Natural Toxins

1. Natural Toxins TODD STEDEFORD, PH.D., DABT

2. OUTLINE • Tetrodotoxin • Saxitoxin • Domoic acid • Scromboid Fish Poison • Ricin • Aflatoxin • Ochratoxin • Trichothecenes

3. Tetrodotoxin • The most lethal seafood toxin • The puffer fish is a common source of tetrodotoxin • The toxin is produced by microorganisms ingested by the puffer fish • The toxin is not found in puffer fish bred in captivity

4. Tetrodotoxin Tetrodotoxin Na+-OH2 • Tetrodotoxin acts as an inhibitor of nerve • conduction through blockade of neural sodium channels • Tetrodotoxin, much larger than the sodium ion, acts like a cork in a bottle, preventing the flow of sodium

5. Tetrodotoxin • Tetrodotoxin is heat stable and unaffected by cooking or freezing • Clinical manifestations usually begin within a few minutes of ingestion, but may be delayed by several hours • Of those dying, most will do so within the first 20 – 30 min • Those surviving the first 24 hours have a good chance of recovery • Nausea, emesis, diarrhea, headache, facial paresthesias, itching and burning of skin, ataxia, and convulsions have all been reported prior to death

6. Ciguatoxin • This is the most common seafood toxin, and poisoning from this is termed ciguatera • Source is from dinoflagellate microorganisms (Gambierdiscus toxicus) that live amidst the microalgae of coral reefs in tropical waters • Death is extremely rare • Ciguatoxin opens sodium pores of excitable membranes by activating voltage-dependent sodium channels in both nerve and muscle cells

7. Ciguatoxin Ciguatoxin Na+-OH2 • Sodium channels open, but fail to close, resulting in spontaneous depolarizations with an increase in intracellular sodium ions and an influx of water

8. Ciguatoxin • The anti-edema properties of mannitol make it a useful compound to block the swelling that occurs at the Nodes of Ranvier by ciguatoxin • Toxin is stable and unaffected by cooking and freezing • Vomiting and diarrhea usually begin within the first 8 h. Headache, myalgias, weakness, and reflex loss usually occur 10 – 18 h after ingestion • Perioral and limb sensory paresthesias, dysesthesias, and pruritus usually appear around 12 – 24 hours after ingestion, and gradually evolve of the next 2 – 5 days into paradoxical sensory disturbances, such as hot feeling cold and vice versa

9. Saxitoxin • Shellfish, particularly mussels, clams, oysters, and scallops may ingest dinoflagellate microorganisms to produce a syndrome known as paralytic shellfish poisoning • These dinoflagellates are from the genus Gonyaulax, which are found in temperate rather than tropical waters • The thermostable saxitoxin, like tetrodotoxin, blocks voltage-dependent neural sodium channels

10. Saxitoxin • Clinically, paralytic shellfish poisoning begins shortly after ingestion of saxitoxin, typically with a gastrointestinal prodrome in the first 30 – 60 min, followed by sensory disturbances of the face and limbs • Paralytic features predominate within a few hours, especially bulbar and respiratory, with areflexic limbs • Mortality rates are about 8 – 10%

11. Domoic Acid • The dinoflagellate microorganism Nitzschia pungens produces domoic acid, which is toxic to both the central and peripheral nervous systems of man • Domoic acid is heat-stable and has been shown to promote synergism between the excitatory amino acid glutamic acid and aspartic acid at the N-methyl-D-aspartate (NMDA) receptor

12. Domoic Acid • Blooms of N. pungens have appeared in late fall in eastern Canada, particularly around Prince Edward Island, and along the Pacific Northwest Coast from Oregon to Canada • Mussels and crabs in these areas can have sufficient quantities of domoic acid in their digestive glands to produce a syndrome in man of amnesia, seizures, coma, and possibly death • Patients experience gastrointestinal symptoms within 24 h of ingestion, and neurologic symptoms within 48 h • Mortality rate is about 3%; post mortem examinations have shown necrosis and neuronal loss in the hippocampus and amygdala

13. Scombroid Fish Poison • Large deep water fish of the Scombridae family include tuna, mackerel, and skipjack • Nonscombroid fish like mahimahi or marlin may also produce the illness • The illness manifests 10 – 30 min after ingestion as a histamine-like reaction • Typical symptoms include flushing, pruritus, headache, urticaria, gastrointestinal hyperactivity, and rarely bronchospasm

