poisonous and venomous marine animals and their toxins

POISONOUS AND VENOMOUS MARINE ANIMALS AND THEIR TOXINS

Findley E. Russell hboratoty of Neurological Research

University of Southern California Los Angeles, California 90033

For the most part, animal venoms are complex mixtures that should be considered products of adaptation in the evolution of the organism. Although we are far from understanding under what circumstances and when the various components of venoms first appeared in the mixture, the more we study the specific biological properties of these poisons the more clearly we begin to see their design in the animal’s armament, and perhaps the more tempting it has become to design chemical characterization studies on the basis of what should be there. This is a distance afield from the popular Tinker Toy approach of gadgetometry and random reaching for any substance or activity that equipment and temptation permit, giving only passing thought to the evolution and design of the venom or to the animal itself. The passing of time has demonstrated that the most successful approach to studies on animal toxins are those initiated within the structure of a reasonable knowledge of the general biology of the animal and an insight into the use to which the animal puts its toxin.

Shortcomings in judgment, if not in fact, were perpetuated by some investigators, who sought to line up all of the biological activities of snake venoms with enzymes, a misconception that, among other things, has led to grave errors in clinical management. A reasonable amount of study of the design of the venom would have indicated that the lethal activity of this poison could not be associated with an enzyme and that perhaps some of the other, more deleterious, activities could be attributed to nonenzymatic fractions, a fact now well demonstrated by the works of Tamiya, Sato, Botes, Karlsson, Jimenez-Porras, Moroz, Dubnoff, and Russell, among others. Equally questionable has been the freehand projection of data on venoms from the humble squid axon through eternities to the not so humble human being. The fascination of “pumps” and “gates” cannot be denied, and indeed the investigations of these phenomena are excellent pieces of work, but they have their limitations in the scheme of nature and we should be willing to recognize this. Finally, in the field of marine toxins, the classification of 1 fish poison as an “anticholinesterase” seems very questionable, and the application of this supposi- tion, again in human beings, has not been very successful.

These matters, among quite a few, should remind us that nature is a pretty good chemist and physiologist and that we should not overlook her hand in the design of those things which have caused the venomous animals to survive, and which have made of venoms one of the most successful, if not the most highly evolved, bits of chemical evolution known to man.

The man we are honoring today was among the first and foremost of American biologists to remind us to take a good look at the animal first, then design the experiment. His teaching and research are testimony to the productivity of this approach and, when all else has been said, the fun of working with the animals themselves. In the field of marine poisons and venoms, Ross Nigrelli has made an outstanding contribution, not the least of which will be remembered as his

58 Annals New York Academy of Sciences

chairmanship of the first symposium on marine toxins, sponsored by the New York Academy of Sciences in 1960. It is a pleasure, and indeed a privilege, to take p5rt in this tribute and to drop a few pearls, or lay a few eggs, in behalf of this scholar.

In general, animal venoms are considered the most complex poisons known to man. Most venoms and animal poisons are mixtures of many different substances. Some venom components may be proteins, including enzymes, polypeptides, or glycopeptides, while others are carbohydrates, aminopolysaccharides, lipids, free amino acids, amines, steroids, alkaloids, furans, and formic acid. A single venom may contain 20 to 30 different components, even though the toxic moiety may be composed of only several or even a single substance, such as the puffer fish toxin tetrodotoxin, an animo perhydroquinazoline. It is obvious that these various components of venoms have evolved and adapted in a remarkable way, in most instances as part of the animal’s offensive or defensive armament; and although there are some fractions of venoms for which we have not yet found a specific biological function that seems related to the design of the venom, there are some synergistic, and perhaps antagonistic, actions of certain whole venoms that are not present when one separates the individual fractions and studies their specific activities. Also, it is quite possible that the biological properties of some venom fractions played an important role in the animal’s posture in eons past and that these characteristics are no longer essential to the function of the venom in its present ecological niche. Finally, the chemistry of venoms appears still in the process of evolution, as are the animals themselves and the animals on which they prey or against which they must defend themselves.

