e e eases. e5 teeduuéN .eE {33.0 . In." 103 £3653qu some 28.54 .25 one use 885983551533 ee .5: oooa e8 deep on: use» see e we 8233.3 .mE A Fig. 29.--Cystadenoma. A section taken from the liverwin Fig. 28 of a rat fed 1000 p.p.m. of dimethylaminoazobenzene for 150 days. H &. E stain: x75. Fig. Sop-Cystadenomf‘ I sectlén‘faken’from a liver of a— rat fed cycad (2%) for 180 days. H 8: E stain: x75. 105 Lb: N: l Slovene: he»: «some sound i i 3 o: o , 1 1 , i ,,_, fleetiicLLii itinfiltfs LEI..- Litiiiti Tiller??? :5: 4 658.. o nee Sec Bea... 3 ed a: e do teen suntan .efl A...__ i infilfioefi 1e 106 {mica _ An .3.— 3 .8»: sin Jen e no he»: on» we eleven} e no .830: auscultmn .wg meme Jinan—330m “NV 3.825. 3 are one nee sac-sebafiefleao we add nNa see at e we tees-tan e: Fig. 34.--Hepatoma. Section taken from the liver in Fig; 32. H & E stain: x187. 818 35-';H°Pat6ma. iSeEtionitaken from the liver in fig. 33 at No. 1. H d E stain: x187. Fig. 36.--Hemngioru. Section taken from a liver of the rat in Fig. 33 at. No. 2. H a: E stain: x75. Figj'fs-Reuculum 6611 'sucbm in £15116» of flint—fa? 1% cycad for 300 days. (1) Dilated liver sinusoids. H a: E Stain: 205. Fig. 38.--Reticulum cell sarcoma in the liver of a rat fed 1% cycad for 300 days. Higher power of Fig. 37. (l) Remains of hepatic cells among the tumorous cells: (2) Remains of portal area of liver lobule. H & E stain: x187. .d' wit a Fig. 39.--Reticulum cell’sarcofia. Same section asiFigI758. Gridley's reticulum stain: x187. . ‘ r Fig. l1'0.---F‘ibroadenoma. on abdominal wall. Rat fed 1% Green for 300 days. Same rat as in Fig. 38. H & E stain: x187. Fig. 'nli-smfién‘afaazn'oma—fi the was gym fat—Ha cycad for 168 days. H & E stain: :05. 112 Fig. h2.--Solid adenoma in the kidney of a rat fed 2i cycad for 168 days. Higher power of Fig. #1. H & E stain: x187. cycad for 2#O days. Section taken at the Junction of adenoma and sarcomatous region. H & E stain: x187. 113 .33. 25.355 23 so .523 loan 2.2.2 663 can now nacho um v0.“ 3.: noun 3.3%: angers-e 5.? £2.33 mo 23.303 35:19.. .mfim seemzw mecm: 'W~. . _ . n62... 4 . e on. ..v id. Fig. u5.--ambryd£;i’fiéph£6;. in a kidney of a rat fed 2% cycad for 2&0 days. Note epithelial sheets in a less dense sarcomatous region. H & E stain: x187. Fig. Ww-Embryonal nephr’oim'er (kidney 61‘ 15(65273“ cycad for 240 days. Anaplastic sarcomatous area. H & E stain: an. kylisno 5.990% 05-3. on I as... non Nu 835 3.05. New. NHo nae. >36: are: oouenfipgwon 05 go «ion. In... 116 2% cycadr— " Section taken from kidney shown in Fig. 47. Fig. h8.--Fibrosarcoma in kidney of a rat fed H & E stain: x187. for 210 days. .esooneeonndu owned 4 5.“: pea .- no .3333 5.35.3 seam posse." afloat: .mE Fig. 50.--Edema of pancreas with vacuolation of cytoplasnr of acinar cells in a rat fed 2% cycad for 8 days. H & E stain: x187. Fig. 51.7--lnterlobulariediena wifiiTeEkbEy€131nr11t§tibn "“ in the pancreas of a rat fed 2% cycad for 18 days. H & E stain: x187. Fig. 52.--Early fibrosis in acinar tissue of the pancreaT of a rat fed 2% cycad for 26 days. H 8: E stain: x187. L, , a, ,,.,,,,,, _. Fig. 53.--Replacsment of acinar tissue by fibrous connective ‘ tissue in the pancreas of a rat fed 2% cycad for 70 days. H 8: E stain: x187. Fig. 5a.--Patchy fibrotic myocarditis of a rat fed 2% cycad for 31 days. H & E stain: x187. Fig.77575.--Herhang7iomaion diaiphragnTof 7a rat Fed 2% cycadwri for 2&0 days. Section taken from the center of the lesion. H & E stain: x187. I- I Fig. 56.--Hemangioma on the diaphragm of arrat Ted 2% cycad for 200 days. Section taken at the periphery of the lesion. where lesion appeared more fibrotic. Same rat as in Fig. 55. H & E stain: x187. Fig. 57.--Hun&xgi’6fiin a chafing. outfit fed 2% 'cycTaE for 200 days. H & E stain: x75. 122 .eflefioaeoesev4 AC “agnofiovceuedmnasom any “asouanom Ad .23. o? .5 osousfiosaofilaafiofle «o .adi 82 e8 53 as €33 a: q «o 5.33.3 .mE . v A ,0 Fig. 59.--Hemangio-endothelioma in the liver ofia rat fed 1000 p.p.m. of dimethylaminoazobenzene for 150 days. Section taken from the liver in Fig. 58. at No. 2. H & E stain: x75. , ‘5;g1§3. . . “‘3.” ‘~A€(.