32
Toxins from Freshwater Cyanobacteria
WAYNE W. CARMICHAEL and NIK A. MAHMOOD
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Department of Biological Sciences and Biomedical Sciences Program, Wright State University, Dayton, OH 45435 Marine and freshwater cyanobacteria produce exotoxins. The main genera responsible for freshwater toxic blooms are Microcystis, Anabaena, Aphanizomenon and O s c i l l a t o r i a . Toxins produced include: 1. anatoxins, alkaloids and peptides of Anabaena; 2. the peptide microcystin and r e lated peptides of Microcystis; 3. aphantoxins, compounds of Aphanizomenon with properties similar to some paral y t i c s h e l l f i s h poisons. Properties of O s c i l l a t o r i a toxin suggest they are peptides s i m i l a r to those of Microcystis. Microcystis toxins are peptides (M.W. approx. 1200) which contain three invariant D-amino acids, alanine, erythro-ß-methyl aspartic and glutamic acids, two variant L-amino acids, N-methyl dehydroalanine and a βamino acid. Individual toxic strains have one or more multiples of this peptide toxin. The one anatoxin charac terized i s a b i c y l i c secondary amine c a l l e d anatoxin-a (M.W.165).The aphantoxin isolated i n our laboratory contains two main toxic f r a c t i o n s . On TLC and HPLC the fractions have the same c h a r a c t e r i s t i c s as saxitoxin and neosaxitoxin. Toxins are produced by several planktonic bloom forming genera of freshwater cyanobacteria. The toxins are primarily a threat to domestic and wild animals which drink from the shores of ponds, lakes and other waters where the surface accumulation of the bloom allows a l o c a l i z e d concentration of toxin both i n the cyanobacteria c e l l s and i n the surrounding water. These surface accumulations of toxins and c e l l s are a p a r t i c u l a r concern i n l a t e spring, summer and f a l l i n the eutrophic water bodies of both southern and north ern temperate l a t i t u d e s . Toxic cyanobacteria were recognized about 100 years ago (1) following death of domestic animals drinking surface blooms containing toxic c e l l s . Since that time research has focused on occurrence and d i s t r i b u t i o n of toxic blooms i n r e l a t i o n to animal death. Direct human contact with toxic blooms has been rare and the present threat to humans i s through drinking water supplies (_2), recreational water (3) and the increasing use 0097-6156/ 84/0262-0377$06.00/0 © 1984 American Chemical Society
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
SEAFOOD TOXINS
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of cyanobacteria as a source of single c e l l protein (4). Since cyanobacteria toxins have fewer vectors than other phyco or mycotoxins, by which they can enter the human body they have not received the same amount of research e f f o r t . While they are not considered to be seafood toxins there are several points of s i m i l a r i t y making i t useful to consider them along with these other toxins. These include: 1) Both seafood toxins and cyanobacteria toxins are waterbased diseases. 2) Both groups of toxins are produced by phytoplankton. 3) While retained within the c e l l s to varying degrees both groups are exotoxins. 4) The toxins produced are fast acting neuro or organ toxins absorbed v i a the oral route. 5) Certain cyanobacteria toxins have structural/functional s i m i l a r i t i e s to certain p a r a l y t i c s h e l l f i s h toxins, especially saxitoxin and neosaxitoxin. Types of Toxic
Cyanobacteria
Toxic cyanobacteria are u n i c e l l u l a r or filamentous i n morphology. Early researchers reported about nine genera of cyanobacteria considered responsible for toxic water blooms (.5,6). The l i s t of confirmed toxic genera, as v e r i f i e d by laboratory culture of toxic i s o l a t e s i s not as large. Toxic strains of Anabaena flos-aquae, Aphanizomenon flos-aquae, Microcystis aeruginosa, O s c i l l a t o r i a agardhii and O s c i l l a t o r i a rubescens are maintained i n laboratory culture c o l l e c t i o n s (7^90% of the toxic a c t i v i t y . The two addi t i o n a l amino acids present occurred as pairs consisting of: 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 pre sent i n nine of the thirteen toxic i s o l a t e s . Botes, et a l . (14) i s o l a t e d and analyzed two toxic Microcystis from South A f r