Not only are the venoms and animal poisons complex in their chemical structure, but they are remarkably diversified and complicated in their modes of action. Some venoms have an effect, either directly or indirectly, on almost every organ system, and few are tissue-specific, although some may have a marked effect on 1 organ or tissue and little on another. For this reason it is best to consider all venoms as capable of producing 1 or more deleterious changes in several organ systems and to sometimes provoke these concurrently. No venom should ever be classified as a “neurotoxin,” a “cardiotoxin,” or a “hemotoxin” and its other pharmacological properties minimized or dismissed. It has been shown that neurotoxins can and do have cardiotoxic or hemotoxic activites, or both; cardiotoxins may have neurotoxic or hemotoxic activites, or both, and the activity of hemotoxins may not be restricted to the blood. More errors in clinical judgment have been made on the basis of oversimplified classifications of the physiopharmacological properties of snake venoms than are generally appreciated.The physician faced with a case of venom poisoning is often confronted with 2 or perhaps 3 or 4 serious disease states. He must give prompt and careful attention to the analysis of a number of important physiological parameters in determining the treatment for his patient.

The effects of venoms are often further complicated by certain autopharmacologi- cal responses. The envenomated organism can produce and release such substances as histamine, bradykinin, and adenosine, which may not only complicate the poisoning but, in themselves, sometimes produce more serious consequences than the venom. For example, it takes of the order of 150 simultaneous bee stings to produce death in a nonsensitized human, but the sting of a single be can be fatal to the sensitized individual.

Venomous animals have sometimes been divided into 2 groups on the basis of the use to which the animal puts it venom. In general, most venoms delivered from the oral pole, usually in association with salivary or maxillary glands, are used by the

Russell: Toxins of Marine Animals 59

animal in offense, as in the gaining of food. The venoms of such animals tend to have a higher enzymatic and lethal index than those delivered from the aboral pole. Venoms delivered from the aboral pole tend to be associated with the defensive posture of the animal and, in general, arise from dermal tissues, as we see in the scorpionfishes and stingrays, and contain few or no enzymatic constituents. As a whole, the aboral venoms are less lethal, but they appear to contain greater amounts or numbers of pain-producing substances. Venomous animals should be differentiat- ed from poisonous animals. The former term is applied to those creatures having a gland or group of highly specialized secretory cells, a venom duct (although this is not a constant finding), and a structure for delivering the venofn. Poisonous animals, as distinguished from venomous animals, have no such apparatus; poisoning by these forms takes place through ingestion. Although the toxin(s) of some poisonous animals may have defensive functions, as in the eggs and spiderlings of the black widow spider, many, if not most such poisons are products of normal metabolism, as in the tetraodon fishes. Ciguatera toxin is a food-chain poison that plays no part in the fish’s offensive or defensive posture.

In the marine animals we have a fascinating and diversified assemblage of toxins. There are 2000 or so species of venomous and poisonous marine animals, and they are widely distributed throughout the marine fauna, from the unicellular protistan Gonyaulax to certain of the chordates. Found in almost all the seas and oceans of the world, they do not usually present a problem to man, except in a few scattered areas, where under certain conditions they sometimes constitute a public health hazard. The toxins of the marine animals are far more varied in structure than those of the reptiles. The brittle star Ophiocomina nigra discharges a viscous substance on stimulation, characterized as a highly sulfated mucopolysaccharide that is acidic and has a pH of 1.0, while the toxins of some of the venomous fishes appear to be proteins having molecular weights approaching 800 OOO; still other marine toxins are amines and lipids.

While the marine toxins as a whole are far more varied in their chemical composition than the venoms of terrestrial snakes or arthropods, there is some degree of consistency within a particular phylum, which is not unlike that seen in the terrestrial venomous animals. Venoms from animals within a single phylum tend to bear some relationship to each other. This is most obvious at the species or subspecies level and least obvious at the class or order level. There are some exceptions, but in general this principle of relationship holds true in all the phyla. Venoms delivered from the oral pole tend to have some properties in common, as do those delivered from the aboral pole. Venoms used in offense, as in the gaining of food, have some similar biochemical and pharmacological properties, and those used in defense also have some common properties. The toxins of venomous animals differ remarkably from the toxins of poisonous animals, even from those of animals within the same phylum; there is no chemical or toxicological relationship, for example, between stingray venom and puffer fish poison. Finally, during recent years it has also been shown that some poisons from one species or genus within a phylum may be similar to or even identical with those from another phylum: the fish poison tetrodotoxin is the same as the newt poison tarichatoxin; tetrodotoxin and saxitoxin have very similar biological activities, although they appear to be chemically distinct.

