PAR2, Keratinocytes, and Cathepsin S Mediate the Sensory Effects of Ciguatoxins Responsible for Ciguatera Poisoning.

Ciguatera fish poisoning is caused by the consumption of fish contaminated with ciguatoxins (CTXs). The most distressing symptoms are cutaneous sensory disturbances, including cold dysesthesia and itch. CTXs are neurotoxins known to activate voltage-gated sodium channels, but no specific treatment exists. Peptidergic neurons have been critically involved in ciguatera fish poisoning sensory disturbances. Protease-activated receptor-2 (PAR2) is an itch- and pain-related G protein‒coupled receptor whose activation leads to a calcium-dependent neuropeptide release. In this study, we studied the role of voltage-gated sodium channels, PAR2, and the PAR2 agonist cathepsin S in the cytosolic calcium increase and subsequent release of the neuropeptide substance P elicited by Pacific CTX-2 (P-CTX-2) in rat sensory neurons and human epidermal keratinocytes. In sensory neurons, the P-CTX-2‒evoked calcium response was driven by voltage-gated sodium channels and PAR2-dependent mechanisms. In keratinocytes, P-CTX-2 also induced voltage-gated sodium channels and PAR2-dependent marked calcium response. In the cocultured cells, P-CTX-2 significantly increased cathepsin S activity, and cathepsin S and PAR2 antagonists almost abolished P-CTX-2‒elicited substance P release. Keratinocytes synergistically favored the induced substance P release. Our results demonstrate that the sensory effects of CTXs involve the cathepsin S-PAR2 pathway and are potentiated by their direct action on nonexcitable keratinocytes through the same pathway.

Transcriptomic signatures in whole blood of patients who acquire a chronic inflammatory response syndrome (CIRS) following an exposure to the marine toxin ciguatoxin.

BACKGROUND: Ciguatoxins (CTXs) are polyether marine neurotoxins found in multiple reef-fish species and are potent activators of voltage-gated sodium channels. It is estimated that up to 500,000 people annually experience acute ciguatera poisoning from consuming toxic fish and a small percentage of these victims will develop a chronic, multisymptom, multisystem illness, which can last years, termed a Chronic Inflammatory Response Syndrome (CIRS). Symptoms of ciguatera CIRS include fatigue, cognitive deficits, neurologic deficits, pain and sensitivity to light. There are few treatment options for ciguatera CIRS since little is known about its pathophysiology. METHODS: This study characterizes the transcriptional profile in whole blood of 11 patients with ciguatera-induced CIRS and 11 normal controls run in duplicate using Agilent one color whole genome microarrays. Differential expression was determined by using a combination of moderated t-test p-value and fold change (FC). Significant genes were subjected to gene ontology, principal component analysis and SVM classification. Seven significant genes found by microarray were validated by PCR. RESULTS: Using a low stringency (p < 0.05 and FC > 1.4) and a high stringency (p < 0.01 and FC > 1.5) filter, the resulting gene sets of 185 and 55, respectively, showed clear separation of cases and controls by PCA as well as 100% classification accuracy by SVM, indicating that the gene profiles can separate patients from controls. PCR results of 7 genes showed a 95% correlation to microarray data. Several genes identified by microarray are important in wound healing (CD9, CD36, vWF and Factor XIII), adaptive immunity (HLA-DQB1, DQB2, IL18R1 and IL5RA) and innate immunity (GZMK, TOLLIP, SIGIRR and VIPR2), overlapping several areas shown to be disrupted in a mouse model of acute exposure to ciguatoxin. Another area of interest was differential expression of long, non-coding sequences, or lncRNA. CONCLUSIONS: Disruptions of innate and adaptive immune mechanisms were recorded at both the genomic and proteomic level. A disruption in the HLA-T cell receptor axis could indicate HLA haplotype sensitivity for this chronic syndrome, as noted in many autoimmune conditions. Taken together, these indicators of illness provide additional insights into pathophysiology and potential therapies.

Ciguatera fish toxicity in French Polynesia: size does not always matter.

