Tissue Distribution and Metabolization of Ciguatoxins in an Herbivorous Fish following Experimental Dietary Exposure to Gambierdiscus polynesiensis.

Ciguatoxins (CTXs), potent neurotoxins produced by dinoflagellates of the genera Gambierdiscus and Fukuyoa, accumulate in commonly consumed fish species, causing human ciguatera poisoning. Field collections of Pacific reef fish reveal that consumed CTXs undergo oxidative biotransformations, resulting in numerous, often toxified analogs. Following our study showing rapid CTX accumulation in flesh of an herbivorous fish, we used the same laboratory model to examine the tissue distribution and metabolization of Pacific CTXs following long-term dietary exposure. Naso brevirostris consumed cells of Gambierdiscus polynesiensis in a gel food matrix over 16 weeks at a constant dose rate of 0.36 ng CTX3C equiv g-1 fish d-1. CTX toxicity determination of fish tissues showed CTX activity in all tissues of exposed fish (eight tissues plus the carcass), with the highest concentrations in the spleen. Muscle tissue retained the largest proportion of CTXs, with 44% of the total tissue burden. Moreover, relative to our previous study, we found that larger fish with slower growth rates assimilated a higher proportion of ingested toxin in their flesh (13% vs. 2%). Analysis of muscle extracts revealed the presence of CTX3C and CTX3B as well as a biotransformed product showing the m/z transitions of 2,3-dihydroxyCTX3C. This is the first experimental evidence of oxidative transformation of an algal CTX in a model consumer and known vector of CTX into the fish food web. These findings that the flesh intended for human consumption carries the majority of the toxin load, and that growth rates can influence the relationship between exposure and accumulation, have significant implications in risk assessment and the development of regulatory measures aimed at ensuring seafood safety.

Ciguatera in the Indian Ocean with Special Insights on the Arabian Sea and Adjacent Gulf and Seas: A Review.

The dinoflagellates of the genus Gambierdiscus are found in almost all oceans and seas between the coordinates 35° N and 35° S. Gambierdiscus and Fukuyoa are producers of ciguatoxins (CTXs), which are known to cause foodborne disease associated with contaminated seafood. The occurrence and effects of CTXs are well described in the Pacific and the Caribbean. However, historically, their properties and presence have been poorly documented in the Indian Ocean (including the Bay of Bengal, Andaman Sea, and the Gulf). A higher occurrence of these microorganisms will proportionately increase the likelihood of CTXs entering the food chain, posing a severe threat to human seafood consumers. Therefore, comprehensive research strategies are critically important for developing effective monitoring and risk assessments of this emerging threat in the Indian Ocean. This review presents the available literature on ciguatera occurrence in the region and its adjacent marginal waters: aiming to identify the data gaps and vectors.

Experimental Evidence of Ciguatoxin Accumulation and Depuration in Carnivorous Lionfish.

Ciguatera poisoning is a food intoxication associated with the consumption of fish or shellfish contaminated, through trophic transfer, with ciguatoxins (CTXs). In this study, we developed an experimental model to assess the trophic transfer of CTXs from herbivorous parrotfish, Chlorurus microrhinos, to carnivorous lionfish, Pterois volitans. During a 6-week period, juvenile lionfish were fed naturally contaminated parrotfish fillets at a daily dose of 0.11 or 0.035 ng CTX3C equiv. g-1, as measured by the radioligand-receptor binding assay (r-RBA) or neuroblastoma cell-based assay (CBA-N2a), respectively. During an additional 6-week depuration period, the remaining fish were fed a CTX-free diet. Using r-RBA, no CTXs were detectable in muscular tissues, whereas CTXs were measured in the livers of two out of nine fish sampled during exposure, and in four out of eight fish sampled during depuration. Timepoint pooled liver samples, as analyzed by CBA-N2a, confirmed the accumulation of CTXs in liver tissues, reaching 0.89 ng CTX3C equiv. g-1 after 41 days of exposure, followed by slow toxin elimination, with 0.37 ng CTX3C equiv. g-1 measured after the 6-week depuration. These preliminary results, which need to be pursued in adult lionfish, strengthen our knowledge on CTX transfer and kinetics along the food web.

