Industrial Applications of Dinoflagellate Phycotoxins Based on Their Modes of Action: A Review

Dinoflagellates are an important group of phytoplanktons, characterized by two dissimilar flagella and distinctive features of both plants and animals. Dinoflagellate-generated harmful algal blooms (HABs) and associated damage frequently occur in coastal areas, which are concomitant with increasing eutrophication and climate change derived from anthropogenic waste and atmospheric carbon dioxide, respectively. The severe damage and harmful effects of dinoflagellate phycotoxins in the fishing industry have been recognized over the past few decades, and the management and monitoring of HABs have attracted much attention, leaving aside the industrial application of their valuable toxins. Specific modes of action of the organisms’ toxins can effectively be utilized for producing beneficial materials, such as Botox and other therapeutic agents. This review aims to explore the potential industrial applications of marine dinoflagellate phycotoxins; furthermore, this review focuses on their modes of action and summarizes the available knowledge on them.


Introduction
Microalgae are photosynthetic microorganisms belonging to diverse phyla [1]. Over the past few decades, several green microalgae, such as Chlorella spp., Scenedesmus spp., and Dunaliella spp., have been recognized as useful bioresources for producing commercial materials, namely cosmetics, pharmaceuticals, dietary supplements, biofuels, and biofertilizers [2][3][4][5][6][7]. In contrast, phycotoxin-producing cyanobacteria, dinoflagellates, and raphidophytes are known to generate frequent harmful algal blooms (HABs), thereby causing severe losses to the fishing industry and aquatic ecosystem [8][9][10][11]. For instance, Lake Erie in the United States is a well-recognized recreational place, but the ecosystem services are under threat owing to cyanobacterial-generated HABs. The annual economic loss of fishing expenditures in Lake Erie was estimated to be approximately USD 2.25-5.58 million during bloom formation [12]. Additionally, massive HABs caused by the dinoflagellate 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].

Dinoflagellate Phycotoxins and Their Modes of Action
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].  [75,93] Prorocentrolides (PC) Acts on nicotinic acetylcholine receptors (nAChRs) [94] Borbotoxin (BTX) Blocks postsynaptic nAChRs [95] Protoceratium reticulatum Yessotoxin (YTX) Activates calcium channel [68,89] Decreases cytosolic adenosine 3 ,5 -cyclic monophosphate (cAMP) levels
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].
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].
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].

