Studies on Aphanizomenon and Microcystis Toxins - ACS Symposium

33

Studies

on

Aphanizomenon

a n d Microcystis

Toxins

1

JOHN J. SASNER, JR., MIYOSHI IKAWA, and THOMAS L. FOXALL

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 6, 2016 | http://pubs.acs.org Publication Date: September 19, 1984 | doi: 10.1021/bk-1984-0262.ch033

Departments of Zoology and Biochemistry, Spaulding Life Science Building, University of New Hampshire, Durham, NH 03824 The aphantoxins from Aphanizomenon flos-aquae (freshwater Cyanobacteria) are s i m i l a r i n chemical c h a r a c t e r i s t i c s and physiological e f f e c t s to p a r a l y t i c s h e l l f i s h poisons from marine sources. Aphantoxin i s o l a t i o n and characterization was done using molecular weight f i l t r a t i o n , solvent separation, column chromatography, electrophoresis, and fluorometry. These methods revealed the presence of the toxic cations neoSTX (90%) and STX (5-10%); and an anionic substance as yet unnamed. The toxins from A. flos-aquae reversibly blocked voltage-dependent Na channels i n crayfish and squid giant axons, s i m i l a r to TTX, STX, and gonyautoxins. Microcystin, from Microcystis aeruginosa, is a family of related peptides which causes hepatomegaly, l i v e r hemorrhaging, destruction of hepatocytes and endothelia, and associated shock in endothermic vertebrates. +

Autotrophic microorganisms are the primary producers which comprise the basis of the food chain and energy budget i n aquatic ecosystems. They u t i l i z e dissolved inorganic nutrients and synthesize new organic materials necessary for the support of animal l i f e at higher trophic levels, i.e., primary, secondary and t e r t i a r y consumers QJ. These primary producers, which play a v i t a l role i n productivity, may also synthesize products (biotoxins) which adversely affect other organisms elsewhere i n the food web. Recent concern about aquatic biotoxins, p a r t i c u l a r l y from microorganisms, stems from an increasing dependency on marine and freshwater environments for food and/or potable water, as a potential source for drugs and other chemicals, and modern applications of genetic engineering (£). There i s an urgent need, therefore, to (a) i d e n t i f y the sources of biotoxins, (b) detect the 7

Current address: Human Nutrition Research Center, New England Medical Center, Boston, M A 02111

0097-6156/ 84/0262-0391 $06.00/ 0 © 1984 American Chemical Society

Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.


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SEAFOOD TOXINS

presence of the toxins i n the food chain, (c) develop accurate and rapid methods f o r the detection, i d e n t i f i c a t i o n , and assay of the toxins, and (d) understand the effects of the toxins on animal systems. These natural products are of more than just academic interest, since they may cause massive animal m o r t a l i t i e s as well as environmental, legal, recreational, and health-related problems. The predominant marine biotoxins are p a r a l y t i c s h e l l f i s h poisons (PSP), tetrodotoxin (TTX), and ciguatera toxin. The l a t t e r two are ichthyosarcotoxins that pose serious health problems i n t r o p i c a l and subtropical countries. Paralytic s h e l l f i s h poisons, on the other hand, are commonly associated with marine molluscs which f i l t e r - f e e d on phytoplankton and act as " b i o l o g i c a l storage depots" for toxin accumulation. Both PSP and ciguatera toxin originate i n several species of d i n o f l a g e l l a t e s — a group of microorganisms that are second i n abundance only to the diatoms as the primary producers i n marine environments (3.)· The predominant freshwater biotoxins are produced by 3 species of cyanobacteria, Aphanizomenon flos-aquae (aphantoxins), Microcystis aeruginosa (microcystin), and Anabaena flos-aquae (anatoxins). Blooms of these organisms have been linked to massive animal k i l l s , including zooplankton, f i s h , l a r v a l amphibians, the poisoning of farm animals, and reduced water q u a l i t y for drinking and recreational uses. Toxicity problems have been reported from a l l of the continents of the world (i). Aphanizomenon flos-aquae and H i c r o c y s t i c aeruginosa occur i n New England, intermittently, i n both toxic and non-toxic forms. Occurrence of Aphanizomenon and Microcystis i n New

