Toxicants Occurring Naturally in Foods. (1966)

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12 J. H. WILLS, JR. Goitrogen Antagonists In addition to these more or less positively goitrogenic components of foods, there are also a few negative ones. These latter are substances that by their absence from foods lead to epithelial hyperplasia of the thyroid gland. Foremost among these is iodine.38:76-78 Avitaminosis A in the presence of a deficient intake of iodine, at least in the rat, brings about marked hyperplasia of the thyroid gland in both sexes,79 8° with later atrophy in the male,’? an abnormal uptake of iodine by the gland,®° and a decrease in formation of thyroxin.8° Thus, the well-known effects on epithelial structures of deficiency of vitamin A appear to extend to the epithelium of the thyroid gland. Vitamin D seems also to have some effect on the response of the thyroid gland to goitrogens. The hyperplasia of the thyroid gland pro- duced ordinarily in animals during winter by administration of aceto- nitrile has been found to be prevented by either exposure of the animals to ultraviolet radiation or administration of vitamin D to the animals.! Another possible factor of this general sort has been identified by Neguib,®2 who reported that in hyperthyroidism about 50 percent of the magnesium of the serum is ionized and that in myxedema all the mag- nesium of the serum is in the ionized form although only about 80 percent is ionized in normal sera. By administration of daily intra- muscular injections of MgCl2 (2.8 g) to three patients with hyper- thyroidism and two patients with nontoxic goiter, he was able to effect a diminution in the size of the thyroid and amelioration of any toxic symptoms that happened to be present. An indirect effect on the thyroid that may be mentioned in a discus- sion of the relation of diet to the production of goiter has been adduced by Van Middlesworth.83 He has shown that reabsorption from the intestinal lumen of the thyroxine excreted in the bile is hampered by the presence in the chyme of indigestible or unabsorbable materials. Such interference with reabsorption of thyroxine may be sufficient to result in enlargement of the thyroid even in the presence of an intake of iodine that would be sufficient ordinarily to ensure an euthyroid status. SUMMARY To sum up, there are several dietary influences of comparatively minor importance that can intensify or even induce enlargement of the thyroid gland. Among these are the presence in the diet of arsenic,

GOITROGENS 13 calcium, cobalt, copper, ergothioneine, noniodine halides, polysulfides, sulfhydryl compounds, and the unidentified goitrogens of soybeans, peanut skins, and liver; the presence of large amounts of indigestible or unabsorbable materials; and the lack in the diet of vitamin A, vitamin D, or readily absorbable magnesium salts. More important dietary factors probably are the cyanoglycosides, the thiocyanates, and the derivatives of 2-thiooxazolidone. The two last are involved in goitrogenic activity by Brassicae and other members of the family of Cruciferae, with 3-indolylacetonitrile and polysulfides contributing apparently to the goitrogenic effect of some Brassicae, at least. Probably the most potent of the naturally occurring goitrogens is ]-5-vinyl-2-thiooxazolidone, found in seeds of many Brassicae and in edible portions of turnip and rutabaga in the form of a precursor. The enzyme that liberates the oxazolidone from its precursor, as well as those involved in the formation of thiocyanates from cyanoglycosides, is destroyed by cooking, so that thoroughly cooked turnip and rutabaga lose much of their goitrogenic potentiality. Much of the goitrogenic activity that passes into certain milks seems to be destroyed by either scalding or freezing of the milk, so that ice cream made from goitro- genic milk is much less active than the milk itself. The same thing is true of custards, puddings, and other cooked dishes prepared from goitrogenic milk. An increased intake of iodine will antagonize, in part at least, the goitrogenic actions of the various known and unidentified goitrogens in foods. In those instances in which the goitrogen interferes not only with the initial uptake of iodine by the thyroid gland but also with its incorporation into iodinated derivatives of tyrosine, an increased intake of iodine will not antagonize completely the goitrogenic action. In using iodides as antagonists of goitrogenic actions by active goitrogens, the fact must be kept in mind that iodine itself can be a goitrogen. REFERENCES 1. M.A. Greer, ‘“‘Nutrition and Goiter,” Physiol. Rev., 30, 513 (1950). 2. M. B. Fertman and G. M. Curtis, ““Foods and the Genesis of Goiter,” J. Clin. Endocrinol., 11, 1361 (1951). F. W. Clements, “Naturally Occurring Goitrogens,”’ Brit. Med. Bull., 16, 133 (1960). A. I. Shtenberg, Yu. I. Okorokova, and Yu. N. Eremin, “‘Alimentarny Faktor i Endemicheskii Zob,”” Usp. Sovrem. Biol., 55, 255 (1963). ¥ >