i c a . A l l toxic monomers were pentapeptides with one residue each of D-glutamic acid, D-alanine and erythro-3-methyl aspartic acid. Methylamine was also present and thought to be a hydrolytic breakdown product of N-methyl dehydroalanine (15). One of the Microcystis i s o l a t e s cultured i n the laboratory contained four toxic monomers labeled BE-2 to BE-5. Configuration assign ments of the amino acids present confirmed that the invariant amino acids were a l l i n the D form while the variant amino acids were i n the L form. The variant L-amino acids for the monomers were Arg and Leu for BE-2, Arg and Tyr for BE-3, Ala and Leu for BE-4 and Ala and Tyr for BE-5 (15). The Australian toxin (12) had the same i n variant amino acids and L-Met and Tyr for the variant amino acids. N-methyldehydroalanine was also found i n the BE-2 to BE-5 monomers but was not described for the Australian toxin. An apolar side chain of about 20 C-atoms was present i n BE-2 to BE-5 but was not reported i n the Australian toxin. The N-methyldehydroalanine and the 20-carbon side chain were postulated as contributing to the high hydrophobicity of the toxin and suggests a possible mechanism of action since i t should have a good a f f i n i t y for c e l l membranes. In a l a t e r publication (16) the BE-4 monomer was analyzed by fast atom bombardment mass spectrometry. The authors concluded that the toxin has a molecular weight of 909 with an estimated molecular weight for the 20-carbon side chain of 313. I t was also f e l t that the toxin was not a l i n e a r peptide as reported e a r l i e r (14,15) but a monocyclic peptide. The 20-carbon side chain was thought to be a novel β-amino acid. Other Peptide Toxin Producing Cyanobacteria. No other cyanobacteria genera have been analyzed for the presence of toxic peptides. Based on t o x i c i t y signs however i t i s thought that peptide toxins are present i n c e r t a i n i s o l a t e s of Anabaena flos-aquae (7) and O s c i l l a t o r i a agardhii (9,17). A l k a l o i d Toxins. Only one a l k a l o i d toxin has been chemically de fined from the cyanobacteria. This i s the secondary amine, 2-acetyl9-azabicyclo (4-2-1) non-2-ene, c a l l e d anatoxin-a. I t i s i s o l a t e d from the filamentous s t r a i n Anabaena flos-aquae NRC-44-1 (18,19).
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
SEAFOOD TOXINS
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Synthesis of antx-a has been done through a ring expansion of co caine (20), from 1, 5-cyclooctadiene (21) and by intramolecular c y c l i z a t i o n between an iminium s a l t and a nucleophilic carbon atom (22). The structure of antx-a i s given i n F i g . 1. Two other suspected a l k a l o i d producing cyanobacteria s t r a i n s , Anabaena flos-aquae NRC-525-17 and Aphanizomenon flos-aquae NH-5, are now being studied. The toxin of An. flos-aquae NRC-525-17 (anatoxin-a(s)) i s thought to have CNS stimulating properties (7) and that of Aph. flos-aquae NH-5 (aphantoxin) i s thought to proHuce the p a r a l y t i c s h e l l f i s h poisons saxitoxin and neosaxitoxin (Fig. 1) (7,23). E a r l i e r research had already suggested that certain blooms °f Aph. flos-aquae could produce p a r a l y t i c s h e l l f i s h poisons. These studies used water blooms collected from Kezar Lake, New Hampshire (25,30). In 1980 Carmichael isolated a neurotoxic s t r a i n of Aph. flos-aquae from a small pond i n New Hampshire. These strains have also been shown to produce toxins s i m i l a r to saxitoxin and neo saxitoxin (23) and are the ones used i n the studies presented here. In our laboratory crude preparations of aphantoxins and anatoxin-a(s) are extracted s i m i l a r l y except at the f i n a l stages of p u r i f i c a t i o n (Fig. 2). A Bio-gel P-2 column (2.2 χ 80 cm) i s used for aphantoxins gel f i l t r a t i o n and a Sephadex G-15 (2.6 χ 42 cm) column for anat o x i n - i s ) . Both toxins are eluted with 0.1 M acetic acid at 1.5 ml/ min. Fractions of aphantoxins from Bio-gel P-2 run are spotted on thin-layer chromatography plates ( S i l i c a gel-60, EM reagents) and developed according to Buckley et a l . (1976) (31). The Rf values for the aphantoxins, saxitoxin and neosaxitoxin standards (Table 1) indicates that two of the aphantoxins ( i . e . I and II) are similar to saxitoxin and neosaxitoxin. The f i r s t toxic peak(s) from the Bio-gel P-2 run (volume r e duced to 1 ml) i s passed through a preparative mini column (Sep-pak C^g) to separate the toxin(s) from yellowish pigments. The eluant i s then subjected to HPLC using a semipreparative column (CN bonded phase 9.4 mm χ 25 cm). F i g . 