*. x. ’. . - ‘ s p u Fig. bO.--Hemangio-endotheliéma in the liVer’ofHa’rat. 7 Higher power of Fig. 59. H & E stain: x187. 124 2332?: “NV “30523053 3U .nhev on." new efiageueesdsedhfiesdv no .sdd 08H can soon. we: no.3: .«eu a no 85.7.23 .mE Fig. 62.--Adenocarcinoma in the liver of a rat fgdiiooo' p.p.m. dimethylaminoazobenzene for 150 days. Section taken from the' liver in Fig. 61. H & E stain: x187. Fig. 63.--Fibrous metaplasia in the adenocarcinoma in the liver of a rat ted 1000 p.p.m. dimethylandnoazobenzene. H a. E stain: x75. Fig. 6#.--Fibrous metaplasia in adenocarcinoma in the liver of a rat. Higher power of Fig. 63. H & E stain: x187. Fig. 65.--Cartilage metaplasia in adenocarcinona in the liver of a rat fed 1000 p.p.m. dimethylaminoazobenzene for 150 days. H & E stain: x75. ‘0. . . ~ ’4 t Fig. 66.--Cartilage netaplasia in adenocarcinoma in"the liver of a rat fed 1000 p.p.m. dimethylaminoazobenzene for 150 days. Higher power of Fig. 65. H & E stain: x187. Fig. 67.--Bile duct adenoma adjacent to apparently normal hepatic cells in the liver of a rat fed 1000 p.p.m. dimethylamino- azobenzene for 150 days. H & E stain: x187. .55.. e5 5 anon—"ea! not: 23 no eon-305 5.3 23 use Keane“; me 83.5.5.8 33.. 38.9 no .38.: no... is. u «o 8333 .5 geese-ea as. Seaside en 332.055 \ ._. 52.. 5 33:2: utzcue D . \ e «sofas: .. . I .324 2. $42.02. oueouamam . x . ; a z .8223 .. 23:35:»; e \ . e. / nae»... Q moeciazauezu .fls 128 PART III. TRANSVISSION OF CYCAD TOXI" FaCiQhS AND CHEMICAL CARCIKOGENS THROUGH THE MAMMARY GLAED AND PLACENTA Experiment I. Transmission Through the Mammary Gland and the Placenta ' INTRODUCTION In Exoeriment I (Part II) it was noted that the young born to .he females fed 2% cycad flour died 2 to u days after birth. while suclling their mothers. Therefore, additional studies were conducted to determine the cause of death. At oarturition the young were destrcved immediately by severing of the spinal cord. The livers were taken and fixed in acetate-buffered 10% formalin. Sections were stained with hematoxylin and eosin and Sudan IV for fat. On histologic examination all sections showed coagulative necrosis of the hepatic cells and occasional fat vacuoles. These lesions were not observed in the young born to mothers fed the basal ration or in voung nursing their mothers and fed the basal ration. These observations suggested the transmission of the toxic factor(s) associated with cycad flour and ssecific carcinogens through the placenta and mammary gland. Two trials were conducted. the first on transfer of factor(s) through_the milk and the second through the placenta. Trial 1. Transmission Through the Hammarz_Gland EXPERIMENTAL PRO EDURE Pregnant females in the latter stages of gestation were used. They were fed the basal ration as previously described until immediately after 129 130 parturition. Approximately 2 days before parturition they were placed in an individual cage. After parturition the young were counted and examined for any gross abnormality. The mothers were then fed various levels of cycad flour (.5. l and 2%) or selected levels of cycasin, dimethylnitrosamine. diethylnitrosamine and dimethylaminoazobenzene. The number of females fed each ration and the level of the specific substance in each diet are given in TABLE 8. The diet containing the required amount of the chemical under test was placed in a container designed to prevent spillage. The feed cup was securely fixed on the top of a platform in the cage. The platform was of such a height that even at weaning the young rats could not get to the diet. The mother could only get at the feed by jumping up on the feeder. Usually, 2 young in each litter were killed at 2, 4, 7, 14 or 21 days of age or when found in a moribund state. The mothers were killed 21 days after parturition. The livers, kidneys. lungs and hearts were fixed in acetate-buffered 10% formalin. Sections were stained with hematoxylin and eosin. Selected tissues were stained with Sudan IV for fat. RESULTS TABLE 8 summarizes the litter size, birth weight and other results. including the histological lesions seen at the various ages and the con- centration of the chemicals used in the diet. Figures 75 to 82 show lthe specific histological lesions. 