From the chemical standpoint, it is thought that fewer than 20 OOO cases of poisoning occur each year following the ingestion of toxic marine animals, and probably less than 200 of these are fatal. However, in only about 3% of all these


cases is the causative factor determined. While these figures are not particularly alarming, they must be viewed with concern, for many food economists see in the present problem of fish poisoning a serious deterrent to progress in the utilization of marine products, not only as food and protein concentrates but also as sources for pharmaceutical and biochemical agents and as sources for fertilizer. Of particular concern to public health workers are paralytic shellfish poisoning, tetraodon poisoning and ciguatera poisoning. The occurrence of the first two of these diseases is predictable. Ciguatera poisoning, however, is not, and it is an important medical entity within a broad circumglobal area. Since almost all of the fishes incriminated in ciguatera poisoning are normally edible, and many are valuable food fishes in some parts of the world, this type of poisoning is not only the most common but also the most treacherous form of fish poisoning.

There are no worldwide compilations of the number of persons stung by venomous marine animals each year. Approximately 5600 persons a year are stung by cnidarians, stingrays, catfishes, and the California sculpin along the coasts of North America. About 5000 persons are stung by cnidarians, stingrays, catfishes, and stonefishes off the Australian beaches each year, and approximately 3000 persons a year in Brazil are stung by venomous marine animals. Incomplete records from fishermen in the North Sea would indicate that during 1958 at least 2000 persons were stung by the weeverfish alone off England, France, Belgium, Holland and Demark. From these and other figures gathered by our laboratory during the past 15 years, it would appear that the number of injuries by venomous marine animals must exceed 40 000 a year. Fortunately, the number of deaths, excluding those due to sea snake venom poisoning, is small; probably less than 75 a year.

It is not possible in this short review to discuss, or even describe the venomous and poisonous marine animals. This has been done in great detail in the compendium by Halstead, as well as in several much lesser works by our group. The toxin of the protistan Gonyaulax, known as paralytic shellfish poison, saxitoxin, mussel poison, or Gonyaulax poison, is stored and concentrated for the most part in the hepatopancreas of certain mussels, the siphon of some clams, or the tissues of several other animals that have been exposed to certain Gonyaulax species. The poison is one of the most lethal substances known. Its chemical properties are summarized in TABLE 1.

Coelenterate or cnidarian venoms, those of the jellyfishes, Portugese man-of-war, sea wasps, sea anemones, and certain corals vary considerably in their structure. The stinging unit is the nematocyst, which within its capsule contains proteins, sulfur-containing amino acids, hydroxyproline, glycine, tyrosine, arginine, proline, alanine, glutamic acid, aspartic acid, a succinoxidase inhibitor, hexosamine, uronic acid, orthodiphenols, mineral salts, alkaline and acid phosphatases, 5 '-nucleotidase, cholinesterase, and 5-hydroxytryptamine. A number of recent studies, with partially purified toxins, indicate that the lethal and certain nerve-muscle effects of cnidarian toxins are due to polypeptides, but their exact nature has not yet been determined.

In the phylum Echinodermata-the starfishes, brittle stars, sea urchins, and sea cucumbers-there are both venomous and poisonous species. Perhaps the most important venomous members of the phylum are certain genera of sea urchins, which have small pincerlike organs, the pedicellariae, distributed over their entire body surfaces between their spines. Extracts from these structures have been shown to contain 8 immunologically distinct proteins, all of which are lethal to mice, and against which specific neutralizing antibodies have been made. Highly lethal materials have been separated by conventional fractionation procedures. The most


TABLE I

Property Mussel poison Saxitoxin G. carenella poison

Molecular formula Molecular weight N-content (Kjehdahl) Diffusion coefficient Specific optical rotation

Molecular extinction of oxidation product Sakaguchi test Benedict-Behre test Jaffe test Aromatic structures Carbonyl groups

Reduction with H2 Lethality

Proposed structure

PKa

CioHi,N701. 2HCI 372 26. I 4.9 x 10-6 130” 8.3, 11.5

6OOo Negative Positive Positive Present None Non-toxic dihydroderi- vative 5300 mouse units/rng. Tetrahydro purine de- rivative

H

C ~ O H I ~ N ~ O ~ . 2HCl 372 26.8 4.9 x 10-6 128” 8.3, 11.5

C I O H ~ T N ~ O ~ . 2HCL 372 26.3 4.8 X 10-6 128“ 8.2. 11.5

Negative Positive Positive Present None Non-toxic dihydroderi- vative 5200 m o w units/mg. Tetrahydro purine de- rivative

Negative Positive Positive Present None Non-toxic dihydroderiva- tive 5100 mouse units/mg. Tetrahydro purine deriv- ative

H Saxitoxin

lethal fraction (LD50 = 2 X 104 mg protein N) behaves like an enzyme. Further purification has resulted in the separation of 5 substances, 3 of which exhibit kininlike activity. The material from 1 has been found to correspond to synthetic bradykinin.