Human Health and Ocean Pollution.

Background: Pollution - unwanted waste released to air, water, and land by human activity - is the largest environmental cause of disease in the world today. It is responsible for an estimated nine million premature deaths per year, enormous economic losses, erosion of human capital, and degradation of ecosystems. Ocean pollution is an important, but insufficiently recognized and inadequately controlled component of global pollution. It poses serious threats to human health and well-being. The nature and magnitude of these impacts are only beginning to be understood. Goals: (1) Broadly examine the known and potential impacts of ocean pollution on human health. (2) Inform policy makers, government leaders, international organizations, civil society, and the global public of these threats. (3) Propose priorities for interventions to control and prevent pollution of the seas and safeguard human health. Methods: Topic-focused reviews that examine the effects of ocean pollution on human health, identify gaps in knowledge, project future trends, and offer evidence-based guidance for effective intervention. Environmental Findings: Pollution of the oceans is widespread, worsening, and in most countries poorly controlled. It is a complex mixture of toxic metals, plastics, manufactured chemicals, petroleum, urban and industrial wastes, pesticides, fertilizers, pharmaceutical chemicals, agricultural runoff, and sewage. More than 80% arises from land-based sources. It reaches the oceans through rivers, runoff, atmospheric deposition and direct discharges. It is often heaviest near the coasts and most highly concentrated along the coasts of low- and middle-income countries. Plastic is a rapidly increasing and highly visible component of ocean pollution, and an estimated 10 million metric tons of plastic waste enter the seas each year. Mercury is the metal pollutant of greatest concern in the oceans; it is released from two main sources - coal combustion and small-scale gold mining. Global spread of industrialized agriculture with increasing use of chemical fertilizer leads to extension of Harmful Algal Blooms (HABs) to previously unaffected regions. Chemical pollutants are ubiquitous and contaminate seas and marine organisms from the high Arctic to the abyssal depths. Ecosystem Findings: Ocean pollution has multiple negative impacts on marine ecosystems, and these impacts are exacerbated by global climate change. Petroleum-based pollutants reduce photosynthesis in marine microorganisms that generate oxygen. Increasing absorption of carbon dioxide into the seas causes ocean acidification, which destroys coral reefs, impairs shellfish development, dissolves calcium-containing microorganisms at the base of the marine food web, and increases the toxicity of some pollutants. Plastic pollution threatens marine mammals, fish, and seabirds and accumulates in large mid-ocean gyres. It breaks down into microplastic and nanoplastic particles containing multiple manufactured chemicals that can enter the tissues of marine organisms, including species consumed by humans. Industrial releases, runoff, and sewage increase frequency and severity of HABs, bacterial pollution, and anti-microbial resistance. Pollution and sea surface warming are triggering poleward migration of dangerous pathogens such as the Vibrio species. Industrial discharges, pharmaceutical wastes, pesticides, and sewage contribute to global declines in fish stocks. Human Health Findings: Methylmercury and PCBs are the ocean pollutants whose human health effects are best understood. Exposures of infants in utero to these pollutants through maternal consumption of contaminated seafood can damage developing brains, reduce IQ and increase children's risks for autism, ADHD and learning disorders. Adult exposures to methylmercury increase risks for cardiovascular disease and dementia. Manufactured chemicals - phthalates, bisphenol A, flame retardants, and perfluorinated chemicals, many of them released into the seas from plastic waste - can disrupt endocrine signaling, reduce male fertility, damage the nervous system, and increase risk of cancer. HABs produce potent toxins that accumulate in fish and shellfish. When ingested, these toxins can cause severe neurological impairment and rapid death. HAB toxins can also become airborne and cause respiratory disease. Pathogenic marine bacteria cause gastrointestinal diseases and deep wound infections. With climate change and increasing pollution, risk is high that Vibrio infections, including cholera, will increase in frequency and extend to new areas. All of the health impacts of ocean pollution fall disproportionately on vulnerable populations in the Global South - environmental injustice on a planetary scale. Conclusions: Ocean pollution is a global problem. It arises from multiple sources and crosses national boundaries. It is the consequence of reckless, shortsighted, and unsustainable exploitation of the earth's resources. It endangers marine ecosystems. It impedes the production of atmospheric oxygen. Its threats to human health are great and growing, but still incompletely understood. Its economic costs are only beginning to be counted.Ocean pollution can be prevented. Like all forms of pollution, ocean pollution can be controlled by deploying data-driven strategies based on law, policy, technology, and enforcement that target priority pollution sources. Many countries have used these tools to control air and water pollution and are now applying them to ocean pollution. Successes achieved to date demonstrate that broader control is feasible. Heavily polluted harbors have been cleaned, estuaries rejuvenated, and coral reefs restored.Prevention of ocean pollution creates many benefits. It boosts economies, increases tourism, helps restore fisheries, and improves human health and well-being. It advances the Sustainable Development Goals (SDG). These benefits will last for centuries. Recommendations: World leaders who recognize the gravity of ocean pollution, acknowledge its growing dangers, engage civil society and the global public, and take bold, evidence-based action to stop pollution at source will be critical to preventing ocean pollution and safeguarding human health.Prevention of pollution from land-based sources is key. Eliminating coal combustion and banning all uses of mercury will reduce mercury pollution. Bans on single-use plastic and better management of plastic waste reduce plastic pollution. Bans on persistent organic pollutants (POPs) have reduced pollution by PCBs and DDT. Control of industrial discharges, treatment of sewage, and reduced applications of fertilizers have mitigated coastal pollution and are reducing frequency of HABs. National, regional and international marine pollution control programs that are adequately funded and backed by strong enforcement have been shown to be effective. Robust monitoring is essential to track progress.Further interventions that hold great promise include wide-scale transition to renewable fuels; transition to a circular economy that creates little waste and focuses on equity rather than on endless growth; embracing the principles of green chemistry; and building scientific capacity in all countries.Designation of Marine Protected Areas (MPAs) will safeguard critical ecosystems, protect vulnerable fish stocks, and enhance human health and well-being. Creation of MPAs is an important manifestation of national and international commitment to protecting the health of the seas.