3 i l l u s t r a t e s the presence of neo saxitoxin (second l a s t peak) and saxitoxin (last peak). A t o t a l of 150 mu (mouse units) was loaded. The p r o f i l e i n F i g . 4 shows only neosaxitoxin (500 mu) i s present because only a portion of the f i r s t toxic peak(s) from the P-2 run was injected. The bottom p r o f i l e i s that of a standard neosaxitoxin (200 mu). The HPLC elution pattern of anatoxin-s(s) i s shown i n F i g . 5 and i s the l a s t peak i n the p r o f i l e . Determination of LD_ gave an approximate value of 40 ug/kg body weight, which i s f i v e times more potent than anatoxin-a. n
Types of Toxic Effects Organtoxic. Of a l l the cyanobacteria toxins, those of the c o l o n i a l Microcystis aeruginosa are the most widespread. They have caused the death of livestock and wild animals i n several countries of the world. While reports on t o x i c i t y of Microcystis have some con f l i c t i n g signs of poisoning the consistent pathological findings i n clude swollen blood-engorged l i v e r with hemorrhagic necrosis and mildly edematous lungs. The v a r i a t i o n i n other signs, such as sur v i v a l time, can be attributed to the dosage, animal species, age and condition of the bloom, presence of other toxic or nontoxic cyano-
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
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32.
CARMICHAEL AND MAHMOOD
Freshwater
anatoxin - a hydrochloride
Cyanobacteria
Toxins
R=H; saxitoxin dihydrochloride R=OH; neosaxitoxin dihydrochloride
F i g u r e 1. S t r u c t u r e of a n a t o x i n - a from Anabaena f l o s - a q u a e NRC44-1, s a x i t o x i n and n e o s a x i t o x i n . S a x i t o x i n and n e o s a x i t o x i n i s produced by c e r t a i n s p e c i e s o f marine a l g a e and by the f r e s h water c y a n o b a c t e r i a Aphanizomenon f l o s - a q u a e NH-5. Freeze Dried Cells (1-2 gm)
I
Acidified Water pH 3.0 (50-100 ml) Sonicate 5' I Centrifuge 4750 g 30'
Repeat 3x
ι Supernatent Volume Reduce (5 ml)
ι Acidified Ethanol pH 3.0 (20-40 ml) I Centrifuge 4750 g 5'
ι Supernatant
I Ultrafiltration 1. Amicon YM-5 Membrane 2. Millipore .45μ Filter I Gel Filtration 1. Anatoxin-A(s) (Sephadex G-15) 2. Aphantoxins (Bio-Gel P-2) Sep-Pak C
1 8
(Sample Prep)
Preparative Η PLC
Extraction and Purification of Anatoxin A-(s) and Aphantoxins F i g u r e 2. Flow diagram f o r the e x t r a c t i o n o f A n a t o x i n - a ( s ) and Aphantoxins.
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
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382
SEAFOOD TOXINS
TABLE 1.
Thin-layer chromatography of Aphanizomenon flos-aquae NH-5 fractions from Bio-gel P-2 gel f i l t r a t i o n
Fractions Aphantoxin I Aphantoxin H , Aphantoxin III Aphantoxin IV Saxitoxin > Neosaxitoxin > a
a
b
a
c
3
c
Flourescence Yellow-green Blue Blue Blue Blue Yellow-green
RF .74 .69 .62 .77 .68 .73
Note: The solvent was pyridine:ethyl acetate:acetic acid:water (75:25:15:30) (24) +
a
Spots are detected by spraying with 1% Hydrogen peroxide. 110°C 15 min. and observed under 366 nm UV. LD
5 Q
Heated
i . p . mouse; 10 yg/kg
Provided by Dr. Sherwood H a l l , Woods Hole Océanographie I n s t i t u t e . b
P o s s i b l y Aphantoxin III i s the B toxin of H a l l , 1980 Aphantoxin IV i s gonyautoxin II of Alam, 1978 (27). ±
(24); and
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
Freshwater
CARMICHAEL AND MAHMOOD
Cyanobacteria
Toxins
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Conditions: Column:
Zorbax CN 9.4 mm χ 25 cm
Sample:
Aphantoxins - Most Toxic Peaks (Bio-Gel P-2) 80/20 10mM N H H P 0 Methanol 4
2
4
Flow Rate:
1 ml/min
Detection:
Beckman 165 UV @ 220 nm 0.1 AUFS
1
i 50
i
40
i 10
— ι i
0
Minutes
F i g u r e 3. HPLC p r o f i l e o f A p h a n t o x i n most t o x i c peak ( l e f t ; corresponds t o s a x i t o x i n ) and the major t o x i c component (secom from l e f t ; corresponds t o n e o s a x i t o x i n ) . A t o t a l of 150 mouse u n i t s were loaded. A mouse u n i t i s e q u a l t o the amount of t o x i n needed t o k i l l a 20 g mouse i n 15 min.
