3. Dinoflagellate Phycotoxins and Their Modes of Action

Table 1 summarizes the reported dinoflagellate-produced phycotoxins. Alexandrium spp. are considered PSP-producing harmful organisms. The causative paralytic toxins of Alexandrium spp. include STX, gonyautoxin (GTX), neosaxitoxin (NSTX), and HL [33,34,35,36]. Among these, STXs are well-known marine algal toxins that block the cellular sodium channel. STX is included in the guanidinium neurotoxin group, sharing the common chemical feature of guanidinium moieties and interacting with voltage-gated sodium channels with high binding affinity and ion flux blockage capacity. This blockage induces the reduced influx of Na⁺ ions into a cell and causes inhibition of the propagation of action potentials in excitable membranes. This process ultimately induces neuromuscular paralysis [37]. The symptoms of PSP induced by STX and its analogue, NSTX, include numbness of the lips and gastrointestinal disorders [35]. Lefebvre et al. [34] reported that measurable levels of STX were detected in both field and cultured Alexandrium spp. using a receptor-binding assay and enzyme-linked immunosorbent assay. The structure and analogues of STX were previously well-described by Wiese et al. [38]. Gymnodinium catenatum and Pyrodinium bahamense produce STXs as well [39,40]. Landsberg et al. [40] reported that the source of STXs detected in pufferfish skin, muscle, and viscera was putatively derived from P. bahamense. In addition, Sako et al. [39] purified and characterized sulfotransferase-specific STX analogues from the cytosolic fraction of G. catenatum. GTXs influence the mammalian nervous system by binding to site 1 of the α-subunit of the voltage-dependent sodium channel in the postsynaptic membrane, thereby preventing synaptic function [41].

Altered hemolytic activity of A. peruvianum under different nutrient ratios indicated the presence of cellular HL [42]. Although the modes of action of dinoflagellate HL are poorly understood and algal species-specific, a possible mechanism could be the hydrolysis of phospholipids and subsequent pore formation in phospholipid bilayers, a mechanism similar to other hemolytic toxins [43,44]. Modes of action of HLs detected in Amphidinium carterae, C. polykrikoides, Heterocapsa circularisquama, K. mikimotoi, and Gonyaulax monilata are described briefly in Table 1 [36,45,46,47,48].

Azadinium spinosum produces azaspiracids (AZA), a group of toxic lipophilic polyether compounds. This toxin caused human intoxication symptoms, such as nausea, vomiting, severe diarrhea, and stomach cramps in a study conducted in the Netherlands [49,50]. AZA was originally believed to be a toxic compound produced by Protoperidinium crassipes [51]. However, it was later demonstrated that the toxins in P. crassipes were a consequence of its feeding on the dinoflagellate A. spinosum, which was in turn reported to be the source of the AZA [52]. This toxin causes damage to the intestinal epithelium, lamina propria, liver, and villi as an acute toxic effect, and causes lung tumors and malignant lymphomas at high concentrations with long-term exposure [53,54,55]. AZAs include more than 30 analogues, and among these, only AZA1, AZA2, and AZA3 are currently regulated in edible shellfish by the European Union through their toxic equivalency factors (TEFs) [56]. It exhibited its action by blocking the human ether-a-go-go-related gene (hERG) potassium channel [57]. AZA interacts with the channel’s central pore (F656) within the S6 transmembrane domain and physically blocks the potassium-conductance pathway of the hERG1 channels [57]. Pelin et al. [58] previously reported that the exposure of immortalized human hepatocyte (IHH) cell line to AZA analogues induced mitochondrial electron transport chain complex-dependent mitochondrial dehydrogenases activity (MDA) in a concentration-dependent manner. The MDA was suppressed in the K⁺, Cl⁻, and Na⁺ free media, and by specific inhibitors of K_ATP (glibenclamide), hERG potassium channels (cisapride), Na⁺/K⁺, ATPase (ouabain), and cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels (CFTR(inh)-172). These results revealed that the AZA-induced MDA is derived from an imbalance of intracellular levels of K⁺ and Cl⁻ ions [58]. The toxic effects, structure, and analogues of AZA were well-described in previous studies [50,59,60,61,62,63].

Another dinoflagellate genus, Coolia, produces cooliatoxin (CTX) and YTX [65,66]. Holmes et al. [65] purified a novel toxin from C. monotis isolated from Australia and named it CTX. This toxin is considered a monosulfate polyether analogue of YTX and caused initial blockage of unmyelinated nerves in vitro, as reported by Holmes et al. [65]. Additionally, sulfated polyether analogues of YTX have been detected in C. malayensis through chemical analysis using NanoLiquid chromatography–mass spectrometry [66]. YTX is a diarrhea-causing toxin and exhibits its toxicity by activating nifedipine and the SKF-96365 sensitive calcium channel [68], and by decreasing cytosolic 3′,5′-cyclic adenosine monophosphate (cAMP) levels [69]. YTX-producing dinoflagellates include Gonyaulax spinifera, Lingulodinium polyedrum, and Protoceratium reticulatum [80,89]. The structure and analogues of YTX were previously well-described by Paz et al. [96].

Karlodinium armiger produces karmitoxin (KTX), which is an amine-containing polyhydroxy-polyene toxin [82]. Although its specific mode of action is not yet identified, ichthyotoxic effects of this toxin toward fish larvae and juveniles have been demonstrated recently [97]. The structure of KTX was previously reported by Rasmussen et al. [82]. Additionally, Karlodinium spp. produces karlotoxin (KmTx), which is structurally similar to amphidinols and is the causative toxin for membrane permeabilization [83]. KmTx is produced by K. armiger and K. veneficum, and its mode of mechanism is the disruption of the cell membrane by specifically binding to cholesterol [82]. The structure and several analogues of KmTx were reported by Van Wagoner et al. [98].

