Toxicants Occurring Naturally in Foods. (1966)

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138 BENJAMIN J. WILSON Russian authors!.119.120 refer to toxigenic species of other genera. Among these are Ustilago longissima, a smut fungus, and Diploidia zeae, a dry rot pathogen, considered etiologically in intoxications of long-horned cattle. Among the most toxic organisms studied, Dendro- dochium toxicum stands out as a causative agent of disease in man and horses. In nature the fungus is most often encountered on substrates containing cellulose. Mucor hiemalis and M. albo-ater were included in the toxigenic fungal flora of grains in Bashkiria, U.S.S.R. Contra- dictory data on the pathogenicity of Trichoderma lignorum have been reported, but it is known that gliotoxin and viridin, both of which are toxic for animals, are synthesized by this organism. Certain species of Alternaria, Rhizopus, and Cladosporium have also been described as toxigenic.!10:119.121.122 Cjadosporium epiphyllum, grow- ing on millet, synthesized a compound called “‘epicladosporic acid,” with the probable formula CH3(CH2)7;CH—CH(CH2)3COSH, and C. fagi formed a related ‘“‘fagicladisporic acid,” which has the suggested formula CH3(CH2)>CH=—CH(CHz2)sCOSH. Although toxic, these compounds did not alter the leucocyte count of blood in experimental animals as did sporofusariogenin and poefusariogenin.!3 In Canada, a neurological incoordination in newborn lambs occurred when the pregnant ewes had been fed mold-contaminated peavine silage for several weeks prior to parturition.!24 The condition has been termed “‘cerebellar ataxia’”’ since it is characterized by lesions of the cerebellum with incoordination and temporary paralyses associated with damage to this part of the brain. A sample of the darkened peavine silage received by this author had a marked tobacco odor and yielded a pure culture of a mold tentatively identified as Oidium sp. Mice were not affected by large amounts of silage extract fed over a period of 5 days. Similarly, negative findings were obtained from examination of molded yams believed responsible for a peculiar trembling disease syndrome of people in western Nigeria. The disease, called ‘‘encepha- litis tremens” by Wright and Morley,!% who first described it, is also locally known as the “‘Ijesha shakes.” In the United States, Ireland, Australia, and other countries, a disease of swine, vulvovaginitis, attributed to estrogen-like stimulation following ingestion of moldy feed, has been reported at widely spaced intervals.!26127 Gibberella zeae (Fusarium graminearum) has been a frequent isolate from grains involved. Stob and co-workers!28 at Purdue were able to reproduce the disease in piglets fed G. zeae grain. The uterotrophic principle was extracted with anhydrous ethanol from

FUNGAL TOXINS 139 the corn-mycelial mixture. The substance was obtained as a white crystalline mass with a melting point range of 161-163°C. Methanolic solutions exhibit a bright greenish-blue fluorescence in ultraviolet light. Other investigators in the United States also are presently studying this problem.!3 Seratostomella fimbriata causes a black rot of sweet potatoes. Affected potatoes are toxic to livestock.!29 This fungus reportedly pro- duces a chemical substance related to ngaione, a hepatotoxin obtained from leaves and seeds of Myoporum laetum (Ngaio tree) of New Zealand.130 SUMMARY Although the filamentous fungi are considered as simple plants phyloge- netically, their importance to man both economically and in relation to infectious diseases has been recognized for a long time. Now it is abundantly evident that molds also play a significant role in the toxic diseases of animals and apparently to a lesser extent in man. Research into this fascinating area was long delayed, and only recently, after seeing the economic and public health implications of extensive live- stock disease outbreaks, has world attention begun to be focused on this area. This research undoubtedly will uncover additional toxigenic organisms along with mycotoxins whose characteristics may prove of considerable interest to many aspects of agricultural and medical science. REFERENCES 1. C.H. Eckles, C. P. Fitch, and J. L. Seal, “Molds in Silage and Their Significance in the Production of Disease Among Livestock,” J. Series, Minn. Univ. Agr. Expt. Sta., No. 417 (1924). 2. S. S. Buckley and W. G. MacCullum, “‘Acute Haemorrhagic Encephalitis Prevalent Among Horses in Maryland,” Am. Vet. Rev., 25, 99 (1901). 3. T. Butler, ““Notes on a Feeding Experiment to Produce Leucoencephalitis in a Horse with Positive Results,” Am. Vet. Rev., 26, 748 (1902). 4. L.H. Schwarte, H. E. Biester, and C. Murray, “A Disease of Horses Caused by Feeding Moldy Corn,” J. Am Vet. Med. Assoc., 90, 76 (1937). 5. J. M. Kingsbury, Poisonous Plants of the United States and Canada, Prentice- Hall, Englewood Cliffs, N.J. (1964), pp. 70-86.