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
384
SEAFOOD TOXINS
Conditions: Column:
Zorbax CN
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9 4 mm χ 25 cm
Minutes
F i g u r e 4. HPLC p r o f i l e of A p h a n t o x i n major peak ( t o p - l e f t ; 500 mouse u n i t s ) and n e o s a x a t o x i n s t a n d a r d (24) ( b o t t o m - l e f t ; 200 mouse u n i t s ) .
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
32.
Freshwater
CARMICHAEL AND MAHMOOD
Cyanobacteria
Toxins
385
Conditions: Column:
Zorbax CN
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9.4 mm χ 25 cm Sample:
Anatoxin - A(S)
Eluant:
90/10
50 mM
CH COONH 3
Flow Rate:
2 ml/min
Detection:
Beckman
4
/ Methanol
165 U V @
220 nm 0.1 AUFS
F i g u r e 5. H P L C p r o f i l e of A n a t o x i n - a ( s ) ; t o x i c peak ( f a r - l e f t ; 2 0 0 mouse u n i t s ) . (LD^Q i . p . mouse equal t o 4 0 ug/kg body w e i g h t ) .
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
SEAFOOD TOXINS
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386
bacteria and amounts of toxin(s) outside the c e l l s . Of special im portance i s the condition of the c e l l s within the bloom and the bloom concentration since the peptides of Microcystis are retained l a r g e l y within the c e l l s u n t i l c e l l l y s i s , either i n the water or i n the animals, has occurred. This leakage of toxin into the water supply means that toxins can be expected to be present even i n finished water when a toxic bloom i s present. In laboratory studies rats and mice injected i v with toxin extracts or i p with toxic c e l l s or extracts die within 1/2 to 3 hours. Death i s preceded by p a l l o r and prostration, with terminal episodes of unprovoked leaping and twitching. Upon necropsy, the animals show grossly enlarged l i v e r s engorged with blood, with the remainder of the carcass being exsanguinated. Liver weight i s i n creased and at death composes about 9 to 10 percent of body weight of mice as opposed to about 5 percent i n controls (32,33). The blood content of l i v e r s of mice poisoned by Microcystis increases from 6.6 ml blood/100 g l i v e r i n controls to 53.4 ml blood/100 g f o r mice k i l l e d 45 min a f t e r toxin i n j e c t i o n (33). H i s t o l o g i c a l examination of the l i v e r reveals extensive cent r i l o b u l a r necrosis with loss of c h a r a c t e r i s t i c architecture of the hepatic cords. TEM examination indicated that both hepatocytes and hepatic endothelial c e l l s were destroyed. The only a l t e r a t i o n s noted p r i o r to c e l l rupture were s l i g h t mitocondrial swelling and s l i g h t c e l l swelling. Damaged c e l l s had extensive fragmentation and v e s i c u l a t i o n of the membrane (34). Gross and h i s t o l o g i c a l examination of i n t e s t i n e , heart, spleen, kidneys and stomach have shown no consistent abnormalities; lungs were mildly congested with occasional patches of debris. P l a t e l e t thrombi have been reported i n the lungs of affected animals (32,33). It has been suggested that these p l a t e l e t thrombi may be a d i r e c t e f f e c t of the toxin and may secondarily cause the l i v e r e f f e c t s by creating s u f f i c i e n t pulmonary-congestion to cause r i g h t heart f a i l ure which, i n turn, could cause blood pooling and congestion i n the l i v e r (32). However, i n time course studies, Falconer et a l . (33) reported that the p l a t e l e t plugs did not appear i n h i s t o l o g i c a l pre parations taken at 15 and 30 minutes a f t e r toxin i n j e c t i o n and were present only i n l a t e r preparations. This evidence, along with other evidence of e f f e c t s on i s o l a t e d hepatocytes and the rapid onset of the l i v e r e f f e c t s i n vivo have l e d other researchers to believe that the l i v e r damage i s a d i r e c t e f f e c t of the toxin on the hepatocyte membrane and that the immediate cause of death i n acutely dosed animals i s hemorrhagic shock (33,35). Occasionally, hemorrhages have been noted i n other organs (17). This could possibly be due to coagulation problems associated with the l i v e r damage. Neurotoxic. Toxins of An. flos-aquae are referred to as anatoxins. Anatoxin-a (LD i . p . mouse, 200 yg/kg) (36,37) produced by An. flos-aquae NRC-^4-1 i s a potent n i c o t i n i c depolarizing neuromuscular blocking agent. Anatoxin-a(s) ( E>Q 1·Ρ· mouse, 40 yg/kg) produced by An. flos-aquae NRC-525-17 has not been pharmacologically charac terized. I t does have neuromuscular blocking action producing opis thotonos and s a l i v a t i o n i n laboratory animals. No e f f e c t of the toxin was seen on i s o l a t e d muscle preparations of frog rectus, chick biventer, rat hemidiaphragm-phrenic nerve or guinea pig ilium. The Q
LD
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
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32.
