[advances in botanical research] advances in botanical research volume 12 volume 12 || algal toxins

Algal Toxins

WAYNE W. CARMICHAEL

Department of Biological Sciences Wright State University Dayton, Ohio, U.S.A.

I. Introduction ............................................................. 47 11. Naming Algal Toxins. ... .. .. ... .. ... .. .. . .. .. . . .. . .. .. .. .. ... . . .... .. .. . . 52 In. Occurrence, Growth, and Toxicity.. . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . 52

A. Chrysophyta ... ...................................................... 52 B. Pyrrophyta. .. . . . . ... .. . .. . . .. . .. . . . . . .. .. .. .. ... . . .. .. .... . .. .. .... . . 59 C. Cyanophyta .......................................................... 64

IV. Isolation, Characterization, and Toxinology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 A. Prymnesium Toxins.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Freshwater Cyanophyte Toxins . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Marine Cyanophyte Toxins ............................................ D. Dinoflagellate Paralytic Shellfish Poisons (PSP) . . . . . . . . . . . . . . . . . . . . . . . , . . E. Dinoflagellate Ciguatera Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Other Dinoflagellate Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V. Environmental Role of Algal Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

67 68 78 82 86 89 91 93

I. INTRODUCTION

Chemicals of secondary biosynthesis are important to the growth and development of all organisms, plant, animal, and microbial. The term “secondary” derives from the observation that these chemicals are not needed for primary metabolism, i.e., respiration or photosynthesis. The term also serves to suit the fact that for many of the chemicals no obvious functional role exists, hence the term secondary would seem to catalog them well. Secondary chemicals were first suggested by Stahl (1888) as

ADVANCES IN BOTANICAL RESEARCH, VOL. 12 47 Copyright 8 1986 by Academic Press Inc. (London) Ltd. All rights of reproduction in any form reserved.

48 WAYNE W . CARMICHAEL

perhaps having evolved for protection against attack by herbivorous animals. Numerous other functions have been suggested for these chemicals, including the following:

1. Regulators of plant growth or biosynthetic activity 2. Storage forms of plant growth regulators 3. Energy reserves 4. Transport facilitators 5. Waste products 6. Detoxification products of environmental poisons (Chew and Rod-

man, 1979)

While some debate still exists concerning the adaptive significance of the secondary chemicals , evidence continues to accumulate to support an ecological role for these metabolites both in interactions among plants and their associated biota and as protective agents against physical environmental stresses (Rhoades, 1979). Secondary chemicals having a marked biological effect, usually negative, are often called biotoxins. While the role of biotoxins for animals is often clear, i.e., prey gathering or defense, the role of microbial biotoxins is not. Although they may act in some capacity as defense chemicals against herbivores or to improve an organism’s competitive advantage for a food source, it is also possible to find nontoxic forms of a potentially toxic species.

Algae responsible for producing toxins are found in three of the eight divisions which make up the algae (Scagel et al., 1982). These are the Chrysophyta (class Prymnesiophyceae), Cyanophyta (cyanobacteria or blue-green algae), and the Pyrrhophyta (class Dinophyceae, also known as the dinoflagellates). In the Chrysophyta the toxic species is the flagel- lated unicellular Prymnesium paruum. It is a euryhaline chrysophyte widespread in brackish and marine habitats, producing toxins best known for causing mass mortalities of fish (Shilo, 1971, 1981). In the Cyanophyta both marine and freshwater toxic species are found. Marine toxic forms are in the filamentous genera Lyngbya, Schizothrix, and Oscillatoria. Toxins produced are of the type responsible for the contact dermatitis called “swimmers itch” (Moore, 1981b, 1984). Toxic freshwater cyanobacteria include the unicellular colonial Microcystis and the filamentous Anabaena, Aphanizomenon, (Figs. 1-3) Nodularia, and Oscillatoria. These freshwater algae can form thick surface accumulations of cells as the water bloom develops within the water body. They can be found in many eutrophic lakes and ponds at temperate latitudes and are responsible for sporadic but widespread outbreaks of wild and domestic animal illness or death (Gentile, 1971; Carmichael, 1981; Carmichael and Mah- mood, 1984; Carmichael and Schwartz, 1984). They are also implicated in

Fig. 1. Scanning electron micrograph of Microcystis aeruginosa. Gelatinous matrix, which holds the cells in a colony form, is embedded with numerous rod-shaped bacteria. Micrograph by M. M. Ecker.

Fig. 2. Scanning electron micrograph of Anabaena circinafis. Numerous rod-shaped bacteria are embedded in the matrix surrounding the individual cells. Anabaena circinafis was recently identified as the probable cause of animal deaths from a toxic water bloom in Illinois (Beasley et al., 1983). Micrograph by M. M. Ecker.

Fig. 3. Scanning electron micrograph of Aphanizornenon flos-aquae from New Hampshire. Cell in center of filament not covered by the sheath is a heterocyst-primary site of nitrogen fixation. Micrograph taken by M. M. Ecker.


human poisonings in certain municipal and recreational water supplies (Carmichael et al., 1985). The Pyrrhophyta contain most of the algae documented as causing outbreaks of algal disease. They are almost exclu- sively from the marine environment. Two major disease types are recognized. The first, paralytic shellfish poisoning (PSP), refers mainly to toxic species of Protogonyaulax (formerly Gonyaulax) (Figs. 4 and 5), which are concentrated by filter-feeding molluscs. Recently the tropical dinoflagellate species Gymnodinium catenatum (Morey-Gaines, 1982) (Fig. 6) and Pyrodinium bahamense var. compressa, and the tropical red alga Jania sp. have also been found to produce PSPs (Oshima et al., 1984). The molluscs are in turn eaten by humans, resulting in yearly episodes of illness and death. The second disease type is called ciguatera seafood poisoning. It refers to the concentration by many reef-feeding fish of toxic dinoflagellates recently identified as belonging to the genera Gambierdis- cus (Fig. 7), Prorocentrum, Gymnodinium, and Gonyaulax. The fish are in turn eaten by humans (Ragelis, 1984).

11. NAMING ALGAL TOXINS

As with most natural toxins, algal toxins have been named from the organisms that produce them or the vectors by which the toxins move through food chains. Only occasionally is a toxin name based on its chemical structure. This seems logical to the investigator doing the research and to his colleagues to whom he must communicate information about the toxin and the organism producing it. Coincidentally, it also reinforces the interest, respect, and image of mystique that the general public ac- cords natural toxins. As more chemical information is known about toxins and when more than one toxic compound is isolated from a given toxic species, the use of this type of nomenclature system has become confus- ing. However, since most toxins are investigated and the findings pub- lished before their structure is entirely understood, the naming of toxins based on the organisms involved will continue. Table I lists the algal toxins and, where known, the origins of their names.

111. OCCURRENCE, GROWTH, AND TOXICITY

A . CHRYSOPHYTA

The chrysophyte Prymnesium parvum was first identified as the causative agent of fish deaths by Carter (1938). Most problems have been reported from brackish water environments in England, Holland, Denmark, Bul-

ALGAL TOXINS 53

Fig. 4. Scanning electron micrograph of Protogonyaulax (Gonyaulax) tarnarensis. This species is the primary offender in outbreaks of PSP along the eastern coast of the U.S.A., Western Europe, and the western Pacific. Micrograph by L. Firtz.

Fig. 5. Scanning electron micrograph of Protogonyaulax (Gonyaulax) catenella, a PSP-producing dinoflagellate from the west coast of the U.S.A. Strands covering the cells are trichocysts extruded by the organism. Micrograph by G . Gaines.

ALGAL TOXINS 55

Fig. 6. Scanning electron micrograph of Gymnodinium catenatum, a paralytic shellfish poison-producing dinoflagellate from Mazatlan, Mexico (Morey-Gaines, 1982). Micrograph by G . Gaines.

TABLE I Algal Toxins-Terms Used and Main Species Responsible

Organism Toxin Chemical group Origin of toxin name Reference

A. Chrysophyta Prymnesium parvum

2 B. Cyanophyta 1. Marine

Lyngbya majuscula

Schizorhrix calcicola Oscillatoria nigrouiridis

Microcystis aeruginosa 2. Fresh water

Anabaena jlos-aquae (different strains of cyanobacteria)

Ichthyotoxin Cytotoxin Hemolytictoxin

Lyngbyatoxin A Debromoaply siatoxin Aplysiatoxin Debromoaplysiatoxin Oscillatoxin A

Microcystin C yanoginosin

Anatoxin-a Anatoxin-b Anatoxin-c Anatoxin-d Anatoxin-b(s) Anatoxin-a(s)

Unknown

Indole alkaloid Phenolic bislactone Phenolic bislactone Phenolic bislactone Phenolic bislactone

Peptide Peptide

Alkaloid Unknown Peptide Unknown Unknown Unknown

Fish toxin Shilo (1981) Cell toxin Red blood cell toxin

Genus Lyngbya

Genus Oscillatoria

Genus Microcystis

Genus Anabaena

Moore (1984)

Carmichael and Mahmood (1984); Botes et al. (1984)

(1978) Carmichael and Gorham

Aphanizomenon Jos-aquae Aphantoxins

Oscillatoria agardhii rubescens

C. Pyrrophyta Gonyaulax (Protogonvaulax)

catenella

tamarensis acatenella phoneus

compressa

(Gymnodinium breve)

fortii acuminata

Pyrodinium bahamense var.

Ptychodiscus brevis

Dinophysis

ul -4

Gambierdiscus toxicus

Oscillatoria toxin

Paralytic shellfish poison (PSP)

Saxitoxin

Neosaxitoxin Gonyautoxin 1-4 Cryptic (B1-2.C14)

Brevetoxins

Dinophysistoxin Pectenotoxin

Ciguatoxin

Alkaloid

Peptide

Alkaloid

Pol ylactone

Okadaic acid-like Polyether lactone

Alkaloid

Genus Aphanizomenon Sasner et a/. ( 1984); Carmichael and Mahmood (1984)

Skulberg ef 01. (1984) Genus Oscillatoria

Hall and Reichardt (1984)

Genus Saxidomas (Alaskan butter clam)

Genus Gonyaulax

Genus P. brevis Baden et al. (1984)

Genus Dinophysis Yasumoto el a/. (1984)

Cigua-from Cuban name Ragelis (1984) for the marine snail Turbo Livona pica

Maitotoxin Scaritoxin

58 WAYNE W. CARMICHAEL

Fig. 7. Scanning electron micrograph of Gumbierdiscus toxicus, collected from the Virgin Islands. Gumbierdiscus toxicus is the primary organism currently identified as being responsible for ciguatera seafood poisoning. Micrograph by G . Gaines.

garia, and especially Israel. The primary concern in Israel comes from the great number of commercial, brackish water fish ponds in which P. paruum was found to grow (Shilo, 1971, 1981). Prymnesium toxin consists of three toxins, hemoly sin, ichthyotoxin, and cytotoxin. Laboratory cultures on modified S50 medium (Shilo, 1967) produced the greatest amount of toxins during the late stages of logarithmic growth and continued at a high level into stationary phase. In contrast to other algal toxins the toxins of P. paruum are secreted into the environment, especially in the late

ALGAL TOXINS 59

stages of growth. Culture conditions can have a significant effect on toxin production. In the absence of light, a marked reduction of toxin production occurs when a carbon-enriched (glycerol) medium is us_ed. Cell multi- plication continues during this time (Shilo, 1967). In contrast a 10- to 20- fold increase in all the toxins is found in phosphate-starved cells with even more toxin than normal being found in the culture medium. Although the greatest production of the toxins occurs under stress conditions which would imply some protective (ecological) role for these secondary chemicals, Shilo (1981) feels that their formation has no direct role or impor- tance in the life of P . paruum.

