30
Toxins from Florida's Red Tide Dinoflagellate Ptychodiscus
brevis
1
1
1
2
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DANIEL G. BADEN , THOMAS J.MENDE ,MARK A.POLI ,and RONALD E. BLOCK 1
University of Miami School of Medicine, Department of Biochemistry, Miami, FL 33101 Papanicolaou Cancer Research Institute, Miami, FL 33136
2
Toxins T17 and T34, isolated from Florida's red t i d e dinoflagellate Ptychodiscus brevis, cause a dose-dependent response i n several in vivo and i n v i t r o b i o l o g i c a l systems. Potent fractions responsible for in s i t u fish l e t h a l i t i e s , respiratory i r r i t a t i o n , and neurotoxic s h e l l f i s h poisoning have been i d e n t i f i e d . The i d e n t i t y of potent fractions from three different research groups as well as the effects of chemical derivatization of the fractions on potency has been determined. Potency has been linked to membrane depolarization through endogenous sodium channels and s p e c i f i c binding to excitable membranes has been observed using t r i t i a t e d toxin. The marine d i n o f l a g e l l a t e Ptychodiscus brevis i s the causative organism i n Florida's red t i d e s ( 1 ) . A similar toxic species occurs i n Spain(_2 ) . The organism i s toxic i n both the f i e l d and i n the laboratory (3) and produces both neurotoxic and hemolytic components. F l o r i d a red t i d e s have a profound effect on the environment, causing extensive f i s h k i l l s and destroying other marine l i f e . This i s the most v i s i b l e consequence of exposure to _P. brevis toxins. Ichthyol e t h a l i t i e s occur as a result of exposure to the neurotoxins produced by the dinoflagellates i n the bloom(3), although there i s some e v i dence that hemolytic components may play a part i n the observed mala d i e s ^ ) . Hemolytic fractions are not potent i n mice(LD >10 mg/kg (5). Besides dead f i s h , which pose enormous economic and sanitation problems, there i s an airborne i r r i t a n t which burns the conjunctivae and mucous membranes and induces persistent non-productive coughing and sneezing(6^,^) . This i r r i t a t i o n i s thought to a r i s e from p a r t i cles of the toxic organism entrapped i n seaspray(3). The neurotoxins may be transmitted to man through bioaccumulat i o n i n an intermediate marine host. Toxic bivalves result from the f i l t e r - f e e d i n g of J>. brevis c e l l s during red t i d e s , and i f consumed result i n neurotoxic s h e l l f i s h poisoning, or NSP. Human o r a l intoxi c a t i o n i s rarely f a t a l . Our laboratory has been interested i n the toxinology of the puri f i e d toxins and we have recently been able to i d e n t i f y potent f r a c qn
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tions responsible for ichthyοlethalities and for o r a l and inhalation intoxication i n humans. We have, through collaborative studies, also begun to determine the mechanism of action of these potent compounds in b i o l o g i c a l systems.
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Summary of Previous Work From our laboratory cultures of P. brevis, we are able to purify to homogeneity and c r y s t a l l i n i t y two toxins, namely T17 and T34(8,9). Both toxins have been subjected to a variety of i n vivo and i n v i t r o test systems i n order to ascribe s p e c i f i c actions to each(10-13). The r e s u l t s are summarized i n Table I below.