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144 89. 91. 92. 93. 94. 95. 96. 97. 98, 100. 101. 102. 103. 104. BENJAMIN J. WILSON K. Uraguchi, T. Tatsuno, F. Sakai, M. Tsukioka, Y. Sakai, O. Yonemitsu, H. Ito, M. Miyake, M. Saito, M. Enomoto, T. Shikata, and T. Ishiko, “‘Isola- tion of Two Toxic Agents, Luteoskyrin and Chlorine-Containing Peptide, from the Metabolites of Penicillium islandicum Sopp, with Some Properties Thereof,” Japan. J. Exptl. Med., 31, 19 (1961). M. Miyake, M. Saito, M. Ushiki, K. Uraguchi, M. Tsukioka, and Y. Ikeda, **Histopathological Studies on the Liver Injury Due to the Toxic Substances of Penicillium islandicum Sopp,” Acta Pathol. Japon., 5, 208 (1955). F. Sakai, “‘Toxic Effect, Particularly on the Kidney, of Yellowed Rice, Polluted by Penicillium citrinum, Thom, as well as of Citrinin, a Pigment Isolated from the Mold,” Folia Pharmacol. Japon., 51, 431 (1955). M. Hori, T. Yamamoto, A. Ozawa, Y. Matsuki, A. Hamaguchi, and H. Soracka, “Studies on a Fungus Species Isolated from Malt Feed which Caused Mass Death of Cows,” Japan J. Bacteriol., 9, 1105 (1954). N. Sakabe, T. Goto, and Y. Hirata, “The Structure of Citreovirdin, a Toxic Compound Produced by P. citreoviride Molded on Rice, Tetrahedron Letters, 27, 1825 (1964). A. M. Ambrose and F. DeEds, ‘“‘Some Toxicological and Pharmacological Properties of Citrinin,” J. Pharmacol. Exptl. Therap., 88, 173 (1946). W. Chu, “Miscellaneous Pharmacologic Actions of Citrinin,” J. Lab. Clin. Med., 31, 72 (1946). W. L. Sippel, J. E. Burnside, and M. B. Atwood, “A Disease of Swine and Cattle caused by Eating Moldy Corn,” Proc. Am. Vet. Med. Assoc., 174 (1953). B. J. Wilson and C. H. Wilson, ‘‘Hepatotoxic Substance from Penicillium rubrum,” J. Bacteriol., 83, 693 (1962). B. J. Wilson and C. H. Wilson, “Extraction and Preliminary Characterization of a Hepato-toxic Substance from Cultures of Penicillium rubrum,” J. Bacteriol., 84, 283 (1962). J. Forgacs and W. T. Carll, “Preliminary Mycotoxic Studies on Hemorrhagic Disease in Poultry,” Vet. Med., 50, 172 (1955). . J. Forgacs, H. Koch, W. T. Carll, and R. H. White-Stevens, “‘Additional Studies on the Relationship of Mycotoxicoses to the Poultry Hemorrhagic Syndrome,” Am. J. Vet. Res., 19, 744 (1958). Yu. I. Rubinshteyn, “Food fusariotoxicoses,” in Mycotoxicoses of Man and Agricultural Animals, V. 1. Bilay, ed. (Engl. Trans.), Office of Tech. Ser., U.S. Dept. of Commerce, Washington, D.C. (1960), p. 89. A. Z. Joffe, ‘““Biological Properties of Some Toxic Fungi Isolated from Over- wintered Cereals,” Mycopathol. Mycol. Appl., 16, 201 (1962). N. V. Perkel’, “Study of the Toxicity of Transbaikal Strains of Fusarium sporotrichiella Bilai in connection with the Etiology of Urov Disease (Kaschin- Beck Disease),” in Mycotoxicoses of Man and Agricultural Animals, V. 1. Bilay, ed. (Engl. Trans.), Office of Tech. Ser., U.S. Dept. of Commerce, Wash- ington, D.C. (1960), p. 117. A. I. Nesterov, “The Clinical Course of Kaschin-Beck Disease,” Arthritis Rheumat., 7, 29 (1964). V. V. Bart, ‘“‘Material on the Study of the Toxicity of Late-Gathered Cereals in the LatvSSR,” in Mycotoxicoses of Man and Agricultural Animals, V. 1. Bilay, ed. (Engl. Trans.), Office of Tech. Ser., U.S. Dept. of Commerce, Washington, D.C. (1960), p. 125.

