Measurement of Brevetoxin Levels by Radioimmunoassay of Blood Collection Cards after Acute, Long-Term, and Low-Dose Exposure in Mice

By Woofter, Ricky; Dechraoui, M-Yasmine Bottein et al. | Environmental Health Perspectives, October 2003 | Go to article overview

Measurement of Brevetoxin Levels by Radioimmunoassay of Blood Collection Cards after Acute, Long-Term, and Low-Dose Exposure in Mice


Woofter, Ricky, Dechraoui, M-Yasmine Bottein, Garthwaite, Ian, Towers, Neale R., Gordon, Christopher J., Cordova, Jose, Ramsdell, John S., Environmental Health Perspectives


We developed a radioimmunoassay (RIA) using a sheep anti-brevetoxin antiserum to evaluate detection of brevetoxin on blood collection cards from mice treated with the brevetoxin congener PbTx-3. The RIA has high affinity for PbTx-3 [half-maximal effective concentration ([EC.sub.50]) [+ or -] SE = 1.2 [+ or -] 0.2 nM; n = 10] and recognizes both type 1 and type 2 brevetoxins, but not ciguatoxin. Direct comparison of the RIA with a radiolabeled [[sup.3]H]-PbTx-3 receptor-binding assay (RBA) revealed excellent sensitivity, congener selectivity, and minimal interference from blood matrix. We first analyzed blood samples from an acute time course exposure, using a maximal nonlethal dose [180 [micro]g/kg body weight (bw)] for 0.5, 1, 2, 4, and 24 hr. Mean blood brevetoxin levels were 36 nM at 30 min and stayed above 20 nM during the 1-4 hr time points. We next analyzed blood brevetoxin levels after longer exposure (0.5, 1, 2, 3, 4, or 7 days). Mean blood brevetoxin levels were 26.0 nM at 0.5 days, decreased to 8.2 nM at 1.0 day, and maintained a significant level (p < 0.05) of 1.3 nM at day 2. We next determined the lowest measurable dose using increasing concentrations of PbTx-3 (10-300 [micro]g/kg bw). Analysis of the blood samples at 60 min revealed a linear relationship between administered and internal doses ([r.sup.2] = 0.993). All doses of brevetoxin administered were detectable at 1 hr, with significant levels found for the lowest administered dose of 10 [micro]g/kg bw--a dose that was 10-fold lower than the lowest observable effect level. This RIA provides an optimal first-tier detection of brevetoxin from blood collection cards and, used in combination with the RBA and liquid chromatography--mass spectrometry, should provide a complete panel of methods to biomonitor brevetoxin exposure. Key words: blood, brevetoxin, radioimmunoassay. Environ Health Perspect 111:1595-1600 (2003). doi:10.1289/ehp.6166 available via http://dx.doi.org/[Online 2 July 2003]

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Red tides have been documented on the Gulf Coast of Florida as early as 1530 (Taylor 1917). They occur nearly annually and often persist for many months (Woodcock 1948). The causative organism for these events, Karenia brevis (formerly Gymnodinium breve and Ptychodiscus brevis) produces a family of neurotoxins, collectively called brevetoxins (Martin and Chatterjee 1969; Steidinger and Joyce 1973). These events are responsible for fish, waterfowl, and marine mammal mortalities (Davis 1948) as well as human intoxication (Lin et al. 1981). The accumulation of brevetoxins in shellfish can lead to the neurotoxic shellfish poisoning syndrome in humans (McFarren et al. 1965), which, before 1993, was believed to be restricted to the southeastern United States. However, in 1993 neurotoxic shellfish poisonings and brevetoxin contamination of shellfish were reported in New Zealand (Mackenzie et al. 1996). The causative organism in the New Zealand waters was reported to be K. brevis or a closely related species (Satake et al. 1996). Subsequently, three different genera of the family Raphidophyceae (Chattonella marina, Fibrocapsa japonica, Heterosigma akashiwo) isolated from Japanese waters have been reported to produce a neurotoxin similar to brevetoxins (Kahn et al. 1995, 1996, 1997). In 2002, brevetoxin was confirmed to be produced by the raphidophytes C. marina and Chattonella antiqua (Haque and Onoue 2002), and samples from mid-Atlantic waters containing Chattonella species have been reported to contain brevetoxin (Bourdelais et al. 2002). The broad distribution of raphidophytes opens the concern of a widespread occurrence of this family of toxins.

Brevetoxins are analyzed largely to assure shellfish safety and on an as-needed basis for marine mammal and human intoxications. In the Gulf of Mexico, substantial monitoring is conducted for the causative organism, K. brevis, to prevent the harvest of contaminated shellfish beds. At present, toxin detection is conducted by the mouse bioassay (Delaney 1985); however, several functional assays are employed as potential alternatives to the mouse bioassay (Dickey et al.

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Measurement of Brevetoxin Levels by Radioimmunoassay of Blood Collection Cards after Acute, Long-Term, and Low-Dose Exposure in Mice
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