Potential Biotechnological Applications of Phycotoxins Based on Their Modes of Action
As shown in Table 2, the aforementioned diverse phycotoxins have extensive biotechnological applications in industry, especially for therapeutics and drug development. As described in the previous section, AZAs block the hERG potassium channel [57]. A variety of potassium channel inhibitors are used for the treatment of cardiac arrhythmias with irregular heartbeat [132]. Therefore, AZAs are potential candidates for the development of antiarrhythmic drugs. BTX and PCs demonstrated nAChRs blocking activity; thus, this mechanism can be a potent target for anticholinergic drugs, including nicotinic antagonists [133]. PbTx is being used for research purposes; this toxin is an activator of the voltage-gated sodium channel and can be further evaluated for the development of post-stroke recovery drugs [134]. Sequeira et al. [134] reported that PbTx treatment in mice decreased the percentage of foot faults with a two-fold increase in dendritic arbor complexity. Additionally, they reported that PbTx-2 enhanced the dendritic arborization and synapse density of the cortical layer V pyramidal neurons in the peri-infarct cortex and improved motor recovery [135]. These results demonstrate that PbTx could be a potential candidate for the development of post-stroke recovery drugs. CTX exhibits inhibition of unmyelinated nerves that are related to pain; hence, it can be potentially used in the development of potent analgesics [65]. Sodium channels are highly associated with pain and depressive disorder symptoms [136,137]. For instance, Manríquez et al. [138] previously performed clinical treatment on five bladder pain syndrome (BPS) patients using NSTX, and all five patients successfully responded to the treatment. Furthermore, the analgesic effect of NSTX lasted for 90 days without adverse effects. Therefore, the sodium channel blockers, namely STX, GTX, and NSTX, can be potential candidates for the development of analgesic and antidepressant drugs.  [102] previously reported that DPX-1 and OA showed tumor-promoting activities through different pathways in a two-stage carcinogenesis experiment in mouse skin. PLTX is considered a novel tumor promoter in mouse skin, which can be used for studying signaling mechanisms involved in carcinogenesis [149]. Although dinoflagellate-derived HL is not a well-defined phycotoxin, the potential use of HLs as anticancer agents has been previously reported in the case of α-HL produced by the bacterium Staphylococcus aureus [143]. Furthermore, Cho et al. [142] reported that the co-culture of dinoflagellate H. circularisquama with green microalga Dunaliella salina showed effective flocculation activity, which is applicable for harvesting industrially available microalgae in biodiesel production. These results indicated that HLs are potential bioflocculation agents. KmTx is regarded as an allopathic compound; it is a type of HL generated by Karlodinium spp., and it exhibited copepod-killing activity [144]. Since KmTx causes cell membrane disruption by binding specifically to cholesterol, this toxin can be a useful component for anticancer or microalgae-harvesting agents, similar to the dinoflagellate HLs, as described above [142,143]. In addition, KTX affects the copepod Acartia tonsa, a natural grazer of Karlodinium spp. Although the mode of action of KTX has not been studied specifically, this toxin showed massive potential as a piscicide and copepod-killing agent owing to its ichthyotoxicity and toxicity in copepods, respectively. MTX has attracted much attention due to its bioactivity, which involves disruption of cellular calcium homeostasis [120]. Reyes et al. [120] previously described in detail the biomedical applications of MTXs, wherein they proposed MTXs as a tool for analyzing ion channels and insulin secretion, interleukin-1β secretagogue and oncotic death induction, and sperm acrosome reaction induction [120]. OA is a selective inhibitor of the protein phosphatases PP1 and PP2A; thus, it can be used as a powerful probe for the study of regulatory mechanisms and neurotoxicity. Therefore, OA cannot only be used as a tumor promoter, but it can also be instrumental in neurodegeneration studies, including Alzheimer's disease [72,146,147]. PLTX can be used in research for anticancer drugs. Görögh et al. [148] previously found that PLTX possesses preferential toxicity for head and neck carcinoma cells, with extensive destruction of xenografted tumors within 24 days. Thus, it seems that PLTX plays contrary dual roles in tumor promotion and suppression. In addition, other phycotoxins including PTX, PCs, and YTXs demonstrated potential anticancer activity. According to a previous review conducted by Kim et al. [150], PTX-2 showed significant cytotoxicity in human cancer cells via downregulation of antiapoptotic Bcl-2, IAP-related proteins, upregulation of Bax protein and ligand (TRAIL)-receptor 1/receptor 2 (DR4/DR5), mitochondrial dysfunction, and apoptosis through suppression of the nuclear factor κB pathway. Additionally, in their recent study, Lee et al. [152] reported that prorocentrolide C from cultured dinoflagellate P. lima could induce human cancer cell death via apoptosis and cell cycle arrest. Furthermore, YTXs represented anticancer activity in a B16F10 melanoma preclinical mouse model, along with antiallergic activity [155].
As described above, many studies have revealed the potential application of the phycotoxins as therapeutic agents. However, more mechanistic and clinical studies are required for their safe use in humans. Although the review does not encompass all the phycotoxins, we expect that this comprehensive summary will provide useful information and garner much attention for the industrial applications of phycotoxins.

Conclusions
Over the past few decades, HAB-forming dinoflagellates have been recognized as an environmental, economic, and health problem owing to their adverse effects on the aquatic ecosystem, fishing industry, and human health. In this review, we described the biotechnological potential of diverse dinoflagellate phycotoxins based on the toxins' intrinsic modes of action. Many studies have reported the potential application of these toxins, especially in medicine. Since algal cells can be easily cultivated in large photobioreactors or open pond systems that sequestrate atmospheric carbon dioxide, the bioproduction of value-added phycotoxins is economically feasible and beneficial to humans and the environment. However, further mechanistic and clinical studies are required for determining the application of dinoflagellate phycotoxins in therapeutics and other fields.