14 10. 11. 12. 13. 14. 15. 16. 17, 18. 19. 21. 22. 24. J. H. WILLS, JR. A. M. Chesney, T. A. Clawson, and B. Webster, “‘Endemic Goitre in Rabbits I. Incidence and Characteristics,” Bull. Johns Hopkins Hosp., 43, 261 (1928). . B. Webster, T. A. Clawson, and A. M. Chesney, Endemic Goitre in Rabbits IT. Heat Production in Goitrous and Non-goitrous Animals,” Bull. Johns Hopkins Hosp., 43, 278 (1928). B. Webster and A. M. Chesney, “Studies in the Etiology of Simple Goiter,” Am. J. Pathol., 6, 275 (1930). D. Marine, E. J. Baumann, and A. Cipra, “Studies on Simple Goiter Produced by Cabbage and Other Vegetables,” Proc. Soc. Exptl. Biol. Med., 26, 822 (1929). E. J. Baumann, A. Cipra, and D. Marine, “Nature of the Goiter Producing Substance in Cabbage,” Proc. Soc. Exptl. Biol. Med., 28, 1017 (1931). C. E. Hercus, and H. D. Purves, “Studies on Endemic and Experimental Goitre,”’ J. Hyg., 36, 182 (1936). M. A. Greer, M. G. Ettlinger, and E. B. Astwood, “Dietary Factors in the Pathogenesis of Simple Goiter,” J. Clin. Endocrinol., 9, 1069 (1949). M. A. Greer and E. B. Astwood, “The Antithyroid Effect of Certain Foods in Man as Determined with Radioactive Iodine,” Endocrinol., 43, 105 (1948). E. B. Astwood, M. A. Greer, and M. G. Ettlinger, “‘/-5-Vinyl-2-thiooxazolidone, an Antithyroid Compound from Yellow Turnip and from Brassica Seeds,” J. Biol. Chem., 181, 121 (1949). C. Y. Hopkins, “‘A Sulfur-Containing Substance from the Seed of Conringia orientalis,” Can. J. Res. B16, 341 (1938). E. B. Astwood, A. Bissell, and A. M. Hughes, “Further Studies on the Chemical Nature of Compounds which Inhibit the Function of the Thyroid Gland,” Endocrinology, 37, 456 (1945). H. D. Purves, “Studies on Experimental Goitre. IV. The Effect of Diiodotyrosine and Thyroxine on the Goitrogenic Action of Brassica Seeds,” Brit. J. Exptl. Pathol., 24, 171 (1943). D. Marine, “Studies on the Etiology of Goitre Including Grave’s Disease,” Ann. Internal Med., 4, 423 (1930). D. Marine, “The Pathogenesis and Prevention of Simple or Endemic Goiter,” J. Am. Med. Assoc., 104, 2334 (1935). W. E. Griesbach, T. H. Kennedy, and H. D. Purves, “‘Studies on Experimental Goitre. III. The Effect of Goitrogenic Diet on Hypophysectomized Rats,” Brit. J. Exptl. Pathol., 22, 249 (1941). . W. E. Griesbach and H. D. Purves, “Studies on Experimental Goitre. V. Pituitary Function in Relation to Goitrogenesis and Thyroidectomy,”’ Brit. J. Exptl. Pathol., 24, 174 (1943). W. E. Griesbach, “Studies on Experimental Goitre. IT. Changes in the Anterior Pituitary of the Rat Produced by Brassica Seed Diet,” Brit. J. Exptl. Pathol., 22, 245 (1941). J. B. Wyngaarden, J. B. Stanbury, and C. H. Du Toit, “On the Mechanism of Iodide Accumulation by the Thyroid Gland,” J. Clin. Endocrinol., 11, 1259 (1951). M. A. Greer, “Isolation from Rutabaga Seed of Progoitrin, the Precursor of the Naturally Occurring Antithyroid Compound, Goitrin (/-5-Vinyl-2-thio- oxazolidone),” J. Am. Chem. Soc., 78, 1260 (1956). J. Sedlak, “Cultivation of Goitrogenous and Nongoitrogenous Cabbage,” Nature, 192, 377 (1961).

GOITROGENS 15 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 41. 42. 43. P. Langer, J. Sedlak, and N. Michajlovskij, ““O strumigennych latkach potravy z hladiska nasich experimentalnych studii,” Bratisl. Lekarske Listy, 42, 330 (1962). P. Langer, “‘Regionalne a sezonne rozdiely hladiny tiokyanidu v_ sere obyvatelstva slovenska a jej vztahy k primijmu strumigennych potravin,” Bratisl. Lekarske Listy, 42, 393 (1962). J. Sedlak, ““Changes in Protein-Bound Sulfhydryl Group Concentration in the Liver of Rats Fed Cabbage,” Experientia, 19, 478 (1963). M. H. Wald, H. A. Lindberg, and M. H. Barker, “The Toxic Manifestations of Thiocyanates,” J. Am. Med. Assoc., 112, 1120 (1939). W. Forster, and E. Muscholl, ““Der Einfluss von BAL, Methionin, und Cystin auf die Schilddriisenhyperplasie nach kurzfristiger Gabe von Methylthiouracil,”’ Arch. Exptl. Pathol. Pharmakol., 222, 388 (1954). A. Kjaer, R. Gmelin, and R. B. Jensen, “‘Isothiocyanstes XVI. Glucoconringiin, the Natural Precursor of 5,5-Dimethyl-2-oxazolidinethione,” Acta Chem. Scand., 10, 432 (1956). A. Kjaer and R. Gmelin, “Isothiocyanates XXVIII. A New Isothiocyanate Glucoside (Glucobarbarin) Furnishing (-) 5-Phenyl-2-oxazolidenethione upon Enzymatic Hydrolysis,” Acta Chem. Scand., 11, 906 (1957). A. Kjaer and R. Gmelin, “Isothiocyanates XXXIII. An Isothiocyanate Glucoside (Glucobarbarin) of Reseda luteola L.”” Acta Chem. Scand., 12, 1693 (1958). R. McCarrison, “‘The Interaction of Food and Sanitary Conditions in the Causation and Prevention of Thyroid Disease,”” Indian J. Med. Res., 20, 957 (1933). M. H. Barker, “The Blood Cyanates in the Treatment of Hypertension,” J. Am. Med. Assoc., 106, 762 (1936). N. Michajlovskij and P. Langer, “Studien iiber Beziehungen zwischen Rhodanbildung und kropfbildender Eigenschaft von Nahrungsmitteln I. Gehald einiger Nahrungsmittel an praformiertem Rhodanid,” Z. Physiol. Chem., 312, 26 (1958). F. W. Clements and J. W. Wishart, “‘A Thyroid-Blocking Agent in the Etiology of Endemic Goiter,”” Metabolism, 5, 623 (1956). R. Greene, H. Farran, and R. F. Glascock, ““Goitrogens in Milk,” J. Endocrinol., 17, 272 (1958). F. W. Clements, ““Thyroid Disease: Public Health Aspects,” Med. J. Australia, ii, 823 (1958). P. Peltola, “Goitrogenic Effect of Cow’s Milk from the Goitre District of Finland,” Acta Endocrinol., 34, 121 (1960). M. Kreula and M. Kiesvaara, “‘Determination of /-5-Vinyl-2-thiooxazolidone from Plant Material and Milk,” Acta Chem. Scand., 13, 1375 (1959). S. Bobek and A. Pelczarska, “‘Thiocyanate-Level in the Serum and Thyroid of Cows from Areas with Different Intensities of Goitre in Human Beings,” Nature, 198, 1002 (1963). E. Wright, “Goitrogen of Milk Produced on Kale,” Nature, 181, 1602, 1603 (1958). P. Langer and N. Michajlovskij, ‘“‘Praformiertes Rhodanid in Nahrungsmitteln als Hauptursache der Rhodanausscheidung im Harn bei Tier und Mensch,” Z. Physiol. Chem., 312, 31 (1958).