Table I. Comparative Potency of _P. brevis Toxins T17 and Test System
T17
In vivo Fish(Gambusia a f f i n i s ) (24 hr L C , m o l e / l i t e r ) 50
1. 40 χ 10"
T34
9
Mouse(Swiss white)(24 hr LD ,mole/Kg) intravenous intraperitoneal oral
•7 1. 05 X 10" •7 1. 89 X 10" •7 10" 5. 80 χ
Guinea Pig bronchoconstriction(equivalent to 0.05 mg/Kg acetylcholine) (mole/Kg intravenous)
1. 10 χ 10"
In v i t r o Rat Phrenic Nerve Hemidiaphragm, neuromuscular block(IC ,mole/liter) tetanus twitch 5Q
Crayfish Giant Axon(ED,_Q, mole/liter) (maximum depolarization)
•9
6 .70 χ
T34 Reference
ΙΟ"
10
(8) (9)
2 .20 χ 10"^ 2 .20 χ 10~' 7 .40 χ 10 6
no effect
(11)
•10 1. 10 χ 10" •9 10" 5. 00 χ
-12 1 .50 χ 10 VL 4 .50 χ 10
•9 1. 50 χ 10' 30 mV
-9 * 10 * 30 mV
(12)
Χ Ζ
(14,2) (14)
Both toxins are potent ichthyotoxins. This i s not surprising as most investigators use a f i s h bioassay to indentify potent f r a c t i o n s during p u r i f i c a t i o n . Thus, these two toxins, i n addition to others which may be present _in s i t u , are l i k e l y responsible for f i s h k i l l s during red t i d e s ( 8 ) . A comparison of potency i n mice by three d i f f e r ent routes of administration i l l u s t r a t e s that only T17 i s acutely toxic i n an o r a l sense, i n d i c a t i n g i t as a l i k e l y agent responsible for neurotoxic s h e l l f i s h poisoning(9). Likewise, only T17 causes a bronchoconstriction i n anesthetized guinea pigs, suggesting i t as a l i k e l y airborne respiratory i r r i t a n t ( 1 1 ) . The differences i n potency are currently regarded by us as deriving from t h e i r respective l i p i d
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
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s o l u b i l i t i e s , discussed i n the following section. We believe that b i o l o g i c a l assays should, whenever possible, be conducted using the route of exposure experienced during normal episodes of i n t o x i c a t i o n . In the phrenic-nerve hemidiaphragm preparation, both T17 and T34 cause a concentration-dependent increase i n resting tension of the muscle, leading to a coneentrâtion-related neuromuscular block. The i n i t i a l contracture observed i s inhibited by tetrodotoxin or d-tubocurarine and i s enhanced by 4-aminopyridine or cholinesterase inhibi t o r s . Inhibitory concentrations are i n the nanomolar to picomolar concentration ranges, with T34 being consistently more potent than T17(12). Using i n t r a c e l l u l a r microelectrode techniques i n the rat phrenic-nerve hemidiaphragm neuromuscular junction, T17 was observed to cause a concentration-related increase i n miniature endplate potent i a l frequency, accompanied by spontaneous endplate p o t e n t i a l s . Persistent sodium channel-mediated nerve depolarization i s postulated to be s u f f i c i e n t to i n h i b i t neuromuscular transmission(13), but the i n crease i n spontaneous m.e.p.p. frequency suggested that perhaps neurotransmitter depletion played a role i n the development of the neuromuscular block. This p o s s i b i l i t y was eliminated by electron microscopic examination of blocked neuromuscular junctions. A complete complement of acetylcholine v e s i c l e s was observed(12). Radioactive acetylcholine tracer experiments support t h i s i n d i c a t i o n . Phrenic-nerve hemidiaphragm preparations were preincubated i n modified Krebs solution containing 1.0 χ 10~6M [methyl-^H]choline ( s p e c i f i c a c t i v i t y ^ 8 Ci/mmole) and 0.5 mM eserine s a l i c y l a t e f o r 30 min, followed by a b r i e f rinse i n eserine-containing modified Krebs solution but no l a b e l . Preparations were then incubated at 37°C i n modified Krebs solution containing eserine for 120 min, taking dup l i c a t e 0.1 ml samples of the