B. PYRROPHYTA

Most reported PSP outbreaks have occurred in northern latitudes. Proto- gonyaulax (Gonyaulax) catenella and P. polyedra (Taylor, 1984) are implicated on the west coast of North America while Protogonyaulax tamarensis var. excauata (Schmidt and Loeblich, 1979) is involved in Northern Europe, Great Britain, Japan, and Western Pacific. These marine dinoflagellates exist in three cell types:

1. A resting zygote (cyst, 25-30 X 45-50 pm) (Dale et al., 1978; Turpin et al., 1978; Anderson, 1984)

2. A baagellate motile cell (35-50 pm in diameter) which is the bloom- forming stage

3. An asexual temporary cell (cyst, 30-40 p m in diameter) called a pelli- cle or ecdysal cyst (Anderson and Wall, 1978; Turpin et al., 1978).

Blooms of these dinoflagellates are generally defined as occurring when cell populations are between 104-106 dm-3. Lower cell concentrations of lo3 dm-3 can occur at any time during the April-October growing season. When cell concentrations reach about lo6 dm-3 the water can be discol- ored and referred to as a “red tide.”

Red tides are not all toxic, however, since (1) red tides can form from the presence of other, nontoxic dinoflagellates (Yentsch et al., 1978); (2) shellfish can accumulate toxins at levels below those necessary to dis- color the water (Hurst and Yentsch, 1981); and (3) not all Protogonyaulax species are toxic when isolated, cultured, and tested (C. M. Yentsch, personal communication). Dense concentrations of toxic marine dinoflagellates can result from several factors (Yentsch and Incze, 1980) in addition to the direct rapid growth of the algal population. These physical factors apparently have a great deal to do with frontal circulation patterns (Seliger et al., 1979). Active phytoplankton growth is situated along or close to these frontal systems that form between well-mixed and season-


ally stratified water masses. Holligan (1979) defines three stages of bloom formation in these frontal areas, as measured by chlorophyll a. The first stage, which depends on upward mixing of nutrient-rich waters, involves growth of phytoplankton at the base of the thermocline. The second stage occurs in the upper part of the thermocline at a depth of about 10 m. The third stage, which often finds the blooms patchy in appearance, may be initiated by upward mixing of the subsurface bloom. While Holligan (1979) does not find evidence to suggest that vertical migration of dinoflagellates occurs, Dugdale (1979) and others find evidence to indicate that migration occurs in response to nutrient gradients, especially nitrates. These studies indicate that dinoflagellates swim downward in the after- noon and evening to take advantage of higher nutrient levels than those near the surface where higher photosynthetic rates have been taking place during the day.

There is also some evidence that toxic dinoflagellates exhibit an annual rhythm. Yentsch and Mague (1980) report that Protogonyaulax excuuatu, cultured under constant conditions over a 5-year period, exhibited a cir- cannual rhythm. Growth rates varied from a maximum of two doublings per day for the months of July and August to 0.10 doubling per day in the month of January. Hydrographical mechanisms are also important in the formation of toxic blooms. These mechanisms require no rapid reproduction and provide a means for delivery of an existing population to an area where biological behavior such as phototaxis can result in dense concentrations. These mechanisms can be triggered by meteorological events such as rainfall and wind or the formation of discontinuities between water masses, called frontal zones. These fronts which result from tide or wind-generated convergences and/or density discontinuities are some of the locations most likely to generate red tides (Yentsch and Incze, 1980).

Blooms and cultures of toxic dinoflagellates can vary in toxicity. When cultured in the laboratory, variations are seen in quantity and type of toxin produced. Points to remember when studying dinoflagellate toxicity include the following:

1 . The mouse bioassay used to detect toxicity has an inherent variability of about k 20%.

2. Toxicity is a measure of total toxins produced by the population in culture and does not distinguish between the different toxin types.

Growth would seem to be the most obvious regulator of toxicity with optimal growth conditions reponsible for optimum toxin production. However, with some dinoflagellates this may not be so. Hall (1982) studied the effects of nutrient deprivation using modified seawater with Guil- lard’s “F/2” enrichment (Guillard and Ryther, 1962), on cell growth and

ALGAL TOXINS 61

toxicity of selected Alaskan coast Protogonyaulax clones. The omission of nitrate and phosphate from the culture medium was tested for its effect on growth and toxicity. Despite growth reductions to a level of 3200 and 3000 cells ~ m - ~ for minus nitrate and minus phosphate, respectively, compared to 16,300 (over 27 days) for controls, the toxicity per cell was slightly better then the control for minus nitrate and over 5 times as much for the minus phosphate cultures. Growth of the clone over a series of temperatures ranging from 6 to 12°C showed a reduction in growth rate at lower temperatures accompanied by an increase in toxin content per cell.

This growth attenuation accompanied by increased toxicity is sup- ported by DuPuy (1968) and Proctor et af. (1975), who concluded that toxin content is inversely related to growth rate. Perhaps this increase in a secondary metabolite under stress conditions can be likened to the production of secondary compounds by higher plants when they are grown under nutrient stress conditions (Janzen, 1974). Hall (1982) also rightfully points out that the conditions that induced higher toxin content, viz., low phosphate and low temperature, are closer to the range of these parame- ters found under natural conditions. Hall (1982) also investigated toxin composition under the attenuated growth conditions which increased overall toxin composition. The results of his limited experiments in this area showed that toxin composition among strains isolated from different regions of the Alaskan coast had different toxin compositions while those from close neighboring areas were uniform through successive culture runs. He found that the sulfamate toxins (Fig. 11) were dominant across the entire geographical range sampled. It should also be said that toxicity of dinoflagellates has been found to decrease as the cultures approach the end of log growth phase (White and Maranda, 1978).

The economic impact of toxic dinoflagellates is felt mainly when the toxins are concentrated by shellfish. Although many different shellfish can accumulate dinoflagellate toxins (Carmichael et af . , 1985) the complex pattern of toxification and detoxification has been best studied using the blue mussel Mytilus edulis. This is based on the organism’s widespread occurrence, ease of collection, and rapid rate of intoxification and detoxification. In the laboratory M. edulis will toxify in proportion to the filtration rate and the number of cells in the water. Along the coast of Maine (U.S.A.) during the summer when optimal cell densities of about lo6 ~ m - ~ are found, M. edulis can toxify at the rate of 10 pg toxin g-I tissue per day. These are the times when Protogonyaulax is in the motile stage (Gilfillan and Hansen, 1975). Under these conditions the lethal levels for humans of 10 mg of toxin are often reached. In a 4-year study (1976-1979) involving shellfish intoxication patterns along the coast of Maine, Hurst and Yentsch (1981) found that the peak toxicity occurred in


1978. Toxin maxima were found in May and ranged from 25 to 114 pg g-' tissue. These high levels of toxin accumulation are possible only because shellfish are not harmed by the toxins they accumulate (Twarog and Yamaguchi, 1975). Detoxification of the mussel tissue depends on the amount of toxin present and the amount of melanin pigment in the tissue (Price and Lee, 1971). Decay constants can in some cases be determined and used to help determine when to reopen contaminated shellfish beds (Hurst and Gilfillan, 1977). Hurst and Yentsch (1981) concluded from their survey that

1. Considerable variability exists for toxicity between stations a few kilometers apart. However, appearance of toxicity is simultaneous over wide areas.

2. Major occurrences of toxicity are during spring or late summer when water temperatures are changing. During this time toxic cysts and motile cells in the water provide the inoculum for a new bloom.

3. Considerable variation between years is found at all stations. Most stations are characterized by one major outbreak per year.

4. Rates of intoxification and detoxification are rapid, being several hundred micrograms of shellfish toxin per 100 g shellfish tissue per day.

Another important factor influencing the apparent toxicity of PSP-producing dinoflagellates is biotransformation by the shellfish (Shimizu and Yoshioka, 1981; Shimizu et al., 1984). By using homogenates of the east coast scallop Placopecten magellanicus, it was shown that gonyautoxins and neosaxitoxin decreased while saxitoxin content increased. Biocon- version of neosaxitoxin to saxitoxin in Mya arenaria and Mercenaria mercenaria was also demonstrated.

Dinoflagellates causing ciguatera disease are found to be most prevalent in the tropics between 35"N and 34"S, especially in the South Pacific islands and the Caribbean. In these areas the disease affects more than 10,000 individuals per year and appears to be increasing in frequency (Halstead, 1967; Ragelis, 1984). Ciguatera has been caused by over 400 species of fish, but especially the herbivorous surgeon fish and parrot fish, and the larger carnivorous reef sharks and red snappers (Banner, 1976). Randall (1958) concluded that all such fish were tied to the coral reef through the food chain. The possibility that ciguatera originates as a result of toxic algae was first mentioned by a British physician, Colin Chisholm, in 1808 (Halstead, 1967). Evidence gathered during the 1960s and 1970s pointed to certain dinoflagellates that grow in close association with macroalgae and/or other bottom structures as progenitors of ciguatera toxins. It was not until a publication by Bagnis et al. (1977) that a causative organism was isolated from the biodetritus layer of waters surrounding

ALGAL TOXINS 63

the Gambier Islands in the South Pacific. It was initially identified as a Diplopsalis sp. by Yasumoto et al. (1977) but later it was decided that this benthic dinoflagellate belonged to a new genus and was named Gambier- discus toxicus (Adachi and Eukuyo, 1979). Other species of dinoflagellates inhabiting the same microhabitats in the Pacific have been found to produce toxins which may also contribute to the ciguatera syndrome. These include Amphidinium carteri, Amphidinium klebsii, Ostreopsis ovata, Ostreopsis siamensis, Prorocentrum concavum (Nakajima, 198 l), and Prorocentrum lima (Yasumoto et al., 1980; Nakajima et al., 1981; Murakami et al., 1982).

Current studies on ciguatera seem to center around those of Tindall and co-workers working in the Caribbean, Yasumoto, Bagnis, and others who are working in the Pacific island area, and Kimura, Hokama, and co- workers in Hawaii. Tindall et al. (1984) surveyed over 70 sites in the Virgin Islands. From these collections 65 strains representing 18 species of dinoflagellates are being cultured at the University of Southern Illinois. These species have all been proved to culture best using the ES medium of Provasoli (1968) to which was added 1-5% soil extract. Initial screening for toxicity showed that all possessed some toxic component and studies to date have concluded that Prorocentrum concavum and Gam- bierdiscus toxicus are the major contributors of ciguatera toxins to these Caribbean reef and inshore ecosystems. These studies also concluded that no one dinoflagellate or toxin is the major cause of ciguatera. It was also found that the major ciguatera toxin found in the contaminated fish, ciguatoxin, was not the major toxin produced by cultured toxic strains. This implies that ciguatoxin may be formed by biotransformation processes in the fish, that laboratory cultures of toxic dinoflagellates produce different toxins in the laboratory than in nature, or that ciguatoxin-producing dinoflagellates have not been successfully isolated and cultured.

A recent study by Carlson et al. (1984) examined the effect of extracts of macroalgae on the growth of G . toxicus, P . concauum, and Prorocen- trum mexicanum. The macroalgae chosen were those commonly found in association with these toxic dinoflagellates in the Caribbean, viz., Chaetomorpha linum (Chlorophyta), Dictyota dichotoma (Phaeophyta), and Turbinaria turbinata (Phaeophyta). Water-soluble extracts from field collections of these macroalgae were aseptically added to axenic cultures of the toxic dinoflagellates. It was found that soil and macroalgal extracts enhanced the growth of bacterized cultures of G . toxicus and P . concauum but inhibited that of bacterized P . mexicanum. Generation times of axenic cultures of G. toxicus and P. concavum were lengthened. Evi- dence was found to indicate that bacteria could contribute to variation in toxin production. Variation of toxicity independent of growth due to bac-


teria has also been demonstrated in freshwater toxic cyanobacteria (Car- michael and Gorham, 1977). The dinoflagellate study concluded that enhanced algal growth in the presence of bacteria was not due to increased C02 production. It was also speculated that hormones such as cytokinins may play a role in the stimulation of growth by the macroalgal extracts.