16 45. 47. 49. 31. 52. 53. 35. 56. 37. 58. 59. 61. 62. 63. 65. J. H. WILLS, JR. E. B. Astwood, “The Chemical Nature of Compounds which Inhibit the Func- tion of the Thyroid Gland,” J. Pharmacol. Exptl. Therap., 78, 79 (1943). W. P. Vander Laan and A. Bissell, “‘Effects of Propylthiouracil and of Potassium Thiocyanate on the Uptake of Iodine by the Thyroid Gland of the Rat,” Endocrinology, 39, 157 (1946). E. J. Baumann and N. Metzger, “Action of Thiocyanates in Producing Goiter,” Federation Proc., 6, 237 (1947). W. T. Salter, R. E. Cortell, and E. A. McKay, “Goitrogenic Agents and Thy- roidal Iodine: Their Pharmacodynamic Interplay upon Thyroid Function,” J. Pharmacol. Exptl. Therap, 85, 310 (1945). R. E. Remington, “Improved Growth in Rats on Iodine Deficient Diets,” J. Nutr., 13, 223 (1937). H. C. Hou, “Thyroid Enlargement Following Liver Feeding in Rats,” Proc. Soc. Exptl. Biol. Med., 43, 753 (1940). G. R. Sharpless, J. Pearsons, and G. S. Prato, “Production cf Goiter in Rats with Raw and with Treated Soy Bean Flour,” J. Nutr., 17, 545 (1939). H. S. Wilgus, Jr., F. X. Gassner, A. R. Patton, and R. G. Gustavson, “The Goitrogenicity of Soybeans,” J. Nutr., 22, 43 (1941). A. W. Halverson, M. Zepplin, and E. B. Hart, “Relation of Iodine to the Goitrogenic Properties of Soybeans,” J. Nutr., 38, 115 (1949). J. A. Ripp, “Soybean-Induced Goiter,” Am. J. Diseases Children, 102, 106 (1961). R. H. Williams, H. Jaffe, and B. Soloman, “Effect of Halides on the Anti- thyroid Action of Thiouracil and Propylthiouracil,’” Am. J. Med. Sci., 219, 1 (1950). D. C. Wilson, “Fluorine in the Aetiology of Endemic Goitre,”’ Lancet, 240, 211 (1941). D. G. Steyn, “Fluorine and Endemic Goitre,” S. African Med. J., 22, 525 (1948). A. K. Shtifanova, “Soderzhanie Ftora v Vode, Pochve 1 rastitelnykh produktakh nekotorykh raionov alma-atinskoi oblasti i ego rol v etiologii kariesa zubov i endemicheskogo zoba,” Zdravookhranenie Kazakhstana, 22 (No. 5), 60 (1962). J. Thompson, “‘Influence of the Intake of Calcium on the Thyroid Gland of the Albino Rat,” Arch. Pathol., 16, 211 (1933). I. Gedalia and N. Brand, “The Relationship of Fluoride and Iodine in Drinking Water in the Occurrence of Goiter,”? Arch. Internat. Pharmacodyn., 142, 312 (1963). E. Hesse, “Die Entgiftung des Schilddriisenhormons durch Metalle und natiirliche Quellen,”’ Klin. Wochschr., 12, 1060 (1933). M. Scott, ““The Possible Role of Arsenic in the Etiology of Goiter, Cretinism and Endemic Deaf-mutism,” Trans. 3rd Intern. Goiter Conf., 34 (1938). G. R. Sharpless and M. Metzger, “‘Arsenic and Goiter,” J. Nutr., 21, 341 (1941). M. G. Kolomiitseva, ‘‘Soderzhanie Kobalta v Pochve, Vode, Pishchevykh Produktakh, Pastbishchnykh v Raione Zobnoi Endemii,” Zravookhranenie Kazakhstana, 22 (No. 6), 55 (1962). M. G. Kolomiitseva, ‘‘Soderzhanie i Sootnoshenie Mikroelementov (Ioda, Kobalta i Medi) v Tkani Normalnoi i Zobno Izmenennoi Shchistovidnoi Zhelezy,” Probl. Endokrinol. Gormonoterap., 7 (No. 6), 63 (1961). A. Lawson and C. Rimington, “Antithyroid Activity of Ergothioneine, A Normal Component of Blood,” Lancet, 252, 586 (1947).

GOITROGENS 17 66. 67. 68. 69. 70. 71. 72. 73. 74, 75. 76. 77. 78. 79. 80. 81. 82. 83. E. B. Astwood and M. M. Stanley, “Antithyroid Activity of Ergothioneine in Man,” Lancet, 253, 905 (1947). A. Lawson and C. Rimington, ‘““Comments (on paper by Astwood and Stanley [66]),”’ Lancet, 253, 906 (1947). R. Pitt-Rivers, ““The Action of Antithyroid Substances on the Formation Jn Vitro of Acetylthyroxine from Acetyldiiodotyrosine,” Biochim. Biophys. Acta, 2, 311 (1948). M. L. Wilson and D. A. McGinty, “Antithyroid Activity of Ergothioneine,” Am. J. Physiol., 156, 377 (1949). V. Srinivasan, N. R. Moudgal, and P. S. Sarma, “Studies on Goitrogenic Agents in Food I. Goitrogenic Action of Groundnut,” J. Nutr., 61, 87 (1957). N. R. Moudgal, E. Raghupathy, and P. S. Sarma, “Studies on Goitrogenic Agents in Food III. Goitrogenic Action of Some Glycosides Isolated from Edible Nuts,” J. Nutr., 66, 291 (1958). H. B. Henbest, E. R. H. Jones, and G. F. Smith, “Isolation of a New Plant- Growth Hormone, 3-Indoleacetonitrile,” J. Chem. Soc., 3796 (1953). L. Jirousek, “Zur Bildung des Rhodanidiones aus Weisskohlinhaltsstoffen in vivo,” Naturwiss., 42, 536 (1955). L. Jirousek, ‘““Zur Frage des Brassica-Faktors und des endemischen Kropfes,” Endokrinol., 33, 310 (1956). L. Jirousek and L. Starka, “Uber das Vorkommen von Trithionen (1,2-di- thiacyclopent-4-en-3-thione) in Brassicapflanzen,”’ Naturwiss., 45, 386 (1958). D. Marine and O. P. Kimball, “The Prevention of Simple Goiter in Man,” J. Am. Med. Assoc., 77, 1068 (1921). N. S. Scrimshaw, A. Cabezas, F. Castillo, and J. Mendez, “Effect of Potassium Iodate on Endemic Goitre and Protein-Bound Iodine Levels in School- Children,” Lancet, 265, 166 (1953). J. A. Maisterrena, E. Tovar, A. Cancino, and O. Serrano, “Nutrition and Endemic Goiter in Mexico,” J. Clin. Endocrinol. Metabol., 24, 166 (1964). H. M. Coplan and M. M. Sampson, “The Effects of a Deficiency of Iodine and Vitamin A on the Thyroid Gland of the Albino Rat,” J. Nutr., 9, 469 (1935). M. B. Lipsett and R. J. Winzler, “Effects of Vitamin A Deficiency on Thyroid Function Studied with Radioactive Iodine,” Endocrinology, 41, 494 (1947). F. Bruman and A. Blomberg, “‘Die Bedeutung des Vitamin D bei der durch Acetonitril erzeugbaren Schilddriisenhyperplasie,”’ Z. Ges. Exptl. Med., 97, 229 (1935). M. A. Neguib, “Effect of Magnesium on the Thyroid,” Lancet, 280, 1405 (1963). L. Van Middlesworth, “Thyroxine Excretion, a Possible Cause of Goiter,”’ Endocrinology, 61, 570 (1957).

