Ciguatoxins (CTXs) contaminate fish worldwide and cause the foodborne illness ciguatera. In the Pacific, these toxins are produced by the dinoflagellate Gambierdiscus toxicus, which accumulates in fish through the food chain and undergoes oxidative modification, giving rise to numerous analogs. In this study, we examined the oxidation of CTXs in vitro with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using reference toxins, and found that CTX4A, CTX4B, and CTX3C, which are produced by the alga, are oxidized to the analogs found in fish, namely CTX1B, 52-epi-54-deoxyCTX1B, 54-deoxyCTX1B, 2-hydroxyCTX3C, and 2,3-dihydroxyCTX3C. This oxidation was catalyzed by human CYP3A4, fish liver S9 fractions, and microsomal fractions prepared from representative ciguateric fishes (Lutjanus bohar, L. monostigumus, and Oplegnathus punctatus). In addition, fish liver S9 fractions prepared from non-ciguateric fishes (L. gibbus and L. fulviflamma) in Okinawa also converted CTX4A and CTX4B to CTX1B, 54-deoxyCTX1B, and 52-epi-54-deoxyCTX1B in vitro. This is the first study to demonstrate the enzymatic oxidation of these toxins, and provides insight into the mechanism underlying the development of species-specific toxin profiles and the fate of these toxins in humans and fish.

Ciguatera fish poisoning (CFP) is one of the most frequently reported seafood poisoning diseases. It is endemic to the tropical region and occurs most commonly in the regions around the Pacific Ocean, Indian Ocean, and Caribbean Sea. The principal toxins causing CFP are ciguatoxins (CTXs). In the Pacific region, more than 20 analogs of CTXs have been identified to date. Based on their skeletal structures, they are classified into CTX1B-type and CTX3C-type toxins. We have previously reported species-specific and regional-specific toxin profiles. In this study, the levels and profiles of CTXs in fish present in the tropical western Pacific regions were analyzed using the liquid chromatography–tandem mass spectrometry (LC–MS/MS) technique. Forty-two fish specimens, belonging to the categories of snappers, groupers, Spanish mackerel, and moray eel, were purchased from various places such as Fiji, the Philippines, Thailand, and Taiwan. Only the fish captured from Fijian coastal waters contained detectable amounts of CTXs. The toxin levels in the fish species found along the coastal regions of the Viti Levu Island, the main island in Fiji, and the toxin profiles were significantly different from those of the fish species present in other coastal regions. The toxin levels and profiles varied among the different fish samples collected from different coastal areas. Based on the toxin levels and toxin profiles, the coast was demarcated into three zones. In Zone-1, which covers the northern coast of the main island and the regions of the Malake Island and Korovau, CTXs in fish were below the detection level. In Zone-2, CTX3C-type toxins were present in low levels in the fish. CTX1B-type and CTX3C-type toxins co-occurred in the fish present in Zone-3. The toxin profiles may have reflected the variation in Gambierdiscus spp.

Ciguatera fish poisoning (CFP) is one of the most frequently encountered seafood poisoning syndromes; it is caused by the consumption of marine finfish contaminated with ciguatoxins (CTXs). The majority of CFP cases result from eating fish flesh, but a traditional belief exists among people that the head and viscera are more toxic and should be avoided. Unlike the viscera, scientific data to support the legendary high toxicity of the head is scarce. We prepared tissue samples from the fillet, head, and eyes taken from five yellow-edged lyretail (Variola louti) individuals sourced from Okinawa, Japan, and analyzed the CTXs by LC-MS/MS. Three CTXs, namely, CTX1B, 52-epi-54-deoxyCTX1B, and 54-deoxyCTX1B, were confirmed in similar proportions. The toxins were distributed nearly evenly in the flesh, prepared separately from the fillet and head. Within the same individual specimen, the flesh in the fillet and the flesh from the head, tested separately, had the same level and composition of toxins. We, therefore, conclude that flesh samples for LC-MS/MS analysis can be taken from any part of the body. However, the tissue surrounding the eyeball displayed CTX levels two to four times higher than those of the flesh. The present study is the first to provide scientific data demonstrating the high toxicity of the eyes.

