Toxicants Occurring Naturally in Foods. (1966)

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TUMORIGENIC AND CARCINOGENIC PRODUCTS 33 SESAMOLIN 0 7 N ee eG ° | CH —CH CHa ofcn Gn T N07 2 “SESMOL -H ! HO—- SAMIN 7°. CHa CH 0 —O0 { Hos CH CH 9 —CH2 0 ae che SESAMIN wz Antithyroid Compounds Thiourea has been detected in the seeds of certain plants in the genus Laburnum™ Thiourea is an antithyroid compound that will produce thyroid adenomas and carcinomas, benign liver tumors, and malignant tumors of the eyelid and ear duct upon administration in the diet to the rat.29:71.72 Another antithyroid compound, /-5-vinyl-2-thiooxazolidone, occurs in turnips, kale, cabbage, and rapeseed.’ Rats fed a diet con- taining 45 percent rapeseed have developed thyroid adenomata.+! Bracken This fern species (Pteridium aquilinum) has been known to damage the bone marrow and intestinal mucosa and to induce polyps in the bladder mucosa of cattle fed the whole plant or extracts thereof. A recent report4 reveals that a diet containing 34 percent of dried bracken fern was active in producing multiple adenocarcinomas of the small intestine in rats. The diet was fed for a total of 74 days and during the following year 10 of 34 rats developed large tumors in addition to numerous smaller tumors in the intestine. An unspecified number of the rats with the small tumors died or were killed. It is of interest that in Japan a similar plant, zen mai (Osmunda japonica) is used as a vegetable and seasoning. The suggestion has been made® that bracken may be confused with zen mai and thus might contribute to the relatively high incidence of stomach cancer found in Japan.

34 JAMES A. MILLER Selenium Derivatives Compounds containing this element, at different levels in the diet, can either be hepatotoxic and hepatocarcinogenic or act as essential nutrients for several mammalian and avian species. Inorganic selenium compounds occur naturally in many soils and the element is incorpo- rated into plant protein, partly as selenomethionine and selenocystine. In some areas the forage contains high amounts of organic selenium compounds and is toxic to livestock.“ In other areas the soil is deficient in selenium compounds and livestock fed on forage grown in these areas develop selenium-deficiency diseases. In several mammalian and avian species selenium compounds, inorganic or organic, act as essential dietary constituents at a level of about 0.01 to 0.1 ppm as Se in the diet.69-82 On the other hand, in the rat, selenium in the form of selenide, selenate, or seleniferous corn or wheat appears to be hepato- carcinogenic when fed in the diet at 5 to 10 ppm for many months.!8.65 Abnormal selenoproteins might be of importance in this carcinogenic process. In view of the newer information on alkylation of nucleic acids, consideration should also be given to the possibility of abnormal methylation of cellular constituents by a compound such as Se-ade- nosylselenomethionine. Other Natural Products The naturally occurring tumorigens and carcinogens. discussed above are active after oral administration to experimental animals. Other natural products have been found to have tumorigenic or carcinogenic properties when administered by a parenteral route, usually sub- cutaneous, to rodents. In many of these cases adequate trials by the oral route have not been reported. Compounds that induce sarcomas at the site of subcutaneous injection(s) include cholesterol,!! 34 oxidation products related to cholesterol,"! parasorbic acid and related lactones,?! and carrageenin, a sulfated polygalactose from certain seaweeds.!’ Various tannins (tannic acids), both hydrolyzable and condensed, ap- pear to induce liver damage and liver tumors in rats‘? and mice“ following repeated subcutaneous injection. Some of the condensed tannins also produced some tumors at the site of injection in mice. Chemically the tannins are poorly characterized substances. SUMMARY It seems likely that an increasing number and variety of natural prod- ucts with tumorigenic and carcinogenic activities will continue to be

TUMORIGENIC AND CARCINOGENIC PRODUCTS 35 found. Some of the naturally occurring dietary carcinogens that are now known, such as the aflatoxins, the pyrrolizidine alkaloids, and the aliphatic azoxy glycosides, have remarkably high carcinogenic activities in rodents. The possible presence of significant amounts of these and other carcinogenic agents in human food deserves the attention of the epidemiologist in considerations on the etiology of cancer in man. A corollary requirement is the need for much more information on the composition and toxicological properties of previously ignored or unknown nonnutritive constituents of human foods. REFERENCES 1. R. Allcroft and R. B. A. Carnaghan, “Toxic Products in Groundnuts: Biological Effects,” Chem. Ind. (London), ii, 50 (1963). 2. R. Allcroft and R. B. A. Carnaghan, “Groundnut Toxicity: An Examination for Toxin in Human Food Products from Animals Fed Toxic Groundnut Meal,” Vet. Record, 75, 259 (1963). 3. A.M. Ambrose, A. J. Cox, and F. DeEds, “Antioxidant Toxicity: Toxicological Studies on Sesamol,” J. Agr. Food Chem., 6, 600 (1958). 4. T. Asao, G. Biichi, M. M. Abdel-Kader, S. B. Chang, E. L. Wick, and G. N. Wogan, “‘Aflatoxins B and G,” J. Am. Chem. Soc., 85, 1706 (1963). 5. L. M. Ashley, J. E. Halver, and G. N. Wogan, ‘“‘Hepatoma and Aflatoxicosis in Trout,” Federation Proc., 23, 105 (1964). 6. L. L. Ashworth, Jr., H. W. Schroeder, and B. C. Langley, ‘“‘Aflatoxins: Environ- mental Factors Governing Occurrence in Spanish Peanuts,” Science, 148, 1228 (1965). 7. E. B. Astwood, M. A. Greer, and M. G. Ettlinger, “‘/-5-Vinyl-2-thiooxazolidone, an Antithyroid Compound from Yellow Turnip and Brassica Seeds,” J. Biol. Chem., 181, 121 (1949). 8. L. L. Barich, J. Schwarz, and D. Barich, “Oral Griseofulvin: A Cocarcinogenic Agent to Methylcholanthrene-Induced Cutaneous Tumors,” Cancer Res., 22, 53 (1962). 9. J. M. Barnes and R. Schoental, ‘Experimental Liver Tumours,” Brit. Med. Bull., 14, 165 (1958). 10. M. Beroza, “The Structure of Sesamolin and Its Stereochemical Relationship to Sesamin, Asarinin, and Pinoresinol,” J. Am. Chem. Soc., 77, 3332 (1955). 11. F. Bischoff, “‘Carcinogenesis through Cholesterol and Derivatives,” Exptl. Tumor Res., 3, 412 (1963). 12. J. E. Burnside, W. L. Sippel, J. Forgacs, W. T. Carll, M. B. Atwood, and E. R. Doll, “‘A Disease of Swine and Cattle Caused by Eating Moldy Corn. II. Experimental Production with Pure Cultures of Molds,” Am. J. Vet. Res., 18, 817 (1957). 13. W. H. Butler and J. M. Barnes, “Toxic Effects of Groundnut Meal Containing Aflatoxin to Rats and Guinea-Pigs,” Brit. J. Cancer, 17, 699 (1963). 14. Ng Ph. Buu-Hoi and F. Zajdela, “‘La luteoskyrine—est elle le principe hepato- toxique du riz jauni?”” Med. Exptl., 6, 29 (1962).