MARTIN STOB Estrogens in Foods Association of changes in the vaginal epithelium with the various stages of the estrous cycle32 led to the development of an assay for estrogens based on cornification of the vaginal mucosa.2° This simple method for detecting female sex hormones led to the accumulation of a vast literature reporting the occurrence of estrogenic activity in a wide variety of substances. An even more widespread distribution of estro- gens was reported following development of a more sensitive means of detection based on uterine hypertrophy.? Some doubt was cast, how- ever, on the validity of assays based solely on uterine hypertrophy, since, by definition, an estrogen is a substance capable of producing growth of the vagina, uterus, mammary gland, and female secondary characteristics.4 In addition, uterine hypertrophy can be elicited by progesterone and androgens; however, the amounts of these com- pounds required to stimulate uterine growth are very much greater than amounts of estrogen capable of producing a similar effect.!5 The term uterotrophic has been used to describe compounds that are able to cause uterine hypertrophy but may not necessarily produce any other physiological effects commonly associated with estrogens.4° Another term, proestrogen, has been suggested to describe compounds that produce vaginal cornification when administered systemically, but not intravaginally, suggesting conversion of a nonestrogenic substance to an estrogenic metabolite.!4 In the following discussion, estrogen will be the term applied to detection of activity associated with either vaginal cornification or uterine hypertrophy or both. 18

ESTROGENS 19 Estrogens in Plant Products Estrogenic activity has been detected in many plants commonly used for food: carrots,!6 soybeans,!9 wheat, rice, oats, barley, potatoes, apples, cherries, plums, garlic, sage leaves, parsley, and licorice root,§ and in wheat bran, wheat germ, rice bran, and rice polish.5 Edible oils in which estrogenic activity has been reported are cottonseed, safflower, wheat germ, corn, linseed, peanut, olive, soybean, coconut, and refined or crude rice bran oil.5 The estrogenic activity of pollené may be responsible for the reported estrogenicity of honey.§ Eleven chemically characterized estrogens have been isolated from plant sources. The identity and source of these compounds are pre- sented in Table 1. Genistein, genistin, and daidzein have been isolated from a plant commonly used for human food—soybeans;* however, these compounds are weak estrogens, as are all the isoflavones, cou- mestrol, and anethole.3-!! Other isoflavones isolated from apple skins,?9 citrus fruits,!2:13 and buckwheat?® are not estrogenic.39 Although corn infected with a common fungal pathogen, Gibberella zeae, may contain an estrogen as yet chemically unidentified,*° it is unlikely that any of the affected grain would be used for food product preparation. TABLE 1 Name, Chemical Classification, and Source of Estrogens Isolated from Plants CHEMICAL REFER- ESTROGEN CLASSIFICATION SOURCE ENCE Genistein (4’ ,5,7-Trihydroxyisoflavone) Isoflavone Soybeans 6 Genistin (Genistein-7-glucoside) Isoflavone Soybeans 6 Biochanin A (5, 7-Dihydroxy-4’-me- Isoflavone Red clover 6 thoxyisoflavone) Prunetin (4’ , 5-Dihydroxy-7-me- Isoflavone Prunus spp. 6 thoxyisoflavone) Daidzein (4’ , 7-Dihydroxyisoflavone) Isoflavone Soybeans 4 Formononetin (7-Hydroxy-4-me- Isoflavone Red clover 27 thoxyisoflavone) Coumestrol (4’, 7-Dihydroxybenzo- Coumarin Alfalfa 2 furocoumarin) Anethole Stilbene Anise oil 6 Estrone Steroid Palm kernel 6 Estriol Steroid Willow catkin 6 Miroestrol Phenanthrene Pueraria sp. 19

20 MARTIN STOB Estrogens in Animal Products Estrogens have not been detected in a wide variety of animal products. Liver may contain estrogen'® because of the role of this organ in hormone metabolism.? Estrogenic activity has been detected in the yolk of eggs in small23 but physiologically active! quantities. Milk may contain some estrogen, particularly the colostrum,2633 which, however, is not channeled into food products. Although the epithelial cells of the bovine mammary gland are relatively impervious to diethylstilbestrol,33 there is some indication that milk produced by pregnant cows may contain increasing amounts of estrogenic activity as pregnancy ad- vances.25 33 This might well be estradiol 178 which is the major ovarian estrogen of the cow6 to which the mammary gland is slightly perme- able.2! Animal fat may contain estrogens* but this is most likely to be the case only after treatment of the animal with sex hormones!735 4! and then only following administration of certain compounds.!7 Normally bovine fat is nonestrogenic.28 3! Hazards of Naturally Occurring Estrogens There seems to be little danger in consuming food products that have been shown to contain some estrogenic activity in spite of the depres- sion of growth in rats22 and inhibition of testicular development in mice fed genistein24 or because of the role of estrogens in neoplasia.® Since estrogens found in plants are very weakly estrogenic,3 consump- tion of foods containing these compounds in quantities sufficient to produce an estrogenic response is a virtual impossibility. The small amounts of estrogens in milk?! or egg yolk? preclude ingestion of these foods in amounts sufficient to elicit a physiological response. Prolonged treatment of women with a very potent estrogen, diethylstilbestrol, in amounts far in excess of that naturally occurring in feeds, has not contributed to any increase in the incidence of mammary or uterine carcinoma.3’ In addition, apparent estrogenic activity reported for many food products may not represent true estrogenic activity since uterine hypertrophy, the basis for many of the assays, may result from compounds other than estrogens!5 or may be caused by fat per se.34 SUMMARY Although estrogenic activity has been detected in a wide variety of foods of plant or animal origin, only three compounds, genistein,