C. CYANOPHYTA

Cyanobacteria producing toxic substances have been reported since the late 1800s (Carmichael, 1981). The first laboratory cultures of toxic strains were obtained from the colonial species Microcystis aeruginosa by Hughes et al. (1958) using a modified Fitzgerald medium (Fitzgerald et al., 1952). Later, cultures of Microcystis utilized the medium ASM (Artificial Seawater McLachlan), which is a modified ASP (Artificial Seawater Pro- vasoli) (McLachlan and Gorham, 1961). Growth experiments on several single-cell isolates (clones) of Microcystis revealed that toxicity varied between clones and that there were toxic and nontoxic cells within a given colony. A natural consequence of this varying toxicity was that only the most toxic clones were retained in culture for future work (Gorham, 1964). The optimal temperature for growth of the toxic clone M . aeruginosa NRC-1 was not the same as that for optimal toxin production. The same toxic clone was grown in 9 dm3 batch cultures for isolation and characterization of the toxic principle (Bishop et al., 1959). Since the late 1960s, however, NRC-1 has not been grown for toxicity studies and when tested in 1971 by this author the toxic characters differed from those when the strain was originally isolated. Recent tests for toxicity in 1980 indicated that overall toxicity was also declining. This has resulted in the use of another toxic strain, M . aeruginosa 7820 (Codd and Carmichael, 1982). It should be noted that over the period of time NRC-1 was cultured, the medium in which it was grown was changed from modified Fitzgerald to ASM to ASM-1 (Gorham et al., 1964), with the current medium being BG- 11 (Stanier et al., 1971). These media have progressively higher levels of nitrate as NaN03. BG-11 has 18 times the amount of nitrate as ASM. Phosphate concentration as orthophosphate is equal in all three media. These culture media were chosen for their growth-promoting potential and only secondary consideration was given to whether toxin production was affected with extended subculturing. In retrospect it is important to ask whether the decline in toxicity is due to the fact that the cells are now in a nutrient-rich medium and no longer “stressed,” resulting in lowered toxin production. This same pattern is emerging with other genera of toxin-producing cyanobacteria similar to those of Microcystis, including Anabaena (Carmichael, unpublished data) and Oscillatoria (0. M.

ALGAL TOXINS 65

Skulberg, personal communication). Sirenko (personal communication) also finds that culture toxicity declines when Microcystis isolated from waters in the U.S.S.R. is maintained in the laboratory over a number of months to years.

In the early 1960s toxic strains of Anabaena flus-aquae were isolated for laboratory studies by Gorham and co-workers (Gorham et al., 1964). The basic ASM medium was modified by doubling the nitrogen, phosphorus, calcium, and iron contents while increasing by four times the boron, zinc, cobalt, and copper contents to produce a medium termed ASM-1, Most studies on the chemistry and toxicology of Anabaena toxins have been done using the clonal isolate NRC-44-1 (Carmichael, 1981). Toxicity has remained quite stable in this strain over the period of 1961 to 1980. At this time, for reasons of convenience, NRC-44-1 was transferred to BG-11 for maintenance in this author’s culture collection. Growth continued to be good in this strain but a routine check of toxicity in 1982 revealed that the strain had become nontoxic. A check of 25 subcultures of NRC-44-1 maintained as backup cultures in the laboratory revealed that only 1 had a level of toxicity detectable by mouse bioassay. Transfer of this toxic subisolate back to ASM-1 medium has restored its normal level of toxicity, i.e., 40-60 mg kg-1 (intraperitoneal injection in mouse). Subcultures maintained by P. R. Gorham (University of Alberta) on ASM-1 medium and 0. M. Skulberg (Norwegian Institute for Water Re- search) on Z-8 medium (Skulberg, 1983), have retained their toxicity throughout this time period. Both ASM-1 and Z-8 media contain the same level of nitrate. Light levels, temperature, and pH were maintained the same throughout this time period. At present, investigations are under- way to see if nontoxic NRC-44-1 returned to ASM-1 medium will regain its former toxicity levels.

Another instance of apparent change in toxicity has been observed in Anabaena flos-aquae S-23-g, a strain isolated by Carmichael from Sas- katchewan, Canada in 1975 (Carmichael and Gorham, 1978). This strain, at the time of initial isolation, produced a toxin, anatoxin-d, with neurotoxic properties. Repeated subculturing of this strain on ASM-1 and then changing to BG-11 medium has resulted in the loss of neurotoxin production and expression of a hepatotoxin like that of Microcystis (Jones, 1984).

It will be important to examine whether cultures of toxic cyanobacteria now being cultured from Norway (Skulberg, 1983; Skulberg et al., 1984) and South Africa (Eloff el al., 1982; Botes et al., 1982a; Siegelman et al., 1984) will also undergo a decrease in toxin production with time when grown on the standard nutrient-rich culture media.

Toxicity of the filamentous c yanobacterium Aphanizomenonflos-aquae


has proved to be the most difficult to study. This is largely due to the difficulty in culturing compared to other toxic cyanobacteria. Phinney and Peek (1961) were among the first to report that blooms of Aph.Jos-aquae could be toxic. Toxicity was not verified, however, by laboratory cultures from the bloom. Gorham (1964) reported some success in culturing Aphanizomenon from sources in Ontario and Saskatchewan but toxicity in the mouse bioassay was negative in all cases. Gentile (1971) reported some success in culturing toxic Aph. Jos-aquae from blooms collected at Klamath Lake, Oregon in 1968. However, no work was done on toxicity or toxicology of the isolates to verify toxicity beyond the standard mouse assay. Toxic isolates were cultured and examined from blooms collected at Kezar Lake, New Hampshire (Gentile and Maloney, 1969). In growth studies toxin production was not increased independently of growth when light or temperature was changed. Toxicity did decrease independently of growth, however, when Aph. flos-aquae was cultured at stress temperatures of 30”C, but not at the more normal environmental temperatures of 15, 20, or 26°C. No experiments were conducted to investigate possible toxin increases by stressing the cells through lowering nitrate or phosphate concentrations. These toxic isolates were subsequently discarded and no toxic Aphanizomenon existed in culture until new isolates from New Hampshire were made by Carmichael in 1980 (Ikawa et al., 1982; Carmichael and Mahmood, 1984). These isolates produce toxins similar to the earlier New Hampshire isolates but as yet no growth studies have been done to investigate possible regulation of toxin production.

Toxic marine cyanobacteria have not been studied in laboratory cultures. Instead, all chemical and toxicological studies have used field collections. Toxic marine cyanobacteria are all found in the Oscillatoriaceae family, occupying benthic habitats in warm water areas such as Hawaii and Okinawa (Moore, 1981a,b). The secondary chemicals produced cause a severe “swimmer’s itch” or contact dermatitis among swimmers who come in contact with the filaments. Most reports of these contact irrita- tions come during the summer months and it is not clear whether toxins are produced at other times of the year, although it would seem that they would be. While direct comparisons between marine and freshwater cyanobacteria toxins are difficult to make, at present there is some indica- tion that freshwater cyanobacteria produce contact irritants. This comes from field observations on certain species of Anabaena, Aphanizomenon, Oscillatoria, and Gloeotrichia in the U S A . and Europe over the past 4 years (Skulberg et al., 1984; Carmichael et al., 1985; Codd and Carmi- chael, unpublished data). When these toxins are characterized and studied it should become clear that the freshwater cyanobacteria are capable of producing secondary chemicals with neurotoxic, hepatotoxic, and dermatotoxic properties.

ALGAL TOXINS 67

Overall there has been little work on the effects of growth on toxin production for any of the toxic algae. Most investigators have been kept busy just maintaining a few strains in culture in order to isolate, purify, and characterize the toxins present. It is possible to outline, however, several studies which could yield information on qualitative and quantitative aspects of toxin production and the role of these secondary chemicals in the ecology of the organism. These include

1. The role of nutrient depletion on toxin production, especially major nutrients such as nitrogen and phosphorus. At least some evidence points to a possible stimulation of toxin production as growth decreases due to a shortage of nitrogen and phosphorus. This should be analyzed over several generations of subculturing.

2. Although the few studies that have been done do not indicate that temperature or light affect toxin production independently of growth, longer term studies could prove useful in examining this possibility.

3. Possible role of zooplankton herbivores through selective feeding of nontoxic over toxic cells. Zooplankton presence could also stimulate production of toxins under herbivore stress, as is now being revealed for higher plants. This possibility does not seem as likely as nutrient stress, however, since it would imply that some chemical communication exists between cells of a bloom.

4. Possible role of extrachromosomal DNA (plasmids) in regulation of toxin production (Hauman, 198 1). Expression of the plasmid regulating toxin production (if present) could be part of nutrient or predator stress.

IV. ISOLATION, CHARACTERIZATION, AND TOXINOLOGY

A. PRYMNESIUM TOXINS

The toxic principles of P. paruum have been purified and characterized by Ulitzur (1969) and Ulitzur and Shilo (1970). The toxin has a broad spectrum of different biological activities in uiuo and in uitro (Shilo, 1971, 1981). It seems that there is a family of compounds having similar composition but different toxic effects, rather than a single compound with different toxic effects. These effects are generally classed as being cyto- toxic, hemolytic, and ichthyotoxic. Since the toxic effects are primarily to fish the main component is often referred to as an ichthyotoxin. Intoxica- tion of fish consists of two stages. Initially there is reversible damage to the gill tissues, resulting in the loss of their selective permeability. The second stage, which leads to death, is the response of the sensitized fish to any of a number of toxicants in the environment including the Prymne-


sium toxin (Shilo, 1981). The gill epithelium of the fish is the primary site of action for the toxin.

Extraction of the toxic compounds indicates that they are acidic, polar lipids. Analysis of the toxin revealed 15 amino acids, a number of fatty acids, 0.47% phosphate, and 10 to 12% hexose sugars (Ulitzur and Shilo, 1970). In its composition of fatty acids together with protein and phosphate, the purified toxin resembles proteolipids (Shilo, 1981). More recently isolation and purification of Prymnesium hemolytic toxin and ichthyotoxin have been done by Kim and Padilla (1977) and Kozakai et al. (1982).

B. FRESHWATER CYANOPHYTE TOXINS

1 . Neurotoxins Known chemical groups of toxins from freshwater cyanobacteria include alkaloids and peptides. The alkaloid toxins are the most rapidly acting. They function as neurotoxins paralyzing peripheral skeletal muscles, then respiratory muscles, with death due to respiratory arrest occurring between a few minutes to a few hours. These toxins are produced by various strains and species of Anabaena and are generally referred to as anatoxins (Table I). Anatoxin-a (antx-a) is the only alkaloid toxin from this group which has been chemically characterized (Fig. 8). It is a potent depolarizing neuromuscular blocking agent (Carmichael et al., 1975, 1979; Spivak et al., 1980). Chemically the compound is the secondary amine, 2-acetyl- 9-azabicyclo[4.2.l]non-2-ene, and is isolated from the filamentous strain Anabaena flos-aquae NRC-44-1 (Huber, 1972; Devlin et al., 1977). Syn- thesis of antx-a has been done through a ring expansion of cocaine (Camp- bell et al., 1977) from 1,5-cyclooctadiene (Campbell et al., 1979), and by intramolecular cyclization between an iminium salt and a nucleophilic carbon atom (Bates and Rapoport, 1979).