Hampshire

Kezar Lake i s a t y p i c a l g l a c i a l ice-scour lake i n North Sutton, N.H. A b r i e f history of t h i s lake i s included here because (a) toxic blooms of Anabaena, Aphanizomenon, and Microcystis have occurred seasonly over the past 20 years, (b) i t has provided aphantoxins and microcystin f o r several research laboratories, (c) i t i s one of r e l a t i v e l y few lakes where extensive physical, chemical, and b i o l o g i c a l data have been collected over extended time periods, and (d) various attempts at experimental corrective measures for c o n t r o l l i n g cyanobacterial blooms have been made here with some success. Kezar Lake covers 182 acres, has a maximum depth of 8.0 meters and an average depth of 3·7 meters. Some 50 years ago, Wadleigh State Park was established on i t s southeastern shore and became a popular recreational area. The town of New London b u i l t a sewage treatment plant i n the early 1930*s and began discharging secondary waste water into Lion Brook, some 5.6 kilometers upstream from the lake. Over time, the secondary effluent from the treatment plant contributed high concentrations of phosphates and nitrates into Lion Brook — the major tributary into the lake. Approximately 70% of the phosphate load i n Kezar Lake was attributed to the effluent from the treatment plant (5.). Cyanobacterial blooms (Anabaena) were f i r s t reported i n the early 1960*s. Water c l a r i t y was reduced, offensive taste and odor occurred, and the f i r s t f i s h m o r t a l i t i e s were noted. Copper sulphate treatment of the lake was successfully accomplished to retard blooms of Anabaena. Within a few years the blooms became



33.

Aphanizomenon and Microcystis Toxins

SASNER ET AL.

393

more intense and included another species, Aphanizomenon. Similar control measures using the algicide were not as e f f e c t i v e on the l a t t e r species and massive f i s h m o r t a l i t i e s resulted — an estimated 11 tons of f i s h i n 1966. The recreational u t i l i t y of Kezar Lake diminished, property values decreased, the N.H. State Tax Commission reduced property values by 30?, and legal action was successfully taken by property owners against the town of New London for remuneration of value lost. In 1968, an attempt was made to a r t i f i c a l l y destratify the lake using on-shore compressors connected to aerators sunk i n the deepest part of the lake. This mixing of the water reduced thermal s t r a t i f i c a t i o n , increased water c l a r i t y , and reduced dense accumulations of cyanobacteria i n the upper surface waters. The positive effects of d e s t r a t i f i c a t i o n were short-lived, however, and were abandoned after a few summers (£). Nutrient stripping and the i n i t i a t i o n of t e r t i a r y treatment at the sewage treatment plant reduced the nutrient discharge levels. However, blooms of Aphanizomenon and Microcystis continued into the mid-1970 s, presumably thriving on residual nutrients. In the summers of the l a t t e r 1970 s and early 1980's, the Aphanizomenon blooms diminished and were replaced by Microcystis blooms. Thus, the recent history of cyanobacterial blooms i n Kezar Lake involved the 3 problem-causing species. In the spring of 1983, approximately 25 acres (14?) of the lake bottom were treated with alum i n an attempt to chelate the nutrients which " f u e l " the i n i t i a t i o n of blooms during the spring turnover. Preliminary observations indicate that the lake improved i n quality, blooms were retarded, water c l a r i t y doubled, and recreation, instead of f i s h mortality, dominated the environment for the f i r s t time i n approximately 20 years. In the mid-1960 s, toxic cyanobacterial blooms i n Kezar Lake, Winnisquam Lake, and Skatutakee Lake i n New Hampshire caused moderate to heavy f i s h m o r t a l i t i e s at these 3 sites. Samples of the dominant microorganisms were i d e n t i f i e d by F. Drouet, Botany Department, Academy of Natural Sciences, Philadelphia, PA and C. M. Palmer, Taft Sanitation Center, Cincinnati, OH, as Aphanizomenon flos-aquae (L.) Ralfs. This species i s also known as A. holsaticum Richt (preferred by Drouet). However, we chose to use Α· f l o s aquae because i t i s more widely noted i n the s c i e n t i f i c l i t e r a t u r e . The e s s e n t i a l l y monospecific blooms of A. flos-aquae (99.5?) occurred as single trichromes (filaments) with 25 to 70 cells/filament. Previous descriptions of this species indicated that filaments were usually bound together i n a "rafted" or f a s c i c u l a t e form. Our samples from these lakes were, apparently, atypical for the species. C e l l dimensions, however, approximate those reported (J) for A flos-aquae c e l l s cultured from the Rideau Canal, Ottawa, Canada i n 1961 and designated NRC 28, 31, and 32. These dimensions were 3 x 5 um for vegetative c e l l s , 5 x 7 urn for heterocysts, and 6 χ 20 um for akinetes. We have not found any fasciculate, toxic blooms of A flos-aquae i n New England. However, both "rafted" and "non-rafted" cultures from Klamath Lake, Oregon were found to be toxic (fL). The t o x i c i t y of Aphanizomenon was f i r m l y established i n 1968 (£). Intermittently, we have sampled almost monospecific blooms of both Aphanizomenon and Microcystis. Samples of M. aeruginosa were f