Accumulation of ciguatoxins (CTXs) in tropical reef fish tissues during their life is responsible of the most prevalent human seafood intoxication in the South Pacific called Ciguatera Fish Poisoning (CFP). It has been assumed for a long time that CTXs are transferred and accumulated along the trophic food chain, and consequently that smaller individuals within a given fish species are safer to eat than larger ones. However, the relationship between toxicity and fish size has been studied for a limited number of species only and the conclusions are often contradictory. The toxicity of 856 fishes from 59 different species sampled in six islands in French Polynesia between 2003 and 2011 was assessed by Receptor Binding Assay. Among them, 45 species × island and 32 families × island for which the number of individuals was ≥6 allowed testing the relationship between toxicity and size. Except for six specimens of Lutjanus bohar caught in Fakarava (P < 0.01; R(2) = 0.854), the 44 remaining species × island showed no significant increase of CTXs concentration with fish total length (TL). Moreover, the proportion of toxic individuals decreased significantly for Epinephelus polyphekadion from Fakarava (n = 24; P < 0.05) and Kyphosus cinerascens from Raivavae (n = 29; P < 0.05), while no significant variation was detected for the other 43 species × island. At the family level, only three positive and three negative relationships between size and CTXs concentration were observed among the 32 family × island analyzed. No relationship between the proportion of toxic fish within a family and the relative total length of individuals were observed. The lack of relationship between toxicity and size observed for most of the species and families from the six islands suggests that fish size cannot be used as an efficient predictor of fish toxicity in French Polynesia. These results highlight the need for improving our knowledge about metabolic processes which may play a role in CTXs bio-accumulation and depuration among the different trophic levels of fishes.

Neurotoxicity and reactive astrogliosis in the anterior cingulate cortex in acute ciguatera poisoning.

Ciguatoxins (CTXs) cause long-term disturbance of cerebral functions. The primary mechanism of neurotoxicity is related to their interaction with voltage-gated sodium channels. However, until now, the neurological targets for CTXs in the brain of intact animals have not been described. In our study, 1 day following oral exposure to 0.26 ng/g of Pacific ciguatoxin 1 (P-CTX-1), we performed in vivo electrophysiological recordings in the rat anterior cingulate cortex (ACC) and identified the increase in spontaneous firings and enhanced responses to visceral noxious stimulation. Local field recordings characterized the P-CTX-1-induced synaptic potentiation and blockage of the induction of electrical stimulation-induced long-term potentiation in the medial thalamus (MT)-ACC pathway. Furthermore, intracerebroventricular administration of P-CTX-1 at doses of 1.0, 5.0, and 10 nM produced a dose-dependent increase in ACC neuronal firings and MT-ACC synaptic transmission. Further studies showed upregulated Na(+) channel expression in astrocytes under pathological conditions. We hypothesized that the astrocytes might have been activated in the ciguatera poisoning in vivo. Increases in glial fibrillary acid protein expression were detected in reactive astrocytes in the rat ACC. The activation of astroglia was further indicated by activation of the gap junction protein connexin 43 and upregulation of excitatory amino acid transporter 2 expression suggesting that glutamate was normally rapidly cleared from the synaptic cleft during acute ciguatera poisoning. However, neurotoxicity and reactive astrogliosis were not detected in the ACC after 7 days of P-CTX-1 exposure. The present results are the first characterization of P-CTX-1-invoked brain cortex neuronal excitotoxicity in vivo and supported the theme that neuron and astroglia signals might play roles in acute ciguatera poisoning.

Evaluation of seafood toxicity in the Australes archipelago (French Polynesia) using the neuroblastoma cell-based assay.

