Effect of Ciguatera-Associated Toxins on Body ... - ACS Publications

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Effect of Ciguatera-Associated Toxins on Body Temperature in Mice PHYLLIS R. SAWYER1, DAVID J. JOLLOW2, PAUL J. SCHEUER3, R I C H A R D YORK4, JOSEPH P. McMILLAN5, N A N C Y W. WITHERS1, H . H U G H FUDENBERG1, and T H O M A S B. HIGERD1,6 Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, SC 29425 2 Department of Pharmacology, Medical University of South Carolina, Charleston, SC 29425 3 Department of Chemistry, University of Hawaii at Manoa, Honolulu, HI 96822 4 Hawaii Institute of Marine Biology, University of Hawaii, Kaneohe, HI 96744 5 Department of Biology and Mathematics, College of the Virgin Islands, St. Thomas, VI 00801 6 National Marine Fisheries Service, N O A A , Southeast Fisheries Center, Charleston Laboratory, Charleston, SC 29412 1

Toxic materials extracted from ciguatoxic f i s h (ciguatoxin) and laboratory cultures of Gambierdiscus toxicus (putative maitotoxin) were chemically d i s t i n c t yet e l i c i t e d similar symptomatology i n mice. Admini s t r a t i o n of either toxin i n t r a p e r i t o n e a l l y to mice evoked a dramatic and prolonged suppression of body temperature. The extent of the depression was dependent on ambient temperature and could be abrogated by elevation of room temperature. However, the artificial maintenance of normal body temperature by r a i s i n g the surrounding temperature of mice treated with the d i n o f l a g e l l a t e toxin had an adverse effect on the surv i v a l rate of the animals. Ciguatera i s a human i l l n e s s that results from the ingestion of c e r t a i n t r o p i c a l and subtropical, c o r a l reef-associated f i s h (l)· The toxin believed responsible for the i l l n e s s was f i r s t i s o l a t e d from the l i v e r s of shark, red snapper and mo ray eel by Scheuer et a l . {2) and named ciguatoxin. Indirect evidence suggests that toxic f i s h accumulate the toxin over a time period v i a t h e i r d i e t . While the source of the toxin remained elusive for many years, current evidence suggests that one source of the toxin i s the benthic dinof l a g e l l a t e , Gambierdiscus toxicus, found i n association with certain macroalgae of c o r a l reefs (_3 ). Several laboratories have been successful i n growing the organism as a unialgal culture under cont r o l l e d conditions. Extracts of laboratory grown cultures of G. toxicus, however, have not yielded a toxin unequivocally i d e n t i f i e d as ciguatoxin. Instead, a more polar toxin s i m i l a r to maitotoxin described i n v i s c e r a l extracts of surgeonfish (h), can be r e a d i l y isolated from these cultures ( 5_) · Maitotoxin s biochemical relationship to ciguatoxin and role i n the pathogenesis of ciguatera remain unknown. During preliminary studies, we noted that the gross 1

0097-6156/ 84/ 0262-0321 $06.00/0 © 1984 American Chemical Society Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