How Do Actinyls Interact with Hyperphosphorylated Yolk Protein Phosvitin?

The development of the nuclear industry has raised multiple questions about its impact on the biotope and humans. Proteins are key biomolecules in cell machinery and essential in deciphering toxicological processes. Phosvitin was chosen as a relevant model for phosphorylated proteins because of its important role as an iron, calcium, and magnesium storage protein in egg yolk. A multitechnique spectroscopic investigation was performed to reveal the coordination geometry of two oxocations of the actinide family (actinyl UVI , NpV ) in speciation with phosvitin. IR spectroscopy revealed phosphoryl groups as the main functional groups interacting with UVI . This was confirmed through laser luminescence spectroscopy (U) and UV/Vis absorption spectroscopy (Np). For UVI , X-ray absorption spectroscopy at the LIII edge revealed a small contribution of bidentate binding present, along with predominantly monodentate binding of phosphoryl groups; for NpV , uniquely bidentate binding was revealed. As a perspective to this work, X-ray absorption spectroscopy speciation of UVI and NpV in the extracted yolk of living eggs of the dogfish Scyliorhinus canicula was determined; this corroborated the binding of phosphorous together with a reduction of the actinyl moiety. Such data are essential to pinpoint the mechanisms of heavy metals (actinyls) accumulation and toxicity in oviparous organisms, and therefore, contribute to a shift from descriptive approaches to predictive toxicology.

Further insights into brevetoxin metabolism by de novo radiolabeling.

