Marine Toxins

Chapter 12

Detection, Metabolism, and Pathophysiology of Brevetoxins Mark A. Poli, Charles B. Templeton, Judith G. Pace, and Harry B. Hines

Downloaded by UNIV OF LIVERPOOL on October 4, 2017 | http://pubs.acs.org Publication Date: January 29, 1990 | doi: 10.1021/bk-1990-0418.ch012

Pathophysiology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21701-5011

M e t h o d s o f detection, metabolism, and pathophysiology o f the brevetoxins, PbTx-2 and PbTx-3, are summarized. Infrared spectroscopy and innovative chromatographic techniques were examined as methods for detection and structural analysis. Toxicokinetic and metabolic studies for i n vivo and i n vitro systems demonstrated hepatic metabolism and biliary excretion. A n i n vivo model o f brevetoxin intoxication was developed i n conscious tethered rats. Intravenous administration o f toxin resulted i n a precipitous decrease i n body temperature and respiratory rate, as well as signs suggesting central nervous system involvement. A polyclonal antiserum against the brevetoxin polyether backbone was prepared; a radioimmunoassay was developed with a sub-nanogram detection limit. This antiserum, when administered prophylactically, protected rats against the toxic effects o f brevetoxin.

R e d tides resulting from blooms o f the dinoflagellate Ptychodiscus brevis i n the G u l f o f M e x i c o have elicited a great deal o f scientific interest since the first documented event over 100 years ago (7). H u m a n intoxications from the ingestion o f contaminated shellfish and the impact o f massive fish kills o n the tourist industry along the G u l f coast o f the U n i t e d States have resulted i n a concerted research effort to understand the genesis o f red tides and to isolate and characterize the toxins o f P. brevis. Research i n this area advanced i n the 1970's as several groups reported the isolation o f potent toxins from P. brevis cell cultures ( 2 - 7 ) . T o date, the structures o f at least eight active neurotoxins have been elucidated (PbTx-1 through PbTx-8) (8). E a r l y studies o f toxic fractions indicated diverse pathophysiological effects i n vivo as well as i n a number o f nerve and muscle tissue preparations (reviewed i n 9 - 1 1 ) . T h e site o f action o f two major brevetoxins, PbTx-2 and PbTx-3, has been shown to be the voltage-sensitive sodium channel (8,12). These compounds b i n d to a specific receptor site o n the channel complex where they cause persistent activation, increased N a flux, and subsequent depolarization o f excitable cells at resting +

This chapter not subject to U.S. copyright Published 1990 American Chemical Society

Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

12. POLI ET A L .

Detection,Metabdism,andPathophyswhgyofBrevetoxim

membrane potentials. A t present, the brevetoxins are the only ligands unequivocally demonstrated to act at this site (neurotoxin receptor site 5), although mounting evidence (13) suggests the toxins involved i n ciguatera intoxication also may bind there. W e are investigating low-molecular-weight toxins o f animal, plant, and microbial origin. O u r goals are to develop methods to detect these compounds i n both environmental and biological samples and to develop prophylaxis and treatment regimens. This chapter summarizes the results o f o u r current investigations o f the brevetoxins. S o m e o f these studies will be published elsewhere i n greater detail.


Chemical Detection and Stability A variety o f chromatographic and spectrographic techniques have been applied to the study o f the brevetoxins. H i g h performance liquid chromatography ( H P L C ) and thin-layer chromatography ( T L C ) were instrumental i n the initial isolation and purification processes. Mass spectrometry ( M S ) , infrared spectroscopy ( I R ) , circular dichroism ( C D ) , nuclear magnetic resonance spectroscopy ( N M R ) , and X - r a y crystallography all played important roles i n structure determinations. A s research efforts expand to include metabolic studies, these techniques become increasingly important for detection and quantification o f exposure as well as structural elucidation o f metabolites.

Chromatography. A number o f H P L C and T L C methods have been developed for separation and isolation o f the brevetoxins. H P L C methods use both C 1 8 reversed-phase and normal-phase silica gel columns (8, 14, 15). Gradient or isocratic elutions are employed and detection usually relies u p o n ultraviolet ( U V ) absorption i n the 208-215-nm range. B o t h brevetoxin backbone structures possess a U V absorption maximum at 208 nm, corresponding to the enal moeity (16,17). In addition, the PbTx-1 backbone has an absorption shoulder at 215 n m corresponding to the 7-lactone structure. W h i l e U V detection is generally sufficient for isolation and purification, it is not sensitive (>1 ppm) enough to detect trace levels o f toxins or metabolites. Excellent separations are achieved by silica gel T L C (14, 15, 18-20). Sensitivity (>1 ppm) remains a problem, but flexibility and ease o f use continue to make T L C a popular technique. Mass Spectrometry. Mass spectrometry holds great promise for low-level toxin detection. Previous studies employed electron impact (EI), desorption chemical i o n ization ( D C I ) , fast atom bombardment ( F A B ) , and cesium i o n liquid secondary i o n mass spectrometry ( L S I M S ) to generate positive o r negative i o n mass spectra (2517, 21-23). F i r m detection limits have yet to be reported for the brevetoxins. Preliminary results from o u r laboratory demonstrated that levels as low as 500 ng PbTx-2 or PbTx-3 were detected by using ammonia D C I and scans o f 500-1000 amu (unpublished data). W e expect significant improvement by manipulation o f the D C I conditions and selected monitoring o f the molecular i o n or the ammonia adduction. T h e success o f the soft ionization techniques ( D C I , F A B , and L S I M S ) presents several possibilities for detection o f brevetoxins i n complex matrices. Positive-ion D C I was used for the analysis o f PbTx-3 metabolites generated i n vitro by isolated rat hepatocytes (see below). U n m e t a b o l i z e d parent was conclusively identified and metabolites were tentatively identified, pending confirmation by alternate methods (see below).

