Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R) in certain labs. A fluorescence based receptor binding assay (RBA(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®- PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R2 = 0.71) with those of the RBA(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F) advantages include the long-term (> 5 years) stability of the BODIPY®- PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample throughput, and is well-suited for routine CTX monitoring programs.

Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Inhalation of brevetoxins aerosolized by wind and wave action can lead to asthma-like symptoms in beach goers. Consumption of either shellfish or finfish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of brevetoxin analogs and competitors to site 5 on these channels has historically been measured using a radioligand competition assay that is fraught with difficulty, including slow analysis time, production of radioactive waste, and cumbersome and expensive methods associated with the generation of radioactive labeled ligands. In this study, we describe the development of a novel fluorescent synaptosome binding assay for the brevetoxin receptor. BODIPY®-conjugated to PbTx-2 was used as the labeled ligand. The BODIPY®-PbTx-2 conjugate was found to displace [3H]-PbTx-3 from its binding site on VSSCs on rat brain synaptosomes with an equilibrium inhibition constant of 0.11 nM. We have shown that brevetoxin A and B analogs are all able to compete for binding with the fluorescent ligand. Most importantly, this assay was validated against the current site 5 receptor binding assay standard, the radioligand receptor assay for the brevetoxin receptor using [3H]-PbTx-3 as the labeled ligand. The fluorescence based assay yielded equilibrium inhibition constants comparable to the radioligand assay for all brevetoxin analogs. The fluorescence based assay was quicker, far less expensive, and did not generate radioactive waste or need radioactive facilities. As such, this fluorescence-based assay can be used to replace the current radioligand assay for site 5 on voltage-sensitive sodium channels and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels.

The regulatory mechanisms of physical activity are postulated to include environmental and biological/genetic factors. In particular, the sex steroids appear to have profound effects on wheel running in rodents. The purpose of this project was to investigate the effects of 17β-estradiol and testosterone on wheel running distance, duration, and speed in male and female C57BL/6J mice. The mice (N=46) were provided free access to running wheels interfaced with computers to track daily running distance, duration, and speed. Activity was assessed at baseline in intact mice, after surgical gonadectomy, and after replacement with either 17β-estradiol or testosterone. Upon removal of the gonads, physical activity levels were significantly reduced in both males and females. Distance (10–30% of baseline) and duration (20–47% of baseline) measures were most affected by the loss of endogenous steroids, while running speed (60–77% of baseline) though significantly reduced-decreased by a much lower magnitude. Testosterone replacement fully recovered running distance, duration, and speed to pre-surgical levels in both sexes (100% of baseline). Distance (30–42% of baseline) and duration (43–47% of baseline) were partially recovered by 17β-estradiol, but not to baseline levels. Speed (100% of baseline) was fully recovered by 17β-estradiol replacement in males and females. This study suggests that physical activity in mice is affected by endogenous steroids and can be altered by exogenous steroid replacement. The differences in the recovery abilities of 17β-estradiol and testosterone suggest that both estrogenic and androgenic pathways may be involved to variable degrees in activity regulation.

Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Consumption of fish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of toxins has historically been measured using a radioligand competition assay that is fraught with difficulty. In this study, we developed a novel fluorescence-based binding assay for the brevetoxin receptor. Several fluorophores were conjugated to polyether brevetoxin-2 and used as the labeled ligand. Brevetoxin analogs were able to compete for binding with the fluorescent ligands. This assay was qualified against the standard radioligand receptor assay for the brevetoxin receptor. Furthermore, the fluorescence-based assay was used to determine relative concentrations of toxins in raw extracts of K. brevis culture, and to determine ciguatoxin affinity to site 5 of VSSCs. The fluorescence-based assay was quicker, safer, and far less expensive. As such, this assay can be used to replace the current radioligand assay and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels.

Gambierdiscus and Fukuyoa dinoflagellates produce a suite of secondary metabolites, including ciguatoxins (CTXs), which bioaccumulate and are further biotransformed in fish and marine invertebrates, causing ciguatera poisoning when consumed by humans. This study is the first to compare the performance of the fluorescent receptor binding assay (fRBA), neuroblastoma cell-based assay (CBA-N2a), and liquid chromatography tandem mass spectrometry (LC-MS/MS) for the quantitative estimation of CTX contents in 30 samples, obtained from four French Polynesian strains of Gambierdiscus polynesiensis. fRBA was applied to Gambierdiscus matrix for the first time, and several parameters of the fRBA protocol were refined. Following liquid/liquid partitioning to separate CTXs from other algal compounds, the variability of CTX contents was estimated using these three methods in three independent experiments. All three assays were significantly correlated with each other, with the highest correlation coefficient (r2 = 0.841) found between fRBA and LC-MS/MS. The CBA-N2a was more sensitive than LC-MS/MS and fRBA, with all assays showing good repeatability. The combined use of fRBA and/or CBA-N2a for screening purposes and LC-MS/MS for confirmation purposes allows for efficient CTX evaluation in Gambierdiscus. These findings, which support future collaborative studies for the inter-laboratory validation of CTX detection methods, will help improve ciguatera risk assessment and management.