CARMICHAEL AND
MAHMOOD
Freshwater
Cyanobacteria
Toxins
387
opistothonos and s a l i v a t i o n could be reversed i n chicks by p r i o r i n j e c t i o n of atropine (7). The neurotoxins isolated from Aph. flos-aquae were shown to have similar chemical and b i o l o g i c a l properties to p a r a l y t i c s h e l l f i s h poisons (PSP) (25,2^,38). Sawyer et a l . i n 1968 (25) were the f i r s t to demonstrate that the crude preparation of aphantoxins behave l i k e saxitoxin, the major p a r a l y t i c s h e l l f i s h poison. They showed that the toxins had no e f f e c t on the resting membrane potent i a l of frog sartorius muscle; blocked action potential on desheathed frog s c i a t i c nerve and also abolished spontaneous contractions i n frog heart. Sasner et a l . (1981) (29) using the lab c u l tured s t r a i n reported similar r e s u l t s . Further confirmation of the s i m i l a r i t i e s i n b i o l o g i c a l a c t i v i t i e s between aphantoxin and PSP was shown by Adelman et a l . (1982) (30). They showed that crude preparations of aphantoxins blocked the Na channel of giant squid axon with equal potency as saxitoxin. +
Summary Cyanobacterial toxins (both marine and freshwater) are functionally and chemically a diverse group of secondary chemicals. They show structure and function s i m i l a r i t i e s to higher plant and a l g a l toxins. Of p a r t i c u l a r importance to this publication i s the production of toxins which appear to be i d e n t i c a l with saxitoxin and neosaxitoxin. Since these are the primary toxins involved i n cases of p a r a l y t i c s h e l l f i s h poisons, these aphantoxins could be a source of PSP standards and the study of their production by Aphanizomenon can provide information on the biosynthesis of PSP*s. The cyanobacteria toxins have not received extensive attention since they have fewer vectors by which they come i n contact with humans. As freshwater supplies become more eutrophicated and as cyanobacteria are increasingly used as a source of single c e l l protein toxic cyanobacteria w i l l have increased importance (39). The study of these cyanob a c t e r i a l toxins can contribute to a better understanding of seafood poisons. Acknowledgments N.A.M. would l i k e to thank the Science University of Malaysia for a research fellowship and the Biomedical Sciences Program at Wright State University f o r travel support. Literature Cited 1. 2.
3.
4.
Francis, G. Nature (London). 1878, 18,11. Sykora, J.L.; K e l e t i , G. In "The Water Environment: A l g a l Toxins and Health"; Carmichael, W., Ed.; Environmental Science Research Vol. 20, Plenum Press: New York, 1981; pp. 285-302. B i l l i n g s , W.H. In "The Water Environment: Algal Toxins and Health"; Carmichael, W., Ed.; Environmental Science Research Vol. 20, Plenum Press: New York, 1981, pp. 243-256. Carmichael, W.W.; Gorham, P.R. In "The Production and Use of Micro-Algae Biomass"; Shelef, G.; Soeder, C.J. Eds.; E l s e v i e r / North Holland: New York, 1980, pp. 437-448.
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
388 5. 6. 7. 8.
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9. 10.
11. 12. 13. 14. 15. 16.
17.
18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
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