FUNGAL TOXINS 145 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. A. F. Tkachenko, “The Problem of Looking for Allergic Methods of Diagnosis of Stachybotryotoxicosis of Horses,” in Mycotoxicoses of Man and Agri- cultural Animals, V. 1. Bilay, ed. (Engl. Trans.), Office of Tech. Ser., U.S. Dept. of Commerce, Washington, D.C. (1960), p. 180. Ya. A. Fialkov and S. Serebryanyy, ‘“‘The Isolation of Toxic Substances from a Culture of Stachybotrys alternans Fungi and the Investigation of Their Chemi- cal Nature,” in A New Fungus Disease of Horses and People, Kiev (1949). J. Forgacs, W. T. Carll, A. S. Herring, and W. R. Hinshaw, “Toxicity of Stachybotrys atra for Animals,” Trans. N.Y. Acad. Sci., 20, 787 (1958). G. Schumaier, H. M. DeVolt, N. C. Laffer, and R. D. Creek, “‘Stachybotryo- toxicosis of Chicks,” Poultry Sci., 42, 70 (1963). D. C. Dodd, “Facial Eczema in Ruminants,”’ in Mycotoxins in Foodstuffs, G. N. Wogan, ed., The M.I.T. Press, Cambridge, Mass. (1965), p. 105. R. Hodges, J. W. Ronaldson, A. Taylor, and E. P. White, ““Sporidesmin and Sporidesmin-B,”” Chem. Ind. (London), 42 (1963). J. D. K. North and J. F. Gwynne, “A Review of Possible Human Implications of Facial Eczema,” New Zealand Med. J., 159, 325 (1960). J. C. Percival and R. H. Thornton, “‘Relationship Between the Presence of Fungal Spores and a Test for Hepatotoxic Grass,’ Nature, 182, 1095 (1958). R. H. Thornton and J. C. Percival, ““A Hepatotcxin from Sporidesmium bakeri Capable of Producing Facial Eczema Diseases in Sheep,” Nature, 183, 63 (1959). R. H. Thornton, “The Identification and Culture of Sporidesmium bakeri Syd.,” Proc. N. Zealand Soc. Animal Prod., 19, 83 (1959). P. H. Gregory and M. E. Lacey, “The Discovery of Pithomyces chartarum in Britain,” Trans. Brit. Mycol. Soc., 47, 25 (1964). R. Hodges, J. W. Ronaldson, A. Taylor, and E. P. White, ‘“‘Sporidesmins. ITI. The Structure of Degradation Products Related to 5-Chloro-6,7-dimethoxy- isatin,” J. Chem. Soc., 5332 (1963). P. H. Mortimer, “The Experimental Intoxication of Sheep with Sporidesmin, a Metabolic Product of Pithomyces chartarum. 1V. Histological and Histo- chemical Examinations of Orally-Dosed Sheep,”’ Res. Vet. Sci., 4, 166 (1963). T. F. Slater and D. B. Griffiths, “Effects of Sporidesmin on Bile Flow Rate and Composition in the Rat,’’ Biochem. J., 88, 60 (1963). N. M. Pidoplichko and V. I. Bilay, “Toxic Fungi which Develop in Food Products and Fodder,” in Mycotoxicoses of Man and Agricultural Animals, V. I. Bilay, ed. (Engl. Trans.), Office of Tech. Ser., U.S. Dept. of Commerce, Washington, D.C. (1960), pp. 3-36. A. V. Vasin, “Fungal Toxicosis of Animals and Its Clinicolaboratorial Diag- nosis,” Veterinariya, 35, 64 (1958). Bloshintsyn, “Illness in Cows and Calves After Eating Corn Cobs Affected by Mold,” Veterinariya, 36, 74 (1959). A. Z. Joffe, “‘Toxin Production by Cereal Fungi Causing Alimentary Toxic Aleukia in Man,” in Mycotoxins in Foodstuffs, G. N. Wogan, ed., The M.I.T. Press, Cambridge, Mass. (1965), p. 77. L. Ye. Olifson, ““‘The Chemical Activity of Some Species of Fungi Which Affect the Grain of Cereals,” in Mycotoxicoses of Man and Agricultural Animals, V. I. Bilay, ed. (Engl. Trans.), Office of Tech. Ser., U.S. Dept. of Commerce, Washington, D.C. (1960), pp. 64-65.

146 124. 125. 126. 127. 128. 129. 130. BENJAMIN J. WILSON F. Whiting, R. Connell, P. J. G. Plummer, and R. D. Clark, “Incoordination (Cerebellar Ataxia) Among Lambs from Ewes Fed Peavine Silage,” Can. J. Comp. Med. Vet. Sci., 21, 77 (1957). J. Wright and D. C. Morley, ‘Encephalitis Tremens,” Lancet, i, 871 (1958). J. S. Koen and H. C. Smith, “‘An Unusual Case of Genital Involvement in Swine Associated with Eating Moldy Corn,” Vet. Med., 40, 131 (1945). B. A. McErlean, ““Vulvovaginitis of Swine,” Vet. Rec., 64, 539 (1952). 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). T. Kubota and N. Ichikawa, *‘On the Chemical Constitution of Ipomeanine, a New Ketone from the Black-Rotted Sweet Potato,” Chem. Ind., 29, 902 (1954). , F. A. Denz and W. G. Hanger, “‘The Liver Toxin in Myoporum laetum,” J. Pathol. Bacteriol., 81,91 (1961).