ESTROGENS 21 genistin, and daidzein, are from a commonly used human food. They are very weak estrogens isolated from soybeans. Estradiol 178 may occur in milk, but evidence to support this is not conclusive. Consump- tion of any food product in quantity sufficient to cause a physiologic effect due to estrogens it contains seems remote. REFERENCES —_ 10. 11. 12. 13. 14. 15. 16, 17. M. Altman and F. B. Hutt, “The Influence of Estrogens in Egg Yolk upon Avian Blood Calcium,” Endocrinology, 23, 793 (1938). E. M. Bickoff, A. N. Booth, R. L. Lyman, A. L. Livingston, C. R. Thompson, and F. DeEds, ““Coumestrol, a New Estrogen Isolated from Forage Crops,” Science, 126, 969 (1957). E. M. Bickoff, A. L. Livingston, A. P. Hendrickson, and A. N. Booth, “Relative Potencies of Several Estrogen-like Compounds Found in Forages,” J. Agr. Food Chem. 10, 410 (1962). J. D. Biggers, ‘‘Plant Phenols Possessing Oestrogenic Activity,”’ in The Pharma- cology of Plant Phenolics, J. W. Fairbairn, ed., Academic Press, New York, (1959), p. 51. A. N. Booth, E. M. Bickoff, and G. O. Kohler, “Estrogen-like Activity in Vegetable Oils and Mill By-products,” Science, 131, 1807 (1960). R. B. Bradbury and D. E. White, ‘““Estrogens and Related Substances in Plants,” Vitamins Hormones, 12, 207 (1954). E. Bilbring and J. H. Burn, “The Estimation of Oestrin, and Male Hormone in Oily Solution,” J. Physiol. (London), 85, 320 (1935). H. Burrows and E. Horning, Oestrogens and Neoplasia, Charles C Thomas, Springfield, Ill. (1952). A. Cantarow, K. E. Paschkis, A. E. Rakoff, and L. P. Hansen, “‘Studies on Inactivation of Estradiol by the Liver,” Endocrinology, 33, 309 (1943). M. W. Carter, W. W. G. Smart, Jr., and G. Matrone, “‘Estimation of Estrogenic Activity of Genistein Obtained from Soybean Meal,” Proc. Soc. Exptl. Biol. Med., 84, 506 (1953). E. W. Cheng, L. Yoder, C. D. Story, and W. Burroughs, “Estrogenic Activity of Some Naturally Occurring Isoflavones,” Ann. N.Y. Acad. Sci., 61, 652 (1955). C. A. B. Clemetson, L. Blair, and A. B. Brown, “Capillary Strength and the Menstrual Cycle,” Ann. N.Y. Acad. Sci., 93, 279 (1962). W. J. Dunlap and S. H. Wender, “‘Purification and Identification of Flavonone Glycosides in the Peel of the Sweet Orange,” Arch. Biochem. Biophys., 87, 228 (1960). C. W. Emmens, *‘Precursors of Estrogens,” J. Endocrinol., 2, 444 (1941). J. S. Evans, R. F. Varney, and F. C. Koch, ““The Mouse Uterine Weight Method for the Assay of Estrogens,” Endocrinology, 28, 747 (1941). R. Ferrando, M. M. Guilleux, and A. Guerrilott-Vinet, ““Oestrogenic Content of Plants as a Function of Conditions of Culture,” Nature, 192, 205 (1961). R. B. Greenblatt and N. H. Brown, “‘The Storage of Estrogen in Human Fat after Estrogen Administration,” Am. J. Obstet. Gynecol., 63, 1361 (1952).

22 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 35. 36. 37. MARTIN STOB J. C. Janney and B. S. Walker, “Estrogenic Substances. I. Apparatus and Methods for Preparation of Stable Extracts from Natural Sources,” Endo- crinology, 14, 101 (1930). H. E. H. Jones and G. S. Pope, “*A Method for the Isolation of Mirestrol from Pueraria mirifica,” J. Endocrinol., 22, 303 (1961). L. C. Kahnt and E. A. Doisy, “The Vaginal Smear Method of Assay of the Ovarian Hormone,” Endocrinology, 12, 768 (1928). T. Lunaas, “Transfer of 178-Estradiol to Milk in Cattle,” Nature, 198, 288 (1963). A. D. Magee, “Biological Responses of Young Rats Fed Diets Containing Genistin and Genistein,” J. Nutr., 80, 151 (1963). H. W. Marlow and D. Richert, ‘Estrogens of the Fowl,” Endocrinology, 26, 531 (1940). G. Matrone, W. W. G. Smart, Jr., M. W. Carter, V. W. Smart, and M. W. Garren, “Effect of Genistin on Growth and Development of the Male Mouse,” J. Nutr., 59, 235 (1956). U. Munch, “Die Ausscheidung von naturlichen Androgenen und Oestrogenen in der Milch,” Milchwissenschaft, 9, 150 (1954). G. S. Pope and J. H. B. Roy, ““The Oestrogenic Activity of Bovine Colostrum,” Biochem. J., 53, 427 (1953). G. S. Pope and H. G. Wright, “‘Oestrogenic Isoflavones in Red Clover and Subterranean Clover,” Chem. Ind. (London), 1019 (1954). R. Preston, E. Cheng, C. D. Story, P. Homeyer, J. Pauls, and W. Burroughs, “The Influence of Oral Administration of Diethylstilbestrol upon Estrogenic Residues in the Tissues of Beef Cattle,” J. Animal Sci., 15, 3 (1956). H. W. Siegelman, “‘Quercetin Glycosides of Grimes Golden Apple Skin,” J. Biol. Chem., 213, 647 (1955). M. Stob, R. S. Baldwin, J. Tuite, F. N. Andrews, and K. G. Gillette, “Isolation of an Anabolic Uterotrophic Compound from Corn Infected with Gibberella zeae,” Nature, 196, 1318 (1962). M. Stob, T. W. Perry, F. N. Andrews, and W. M. Beeson, “Residual Estrogen in the Tissues of Cattle Treated Orally with Diethylstilbestrol, Dienestrol, Hexestrol, and Chlortetracycline,’”’ J. Animal Sci., 15, 997 (1956). C. R. Stockard and G. N. Papanicolaou, ““The Existence of a Typical Estrous Cycle in the Guinea Pig with a Study of Its Histological and Physiological Changes,” Am. J. Anat., 22, 225 (1917). C. W. Turner, “Estrogen Content of Colostrum and Milk of Dairy Cattle,” J. Dairy Sci., 41, 630 (1958). . E. J. Umberger and G. H. Gass, “The Effect of Dietary Fat on the Uterine Weight Response of Immature Mice to Oral Stilbestrol,” Endocrinology, 63, 801 (1958). E. J. Umberger, G. H. Gass, K. J. Davis, J. M. Curtis, and C. G. Durbin, **Estrogenic Residues in the Edible Tissues of Stilbestrol-Fattened Chickens,” Poultry Sci., 38, 118 (1959). W. Velle, ‘‘Metabolism of Estrogenic Hormones in Domestic Animals,” Gen. Comp. Endocrinol., 3, 621 (1963). S. Wallach and P. H. Henneman, ‘Prolonged Estrogen Therapy in Post- menopausal Women,” J. Am. Med. Assoc, 171, 1637 (1959).