f

f



394

SEAFOOD TOXINS

collected from several sources. Kezar Lake and Marsh Pond provided both toxic and non-toxic clones which were i d e n t i f i e d by A. Baker, Botany Department, university of New Hampshire. C e l l concentrations generally exceeded 5 x 10 /ml, and during dense blooms were > 10 cells/ml. An e f f e c t i v e method of c o l l e c t i n g large amounts of A. flos-aquae employed DeLaval separators, at lakeside or i n the laboratory, to spin c e l l s from large volumes of water. To c o l l e c t Microcystis i n the f i e l d , p l a s t i c storage containers were used to skim surface colonies. Samples were placed i n large separatory funnels and the f l o a t i n g c e l l masses were concentrated by f l o t a t i o n and removal of water. Crude materials from both species were stored, either wet or lyophilized, i n the f r o z e n state. In order to v e r i f y the s p e c i f i c sources of toxins and ensure large quantities of working material, i t was also necessary to maintain mass cultures of A. flos-aquae and M. aeruginosa. Monospecific cultures of the l a t t e r species (NRC-1, SS-17 strain) was obtained from other laboratories and maintained i n mass culture for several years. Unialgal i s o l a t e s of A. flos-aquae were prepared from Kezar Lake and maintained i n our laboratory from 1968 to 1978. The NH-1 s t r a i n of t h i s species was isolated at Wright State University, by W. Carmichael, i n 1980, from a bloom i n a farm pond near Durham, NH. Laboratory cultures of both species (Α· flos-aquae and Μ· aeruginosa) were grown i n ASM-1 medium (10) buffered with T r i s (pH 8.2) and containing 1% s o i l extract. Test tube cultures were expanded to 20 1 glass carboys, then to 160 1 p l a s t i c tanks. In mass culture the s o i l extract was omitted. A l l cultures were maintained under constant i l l u m i n a t i o n (ca. 4000 f.c.) at 21 ± 2°C. They were aerated with a i r passing through s t e r i l e cotton f i l t e r s . Cultures were harvested after approximately 2 weeks, using a continuous flow DeLaval Separator, as with the samples from natural blooms. The c e l l s were stored frozen as wet or l y o p h i l i z e d samples. Periodic mouse bioassays were used to check on the potency of the material. Cyanobacterial samples retained potency under these conditions for several years. Physiological E f f e c t s - A p h a n t o x i n s The e f f e c t s displayed by f i s h and mammals challenged with aphantoxins were q u a l i t a t i v e l y s i m i l a r to symptoms reported f o r p a r a l y t i c s h e l l f i s h poisons from marine sources. In mice, the c h a r a c t e r i s t i c symptoms include coordination loss, i r r e g u l a r v e n t i l a t i o n , spastic twitching, gaping mouth, and death by respiratory f a i l u r e . Plankton samples collected during or shortly after A. flos-aquae blooms were usually devoid of common zooplankters. In the laboratory, Daphnia magna were affected when placed i n aerated cultures or media containing extracts of Α· f l o s aquae. Within a short time, the c h a r a c t e r i s t i c movements of the second antennae were e r r a t i c or stopped, causing the animals to s e t t l e to the bottom. Daphnia removed to clean water recovered while those retained i n toxin-containing media perished i n from 2 to 24 hours. Other species among the primary consumers are also sensitive to the presence of aphantoxins (&, 11). The freshwater bivalves, E l l i p t i o camplanatus and Corbicula fluminea, stored small