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symptoms of mice treated with maitotoxin were similar to those of ciguatoxin-treated mice. This manuscript reports our observations on the apparent hypothermia e l i c i t e d by both toxins and postulates that these toxins possess an i n t e r r e l a t i o n s h i p not previously described. When mice receive sub-lethal intraperitoneal injections of the f i s h toxin, they exhibit symptoms of generalized malaise but display no gross distinguishing b i o l o g i c a l response useful i n defining or i d e n t i f y i n g the responsible toxin. E a r l i e r reports by Doorenbos et a l . (6)and Hoffman et a l . {j) demonstrated that mice receiving crude extracts of f i s h toxin manifested depressed body temperature. The i n i t i a l purpose of t h i s study was to exploit t h e i r observation as a useful t o o l to help distinguish various toxins. Instead, our results showed that the d i n o f l a g e l l a t e toxin (putative maitotoxin) also evokes t h i s unusual and dramatic response of decreased body temperature previously thought to be unique to ciguatoxin. In addition, t h i s study evaluates the b i o l o g i c a l nature of the temperature depression e l i c i t e d by these two ciguatera-associated toxins. Methods and Materials Gambierdiscus toxicus culture. G. toxicus, Adachi and Fukuyo (8) was collected from Dictyota acutiloba growing at a depth of about 3 meters on the west of Tern Island, Northwest Hawaiian Island Chain. Single dinoflagellate c e l l s isolated by micropipetting were washed exhaustively i n s t e r i l e seawater. In order to diminish the contamination by diatoms, Ge02 vas added to 80 ppm. The single c e l l i s o l a t e eventually selected for these studies, clone T-39> was cultured i n F/2-t medium (9) supplemented with a seaweed extract (an autoclaved, aqueous extract of blended Acanthophora s p i c i f e r a and Sargassum sp.; 10). The 10 ml tubes used for the i n i t i a l cultures were maintained at 25°C to 27°C at a continuous l i g h t intensity of hO u einsteins/m/sec without aeration. The d i n o f l a g e l l a t e c e l l s for t h i s study were mass cultured and harvested from 100 l i t e r culture vats by f i l t r a t i o n through a 36 u mesh screen. Extraction of cultured G. toxicus. To prepare the harvested d i n o f l a g e l l a t e c e l l s (about 1 χ 1θ' c e l l s ) for shipment from Hawaii to South Carolina, absolute methanol was added to effect a f i n a l concentration of approximately 25% (v/v). Upon receipt of the culture, additional methanol was added to a t t a i n a f i n a l concentra t i o n of 80% (v/v). After extraction at room temperature for 7 days, the suspension was c l a r i f i e d by centrifugation. The supernatant was dried and the resultant solids were weighed and resuspended i n absolute methanol. The suspension was f i l t e r e d and the f i l t r a t e designated as the crude d i n o f l a g e l l a t e extract used i n t h i s study. Subsequent fractionation of t h i s extract by HPLC (Higerd et a l . , manuscript i n preparation) showed that the material responsible for t o x i c i t y eluted i n a f r a c t i o n well removed from the f r a c t i o n that exhibited t o x i c i t y when extracts of either P a c i f i c moray eel or Caribbean f i s h were chromâtographed. In a l l cases, the body temperature depression effect was evident only i n those fractions which also exhibited t o x i c i t y . Source of Extracted Fish Toxin. Two different extracts of ciguatoxic f i s h were used i n t h i s study. P a r t i a l l y p u r i f i e d ciguatoxin was derived from the l i v e r and viscera of the P a c i f i c moray eel (Gymnothorax j avanicus) and was a side f r a c t i o n of the

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


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chromatographically p u r i f i e d ciguatoxin described by Tachibana (ll)· Crude f i s h toxin of Caribbean o r i g i n was partitioned extract derived p r i m a r i l y from the b l a c k f i n snapper (Lutj anus buccanella) as reported previously ( 7_) · Each was stored at k°C i n absolute methanol· T o x i c i t y Tests. Assays for t o x i c i t y were conducted on ICR female mice weighing approximately 2 0 g each. Animals were maintained on Wayne Laboratory Animal diets (Lab-Blox) and water, ad l i b i t u m . A known quantity of toxic extract was dried under a stream of dry nitrogen and placed i n a vacuum dessicator overnight. The dried extracts were dissolved i n phosphate buffered saline containing 5 % Tween 8 0 and administered i n t r a p e r i t o n e a l l y . Control animals received an equal volume of the v e h i c l e . L e t h a l i t y was assessed at 1*8 h. Body Temperature Determination. Unless otherwise noted, experiments were c a r r i e d out at 2U°C (room temperature) and no attempt was made to i n d i v i d u a l l y cage the treated or control animals. For the elevated temperatures of 2 8 ° C , 3**°C and 37°C, animals were acclimated to the desired temperature for 2k h. Temperatures of the mice were taken using a YSI temperature probe inserted approximately 2 0 mm into t h e i r rectum. The i n i t i a l temperatures (recorded as zero time) were taken immediately before administration of the extracts.

Results A pronounced drop i n the body temperature of mice was observed following the i n j e c t i o n of a methanol extract of G. toxicus. This extract was used throughout the study and had an estimated LD50 dose of 2 0 mg/kg for mice (Figure l ) . To quantitate the change i n body temperature, eight mice were injected with the d i n o f l a g e l l a t e extract (k3 mg/g). Control groups of eight mice received the c a r r i e r alone and were maintained with the toxin-treated animals at room temperature (Figure 2 ) . The r e c t a l temperatures of the treated and control mice were measured intermittently for 2k h. The average body temperature of the toxin treated mice was s i g n i f i c a n t l y lower than the control group at the end of the f i r s t hour. In a few mice, body temperatures as low as 2U°C were recorded p r i o r to death. In addition, the duration of t h i s apparent hypothermia was s t r i k i n g ; a few animals i n the population exhibited lowered body temperature for kQ h or longer. The response of depressed body temperature i n mice treated with d i n o f l a g e l l a t e extract i s dose dependent (Figure 3)· When doses as low as 3 mg/kg or 6 mg/kg (15% and 30% of the LD^o* respectively) were administered, no drop i n body temperature was noted. With injections of extract containing $0% or more of the L D ^ Q dose, however, an apparent hypothermia resulted, and the rate at which the temperature dropped was d i r e c t l y dependent on the quantity of extract administered. To ascertain the b i o l o g i c a l nature of t h i s response, groups of toxin-treated and control animals were maintained at elevated ambient temperatures (Figure U). Animals that had received j6% of an L D 5 0 dose and were held at room temperature exhibited the