4

Fig. 8. Structure of anatoxin-a hydrochloride, a depolarizing neuromuscular blocking agent produced by the filamentous cyanobacterium Anabaena flos-aquae clone NRC-44-1.

ALGAL TOXINS 69

Poisoning of domestic and wild animals by anatoxins comes from ingestion of the toxic cells and extracellular toxin from a water bloom. Given the LD5o for intraperitoneal bioassay in mouse for purified anatoxin-a as 200 pg kg-I body weight and the approximate oral lethal dose for certain animals (Carmichael et af . , 1977; Carmichael, 1981), it is estimated that a lethal bolus of water bloom ranges from a few cubic centimeters to a few cubic decameters, depending upon animal species, toxicity of the bloom, and amount of food material in the animal’s gut. The presence of more than one toxin in the bloom can also result in different signs of poisoning and survival times.

Known toxins of Aphanizomenon Jos-aquae are also neurotoxic alkaloids, generally referred to as aphantoxins. Sasner and his colleagues presented evidence that aphantoxin from water blooms of Aph. Jos- aquae was similar to the paralytic shellfish poisons saxitoxin and neosaxitoxin (Sawyer et al., 1968; Jackim and Gentile, 1968; Alam et af . , 1973, 1978; Alam and Euler, 1981; Sasner et af . , 1981; Adelman et al., 1982). Current research with aphantoxin is concerned with a toxic strain isolated from a small pond near Durham, New Hampshire in 1980 by Carmichael, referred to as NH-1 and NH-5 (Carmichael, 1982; Ikawa et a f . , 1982). The toxins of NH-5 are primarily neosaxitoxin (80%) and saxitoxin (15%). Depending on extraction procedure it appears that aphantoxin also contains some precursors to neosaxitoxin and saxitoxin (Carmichael and Mahmood, 1984; Sasner et al., 1984). At present the structures of the aphantoxins have not been determined but assuming that they are the same as PSPs, and all evidence to date supports this, they will have structures like those in Fig. 1 1 .

Detection methods for these toxins in water supplies have not been adequately developed but high-performance liquid chromatography (HPLC) is being used in some cases. Both Astrachan and Archer (1981) and Wong and Hindin (1982) have used HPLC for the detection of anatoxin-a. In our laboratory we routinely use HPLC to purify other neurotoxins of Anabaena and Aphanizornenon (Carmichael and Mahmood, 1984). These methods could be modified to detect low levels of the toxins in water reservoirs or recreational waters.

2. Hepatotoxins The peptide toxins of the freshwater cyanobacteria are primarily found in various strains of Microcystis aeruginosa. These toxins are responsible for most poisonings by cyanobacteria since M. aeruginosa is more common in its worldwide distribution than the other toxigenic cyanobacteria. Although other cyanobacteria, including Anabaena, Aphanizomenon, and Oscilfatoria (Carmichael, 1981, 1982; Skulberg et af . , 1984) are


thought to produce peptide toxins, Microcystis peptides have been studied most. Toxic blooms have been found in ponds and lakes thoughout the world including the U.S.A., Canada, the U.S.S.R., Europe, South Africa, India, Japan, the Middle East, and Australia. In temperate zones most toxic blooms occur in mid to late summer (Carmichael et al., 1985).

Rats and mice injected either intravenously or intraperitoneally with acutely toxic doses of the cells or toxin extract (LDso 25-100 pg/kg, intraperitoneally in mouse) die within 1 to 3 hr. Death is preceded by pallor and prostration, with episodes of unprovoked leaping and twitch- ing, Upon necropsy, the animals show grossly enlarged livers engorged with blood, with the remainder of the carcass being exsanguinated. Liver weight is increased, and at death composes about 8 to 10% of body weight of mice as compared to about 5% in controls (Slatkin et al., 1983; Fal- coner et al., 1981; Theiss, 1984). The blood content of livers of mice poisoned by Microcystis increases from 66 mm3 g-' liver in controls to 534 mm3 g-l for mice killed 45 min after toxin injection (Falconer et al., 1981).

Histological examination of the liver reveals extensive centrilobular hemorrhagic necrosis with loss of characteristic architecture of the hepatic cords. Transmission electron microscopic examination indicates that both hepatocytes and hepatic endothelial cells are destroyed. The only alterations noted prior to cell rupture are slight mitochondria1 and cell swelling. Damaged cells have extensive fragmentation and vesicula- tion of the membrane (Runnegar et al., 1981).

Gross and histological examination of intestine, heart, spleen, kidneys, and stomach show no consistent abnormalities; lungs are mildly con- gested with occasional patches of debris. Thrombi thought to contain platelets are found in the lungs of affected animals (Slatkin et al., 1983; Falconer et al., 1981). It is suggested that these thrombi may be a direct effect of the toxin and may secondarily cause the liver effects by creating sufficient pulmonary congestion to cause right heart failure which, in turn, could cause blood pooling and congestion in the liver (Slatkin et al., 1983). However, in time course studies, Falconer et al. (1981) reported that the pulmonary thrombi did not appear in histological preparations taken at 15 and 30 min after toxin injection and were present only in later preparations (while liver damage was noted as early as 15 min). This evidence, along with other evidence of effects on isolated hepatocytes and the rapid onset of the liver effects in uiuo, have led other researchers to believe that the liver damage is a direct effect of the toxin on the hepatocyte membrane and that the immediate cause of death in acutely dosed animals is hemorrhagic shock (Falconer et al., 1981; Runnegar and Falconer, 1981; Theiss, 1984). Occasionally, hemorrhages are noted in organs other than the liver (Ostensvik et al., 1981). This could possibly be due to coagulation problems associated with the liver damage.

ALGAL TOXINS 71

The action of Microcystis toxin on isolated rat hepatocytes was investigated by Runnegar et al. (1981), Runnegar and Falconer (1981), and Fox- all and Sasner (1981). In cell suspension, normal hepatocytes viewed under the scanning electron microscope show a rounded appearance with a surface covered with microvilli. Within 5 min of incubation with the toxin, obvious lumps can be seen on the cell surface, which increase with time to present a distorted lumpy cell without microvilli. These changes occur only in live cells and are found to be dose dependent, with the minimum concentration of toxin causing deformation being directly related to the LDso for the toxin. There is no difference in trypan blue exclusion by hepatocytes incubated with or without the toxin, the toxin causes no cell lysis, and there is no release of aspartate amino transferase (ASAT) into the medium. The toxin appears to be transported into the cell via the bile acid transporters in the cell membrane. Deformation of the cells by Microcystis toxin is blocked by addition of sodium deoxycholate to the medium. The blocking effect also shows a dose response, with increasing concentration of toxin requiring a higher bile acid concentration.

In another study by Grabow et al. (1982) the effect of Microcystis toxin was tested on isolated liver, lung, cervix, ovary, and kidney cell cultures. Cells of all cultures were damaged or disintegrated after overnight incubation in the presence of Microcystis toxin. While no mechanism of action was proposed it was suggested that the toxin may act on cell membranes.

The effect on isolated hepatocytes of the presence and absence of calcium in the medium was also investigated (Runnegar and Falconer, 1982). It has been suggested by Farber and his co-workers that calcium entry into the cell is the final common step in the death of cells injured by membrane toxins and that cells can be protected by excluding calcium from the medium (Farber, 1981). Conversely, it has been reported by other researchers that toxic injury to hepatocytes is not dependent on calcium and in some cases calcium in the medium actually protects cells from injury by various toxins (Smith et al., 1981). Tests with Microcystis toxin and isolated hepatocytes revealed that the presence or absence of calcium in the medium makes no difference to the toxic effect (Runnegar and Falconer, 1982).

It was reported that toxic extracts from algal blooms would agglutinate red blood cells (Carmichael and Bent, 1981). However, Runnegar and Falconer reported no red blood cell agglutination with extracts isolated in their laboratory (Runnegar and Falconer, 1982).

The effects of the toxin on mouse liver slices, isolated mitochondria, and microsomes were investigated and no specific effects were noted (Runnegar and Falconer, 1981). The toxin did not significantly affect the incorporation of labeled leucine, uridine, or methylthymidine into trichlo-


roacetic acid-insoluble precipitates from incubated mouse liver slices, indicating that it exerts no major effect on protein, RNA, or DNA synthe- sis. The toxin did not affect oxygen consumption of liver slices and only slightly increased that of isolated mitochondria. Measurements of glycogen degradation in liver slices incubated with toxin showed a variable increase in glycogen loss and glucose appearance in the incubation medium (Runnegar and Falconer, 1982).

Runnegar and Falconer (1981) noted similarities in both structure and gross pathological effects of Microcystis toxin and phalloidin, a bicyclic peptide from the poisonous mushroom Amanita phalloides. Phalloidin produces a hepatic hemorrhagic necrosis similar to that produced by Mi- crocystis, but these similarities do not extend to the cellular level. Phal- loidin produces its toxic effects by inhibiting the depolymerization of F actin to actin; Microcystis does not. The molecular mechanism of action of Microcystis toxin is, at this time, unknown.

Time course studies of dosed rats and mice have been done by several researchers (Slatkin et al., 1983; Ostensvik et al., 1981; Runnegar and Falconer, 1981). Liver damage can be noted in histological sections as early as 15 min after dosing, with the centrilobular hemorrhagic necrosis steadily progressing outward with time. Platelet counts taken during the course of the toxic reaction show a steady decrease in the circulating platelet count that is inversely proportional to the increase in liver weight (Slatkin et al., 1983; Jones, 1984).

Runnegar and Falconer (1981) noted changes in aspartate amino transferase (ASAT) and lactic dehydrogenase at 15 min, and by 30 min had increased to 50 times control levels. Ostensvik et al. (1981) found changes in ASAT commencing at 30 min, but no changes in alanine amino transferase (ALAT) nor bilirubin during the course of toxicity.

Ostensvik et al. (1981) monitored the effect of an extract of a predomi- nantly Microcystis water bloom on blood pressure of rats. The blood pressure decreased markedly immediately after intravenous administration, but began increasing at 1.5 min and was back to normal levels within 10 min of injection. During the next 40 min, the blood pressure decreased slowly to very low levels consistent with hemorrhagic shock and remained constant at these low levels until the rats died at about 90 min. Theiss (1984), using purified toxic peptide of M. aeruginosa strain 7820, also found that blood pressure responses, both arterial and venous, were indicative of a direct hepatotoxin which caused death by hemorrhagic shock.

Jackson et al. (1984) inoculated sheep intraruminally with M. aeruginosa water bloom suspension. The majority of the lethally poisoned sheep died within 18 to 23 hr. The carcasses of the sheep showed that the

ALGAL TOXINS 73

primary site of toxicity was the liver, which had centrilobular to near massive hepatocyte necrosis. Small hemorrhages were also noted in many other areas of the body. Lungs were mildly edematous, but other organs were normal except for the hemorrhages. It was noted that neutrophils were attracted into the lumen of bile ductules without a cholangitis. This has been noted in other animals poisoned with Microcystis and may have diagnostic significance in differentiating algal poisoning from other plant hepatotoxicities. These sheep experiments revealed a sharp dose-response curve in that up to 90% of the lethal dose of bloom could be ingested in a single administration without measurable effect. From the animal response it was estimated that the 27-kg sheep used would have to ingest about 650 cm3 of the thick algal bloom as it occurred on the pond to provide the approximately 30 g of dried cells necessary to cause acute lethal toxicity.

Foxall and Sasner (1981) reported that cladocerans, amphibians, crus- tacea, and teleosts were not affected by the toxin, but all mammals and birds tested to date are sensitive to it. Some effects have been reported on cardiac muscle and blood hemostasis (Kirpenko and Kirpenko, 1980). No effect has been reported on skeletal muscle or isolated nerve preparations.