33. SASNER ET AL.


395

amounts of toxin after feeding on A. flos-aquae cultures. Mouse bioassay of entire bivalve meat produced symptoms s i m i l a r to those described for c e l l extracts or PSP. However, the bivalves stored approximately only 40 to 60 micrograms of toxin (STX equivalents) per 100 grams of meat before they were affected by the aphantoxins. E a r l i e r work (12.) showed that nerve preparations from E l l i p t i o were more sensitive to STX and TTX than nerves from several marine bivalves. We have fed marine mussels (Mytilus edulis) on concentrated c e l l masses from laboratory cultures of A. flos-aquae and found s i g n i f i c a n t l y higher toxin accumulations (200 to 300 micrograms/100 grams meat) than occurred i n the freshwater bivalve tissues. The freshwater f i l t e r feeders displayed a f l a c c i d paralysis of the foot and mantle after 2-3 days i n A. flos-aquae cultures, which was reversed upon return to non-toxic water. Isolated tissue and c e l l preparations were used to determine the s i t e and mode of action of the toxin. Lyophilized Aphanizomenon c e l l s were suspended i n d i s t i l l e d water, sonicated, and centrifuged to remove particulate material. Molecular weight f i l t e r s were used to "clean up" the crude material. The supernatant was passed through a 10 Κ dalton M i l l i p o r e f i l t e r , a 500 dalton Amicon f i l t e r , and the f i n a l f i l t r a t e lyophilized. The l a t t e r was reconstituted i n appropriate Ringers or a r t i f i c i a l seawater for testing on giant axons of crayfish (Cambarus sp.) or the squid (Loligo pealiei). The c r a y f i s h ventral nerve cords were excised, the medial and l a t e r a l giant axons p a r t i a l l y desheathed. and the preparations placed i n a chamber divided into 3 (1 cnH) sections. Each section was separated by petroleum j e l l y . Experiments were performed at room temperature (20-22°C). The c r a y f i s h Ringers was composed of ( i n mM/1) NaCl (146); KC1 (4); C a C l (8); M g C l (4); and T r i s maleate buffer, pH 4.0, (10 mM/1). In one series of experiments, stimulation of the axons occurred i n the f i r s t section of the chamber, perfusion i n the second, and i n t r a c e l l u l a r recording was from the t h i r d section. Aphantoxins (0.4 to 10 ug/ml) reversibly blocked the conduction of action potentials i n the crayfish giant axons. Following the addition of toxin, the latency between the stimulus, and action potential increased. When t h i s latency period increased by 1 msec (25 to 30$), then conduction was blocked. The block time, after addition of toxin, and the recovery time, after Ringer wash, was dose-dependent. In other experiments, bathing solutions containing aphantoxin, STX, and TTX were adjusted to give s i m i l a r and reproducible block times. The subsequent recovery times after Ringer wash was measured (to 90$ control). The recovery times f o r aphantoxin and STX blocked axons were s i m i l a r and s i g n i f i c a n t l y shorter than for TTX, Table I. 2

