Seafood Toxins - American Chemical Society

22 Lipid-Extracted Toxins from a Dinoflagellate, Gambierdiscus toxicus

Downloaded by TUFTS UNIV on June 4, 2018 | https://pubs.acs.org Publication Date: September 19, 1984 | doi: 10.1021/bk-1984-0262.ch022

DONALD M. MILLER, ROBERT W. DICKEY, and DONALD R. TINDALL Department of Physiology and Pharmacology, School of Medicine, and Department of Botany, Southern Illinois University, Carbondale, IL 62901 Crude and three diethyl ether extracted, acetone treated, fractions were isolated from large-scale cultures of Gambierdiscus toxicus. Crude extracts at .04 mg/ml inhibited the histamine contraction response in smooth muscle of the guinea pig ileum. Three semi purified fractions at 5 ng/ml, effectively inhibited the guinea pig ileum preparation. Two of these fractions followed Michaelis-Menten kinetics for a competitive inhibition. The third fraction inhibited in a non-reversible manner. This study has established the presence of three lipid extracted toxins in G. toxicus, outlined a method for their assay in small quantities, and identified at least two of the effects of these toxic extracts in animals. Ciguatera was the name given to a food poisoning syndrome which affected Spanish people who s e t t l e d in Cuba during the exploration of the American continents (1-3). The name was derived from the Spanish word "cigua" f o r a species of turban s h e l l which they thought responsible f o r the i l l n e s s . Later t h i s same name was applied to a s i m i l a r syndrome which occurs in the P a c i f i c . It has come to be defined as a form of food poisoning caused by the ingestion of any one of a number of marine fishes in t r o p i c a l and subtropical areas. The symptomology in humans has been described Scheuer and coworkers (9) i s o l a t e d substances from Lutjanus bohar (red snapper), Carcharhinus menisorrah (shark), and Gymnothorax jayanicus (moray eel) which they c a l l e d " c i g u a t o x i n " and imp!icated in the ciguatera syndrome of the P a c i f i c . The wealth of pharmacological and other data accumulated on the ciguatera syndrome has indicated that more than one toxin i s involved (£»1£ 11). Thus, at present there i s no certainty which toxin (e.g., ciguatoxin, maitotoxin, s c a r i t o x i n , e t c . ) plays the major role in the ciguatera syndrome. Indeed, not only may the 5

0097-6156/ 84/ 0262-0241 $06.00/0 © 1984 American Chemical Society

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


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causative agent be d i f f e r e n t in the Caribbean and the P a c i f i c , but some evidence indicates that i t may be d i f f e r e n t from island to island within the same area (12). A d i n o f l a g e l l a t e , Gambierdiscus toxicus has been reported to be the l i k e l y progenitor of ciguatoxin in the South P a c i f i c (13-15). Recently, T i n d a l l , Q 6 ) , M i l l e r , Q7) and Carlson, (J8) reported on t o x i c extracts from G. toxicus and two species of Prorocentrum from the Caribbean. " The physiological action of t o x i c extracts from G. toxicus were found to be s i m i l a r to those induced by t o x i c extract from Scomberomorus cavalla (Kingfish) from the same region (19). Attempts to delineate the active components in the genesis of t o x i c f i s h and to e s t a b l i s h prophylactic measures f o r ciguatera have been hampered by; the lack of adequate amounts of toxin f o r t e s t i n g and p u r i f i c a t i o n , the lack of a c l e a r d e f i n i t i o n of whether the t o x i c material was a single e n t i t y or multiple f a c t o r s , and the lack of discriminating tests which u t i l i z e d small amounts of toxin to characterize each t o x i c component. With these aspects in mind, we have begun a systematic program which i n cludes: (1) the growth, in large-scale culture of eighteen species of "Ciguatera-community" d i n o f l a g e l l a t e s including G. toxicus, (2) the extraction and p u r i f i c a t i o n of large quantities of t o x i c materials from these large-scale c u l t u r e s , and (3) the establishment of assay procedures f o r each t o x i c component and characterization of each using specific physiological model-systems. This paper i s a report on the l i p i d extracted toxins from G. toxicus and t h e i r e f f e c t on i s o l a t e d guinea pig ileum preparat i o n s . This paper also proposes the use of t h i s preparation f o r q u a n t i f i c a t i o n of two of the l i p i d extracted toxins. Materials and Methods Culture of D i n o f l a g e l l a t e . The i s o l a t e of G. toxicus (SIU 350) used in t h i s study was obtained from the South Sound of V i r g i n Gorda, B r i t i s h V i r g i n Islands. The species was brought into unialgal culture and maintained in enriched sea water (20) under continuous l i g h t (3200 lux cool white fluorescent) at 27.0° C. The development of large-scale cultures involved t r a n s f e r r i n g c e l l s from stock cultures to a series of two l i t e r Fernbach f l a s k s containing enriched seawater medium. A f t e r the early stationary phase of growth had been reached (approximately 15-20 days) each of these cultures were used to innoculate 18 l i t e r s of the same medium in 20 l i t e r carboys. Large-scale cultures were grown under continuous l i g h t (4300 lux cool white fluorescent) at 27.0° C. C e l l s were harvested by centrifugation a f t e r cultures reached the early stationary phase of growth (30-35 days). Extraction and P u r i f i c a t i o n of Toxins. Two large-scale cultures were examined: (1) 350F and (2) 350G. Culture F consisted of 53 l i t e r s and yielded 13.1 g fresh weight (2.62 g dry mass) of G. toxicus c e l l p e l l e t . Culture G consisted of 130 l i t e r s which yielded 49.5 g fresh weight (9.9 g dry mass). The c e l l s were