The toxic dinoflagellate Karenia brevis, responsible for early harmful algal blooms in the Gulf of Mexico, produces many secondary metabolites, including potent neurotoxins called brevetoxins (PbTx). These compounds have been identified as toxic agents for humans, and they are also responsible for the deaths of several marine organisms. The overall biosynthesis of these highly complex metabolites has not been fully ascertained, even if there is little doubt on a polyketide origin. In addition to gaining some insights into the metabolic events involved in the biosynthesis of these compounds, feeding studies with labeled precursors helps to discriminate between the de novo biosynthesis of toxins and conversion of stored intermediates into final toxic products in the response to environmental stresses. In this context, the use of radiolabeled precursors is well suited as it allows working with the highest sensitive techniques and consequently with a minor amount of cultured dinoflagellates. We were then able to incorporate [U-¹4C]-acetate, the renowned precursor of the polyketide pathway, in several PbTx produced by K. brevis. The specific activities of PbTx-1, -2, -3, and -7, identified by High-Resolution Electrospray Ionization Mass Spectrometer (HRESIMS), were assessed by HPLC-UV and highly sensitive Radio-TLC counting. We demonstrated that working at close to natural concentrations of acetate is a requirement for biosynthetic studies, highlighting the importance of highly sensitive radiolabeling feeding experiments. Quantification of the specific activity of the four, targeted toxins led us to propose that PbTx-1 and PbTx-2 aldehydes originate from oxidation of the primary alcohols of PbTx-7 and PbTx-3, respectively. This approach will open the way for a better comprehension of the metabolic pathways leading to PbTx but also to a better understanding of their regulation by environmental factors.

Identification of ciguatoxins in Hawaiian monk seals Monachus schauinslandi from the Northwestern and Main Hawaiian Islands.

Ciguatoxins are potent algal neurotoxins that concentrate in fish preyed upon by the critically endangered Hawaiian monk seal (Monachus schauinslandi). The only report for Hawaiian monk seal exposure to ciguatoxins occurred during a 1978 mortality event when two seal liver extracts tested positive by mouse bioassay. Ciguatoxins were thus proposed as a potential threat to the Hawaiian monk seal population. To reinvestigate monk seal exposure to ciguatoxins we utilized more selective detection methods, the Neuro-2A cytotoxicity assay, to quantify ciguatoxin activity and an analytical method LC-MS/MS to confirm the molecular structure. Tissue analysis from dead stranded animals revealed ciguatoxin activity in brain, liver, and muscle, whereas analysis of blood samples from 55 free-ranging animals revealed detectable levels of ciguatoxin activity (0.43 to 5.49 pg/mL P-CTX-1 equiv) in 19% of the animals. Bioassay-guided LC fractionation of two monk seal liver extracts identified several ciguatoxin-like peaks of activity including a peak corresponding to the P-CTX-3C which was confirmed present by LC-MS/MS. In conclusion, this work provides first confirmation that Hawaiian monk seals are exposed to significant levels of ciguatoxins and first evidence of transfer of ciguatoxin to marine mammals. This threat could pose management challenges for this endangered marine mammal species.

Toxicokinetics of the ciguatoxin P-CTX-1 in rats after intraperitoneal or oral administration.

Ciguatoxins are voltage-gated selective algal toxins responsible for ciguatera fish poisoning. In this study we evaluate the toxicokinetics of one of the most common ciguatoxins found in the Pacific, the P-CTX-1, in rat after an oral or intraperitoneal (ip) dose of 0.26 µg/kg body weight. We report levels of ciguatoxin activity assessed over time in blood, urine and feces, and at 4 days in liver, muscle and brain, using the functional in vitro N2A cytotoxicity assay. Following exposure, the ciguatoxin activity exhibited a rapid systemic absorption that was followed by a bi-exponential decline, and data best fit a two-compartment model analysis. Maximum blood concentrations were reached at 1.97 and 0.43 h after the oral and ip dose, respectively. Ciguatoxin elimination from blood was slow with terminal half lives (t(½)ß) estimated at 82 h for oral and 112 h for ip dosing. Ciguatoxin activity remained in liver, muscle and brain 96 h after ip and oral administration. While smaller amounts appeared in the urine, the main excretion route was feces, with peak rates reaching > 10 pg P-CTX-1 equivalents/h in both routes of administration. Assay guided fractionation showed the presence in the feces and liver of peaks of activity corresponding to the P-CTX-1 and to other less polar metabolites. In conclusion, biologically active ciguatoxins are detectable in blood, liver, muscle and brain, and continued to be excreted in urine and feces 4 days following exposure. Blood, as well as urine and feces may be useful matrices for low-invasive testing methods for ciguatera clinical cases.