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TUMORIGENIC AND CARCINOGENIC PRODUCTS 37 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 49. SI. 32. 53. 35. 56. 37. F. Homburger, T. Kelley, Jr., G. Friedler, and A. B. Russfield, ““Toxic and Possible Carcinogenic Effects of 4-Allyl-1,2-methylenedioxybenzene (Safrole) in Rats on Deficient Diets,” Med. Exptl., 4, 1 (1961). W. C. Heuper and W. D. Conway, Chemical Carcinogenesis and Cancers, Charles C Thomas, Springfield, Ill. (1964). E. W. Hurst and G. E. Paget, “Protoporphyrin, Cirrhosis and Hepatomata in the Livers of Mice given Griseofulvin,” Brit. J. Dermatol., 75, 105 (1963). H. de Iongh, R. K. Beerthuis, R. O. Vles, C. B. Barrett, and W. O. Ord, “In- vestigation of the Factor in Groundnut Meal Responsible for ‘Turkey X Disease’,”” Biochim. Biophys. Acta, 65, 548 (1962). H. de Iongh, R. O. Vles, and J. G. van Pelt, “‘Milk of Mammals Fed an Afla- toxin-Containing Diet,”’ Nature, 202, 466 (1964). K. S. Kirby, “Induction of Tumours by Tannin Extracts,” Brit. J. Cancer, 14, 147 (1960). G. Klein and E. Farkass, ‘‘Die mikrochemische Nachweis der Alkaloide in der Pflanze. XIV Cystine.”’ Oesterr. Botan. Z., 79, 107 (1930). A. Kobayashi and H. Matsumoto, “Studies on Methylazoxymethanol, the Aglycone of Cycasin,” Arch. Biochem. Bioplhys., 110, 373 (1965). Y. Kobayashi, K. Uraguchi, F. Sakai, T. Tatsuno, M. Tsukioka, Y. Noguchi, H. Tsunoda, M. Miyake, M. Saito, M. Enomoto, T. Shikata, and T. Ishiko, ‘Toxicological Studies on the Yellowed Rice by P. islandicum Sopp. III. Experi- mental Verification of Primary Hepatic Carcinoma of Rats by Long Term Feeding with the Fungus-Growing Rice,” Proc. Japan Acad., 35, 501 (1959). B. Korpassy, ““Tannins as Hepatic Carcinogens,” Progr. Exptl. Tumor Res., 2, 245 (1961). . M. C. Lancaster, F. P. Jenkins, and J. McL. Philp, “Toxicity Associated with Certain Samples of Groundnuts,”” Nature, 192, 1095 (1961). G. L. Laqueur, “Carcinogenic Effects of Cycad Meal and Cycasin, Methyl- azoxymethanol Glycoside, in Rats and Effects of Cycasin in Germ-Free Rats,” Federation Proc., 23, 1386 (1964). G. L. Laqueur, O. Mickelsen, M. G. Whiting, and L. T. Kurland, “Carcinogenic Properties of Nuts from Cycas circinalis L. Indigenous to Guam,” J. Natl. Cancer Inst., 31,919 (1963). E. Le Breton, C. Frayssinet. and J. Boy, “Sur l’apparition d’hepatomes spontanes chez le rat wistar. Role de la toxine de Il’ Aspergillus flavus. Interet en pathologie humaine et concerologie experimentzle,”* Compt. Rend., 225, 784 (1962). K. Y. Lee, W. Lijinsky, and P. N. Magee, “‘Methylation of Ribonucleic Acids of Liver and Other Organs in Different Species Treated with C4 and H3 Dimethylnitrosamines Jn Vivo,” J. Natl. Cancer Inst., 32, 65 (1964). B. Levenberg, “An Aromatic Diazonium Compound in the Mushroom Agaricus bisporus,” Biochim. Biophys. Acta, 63, 212 (1962). B. Levenberg, “Isolation and Structure of Agaritine, a y-Glutamyl-Substituted Arylhydrazine Derivative from Agaricaceae,” J. Biol. Chem., 239, 2267 (1964). E. L. Long and P. M. Jenner, “‘Esophageal Tumors Produced in Rats by the Feeding of Dihydrosafrole,”’ Federation Proc., 22, 275 (1963). E. L. Long, A. A. Nelson, O. G. Fitzhugh, and W. H. Hansen, “Liver Tumors Produced in Rats by Feeding Safrole,”’ Arch. Pathol., 75, 595 (1963). P. N. Magee, “Cellular Injury and Chemical Carcinogenesis,” in Cancer. Progress Volume, R. W. Raven, ed., Butterworths, London (1963), pp. 56-66.