Table I. Comparative R e v e r s i b i l i t y :

Toxin Aphantoxin STX TTX

No. of Preos 5 5 5

Conc.c/ml 4.0 χ 10"^ 3.5 x 10"° 2.5 x 10"°

2

Aphantoxin versus STX and TTX Block Time (sec.) 20 + 2 28 ± 8 24 + 5

Recovery Time (sec.) 115 + 20 95 + 19 257 + 34



396

SEAFOOD TOXINS

The r e l a t i v e recovery times after Ringer wash suggest that aphantoxins acted more l i k e STX than TTX i n c r a y f i s h l a t e r a l and medial giant axons (11, 1λ). In order to examine the e f f e c t of aphantoxin on the generation of the action potential and the membrane resistance, i n t r a c e l l u l a r recordings were obtained from desheathed nerve cords. When the toxin (0.8 ug/ml) and recording microelectrode were placed i n the same chamber, the spike amplitude gradually decreased to t o t a l block i n 25 min. The r i s e time from baseline to peak amplitude increased 3 to 5 f o l d before complete block occurred, and the slope of the f a l l i n g or recovery phase remained unchanged. In Figure I, for example, the r i s e time increased from 0.5 msec to 1.8 msec, while the f a l l i n g phases were approximately 1.3 msec i n a l l traces. Toxin a p p l i c a t i o n did not a l t e r the transmembrane r e s t i n g potential (-80 mV) or the membrane resistance (11, 12). The Na dependency of the crayfish preparations was v e r i f i e d by challenging them with Na -free Ringers (choline chloride substitution). Records s i m i l a r to Figure I, with toxin were obtained, showing r e v e r s i b l e blocking of action potential generation. On the other hand, calcium-free Ringers only s l i g h t l y affected the waveform and the subsequent addition of toxin decreased the r i s e rate, just as i n the normal Ringers with toxin. The r i s i n g or depolarizing phase of the action potential i s associated with a transient increase i n Na conductance (14). The data shows that aphantoxins blocked the Na dependent, depolarizing phase without affecting the K -dependent r e p o l a r i z a t i o n phase. +

+

+

+

+

V e r i f i c a t i o n of t h i s i n t e r p r e t a t i o n came from voltage-clamp studies using squid giant axons. Aphantoxin samples were prepared by molecular weight separation, using u l t r a f i l t r a t i o n membranes, as described above for the extracts used on crayfish. Squid axons were voltage clamped to a series of depolarizing potentials and the corresponding membrane currents were recorded. The bathing medium was changed to aphantoxin/seawater solutions and the series of depolarizing potentials was repeated (Figure II). The voltageclamp data shows aphantoxins are very potent and s p e c i f i c i n h i b i t o r s of voltage-de pendent Na channels i n the squid axon. There was no effect on the K* conductance, and the blocking of e x c i t a b i l i t y was completely reversible. The dose-response r e l a t i o n s h i p of the transient peak current was plotted as a function of toxin concentration, expressed as STX equivalents i n a r t i f i c i a l seawater. The data points f e l l on a curve which was representative of 1:1 stoichiometry (15). The d i s s o c i a t i o n constant from this r e l a t i o n s h i p indicated that 3.47 nM aphantoxin (as STX equivalents) blocks 50? of the membranes Na channels i n the squid axon (16). Aphantoxin was found to contain Neo-STX and STX i n an approximate r a t i o of 9:1 (17). These two toxic components were found to be equipotent when tested on r i s e rate of action potentials and current-voltage r e l a t i o n s i n amphibian sartorius muscle f i b e r s (18). The equipotency of STX and Neo-STX was also determined i n voltage-clamped squid giant axons (19). The s p e c i f i c i t y of Neo-STX f o r the Na channel was established, as w e l l as the i d e n t i f i c a t i o n of the 7,8,9 guanidinium moeity as the active group involved i n binding/blocking the channels (18). +

1

+

+



33. SASNER ET AL.

Figure I.


