Measurement of brevetoxin levels by radioimmunoassay of blood collection cards after acute, long-term, and low-dose exposure in mice.

Abstract

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(50)) +/- SE = 1.2 +/- 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 [(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 microg/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(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.

Co-occurrence of multiple classes of harmful algal toxins in bottlenose dolphins (Tursiops truncatus) stranding during an unusual mortality event in Texas, USA

“…Samples were analyzed for brevetoxins by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), utilizing methods described by Woofter et al (2003) and Maucher et al (2007), respectively. The RIA was performed using sheep antisera prepared against a PbTx-2-fetuin conjugate.…”

Brevetoxin in two planktivorous fishes after exposure to Karenia brevis: implications for food-web transfer to bottlenose dolphins

“…Radioimmunoassays were performed using sheep antisera prepared against a PbTx-2-fetuin conjugate (Garthwaite et al 2001, Woofter et al 2003. Radioimmunoassays were run in 12 × 75 borosilicate glass tubes in amended phosphate-buffered saline (PBS) containing 137 mM NaCl, 8 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 2.7 mM KCl, and 0.01% Emulphor-EL (GAF).…”

“…The second study utlized a higher brevetoxin dose exposure of Atlantic menhaden via K. brevis culture followed by a 1 mo depuration. Brevetoxin content was analyzed in 3 isolated whole body components using a brevetoxin radioimmunoassay (Woofter et al 2003). We utilized these data (with consideration given to the timing of retention of toxin in different tissues) to evaluate a biological mechanism involving consumption of K. brevis by striped mullet and Atlantic menhaden.…”

Dried blood spots in toxicology: from the cradle to the grave?

“…In coastal waters in the Gulf of Mexico, this approach has been used for monitoring the exposure of bottlenose dolphins to the marine algal biotoxins domoic acid and brevetoxins, respectively produced by members of the diatom genus Pseudo-nitzschia and by the dinoflagellate Karenia brevis (Twiner et al, 2011). Both biotoxins have been measured in DBS, obtained by spotting filter paper cards with 100 l of blood, obtained from either exposed laboratory test animals (mice, rats or the fish species striped mullet) or from free-living dolphins (Fairey et al, 2001, Woofter et al, 2003, Woofter et al, 2005, Schwacke et al, 2010, Twiner et al, 2011. Toxin detection in DBS extracts has been performed using receptor-binding assays (Fairey et al, 2001) and radio immuno-assay (RIA, for brevetoxins) (Woofter et al, 2003) and, more recently, by competitive ELISA, either detecting brevetoxins and their metabolites, or domoic acid Ramsdell, 2005, Maucher et al, 2007).…”

“…Both biotoxins have been measured in DBS, obtained by spotting filter paper cards with 100 l of blood, obtained from either exposed laboratory test animals (mice, rats or the fish species striped mullet) or from free-living dolphins (Fairey et al, 2001, Woofter et al, 2003, Woofter et al, 2005, Schwacke et al, 2010, Twiner et al, 2011. Toxin detection in DBS extracts has been performed using receptor-binding assays (Fairey et al, 2001) and radio immuno-assay (RIA, for brevetoxins) (Woofter et al, 2003) and, more recently, by competitive ELISA, either detecting brevetoxins and their metabolites, or domoic acid Ramsdell, 2005, Maucher et al, 2007). Ciguatoxins are another class of highly potent neurotoxins, sharing with brevetoxins the binding site 5 on the α-subunit of voltage-gated sodium channels as effector site (Wang, 2008).…”