38 58. 59. 61. 62. 63. 65. 67. 69. 70. 71. 72. 73. 74, 75. 76. JAMES A. MILLER P. N. Magee and E. Farber, “Toxic Liver Injury and Carcinogenesis. Methyla- tion of Rat-Liver Nucleic Acids by Dimethylnitrosamine Jn Vivo,” Biochem. J., 83, 114 (1962). P. N. Magee and R. Schoental, “Carcinogenesis by Nitroso Compounds,” Brit. Med. Bull., 20, 102 (1964). H. Matsumoto, T. Nagahama, and H. O. Larson, “Studies on Methylazoxy- methanol, the Aglycone of Cycasin: A Synthesis of Methylazoxymethyl Acetate,” Biochem. J., 95, 13C (1965). H. Matsumoto and F. M. Strong, ““The Occurrence of Methylazoxymethanol in Cycas circinalis L.,” Arch. Biochem. Biophys., 101, 299 (1963). J. A. Miller and E. C. Miller, ““Natural and Synthetic Chemical Carcinogens in the Etiology of Cancer,” Cancer Res., 25, 1292 (1965). M. Miyake, M. Saito, M. Enomoto, K. Takahashi, H. Shimada, and E. Itakura, “On the Tumors of the Liver and Other Tissues of Mice (dd Strain) Induced by Long-Term Feeding of Luteoskyrin and Chlorine-Containing Peptide from Penicillium islandicum,” Trans. Soc. Pathol. Japan, 53, 124 (1964). O. H. Muth and W. Binns, “Selenium Toxicity in Domestic Animals,” Ann. N.Y. Acad. Sci., 111, 583 (1964). A. A. Nelson, O. G. Fitzhugh, and H. O. Calvery, “Liver Tumors Following Cirrhosis Caused by Selenium in Rats,” Cancer Res., 3, 230 (1943). A. A. Nelson, O. G. Fitzhugh, H. J. Morris, and H. O. Calvery,**Neurofibromas of Rat Ears Produced by Prolonged Feeding of Crude Ergot,” Cancer Res., 2, 11 (1942). B. Nesbitt, J. O’Kelly, K. Sargeant, and A. Sheridan, ‘‘Toxic Metabolites of Aspergillus flavus,” Nature, 195, 1062 (1962). . P. M. Newberne, W. W. Carlton, and G. N. Wogan, “‘Hepatomas in Rats and Hepatorenal Injury in Ducklings Fed Peanut Meal or Aspergillus flavus Extract,” Pathol. Vet. (Basel), 1, 105 (1964). J. E. Oldfield, J. R. Schubert, and O. H. Muth, “Implications of Selenium in Large Animal Nutrition,” J. Agr. Food Chem., 11, 388 (1963). G. E. Paget, ““Exudative Hepatitis in Guinea-Pigs,” J. Pathol. Bacteriol., 67, 393 (1954). H. D. Purves and W. E. Griesbach, “Studies on Experimental Goitre. VIII. Thyroid Tumours in Rats Treated with Thiourea,” Brit. J. Exptl. Pathol., 28, 46 (1947). A. Rosin and H. Ungar, “‘Malignant Tumors in the Eyelids and the Auricular Region of Thiourea-Treated Rats,” Cancer Res., 17, 302 (1957). J. Sakshaug, E. Sognen, M. A. Hansen, and N. Koppang,‘*Dimethylnitrosamine, Its Hepatotoxic Effect in Sheep and Its Occurrence in Toxic Batches of Herring Meal,” Nature, 206, 1261 (1965). W. D. Salmon and P. M. Newberne, “Occurrence of Hepatomas in Rats Fed Diets Containing Peanut Meal as a Major Source of Protein,” Cancer Res., 23, 571 (1963). K. Sargeant, R. B. A. Carnaghan, and R. Allcroft, “Toxic Products in Ground- nuts. Chemistry and Origin,” Chem. Ind. (London), ii, 53 (1963). R. Schoental, “Liver Lesions in Young Rats Suckled by Mothers Treated with Pyrrolizidine (Senecio) Alkaloids, Lasiocarpine, and Retrorsine,” J. Pathol. Bacteriol., 77, 485 (1959).

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IRWIN E. LIENER Lathyrogens in Foods Lathyrism, a disease in man that is characterized by muscular weakness and paralysis of the lower legs, has been recognized since the time of Hippocrates. Sharma! has written an interesting review of the early historical background of lathyrism. In more recent times, this disease has afflicted some segments of the population in India and the Medi- terranean area, and is generally associated with the consumption of certain peas of the genus Lathyrus, notably L. sativus (chickling vetch), L. cicera (flat-podded vetch), and L. clymenum (Spanish vetchling). The precise etiology of human lathyrism has not been easy to elucidate because of the difficulty with which this disease can be reproduced in animals. The aforementioned species of lathyrus that have been impli- cated in human lathyrism are relatively nontoxic to most animals? although two other species of lathyrus—L. Jatifolius (perennial sweet pea) and L. sylvestris Wagneri (flat pea)— produce neurological symp- toms in rats that resemble those of human lathyrism.3+6 Rats fed L. odoratus (sweet pea), L. pusillus (singletary pea), or L. hirsutus (Caley pea) develop marked skeletal deformities that are quite different in symptomology from human lathyrism.3-78 It is evident therefore that the concept of what actually constitutes lathyrism is not a simple one. Some clarity is achieved if one accepts the conclusion of Selye® that the consumption of lathyrus can produce two distinctly different types of disease, a “neurolathyrism”’ which involves damage to the central nervous system, and an “‘osteolathyrism” which affects bone and connective tissue. The clinical characteristics of neurolathyrism and osteolathyrism and the lathyrus species associated with each have been reviewed.?—!! 40