supernatant f l u i d at 10 min i n t e r v a l s . At 60 min, 1 χ 1 0 " T 1 7 was added to the bath. Samples taken both before and a f t e r T17 addition were subjected to high voltage paper electrophoresis to separate [-^H]choline from [ % ] a c e t y l c h o l i n e . Fractions were v i s u a l i z e d with iodine vapor, and were cut out and placed i n s c i n t i l l a t i o n v i a l s with 10 ml Aquasol. Radioactivity was estimated using l i q u i d s c i n t i l l a t i o n techniques. Of the t o t a l radio a c t i v i t y present i n the preparations following preincubation, T17 caused the release of 15 to 25% of the l a b e l . The release was immed iate and complete within 10 t o 20 min of application. As can be seen from Figure 1, [^H]choline i s slowly released from the preparation during the entire experiment, except for the 10 to 20 min following T17 addition. Conversely, [^H]acetylcholine i s not released u n t i l T17 i s added to the bath f l u i d , and then i t s release i s only trans ient and does not continue, even i n the presence of T17. This pat tern of release i s consistent with the results we have presented pre viously, i . e . the transient increase i n resting tension of the muscle upon toxin addition. The slowed rate of release of [^H]choline upon T17 addition cannot be explained at t h i s time. However, with 75 to 85% of the [3H]acetylcholine s t i l l associated with the preparation at the termination of the experiment, we are c e r t a i n that neurotrans mitter depletion does not take place under these conditions. The neurotoxic actions of T17 on membrane e x c i t a b i l i t y were ex amined i n squid giant axon i n i t i a l l y and i n more d e t a i l using cray f i s h giant axon and i n t r a c e l l u l a r microelectrode techniques(14). De t a i l e d studies u t i l i z i n g T34 are not a v a i l a b l e due to t e c h n i c a l prob lems associated with i t s extreme hydrophobicity and resulting d i f f i -
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
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c u l t i e s i n removing i t from Plexiglass t e s t i n g chambers. The toxins are e f f e c t i v e when applied either externally or i n t e r n a l l y . When ap p l i e d externally, T17 caused a dose-dependent depolarization and a depression of the amplitudeand rate of r i s e of the action p o t e n t i a l . At 1 χ 10"6M, a complete block of e x c i t a b i l i t y occurred. T17-induced depolarization was e f f e c t i v e l y reversed by 3 χ 10" ?M tetrodotoxin or 1 χ 10"3M external sodium solution. Pretreatment with tetrodotoxin completely protected the axon from T17 s e f f e c t s but depolarization occurred upon removal of tetrodotoxin from solution. Anthopleurin A enhances the effects of T17. These r e s u l t s indicate that T17 a c t i vates sodium channels by binding at a s i t e separate from that of tetrodotoxin or Anthopleurin A.
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f
Correlation of Potent Fractions Several potent fractions isolated from laboratory cultures of _P. brevis have been prepared by a number of research groups, but due to a lack of standardized nomenclature, the number of toxins produced by the organism i s uncertain. The structures f o r three "brevetoxins" have now been reported(15-17). BTX-B, the major toxin reported by Lin et al.(15)(Figure 2a), i s thought to be i d e n t i c a l to GB-2 i s o lated e a r l i e r by Shimizu(16). Likewise, T34 isolated from our labor atory cultures of the organism(3), i s also thought to be i d e n t i c a l to GB-2. GB-3, isolated by Chou and Shimizu(17), was i d e n t i f i e d as F i g ure 2b and sodium cyanoborohydride reduction of GB-2 yielded a toxin containing an hydroxymethylene function i n place of the aldehyde group, %-nmr spectra of GB-3 and reduced GB-2 confirmed t h e i r iden t i c a l nature(17). BTX-C, an halogenated brevetoxin(Figure 2c) has also been reported(18). BTX-C i s not present i n our cultures, nor i s i t present i n the cultures of Shimizu(Y. Shimizu, personal communica t i o n ) . From our cultures of P_. brevis, we have p u r i f i e d T34 i n a y i e l d of 5.6±0.7 mg/10 cells; T17 i n a y i e l d of 1.5±0.3 mg/lO^cells. 9