Chronic respiratory diseases, including chronic obstructive pulmonary disease (COPD), cystic fibrosis, and asthma, are some of the leading causes of illness and fatalities worldwide. The search for novel treatments led to the exploration of marine natural products as drug candidates to combat the debilitating effects of mucus accumulation and chronic inflammation. Previous research showed that an alga-derived compound, brevenal, could attenuate the effects of inflammatory agents, but the mechanisms by which it exerted its effects remained unclear. We investigated the effects of brevenal on lipopolysaccharide (LPS) induced cytokine/chemokine production from murine macrophages and human lung epithelial cells. It was found that brevenal reduces proinflammatory mediator secretion while preserving anti-inflammatory secretion from these cells. Furthermore, we found that brevenal does not alter cell surface Toll-like receptor 4 (TLR4) expression, thereby maintaining the cells’ ability to respond to bacterial infection. However, brevenal does alter macrophage activation states, as demonstrated by reduced expression of both M1 and M2 phenotype markers, indicating this putative anti-inflammatory drug shifts innate immune cells to a less active state. Such a mechanism of action would be ideal for reducing inflammation in the lung, especially with patients suffering from chronic respiratory diseases, where inflammation can be lethal.

Brevetoxins are a family of ladder-framed polyether toxins produced during blooms of the marine dinoflagellate, Karenia brevis. Consumption of shellfish or finfish exposed to brevetoxins can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are believed to be due to the activation of voltage-sensitive sodium channels in cell membranes. The traditional cytotoxicity assay for detection of brevetoxins uses the Neuro-2A cell line, which must first be treated with the neurotoxins, ouabain and veratridine, in order to become sensitive to brevetoxins. In this study, we demonstrate several drawbacks of the Neuro-2A assay, which include variability for the EC50 values for brevetoxin and non-linear triphasic dose response curves. Ouabain/veratridine-treated Neuro-2A cells do not show a typical sigmoidal dose response curve in response to brevetoxin, but rather, have a polynomial shaped curve, which makes calculating EC50 values highly variable. We describe a new fluorescence live cell imaging model, which allows for accurate calculation of cytotoxicity via nuclear staining and additional measurement of other viability parameters depending on which aspect of the cell is stained. In addition, the SJCRH30 cell line shows promise as an alternative to Neuro-2A cells for testing brevetoxins without the need for ouabain and veratridine.

Paralytic shellfish poisoning (PSP) is precipitated by a family of toxins produced by harmful algae, which are consumed by filter-feeding and commercially popular shellfish. The toxins, including saxitoxin, neosaxitoxin, and gonyautoxins, accumulate in shellfish and cause intoxication when consumed by humans and animals. Symptoms can range from minor neurological dysfunction to respiratory distress and death. There are over 40 different chemical congeners of saxitoxin and its analogs, many of which are toxic and many of which have low toxicity or are non-toxic. This makes accurate toxicity assessment difficult and complicates decisions regarding whether or not shellfish are safe to consume. In this study, we describe a new antibody-based bioassay that is able to detect toxic congeners (saxitoxin, neosaxitoxin, and gonyautoxins) with little cross-reactivity with the low or non-toxic congeners (decarbamoylated or di-sulfated forms). The anti-saxitoxin antibody used in this assay detects saxitoxin and neosaxitoxin, the two most toxic congers equally well, but not the relatively highly toxic gonyautoxins. By incorporating an incubation step with L-cysteine, it is possible to convert a majority of the gonyautoxins present to saxitoxin and neosaxitoxin, which are readily detected. The assay is, therefore, capable of detecting the most toxic PSP congeners found in commercially relevant shellfish. The assay was validated against samples whose toxicity was determined using standard HPLC methods and yielded a strong linear agreement between the methods, with R2 values of 0.94–0.96. As ELISAs are rapid, inexpensive, and easy-to-use, this new commercially available PSP ELISA represents an advance in technology allowing better safety management of the seafood supply and the ability to screen large numbers of samples that can occur when monitoring is increased substantially in response to toxic bloom events