LATHYROGENS 41 The whole problem of the etiology of lathyrism has been considerably clarified in recent years due to the isolation and characterization of what appear to be the causative principles of these two forms of lathyrism. In 1954, an active principle capable of reproducing the symptoms of osteolathyrism in rats was isolated from L. pusillus by Dupuy and Lee!2 and from L. odoratus by McKay et al.!3 This compound was identified as B-N-(y-L-glutamyl)aminopropionitrile!4 although it was subsequently established that the active portion of the molecule is 8-aminopropioni- trile.!5.16 The feeding of this compound to rats at levels as low as 0.1 to 0.2 percent of diet produced all the symptoms associated with osteo- lathyrism. In addition to being found in L. odoratus, B-aminopropioni- trile was also found in L. pusillus, L. hirsutus, and L. roseus but could not be detected in L. sativus, L. sylvestris Wagneri, L. cicera, L. latifolius, L. strictus, L. splendus, or in a variety of other legume seeds including soybeans, edible garden peas, navy beans, cow peas, mung beans, velvet beans, fava beans, white legume, hairy vetch, red clover, or alfalfa.!7.18 It will be noted from this listing that 8-aminopropionitrile is notably absent from those species of lathyrus that have been impli- cated in human lathyrism or produce neurological symptoms in rats. The ease with which osteolathyrism can be produced in animals by the administration of a simple organic compound has provided a valuable experimental tool for investigating such diseases of the con- nective tissue as dissecting aneurysm of the aorta, degenerative changes in growth cartilages, hernias, and fragility and thinning of the skin (for reviews of the literature see references 9, 19, and 21). The underlying biochemical lesion observed in such studies appears to involve severe and dramatic changes in the mesenchymal tissues that compose the skin, bone, and blood vessels. Gross and his co-workers?9.22:23 are of the opinion that the principal defect induced by lathyrogenic agents is an interference with the normal intramolecular cross-linking of collagen fibrils, which is essential to the normal maturation of connective tissue. In 1961, Ressler et al.24 reported the isolation of L-a-y-diamino- butyric acid (see Figure 1) from L. Jatifolius and L. sylvestris Wagneri. The feeding of this compound to rats produced neurotoxic symptoms similar to those that had been previously described by Lewis and co-workers? 56 for rats on diets containing these same species of lathyrus. Arscott and Harper? observed that chicks fed a diet containing 1 per- cent of a, y-diaminobutyric acid grew poorly and developed symptoms of blindness. Bell? showed L-a-y-diaminobutyric to be present in 10 other species of lathyrus as well (see Table 1). It is of interest to note,

42 IRWIN E. LIENER BB —- aminopropionitrile * COs 0 i ° NH» ‘oN CH, — NHo | CHo — H20 CHo + Ho CHo | —_—_—_—_—-> ————> | CH~NH> CH—NHo CH—NH9 COOH COOH COOH asparagine 8 - cyano— a, y— diamino— L— alanine butyric acid Hoffman degradation i CH, —NH _ -—-C— 2 2 oxolie CHa —NH —C—COOH | acid | COOH COOH a, 8 -diamino— BB -N ~oxaly!— a, 6-diamino- propionic acid -proplonic acidt 4 Osteolathyrogen Tt Neurolathyrogen FicurE 1 Possible metabolic interrelationship of various lathyrogenic compounds. however, that this compound could not be detected in those species —L. sativus, L. cicera, and L. clymenum—that have been implicated in human lathyrism. A new amino acid, homoarginine (a-amino-e guanidinocaproic acid), has been isolated from L. cicera® and L. sativus?’ but this compound appears to be nontoxic, at least to chicks.%

LATHYROGENS 43 TABLE 1 Classification and Lathyrogenic Content of Seeds SEED CONTENT, 7% SEED CONTENT, % OSTEOLATHYROGENS NEUROLATHYROGENS 8-Aminopropionitrile a,7-Diaminobutyric Acid L. odoratus* 0.05-0. 16 L. aurantius* >] L. pusillus« 0.062 L. cirrhosus >I L. hirsutus 0.021 L. gorgoni >1 L. roseus* L. grandiflora >1 L. heterophyllus >1 NEUROLATHYROGENS L. laevigatus >1 6-Cyano-L-alanine L. lutus >I V. sativae 0.15 L. multiflora >i V. angustifolia¢ 0.09-0.12 £- rotundifolius >1 L. tuberosus >1 a, y-Diaminobutyric Acid L. latifolius¢ 0.51-0.67 8-N-Oxalyl-a,8-diaminopropionic Acid L. sylvestris W.4 1.4 L. sativus! 0.1 « Garbutt and Strong.” + Detected by paper chromatography onty by Bell. ¢ Ressler.™ ¢ Ressler ef al.™ * Detected in this species, and all those that follow, by paper chromatography by BelF* who states that a,7-diaminobutyric is present at a level of at least 1 percent of the dry weight of the seed. J Adiga et l; Murti et a/.8 Ressler>° has more recently isolated a neurotoxic principle from Vicia sativa (common vetch), a leguminous seed that is a common contaminant of L. sativus and which itself is toxic to certain ani- mals.2-531 This compound, identified as 8-cyano-L-alanine (see Figure 1), was also shown to be present in Vicia angustifolia (narrow-leaf vetch), but not in L. sativus2° When fed to rats at a level of 1 percent, B-cyano-L-alanme caused hyperirritability, tremors, and convulsions within 3 to 5 days. A dose of 15 mg/100 g of body weight administered by stomach tube caused temporary tremors, convulsions, and rigidity from which the animal recovered in 4 hours, whereas a dose of 20 mg/100 g injected subcutaneously caused convulsions, rigidity, prostra- tion, and eventual death. Arscott and Harper also noted that a 30 percent level of a crude preparation of 6-cyano-L-alanine from L. sativus was highly toxic to chicks. Ressler>° pointed out that there is no certainty that the biological effects produced in rats by 8-cyano-L-alanine is the same as those that accompany human lathyrism nor is the presence of other neurotoxins