Foxall and Sasner (1981) reported that young hepatocytes in uitro are not affected and neither are young mice nor rats. These mice were not affected by lethal doses of the toxin until they had reached the age of approximately 20 days. This would suggest a possible activation of the toxin by the liver enzyme systems, but to date no one has investigated this possibility. Female mice were slightly more sensitive to the toxin than male mice.

Foxall and Sasner (1981) reported no antibiotic activity against green algae, yeast, or bacteria and no toxicity to certain zooplankton, crayfish, amphibians, and teleosts. However, Grigor’yeva et al. (1977) reported a wide spectrum of antimicrobial activity against Escherichia coli, Shigella jlexneri, Salmonella typhimurium, Staphylococcus aureus, Enterococ- cus, and Candida. The mechanism involved was a decreased thiamine content and inhibition of dehydrogenase activity. Kirpenko et al. (1982) found that Microcystis toxin extracts inhibited the development of sa- prophytic microflora in artificial ponds.

Kirpenko et al. (1981) found that toxic Microcystis caused embryo- lethal, teratogenic, and gonadotoxic effects in the rat. Mutagenesis involving anomalies of chromosome and chromatid apparatus was also reported. However, Runnegar and Falconer (1982) reported no mutagenicity when a purified extract was tested by the Ames Salmonella assay.


Reports on Microcystis poisoning are often conflicting. The only consistent pathological findings in Microcystis toxicity are the swollen, blood-engorged liver with hemorrhagic necrosis and the mildly edematous lungs. These conflicting reports may be attributed to possible differences in the effects of different strains of toxic Microcystis, the use of extracts from mixed blooms that contain other algal or bacterial species, or differences in research technique.

Microcystis has been linked to an outbreak in 1979 of hepatoenteritis among an aboriginal population on Palm Island (Queensland, Australia) (Bourke et al., 1983). An epidemiological investigation revealed that 139 children and 10 adults were affected shortly after a copper sulfate treatment of a dense algal bloom in Solomon Dam, the source of the island’s reticulated water supply. All of the affected individuals were using water from Solomon Dam and no incidence of disease was reported among persons not receiving water from this supply. The disease had three well- defined phases which presented in the following order: hepatitis phase-2 days; lethargic phase with severe electrolyte derangement-1 to 2 days; diarrheal phase-5 days. Repeated sanitary assessments of the environment of Palm Island failed to uncover any other possible cause for the outbreak.

Falconer et al. (1983a) examined the results of routine assays for hepatic enzymes in plasma of persons who obtained drinking water from a reservoir (Malpas Dam, Armidale, New England, Australia) containing a heavy bloom of toxic M . aeruginosa during periods before, during, and after the algal bloom. These results were compared with corresponding assays from an adjacent population which did not use water from this source. The residents supplied with water from the bloom-infested Malpas Dam reservoir showed a significant rise in y-glutamyltransferase (GGT) during the bloom period, while no such increase occurred in residents not receiving their water from the Malpas Dam. ALAT also showed an increase during this period, but it was not statistically significant (p I 0.10). ASAT and alkaline phosphatase showed no significant increases. GGT is characteristically released after alcohol or toxin damage to liver cell membranes and is a more sensitive indicator of liver damage than alkaline phosphatase or ASAT.

The toxin of M. aeruginosa is normally contained within the algal cells and is released only when the cell is damaged, either by poisoning the algae with copper sulfate, by mechanical rupture of the cell, by break- down in the stomach, or age-related death of the cell. Damage could occur to the cell when algae in a reservoir are transported through a municipal water system; therefore presence of the blooms in a water supply reser-

ALGAL TOXINS 75

voir might be expected to result in the presence of the toxin in drinking water. Some toxins and odor and flavor organics associated with algal blooms can be removed by filtration of reservoir water through sand topped by a layer of granular activated carbon (Falconer et al., 1983b).

The exact composition and structure of the Microcystis toxin have remained elusive, despite the efforts of many scientists since the attempts of Louw (1950) to identify the toxic principle from a Microcystis bloom in South Africa. Considerable variation exists in the toxins from different strains of the algae. The toxin also appears to have an unusual structure which does not lend itself to classical techniques. Working with different strains and utilizing different procedures for purification, most workers now agree that the toxin is a pentapeptide, with some common and some variable amino acids. On hydrolysis the toxin is found to contain five amino acids and methylamine in approximately equimolar amounts. Amino acids isolated from all various toxin preparations invariably include aspartic acid (or P-methylaspartic acid), glutamic acid, and alanine. Glutamic acid and alanine, the invariant amino acids of Runnegar and Falconer (1981) and Elleman et al. (1978), and methionine, have no free carboxyl groups while the a-carboxyls of glutamic and P-methylaspartic acid were shown to be free. The toxin does not react with ninhydrin, nor were any amino groups dansylated in the intact toxin, indicating the absence of any free amino groups (Bishop et al., 1959; Murthy and Capin- dale, 1970; Rabin and Darbre, 1975; Toerien et al., 1976; Elleman et al., 1978; Botes et al., 1982a; Eloff et al., 1982; Santikarn et al., 1983).

From amino acid analysis, a minimum molecular weight of 654 was derived by Runnegar and Falconer (1981). Other proposed molecular weights, derived by different researchers from various strains and utilizing different techniques, have also been reported (Runnegar and Fal- coner, 1981). The existence of the toxin as dimers, trimers, or even larger groups of identical or similar subunits could explain the wide variability of proposed molecular weights of the toxin (Eloff et al., 1982). The lack of free amino groups has led to the speculation that the toxin is cyclic (Bishop et al., 1959; Santikarn et al., 1983; Williams, 1983; Botes et al., 1984) and/or has a blocking group on the terminal amide group (Botes et al., 1982a,b).

The extreme hydrophobicity of Microcystis toxins, as exemplified by their chromatographic behavior on paper, cannot be accounted for in terms of their peptide composition and could logically reside in the properties of such a blocking group. The UV spectrum of the toxin shows an absorption maximum at 240 nm; again, this cannot be accounted for by the peptide portion of the molecule, since Botes et al. (1982a) found no


aromatic amino acid present. They do suggest that this absorbance peak could be due to the presence of a conjugated diene chromophore in the blocking group.

Eloff et al. (1982) found that the toxins present in different strains were very similar, but variations in toxin composition were found. In some organisms, as many as six different toxins were obtained, but generally one or two toxins accounted for >90% of the toxin in a single isolate. In all cases the toxin contained P-methylaspartic acid, glutamic acid, alanine, methylamine, and two other amino acids in equimolar ratios. The additional amino acids present in major toxins were as follows:- leucine and arginine, leucine and alanine, tyrosine and arginine, methionine and arginine, leucine and tyrosine, alanine and tyrosine, or arginine and arginine. The peptide containing leucine and arginine was present in 9 of the 13 toxic isolates.

Botes et al. (1982a) isolated four toxin variants (BE-2 to BE-5) from a laboratory strain of Microcystis aeruginosa (WR-70). The strain was cultured in a modified Volk and Phinney medium (1968) with the trace ele- ment mix of Stanier et al. (1971). All variants were composed of five amino acid residues with one residue each of P-methylaspartic acid, glutamic acid, and alanine common to all. For the remaining two residues combinations of leucine and arginine, tyrosine and arginine, leucine and alanine, and tyrosine and alanine were found. Methylamine was detected in acid hydrolysates in all cases. Configuration assignments (Botes et al., 1982b) of the a-carbon atom of the amino acid residues have been made by stereospecific enzymatic transformations, showing that the constant residues are in the D-form, whereas the L-configuration could be assigned to all the variant residues. The relative configuration of the @carbon atom of P-methylaspartic acid could be made by comparison of the electropho- retic mobility of the toxin-derived residue with literature values for the authentic compound. The presence of N-methyldehydroalanine, which gives rise to methylamine upon acid hydrolysis, has been confirmed by identifying N-methylalanine in the acid hydrolysates of the toxin variants, after reduction of the toxin with sodium borohydride. The use of 400 MHz proton NMR spectroscopy showed the toxins to be more complex than suggested by amino acid analysis alone. Apart from the unambiguous assignment of the amino acid residues, an apolar side chain of 20 carbon atoms was demonstrated. The toxin is thought to exist in dimers, trimers, and other larger aggregates with each subunit consisting of a pentapeptide plus an apolar side chain. In this study the molecular weight of each subunit was estimated at 909.

Based on mass spectrometry, using the BE-4 toxin, Santikarn et al. (1983) and Williams (1983) proposed that the blocking group is a highly

ALGAL TOXINS 77

unsaturated hydrocarbon with a molecular weight of approximately 3 13. This would be in addition to the 909-Da subunit of BE-4 toxin. The structure of this side group was reported to be a novel p-amino acid.

There is some new evidence that the peptide may be cyclic. This would explain the resistance of the intact toxin to Edman degradation and degradation by proteolytic enzymes. Neither fast atom bombardment mass spectrometry nor the electron impact mass spectrum of methylated Mi- crocystis toxin BE-4 shows evidence of the sequence of ions normally observed in the spectra of linear peptides (Santikarn et al., 1983; Wil- liams, 1983). Botes et al. (1984) showed that the p-amino acid residue was a part of the linear amino acid sequence. The molecular weight of the entire toxin was concluded to be 909. They also proposed the term cyanoginosin for the monocyclic heptapeptides. To designate the variable L-

amino acids they propose a two-letter suffix after cyanoginosin. Thus the BE-4 toxin becomes cyanoginosin-LA (Fig. 9).

A low-toxicity strain of M. aeruginosa collected by Watanabe and Oishi (1980, 1982) and Watanabe et al. (1981) from Lake Suwa in Japan, in contrast to reports of toxins obtained from other world-wide sources, was ninhydrin positive, inactivated by proteases, and not toxic when adminis- tered orally to mice. The main amino acids composing this toxin were glutamic acid, aspartic acid, alanine, glycine, arginine, and leucine. The molecular weight was reported to be approximately 2950, with a minimum of 770, as determined by HPLC. There have been no reported livestock deaths from Microcystis in Japan.

Siegelman et al. (1984) presented a method for microdetection of Mi- crocystis toxins. This involves extraction of 20 mg of lyophilized cells with 1 cm3 of 38% ethanol, 5% n-butanol, 50 mM ammonium acetate for 1 hr, followed by centrifugation for 5 min at 12,000 g . One cm3 of n-butanol followed by 1 cm3 of water are added, with vortexing, to the supernatant. The sample is then centrifuged (10 min at 500 g), and the upper phase of n- butanol is collected and dried by evaporation. The residue is extracted with I cm3 of 26% acetonitrile, 500 mM ammonium acetate, stored at -10°C for 16 hr, and then centrifuged for 10 min at 12,000 g . These partially purified extracts are stable for several months when stored at - 10°C.

1 o-Ala-m-Masp-B-Adda - o-Glu- Mdha r Fig. 9. Proposed general structure for cyclic heptapeptide toxins of Microcystis aeru-

ginosa (Botes er a/., 1984). X and Y, variable L-amino acids which can differ between strains. Masp, P-methylaspartic acid; Adda, p-amino acid residue of 3-amino-9-methoxy- 2,6,8-trirnethyl-10-phenyldeca-4,6-dienoic acid; Mdha, methyldehydroalanine.