Ciguatera fish poisoning (CFP), a disease caused by consuming fish that have accumulated ciguatoxins (CTXs) in their tissue, is regarded as the most prevalent form of intoxication in French Polynesia. Recently, the Australes, one of the least affected archipelago until the early 1980s, has shown a dramatic increase in its incidence rates in 2009 with unusual CFP cases. In the present work, potential health hazards associated with the proliferation of various marine phytoplankton species and the consumption of fish and marine invertebrates highly popular among local population were assessed in three Australes islands: Raivavae, Rurutu and Rapa. Extracts from the marine dinoflagellates Gambierdiscus, Ostreospis and mat-forming cyanobacteria as well as fish, giant clams and sea urchin samples were examined for the presence of CTXs and palytoxin (PLTX) by using the neuroblastoma cell-based assay (CBA-N2a). Cytotoxic responses observed with both standards (Pacific CTX-3C and PLTX) and targeted marine products indicate that CBA-N2a is a robust screening tool, with high sensitivity and good repeatability and reproducibility. In Rurutu and Raivavae islands, our main findings concern the presence of CTX-like compounds in giant clams and sea urchins, suggesting a second bio-accumulation route for CFP toxins in the ciguatera food chain. In Rapa, the potential CFP risk from Gambierdiscus bloom and fish was confirmed for the first time, with levels of CTXs found above the consumer advisory level of 0.01 ng Pacific CTX-1B g(-1) of flesh in three fish samples. However, despite the presence of trace level of PLTX in Ostreopsis natural assemblages of Rapa, no sign of PLTX accumulation is yet observed in tested fish samples. Because this multi-toxinic context is likely to emerge in most French Polynesian islands, CBA-N2a shows great potential for future applications in the algal- and toxin-based field monitoring programmes currently on hand locally.

Normal axonal ion channel function in large peripheral nerve fibers following chronic ciguatera sensitization.

Although the acute clinical effects of ciguatera poisoning, due to ingestion of ciguatoxin, are mediated by activation of transient Na+ channels, the mechanisms underlying ciguatera sensitization remain undefined. Axonal excitability studies were performed by stimulating the median motor and sensory nerves in two patients with ciguatera sensitization. Excitability parameters were all within normal limits, thereby arguing against dysfunction of axonal membrane ion channels in large-diameter fibers in ciguatera sensitization.

Intoxicación por ciguatera. Estudio de 227 pacientes durante el periodo 1999 al 2005 / Ciguatera poisoning: Study of 277 patients in the 1999-2005 period

La ciguatera es la intoxicación alimentaría causada por el consumo de pescados ciguatóxicos, cuyos efectos sobre el ser humano pueden ser graves y conducir a la muerte. Se describieron las principales características epidemiológicas, clínicas y del tratamiento en los pacientes con ciguatera. La investigación incluyó 227 pacientes con ciguatera estudiados de forma prospectiva en el Hospital Clínico-Quirúrgico Universitario Comandante Manuel Fajardo de Ciudad de La Habana entre los años 1999 al 2005. Para la recolección de la muestra se elaboró una encuesta que recogió los datos demográficos, identidad personal, manifestaciones clínicas, tratamiento empleado en los pacientes, y la especie que causó la intoxicación. Se encontró que existe un incremento significativo en la incidencia de pacientes con ciguatera que acuden al hospital en los últimos 3 años. Los meses de abril a septiembre son los de mayor incidencia de la enfermedad. Hubo predominio de las mujeres sobre los hombres. Los pacientes comprendidos entre las edades de 21 y 50 años son los más afectados. El promedio de edad de los enfermos es de 39,2±10 años. Las especies barracuda y gallego fueron las que más frecuentemente provocaron la intoxicación en 74 y 38 pacientes, respectivamente y 73 intoxicados desconocían la especie ingerida. Los síntomas referidos con mayor frecuencia por los pacientes fueron la fatiga muscular, los trastornos gastrointestinales y las manifestaciones sensitivas. Los hallazgos más frecuentes al examen físico fueron los sensitivos, los signos de deshidratación y la debilidad muscular. El tiempo transcurrido entre la ingestión del pescado y el inicio del cuadro clínico fue menor o igual a las 12 horas en 70,8 por ciento de los casos. Los medicamentos que con más frecuencia se emplearon en los enfermos fueron los antieméticos, vitaminas, antihistamínicos y el manitol al 20 por ciento resultando este último la única terapia que revierte los síntomas en los pacientes con ciguatera.