44 IRWIN E. LIENER in V. sativa excluded. Although the consumption of V. sativa and V. angustifolia is not usually associated with human lathyrism, it is nevertheless important to note that these seeds have been reported as contaminants in ZL. sativus? and wheat32 consumed during several outbreaks of lathyrism in India. One is therefore faced with the question whether 6-cyano-L-alanine present to the extent of only 0.1 percent in Vicia seed, which itself occurs as a contaminant of nontoxic seeds, is present in high enough concentration to be toxic to man. More recently, two groups of workers in India28.29.33 have reported the isolation from L. sativus itself of a neurotoxic principle that was identified as $-N-oxalyl-a,8-diaminopropionic acid (see Figure 1). Intraperitoneal injections of 20 mg into chicks produced neurotoxic symptoms manifested by an inability to stand, head retraction, paralysis of legs, and convulsions. a ,8-Diaminopropionic acid itself proved non- toxic, thus showing the essentiality of the oxalyl group for toxicity. A final judgment as to whether 6-N-oxalyl-a ,@-diaminopropionic acid is indeed the long sought-for causative principle of human lathyrism must await further definitive studies. In Table 1 have been collected the data thus far available on the lathyrogenic content of various species of Lathyrus and Vicia. An examination of the chemical structures of the various lathyrogens (Figure 1) reveals an interesting possible metabolic relationship. The compound, #-cyano-L-alanine, presumably derived from asparagine by dehydration, could serve as a biological intermediate that is either decarboxylated to B-aminopropionitrile or reduced to a, y-diamino- butyric acid. Murti et al.33 have suggested that B-N-oxalyl-a, 6-diamino- propionic acid may also be derived from asparagine by a sequence of steps involving the replacement of —CONH)2 of asparagine by —NH2 (analogous to the Hoffman reaction) to give a ,8-diaminopropionic acid. Subsequent acylation of the B-amino group with oxalic acid would produce the neurotoxin, 8-N-oxalyl-a,8-diaminopropionic acid. One may speculate that the lathyrogen that is present in a particular seed is determined by the manner in which asparagine is metabolized, and this in turn is a consequence of the enzymic constitution of the plant. At any rate it is indeed curious that such slight structural modifications can impart such profound differences in physiological effects in man and animals. REFERENCES 1. D.N. Sharma, “‘Lathyrism: The Old and New Concepts,” J. Indian Med. Assuc.., 36, 299 (1961).

LATHYROGENS 45 2. 3. 10 11 12 13. 14. 15. 16. 17. 18. 19. 21. L. A. P. Anderson, A. Howard, and J. L. Simonsen, “Studies on Lathyrism,” Indian J. Med. Res., 12, 613 (1925). H. B. Lewis, R. S. Fajans, M. B. Esterer, C. Shen, and M. Oliphant, “The Nutritive Value of Some Legumes. Lathyrism in the Rat. The Sweet Pea (Lathyrus odoratus), Lathyrus sativus, Lathyrus cicera and some other Species of Lathyrus,” J. Nutr. 36, 537 (1948). D. K. Dastur, “‘Lathyrism: Some Aspects of the-Disease in Man and Animals,” World Neurol., 3, 721 (1962). H. B. Lewis and A. R. Schulert, ‘‘Experimental Lathyrism in the White Rat and Mouse,” Proc. Soc. Exptl. Biol. Med., 71, 440 (1949). A. R. Schulert and H. B. Lewis, “Experimental Lathyrism,” Proc. Soc. Exptl. Biol. Med., 81, 86 (1952). B. J. Geiger, H. Steenbock, and H. T. Parsons, ““Lathyrism in the Rat,” J. Nutr., 6, 427 (1933). J. G. Lee, “Experimental Lathyrism Produced by Feeding Singletary Pea (Lathyrus pusillus) Seed,” J. Nutr., 40, 587 (1950). H. Selye, “‘Lathyrism,” Rev. Can. Biol., 16, 1 (1957). F. M. Strong, “‘Lathyrism and Odoratism,” Nutr. Rev., 14, 65 (1956). A. F. Gardner, ‘Experimental Lathyrism: Review of the Literature,” Am. J. Clin. Nutr., 7, 213 (1959). H. P. Dupuy and J. G. Lee, “‘The Isolation of a Material Capable of Producing Experimental Lathyrism,” J. Am. Pharm. Assoc. Sci. Ed., 43, 61 (1954). G. F. McKay, J. J. Lalich, E. D. Schilling, and F. M. Strong, “A Crystalline ‘Lathyrus factor’ from Lathyrus adoratus,” Arch. Biochem. Biophys., 52, 313 (1954). E. D. Schilling and F. M. Strong, “Isolation, Structure and Synthesis of a Lathyrus Factor frcm L. odoratus,” J. Am. Chem. Soc., 76, 2848 (1954). W. Dasler, “Isolation of Toxic Crystals from Sweet Peas (Lathyrus odoratus),”’ Science, 120, 307 (1954). S. Wawzonek, I. V. Ponseti, R. S. Shepard, and L. G. Wiedenmann,“Epiphyseal Plate Lesions, Degenerative Arthritis, and Dissecting Aneurysm of the Aorta Produced by Aminonitriles,” Science, 121, 63 (1955). J. T. Garbutt and F. M. Strong, “Colorimetric Determination of 8-Amino- propionitrile in Mature Legume Seeds,” J. Agr. Food Chem., 5, 367 (1957). E. A. Bell, “Associations of Ninhydrin-Reacting Compounds in the Seeds of 49 Species of Lathyrus,” Biochem. J., 83, 225 (1962). A. F. Gardner, W. Dasler, and J. P. Weinmann, ““Masticatory Apparatus of Albino Rats in Experimental Lathyrism,” J. Dental Res., 37, 492 (1958). ‘C. I. Levene and J. Gross, “‘Alterations in State of Molecular Aggregation of Collagen Induced in Chick Embryos by §-aminopropionitrile (Lathyrus Fector),” J. Exptl. Med., 110, 771 (1959). M. J. Karnovsky and M. L. Karnovsky, “Metabolic Effects of Lathyrogenic Agents on Cartilage In Vivo and In Vitro,” J. Exptl. Med., 113, 381 (1961). G. R. Martin, J. Gross, K. A. Piez, and M. S. Lewis, “On the Intramolecular Cross-linking of Collagen in Lathyritic Rats,” Biochim. Biophys. Acta, 53, 599 (1961). J. Gross, “An Intermolecular Defect of Collagen in Experimental Lathyrism,” Biochim. Biophys. Acta, 71, 250 (1963). C. Ressler, P. A. Redstone, and R. H. Erenberg, “Isolation and Identification of a Neuroactive Factor from Lathyrus latifolius,” Science, 134, 188 (1961).