78 W A Y N E W . CARMICHAEL

Aliquots of 5 to 25 mm3 of these extracts are examined by HPLC as follows :

Precolumn-pellicular octadecyl (C-18) beads (SynChropak, RSC) (0.4

Column-3-pm octadecyl (C-18) silica (Hypersil, Shandon) (0.46 x

Solvent-26% acetonitrile, 500 mM ammonium acetate, pH 6.0

x 5.0 cm)

5.0 cm)

Flow- 1 .O cm3 min-I Detection-238 nm at 0.04 AUFS (absorbance units full scale)

In our laboratory we are using a modification of this procedure to purify and compare toxic peptides of Microcystis, Anabaena, and Oscillatoria. These modifications employ C- 18 semipreparative columns, Sephadex G-25 gel filtration columns, and Bond Elut C-18 preparative cartridges (Theiss, 1984; Carmichael, unpublished data). Kirpenko et al. (1976) have reported on the use of an enzyme photometric assay to detect Microcystis toxins. The method uses cholinesterase and acetylcholine in the presence of the toxin to yield a product which can be measured with the color indicator bromothymol blue.

C. MARINE CYANOPHYTE TOXINS

Marine cyanobacteria have not presented as many problems as the freshwater cyanobacteria, but several species of the family Oscillatoriaceae (unbranched filamentous, lacking heterocysts) produce toxic compounds that cause dermatitis in man and animals and promote tumors in laboratory animals. These toxins may also have toxic affects on some forms of marine life (Moore, 1981b).

The most common and best documented toxic reaction to marine cyanobacteria is a severe contact dermatitis known as “swimmer’s itch.” This is a cutaneous inflammation characterized by erythema, followed by blisters and deep desquamation within 12 hr of exposure to the algae. The dermatitis is induced by exposure to two compounds, debromoaplysiatoxin and lyngbyatoxin A (Fig. lo), which have been isolated from Lyngbya majuscula (Moikehai and Chu, 1971; Moikehai et al., 1971; Kato and Scheuer, 1975; Hashimoto et al., 1976; Mynderse et al., 1977; Cardel- h a et al., 1979). Debromoaplysiatoxin has been isolated from deep water strains of L . majuscula; while lyngbyatoxin A is found in a Hawaiian shallow water variety of L . majuscula. The dermatitis produced by lyngbyatoxin A is the same as that from debromoaplysiatoxin. However, the shallow water variety of Lyngbya majuscula has never been impli-

ALGAL TOXINS 79

cated in an outbreak of swimmer’s itch, mainly because it grows on the leeward side of Oahu and swimmers rarely come into contact with the broken filaments of the alga in the water, since the normal tradewinds blow them out to sea rather than toward the shore.

Debromoaplysiatoxin, along with several closely related compounds, has also been isolated from other Oscillatoriaceae (Mynderse and Moore, 1978; Moore, 1981b). Debromoaplysiatoxin and oscillatoxin A, for example, are major toxic constituents of a mixture of cyanobacteria from Ene- wetak identified as Schizothrix calcicola and Oscillatoria nigroviridis. Recent work (Moore et al., 1984) shows that oscillatoxin A has a structure similar to the aplysiatoxins.

Solomon and Stroughton (1 978) reported reactions to debromoaplysiatoxin occurring in humans at concentrations of 5.6 mg ~ m - ~ and in animals at 0.5 pg making it one of the most potent dermatotoxins known. Since swimmers who come in direct contact with toxic strains of the algae develop erythema, followed by blisters and deep desquamation, within 12 hr of exposure, the active agent is classed as a primary irritant rather than an allergen. During animal testing, a second application of the debromoaplysiatoxin produced a similar reaction to the first, which is not suggestive of sensitization.

Debromoaplysiatoxin, a phenolic substance, has been isolated from the midgut gland of the sea hare (Stylocheilus Eongicauda) along with a bro- mine-containing analog bromoaplysiatoxin (Kato and Scheuer, 1976). Lyngbya majuscula is a favorite food of the sea hare, and the digestive tract of this animal appears to be unaffected by the toxin. The sea hare, in fact, utilizes an unknown metabolite in the algae for metamorphosis (Switzer-Dunlap and Hadfield, 1977). Hashimoto et al. (1976) observed rabbitfish (Siganus fuscescens) feeding on sea grass entangled with L . majuscula, but observed no harm to the digestive tract of the fish. How- ever, he suggested that ingestion of L . majuscula might be related to an epidemic of rabbitfish poisoning among the natives in the Ryukyus Islands in 1969, but no further study was made of this.

Other marine life infesting toxic Lyngbya may not be so fortunate as the sea hare and the rabbitfish. Lightner (1978) suggested that Lyngbya spp. might be responsible for mass mortalities of raceway-reared blue shrimp (Penaeus stylirostris) in the large shrimp culture facility in Penasco, Mex- ico. The affected shrimp had severe necrosis in the epithelia lining the midgut, dorsal cecum and hindgut gland, along with subsequent hemo- cytic enteritis. However, Moore (1981a) could detect no mouse toxicity or activity against P-388 lymphocytic mouse leukemia in lipophilic extracts of a sample of the toxic algae obtained from Lightner’s laboratory in 1979. The appearance of the necrosis in the shrimp was slow and Moore sug-


gests that perhaps a very small contaminant in the algae that normally grow in the rearing tanks may have been responsible.

Debromoaplysiatoxin and lyngbyatoxin A, isolated from the lipophilic extract of Lyngbya majuscula, show activity against P-388 lymphocytic mouse leukemia at sublethal doses (Mynderse et al., 1977; Cardellina et al., 1979), as do the aqueous extracts of some marine Oscillatoriaceae (Kashiwagi et al., 1980). Water-soluble toxins are also present in several other cyanobacteria belonging to the Oscillatoriaceae, but none of these has been isolated and characterized (Moore, 1981b). Cooper (1964) reported that the natives in Marakei atoll associate toxic fish with an alga they called “tan-tan.” This cyanobacteria was identified as Schizothrix calcicola, which contains both lipid- and water-soluble toxins which had a small amount of anticholinesterase activity (Banner, 1967). Lyngbya majuscula also possess water-soluble toxins (Kashiwagi et al., 1980) but little is known of these toxins at the present time.

The gross structure of lyngbyatoxin A, as determined by chemical and spectral data, is the same as that of teleocidin A (Fig. lo), a poisonous substance associated with several strains of Streptomyces (Cardellina et al., 1979; Sakabe et al., 1966). Teleocidin was first isolated from the mycelia of Streptomyces mediocidicus (Takashima and Sakai, 1960). It is now known that teleocidin is composed of two compounds, teleocidin A (M, = 437) and teleocidin B ( M , = 451) (Fujiki et al., 1982a, 1984). Te- leocidin A produces dermatotoxic effects that are the same as those of lyngbyatoxin A and both are highly toxic to certain types of fish.

Lyngbyatoxin A, bromoaplysiatoxin, debromoaplysiatoxin, and teleocidin are also reported to be tumor promoters in mice (Fujiki et al., 1981, 1983, 1984). The in uitro effects of these substances are identical to tetradecanoylphorbal 13-acetate (TPA), the tumor-promoting constituent of croton oil. To screen for the tumor-promoting activity of these compounds a three-test procedure was used. The first test is on irritation of mouse ear, the second on induction of ornithine decarboxylase (ODC) in the skin of the back of mice, and the third on adhesion of human promyelocytic leukemia cells (HL-60) (Fujiki et al., 1984). Using these tests it was shown that lyngbyatoxin A, bromoaplysiatoxin, and teleocidin were all potent promoters, but debromoaplysiatoxin was much weaker.

In addition to the above, lyngbyatoxin A and teleocidin show a number of effects on mammalian cells in culture that are similar to those of TPA. These include induction of differentiation of human promyelocytic leuke- miacells (HL-60) (Nakayasu et al., 1981; Fujiki et al., 1982b), aggregation of human lymphoblastoid cells (Hoshino et al., 1980), and stimulation of prostaglandin production and choline turnover in HeLa cells. All of these effects are similar to those produced by the structurally dissimilar phorbol ester tumor promoter TPA (Sakamoto et al., 1981).

ALGAL TOXINS 81

OH bh

B

Fig. 10. (A) Debromoaplysiatoxin and bromoaplysiatoxin (R = Br), a dermatotoxic phenolic bislactone produced by several genera of marine cyanobacteria. (B) Lyngbyatoxin A, a dermatotoxic indole alkaloid, is produced by Lyngbya majuscula. This toxin has the same structure as teleocidin A, which is produced by strains of the filamentous bacterium Streprornyces.

The discover of these tumor-promoting properties suggests that, in addition to the problem posed by the dermatotoxicity of L y n g b y a majuscula, the toxins produced by these cyanobacteria could conceivably be involved in the development of human cancer, even though there is no direct evidence that L y n g b y a majuscula has caused human cancer.

Freshwater cyanobacteria have not been investigated for the production of these dermatitis toxins with tumor-promoting activity. Some evidence exists that they are being produced however. This includes reports of contact irritation from freshwater recreational waters (Billings, 1981 ; Carmichael et al., 1985; Carmichael and Codd, unpublished data). It appears that freshwater cyanobacteria are capable of producing three


groups of toxins: neurotoxic alkaloids, hepatotoxic peptides, and the as yet unidentified dermatitis toxins which may have some similarity to marine cyanobacteria toxins. These three groups are in addition to the presence of lipopolysaccharide endotoxins, which are a part of the cyanobac- terial cell wall and do not apparently contribute to their environmental toxicity (Raziuddin et al., 1983).

D . DINOFLAGELLATE PARALYTIC SHELLFISH POISONS (PSP)

PSP has been known as mussel or clam poison depending on the source of extracts. Occasionally it has been called plankton poison and mytilotoxin. The poison was later named saxitoxin (STX), a name derived from Sax- idomus gigenteus (Alaskan butter clam), which was then thought to be the only toxic material responsible for all PSP along the Pacific coasts (Schuett and Rapoport, 1962).

From the early work of Meyer et al. (1928) and Muller (1939), it was observed that the poison was water soluble, acid stable, and alkaline labile. Using these observed properties, Schantz et al. (1957) was able to purify saxitoxin using carboxylate cation exchange resins. Further analysis of the purified poison as the dihydrochloride. salt (white hygroscopic powder) indicated a molecular weight of 300 to 400 (diffusion coefficient of 4.8 X cm2 sec-I; pK, of 8.2 and 11.6; specific optical rotation of 130 * 2"). Saxitoxin was also found to be insoluble in lipid solvents, could be detoxified by hydrogen reduction, and showed no absorption above 220 nm (Schantz et al., 1957, 1966; Schantz, 1960). The correct structure of saxitoxin (Fig. 11) was shown to be an alkaloid plus a purine (Schantz et al., 1975).

Schantz (1960) and hvans (1970) found out that a large component of toxins isolated from North Atlantic mussels was not being retained by the carboxylate cation exchange resins. With modifications in the purification method involving gel filtration on polyacrylamide gel (Shimizu et al., 1975) and TLC (Buckley et al., 1976), they were able to identify the unretained toxins as derivatives of saxitoxin. It was obvious then that saxitoxin was not the sole cause of toxicity.

Ghazarossian et al. (1974) suggested that the saxitoxin derivatives were n-oxides. Subsequently Shimizu et al. (1978) and Boyer et al. (1978) demonstrated structurally that the derivatives are n-1-hydroxysaxitoxin and 1 1-hydroxysaxitoxin sulfates, which are commonly known as neosaxitoxin and gonyautoxins.

Hall et al. (1980) and Koehn et al. (1982) isolated and identified other toxins including saxitoxin from Pacific isolates of cultured Protogo- nyaulax spp. This array of 12 toxins can be divided into 2 groups (Fig. 11):

ALGAL TOXINS 83

Carbamate - R 4 (H)

H H H STX H H OSO, GTXz H OS03- H GTX 3

OH H H neoSTX H OH H OSO, GTXl OH OSO, H GTX4

&l&

N - Sulfocarbamoyl - Rg (SO3-)

R 1 R 2 b H H H B 1 (GTX5)

H OS03- H C2(GTXe) OH H H BP(GTX6)

H H OSO, C1

OH H OSO, C3 OH OSO, H c 4

Fig. 1 1 . Structure of paralytic shellfish poisons produced by several genera of marine dinoflagellates. At least some of these toxins have been found in species of Protogonyuulux, Pyrodinium, the red alga Juniu, and the freshwater cyanobacterium Aphanizomenon (Car- michael er al., 1985).