Using authentic samples of BTX-B, we subjected i t and T34 to chemical reduction akin to that employed by Chou and Shimizu to re duce GB-2 to GB-3. Employing equimolar sodium borohydride i n aceton i t r i l e solution at room temperature f o r 3.5 min, both T34 and BTX-B were reduced to i d e n t i c a l f r a c t i o n s which comigrated with T17 i n three s i l i c a gel thin-layer chromatographic systems, possessed the same ^H-nmr spectra as T17 and GB-3, and were a l l equipotent i n mouse bioassay. Chemical oxidation of BTX-B and T34 using argentic oxide in the presence of sodium cyanide i n dry methanol(19) resulted i n a single homogeneous but e s s e n t i a l l y non-toxic f r a c t i o n upon thin-layer chromatography(Figure 2d)(Table I I ) . The 100 MHz J-H-nmr spectra of (a)-(d) are v i r t u a l l y i d e n t i c a l and (a)-(c) have been reported previously(15,16,18). The ^H-nmr spec tra of BTX-B, T34, and GB-2 are i d e n t i c a l to one another(_2), and the respective spectra of reduced T34, T17 and GB-3 are also i d e n t i c a l to one another. Thus, i n T17 and T34 spectra, there are seven methyl signals, each corresponding to signals present i n GB-3 and GB-2(BTX-B) respectively(16,18). T17 and T34 d i f f e r from one another i n the lower f i e l d region of the ^-H-nmr spectrum. In Tl7(or reduced T34) , the aldehyde signal of Τ34(ΰΌΰ1 ) at (69.50) i s absent and i s r e placed by a 2 proton singlet(64.06). There i s a simultaneous s h i f t of the terminal methylene protons i n T34(66.06, 6.29) to upper f i e l d s in T17(64.94, 5.10). The remaining o l e f i n i c protons i n t h e i r respec3
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
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30.
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Red Tide Dinoflagellate
Ti me
Ptychodiscus brevis
(min)
F i g u r e 1. The e f f e c t o f t o x i n T17 a d m i n i s t r a t i o n on the r e l e a s e of [ 3 ] c h o l i n e (•) and [ H ] a c e t y l c h o l i n e (·) from r a t p h r e n i c n e r v e hemidiaphragm p r e p a r a t i o n s . 3
H
F i g u r e 2. The s t r u c t u r e s o f the b r e v e t o x i n s . Key: a=T34, BTX-B, GB-2; b=T17, GB-3; c=BTX-C; d e o x i d i z e d T34.
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t i v e spectra 6(CDC1 ): 5.71(m,lH), 5.75(m,2H), 6.29(d,lH) do not s h i f t upon reduction of the aldehyde function. The chemical s h i f t s described above reproduce the spectral changes which accompany the reduction of GB-2 to GB-3(18). iH-nmr spectra of Figure 2d are ident i c a l to those of Figure 2a with the exception of the aldehyde proton (69.50), which i s replaced by an acid proton(broad, 611.2). In addition, the terminal methylene proton signals i n Figure 2a (66.08, 6.31) are shifted u p f i e l d i n Figure 2d (64.75,4.93).
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3
Table I I . S i l i c a Gel Thin-Layer Chromatographic Analysis of Brevetoxins and Their Derivatives Compound T34, BTX-B T17,
reduced T34,
Oxidized T34,
reduced BTX-B
oxidized BTX-B
System 1
System 2
System 3
0.34
0.79
0,.41
0.17
0.66
0,.22
0.74
0.82
0,.69
system 1= acetone/light petroleum (30/70); system 2= chloroform/metha n o l / t r i f l u o r o a c e t i c acid (100/10/1); system 3= ethyl acetate/light petroleum (70/30).