ESTROGENS 23 38. J. E. Watkin and A. C, Neish, “‘Biosynthesis of Quercetin in Buckwheat. Part III,” Can. J. Biochem. Physiol., 38, 559 (1960). 39. D. G. Wenzel and P. Rosenberg, “‘Estrogenic Activity of Some Flavonoids,” J. Am. Pharm. Assoc. Sci. Ed., 45, 367 (1956). 40. W. C. Young, ed., Sex and Internal Secretions, Williams & Wilkins, Baltimore, Md. (1961). 41. C. Zomzely, R. Astt, and J. Mayer, “‘Storage of Steroid Hormones by Adipose Tissue in Two Experimental Obesities,”’ Science, 129, 1546 (1959).

JAMES A. MILLER Tumorigenic and Carcinogenic Natural Products In the past three decades a large number and variety of synthetic chem- ical carcinogens have been discovered by accident and design.!9 32.39.83 The existence of these agents, the present knowledge of chemical carcinogenesis in man and experimental animals,!9:39.62 and the neces- sary wide and increasing use of chemicals in the modern world have engendered both concern about and legislation on the carcinogenic hazards to man of the chemicals in his modern environment. Similarly, increasing attention!6 30.84 has been focused on the detection and interpretation of carcinogenicity as important aspects of chronic toxicity tests performed on the chemicals proposed for use in our environment. Until recently little attention has been given to the possibility that various forms of life might produce tumorigens (agents giving rise to benign tumors) and carcinogens (agents giving rise to benign and malignant tumors) for other living systems. Today, we are aware of a small but increasing number of such agents. Most of these naturally occurring compounds are hepatocarcinogenic in rodents and some are of remarkably high activity. Some of these agents could find their way into certain human foods and medicines and into animal feeds, and indeed this has occurred in some instances. However, the role played by these carcinogens in the occurrence of certain cancers in man in regions where these agents are found is still unknown. An informed approach to the hazards for these agents and the synthetic chemicals in our environment is necessary. Unfounded fears and unconcern are both to be condemned. This point of view must be kept in mind in any consideration of the naturally occurring carcinogens described in the following paragraphs. 24

TUMORIGENIC AND CARCINOGENIC PRODUCTS 25 Ergot* Considering the well-known ability of fungi to synthesize a great variety of compounds, it is perhaps not surprising to find tumorigens and carcinogens among these products. One of the first intimations of this was observed some years ago by Nelson ef al.® in studies with ergot. Ergot is the dried sclerotia of the fungus Claviceps purpurea, which is parasitic on rye; it is known to contain many physiologically active factors. When crude ergot was fed to rats at 5 percent of the diet for 2 years about one half of the rats developed multiple neuro- fibromas of the ears. Most of the tumors regressed and disappeared after the administration of the ergot was stopped, but reappeared upon further ingestion of ergot. This appears to be an instance of a benign tumor whose growth and existence is dependent on a foreign factor; unfortunately, this observation has not been further investigated. Yellow Rice* Another instance of mold metabolites with the ability to cause atypical growth was found in Japan shortly after World War II. At this time large amcunts of rice had to be imported and some of the rice ship- ments were found to be contaminated with a strain of Penicillium islandicum. The rice became yellow and bitter and was apparently little used for human consumption. The yellow rice was found to be hepato- toxic for rats and mice and when it was administered to rats in the diet at a high level for a long time, a low incidence of benign and malignant liver tumors resulted.46 Investigators at the University of Tokyo have isolated two hepatotoxic substances from the contaminated rice. One of these compounds appears to be luteoskyrin, a dimeric polyhydroxy dihydroanthraquinone of known structure. However, since a sample of this compound has been reported to be relatively nontoxic,!4 the identity of this hepatotoxic constituent is uncertain. The other hepato- toxic constituent of yellow rice appears to be a chlorine-containing peptide of unknown composition. Both hepatotoxic substances have been reported to induce liver tumors in mice. Griseofulvin The antibiotic griseofulvin (1), formed by another Penicillium species, P. griseofulvum, appears to have some cocarcinogenic and tumorigenic * Other aspects are discussed in the chapter on fungal toxins, page 126.

26 JAMES A. MILLER properties. Barich et al. found that orally administered griseofulvin increased the incidence of skin tumors in mice following topical appli- cations of 3-methylcholanthrene. When 0.5 to 1 percent of this anti- biotic was fed in the diet to a pathogen-free strain of mice considerable incidences of liver hypertrophy, biliary cirrhosis, and hepatomas resulted.4° Aflatoxins* At about the time the yellow rice problem occurred in Japan, out- breaks of an unusual disease involving hepatotoxicity were becoming prominent in poultry and other livestock in England. This disease became known as “turkey X disease” after severe losses of turkey poults occurred in 1960. Subsequent investigation soon showed! ‘5 that the disease in poultry resulted from the inclusion of certain lots of Brazilian groundnuts (peanuts) in the rations. Further work has shown that toxic peanut meals have also originated in East Africa! and in the United States.23:58:74 A considerable literature on the effects of toxic peanut meals in other livestock (cf. reference 1) and in experimental animals! 5.23.48.51.70.74.75 has developed. In 1961, the cause of the poisoning was found to reside in the infec- tion of the peanuts by a toxin-producing strain of Aspergillus flavus.75 The toxic factor was termed “aflatoxin,” and further studies*! 67.86.94 have shown that several toxic substances are produced by this mold. Two highly hepatotoxic compounds, aflatoxin B, and aflatoxin G; (also known as aflatoxins B and G, the letters denoting their blue and green fluorescence, respectively, in ultraviolet light), and their less toxic dehydro derivatives (Bz and G2) have been isolated from the toxic mold. Aflatoxins B; and G; have been characterized as complex difurano- coumarins‘)! (JI); aflatoxins Bz and G2 lack the isolated double bond in the difurano group. These compounds and the toxic peanut meals injure the liver in a variety of species with the development of charac- * Other aspects are discussed in the chapter on fungal toxins, page 126.