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F i g u r e 1. Dose-response curve o f the l e t h a l i t y o f the t h r e e d i f f e r e n t e x t r a c t s used i n t h i s s t u d y . The e x t r a c t s were a d m i n i s t e r e d t o mice i . p . i n PBS w i t h 5% Tween-80. Lethality was a s s e s s e d a t 48 h. f o r a n i m a l s m a i n t a i n e d a t 24 °C. E x t r a c t s were Gambierdiscus t o x i c u s , f i s h of C a r i b b e a n o r i g i n , and p a r t i a l l y p u r i f i e d moray e e l c i g u a t o x i n . U 0

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F i g u r e 2. Body temperature response o f mice r e c e i v i n g the e x t r a c t of c u l t u r e d Gambierdiscus t o x i c u s o r the v e h i c l e a l o n e . Each p o i n t r e p r e s e n t s the mean ± S.E. o f the r e c t a l temperature measurements on 8 mice.


Effect of Ciguatera


27. SAWYER ET AL.

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Time

F i g u r e 3. Body temperature response of mice r e c e i v i n g v a r i o u s q u a n t i t i e s of c u l t u r e d Gambierdiscus t o x i c u s e x t r a c t . Each p o i n t r e p r e s e n t s the mean ± S.E. o f the r e c t a l temperature o f 8 animals.

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F i g u r e 4. Body temperature o f mice a d m i n i s t e r e d an e x t r a c t o f Gambierdiscus t o x i c u s o r the v e h i c l e a l o n e , and m a i n t a i n e d a t 24 °C, 28 °C, 34*" C, o r 37 °C. Each p o i n t r e p r e s e n t s the mean - S.E. o f the r e c t a l temperature o f 8 mice. U


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expected drop i n body temperature. On the other hand, as the ambient temperature was increased to 2 8 ° C , 3h°C and 3 7 ° C , the body temperature difference between the treated and control mice diminished. When treated mice were held at 37°C, no s i g n i f i c a n t a l t e r a t i o n of body temperature was observed. Since the extent of the observed hypothermia was dependent on ambient temperature, the r e s u l t i n g temperature response suggested an impairment i n the animals a b i l i t y to control normal body temperature. Since crude extracts of ciguatoxic f i s h have been reported to produce hypothermia i n mice (§_,j), i t was of interest to confirm these e a r l i e r reports with well-characterized toxin preparations and t o measure the extent and duration of any temperature a l t e r a t i o n i n l i g h t of our results with G. toxicus extracts. Crude extracts of ciguatoxic f i s h from the Caribbean subjected to l i q u i d - l i q u i d p a r t i t i o n and to chromatography on s i l i c i c acid ( 7 ) were used i n t h i s study. The L D ^ Q of these crude extracts was approximately 2 5 0 mg/kg (Figure l ) . As was the case with the d i n o f l a g e l l a t e extract, the extent and duration of the decrease i n body temperature was dependent on dose of administered f i s h extract (Figure 5 ) · Furthermore, both extent and duration of the observed hypothermia produced by the crude f i s h toxin were s i m i l a r to that recorded with the dinof l a g e l l a t e extract. Since only a l i m i t e d amount of f i s h toxin was a v a i l a b l e , two temperatures {2k°C and 3U°C) were used to assess the influence of ambient temperature on the apparent hypothermia induced by crude f i s h t o x i n . The hypothermia was dependent on ambient temperature and experimentally demonstrated that mice treated with f i s h toxin also l o s t t h e i r a b i l i t y to maintain body temperature (Figure 6 ) . To determine the effect of lowered body temperature on mortality, groups of toxin-treated mice were maintained at several elevated temperatures and t h e i r survival rates monitored. Because of the l i m i t e d quantity of f i s h t o x i n , only the d i n o f l a g e l l a t e toxin could be administered to a s i g n i f i c a n t number of animals held at various temperatures. Groups of eight mice treated with one-half of an L D 5 0 dose were placed at four different ambient temperatures. The percentage of mice i n each group that survived a f t e r f i v e hours was 1 0 0 % , 7 5 % , 6 2 % and 2 5 % when maintained at 2k°C, 2 8 ° C , 3h° C and 3 7 ° C , respectively. A similar result was observed when crude f i s h t o x i n was administered to a group of three mice. I t would appear, therefore, that holding animals at higher temperatures, while ameliorating the drop i n body temperature, had an adverse effect on survival.