Dinophysis is a medium-sized dinoflagellate that produces DSP toxins, including okadaic acid (OA), pectenotoxin (PTX), and dinophysistoxin (DPX) [36,73]. OA is a polyether fatty acid, and its structure is highly similar to that of acanthifolicin [71]. DPXs are considered analogues of OA, whereas PTXs are a type of polyether lactones [99,100]. The mode of action of OA and DPXs is an inhibition of the serine/threonine (Ser/Thr) phosphatases that further induces tumor growth promotion and neuronal cell death [71,72,101,102]. The structures and analogues of OA and DPX were previously reported by Uchida et al. [103] and Fernandez et al. [104]. PTXs demonstrate diverse physiological functions, including inhibiting Ser/Thr phosphatases, depolymerizing actin filaments, and disrupting the actin cytoskeleton [35,36,76,77,78]. According to Espiña et al. [105], marked depolymerization of F-actin, associated with an improved G-actin level in hepatocyte cell line by PTX-1, PTX-2, and PTX-11 (1–1000 nM) treatments, was observed via confocal image analysis. However, no activity was observed by treatment with PTX-2 seco acid (PTX-2 SA), which is an enzymatically digested derivative of PTX-2 [105]. PTXs were initially classified into the DSP-producing group; however, mice toxicity tests confirmed that this toxin does not induce diarrheic symptoms but causes severe hepatotoxicity [106]. Specifically, Miles et al. [107] previously developed an effective method to isolate pectenotoxins from dinoflagellate cells, and they showed isolated PTX-2 caused acute toxicity in mice, whereas its derivative, PTX-2 SA, had no effect at 5000 µg/kg. Additionally, no diarrhea was observed in mice receiving either PTX-2 or PTX-2 SA treatments [107]. The structures and analogues of PTXs were previously described by Allingham et al. [76] and Wilkins et al. [108]. OA can be produced by dinoflagellates D. acuta, D. acuminate, D. fortii, Prorocentrum concavum, P. rhathymum, P. belizeanum, P. lima, and P. arenarium [109,110,111,112]. DPXs are produced by D. acuta, D. acuminate, P. foraminosum, and P. lima [93,113,114,115], whereas PTXs are produced by D. fortii, D. acuta, D. acuminate, and D. caudata [109,116,117].

Gambierdiscus toxicus produces ciguatera-inducing maitotoxin (MTX) [118]. Holmes and Lewisk purified G. toxicus-derived distinct MTXs using high-pressure liquid chromatography and reported that these compounds caused contractile responses of the muscle [108]. MTX is considered one of the largest natural products (3422 Da) that can activate cellular calcium channels [79,119]. Takahashi et al. [79] showed the association of increased calcium influx and calcium-dependent release of [3H] norepinephrine in a pheochromocytoma cell line. Their findings indicated that MTX’s mode of action is the activation of cellular calcium channels. The structures and analogues were previously described by Reyes et al. [120].

Karenia spp. dinoflagellates, including K. brevis and K. mikimotoi, are well-recognized as harmful algae in Japan and the USA [113,114]; these species produce brevetoxin (PbTx) and gymnocin (GC), respectively [85,86,87]. PbTx activates mammalian voltage-gated sodium channels, thereby causing NSP [20,84,121]. Further, aerosolized PbTx in sea spray causes reduced respiratory function and asthma [116]. The structures, analogues, and toxicity of PbTx were previously well-elucidated by European Food Safety Authority (EFSA) panels [122]. GCs are polyether toxins that include GC-A and GC-B [86,87]. Although their modes of action are still poorly understood, GCs are carboxylic acids and show moderate cytotoxicity activity against mouse lymphoid P388 cells [86,87]. The structures of GC-A and gymnocin-B were determined by Satake et al. [86,123]. Additionally, Tanaka et al. [87] determined the structures of GC analogues, including GC-A carboxylic acid and GC-A2.

Ostreopsis spp. are well-recognized, harmful algae worldwide due to their spread to many tropical and temperate regions. They produce aerosolized palytoxin (PLTX) along with its analogues, which have caused myalgia, respiratory problems, impairment of the neuromuscular apparatus, and abnormalities in cardiac function [124,125]. PLTX is considered one of the most lethal marine toxins, and its mode of action is unique wherein it causes the Na⁺/K⁺ pump to turn into a shape that allows the passive transport of sodium and potassium ions [35,91]. PLTXs and their analogues can be produced by the dinoflagellates O. siamensis, O. ovata, and O. mascarenensis [126,127,128]. The structure, analogues, and toxicity of PLTXs were previously represented by Ramos and Vasconcelos [129].

Prorocentrum spp. produce species-specific diverse phycotoxins, such as OA, DPX, prorocentrolides (PC), and borbotoxin (BTX) (Table 1). PCs are a member of the cyclic imine phycotoxins family produced by P. lima and P. maculosum, and they act on both muscle and neuronal nicotinic acetylcholine receptors (nAChRs) [94,130,131]. P. borbonicum produces BTX-A that was purified by Ten-Hage et al. [95] and has a similar mode of action on nAChRs. The general structure, analogues, and modes of action of PCs and BTX were reported by Amar et al. [94] and Ten-Hage et al. [95].