Ciguatera is food poisoning caused by ingestion of ciguatoxic fish whose effects on the human beings could be severe and lead to death. The main epidemilogical, clinical and therapeutical characteristics in patients with ciguatera were described. The research covered 227 patients with ciguatera, who were prospectively studied at Comandante Manuel Fajardo clinical-surgical university hospital from 1999 to 2005. For sample collection, a survey was administered to gather demographic data, personal identity, clinical manifestations, treatment and the species causing the poisoning. It was found that the incidence of patients with ciguatera going to the hospital in the last three years had significantly increased. April to September was the most incidental period of the disease. Females predominated, the 21-50 years age group was the most affected and the average age of patients was 39,2±10 years. Barracuda and gallego were the main responsible for poisoning in 74 and 38 patients respectively. Seventy three poisoned persons did not know the fish species that they had eaten. The most frequent symptoms were muscular fatigue, gastrointestinal disorders and sensitive manifestations. On physical exam, the most common findings were sensitive manifestations, dehydration signs and muscle weakening. The lenght of time from fish ingestion to the onset of poisoning clinical picture was 12 hours or less in 70,8 percent of cases. The most used drugs were antiemetics, vitamins, antihystamines and 20 percent manitol, being the latter the only therapy capable of reversing symptoms in patients with ciguater.

Intoxicación por ciguatera. Estudio de 227 pacientes durante el periodo 1999 al 2005 / Ciguatera poisoning: Study of 277 patients in the 1999-2005 period

La ciguatera es la intoxicación alimentaría causada por el consumo de pescados ciguatóxicos, cuyos efectos sobre el ser humano pueden ser graves y conducir a la muerte. Se describieron las principales características epidemiológicas, clínicas y del tratamiento en los pacientes con ciguatera. La investigación incluyó 227 pacientes con ciguatera estudiados de forma prospectiva en el Hospital Clínico-Quirúrgico Universitario Comandante Manuel Fajardo de Ciudad de La Habana entre los años 1999 al 2005. Para la recolección de la muestra se elaboró una encuesta que recogió los datos demográficos, identidad personal, manifestaciones clínicas, tratamiento empleado en los pacientes, y la especie que causó la intoxicación. Se encontró que existe un incremento significativo en la incidencia de pacientes con ciguatera que acuden al hospital en los últimos 3 años. Los meses de abril a septiembre son los de mayor incidencia de la enfermedad. Hubo predominio de las mujeres sobre los hombres. Los pacientes comprendidos entre las edades de 21 y 50 años son los más afectados. El promedio de edad de los enfermos es de 39,2±10 años. Las especies barracuda y gallego fueron las que más frecuentemente provocaron la intoxicación en 74 y 38 pacientes, respectivamente y 73 intoxicados desconocían la especie ingerida. Los síntomas referidos con mayor frecuencia por los pacientes fueron la fatiga muscular, los trastornos gastrointestinales y las manifestaciones sensitivas. Los hallazgos más frecuentes al examen físico fueron los sensitivos, los signos de deshidratación y la debilidad muscular. El tiempo transcurrido entre la ingestión del pescado y el inicio del cuadro clínico fue menor o igual a las 12 horas en 70,8 por ciento de los casos. Los medicamentos que con más frecuencia se emplearon en los enfermos fueron los antieméticos, vitaminas, antihistamínicos y el manitol al 20 por ciento resultando este último la única terapia que revierte los síntomas en los pacientes con ciguatera(AU)

Ciguatera is food poisoning caused by ingestion of ciguatoxic fish whose effects on the human beings could be severe and lead to death. The main epidemilogical, clinical and therapeutical characteristics in patients with ciguatera were described. The research covered 227 patients with ciguatera, who were prospectively studied at Comandante Manuel Fajardo clinical-surgical university hospital from 1999 to 2005. For sample collection, a survey was administered to gather demographic data, personal identity, clinical manifestations, treatment and the species causing the poisoning. It was found that the incidence of patients with ciguatera going to the hospital in the last three years had significantly increased. April to September was the most incidental period of the disease. Females predominated, the 21-50 years age group was the most affected and the average age of patients was 39,2±10 years. Barracuda and gallego were the main responsible for poisoning in 74 and 38 patients respectively. Seventy three poisoned persons did not know the fish species that they had eaten. The most frequent symptoms were muscular fatigue, gastrointestinal disorders and sensitive manifestations. On physical exam, the most common findings were sensitive manifestations, dehydration signs and muscle weakening. The lenght of time from fish ingestion to the onset of poisoning clinical picture was 12 hours or less in 70,8 percent of cases. The most used drugs were antiemetics, vitamins, antihystamines and 20 percent manitol, being the latter the only therapy capable of reversing symptoms in patients with ciguater(AU)