131 chad Eloug Lesions Liver. The earliest lesion seen in a litter suckling a mother fed 2% cycad flour was a vacuolation of the hepatic cells. This was first observed on the nth day of suckling. On the 7th day the vacuolation was very marked in the livers from the rats in some litters. The vacuolates varied as fine vacuolates up to the size of hepatic cell nuclei. Young killed at l“ or 21 days had various hepatic cell changes. These varied from fatty infiltration to coagulative necrosis. Signifi- cant variations were noted in different litters and also on lobes of the same livers. In some litters, even after 21 days of suckling, no lesions were evident. The litters of the females fed 2% cycad flour usually died between 6 and 18 days of age, with the exception of one litter in which 5 were weaned. These. after weaning, were fed the control diet. In the pups suckling mothers fed the diet containing 1% cycad flour, the earliest lesion was seen after 7 days. This was fatty infiltration of the hepatic cells. At lfi days. necrosis of hepatic cells was demon- strated in some of the livers. Fourteen young were weaned from the litters of the mother fed 1% cycad flour. These were also fed the con- trol diet at weaning. They were all killed after 6 months or were killed when found in a moribund state. chasin Lesions Liver. In the livers of the young suckling 2 mothers fed 400 p.p.m. cycasin, the earliest lesions were seen on the 5th day. In one of these litters all the pups except 1 were dead by the 5th day. Histologically. 132 there were varying degrees of hemorrhagic necrosis of hepatic lobules. It was very extensive in some animals and only slight in others. Two of the 3 litters from the mothers fed the 600 p.p.m. cycasin and the litter from a mother fed 400 p.p.m. cycasin died between 5 and 21 days of age and histologically there were lesions of the hepatic cells varying from fatty infiltration to coagulative necrosis with hemorrhagic components. Kidney. There was a slight degeneration of the epithelium of both the proximal and distal convoluted tubules varying from cloudy swelling to fatty infiltration with coagulative necrosis. The lesions were seen only in the young after 14 days of suckling. The other litter from a mother fed 600 p.p.m. cycasin had 11 pups but only 3 survived until weaning. They were then fed the basal ration. In the livers of pups suckled by mothers fed rations containing 300 p.p.m. cycasin, only fatty infiltration could be demonstrated after 14 days of suckling. and no lesions were noted in the kidney. Most of the litters survived until weaning and were then changed to the control diet. Dimethylnitrosamine Lgsiogs Liver. Pups suckling mothers fed 150 or 200 p.p.m. of dimethylnitrosamine died between 8 and 21 days of age. Those suckling mothers fed 100 p.p.m. lived to weaning. The first histological lesion seen in the liver of these rats was after suckling for 7 days. This consisted of fatty infiltration and centrolobular hemorrhage. The rats killed or dying between 8 and 21 days had histological lesions Varying from slight fatty infiltration to extensive necrosis with blood lagoons. The variation here 133 was very great, in that very extensive lesions could be detected in all lobes of the liver. while others had only slight fatty infiltration. One mtstanding lesion was noted in a rat killed after 8 days of suckling. In this rat the right lateral lobe appeared dark purple in color. It was firm to the touch, while the rest of the lobules had an apparently normal appearance. Histologically. 