1 . The carbamate toxins-H is the substituted group at &. This group includes saxitoxin, neosaxitoxin, gonyautoxins 1 , 2, 3 , and 4.

2. The N-sulfocarbamoyl toxins-sulfite (SO3-) is the substituted group at R4 and includes B1 (gonyautoxin 5 ) , B2 (gonyautoxin 6), C1, C2 (gonyautoxin 8), C3, and C4.

Another naturally occurring toxin that had been reported by Hsu et af . (1979) and Wichmann et al. (1981) (as an unknown) is gonyautoxin 7. A similar array of toxins has been reported from sources found in subtropi- cal and tropical waters (Yasumoto et af . , 1983). Oshima et af . (1984) demonstrated that the tropical marine dinoflagellate Pyrodinium bahamense var. compressa can produce STX, neoSTX, GTXS, GTX6, and decarbamoylsaxitoxin (dcSTX). In addition they showed that the red algal genus Jania produced gonyautoxins which were ingested by coral reef crabs and gastropods. In preliminary tests they found that these gonyautoxins could be bioconverted in the digestive organs of these crabs to STX. In addition, Harada et al. (1982) reported the presence of unidentified new toxins from turban shells (actually top shells) which were desig- nated as TST (turban shell toxin).

The relative potencies of these toxins were found to be different. Sax-


itoxin at an LDso of 10 pg kg-' or 5000 MU mg-' (1 MU is defined as the amount of toxin needed to kill a 20 g mouse in 15 min) is the most potent (Schantz et al., 1966). Hall (1982) and Proctor et al. (1975) showed that the potencies of some of these toxins change upon hydrolysis (0.2 M HCl, 100°C for 5 min) indicating transformations. Toxins B1, C1, C2, B2, C3, and C4 after hydrolysis were found to convert to STX, GTX2, GTX3, neoSTX, GTXI, and GTX4, respectively, indicating hydrolysis of the R4 group (SO3-) (Hall et al., 1980). Neosaxitoxin is as potent as saxitoxin (Shimizu et al., 1978; Hsu et al., 1979; Hall, 1982) even though Genenah and Shimizu (1981) reported a much lower value (2363 ? 101 MU mg-I). Potencies, according to Hall (1982), are STX and neoSTX > GTX3 > C2 > B2 > B1 > C1 (no data given for GTX,, C3, GTX4, and C4; while according to Genenah GTX3 was > STX > GTXl > neoSTX > GTX2 >

As mentioned earlier, characterization studies of STX indicated two dissociable groups with pK, values of 8.3 and 11.6 -+ 0.1. Rogers and Rapoport (1980) reported values of 8.22 and 11.28. A similar titration of neoSTX by Shimizu et al. (1978) revealed a third dissociable group with a pK, of 6.75. These pK, values correspond to the dissociation of N-l- hydroxyl (6.75), C-8 guanidium (8.5), and C-2 guanidium (1 1.6). The observed differences in the binding to carboxylate cation exchange resins are due to the presence of charges under the acidic condition. STX and neoSTX have a net charge of + 2 whereas GTXs (1 through 4) have a net charge of + 1 (Boyer et al., 1978). Hence it was once postulated that the net charge was the sole determinant of potency.

However, structural variation studies on potency of synthetically derived material indicated otherwise. Manipulations of C-I2 OH groups (Fig. 11) made the molecule completely devoid of activity (Kao et al., 1981; Kao and Walker, 1982). Removal of the carbamoyl group (-CONHz) reduces potency to less than 70% of that of STX (Kao and Walker, 1982). Also effective in reducing potency (in uiuo by 15 times that of STX) is the substitution of the carbamate group with a sulfo group (SO3-) (Hall, 1982).

Symptoms of paralytic shellfish poisoning are primarily neurologic and appear rather quickly, often within 30 min after ingestion of contaminated shellfish. Most deaths occur from respiratory paralysis within 12 hr of illness. The prognosis is considered good if one survives after 24 hr (Schantz, 1971). The amounts of toxins ingested to cause illness vary with the individual but ranges from 3000 to 20,000 MU.

The illness is characterized by paresthesia of lips, mouth, face, and extremities, often followed by nausea, vomiting, weakness, and incoher- ence. As the illness progresses, respiratory distress and muscular paraly-

GTX4 > B1 (GTX5).

ALGAL TOXINS 85

sis become more severe and death results from respiratory paralysis. In a large outbreak of PSP in England in 1968, the common symptoms reported were paresthesia, weakness of extremities, lightheadedness, and floating sensation (McCollum et al., 1968).

Since the toxins leave the system quickly, resuscitation is recom- mended for treatment. But in severe cases, treatment should consist of a cathartic or enema to remove the unabsorbed toxins from the intestinal tract. Gastric lavage should be considered if vomiting has not occurred (Hughes and Merson, 1976). Artificial respiration should be supplied if respiratory difficulties appear (Schantz, 1971). Evans (1965) and Kao (1975) reported some hypotensive effects of the toxins with high doses.

Potency varies widely depending on the recipient and routes of administration. From experiments with mice, rats, and rabbits, intraperitoneal (ip) or intravenous (iv) administration of STX is far more potent than oral administration, ranging from 10 to 50 times in potency (Evans, 1972). Thus substances that increase intestinal absorption or peripheral circulation will definitely intensify the symptoms of illness.

The mechanism of action for saxitoxin is interference of the transmission of electrical impulses along the nerve and muscle membranes. The toxin causes a reversible blockage of the permeability to sodium ions without affecting permeability to potassium ions. Thus the propagation of nerve-muscle action potentials is blocked (Evans, 1964, 1970; Kao and Nishiyama, 1965). Kao and Walker (1982), using analogs of STX in their studies of Na+ channel blocking, showed that active groups of STX were the C-12 OH groups, the 7,8,9 guanidinium and carbamyl group, but not the N-1 of 1,2,3 guanidinium. They proposed that STX binds the receptor located in the outside of the membrane very close to the opening of the Na+ channels. The guanidinium group is then thought to be electrostatic- ally attracted by the fixed anionic charges around the opening of the Na+ channel and the bulk of the molecule (STX) covers up the channel.

The idea of having a toxin-binding site distinct from the Na+ channel was recognized by Twarog and Yamaguchi (1975). Later Spaulding (1980) showed that frog muscle fibers subjected to trimethyloxonium ion (which derivates carboxylate groups) became less susceptible to STX blockade. Cohen and Barchi (1981) noted the binding sites to have glycoprotein characteristics.

Detection of paralytic shellfish poisons has been a concern for public health and fisheries people for many years. The officially employed method of detection, worldwide, is the mouse bioassay (Horwitz, 1980; Houser, 1965). Because of the variable nature of this assay method and the inability to distinguish types of toxins present other methods are being developed. Carlson et al. (1984) have developed a radioimmunoassay for


saxitoxin but it does not bind to neosaxitoxin. Other immunoassay methods are also being developed including a rabbit antiserum which has successfully been used as a therapeutic agent in mice to prevent death from saxitoxin (Davio and Pickering, 1983). HPLC is also proving a powerful tool in the assay of PSPs. This method uses the fluorescence property of the PSP derivatives when oxidized under alkaline conditions. Initially problems existed in the detection of neosaxitoxin and gonyautoxin 1 and 4 due to its poor fluorescence (Sullivan and Iwaoka, 1983; Sullivan et al., 1983). These problems have been corrected, making it possible to detect by fluorescence all of the known carbamate and N-sulfocarbamoyl toxins of PSP (Sullivan and Wekell, 1984).

E. DINOFLAGELLATE CIGUATERA TOXINS

Ciguatoxin (CTX) is the name given to the first active component isolated which was felt to be responsible for ciguatera poisoning. It is a lipid- soluble neurotoxin. It imparts no different taste or color to the fish and is not affected by cooking or freezing. It was isolated from contaminated red snapper (Lutjanus), shark (Carcharhinus), and the moray eel (Gymnotho- rux) by a series of organic solvents, liquid-liquid partition, and chromatography. The isolated toxin is a transparent, yellow oil. This final product had an MLD (minimum lethal dosage) of 0.5 mg kg-' and was identified as having the molecular formula CuH65NOs, with the nitrogen being quaternary (Scheuer et al., 1967). Since then it has been further purified and found to have a polyether structure similar to okadaic acid or brevetoxin C, with a molecular weight of about 11 11 (Nukina et al., 1983), a crude extract LD5o of 28.1 mg kg-', and a purified toxin LDw of 0.25 pg kg-' intraperitoneally in mouse (Hokama et al., 1984).

Research on ciguatera toxin at the University of Southern Illinois found that there is more than one ciguatoxin-like compound. From cultures of Gambierdiscus toxicus grown in the laboratory, three distinct lipid toxins, GT-1, GT-2, and GT-3, were isolated, which all act as competitive antago- nists against acetylcholine and histamine sites on guinea pig ileal smooth muscle, and whose effects are not offset by calcium (Miller et al., 1984). It may be that these toxins are individual cyclic polyether residues similar to okadaic acid isolated by Tachibana et al. (1981).

Another toxin thought to contribute to ciguatera poisoning is the water- soluble substance known as maitotoxin (MTX), which is found in the liver and gut of affected fish (Yasumoto et al., 1971) and from laboratory cultures of G . toxicus (Dickey et al., 1984). It has been suggested that MTX is a precursor of CTX (E. P. Ragelis, personal communication). On the isolated guinea pig atrium, MTX shows positive inotropic effects similar

ALGAL TOXINS 87

to CTX, but only at low concentrations; and a decrease in contactibility leading to complete suppression with higher concentrations (Miyahara et al., 1979). In anesthetized cats, MTX induced hyperventilation, then respiratory depression, slight bradycardia, with hypertension at low doses and cardiac arrest prior to respiratory arrest. MTX seemed to be more potent in these experiments than CTX (Legrand et al., 1982). MTX has been reported as having a lethal dose ranging from an MLD of 0.2 pg kg-l (Takahashi, 1982) to 1.1 mg kg-' (Dickey et al., 1984). It has also been demonstrated to cause an increase in calcium ion influx into cells, a release of norepinephrine (Takahashi et al., 1982), and to inhibit Na+- and K+-ATPase from microsomes of cat and human kidneys (Bergmann and Nechay, 1982). MTX has been isolated from cultures of G. toxicus and following a latent period it produces irreversible contraction of the guinea pig ileum (Dickey et al., 1984).

A third, and less well-studied, toxin thought to contribute to ciguatera poisoning was isolated from the parrotfish (Chungue ef al., 1977) and termed scaritoxin (Bagnis et al., 1974). Individuals having eaten this toxic fish showed initial symptoms similar to ciguatera poisoning but then after 5 to 10 days progressed into a second phase which persisted for more than a month, consisting of equilibrium disorders and kinetic tremors. Like CTX, it is lipid soluble and distinguished from it by its chromatographic properties. Likewise, many of its physiological properties are similar and it has been suggested that it might be a metabolite of CTX.