Therefore, by preparing i d e n t i c a l reduction products of BTX-B and T34 and by determining t h e i r r e l a t i v e migrations i n three TLC systems, we could t e n t a t i v e l y conclude that BTX-B and T34 were equivalent compounds. Further, since the ^H-nmr spectrum of reduced T34 i s i d e n t i c a l to that of GB-3, we could conclude that GB-2 and T34 were i d e n t i c a l compounds. The EI mass spectrum of T17 (and reduced T34) at 16 eV also confirms that GB-3 and T17 are i d e n t i c a l . Thus, the major fragment ions obtained were: m/z 725(1%), 681(50%), 349 (13%), 291(15%), 109(46%0, and 44(100%). The oxidation of both T34 and BTX-B to the corresponding acid, and determination of comigration in the three TLC systems i s a further confirmation of t h e i r equivalence. Potency of Figure 2 a,b,and d i n intraperitoneal mouse bioassay i l l u s t r a t e s that the character of the chemical substituent at carbon 40 i n part determines potency. Thus, T17 (GB-3; LD5Q=0.17 mg/Kg body weight) i s more potent than T34 (BTX-B,GB-2; LD5Q=0.20 mg/Kg) and both are more potent than oxidized T34 (oxidized BTX-B; LD5Q=1.50 mg/ Kg. It should be noted that a l l three are l e t h a l , however, and so the acute t o x i c i t y of these compounds i s not e n t i r e l y due to C-40 substituent e f f e c t s . Potency does follow the oxidation series from alcohol to aldehyde to acid i n vivo, suggesting that perhaps these substituents influence the degree of a c c e s s i b i l i t y of each l i p i d - s o l vent soluble toxin to i t s membrane s i t e of action. Being that the toxins i n t h e i r natural forms are so soluble i n non-polar solvents, and tend to bind to or s o l u b i l i z e i n the l i p i d components of membrane
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i t i s not surprising that the acid derivative i s the least potent of the three. The exact mechanism by which these toxins exert t h e i r l e t h a l e f f e c t s i s not known with c e r t a i n t y . Based on e a r l i e r work of McFarren et al.(20), incubation of brevetoxins i n d i l u t e a l k a l i t o t a l l y destroys a c t i v i t y . The potent portion of the molecules may therefore be postulated to reside i n the α,β-unsaturated lactone of these toxins and may hence act i n a manner similar to d i g i t a l i s ( 2 1 ) . We are continuing chemical modification studies, which w i l l further delineate substituent e f f e c t s on the potency of t h i s new c l a s s of polyether toxins. The potency of the brevetoxins i n b i o l o g i c a l systems leads us to believe that s p e c i f i c i n t e r a c t i o n s , presumably associated with the voltage-dependent sodium channel, take place. Thus, i t may be ex pected that a s p e c i f i c s i t e or s i t e s on excitable membranes bind(s) the brevetoxins i n a concentration-dependent manner. Membrane-bind ing assays, performed according to the methods of C a t t e r a l l et a l . (22), show a s p e c i f i c binding of T17 to rat brain synaptosomes i n a dose-dependent manner(Figure 3). The radioactive probe, [3H]T17, was prepared by NaB^ïfy reduction of T34, and yielded a s p e c i f i c act i v i t y of 15-20 Ci/mmole. I t was necessary to add an emulsifying agent to the binding medium to prevent micelle formation when T17 reached higher concentrations. The Kp observed (a preliminary observation) with (6.6 χ 10"7M) or without(2.3 χ 10"7M) emulsifier d i f f e r s by about three-fold, a result we a t t r i b u t e to differences i n T17 s o l u b i l i t y . Our studies are continuing i n t h i s d i r e c t i o n to ob t a i n a t o t a l Scatchard analysis of brevetoxin binding to syaptosomes and to determine the r e l a t i o n s h i p of i t s binding to the binding of other sodium channel-perturbing toxins. Acknowledgments The authors thank Prof. K o j i Nakanishi for h i s generous g i f t of BTX-B and to Prof. Yuzuru Shimizu for 500 MHz spectra of GB-2. Work from our laboratory was funded by NIH grant number ES-02299 and ES-02651, DHHS. Legend of Symbols LD50 LC5Q IC^Q ED^Q
l e t h a l dose for 50% of the experimental population. l e t h a l concentration ( f i s h assay) for 50% of the experimental population. concentration required to i n h i b i t action (muscle twitch or tetanus strength) to 50% of i t s control l e v e l . concentration required to effect a response of 50% of the maxi mal response(squid/crayfish giant axon depolarization).