80% of the right lateral lobe was destroyed and consisted of bloody cysts filled with erythro- cytes and necrotic debris. The liver capsule was intact. so that the blood appeared as if inside a pocket formed by the liver capsule. The rest of the hepatic cells surrounding the hemorrhagic area had only vacuolation of the cytOplasm. At the portal areas there was leukocytic infiltration with bile duct proliferation. The other lobes had slight to heavy fatty infiltration without necrosis. Diethylnitrosamine Lesions Liver. The pups suckling the mother fed 200 p.p.m. of diethylnitrosamine all died on the 4th day of suckling. No histological lesions could be demonstrated. The pups from a dam fed 150 p.p.m. diethylnitrosamine had fatty infiltration of the liver after 14 days of suckling. No necrosis was demonstrated, even at 21 days. Those not killed lived to weaning and were then fed the control diet. Dimethylaminoazobenzene Lesions liver. The litter suckling the mother fed lOOO p.p.m. of dimethylamino- azobenzene died between 8 and 12 days. Those from dams fed 750 p.p.m. dimethylaminoazobenzene died between 6 and 21 days. Those from dams 134 fed SOO p.p.m. of dimethylaminoazobenzene, when not killed, lived to weaning and were fed the control diet. After 6 days the rats suckling mothers fed rations containing either 750 or 1000 p.p.m. dimethylaminoazobenzene had livers which had fatty infiltration and cloudy swelling. Between 14 and 21 days fatty infil- tration and rarification of the hepatic cell cytOplasm were seen. In a few cases the cytoplasm had eosinOphilic staining characteristics but no nuclear changes. indicating necrosis was present. No lesions were seen in pups suckling dams fed SOO p.p.m. of dimethylaminoazobenzene. Lactatigg Mothers at 21 Days At weaning the mother rats fed diets containing various chemicals while suckling their young were killed. Their livers were processed in the same manner as the livers of the young. The histological lesions of the liver were compared with nonlactating rats fed diets containing similar chemicals and killed after 21 days of feeding. The lesions in the livers of lactating dams were mild in some cases, while in others they were similar to those of nonlactating rats of the same age and sex. Trial 2. Transmission Through the Placenta EXPERIMENTAL PROCEDURE To obtain information on the transmission of the toxic factor(s) in cycad flour, 13 mature female rats were used. Some were from Experi- ment I. Part 2, and some were purchased. The procedure was to place a male with 3 or 4 females and then feed them all the cycad-containing ‘ ration. When it was evident the female was pregnant. she was placed in an individual cage and continued on the same ration. After parturition 135 the female was fed the basal ration. Sixty pups, randomly selected from 13 litters, were used to study the pathologic effects of the cycad flour on the newborn fetus. To study the transmission of specific carcinogens through the pla- centa, the results of the histopathologic examination of the young of mothers each fed a specific chemical were used. “ESULTS The results of feeding the different levels of cycad flour are summarized in the introduction to this experimen . The carcinogen, the level fed and number of young examined are summariZed in TABLE 9. None 'f the substances at the levels fed interfered in conception or caused abortions. Figures 69 throurh 74 show the specific liver sections. All livers of the pups destroyed immediately after birth from dams fed the various chemicals had necrotic hepatitis. There was a disappearance of the hepatic cell cytoplasm. The cell membranes and walls of the blood vessels were intact. Liver lobules in some areas appeared as a sheet with well demarcated empty spaces outlined by cell membranes. Erythrocytes within the blood vessels were normal. and no hemolysis was detected. Experiment I I . LongiTerm Effects of Exposing Rats During Sucklinggt o Carcinogens Transmitted Through Their Dam's Milk INTQODUCTION This experiment was conducted to see whether the rats that survived after suckling dams fed diets containing various chemicals would develop tumors if kept on a control diet for a long time. 136 EXPERIKENTAL PROCEDURE After weaning, the rats that survived suckling mothers fed diets containing various chemicals (Experiment I. Part 3) were fed the control ration for 26 weeks. Their weights were recorded weekly. Those killed or found dead were examined according to standard necropsy procedures. Liver, kidney and pancreas were routinely