TUMORIGENIC AND CARCINOGENIC PRODUCTS 27 teristic lesions;!-* the duckling, guinea pig, and rainbow trout are especially sensitive.!5-13 The toxic peanut meals induce considerable incidences of carcinomas in the livers of rats,1348-58-74 duck,5-¢ and rainbow trout.5 Kidney adenomas’ and some stomach adenocarci- nomas!3 were also observed in the rat. The dietary intake of aflatoxin and the time of exposure for carcinogenesis in the rat was estimated in one study as | to 6 mcg/day for 6 months.® In another study® the diets contained up to about 1 ppm and were fed for 10 to 12 months. The purified aflatoxins appear to be carcinogenic in the livers of rainbow trout at levels of about 0.08 ppm in the diet,5 and they induce sarcomas in rats upon the subcutaneous injection of total doses of 2 to 8 mg.~ The high toxicity and carcinogenicity of the aflatoxins and their sporadic occurrence in a staple food used for livestock and humans have given rise to considerable concern (cf. reference 88). While some toxin appears in milk from cows fed the toxic meals, this toxin has not been detected so far in bulk milk supplies possibly because of the great dilution.2 Evidence was recently presented that the milk toxin is an altered aflatoxin.42 Trace amounts of aflatoxins are reported to have * Jn relation to man (see Pyrrolizidine Alkaloids, page 29) it is of interest that oral administration of high doses of aflatoxin to young rhesus monkeys quickly induces fatty liver and hepatic fibrosis and cirrhosis in these primates [T. F. Madhaven, P. G. Tulpule, and C. Gopalan, “‘Aflatoxin-Induced Hepatic Fibrosis in Rhesus Monkey,” Arch Pathol., 79, 466 (1965)]. t Carnaghan has recently reported in detail on the carcinogenicity of a toxic peanut meal in ducks [R. B. A. Carnaghan, ‘“‘Hepatic Tumors in Ducks Fed a Low Level of Toxic Groundnut Mea!, Nature, 208, 308 (1965)]. A diet containing 0.5 percent of a toxic meal was fed for 14 months. Eleven of 37 ducks survived for this period. Eight of the survivors developed hepatic tumors whereas none was noted in 10 survivors of 16 ducks fed the diet without the toxic meal for the same period. The toxic diet was shown to contain only 0.03 ppm of aflatoxin (assayed as aflatoxin By).

28 JAMES A. MILLER been detected in some peanut butters ;88 however, peanut oils are free of the aflatoxins because the alkaline treatment used in processing the oils destroys these lactones.88 The aflatoxins are heat stable.2 Although the infection of the peanuts with the toxic strain of A. flavus appears to occur during improper harvesting and storage of the nuts, it is possible that some infection also occurs in nuts with cracked shells underground before harvest. In any event, prompt lowering of the moisture content after harvest and during storage greatly inhibits growth of the mold.6 Toxic maize or corn has also been reported,! and in the United States there have been cases of livestock toxicity from moldy feed.!2 In one instance involving moldy corn only one of the nine isolates of A. flavus produced toxic metabolites.!2 A toxic strain of Penicillium rubrum was also isolated and recent work on this organism} has shown that it elaborates a hepatotoxic factor with the properties of an organic acid containing carbonyl groups. In this regard it is of interest that a strain of Penicillium puberulum was recently isolated from a sample of moldy peanuts and was found to form aflatoxin.36 Other Mold Metabolites In 1960, an unusual mold metabolite, p-formyl-N-nitroso-N-methyl- aniline was isolated.33 This is of interest in view of its structural simi- larity to N-nitroso-N-methylaniline, a known synthetic carcinogen for the esophagus of the rat.26 Similarly, two unusual compounds have recently been found in a common commercial edible mushroom.53-54 These are agaritine or the p-hydroxymethylphenylhydrazide of L-glu- tamic acid and its oxidative acid degradation product p-hydroxymethyl- benzene diazonium ion. The latter compound only remotely resembles the known carcinogenic aminoazo dyes, but it would be interesting to test the effects in mammals of these unusual fungal metabolites. Ethionine Ethionine, the S-ethyl analog of methionine, was discovered as a synthetic carcinogen.2’? Recent tracer studies have shown ethionine to be a metabolite of several bacteria including Escherichia coli grown in a salts-glucose medium containing sulfate ion or methionine.28 Algal, yeast, or tumor cells grown under the same conditions did not form ethionine. The bacterially synthesized ethionine occurred in the free form in the cells and medium and was not incorporated into the bac- terial protein. In the rat?” ethionine is incorporated into protein in

TUMORIGENIC AND CARCINOGENIC PRODUCTS 29 several tissues in vivo, presumably through the usual pathway which requires activation of the carboxyl group with adenosine triphosphate (ATP). Activation at the sulfur atom in ethionine with ATP also occurs in the liver of the rat, and the resulting S-adenosyl ethionine appears to ethylate some of the nucleic acids in this organ.2789 If abnormal nucleic acids so formed give rise to the hepatomas found in the rat after ethionine administration, S-adenosyl ethionine would be a proximate carcinogen in this case. The extent to which mammals are exposed to bacterially synthesized ethionine produced in the caecum and large intestine is not known. In the rodent, for example, coprophagy would probably increase the amount obtained. A search for ethionine in the rumen contents and milk of ruminants might also be instructive. Pyrrolizidine Alkaloids Some carcinogens and tumorigens are also found among the products of metabolism of plants. The numerous pyrrolizidine alkaloids, elabo- rated by the widespread species in the Senecio, Crotolaria, and Helio- tropium genera, have received considerable attention in this respect. The pyrrolizidine alkaloids that contain a double bond in the nucleus and a branched aliphatic R group in the ester function are highly hepatotoxic (III).2°8! In the rat several of these alkaloids have been found to be both highly hepatotoxic and hepatocarcinogenic,”7:78 even after one or a few small doses.’? The alkaloids are secreted into the mother’s milk in the rat, and severe liver damage can thus be induced in the highly susceptible young suckling rat.76 Culvenor et al.2° showed that the toxic pyrrolizidine alkaloids can act as alkylating agents because of the sterically hindered allylic ester grouping in which fission of the CH2—O bond occurs (IJ). Thus this is another example of alkylating activity in a carcinogen. R'O CH-O-G-R Ty