25. 26. 29. 30. 31. 32. 33. IRWIN E. LIENER G. H. Arscott and J. A. Harper, “Relationship of 2,5-Diamino-4,6- diketopyrimidine, 2,4-Diaminobutyric Acid, and a Crude Preparation of 8-Cyano-L-alanine to the Toxicity of Common and Hairy Vetch Seed Fed to Chicks,” J. Nutr. 80, 251 (1963). E. A. Bell, a, y-Diaminobutyric Acid in Seeds of Twelve Species of Lathyrus and Identification of a New Natural Amino Acid L-Homoarginine in Seeds of other Species Toxic to Man and Domestic Animals,” Nature, 193, 1078 (1962). S. L. N. Rao, L. K. Ramachandran, and P. R. Adiga, “The Isolation and Characterization of L-Homoarginine from Seeds of Lathyrus sativus” Bio- chemistry (USSR) (English Transl.), 2, 298 (1963). . P. R. Adiga, S. L. N. Rao, and P. S. Sarma, “Some Structural Features and Neurotoxic Action of a Compound from Lathyrus sativus Seeds,” Current Sci. (India), 32, 153 (1963). S. L. N. Rao, P. R. Adiga, and P. S. Sarma, ‘‘The Isolation and Characterization of B-N-oxalyl-a, 8-diaminopropionic Acid, a Neurotoxin from the Seeds of Lathyrus sativus,” Biochemistry (USSR) (English Transl.), 3, 432 (1964). C. Ressler, “‘Isolation and Identification from Common Vetch of the Neurotoxin B-Cyano-L-alanine, A Possible Factor in Neurolathyrism,” J. Biol. Chem., 237, 733 (1962). J. A. Harper and G. H. Arscott, “Toxicity of Common and Hairy Vetch Seed for Poults and Chicks,” Poultry Sci., 41, 1968 (1962). S. R. A. Shah, “A Note on Some Cases of Lathyrism in a Punjab Village,” Indian Med. Gaz., 74, 385 (1939). V. V. S. Murti, T. R. Seshadri, and T. A. Venkitasubramanian, “‘Neurotoxic Compounds of the Seeds of Lathyrus sativus,” Phytochemistry, 3, 73 (1964).

IRWIN E. LIENER Favism Hemolytic anemia, a characteristic syndrome of favism, frequently accompanies the ingestion of the fresh or uncooked broad or fava bean, Vicia faba, and occurs quite commonly in some Mediterranean countries.! Sporadic cases of favism have been observed in the United States, and these case histories have been compiled and reviewed by McPhee.2 Individual susceptibility to this disease is believed to be hereditary and is transmitted by a sex-linked gene of intermediate dominance.3 An attack is frequently precipitated simply by exposure to the pollen of the blossoms of this legume.! A considerable amount of work has been conducted on some of the biochemical abnormalities of the erythrocytes of people who are sus- ceptible to, or actually suffering from, favism. Thus, the glutathione content of cells from susceptible individuals is reduced,‘—* the activity of the enzyme glucose-6-phosphate dehydrogenase is markedly dimin- ished,3:7-9 and the red blood cells readily undergo spontaneous glu- colysis.!° Bonsignore et al.!! have reported elevated levels of transketo- lase and transaldolase activities in the blood of susceptible individuals. These authors believe that this biochemical abnormality represents a compensatory mechanism whereby the body seeks an adequate level of pentose phosphate to bypass the normal oxidative mechanism that utilizes glucose-6-phosphate dehydrogenase. In spite of the rather extensive studies that have been made on the clinical manifestations of favism, the causative principle of this disease has not been identified with any degree of certainty. One of the diffi- culties is the fact that it has thus far been impossible to reproduce the disease in experimental animals. Although Borchers and Ackerson!2 47