fixed in 10% acetate-buffered formalin. Sections were stained with hematoxylin and eosin. TABLE 10 gives the growth rates of the rats exposed to various chemicals during nursing. There was no obvious decline in the normal growth rate in any animals except those which had suckled dams fed dimethylnitrosamine and 300 p.p.m. cycasin. One rat from a dam fed BOO p.p.m. cycasin died in 4 days. One that had nursed a mother fed dimethylnitrosamine died at 8 weeks. Lesions Liver. Microscooic lesions were present in 2 pups from dams fed dimethylnitrosamine. In the rat that died 8 weeks postweaning. the liver parenchymal cells had fatty vacuolation and the sinusoids were very congested. In the other rat killed at 26 weeks postweaning, the liver parenchyma had patchy areas of vacuolated cytoplasm. In the rats that had suckled mothers fed GOO p.p.m. cycasin some lesions were noted. In the rat which died 4 days postweaning, the liver was grossly congested. Microsc0pically. the hepatic cells were at different stages of degeneration. Fatty infiltration and necrosis of hepatic cells were noted.‘ The sinusoids were congested. 137 Kidney. In the 2 rats that survived while fed the control diet for 26 weeks, one had bilateral tumorous masses in the kidneys. The right kidney had a whitish mass oval in shape, about 1 cm. in diameter, at the junction of the cortex and medulla. The left kidney had 2 different nodules whitish in color. One extended from the cortex of the kidney to the pelvis, and the other was a small nodule 3 mm. in diameter. situated in the cortex. The other rat had a very small nodule in the right kidney which was only visible during the trimming of the tissue. It was situated in the renal cortex. All the tumors microscOpically were similar. They had sarcomatous characteristics. The cellular components were elongated with oval nuclei. Mitotic figures were very common. In general these tumors resembled the sarcomatous areas of the tumors described as embryonal nephroma in weanling rats fed 2% cycad but they did not have adenomatous components. In some areas there were many cysts (Figure 95). as described previously in embryonal nephroma. The tumors were diagnosed as undifferentiated cell sarcoma. The unin- volved part of the kidney had chronic interstitial nephritis with dilated tubules. Experiment III. Transmission of Toxic Factor(s) in the Milk of Swine and Cattle INTRODUCTION This experiment was designed to determine whether cycad toxic factor(s) would be transmitted through the milk of other species besides the rat. 138 EXPERIMENTAL PROCEDURE A lactating cow with a lO-day-old calf and a lactating sow with a litter of 9 newborn pigs were used in this experiment. The cow was fed 0.5 pound of a mixture of oats and ground shelled corn per 100 pounds body weight with 2% unwashed cycad flour incorporated into it. This ration was fed for 76 days. At the end of this time, the leval of cycad was increased to hfi and maintained at this level for another On days. Blood samples were collected each week from the jugular vein and analyses were carried out as in Experiment I. Both the calf and the cow were weighed weekly. After 1&9 days of the experiment. both the cow and the calf were killed. NecrOpsy was done according to stan- dard procedures. The sow was fed a standard pig ration to which was added 2% unwashed cycad flour. This ration was started 4 days after parturition. After 77 days, while still nursing the sow. 3 pigs (2 males and l female) were killed. The sow was killed 21 days later. NecrOpsy was performed in all animals according to standard procedures. At no time during the experiment were the suckling pigs permitted access to the damfs feed. Of the 6 remaining pigs, 3 males were killed at 22% days and the remaining 3 females were killed at 254.days after each had a litter of normal pigs. TAQLE 11 gives the weekly serum 36 T. SGPT. bilirubin, hemoglobin and packed cell volume values of the cow and the calf. Figures 83 to 92 show the lesions present in the cow and calf. A steady rise of serum SGOT appeared after 7 days on the experiment in both the cow and