J. H. WILLS, JR. Seafood Toxins This discussion is limited to intoxications that follow ingestion of animals that live in the seas, and thus does not concern venoms of such creatures. Insofar as the present state of knowledge allows, poisons formed by bacterial decomposition of the tissues of animals taken from the oceans will not be considered. The principal recognized types of poisoning by seafoods and brief statements about their characteristics follow. The first four sorts of poisoning are the most important from a quantitative point of view. Seafood poisons are particularly important from the standpoint of public health in Japan, where marine organisms contribute about 10 percent of the total food supply. There, poisoning by seafoods has been estimated to be responsible for 60 to 70 percent of all food poisoning. Ciguatera Poisoning This kind of seafood poisoning was first reported from the West Indies by Peter Martyr in 1555.! The first symptoms of the poisoning may be 2 tingling of the lips, tongue, and throat, followed by numbness of the same areas. In other cases, the initial symptoms consist of nausea, vomiting, metallic taste, dryness of the mouth, abdominal cramps, painful spasm of the anal sphincter, and diarrhea, followed by perioral tingling and numbness. Headache, anxiety, malaise, prostration, dizziness, pallor, cyanosis, insomnia, chilliness, fever, profuse sweating, rapid and weak pulse, weight loss, muscular pain, and joint pain are commonly present. Weakness may become progressively worse until the intoxicated person is unable to walk. The pupils of the eyes are 147

148 J. H. WILLS, JR. usually dilated, so that photophobia, blurred vision, scotomata, and even temporary blindness are common. In severe intoxications, pares- thesia and paradoxical temperature sensations are common. Ataxia and motor incoordination become progressively more severe and paral- ysis develops. Death is the result of respiratory paralysis and occurs in from 2 to 3 percent* to about 7 percent of cases of this type of poison- ing3 Moray Eel Poisoning This poisoning resembles ciguatera poisoning in many respects. It begins usually3 5 with tingling and numbness of the lips, tongue, hands, and feet, with a sensation of heaviness of the extremities. Other signs and symptoms of this intoxication are nausea, vomiting, diarrhea, abdominal pain, malaise, metallic taste, sore throat, laryngospasm and laryngeal paralysis, stiff neck, excessive production of mucus with foaming at the mouth, marked sweat production, increased body temperature, paralysis of thoracic musculature, ataxia, motor incoordination, violent convulsions, purposeless movements, areflexia, coma, and death. Death is caused probably by paralysis of the diaphragm and is estimated to occur in about 10 percent of the cases.2 In nonfatal cases, areflexia may persist for 2 or more months after the poisoning.5 Scombroid Poisoning This type of poisoning may be due‘ to products of bacterial action in the flesh of scombroid fishes (tuna, bonito, mackerel, and skipjack, for example). The toxic agent has been considered to be a histamine-like substance, released from a peptamine by bacterial action after the death of the fish.7 Kawabata and his associates have reported®:9 the isolation of an orally active substance from Scombresox saurus. They have given the name saurine to this material and believe that it is formed in the tissues of the fish by bacterial action. Saurine seems to be a potent vagal stimulant; its discoverers believe that it is the cause of scombroid poisoning, and that its vagal stimulant action is its principal mechanism of toxicity. Poisoning by eating scombroid fishes resembles that by histamine and differs from the two preceding types in having no signs of neurotoxicity. The principal symptoms? are nausea, vomiting, facial flushing, intense headache, epigastric pain, burning of the throat, thirst, difficulty in swallowing, labial edema, itching of the skin, and urticaria.

SEAFOOD TOXINS 149 Death occurred in 17 of 2,159 persons poisoned by scombroid fishes.!° The symptoms of nonfatal poisoning usually subside within 12 hours. Puffer Fish Poisoning This poisoning frequently begins? with a tingling or prickling sensation of the fingers and toes and is rapidly fulminating. Malaise, dizziness, pallor, numbness of the lips, tongue, and extremities, and ataxia may develop within 10 to 45 minutes after ingesting the fish. On the other hand, symptoms may not develop for 3 or more hours. Nausea, vomit- ing, diarrhea, and epigastric pain may appear, but they are not constant components of the syndrome. As the intoxication deepens, the eyes become fixed and the pupillary and corneal reflexes are lost. Res- piratory distress—increased rate of breathing and diminution in depth of breathing—is seen frequently. Muscular twitching, tremor, and incoordination become progressively more marked, terminating in extensive muscular paralysis with intense cyanosis. Terminal con- vulsions may occur. According to Japanese statistics,!! 2,090 people died out of 3,106 poisoned between 1888 and 1909 (mortality rate above 67 percent). A more recent collection!? of Japanese mortality data shows that, during 1956-1958, death occurred in 420 of 715 people who were recognized to have been poisoned by the puffer fish toxin. Cephalopod Poisoning Squid and octopus have been responsible for sickness among the Japanese.!° The predominant symptoms were nausea, vomiting, diarrhea, abdominal pain, mild fever, headache, chills, and weakness. Some patients developed paralysis or convulsions. Most of the patients recovered within about 48 hours. In one study of the period 1952-1955, death occurred in 10 of 2,649 people who had eaten squid. In an out- break during 1955, there were no fatalities among 210 people who had eaten octopus, although 21 became sick. In an experiment during July 1955, 50 people ate squid and 15 became sick.!° No organisms known to be causative agents of any of the ordinary types of bacterial food poisonings could be isolated from either the toxic or the nontoxic squid in this experiment. Chimaeroid Poisoning Pellegrin!3 and Phisalix!4 made passing mention that certain chimaeroid fishes (ratfish) produce neurotoxic effects. Detailed symptomatology