48 IRWIN E. LIENER noted that heating improves the nutritive value of Vicia faba as mea- sured with rats, no mention was made of any pathological disturbances resembling favism in man when animals were fed the raw bean. It may be significant to note that extracts of Vicia faba agglutinate red blood cells in vitro'3—!7 and will cause hemolysis when injected into rabbits,'6 but the exact relationship that these properties bear to the previously noted biochemical defects of favism remains obscure. In a recent series of papers, Lin and Ling!8-29 claim that the nucleo- side, vicine (structure shown in Figure 1), is the active principle of Vicia faba responsible for favism. Their conclusion is based on the observations that vicine fed at a level of 0.6 percent of the diet retards the growth of rats, and its administration to dogs by stomach tube at a level of 0.2 g/kg of body weight induces mild hemoglobinuria.!9 Vicine also inhibited the in vitro activity of glucose-6-phosphate dehydrogenase,?° an observation that is consistent with one of the observed biochemical defects of favism in man (see above). CH OH N—C=0 Ht | | acid H ———> + HaN~c HC—NHo | OH H OH hydrolysis }O\OH ° HN- C=0 Vicine D - Glucose Divicine (2,5 - diamino- 4,6 ~ diketopyrimidine) Figure 1 Structure of vicine, a nucleoside isolated from Vicia faba by Lin and Ling.!8 It is a curious fact that vicine was one of the first nucleosides to be discovered in plant materials having been isolated from the seeds of the vetch, Vicia sativa, and the garden pea, Pisum sativum, as early as 1891.2! Vicia sativa has been estimated to contain as much as 0.3 per- cent of vicine.22 The latter is the only nucleoside in nature known to contain a glucose residue.?3 As shown in Figure 1, mild acid hydrolysis releases glucose as well as 2,5-diamino-4,6-diketopyrimidine, also known as divicine. Although divicine is toxic when injected into experi- mental animals,?2:24 and has in fact been suggested as the causative agent of human lathyrism,22 feeding experiments have shown divicine to produce no harmful effects other than an inhibition of growth at a level of 1 percent of the diet of rats? and chicks.26 In contrast to the

FAVISM 49 toxic effects of injected divicine, the parent nucleoside, vicine, is rela- tively nontoxic by the same mode of administration,2”23 but does inhibit the growth of rats when fed.!9 If growth inhibition is indeed the only outward manifestation of the effects of the oral ingestion of divicine or vicine in experimental animals, it is not surprising that the physiological significance of these substances as a possible causative factor of favism has escaped the notice of earlier investigators. REFERENCES 1. 2. 3. 10. 11. 12. 13. 14, A. Luisada, ““Favism: Singular Disease Chiefly Affecting Red Blood Cells,” Medicine (Tokyo), 20, 229 (1941). W. R. McPhee, “*Acquired Hemolytic Anemia Caused by Ingestion of Fava Beans,”’ Am. J. Clin. Pathol., 26, 1287 (1956). W. H. Zinkham, R. E. Lenhard, Jr., and B. Childs, “‘A Deficiency of Glucose-6- phosphate Dehydrogenase Activity in Erythrocytes from Patients with Favism,” Bull. Johns Hopkins Hosp., 102, 169 (1958). A. Szeinberg and A. Chari-Bitron, “Blood Glutathione Concentration after Hemolytic Anemia Due to Vicia faba or Sulphonamides,” Acta Haemazol., 18, 229 (1957). . P. Brunetti and F. Grignani, “The Glutathione Stability Test in Red Cells of Patients with Favism Long after the Hemolytic Crisis,” Rass. Med. Sarda, 60, 387 (1958). D. G. Walker and J. E. Bowman, “*In vitro Effect of Vicia faba Extracts upon Reduced Glutathione of Erythrocytes,” Proc. Soc. Exptl. Biol. Med., 103, 476 (1960). J. E. Bowman and D. G. Walker, “Action of Vicia faba on Erythrocytes: Possible Relationship to Favism,” Nature, 189, 555 (1961). G. Sansone and G. Segni, ““New Aspects of the Biochemical Changes of Red Cells in Favism: Almost Complete Absence of Glucose-6-phosphate De- hydrogenase,” Boll. Soc. Ital. Biol. Sper., 34, 327 (1958). S. Ventura, P. Brunetti, and F. Grignani,“Bioenzymic Research on Relatives of Patients with Favism,” Rass. Med. Sarda, 60, 449 (1958). P. Brunetti, “Spontaneous Glucolysis of Erythrocytes in Favism,” Boll. Soc. Ital. Biol. Sper., 34, 102 (1958). A. Bonsignori, G. Fornaini, G. Segni, and A. Seitun, ‘““Transketolase and Transaldolase Reactions in the Erythrocytes of Human Subjects with Favism History,” Biochem. Biophys. Res. Commun., 4, 147 (1961). R. Borchers and C. W. Ackerson, ““The Nutritive Value of Legume Seeds. X. Effect of Autoclaving and the Trypsin Inhibitor Test for 17 Species,” J. Nutr., 41, 339 (1950). M. Krupe, “‘Hamagglutinine mit spezifischen Affinitaten in Samen von Papilio- naceen,” Biol. Zentr., 72, 424 (1953). W. C. Boyd and R. M. Reguera, ‘“‘Hemagglutinating Substances for Human Cells in Various Plants,” J. Jmmunol., 62, 333 (1949).