the calf. This rise in serum SGOT continued for more than 35 days in the ‘5 all. I ill! I. II} If L.) 13, \ calf and for 63 days in the cow and then started to decrease. After a steady decrease of serum SGOT values there was slight rise of serum SGPT and bilirubin values. Bilirubin Values increased steadily until the termination of the experiment. In both the calf and cow, the values I were higher at the termination of the experiment than at the beginning. Lesions - Cow Liver. This organ had small pale gray depressed lesions on the surface. The lesions varied in size from 1 to 5 mm. in diameter and appeared as necrotic focal lesions (Figure 82). The grossly depressed areas. histologically, were extensive necrotic foci. where many of the hepatic cells had been turned into a homogeneous eosinophilic debris. Within the necrotic areas was leuko- cytic infiltration. mainly eosinophils. monocytes and a few neutrophils. These lesions involved more than one lobule. and it was difficult to judge whether they were principally more of the portal or centrolobular lesions. Away from these lesions the portal areas showed chronic pro- liferation of connective tissue with m rked leukocytic infiltration. In some areas bile duct proliferation was quite extensive. while in other areas it was only slight. Around the large bile ducts there was exten- sive fibrosis, and the epithelial lining of the ducts had necrotic changes. Some of the bile duct columnar epithelium contained intracytoplasmic hyaline drOplets. These hyaline bodies were clearly demarcated from the rest of the cytoplasm by hollow spaces. In the rest of the liver lobules, hepatic cells showed different stages of degeneration. The cytoplasm of the hepatic cells appeared vacuolated. 1:30 Kidney. No apparent gross lesion was noted in the kidney. MicroscOpi- pally. some degree of interstitial nephritis with mononucleated cell infiltration was noted. Peritoneum. There was about 200 ml. of serous fluid in the abdominal cavity. Pancreas. In the interstitial areas. fat necrosis was seen between lobules of the pancreas. The acinar tissue had vacuolation of the cyto- plasm. A few cells had a complete loss of the cytOplasm which had been replaced by vacuoles. The vacuolation only occurred at the periphery I of the pancreatic lobules. A few lobules had leukocytic infiltration in the interstitial areas. Lungs. Several ecchymotic hemorrhages were noted on the surface of the lungs. Patchy areas of consolidation were seen on the apical, cardiac. intermediate and anterior part of the diaphragmatic lobes of the lungs. This involved about 40% of the lung tissue. The consolidated areas were noncrepitating on pressure and were dark brown in color, with patchy purplish-red areas. The consolidated areas were associated with a few emphysematous lobules. On sectioning the involved tissue bulged out. It appeared coarsely lobulated, the interlobular septum being thickened and edematous. Microscopically the interlobular septa were edematous and had heavy leukocytic infiltration. The leukocytes were mainly eosinOphils and lymphocytes. There was peribronchial pneumonia with rings of lympho- cytes, eosinOphils and mononucleated leukocytes surrounding the terminal bronchioles. The alveoli were distended and filled with cellular lul exudate. which stained pink with hematoxylin and eosin. Occasionally. dense fibrinoid material filled the alveoli and some alveoli had fibrin strands within the exudate. Several lung arterioles had thickened walls as a result of intimal proliferation. In a few instances the arterial walls were infiltrated with inflammatory cells. Spleen. There was both capsular and parenchymal edema in the spleen. Heart muscle. Heart muscle had vacuolation of muscle bundles. A few bundles showed hyaline degeneration. These lesions were very patchy in distribution. In a few areas there were Anitschkow cells. Gastrointestinal tract. The serosal surface of the abomasum. omasum and omentum had ecchymotic hemorrhages. In the fundic region of the abo- masum the mucosa appeared thickened and rough. There was catarrhal enteritis in the