1

Discussion S u f f i c i e n t quantities of these toxins have not yet been p u r i f i e d to t e s t the hypothesis that both poikilothermia and t o x i c i t y are prope r t i e s of the same molecule. Recently, however, the toxic components i n both the d i n o f l a g e l l a t e and the Caribbean f i s h have been p a r t i a l l y p u r i f i e d by HPLC on a reversed phase o c t y l s i l a n e column using a methanol-water gradient (Higerd et a l . , manuscript i n prepar a t i o n ) . In every instance; the mouse t o x i c i t y could not be separated from the hypothermia e l i c i t i n g property. In addition, a side f r a c t i o n obtained during the p u r i f i c a t i o n of ciguatoxin from moray


SAWYER ET AL.

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3Θ r


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F i g u r e 5. B o d y t e m p e r a t u r e r e s p o n s e s o f m i c e r e c e i v i n g v a r i o u s amounts o f c r u d e C a r i b b e a n c i g u a t o x i c f i s h e x t r a c t . Each p o i n t r e p r e s e n t s t h e mean - S.E. r e c t a l t e m p e r a t u r e o f 6 a n i m a l s .

υ

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F i g u r e 6. Body t e m p e r a t u r e o f mice r e c e i v i n g t h e c r u d e f i s h e x t r a c t o r t h e v e h i c l e a l o n e . Mice were m a i n t a i n e d a t e i t h e r 24 °C o r 3 4 °C. E a c h p o i n t r e p r e s e n t s t h e mean - S.E. o f t h e r e c t a l temperature of 6 animals.



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eel l i v e r described by Scheuer (2) was assessed for r e l a t i v e t o x i c i t y (Figure l ) . When small amounts of this toxin were administered t o mice, a marked body temperature depression was observed. I t would appear, therefore, that the b i o l o g i c a l response of apparent hypothermia i s a c h a r a c t e r i s t i c property of the toxins and not due t o some contaminating component of the crude extract. At least two toxins have been associated with ciguatera, namely ciguatoxin present i n toxic f i s h extracts, and maitotoxin, present i n cultured c e l l s of the d i n o f l a g e l l a t e implicated as the b i o l o g i c a l source of ciguatoxin in nature. Despite differences i n chemical structure ( 1 1 , 1 2 ) , both toxins affect the a b i l i t y of mice to regulate t h e i r body temperature i n such a dramatic manner that t h i s property appears to be singular among pharmacologically active compounds. The uniqueness of t h i s response and the ease of i t s measurement may make t h i s quantifiable b i o l o g i c a l response a useful t o o l in d i f f e r e n t i a t i n g various marine toxins, p a r t i c u l a r l y those associated with ciguatera, from other pharmacologically active toxins which otherwise e l i c i t non-discriminatory symptoms in mice. The underlying mechanism by which these toxins lower body temperature i s unclear. Increased peripheral blood flow with augmented convective and radiant heat loss are generally believed to be important factors leading to hypothermia. Additional p o s s i b i l i t i e s mediating the f a l l i n body temperature include decreased metabolic heat production or increased respiratory evaporative heat l o s s . It may be s i g n i f i c a n t that mice injected with either of these toxins display decreased respiratory rates and no gross evidence of vascul a r change (including t a i l vein vasodilation). These toxins could have a direct action on peripheral temperature sensors or on the thermoregulatory f o c i of the hypothalamus. A d d i t i o n a l l y , they may exert an indirect effect on neurotransmitters involved i n the cent r a l thermoregulatory pathways. Obviously, we can only speculate on the mechanism(s) by which these toxins exert t h i s e f f e c t . Recent hypotheses in thermoregulation focus on the set-point concept which describes some reference l e v e l around which body temperature i s regulated. Since the lowered temperature response can be reversed by changing the ambient temperature from low to high, i t would appear that the response i s not the result of an altered set-point. Since both ciguatoxin and maitotoxin have an effect on transportation of calcium ions across membranes ( 1 2 , 1 3 ) , i t may be s i g n i f i c a n t that several investigators ( I k , 1 5 , 1 6 , 1 T ) have reported prolonged hypothermia in various animals following repeated microperfusions with excess calcium. Our knowledge of the physiological mediators of hypothalamus a c t i v i t y i s cursory; with further studies, these toxins may become useful as b i o l o g i c a l probes to help define the biochemical basis of thermoregulation. I t i s generally accepted that ciguatoxin, as extracted from toxic f i s h , i s not synthesized i n s i t u but i s the result of dietary intake. Although toxicus may be one source of ciguatoxin in the d i e t , i t i s d i f f i c u l t to c o l l e c t large enough numbers of wild c e l l s in order to extract s u f f i c i e n t amounts of toxin for an unequivocal chemical assessment of ciguatoxin content. Alternately, both wild and cultured G_._ toxicus c e l l s produce a toxin that i s chemically d i s t i n c t from ciguatoxin ( 1 2 ) . As reported in this study, cultured d i n o f l a g e l l a t e t o x i n , putatively maitotoxin, has b i o l o g i c a l proper-