397

Action Potentials from crayfish giant axons. Control = Top Trace. Toxin (0.8 ug/ml) reduced r i s e rate and amplitude after 5, 10, 15, 20, and 25 mln. Reversible. Stlm. = 2 V; 0.25 msec pulse width, (from Sasner et a I., 1981)

cm

I— 2 msec

Figure II.

Membrane currents In voltage-clamped squid axons. Current traces from holding potentials of -60, -30, 20, -10, and 0 mV In (A), (C), and (E). (A) = c o n t r o l ; (C) = 30 η M aphantoxin (STX e q u î v . ) ; (E) = recovery a f t e r wash (10 mln). (B), (D), and (F) = 20, and 40 mV. (B) = c o n t r o l ; (D) = t o x i n ; (F) = recovery. Temp. = 6.2°C. Reproduced with permission from Ref. 16. Copyright 1982, Pergamon Press, Ltd.


SEAFOOD TOXINS

398


Physiological E f f e c t s - Microcystin Three d i f f e r e n t toxins have been associated with Microcystis aeruginosa. The most common of these i s a fast death factor (FDF, 1 hr), which i s a family of related peptides c o l l e c t i v e l y c a l l e d microcystin. A slow death factor (SDF, 24-48 hrs) may also be present i n some strains, as w e l l as a diarrheagenic factor (DF). The l a t t e r 2 originate i n bacteria co-existing with the Microcystis (20-22), and are not always present or measurable by bioassay i n the presence of microcystin. The complex of peptides (microcystin) are secondary metabolites (23) which mainly affect endothermic vertebrates (birds and mammals) a f t e r ingestion of toxin by drinking from waterblooms. Ectothermic vertebrates ( f i s h and amphibians) and several invertebrates were not affected by c e l l extracts (i.p. i n j e c t i o n or immersion) (24). When small mammals were challenged (i.p.) with reconstituted l y o p h i l i z e d c e l l u l a r material (50 mg/Kg), s u r v i v a l times were aproximately 1 hour. Characteristic symptoms included p a l l o r of the extremities, lethargy, labored v e n t i l a t i o n and non-violent death. A h i s t o l o g i c a l survey of tissues from the major organ systems of mice revealed d r a s t i c changes only i n the l i v e r . Hepatocyte degeneration and necrosis progressed outward from the c e n t r i l o b u l a r regions i n poisoned animals. Hepatocytes became progressively swollen over time, and showed a cloudy, e o s i n o p h i l i c cytoplasm. Hepatic sinusoids became distended, c e l l s ruptured, the i n t e g r i t y of the parenchymal cord structure was disrupted, and massive hemorrhaging into the l i v e r was routinely observed (Figure III). The progressive l i v e r damage was examined at the u l t r a s t r u c t u r a l l e v e l over the course of 1 hour after i.p. treatment, with a l e t h a l dose of microcystin (50 mg/Kg). After 10 minutes, membrane fragments were noted i n the sinusoids. Within 20 minutes, b i l e c a n a l i c u l i and m i c r o v i l l i i n the space of Disse were distorted, and endothelia were either swollen or fragmented. Within 40 minutes, hepatocyte disruption was seen i n c e n t r i l o b u l a r regions, and the pooling of RBC s and c e l l organelles characterized the d i s i n t e g r a t i o n of l i v e r structure and the massive hemorrhaging associated with microcystin poisoning. Within 60 minutes, c e l l degeneration and necrosis was extensive i n the c e n t r i l o b u l a r regions and extended outward into the parenchymal cords, (24, £fl). Table II i l l u s t r a t e s the weight and volume changes i n mouse l i v e r at 60 minutes post i n j e c t i o n with l y o p h i l i z e d samples of microcystin (50 mg/Kg). Using the F test for significance, (a) no s i g n i f i c a n t difference was observed between control and experimental body weights and (b) a highly s i g n i f i c a n t difference was noted between control and experimental l i v e r weights a f t e r 1 hour (99$ confidence interval). In adult mice, the t o t a l c i r c u l a t i n g blood volume i s estimated at 77.8 to 86.4 cc/Kg body weight (26). For the 20 gram animals used i n our study, t h i s calculates to 1.6 to 1.7 cc blood/animal. The increase i n l i v e r weight and volume and the observed hemorrhaging over time suggests a 36$ decrease i n c i r c u l a t i n g blood volume. The 70$ increase i n l i v e r volume also must be viewed as a s i g n i f i c a n t decrease i n c i r c u l a t i n g blood volume leading to cardiovascular shock. f