anterior part of the small intestine. Microscopically, the fundic glands appeared hyperplastic. A few had several layers of epithelial lining. There appeared to be an excess amount of fibrous connective tissue with the lamina propria associated with leukocytic infiltration in the edematous areas. In the duodenum there was mucinous degeneration of the epithelial lining and an increase of fibrous connective tissue in the lamina propria. Brunner's glands showed some atrOphy and were being replaced by rings of proliferating connective tissue and infiltrating leukocytes. These lesions within the lamina propria extended into all parts of the intestinal tract. The submucosa was edematous the whole length of the intestine. 1&2 Lgéigns - Calf The gross pathology of the suckling calf resembled very much that of the dam. The specific lesions in the cow and calf are shown in Figures 83 to 92. In the liver the necrotic lesions were smaller in the calf than in the dam, as shown in Figures 82 and 83. The heart appeared enlarged and flabby. MicrOSCOpically, the lesions were similar to those of the mother. In some instances the lesions were more marked in the calf than in the dam. In the lung of the calf the lesions were more exten- sive and more chronic. The pink staining exudate was very extensive in every alveolusand it appeared as an eosinOphilic membrane. Associated with the exudate was a heavy leukocytic infiltration. Thromboses and periarteritis were more extensive in the lung arterioles of the calf. Interlobular bronchi had mucous degeneration with inflammatory exudate in the bronchiole lumen. The areas between the lining epithelium and Reisseisen's muscle were edematous and extended by leukocytic infiltra- tion. Fibrotic patchy lesions were seen on the heart muscle of the calf which were not seen in the cow. Lesions - Sow Liver. Grossly. the liver appeared firm in consistency. Microscopically. there was a slight increase of fibrous connective tissue in the portal area. as demonstrated with Heidenhain's aniline blue stain. In several lobules of the liver centrolobular fibrosis was present. Kidney. No lesion was detected grossly. Microscopically. nephritis with proliferation of connective tissue was noted. Some renal tubules had indications of hyperplasia characterized by formation of several 143 layers thick of cellular sheets. These epithelial sheets had very closely packed cells. and their cytOplasm was either clear or ba50philic in staining characteristics. Some tubules were dilated and were filled with hyaline casts. Gastrointestinal tract. MicroscOpically. the epithelium of both the stomach and intestines had mucinous degeneration. The part of the lamina propria adjacent to the epithelial lining had extensive lymphocytic infiltration associated with a few neutrophils and eosinOphils. In the duodenal area the lamina prepria appeared hyperplastic with an increase of glands of Lieberkuhn. Lesions - Pigs Liver. In general, grossly there were many necrotic foci varying between 3 and 6 mm. in diameter. These were seen on the livers of all the pigs that suckled the mother while fed the cycad diet. The number and size varied from one pig to the other. Microscopically, the liver had extensive portal fibrosis with large bands of connective tissue dividing the lobules into pseudolobules and extending to the central veins. A few lobules had a well established fibrosis in the central vein areas. The grossly apparent necrotic lesions in histological sections were areas with most of the hepatic cells destroyed and being replaced by proliferating fibroblasts with leukocytic infiltration. Normally, these lesions involved morethan one lobule. Six of the pigs were kept on natural grain ration after weaning and were maintained on this ration for ZZO—ZSQ days at which time they 'were killed and necropsy performed according to standard procedures. lab Grossly. no lesions were seen in any of the pigs. Microsc0pically. in all the pigs there appeared to be more collaginous fibers than normal in the portal area.