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
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BOUND
Τ 17
( CPM χ 10~ D ο F i g u r e 3. The s p e c i f i c b i n d i n g of [ H]T17 t o r a t b r a i n synaptosomes i n the absence ( t o p p a n e l ) or presence (bottom p a n e l ) of emulsifier. 3
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Ptychodiscus brevis
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Davis, C.C. Bot. Gaz. 1947, 109, 358. Baden, D.G. Int. Rev. Cytol. 1983, 82, 99. Steidinger, K.A.; Burklew, M.A.; Ingle, R.M. In "Marine Pharm acognosy"; Martin, D.F.; P a d i l l a , G.M., Eds.; Academic Press: New York, 1973; p. 179. 4. Quick, J.Α.; Henderson, G.E. Int. Conf. Tox. Dinoflagellate Blooms, 1st, 1975, p. 413. 5. T r i e f f , N.M.; Ramanujam, V.M.S.; Alam, M.; Ray, S.M. Int. Conf. Tox. Dinoflagellate Blooms, 1st, 1975, p. 309. 6. Music, S.L.; Howell, J.T.; Brumback, C.L. J. F l a . Med. Assoc. 1973, 60, 27. 7. Weech, A.A. J . F l a . Med. Assoc. 1976, 63, 409. 8. Baden, D.G.; Mende, T.J.; Lichter, W.; Wellham, L. Toxicon. 1982, 19, 455. 9. Baden, D.G.; Mende, T.J. Toxicon. 1982, 20, 457. 10. Baden, D.G.; Mende, T.J.; Block, R.E. In "Toxic Dinoflagellate Blooms"; Taylor, D.L.; Seliger, H.H., Eds.; Elsevier:Amsterdam. 11. Baden, D.G.; Mende, T.J.; Bikhazi, G.M.; Leung, I. Toxicon. 1982 20, 929. 12. Baden, D.G.; Bikhazi, G.M.; Decker, S.J.; Foldes, F.F.; Leung, I. Toxicon. i n press. 13. Vogel, S.M.; Atchison, W.D.; Narahashi, T. Fed. Proc. Am. Soc. Exp. B i o l . 1982, 41, 8487(Abstr.). 14. Huang, J.M.; Wu, C.H.; Baden, D.G. J . Pharm. Exp. Ther. i n press. 15. L i n , Y.Y.; Risk, M.; Ray, S.M.; Van Engen, D.; Clardy, J . ; Golik, J.; James, J.C.; Nakanishi, K. J . Am. Chem. Soc. 1981, 103, 6773. 16. Shimizu, Y. Pure Appl. Chem. 1982, 54, 1973. 17. Chou, H.N.; Shimizu, Y. Tetrahedron L e t t . 1982, 23, 5521. 18. Golik, J.; James, J.C.; Nakanishi, K.; L i n , Y.Y. Tetrahedron Lett. 1982, 23, 2535. 19. Corey, E.J.; Gilman, N.W.; Ganem, B.E. J. Am. Chem. Soc. 1968, 90, 5616. 20. McFarren, E.F.; Tanabe, H.; S i l v a , F.J.; Wilson, W.B.; Campbell, J.E.; Lewis, K.H. Toxicon 1965, 3, 111. 21. Campbell, S.F.; Danilewiez, J.C. Ann. Rep. Med. Chem. 1978, 13, 92. 22. C a t t e r a l l , W.A.; Morrow, C.S.; Hartshorne, R.P. J. B i o l . Chem. 1979, 254, 11379. RECEIVED February 6, 1984
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