150 J. H. WILLS, JR. was not given. Halstead and Bunker'5 reported that an extract of ovary from a ratfisin was toxic upon intraperitoneal injection into mice. Cyclostome Poisoning The mucus and flesh of some lampreys and hagfishes have been re- ported3-!6-18 to produce gastrointestinal upset, with nausea and vomit- ing. The signs and symptoms of this poisoning have not been described carefully. Lamprey is said!! to be wholesome if strewn with salt while still alive and carefully freed of mucus. Elasmobranch Poisoning Poisonings from eating portions of shark, dogfish, or ray are charac- terized!9-22 by nausea, vomiting, abdominal pain, oily stool, pallor, headache, burning and tingling of lips, tongue, and throat, visual disturbances, pain and sensation of heaviness of the limbs, chest pain, and generalized itching. Coma and death may occur several days after ingestion. The most severe poisoning results from ingestion of the liver of the elasmobranches.!! The mortality from this kind of poisoning is not known. Gempylid Poisoning The flesh of some of the snake mackerels contains a purgative oil that has a pronounced effect when ingested. Diarrhea develops rapidly but is not accompained by cramping or abdominal pain or by any other signs or symptoms. Hallucinogenic Fish Poisoning Helfrich has reported”? that fishes belonging for the greatest part to species that can cause the ciguatera type of intoxication, with pares- thesia as its most striking neurological component, sometimes cause a syndrome of dizziness, loss of equilibration, ataxia, hallucination, and mental depression. The intoxication seems to be generally not fatal. Clupeoid Poisoning Phisalix has reported!4 a disease caused by eating certain herring-like fishes of the Pacific; it begins with nausea, vomiting, and abdominal

SEAFOOD TOXINS 151 pain very soon after eating the toxic fish and may progress rapidly through tachycardia, cold sweat, dyspnea, cyanosis, coma, and con- vulsion to death. Death may occur within 30 minutes after the fish is eaten.22 The poison is stable to the heat of cooking. Porpoise Poisoning Macgowan reported in 188424 that although porpoise is highly esteemed by the Chinese as food, they have known for a long time that porpoises that go far inland along the Yangtze become poisonous. A Chinese proverb says, “Eat porpoise if you wish to discard life.’”” Macgowan reports further that, according to the Chinese, the blood and liver of the porpoise are generally poisonous, the fat causes swelling and numbness of the tongue, and the eyes produce dimness of vision. The entrails are poisonous also. He reported 11 deaths at Yangchow from eating portions of porpoises; later, he reported% that five persons died at Anch’ing in April of 1883 from eating porpoise. The symptoms and signs of the poisoning were pain, marked swelling of the abdomen, a purplish discoloration of the skin (cyanosis?), numbness, and dribbling of greenish saliva from the mouth. Turtle Poisoning This is another little-studied poisoning, first reported by Chevallier and Duchesne!‘ in 1851, caused by eating the flesh of such animals as Chelonia japonica, Dermochelys coriacea, and Eretmochelys imbricata. It is characterized by nausea, vomiting, diarrhea, weakness, sore lips and throat, hallucinations, coma, and death. Death may occur within 12 hours after ingestion or may be delayed for as long as 2 weeks after ingestion. Paralytic Shellfish Poisoning Poisoning after eating mussels and clams is noted first? as paresthesia of the lips, tongue, and fingertips, followed by ataxia and muscular incoordination. There is an ascending weakness and paralysis, involving first the legs, then the thorax, the arms, the neck, and the face. Death is due to cardiovascular collapse and respiratory failure. In the collection of 243 cases of this poisoning made by Meyer et al. in 1928,26 37 (15.2 percent) died. In the more extensive series collected by McFarren et al. in 1960,27 173 died out of 792 people poisoned (21.8 percent mortality).

152 J. H. WILLS, JR. Venerupin Poisoning This poisoning 1s due to the ingestion of the clam, Venerupis semidecussata Reeve, that grows in Japanese waters. The initial symp- tom of the poisoning is gastrointestinal upset.28 There may be acute yellow or red atrophy of the liver and hemorrhagic effects. The toxin differs from that of paralytic shellfish poisoning in not being inactivated by Reinecke acid.?9 Oyster Poisoning Poisonings from eating various oysters have been noted.!6.24.28.29 Jtai and Kamiya reported29 that, by paper chromatography of the methanolic extract of toxic oysters from Lake Hamana, they obtained a white product that decomposed at high temperature, migrated to the cathode in an electric field, and killed mice after interperitoneal injection of 0.5 mg of the material. Abalone Liver Poisoning In 1899, Takenaka et al.3° described a peculiar dermatitis observed in Japanese fishermen engaged in collecting abalones. The patients experienced sudden onsets of sensations of stinging and burning over the entire body; these initial effects were followed by pricking, itching, edema, and possible ulceration of the skin in the affected area. Etiology of Ciguatera Poisoning With comparatively few exceptions, the fishes responsible for ciguatera poisoning are not uniformly and consistently poisonous. For example, only 7 of 20 examples of Cephalopholis argus Bloch and Schneider collected near Palmyra Island during April 1953 were toxic.3! Also, most poisonous fishes seem to be taken near shores rather than from deep waters. Furthermore, toxicity is not species specific. Table 1 presents a list, compiled largely from those given by Fish and Cobb?2 and by Halstead, of families of fishes that have been reported to be poisonous. With the exception of the freshwater species, which cause a disease with colic, vomiting, cold sweat, hypertension, dizziness, faintness, tremor, distended abdomen, diarrhea, dilated pupils, and occasional death,!4 and those related to other types of poisoning specified above, these families of fishes are not always poisonous. Fish and Cobb*2 have pointed out the close correspondence between the distributions in the oceans of toxic fish and coralline growth.