399


33. SASNER ET AL.

Figure

ill.

Scanning Electron Micrographs of Mouse Liver. A = Control (440X); Β = 30 min. after microcystin (790X). Note RBC's and damaged hepatocytes.


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SEAFOOD TOXINS

Table II.

Mouse L i v e r Weight and Volume Changes a f t e r Microcystin


Ν Controls (X) 15 Expérimentais (X) 15 Percent Change —

Body Wt. (β) 20.66 19.28

Liver Wt. (z) 1.05 1.51

$ Liver/ Bodv Wt. 5.02

Liver Vol. fee) 0.86

7.77

+44$

+55$

1.47 +71$

Other workers (27) measured a 48$ increase i n hepatic blood volume within 45 minutes post i n j e c t i o n with microcystin. This i s consistent with our estimates of the extensive l i v e r damage. Blood pressure data from rats treated with l e t h a l doses of microcystin (50 to 200 mg/Kg body weight) (2&) f e l l to l e s s than h a l f the i n i t i a l or control pressure within 45 minutes. In addition, concentrations of l i v e r enzymes increased markedly i n the blood, thereby lending chemical support to the anatomical observations (29). The current h i s t o l o g i c a l , u l t r a s t r u c t u r a l , blood pressure, and blood chemistry information supports the view that extensive and rapid l i v e r damage with massive hemorrhaging i n t o t h i s organ i s symptomatic of microcystin poisoning. Other recent work (30) does not agree with t h i s i n t e r p r e t a t i o n and suggests that the toxin causes pulmonary p l a t e l e t thrombi formation. This would lead to hypoxemia, heart f a i l u r e and shock, and the l i v e r damage described above i s viewed as a secondary e f f e c t after a cascade of pulmonary and cardiovascular anomalies triggered by the p l a t e l e t thrombi. To resolve this controversy, c a r e f u l l y controlled experiments must be done to e s t a b l i s h the s p e c i f i c timing of events a f t e r poisoning. It i s important to resolve whether primary s t r u c t u r a l damage i n the l i v e r causes subsequent lung and c i r c u l a t o r y e f f e c t s or whether primary pulmonary blockage i s the cause of the c i r c u l a t o r y e f f e c t s and l i v e r damage. Chemistry - Aphantoxins The physiological e f f e c t s of aphantoxins from toxic Aphanizomenon noted above, were s i m i l a r to those described f o r p a r a l y t i c s h e l l f i s h poisons (PSP) from the marine d i n o f l a g e l l a t e , Gonyaulax tamarensis. In addition, certain chemical s i m i l a r i t i e s were also found early, i.e., IR spectra and color reactions, using stored materials from Kezar Lake (3JJ. Subsequent chemical data suggested the presence of 4 toxic components from A. flos-aquae — one of which was saxitoxin (STX) Π 2 ) . Using stored c e l l samples collected from natural blooms and laboratory cultures, we have studied the chemical properties, methods of detection and quantitation, and s t a b i l i t y of aphantoxins. This work was a "spin off" from our i n i t i a l goal of developing chromatographic and fluorometric procedures for the detection, quantitation, and i s o l a t i o n of STX-related toxins from crude extracts of marine b i v a l v e t i s s u e s (33.-3ÊJ. Weak cation exchange resins (carboxylic acid type) have been widely employed for the i s o l a t i o n and detection of marine PSP's. However, the l e s s basic gonyautoxins (GTX's) tended to elute with the solvent front with these resins — e s p e c i a l l y when very crude f