‘ In one male pig, portal and centrolobular fibrosis was noted in a few liver lobules. The fibrosis was less in extent than that noted in pigs killed after 77 days. GENERAL DISCUSSION OF TRANSMISSIOJ EXPERIMENTS The transmission of these carcinogens, including the toxic factor in cycad flour through placental and mammary membranes, is considered a significant finding. The transmission through the mammary gland was not as definite in the case of dimethylaminoazobenzene and diethylnitrosa- mine. as determined from histological changes of the livers of the rat pups. However. mortality occurred in the pups at the higher dosage, ‘which hardly affected the lactating animal. With cycasin. cycad flour and dimethylnitrosamine pronounced changes were observed in livers of the pups, but variations were marked between litters, between pups in the same litter. and between different lobules of the same liver. In recent years considerable interest has been expressed in the presence of insecticides in bovine milk. Marth gt 3;. (1959) have characterized insecticide products in milk and in milk products. Davson 23 al. (l9h3) were the first to hypothesize that certain com- pounds may penetrate membranes in their unionized lipid soluble state. Schanker (1962) summarized the passage of drugs across the membrane in accordance with their lipid solubility. Rasmussen (1953 and 1959), in studies with cows and goats, demonstrated that mammary gland epithelium was permeable to the unionized form of drugs and almost impermeable to the ionized forms. Sisodia gt al. (l95h) stated that in essence. the 1&5 concept of passive diffusion was that the unionized fraction of many compounds crosses the biological membrane in consonance with their lipid and water solubility coefficient. The rate of crossing in the case of mammary glands depends on PKa and milk pH, according to Sisodia _t _l. (l9é4). Rasmussen's studies demonstrated that basic compounds appeared in milk in a higher concentration than in plasma. An increase in the pH of the milk increased the concentration of the drug in the milk. The passage of drugs across the placenta has been shown to depend to a certain degree upon lipid solubility of the drugs. The compounds which have been detected in fetal blood or tissues shortly after administra- tion to the mother include all the anesthetic gases and vapors, many barbiturates. sulfonamides. salicylates, quinine. methadone and meperidine. all of which have moderate solubility in lipids (Argar gt gl., 1960, Baker. 1960, and Marx. 1961). Dancis (1953) showed that esteriol and the lipid-soluble metabolites of estradiol readily traverse the placenta in either direction. while the lipid-insoluble glucomides of these compounds hardly cross at all. All the carcinogens used in these studies are lipid-soluble to a varying degree, and their passage across the placenta] and mammary barriers may be in the same manner as other lipid-soluble unionized acidic and basic compounds. . cycad plants are widely distributed in semitropical areas and are widely used as food by man and animals. They are also used as a medicine for both adults and children. The present studies suggest that both the dietary and medicinal use of cycad and its products may have deleterious effects. This is based on the finding that in all these species of animal studies, the 1&6 toxic cycad factors were transmitted to the young both during fetal develOpment and during nursing. The situation among human beings differs from the present study in that cycad nut is cooked in water prior to consumption. This pro- cedure inactivates the toxic factor and renders the cycad flour innocuous. However, there is a possibility that milk cows in those areas of the world where cycad grows may ingest some parts of the plant. The toxic substance in these plants may be transmitted to the infants fed this milk. FUrther studies should be carried out to determine the potential danger associated with this possibility. The present studies suggest that the toxic substances present in cycad may be eliminated through the milk and can also pass through the placental tissue. 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