Gymnodimine-A (GYM-A) is a fast-acting microalgal toxin and its production of certified materials requires an efficient harvesting technology from the large-scale cultures of toxigenic microalgae. In this study the recoveries of GYM-A were compared between several liquid-liquid extraction (LLE) treatments including solvents, ratios and stirring times to optimize the LLE technique for harvesting GYM-A from Karenia selliformis cultures, of which the dichloromethane was selected as the extractant and added to microalgal cultures at the ratio 55 mL L⁻¹ (5.5%, v/v). The recovery of GYM-A obtained by the LLE technique was also compared with filtration and centrifugation methods. The stability of GYM-A in culture media were also tested under different pH conditions. Results showed that both the conventional filter filtration and centrifugation methods led to fragmentation of microalgal cells and loss of GYM-A in the harvesting processes. A total of 5.1 µg of GYM-A were obtained from 2 L of K. selliformis cultures with a satisfactory recovery of 88%. Interestingly, GYM-A obviously degraded in the culture media with the initial pH 8.2 and the adjusted pH of 7.0 after 7 days, but there was no obvious degradation in the acidic medium at pH 5.0. Therefore, the LLE method developed here permits the collection of large-volume cultures of K. selliformis and the high-efficiency extraction of GYM-A. This work provides a simple and valuable technique for harvesting toxins from large-scale cultures of GYM-producing microalgae.

When humans consume seafood contaminated by lipophilic polyether phycotoxins, such as azaspiracids (AZAs), the toxins are mainly leached and absorbed in the small intestine, potentially causing intestinal damage. In this study, human intestinal epithelial Caco-2 cells were used to investigate the adverse effects of azaspiracid-2 (AZA-2) on human intestinal epithelial cells. Cell viability, apoptosis, oxidative damage and mitochondrial ultrastructure were investigated, and ribonucleic acid sequence (RNA-seq) analysis was applied to explore the potential mechanisms of AZA-2 toxicity to Caco-2 cells. Results showed that AZA-2 significantly reduced the proliferation of Caco-2 cells in a concentration-dependent response, and the 48 h EC₅₀ of AZA-2 was 12.65 nmol L⁻¹. AZA-2 can induce apoptosis in Caco-2 cells in a dose-dependent manner. Visible mitochondrial swelling, cristae disintegration, membrane rupture and autophagy were observed in Caco-2 cells exposed to AZA-2. Reactive oxygen species (ROS) and malondialdehyde (MDA) content were significantly increased in Caco-2 cells after 48 h of exposure to 1 and 10 nmol L⁻¹ of AZA-2. Transcriptome analysis showed that KEGG pathways related to cellular oxidative damage and lipid metabolism were affected, mainly including mitophagy, oxidative phosphorylation, cholesterol metabolism, vitamin digestion and absorption, bile secretion and the peroxisome proliferator-activated receptor signaling pathway. The cytotoxic effects of AZA-2 on Caco-2 cells may be associated with ROS-mediated autophagy and apoptosis in mitochondrial cells. Results of this study improve understanding of the cytotoxicity and molecular mechanisms of AZA-2 on Caco-2 cells, which is significant for protecting human health.

Harmful algal blooms (HABs) formed by toxic microalgae have seriously threatened marine ecosystems and food safety and security in recent years. Among them, Karenia selliformis has attracted the attention of scientists and society due to its acute and rapid neurotoxicity in mice. Herein, the growth and gymnodimine A (GYM-A) production of K. selliformis were investigated in diverse culture systems with different surface-to-volume (S/V) ratios and nitrogen/phosphorus concentrations. The results showed that the specific growth rates (μ), maximal cell yields, and GYM-A production levels of K. selliformis increased with higher S/V, but no significant differences were observed under different culture volumes with the same S/V, which indicated that light penetration and gas exchange in the seawater culture systems actually influenced the growth and GYM-A production of K. selliformis. The maximum cell density and photosynthetic efficiency of K. selliformis decreased under nitrogen (N) and phosphorus (P) deficiency, suggesting that the growth of K. selliformis was significantly inhibited by the deficiency in N or P. Both N and P limitation conditions, especially P deficiency, promoted the cellular GYM-A quotas of K. selliformis. In this study, a scientific basis is provided for understanding the effects of culture systems and nutrient concentrations on the growth and toxin production of K. selliformis.

Gymnodimines (GYMs) are a group of fast-acting phycotoxins and their toxicological effects on human beings are still unclear due to the lack of sufficiently well-characterized large quantities of purified toxins for toxicology studies. In this study, a certified reference material (CRM) of GYM-A was prepared from the dinoflagellate Karenia selliformis, followed by multi-step chromatography separation and purification. Subsequently, the stability of GYM-A in methanolic media was evaluated at different temperature (−20, 4, and 20 °C) and pH (3, 5, and 7) conditions for 8 months, and the conversion products of GYM-A were explored by liquid chromatography–high resolution mass spectrometry (LC-HRMS). The results show that the stability of GYM-A decreased with increasing temperature and pH values. The GYM-A was stable during storage at −20 °C regardless of pH, but it decreased rapidly (81.8% ± 9.3%) at 20 °C in pH 7 solution after 8 months. Moreover, the concentrations of GYM-A did not significantly change at all temperatures in solutions with pH 3 (p > 0.05). It is recommended that GYM-A should be stored at low temperature (≤−20 °C) and pH (≤3) conditions for long-term storage in aqueous methanolic media. In addition, two conversion products of GYM-A, tentatively named as GYM-K (m/z 540) and GYM-L (m/z 524), were identified in the samples stored at high levels of pH and temperature. Based on the LC-HRMS data, the hypothetical chemical structures of both converting derivatives were proposed. A useful strategy for long-term storage of GYM-A CRM in aqueous methanolic media was suggested and two hypothesized conversion products of GYM-A were discovered in this study.