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extracts were used. We currently use strong cation exchange resins (sulfonic acid type) for the separation of the toxic components i n PSP and Aphanizomenon (17, 3 D · The fluorescence generated on oxidation of the STX group of toxins (STX, GTX2, and GTX3) was used for t h e i r detection. Unfortunately, the neosaxitoxin (neo-STX) group (neo-STX, GTX1, and GTX4) did not fluoresce, under the conditions of the tube assay, and was routinely estimated using the standard mouse bioassay. Figure (IV) shows the fluorescence and t o x i c i t y p r o f i l e s of Α· flos-aquae extracts from Kezar lake. The p r o f i l e s show a close c o r r e l a t i o n between t o x i c i t y and fluorescence. The early eluting peak i s of unknown nature while the l a t e r peak was coincident with STX standards. The early peak suggested the possible presence of gonyautoxins which were routinely found i n marine s h e l l f i s h samples contaminated with Gpnyaulax tamar-ensjs (Figure V B). However, hot acid treatment (0.02 M HC1) of the A. flos-aquae extract caused t h i s early peak to disappear. Similar acid treatment of the gonyautoxins from Mytilus showed them to be more stable i n acid than the Kezar aphantoxin. The r e s u l t s showed that the earlyeluting peak from Kezar lake samples was very l a b i l e , but not very potent and, therefore, d i f f e r e n t from gonyautoxins II and III (38). It i s possible that t h i s early eluting peak could be a c r y p t i c form of PSP such as B-^ (3SJ> which i s r e l a t i v e l y non-toxic but highly fluorescing, and which y i e l d s STX on mild acid treatment. However, no p a r t i c u l a r increase i n STX was observed upon acid treatment. The toxin p r o f i l e was d i f f e r e n t for the Kezar Lake and NH-1 strains or clones of A. flos-aquae (Figures IV and V A). When compared with e l u t i o n p r o f i l e s from Mytilus. the NH-1 s t r a i n showed only one fluorescent toxic peak (fractions 70 to 80) which corresponded to the Mvtilus peak for STX, and a non-fluoréscent but toxic peak (fractions 60 to 70) which corresponded to neo-STX (Figure V A & B). Samples of the NH-1 s t r a i n of Α· flos-aquae were p u r i f i e d on a Bio-Gel P-2 column (Bio Rad Laboratories). The c e l l extracts were pre-treated with ammonium sulphate and/or molecular f i l t r a t i o n (10 Κ dalton, M i l l i p o r e Corp.) to remove i n t e r f e r i n g proteins and pigments. The mobile phase of the P-2 column was changed from dist. H 0 to 0.1 M acetic acid prior to e l u t i o n of the second peak. The t o x i c i t y p r o f i l e (Figure VI) from the P-2 column procedure, showed an early, toxic, water eluting peak, and a larger (4X) l a t e r peak that eluted with the acid fractions. Fractions representing the larger (acid) peak were pooled and chromatographed on a Bio-Rex 70 (H+) column (0.8 χ 60 cm) using a l i n e a r acetic acid gradient. The gradient was 0.0-0.3 M acetic acid to tube 40, and 0.3-1.3 M acetic acid to tube 80. The presence of gonyautoxins i n A. f l o s aquae NH-1 was not detected, since no measurable t o x i c i t y or fluorescence was seen at low acetic acid concentrations (Figure VII). At higher acetic acid concentrations, a toxic but nonfluorescent peak immediately preceeded a toxic fluorescent peak. This p r o f i l e was s i m i l a r to the neo-STX - STX pair reported for scallop extracts (40). The early, toxic, water-eluted peak from the P-2 column (Figure VI) showed the presence of two new toxic components i n the Α· flos-aquae, NH-1 strain. These compounds behaved as anionic substances on electrophoresis at pH 8.6 (JUL). 2


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Studies on Aphanizomenon and Microcystis Toxins - ACS Symposium

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