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t i e s that are indistinguishable from the f i s h toxin, ciguatoxin. Both toxins exhibit similar dose-response p r o f i l e s and both toxins e l i c i t the apparent hypothermia to the same extent and for the same duration. This response, whether promoted by the f i s h toxin or the d i n o f l a g e l l a t e t o x i n , i s reversed by increasing ambient temperature. In addition, both toxins evoke equivalent gross symptoms of malaise. These s i m i l a r i t i e s in b i o l o g i c a l t r a i t s suggest that more than a casual relationship exists between ciguatoxin and maitotoxin.


Acknowledgments We thank Marilyn Orvin for her technical assistance. This report i s publication no. 635 from the Department of Basic and C l i n i c a l Immunology and Microbiology, Medical University of South Carolina. Research supported i n part by Grant Na-80-AA-D-00101 from the National Oceanic and Atmospheric Administration. Literature Cited 1. 2. 3. 4. 5.

6. 7. 8. 9.

10.

11. 12. 13. 14. 15. 16. 17.

Withers, N. W. Ann. Rev. Med. 1982, 33, 97-111. Scheuer, P. J . ; Takahashi, W.; Tsutsumi, J . ; Yoshida, T. Science 1967, 155, 1267-8. Yasumoto, T.; Nakajima, I.; Bagnis, R.; Adachi, R. B u l l . Jpn. Soc. S c i . F i s h . 1977, 43, 1021-6. Yasumoto, T.; Bagnis, R.; Vernoux, P. Nippon Suisan Gakkaishi 1976, 42, 359-65. Yasumoto, T.; Nakajima, I.; Oshima, Y.; Bagnis, R. In "Toxic Dinoflagellate Blooms"; Taylor, L.; Seliger, H. H. Ed.; S e l i g e r , North Holland, 1979; pp. 65-70. Doorenbos, N. J . ; Granade, H. R.; Chen, P. C.; Morgan, J. M. In "Food-Drugs from the Sea"; Mar. Tech. Soc., 1976; p. 414. Hoffman, P. Α.; Granade, H. R.; McMillan, J. P. Toxicon 1983, 21, 363-9. Adachi, R.; Fukuyo, Y. B u l l . Jpn. Soc. S c i . F i s h . 1979, 45, 67-71. G u i l l a r d , R. R. L. In "Culture of Marine Invertebrate Animals"; Smith, W. L.; Chanley, M. H. Ed.; Plenum Publication Corp., New York, 1975; pp. 29-60. Withers, N. W. In "Symposium on Northwestern Hawaiian Islands"; Sea Grant Program Publication, Honolulu, 1983; i n press. Tachibana, K. Ph.D. Dissertation, U. Hawaii, Honolulu, 1980. Takahashi, M.; Ohizumi, Y.; Yasumoto, T. J . B i o l . Chem. 1982, 257, 7287-9. Rayner, M. D. Fed. Proc. Am. Soc. Exp. B i o l . 1971, 31, 1139-45. Feldberg, W.; Saxena, P.N. J . Physiol. (London) 1970, 211, 245-61. Myers, R. D.; Veale, W.L.; Yaksh, T.L. i b i d . 1971, 217, 381-92. Myers, R. D.; Brophy, P. D. Neuropharmacology 1972, 11, 351-61. Myers, R. D.; Buckman, J. E. Am. J . Physiol. 1972; 226, 1313-8.

R E C E I V E D March 2, 1984


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