30 JAMES A. MILLER Extracts of common plants containing the toxic pyrrolizidine alka- loids have been used in many parts of the world, e.g., Africa and India, as folk medicines and in rituals.2 Many livestock losses have been attributed to these toxic alkaloids, especially to their N-oxides, which are not bitter to the taste.!8:85 The high hepatotoxicity and hepato- carcinogenicity of the pyrrolizidine alkaloids have given rise to the suspicion that such beverages as bush-teas containing these alkaloids may have contributed to the high incidences of liver disease and hepatocellular tumors in natives in certain areas such as the part of Africa south of the Sahara. The situation in these areas is obviously complex, for other factors such as the consumption of moldy corn (containing aflatoxins?), the occurrence of protein deficiency disease or kwashiorkor, and viral hepatitis also must be considered. No single known factor or combination of factors yet accounts adequately for the high incidence of liver tumors seen in man in certain parts of the world. Higginson35 has recently provided a valuable review of this subject and has proposed an interesting two-stage theory of its patho- genesis. Cycads and Cycasin A fascinating new chapter in experimental carcinogenesis has arisen recently from studies on the toxic cycads.°°-92 The cycads are an ancient family of palm-like trees that may represent an evolutionary step from fern to flowering plant. Many species still inhabit tropical and sub- tropical regions and are able to survive drought and hurricanes. Cycads have provided emergency and staple food and medicines for natives of these regions for a long time. The cycads have long been recognized to contain principles toxic to man and livestock, but, as in the preparation of edible starch from cycad nuts, the toxins can be extracted with water. Studies from 1941 to 1959 in Australia and Japan (cf. references 61 and 92) on the isolation and characterization of the toxic factors in the cycads showed that these consist of a series of glycosides, of which the most common appears to be cycasin (IV). Methylazoxymethanol, the common aglycone of the glycosides (cf. Table 6 in reference 92) has recently been isolated*5.6! and synthesized. This is the primary toxic substance and is released by 6-glycosidases in plants and bacteria.45.6! The toxic cycad nuts, cycasin, and methyl- azoxymethanol cause many untoward symptoms°°.592 but prominent among these is moderate to severe liver damage.

TUMORIGENIC AND CARCINOGENIC PRODUCTS 31 CYCASIN DIMETHYLNITROSAMINE x CH H > \n-No 7 HO cH 3 v HOH LIVER MICROSOMES 0 2 + TPNH + 09 5 - GLUCOSIDASE (PLANTS, BACTERIA) C"H, . CH,~N=N—CH,OH N-NO | + HC*HO v H” 0 x NS es = 7] + Heo |erH Ne on | | chy No ] NO ft [ers | +N, | DNA, RNA, PROTEIN C*H3R (7-N OF GUANINE, RING-N OF HISTIDINE) Tw Recent interest in the cycads and their toxic principles developed from the possible relationship of these plants as sources of foods and medicines to the high incidence of certain neurological diseases in areas where they are used by the natives for these purposes.59.52 Examination of this possibility at the National Institute of Neurological Diseases and Blindness in the United States has not revealed much support for this relationship. However, these studies revealed a quite unexpected result. When rats were fed the toxic cycad nut meals at several percent of the diet for several months a high percentage of the animals developed’ primary tumors in the liver and kidney.5° Later studies showed that the toxic cycad nut meal is hepatocarcinogenic in the guinea pig®’ and that cycasin is carcinogenic in the rat.49 It is highly probable that these effects are due to the toxic aglycone. Methylazoxymethanol easily decomposes to formaldehyde, methanol, and nitrogen,§! and it bears an obvious similarity to the synthetic carcinogen, dimethylnitrosamine, which induces similar tumors in the rat.5’ There is now considerable evidence that dimethylnitrosamine is metabolized in the rat liver to diazomethane or to reactive CH;+ groups and that these alkylating agents then methylate certain of the nucleic acids and proteins in this

32 JAMES A. MILLER organ.52.57.58.59 It seems very likely that dimethylnitrosamine and methylazoxymethanol yield the same proximate carcinogenic agents in vivo (IV). Diazomethane itself has been found to be carcinogenic®°® and the possibility that these alkylating agents yield abnormal nucleic acids in vivo27-8 that play important roles in the carcinogenic responses is very attractive. Although first known as a synthetic carcinogen, dimethylnitrosamine has recently been found in batches of herring meal preserved with sodium nitrite.73 These meals had proved to be hepato- toxic to sheep. Safrol and Related Compounds Still other types of tumorigens and carcinogens have been found in plants. Safrol or p-allylmethylenedioxybenzene (V) has recently been found to produce liver tumors in rats when administered in the diet for several months at the rather high level of 0.5 percent.37386This compound is the chief constituent of oil of sassafras and a very minor constituent of several spices. Until the recent reports on its carcinoge- nicity, safrol was used in small amounts to flavor certain soft drinks in the United States. A good example of the effect of structure on carcinogenicity is shown in the subsequent finding that diets containing similar amounts of dihydrosafrol (V), in which the allyl group is saturated, proved carcinogenic for the esophagus of the rat but appar- ently not for the liver of this species.55 The methylenedioxybenzene grouping is also found in several of the minor constituents of sesame oil! (VI), a widely used edible oil. One of these compounds, sesamol, has been administered to rats at a level of 1 percent in the diet for many months. No effect on growth, mortality, or blood morphology was noted but, as compared with the control rats or with rats receiving lower levels of sesamol, a moderate increase in benign “proliferative lesions” appeared to result from ingestion of this relatively large amount of sesamol.3 —CHo i CHa —CHo—CH3 Z