Several attempts have been made to find more reliable methods of detecting the presence of ciguatoxin than the sometimes used cat, mon- goose, or mouse bioassays (Halstead, 1967). A radioimmunoassay (RIA) using CTX-human serum albumin to produce anti-CTX in sheep was developed by Hokama et al. (1977). Testing has shown that this method is relatively accurate but economically feasible only in fish weighing greater than 9 kg (Kimura et al., 1982a). This RIA method shows close correla- tion with mouse bioassay and the in uitro guinea pig atrium assay (Miya- hara et al., 1979), and cross-reacts with okadaic acid, a toxic component of sponge (Kimura et al., 1982b). The mouse bioassay involves injecting a fish extract intraperitoneally into mice and watching symptoms for a 48-hr period. Symptoms have been shown to be dose dependent and include lowered rectal temperature, reduced pain reflexes, reduced grip and tight- rope responses, reduced locomotor activity, and death (Hoffman et al., 1983). An enzyme immunoassay has been developed and is being used in parts of Hawaii to survey fish (Hokama et al., 1983). This enzyme immunoassay is reported to be as sensitive as the radioimmunoassay but is easier to run, economically feasible for screening all sizes and varieties of fish, and could be used for testing liver in addition to flesh samples. The


enzyme assay uses the sheep anti-ciguatoxin used for the radioimmunoassay but instead of iodine employs horseradish peroxidase to couple with the antibody (Hokama et al., 1984).

The symptoms of ciguatera disease begin within minutes of ingestion to 30 hr afterward. As there are no definitive tests used to diagnose ciguatera poisoning, it is made solely from symptomatology and history. It is often differentiated from other types of poisoning by the presence of paresthesia of the extremities. The first symptoms present are usually gastrointestinal, lasting a few hours; followed by neurological disturbances which last up to several weeks or even months (Halstead, 1967). Additional symptoms can be dermatological and neuromuscular (Morris et al., 1982; Lawrence el al., 1980). Ten to 12% of the intoxications lead to death by respiratory paralysis (Rayner, 1972). With severe poisoning, bradycardia was observed and an electrocardiogram showed inverted T waves in some patients, indicating myocardial ischemia, which was reversible upon re- covery (Hanno, 1981). Symptoms have been known to vary in different locations and with ingestion of different fish (Chretien et al . , 1981). An unexplained sensitization has often been observed after ciguatera poisoning and patients often experience the same symptoms of poisoning after the ingestion of nontoxic fish or after alcohol ingestion (Barr, 1982). Ban- ner (1967), however, has suggested that this may be psychosomatic. Treatment mostly involves supportive care, but may include gastric lavage, cathartics, atropine, and protopam chloride (Dembert and Pearn, 1982; Bagnis, 1967). Alkaloids from the plant Duboisia myoporoides have been used by natives of New Caledonia as an antidote, and are thought to contain atropine, nicotine, hyoscyamine, and scopolamine (Dufva et al., 1976).

Ciguatoxin has been shown to have anticholinesterase activity in uitro (Li, 1965) but not in uiuo (Rayner, 1968). Kosaki and Anderson (1968) observed depressed respiration, a fall in arterial pressure, bradycardia, arrhythmias, and neuromuscular responses in rats after administration of lethal doses of ciguatoxin extracted from various fish, and suggested that the toxicity may be due to demyelination of peripheral and central ner- vous tissue. In anesthetized cats, ciguatoxin caused respiratory depression, leading to respiratory arrest with bradycardia. An ECG indicated ventricular extrasystole and idioventricular rhythm (Legrand et al . , 1982).

Setliff et al. (1971) demonstrated that ciguatoxin increases the passive permeability of the cell membrane of frog skin, thus increasing the influx of sodium ions by competitively inhibiting calcium binding at receptor sites which regulate steady state sodium ion permeability. This suggested that possibly ciguatoxin inhibited Na+,K+-ATPase activity. However, when highly purified extrakts of ciguatoxin were used, no inhibition of

ALGAL TOXINS 89

Na+ ,K+-ATPase was seen, indicating that impurities may have had an effect on some previous results (Rayner and Szekerczes, 1973).

A biphasic response is observed on isolated atria from rats and rabbits, with an initial inhibition followed by stimulation of atrial contraction, indicating both cholinergic and adrenergic action and possibly the release of catecholamines (Ohshika, 1971). CTX was later shown to exhibit both positive inotropic and chronotropic effects on the isolated atria of the guinea pig (Miyahara et al., 1979). CTX has also been shown to induce contraction of the guinea pig vas deferens, by releasing stores of nore- pinephrin, which is probably due to the increased permeability to sodium ions (Shizumi et al., 1981).

Possible effects of the ciguatera disease on an unborn infant were observed by Pearn et al. (1982). A cesarean section was performed 2 days after a full-term gravid woman became poisoned with ciguateric fish. The woman experienced fetal movements which began simultaneously with her own symptoms. The infant was born with left-sided facial palsy, but with no heart abnormalities. This suggest that the toxin can cross the placenta, which would not be surprising due to its small molecular size.

F. OTHER DINOFLAGELLATE TOXINS

While several other dinoflagellate genera have some species producing various toxic factors, the main genera other than Protogonyaulax (Go- nyaulax) involved in poisonings are Ptychodiscus (formerly in the genus Gymnodinium) and Dinophysis (Taylor, 1984).

The best known toxin producer in Gymnodinium is the fish killer G . breve. Recently it was transferred to the genus Ptychodiscus by Steidinger (1979). Baden et al. (1984) was able to isolate and purify two toxins from laboratory cultures of P . breuis termed T34 and T17. Both polyether toxins are neurotoxic and ichthyotoxic. One of the toxins also caused bronchioconstriction in anesthetized guinea pigs, which suggests it is responsible for the airborne respiratory irritant in humans associated with these toxic blooms along the Florida coast. The structure of these two “brevetoxins” is given in Fig. 12.

The Dinophysis toxins are collectively referred to as diarrhetic shellfish poisoning (DSP). While the difficulty in culturing these dinoflagellates has slowed definitive studies it is thought that D . fortii and D . acuminata are the main toxic species (Taylor, 1984; Yasumoto et al., 1984). DSP produces gastrointestinal disturbances but not fatalities in humans ingesting various shellfish, i.e., Mytilus, which have concentrated the dinoflagellate. DSP toxins isolated from shellfish in Japan are all polyether-based compounds referred to as okadaic acid, dinophysistoxin, and pecteno-

90 W A Y N E W . CARMICHAEL

a b

Key: a=T34 b=T17

Fig. 12. Structure of brevetoxins, T34 and T17, produced by the Florida red tide dinoflagellate Ptychodiscus brevis (Baden et al., 1984).

okadaic acid : R1=H, R,=H dinophysistoxin- 1 : R, = H, R, = CH, dinophysistoxin- 3 : R, = acyl, R, = CH,

R 47

40

pectenotoxin-1 : R =OH pectenotoxin-2 : R = H

Fig. 13. Structure of polyether lactone toxins involved in cases of diarrhetic shellfish poisoning (DSP). The toxins are produced by the dinoflagellate Dinophysis acuminata found in the shellfish areas of northern Japan (Yasumoto et a / . , 1984).

ALGAL TOXINS 91

toxin (Yasumoto er al., 1984). Figure 13 illustrates the structure of these DSP toxins.

V. ENVIRONMENTAL ROLE OF ALGAL TOXINS

Since so little is known about the ecological or metabolic roles of algal toxins it is important to consider the possible roles in the light of what is known about plant toxins. Since the 1960s evidence has been accumulating to show that vascular plant secondary chemicals have distinct ecological roles (Fraenkel, 1959, 1969). These plant secondary chemicals provide predation protection throughout the growing and dormant seasons. Evi- dence is now accumulating which shows that chemical protection can be regulated and their production quite rapid. For example, leaves of red oak trees defoliated by gypsy moth larvae show higher secondary chemical production in subsequent years (Schultz and Baldwin, 1982). Responses have also been observed in which the length of time for chemical response has been days to months. Time response can be even more rapid, however, as in the studies of Carroll and Hoffman (1980). They found that damaged leaves of Cucurbita moschata mobilize substances to the damaged region within 40 min. There is even some evidence that damaged plants can chemically communicate the need for increased chemical defense to undamaged plants within the nearby area (Baldwin and Schultz, 1983). This indicates the presence of two broad classes for chemical de- fenses of plants: those present before herbivore attack and those that change in response to herbivore attack (Levin, 1976). In these cases of secondary chemical production it has been in response to environmental stress. This stress can come from predation pressures as just presented, or from growth pressures such as would be provided by secondary chemical production in response to low soil nutrients (Janzen, 1974; McKey, 1979; Chew and Rodman, 1979). Central to the concept of an ecological role for secondary chemicals is the concept of cost and benefit for the defense of the organisms.

If these secondary chemicals are serving an ecological rather than a metabolic (i.e., nutrient storage) role, then the energy cost involved in producing them should be less than the protective value obtained from their presence. While hypotheses abound supporting this idea within secondary plant chemicals, there are no careful quantitative models of costs and benefits (McKey, 1979).

With all of these concepts in mind, some points worth considering if algal toxins are to be considered as having an ecological role include the following:

92 W A Y N E W. CARMICHAEL

1, Evidence is accumulating which shows that zooplankton herbivores of algae do respond to the toxins (Snell, 1980; Lambert, 1981; Ransom, 1978; Porter and Orcutt, 1980). What is not clear is just how much, if any, avoidance of toxic cells or filaments occurs relative to nontoxic cells or filaments. If there is a selective advantage to being toxic then it should be possible to show this in laboratory experiments. An important need in the testing of this idea will be the development and use of sensitive assays for toxicity of cells. This would also make it possible to check for increases or changes in toxin production due to predation pressures. Since these algae exist largely as single cells, or in some cases as filaments, there is a need to show chemical communication between the cells if production of toxins as opposed to simple selection of toxic cells is to be demonstrated. For the present it is perhaps too much to envisage that toxin production could be influenced by such mechanisms. At present it is hard to see why many water blooms of cyanobacteria and even dinoflagellates are apparently nontoxic if predation pressures are to have a significant influence on toxin production.

2. What is more tempting to envisage is the possible role of growth conditions on toxin production. This includes the possibility that nutrient composition, pH, temperature, or light may independently or in combina- tion influence toxin production. The algae may in turn produce higher levels or different kinds of toxins in order to minimize predation pressure or to store the toxins for some metabolic need. This seems to be sup- ported by the preliminary work of Hall (1982), who demonstrated higher specific toxin levels per cell in dinoflagellates grown on decreased nitrogen or phosphorus. It also could explain decreases observed in toxin producticin in our laboratory for almost all toxic cyanobacteria when they are grown in nutrient-rich media. This reduction in toxin production is most common among the peptide toxin-producing strains and almost never observed among the alkaloid toxin-producing strains. The decrease in toxin production is also slow, requiring months or more of repeated subculturing before significant reductions are observed. This implies that if toxin production is to be influenced by growth conditions it will take more than a few subcultures. Just as important, however, could be the qualitative changes in toxin production that growth changes could influence.

3. Finally, it has been pointed out that the costs of storing some kinds of secondary compounds are higher than those of others (McKey, 1979). Substances such as tannins and saponins exert their toxic effect by reducing digestion of proteins in the gut of the herbivore. Other toxins such as alkaloids are absorbed from the gut and are active against specific metabolic processes. Rhoades and Cates (1976) argue that the digestion inhibi-

ALGAL TOXINS 93

tors impose higher costs on the plants since they can influence plant processes and thus must be segregated from other cellular components. The more specific metabolic toxins such as alkaloids are effective against herbivores in lower concentrations and are less likely to influence plant metabolism. This perhaps is part of the reason why algae produce the more toxic, more metabolically specific alkaloids, peptides, and related toxins.

Whatever the role for algal toxins, it should be evident that (1) they are diverse groups of physiologically potent secondary chemicals that are more chemically related to higher plant toxins than they are to bacterial or animal toxins; (2) their production should not be considered fortuitous or accidental but rather part of evolutionary processes by which the organisms have adapted to particular needs and requirements of their environment; and (3) studies on production of the toxins including possible environmental and genetic influences will serve to aid an overall under- standing of secondary chemical production.

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