Chemical Stability and Decontamination. T h e stability o f the brevetoxins is o f great interest from the standpoints o f detection, metabolism, and safety. PbTx-2 and PbTx-3 have been investigated i n o u r laboratories i n order to design rational safety


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protocols for toxin handling and disposal o f contaminated waste (24; R . W . W a n namacher, unpublished data). These compounds were stable for months when stored i n the refrigerator either dry o r i n organic solvents such as ethanol, methanol, acetone, o r chloroform. However, toxins w i t h the PbTx-1 backbone have been reported to be unstable i n alcohol (75). PbTx-2 and PbTx-3 were unstable at p H values less than 2 and greater than 10, i n the presence o f 50 p p m chlorine, and at temperatures greater than 300 * C . F o r decontamination o f laboratory glassware and surfaces, greater than 9 9 % o f the detectable brevetoxin was destroyed by a 10-min exposure to 0.1 N N a O H . Disposable waste can be incinerated i f the combustion chamber temperature reaches at least 300 * C . A u t o c l a v i n g was shown to be ineffective for decontamination (24).


Distribution and Metabolic Fate—In Vivo and In Vitro Studies O f particular interest i n brevetoxin research are the diagnosis o f intoxication and identification o f brevetoxins and their metabolites i n biological fluids. W e are investigating the distribution and fate o f radiolabeled PbTx-3 i n rats. Three model systems were used to study the toxicokinetics and metabolism o f PbTx-3: 1) rats injected intravenously with a bolus dose o f toxin, 2) isolated rat livers perfused with toxin, and 3) isolated rat hepatocytes exposed to the toxin i n vitro. In the first study, male Sprague-Dawley rats (300-350 g) were given an intravenous bolus o f [ % ] P b T x - 3 (9.4 C i / m m o l , 6 /ig/kg body weight) via the penile vein (25). T h e plasma concentration curve (Figure 1) was bi-exponential w i t h a rapid distribution phase (half-life approx. 30 sec) and a slower e l i m i n a t i o n phase (half-life = 112 m i n ) . T o x i n clearance (dose administered/area under the plasma concentration curve) i n the w h o l e animal was 0.23 ml/min/g liver. In a 325-g rat, hepatic b l o o d flow ( Q ) is 13.2 m l / m i n (26), and, assuming hepatic clearance was equal to mean total clearance (CI), the calculated i n vivo extraction ratio ( C l / Q ) was 0.55. W i t h i n 1 m i n , 94% o f the administered toxin had distributed to the tissues. A f t e r 30 m i n , the liver contained 16%, skeletal muscle 70%, and the gastrointestinal tract 8% o f the administered radioactivity. T h e heart, kidneys, testes, brain, lungs, and spleen each contained less than 1.5%. B y 24 hr, radioactivity i n skeletal muscle decreased to 2 0 % o f the total administered dose. Over the same period, radioactivity remained constant i n the liver and increased i n the stomach, intestines, and feces, suggesting biliary excretion was an important route o f toxin e l i m i n a t i o n . B y day 6, 8 9 % o f the total radioactivity had been excreted i n the urine and feces i n a ratio o f 1:5. T L C analysis o f urine (Figure 2) and feces indicated that the parent toxin had been metabolized to several m o r e polar compounds. T o further investigate the role o f the liver i n brevetoxin metabolism, PbTx-3 was studied i n the isolated perfused rat liver model (27, 28). Radiolabeled PbTx-3 was added to the reservoir o f a recirculating system and allowed to mix thoroughly w i t h the perfusate. Steady-state conditions were reached w i t h i n 20 m i n . A t steady-state, 5 5 - 6 5 % o f the delivered PbTx-3 was metabolized and/or extracted by the liver; 2 6 % remained i n the effluent perfusate. U n d e r a constant liver perfusion rate o f 4 m l / m i n , the measured clearance rate was 0.11 ml/min/g liver. T h e calculated extraction ratio o f 0.55 was i n excellent agreement w i t h the i n vivo data. Radioactivity i n the bile accounted for 7% o f the total radiolabel perfused through the liver. PbTx-3 was metabolized and eliminated into bile as parent toxin plus four more-polar metabolites (Figure 3). Preliminary results o f samples stained with 4-(p-nitrobenzyl)-pyridine (29) indicated the most polar metabolite was an epoxide. 3

In vitro metabolism o f [ H ] P b T x - 3 was studied i n isolated rat hepatocytes (25). Hepatocyte monolayers cultured i n 6-well plates containing 1 m l modified W i l l i a m s E m e d i u m were incubated with 0.1 fig radiolabeled toxin at 37 ° C for 24 hr. T h e


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POLI ET AL.

Detection, Metabolism, and Pathophysiology of Brevetoxins

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100.0 r 30.0


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Minutes Figure 1. Semilogarithmic plot o f brevetoxin (/iCi) i n plasma over time after an intravenous injection o f tritium-labeled PbTx-3. T 1/2 alpha = 30 sec; T 1/2 beta = 112 m i n .

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MIGRATION (cm) Figure 2. Thin-layer radiochromatogram o f urine (100 u\) from rats injected w i t h labeled PbTx-3. T L C plates were developed i n two sequential solvent systems: chloroform:ethyl acetate:ethanol (50:25:25; 80:10:10). Radioactive zones were scraped and counted i n a l i q u i d scintillation counter. Native PbTx-3 runs at 13 c m .


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MARINE TOXINS: ORIGIN, STRUCTURE, AND MOLECULAR PHARMACOLOGY


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Marine Toxins

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