The most active toxin in extracts of certain sea cucumbers is holothurin A. The formula is C5”52Hs135025-26’, and the molecular weight has been calculated to be 1155. The provisional structure is:


& Me Me

I Me Me

R COMPOUND - HOLOTHURIN -0SO;Na'

SUGAR SYMBOL

R[i G-OMe De H -H

D-GLUCOSE G D-XY LOSE X D-QUINOVOSE Q

Q

3-0-METHYLGLUCOSE G 4 M

Fish toxins can be divided into 2 major groups: toxins from poisonous fishes and toxins from venomous fishes. Fish poisoning, or ichthyotoxism, can be further subdivided into: (a) ichthyosarcotoxism (fish whose toxin is within their muscula- ture, viscera, or skin), (b) ichthyootoxism (fish whose gonads or roe are toxic or in which there is a relationship between gonadal activity and the production of the toxin), (c) ichthyohemotoxism (fish that have a toxin in their blood), and (d) ichthyocrinotoxism (fish that produce a toxin by glandular secretion from their skin but otherwise lack a true venom apparatus). The toxins of these poisonous fishes vary remarkably, and there does not appear to be any common structural basis on which they might be compared. Tetrodotoxin, from the puffer fish, has the formula CllHllN308 and the structure

0- 1

while the toxic substance released from the skin of the boxfish Ostracion Ientiginosur has the formula C23H4604NC1 and the structure

H I I

CH~-(CHZ)~~-C-CH~-CO~-(CH~)-~~(CH~)~ Cl-

OCOCH3


Both of these toxins have interesting pharmacological properties that have already been put to use as tools in biology and medicine.

The venoms of the stingrays, scorpionfishes, lionfishes, and stonefishes appear to be somewhat related chemically and pharmacologically, and to a lesser extent so do the venoms of the weever fishes and most of the other venomous piscines. A common property to almost all fish venoms is their relative instability. Most of the lethal property, as well as certain of the other deleterious activities, is lost on heating. Until very recently, studies on these toxins have been greatly hampered by difficulties in obtaining a stabilized product. Preliminary studies now show that the toxic fractions of fish venoms are protein having molecular weights betyeen 50 OOO and 800 OOO, that they are relatively free of enzymes, and that the pain-producing fraction is much more stable than the more deleterious components and may be a kininlike material.

These are only a few of the many thousands of marine toxins. I wish it were possible to review these interesting substances in more detail, but that has been done elsewhere.

DISCUSSION

DR. JOSEPH GENNARO (New York University, New York, N.Y.): I particularly wanted to be able to speak after Russell’s very nice summary of the problem that one has to deal with when one works with venom. Intricacies of the venomous animal’s adaptation are almost insurmountable. It is extremely difficult to understand the point of view of the venomous animal. At the onset, it’s difficult even to determine whether the animal carrying the venom does so for defense or for offense; that is, uses venom as a warning device and weapon or as a prehensile tool for obtaining prey. When Fin spoke about the great variation of venom-dose delivered by a snake, it occurred to me to mention some data in that area which we developed in our laboratory, especially since Ross Nigrelli was important in contributing to our understanding of its significance. When we were working on this problem, we had data that did not form the kind of graph we would have liked to have. It looked similar to shotgun patterns on a wall and indicated, we thought, that the amount of venom injected by a snake into a mouse was somewhat less than the amount of venom injected into a guinea pig or a rat, and that even less venom was injected into a rabbit. The only thing that prevented us from disregarding the graph altogether was the very significant difference in the doses delivered to the mouse compared with the rabbit. In no way could this difference be attributed to chance.

At that time we thought less about the mechanism than about the significance of the data. When I spoke to Dr. Nigrelli about it, he told me of some experiments which he had done using the electric eel. He told me that ordinary Fundulus fed to the eel were shocked to the point of unconsciousness but eaten alive. The eel doesn’t vary the voltage but varies the number of shocks. When instead of feeding Fundulur, goldfish were substituted, fish which are more resistant to electricity, probably because of the gold, the eel quickly learned to give more shocks to stun the fish. When the eel was again fed Fundulus later, the fish were killed until the eel learned to give fewer shocks.

I thanked my good fortune at having a person of Ross Nigrelli’s breadth and experience available for consultation. After his narrative, I grasped my data with new faith feeling that the control exhibited by the eel could certainly be attributable to an organism from a different world using venom instead of electricity as a status symbol.

DR. RUSSELL: Actually this work that I was speaking about on the rattlesnake

poisonous and venomous marine animals and their toxins

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