Ingesting fish contaminated with ciguatoxins (CTXs) originating from epibenthic dinoflagellates causes ciguatera fish poisoning (CFP). CFP occurs mainly in the tropical and subtropical Indo–Pacific region and the Caribbean Sea. Furthermore, it occurs sporadically in Japan, especially in the Ryukyu Islands between Taiwan and Kyushu, Japan. Variola louti is the most frequently implicated fish with a suggested toxin profile, consisting of ciguatoxin-1B and two deoxy congeners. Therefore, using the liquid chromatography–tandem mass spectrometry (LC-MS/MS), we analyzed CTXs in the flesh of 154 individuals from various locations and detected CTXs in 99 specimens (64%). In 65 fish (43%), CTX levels exceeded the Food and Drug Administration (FDA) guidance level (0.01 µg/kg). Furthermore, in four specimens (3%), the guideline level in Japan (>0.18 µg/kg) was met. Additionally, although the highest total CTX level was 0.376 µg/kg, the consumption of 180 g of this specimen was assumed to cause CFP. Moreover, only CTX1B, 52-epi-54-deoxyCTX1B, and 54-deoxyCTX1B were detected, with the relative contribution of the three CTX1B analogs to the total toxin content (35 ± 7.7 (SD)%, 27 ± 8.1%, and 38 ± 5.6%, respectively) being similar to those reported in this region in a decade ago. Subsequently, the consistency of the toxin profile in V. louti was confirmed using many specimens from a wide area. As observed, total CTX levels were correlated with fish sizes, including standard length (r = 0.503, p = 3.08 × 10⁻¹¹), body weight (r = 0.503, p = 3.01 × 10⁻¹¹), and estimated age (r = 0.439, p = 3.81 × 10⁻⁷) of the specimens. Besides, although no correlation was observed between condition factor (CF) and total CTX levels, a significance difference was observed (p = 0.039) between the groups of skinnier and fattier fish, separated by the median CF (3.04). Results also showed that the CF of four specimens with the highest CTX level (>0.18 µg/kg) ranged between 2.49 and 2.87, and they were skinnier than the average (3.03) and median of all specimens.

Microcystins (MCs) are a group of cyclic heptapeptide hepatotoxins produced by Microcystis and several other genera of cyanobacteria. Many structural variants have been characterized using various methods such as liquid chromatography–mass spectrometry (LC-MS) analysis, enzyme-linked immunosorbent assay (ELISA) and protein phosphatase 2A (PP2A) inhibition assay. The representative MC, MC-LR, and related cyanobacterial toxins strongly inhibit PP2A activity and can therefore be assayed by measuring the extent of PP2A inhibition. However, these methods require reference toxin standards for the quantification and identification of known MCs. To obtain various MC-producing cyanobacterial strains, we surveyed and collected MC-producing cyanobacteria from environmental sources of water in Okinawa, Japan. Using a dual assay (LC-MS analysis and PP2A inhibition assay), we identified and isolated Microcystis strains producing five MC variants (MC-LR, -RR, -LA, -FR and -WR). Approximately 4 mg of MC-WR and -FR toxins were purified from the laboratory culture of the Microcystis isolate NIES-4344. Pure MC-WR and -FR variants were prepared for future use as toxin standards in LC-MS analysis. Phylogenetic analysis based on ftsZ revealed that the NIES-4344 strain belongs to the identified groups in Microcystis aeruginosa. This is the first report of Microcystis strains producing mainly MC-WR and -FR toxins in Japan.

Ciguatera fish poisoning (CFP) refers to an illness caused by ingesting fish that have accumulated ciguatoxins (CTXs). CFP frequently occurs in the tropical and subtropical Indo-Pacific Ocean and the Caribbean Sea. In Japan, CFP occurs sporadically but constantly in Okinawa and the Amami Islands. The grouper Variola albimarginata is regarded to be safe for consumption. To assess the real risk of V. albimarginata, we analyzed 133 specimens of the fish in Okinawa using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Ciguatoxin-1B, 54-deoxyciguatoxin-1B, and 52-epi-54-deoxyciguatoxin-1B were detected in 28 specimens (21%). In 11 of these specimens (8%), the CTX levels exceeded the US FDA guidance level (0.01 µg/kg CTX1B equivalent). However, only one fish (<1%) was found to have levels above the recommended level in Japan (0.175 μg/kg CTX1B equivalent). The amount of CTXs in the flesh (280 g) of the most toxic specimen (0.225 μg/kg) did not reach the level needed to cause illness. The CFP risk due to the consumption of this species was thus considered to be low in Okinawa, supporting local belief. The CTX levels in the flesh were positively correlated with standard length, body weight, and age. The total CTX levels significantly fluctuated between the male and the female of the species. The estimated annual catch of V. albimarginata in Okinawa and Yaeyama Islands was 4909 kg or 13,636 fish. As many as 1227 fish had levels over the US FDA guidance level, but only 136 fish had levels above the Japanese recommendation. Risk management based on the Japanese recommendation level seems to be effective in protecting public health and enabling appropriate exploitation of fishery resources.