SEAFOOD TOXINS 153 TABLE 1 Fish Families Reported to be Toxic Acanthuridae surgeonfishes Molidae ocean sunfish Acipenseridae sturgeons Monacanthidae _filefishes Ageneiosidae catfishes Mugilidae mullets Albulidae bonefishes Mullidae goatfishes Anarhichadidae wolffishes Muraenidae moray eels Anguillidae freshwater eels Myliobatidae eagle rays Antennariidae frogfishes Myxinidae hagfishes Ariidae sea catfishes Notidanidae cow sharks Aulostomidae trumpetfishes Ogcocephalidae batfishes Balistidae triggerfishes Ophichthyidae __ snake eels Batrachidae toadfishes Osmeridae smelts Belonidae needlefishes Ostraciontidae _ trunkfishes Bothidae left-eyed flounders Pempheridae deep-water catalufas Callionymidae —_dragonets Petromyzontidae lampreys Canthigasteridae sharp-nosed puffers Pleuronectidae __right-eyed flounders Carangidae jacks and pompanos__| Plotosidae Oriental catfishes Carcharhiidae sand sharks Pomacentridae damselfishes Cepolidae bandfishes Priacanthidae catalufas Chaetodontidae butterfly fishes Rajidae skates Chimaeridae chimaeras Rhinodontidae — whale sharks Chonerhinidae puffers Salmonidae salmon, trout Clupeidae herrings Scaridae parrotfishes Congridae conger eels Scatophagidae __ scats Coryphaenidae __ dolphins Sciaenidae croakers Cottidae sculpins Scombresocidae sauries Cybiidae Spanish mackerels Scombridae mackerels Cyprinidae minnows Scorpaenidae scorpionfishes Cyprinodontidae killifishes Scorpidae halfmoonfishes Dalatiidae Greenland sharks Scyliorhinidae cat sharks Dasyatidae rays Scyllidae dogfish Diodontidae burrfishes Serranidae groupers, basses Dorosomidae gizzard shads Siganidae rabbit fishes Engraulidae anchovies Siluridae catfishes Esocidae pikes Sparidae porgies Gadidae cods Sphyraenidae barracudas Gasterosteidae __ sticklebacks Sphyrinidae hammerhead sharks Gempylidae snake mackerels Squatinidae angel sharks Gerridae silver perch Syngnathidae pipefishes Gobiidae gobies Synodentidae lizardfishes Hemirhamphidae halfbeaks Tetragonuridae _squaretails Holocentridae squirrelfishes Tetraodontidae __ puffers Isuridae mackerel sharks Thunnidae tunas Katsuwonidae skipjacks Torpedinidae electric rays Labridae wrasses Triacanthidae spikefishes Lagocephalidae _ puffers Triakidae smooth hound sharks Lepisosteidae gars Triglidae poachers Liparidae snailfishes Trichodontidae — sandfishes Lophiidae anglers Xiphiidae swordfishes Lutjanidae snappers Zanclidae Moorish _ idolfishes

154 J. H. WILLS, JR. Dawson* has found that the flora of Palmyra atoll, following its occupation by U.S. forces during World War II, changed in such a way that the growth of the toxic alga Lyngbya majuscula on the reef flats was favored. Furthermore, the recognition of fragments of Lyngbya in the contents of the guts of poisonous fish from this atoll strengthens the suspicion that the occurrence of toxic fish in this area, at least, may be correlated with ingestion of the toxic Lyngbya. Other fish of the same species that had not ingested Lyngbya would not be toxic according to this theory. Randall,*5 from a worldwide review of ciguatera poisoning, had concluded that the ultimate source of the toxic material involved in this type of poisoning by fishes is probably a blue-green alga. Banner et al.36 have established that the vesicant material in some samples of Lyngbya majuscula differs in solubility characteristics from the toxic substances isolated from flesh of toxic Lutjanus bohar and conclude that there is no evidence that the toxins from the alga and the fish are related. Hessel has described3? a method for partial purification of a toxic material from toxic Lutjanus bohar by extraction of the fish with warm methanol and subsequent fractionation by chromatography on a column of silicic acid. The most toxic fraction, a yellow oil, killed mice after interperitoneal injection of 13 mcg/kg, the animals dying within about 3 hours after the injection.38 Halstead and Hessel?? have used the same method to isolate similar toxic fractions from both toxic fish and Lyngbya majuscula. This last finding appears to furnish strong support for the idea that fish causing ciguatera intoxication become toxic by ingesting the toxin. Furthermore, Helfrich and Banner“ found that the flesh of poisonous Lutjanus bohar, without apparently affecting the health of the surgeon fish (Acanthurus xanthopterus) to which it was fed, could cause the flesh of the latter fish to become toxic. The toxin of ciguatera appears, therefore, to be capable of being passed through a food chain without losing its lethal activity and without apparent harm to the carrier. We have, then, the general picture that the fishes that cause ciguatera poisoning are not inherently toxic but that they become toxic after feeding on toxic algae or other toxophoric materials present on coralline reefs or other shallows surrounding islands or continents in tropical or subtropical seas. The exact chemical nature of the toxin responsible for this poisoning is unknown, although it may be a lipid other than a phospholipid. Li has found‘! that extracts of the flesh of Lutjanus bohar, Gymnothorax javanicus, or Epinephelus fuscoguttatus or of the liver of Carcharhinus menisorrah all have similar toxic effects, resembling