50 15. 16. 17. 18. 19. 20. 21. 24. 25. 26. 27. 28. IRWIN 8. LIBNER W. P. Creger and H. Gifford, “Some Interrelationships of Blood and Fava Bean Principle in vitro,” Blood, 7,721 (1952). E. Cugudda, C. Gigli, and S. Massenti, “Pathogenesis of Favus. Hemagglutina- tion and Hemolytic Activity of Some Chemical Constituents,” Minerva Med., 44, I, 140 (1953). K. L. Roth and A. M. Frumin, “Studies on the Hemolytic Principle of the Fava Bean,” J. Lab. Clin. Med., 56, 695 (1960). J. Y. Lin and K. H. Ling, “Studies on Favism. I. Isolation of an Active Principle from Faba Beans (Vicia faba),”’ J. Formosa Med. Assoc., 61, 484 (1962). J. Y. Lin and K. H. Ling, “Studies on Favism. II. Studies on the Physiological Activities of Vicine in vivo,” J. Formosa Med. Assoc., 61, 490 (1962). J. Y. Lin and K. H. Ling, “‘Studies on Favism. III. Studies on the Physiological Activities of Vicine in vitro,” J. Formosa Med. Assoc., 61, 579 (1962). E. Schulze, “Basic Nitrogen-Containing Compounds from the Seeds of Vicia sativa and Pisum sativum,” Z. Physiol. Chem., 15, 140 (1891). L. A. P. Anderson, A. Howard, and J. L. Simonsen, “Studies on Lathyrism,” Indian J. Med. Res., 12, 613 (1925). P. A. Levene and L. W. Bass, Nucleic Acids, The Chemical Catalog Co., New York (1931), p. 161. I. S. Kleiner, ““The Physiological Action of Some Pyrimidine Compounds of the Barbituric Acid Series,” J. Biol. Chem., 11, 443 (1912). J. G. Lee, “Experimental Lathyrism Produced by Feeding Singletary Pea (Lathyrus pusillus) Seed,” J. Nutr., 40, 587 (1950). G. H. Arscott and J. A. Harper, “Relationship of 2,5-Diamino-4,6-diketo- pyrimidine, 2,4-Diaminobutyric Acid and a Crude Preparation of 8-Cyano-L- alanine to the Toxicity of Common and Hairy Vetch Seed Fed to Chicks,” J. Nutr., 80, 251 (1963). H. Herissey and J. Cheymol, “Physiologic Action of Vicioside (Vicin),” Bull. Soc. Chim. Biol., 16, 1176 (1934). O. Fléssner, “The Physiological Effects of Nucleic Acids and Their Derivatives,” Arch. Exptl. Pathol. Pharmakol., 174, 215 (1934).

IRWIN E. LIENER Hemagglutinins in Foods* The presence of substances in legumes that have the ability to aggluti- nate the red blood cells from various species of animals has long been recognized.2-7 These hemagglutinins are protein in nature and are sometimes referred to as phytoagglutinins or lectins;® their distribution among the legumes is shown in Table 1. Much of the work dealing with the reaction of the seed agglutinins with red blood cells has involved the use of crude aqueous or saline extracts of the ground seed or, in some cases, the use of concentrates partially purified by salt fractiona- tion. The only seed hemagglutinins, however, that have been purified to a sufficient extent to permit meaningful measurements of their chemical and physical properties are those of the castor bean, jack bean, soybean, and beans belonging to the species Phaseolus vulgaris. Table 2 presents a summary of the physical constants of the hemag- glutinins that have been purified and for which such data are available. Little information is available concerning the concentration of hemag- glutinins in the seeds of legumes except that the soybean hemagglutinin has been reported to comprise about 3.2 percent of the defatted soy- bean meal,37 and ricin, the hemagglutinin of the castor bean (not a legume), reportedly constitutes about 1.5 percent of the oil-free meal.38 Perhaps the most familiar of the hemagglutinins is the highly toxic protein, ricin, derived from the castor bean. Because of its extreme toxicity, castor bean meal must be thoroughly detoxified by heat before it can be used as a source of protein for animals.39—*2 It is perhaps no coincidence that all of the edible legumes that are known to contain hemagglutinins (see Table 1) are also toxic or have poor nutritive value * This subject has recently been reviewed by the author in more detail.! 51

52 IRWIN E. LIENER TABLE 1 Distribution of Hemagglutinins among the Legumes BOTANICAL NAME COMMON NAME REFERENCES Arachis hypogaea Peanut 9 Groundnut Canavalia ensiformis Jack bean 10-12 Canavalia gladiata Sword bean _ 13 Dolichos lablab Horse gram 8, 14, 15 Horse bean Field bean Hyacinth bean Glycine max Soybean 2, 16, 17 Lathyrus odoratus Sweet pea 18 Lens esculenta Lentil 3 Phaseolus aureus Mung bean 18 Green bean Phaseolus lunatus Lima bean 19-21 Phaseolus vulgaris Navy bean 2, 4, 18, 22-25 Kidney bean Pinto bean French bean Black bean White bean Ricinus communis® Castor bean 26 Vicia faba Fava bean 17, 18, 27-29 Horse bean Broad bean Vicia sativa Common vetch 3, 18, 30 « The castor bean is not a legume. unless subjected to heat treatment.* Legumes constitute an important source of dietary protein for large segments of the world’s population, and a number of reports exist in the literature describing manifesta- tions of toxicity in subjects who have eaten insufficiently cooked legumes.48—50 * A comprehensive treatment covering the subject of the effect of heat treatment on the nutritive value of legumes may be found in references 43 and 44. Phaseolus vulgaris and Dolichos lablab require preliminary soaking prior to cooking to eliminate completely the toxicity of these legumes.45—47

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