14. Scombroid Fish Poison • Poorly refrigerated deep water fish can result in an accumulation of histamine in the tissues • Bacterial degradation of the fish flesh can result in bacterial decarboxylation of histidine to histamine • The manifestations are usually self-limiting over 3 – 6 h, but can be ameliorated quickly by antihistamines (both H1- and H2-receptor antagonists)

15. Summary

16. Ricin • Ricin is a cytotoxic lectin produced in seeds of the castor oil plant (Ricinus communis) • Within a few hours of ingestion, the symptoms are abdominal pain, vomiting, diarrhea (sometimes bloody). • Within several days, there is severe dehydration, decrease in urine, and a decrease in blood pressure • If death does not occur in 3 – 5 days, the individual • usually recovers

17. Ricin • The holotoxin contains a ribosome-inactivating A chain disulphide linked to a galactose-binding lectin B chain • The ricin A portion of the heterodimer binds and depurinates a specific adenine on the 28s rRNA • The adenine (A) ring of the ribosome becomes sandwiched in the catalytic cleft of ricin A and is hydrolyzed

18. Mycotoxins • Aflatoxins • Ochratoxins • Trichothecenes

19. Aflatoxins • Produced mainly by Aspergillus flavus and • A. parasiticus • Over a dozen known aflatoxins • The liver is the primary target organ • Causative toxins in Turkey X disease

20. Aflatoxin B1 Aflatoxin G1 Aflatoxin B2 Aflatoxin G2 Aflatoxins • Four major aflatoxins • Aflatoxin B1 is the most intensely studied

21. Aflatoxin B1 Aflatoxin M1 (Less Toxic) Aflatoxins Aflatoxins M1 and M2 are the hydroxylated metabolites of B1 and B2 that may be found in milk and milk products

22. O O O O O OCH3 Aflatoxin B1 Aflatoxins Metabolism of aflatoxin B1

23. O O O O O O OCH3 Aflatoxin B1-8,9-epoxide Aflatoxins CYP-catalyzed oxidation of aflatoxin B1 Primarily by CYP3A4

24. O dR N N O O HO O O O OCH3 HN H2N N Aflatoxins Bioactivation Aflatoxin B1-8,9-epoxide binds to proteins and the N7 position of guanines in DNA

25. O O O HO O O OCH3 HO Aflatoxins Detoxification Glutathione S-transferase detoxifies aflatoxin B1-8,9-epoxide via glutathione conjugation

26. Ochratoxins • Ochratoxin A, was isolated in 1965 from a culture of Aspergillus ochraceus • The kidney is the primary target organ; nephrotoxic to all animal species studied • Ability to be carried through the food chain

27. Ochratoxin A Serum half-life: Rats 55 – 120 hrs Human 35 days Macaques ~21 days Pigs 72 – 120 hrs Mice 24 – 39 hrs 0 hrs 40 days

28. O O O O C OH C OH OH O O NH2 O N H2C H H H2C CH3 Cl Ochratoxin A Toxicity generally attributed to its inhibitory effects on the aminoacyl-tRNA synthetase (ARS) Ochratoxin A Phenylalanine

29. O O C O P NH2 H2C 2 P Phenylalanine Ribose Adenine tRNA tRNA ATP HO Ochratoxin A ARS

30. Trichothecenes • The first compound was isolated from Trichothecium roseum in 1948 • Over 60 compounds • T-2 appears to be the most potent in animal studies

31. Trichothecenes • Fusarium is the most important worldwide fungus that produces them • Potent inhibitors of protein synthesis • Probable causative agents of alimentary toxic aleukia (ATA) and stachybotryotoxicosis

32. T-2 Toxin HT-2 toxin (less toxic) T-2 toxin

33. H2C O C O NH2 Peptidyl-tRNA O O tRNA C NH2 H2C H2C O C O H2C NH Peptidyl transferase O C O NH2 New Peptidyl-tRNA molecule with growing polypeptide chain Trichothecenes Aminoacyl-tRNA HO tRNA cleaved from growing polypeptide chain

34. Summary • Ricin exerts its cytotoxic effects by disrupting protein synthesis via inactivation of the 28s ribosomal subunit • Aflatoxin is potentially toxic following bioactivation to its reactive epoxide metabolite • Ochratoxin A is structurally similar to phenylalanine and can disrupt protein synthesis by inactivating aminoacyl-tRNA synthetase • Trichothecenes can inhibit peptidyl transferase thereby disrupting protein synthesis

35. QUESTIONS?