Gene expression profiling in brain of mice exposed to the marine neurotoxin ciguatoxin reveals an acute anti-inflammatory, neuroprotective response.

BACKGROUND: Ciguatoxins (CTXs) are polyether marine neurotoxins and potent activators of voltage-gated sodium channels. This toxin is carried by multiple reef-fish species and human consumption of ciguatoxins can result in an explosive gastrointestinal/neurologic illness. This study characterizes the global transcriptional response in mouse brain to a symptomatic dose of the highly toxic Pacific ciguatoxin P-CTX-1 and additionally compares this data to transcriptional profiles from liver and whole blood examined previously. Adult male C57/BL6 mice were injected with 0.26 ng/g P-CTX-1 while controls received only vehicle. Animals were sacrificed at 1, 4 and 24 hrs and transcriptional profiling was performed on brain RNA with Agilent whole genome microarrays. RT-PCR was used to independently validate gene expression and the web tool DAVID was used to analyze gene ontology (GO) and molecular pathway enrichment of the gene expression data. RESULTS: A pronounced 4°C hypothermic response was recorded in these mice, reaching a minimum at 1 hr and lasting for 8 hrs post toxin exposure. Ratio expression data were filtered by intensity, fold change and p-value, with the resulting data used for time course analysis, K-means clustering, ontology classification and KEGG pathway enrichment. Top GO hits for this gene set included acute phase response and mono-oxygenase activity. Molecular pathway analysis showed enrichment for complement/coagulation cascades and metabolism of xenobiotics. Many immediate early genes such as Fos, Jun and Early Growth Response isoforms were down-regulated although others associated with stress such as glucocorticoid responsive genes were up-regulated. Real time PCR confirmation was performed on 22 differentially expressed genes with a correlation of 0.9 (Spearman's Rho, p < 0.0001) with microarray results. CONCLUSIONS: Many of the genes differentially expressed in this study, in parallel with the hypothermia, figure prominently in protection against neuroinflammation. Pathologic activity of the complement/coagulation cascade has been shown in patients suffering from a chronic form of ciguatera poisoning and is of particular interest in this model. Anti-inflammatory processes were at work not only in the brain but were also seen in whole blood and liver of these animals, creating a systemic anti-inflammatory environment to protect against the initial cellular damage caused by the toxin.

Bioassay methods for detection of N-palmitoylbrevetoxin-B2 (BTX-B4).

Brevetoxins (BTXs) are a class of cyclic polyether toxins produced by the dinoflagellate Karenia brevis. These substances are subject to extensive conjugative metabolism in shellfish. BTX-B forms a conjugate with cysteine and is oxidized and reduced to yield BTX-B2, which is further modified by fatty acid addition via cysteine amide linkage to give biologically active brevetoxin metabolites. In this study, we evaluated the commonly used in vitro (ELISA, radioimmunoassay, receptor binding assay and N2A cytotoxicity assay) and in vivo mouse brevetoxin bioassays for the detection of the brevetoxin fatty acid conjugate N-palmitoylBTX-B2, and compared the results to those for dihydroBTX-B and BTX-B2. The receptor binding assay for N-palmitoylBTX-B2 showed comparable sensitivity to that for dihydroBTX-B, and an 11-fold higher sensitivity than for BTX-B2. Although the ELISA showed similarly high sensitivity to dihydroBTX-B and BTX-B2, with EC(50) values of ca. 0.26 ng/ml, it was 23 times less sensitive to N-palmitoylBTX-B2. On the other hand, the N2A cytotoxicity assay was highly sensitive to N-palmitoylBTX-B2, with an EC(50) of 0.15 ng/ml, but was 12- and 40-fold less sensitive to dihydroBTX-B and BTX-B2, respectively. The relative sensitivity of the N2A cytotoxicity assay for each of these metabolites paralleled that of the mouse bioassay (relative LD(50) values 1:20:30 for N-palmitoylBTX-B2:dihydroBTX-B:BTX-B2). We conclude that the most sensitive bioassay for dihydroBTX-B and BTX-B2 is the ELISA, whereas the N2A cytotoxicity assay is most sensitive for N-palmitoylBTX-B2.