22.

MILLER ETAL.

Lipid-Extracted

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extracted by r e f l u x i n g in b o i l i n g aqueous methanol. The methanol extracts were concentrated and subjected to l i q u i d - l i q u i d p a r t i t i o n i n g followed by cold acetone treatment of the t o x i c components (Figure 1). Totals of 103.98 and 485.21 mg DM of crude ether soluble acetone p r e c i p i t a t e s (ESAP) were i s o l a t e d from 350F and 350G, respectively ( s i x t h and seventh successive large scale c u l t u r e s ) . The major portion of the ESAP f r a c t i o n from 350G was subjected to further p u r i f i c a t i o n by sequential treatment on columns of s i l i c i c a c i d , basic aluminum oxide and sephadex G-15. Eluting solvents and t h e i r ratios are detailed in the results section. Thin Layer Chromatography Procedures. S i l i c a gel G (Suppelco, Redi-coat, 5 X 20 cm X 0.25 mm) plates were activated by heating in an oven f o r 1 hour at 110°C. Toxic products were spotted onto duplicate plates at concentrations corresponding to t h e i r previously determined L D l e v e l s . The plates were developed to 14 cm with chloroform, methanol and (6N) ammonium hydroxide (90:9.5:0.5) or chloroform, methanol, and water (60:35:8). Plates were developed and v i s u a l i z e d by spraying with 50% aqueous s u l f u r i c acid and charring. Undeveloped plates were scraped, the s i l i c a gel fines removed and the extracts concentrated to a residue under a nitrogen gas stream. 99

Mouse Assays. Outbred female Swiss mice (Harlan Sprague Dawley ICR "BR") weighing 19 to 21 g were used. Doses of t o x i c extract were suspended in 0.5 ml of 0.1% Tween 60 in 0.15 M NaCl and administered by i . p . i n j e c t i o n . Mice were observed f o r a period of 48 hours. Guinea Pig Ileum Preparation. Guinea pigs (350-600 gm) were s a c r i f i c e d by a c e r v i c a l d i s l o c a t i o n . A 2-3 cm segment of ileum was removed and placed in physiological saline solution at 37°C. The saline solution consisted of the following (mM): NaCl, 136.9; KC1, 2.68; C a C l , 11.84; MgCl , 1.03; NaHC0 , 11.9; KI-^POi,, 0.45; glucose, 5.55. If required, the i n t e s t i n a l lumen was evacuated by slowly flushing with saline s o l u t i o n . The dissected ileum segment was attached by a wire hook to a glass rod which extended to the bottom of an organ bath. The other end of the ileum was attached by a s i l v e r chain to the lever of the transducer. The apparatus u t i l i z e d f o r studying the responses of the ileum to toxin consisted of a: water-jacketed tissue bath, Bionix myograph transducer, Beckmann physiograph, Beckmann temperature bath, and an a i r pump. Dose response relationships f o r phasic contractions of i l e a l segments were determined following the method of van Rossum (21). Time dependent assays were conducted by subjecting the preparation to a single concentration of toxin and then challenging the preparation with the agonist concentration at progressive time periods. 2

2

3

Calcium Substitution Experiments. Saline solutions u t i l i z e d in t h i s series of experiments contained calcium chloride at 0.5X, 2X,


SEAFOOD TOXINS

L A R G E SCALE CULTURE

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Seafood Toxins - American Chemical Society

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