SEAFOOD TOXINS 155 those caused by inhibitors of cholinesterase. The extract of shark’s liver was found to inhibit the cholinesterase of human red blood cells, a concentration of 2 mcg/ml of pure toxin being estimated to inhibit in vitro about 50 percent of the cholinesterase. Atropine and the oxime 2-PAM Cl were found to antagonize the toxic actions of ciguatera toxin in the mouse and the rat. Etiologies of Other Fish Poisonings Moray eel poisoning follows ingestion of various species of the genus Gymnothorax. The toxic principle or principles may occur in the eel in the flesh, the blood, or both. The poison in the flesh appears to be a substance of low molecular weight, but its exact composition is not known. Recent attempts to isolate and identify the toxic component of eel’s blood42 have revealed that the toxin can be precipitated from the serum of the blood with ammonium sulfate and ethyl alcohol, that the toxic activity of the separated fraction is destroyed by incubation with trypsin, and that the toxic fraction, after injection into rabbits, induces clonic convulsions, paralysis, ventricular fibrillation, and death when 0.2 mg of the protein per kilogram was administered. The toxic protein contains three components, the most abundant making up about 70 percent of the mixture. As will be noted from the signs listed above, the purified protein from eel’s blood does not reproduce all the effects of injection of crude serum; in particular, it does not produce hemolysis, abdominal pain and vomiting, hypesthesia, salivation, and dyspnea. This suggests that the protein mixture isolated by Ghiretti and Rocca is not the only com- ponent of eel’s blood contributing to its toxic actions. Jaques has adduced43 evidence that the gut-stimulating principal of serum may be serotonin; the hemolytic factor in the serum has been reported“ to be a salt of a higher fatty acid. Treatment for poisoning from eating the moray eel has to be sympto- matic at present. Intravenous injection of calcium gluconate is reported to have benefited five of six patients with this poisoning. Scombroid poisoning is caused by eating such fish as Scomber japonicus, Katsuwonus pelamis, and Thunnus thynnus. There is some evidence that the scombroid fishes are not toxic when cooked im- mediately upon being caught, but that the poison is formed very rapidly after death of the fish. The precise chemical nature of this poison seems to be unknown. The “saurine” mentioned previously may be an amine somewhat similar to histamine; the poison produces

156 J. H. WILLS, JR. a group of effects very similar to that produced by histamine. Anti- histaminics have been recommended?-4’ as treatment for this poisoning. Puffer fish poisoning is one of the most thoroughly studied of the poisonings resulting from eating fishes. There are at least 40 toxic species among 10 genera.45 The genera having the highest numbers of species known to be toxic are Sphoeroides, Arothron, Lagocephalus, and Tetraodon. Toxic puffers have been collected from practically all the seas of the world. Toxicity of puffers seems‘ to be greatest during spring and the early summer and to reside particularly in the gonads and associated viscera. According to Nichols and Bartsch,*? the flesh of toxic puffers can be made edible by thorough washing when the fish has just been caught. The toxin of puffers has been purified5° and has been identified as a zwitterion form of an internal ortho-ester including several fused rings.5! The empirical formula for the compounds is Cy,Hi7N30g. The crystalline toxin is not precipitated by protein or alkaloidal precipitating agents and is fairly stable to boiling except when in an alkaline solu- tion.5! The intraperitoneal LDso of crystalline material for mice is 8 to 20 mcg/g; the intravenous and intraperitoneal LDso’s for the cat are less than 10 mcg/kg. Many details of the functional changes induced by this poison have been presented.5! 53-68 An important facet of the action of the puffer poison seems to be a decrease in the release of acetylcholine from cholinergic nerve endings,® but this action may not be the only one exerted by the compound.®2 The poison appears to stimulate 67 the chemoreceptive trigger zone of the area postrema and to decrease the responsiveness of skeletal muscle to direct electrical stimulation, as examples. The toxin of puffer fish has been shown recently to be identi- cal to that in the egg masses of newts of the genus Jaricha,5' so that information about the latter toxin applies also to the former. Etiologies of Poisonings by Miscellaneous Forms The causative agent or agents in outbreaks of cephalopod poisoning from eating squid or octopus have not been identified definitely. Ogasawara et al.69 have reported isolating from toxic squid three amines and a protein that were toxic and that stimulated the auricle and the intestine of the guinea pig. One of these amines was identified tentatively as agmatine. Nothing is known about the nature of the toxic agents responsible for chimaeroid, cyclostome, elasmobranch, clupeoid, porpoise, and turtle

SEAFOOD TOXINS 157 poisonings or hallucinogenic fish poisoning, except that Li has ob- tained evidence‘! that the toxic substance in elasmobranch poisoning may be the same as that in ciguatera poisoning. The active material that induces purgation in gempylid poisoning has been found to be a component of the nonsaponifiable fraction of oil from the gempylid fish Ruvettus putiosus.’° Cetyl acetate was identified as the most active component of the nonsaponifiable fraction, producing its laxative action without significant irritation of the intestinal tract. It may be an active cathartic simply be acting as a wetting agent, in an action similar to that of dioctyl sodium sulfosuccinate.7! Etiologies of Shellfish Poisonings The toxin of paralytic shellfish poisoning has been concentrated72 to an apparently maximal extent and is thought to be essentially pure. The toxins picked up from their environment by various shellfish (oysters, clams, and mussels, for example) are thought to have a common source in the dinoflagellates, of which Gonyaulax catanella seems to be an important representative on the Pacific Coast of the United States.73 The poison responsible for the other type of intoxication by pelecypods, venerupin poisoning, also has been purified.?74 Hashimoto and Migita,”4 have proposed that venerupin, which they believe to be an amine and which is known to become more toxic to mice after treatment with acidulated alcohol, may be a precursor or prototype of the toxin of paralytic shellfish poisoning. Against their theory is the fact that their preparation of venerupin is less than 1/1,500th as lethal as the best preparation of the poison isolated from poisonous mussels and clams. Although the source of the toxin of paralytic shellfish poisoning is not known conclusively, there is a strong presumption that it is in- gested by shellfish as a component of dinoflagellates’? on which they feed without significant intoxication except when the concentration of the dinoflagellates in sea water becomes very high. Even then, death of mollusks may result from a physical (smothering) effect rather than from a chemical one.75:76 The protozoa responsible for paralytic shellfish poisoning have been considered to be Gonyaulax catenella on the Pacific Coast of North America, Gonyaulax tamarensis on the northern Atlantic Coast of North America, Gymnodinium brevis on the southern coasts of North America, and Pyrodinium phoneus on the Belgian Coast. The poison in Gonyaulax catenella has been found to be identical with that isolated from toxic mussels from the Pacific Coast.73:77 The toxin has an empirical formula of CioHiz7N7O4 and appears to contain a tetrahydropyrrolopyrimidine structure.

158 J. H. WILLS, JR. Although the chemical identity of the poison of paralyzing shellfish is not known, pharmacologic study of the presumably pure poison has shown that both central and peripheral effects contribute to the res- piratory arrest and cardiovascular collapse that characterize poisoning by this compound in experimental animals.® There is a direct effect on myocardium that adds to the centrally mediated hypotension. The effect of the poison on activation of skeletal muscle by motor impulses originated through reflex arcs is greater than that on activation by electrical stimulation of the motor nerve, but there is still a significant curaremimetic action in the periphery. Conduction by sensory path- ways is altered also.78 Nothing seems to have been done to identify the causative agents in oyster poisonings beyond the work already mentioned on the oyster from Lake Hamana. Whether a substance similar to that found by Itai and Kamiya29 in the Lake Hamana oyster occurs in other toxic oysters appears to be unknown. The dermatitis of abalone poisoning appears not to be a directly cliemical effect, but to result from chemical sensitization of the skin to sunlight. The first indication of this mechanism of action came from the observation?° that the superficial lesions of the disease are confined to areas of skin exposed to sunlight. This was followed by evidence’? that ' ingestion of the raw liver of two species of abalone by mice, rats, a rabbit, and cats sensitized the animals to sunlight transmitted through ordinary window glass. Hashimoto and Tsutsumi®° reported the isola- tion from the liver of Haliotis discus hannai of a fluorescent pigment capable of sensitizing the rat to light. This pigment has an absorption spectrum similar to, but not identical with, that of pheophorbide a.8! 82 In summary, there are in seafoods a number of probably distinct toxic substances. The most potent of these substances in pure form (paralytic mussel or clam poison) has an intravenous LDg9 of about 3 mcg/kg. Most of these intoxications may be characterized as neurotoxic diseases, the principal exception to this being gempylid diarrhea. This seems to depend on the presence of the active material inside the gastrointestinal lumen and to disappear rapidly following emptying of the gastrointestinal tract. No specific treatment is available for any of these poisonings, except that Li’s finding*! of therapeutic activity by 2-PAM Cl given with atropine in experimental ciguatera poisoning of mice and rats may furnish a specific treatment for human poisonings by this type of sea- food poison. Removal of toxic food from the gastronintestinal tract may be useful when vomiting does not occur as a part of the poisoning