Effects of the plastic additive 2,4-di-tert-butylphenol on intestinal microbiota of zebrafish.

Plastic additives such as the antioxidant 2,4-di-tert-butylphenol (2,4-DTBP) have been widely detected in aquatic environments, over a wide range of concentrations reaching 300 µg/L in surface water, potentially threatening the health of aquatic organisms and ecosystems. However, knowledge of the specific effects of 2,4-DTBP on aquatic vertebrates is still limited. In this study, adult zebrafish were exposed to different concentrations of 2,4-DTBP (0, 0.01, 0.1 and 1.0 mg/L) for 21 days in the laboratory. The amplicon sequencing results indicated that the diversity and composition of the zebrafish gut microbiota were significantly changed by 2,4-DTBP, with a shift in the dominant flora to more pathogenic genera. Exposure to 2,4-DTBP at 0.1 and 1.0 mg/L significantly increased the body weight and length of zebrafish, suggesting a biological stress response. Structural assembly defects were also observed in the intestinal tissues of zebrafish exposed to 2,4-DTBP, including autolysis of intestinal villi, adhesions and epithelial detachment of intestinal villi, as well as inflammation. The transcriptional expression of some genes showed that 2,4-DTBP adversely affected protein digestion and absorption, glucose metabolism and lipid metabolism. These results are consistent with the PICRUSt2 functional prediction analysis of intestinal microbiota of zebrafish exposed to 2,4-DTBP. This study improves our understanding of the effects of 2,4-DTBP on the health of aquatic vertebrates and ecosystems.

Emergency response to ecological protection in maritime phenol spills: Emergency monitor, ecological risk assessment, and reduction.

Recently, hundreds of maritime accidental spills of hazardous chemicals have raised public concerns, especially for phenol due to its potential of spills and highly toxicity. Therefore, for marine ecological protection, this article prepared specific strategies of emergency response to phenol spills. Through the identification for phenol behavior at sea, migration prediction, emergency monitor, as well as their new methods were reviewed. Further, ecological risk assessment and seawater quality criteria were conducted by using a species sensitivity distribution (SSD) approach, wherein, risk quotient (RQ) indicated phenol of simulated marine spills posed a high risk (RQ > 1) in 30 days. The method with eco-friendliness and high-efficiency for phenol reduction was constructed by combination of dredging equipment such as pneumatic dredgers (Airlift) and bioremediation, where marine microorganisms that degraded phenol were summarized, as well as future research needs. This study provided a guidance for emergency response and policy development of phenol spills.

Detection and dietary risk of per- and polyfluoroalkyl substances in shellfish products from the coasts of Bohai Sea and South China Sea.

Artificial per- and polyfluoroalkyl substances (PFASs) are widely distributed in the environment and are potentially harmful to human health. This study assessed the matrix effect of different shellfish on LC-MS analysis and the recoveries of PFASs in purified extracts purified by adding ENVI-Carb graphitized carbon black. Total 76 samples were collected from coastal cities of the Bohai Sea and South China Sea in China. Results showed that the signal response of perfluorocarboxylic acid increased with the length of fluorocarbon chains. ENVI-Carb can mitigate the shellfish matrix effects for analysis of PFASs. Ten PFASs components were detected in shellfish samples at concentrations ranging from 1.3 to 8.5 ng/g wet weight. The PFOA and PFHxS were the dominant components, and PFOA, PFTrDA and PFNA were detected at high rates of 58-93%. The highest levels of ∑PFASs were accumulated in clams, while the lowest levels were found in mussels. The dietary risk assessment indicated that PFASs potentially threaten human health via consumption of clam products in the Bohai Sea region. This study will improve the understanding of the contamination status and the dietary risk of PFASs in shellfish products along the coasts of Bohai Sea and South China Sea in China.

How does the neurotoxin ß-N-methylamino-L-alanine exist in biological matrices and cause toxicity?

The neurotoxin ß-N-methylamino-L-alanine (BMAA) has been deemed as a risk factor for some neurodegenerative diseases such as amyotrophic lateral sclerosis/parkinsonism dementia complex (ALS/PDC). This possible link has been proved in some primate models and cell cultures with the appearance that BMAA exposure can cause excitotoxicity, formation of protein aggregates, and/or oxidative stress. The neurotoxin BMAA extensively exists in the environment and can be transferred through the food web to human beings. In this review, the occurrence, toxicological mechanisms, and characteristics of BMAA were comprehensively summarized, and proteins and peptides were speculated as its possible binding substances in biological matrices. It is difficult to compare the published data from previous studies due to the inconsistent analytical methods and components of BMAA. The binding characteristics of BMAA should be focused on to improve our understanding of its health risk to human health in the future.

Responses of the intestinal microbiota to exposure of okadaic acid in marine medaka Oryzias melastigma.

It is still limited that how the microalgal toxin okadaic acid (OA) affects the intestinal microbiota in marine fishes. In the present study, adult marine medaka Oryzias melastigma was exposed to the environmentally relevant concentration of OA (5 µg/L) for 10 days, and then recovered in fresh seawater for 10-days depuration. Analysis of taxonomic composition and diversity of the intestinal microbiota, as well as function prediction analysis and histology observation were carried out in this study. Functional prediction analysis indicated that OA potentially affected the development of colorectal cancer, protein and carbohydrate digestion and absorption functions, and development of neurodegenerative diseases like Parkinson's disease, which may be associated with changes in Proteobacteria and Firmicutes in marine medaka. Significant increases of C-reactive protein (CRP) and inducible nitric oxide synthase (iNOS) levels, as well as the changes of histology of intestinal tissue demonstrated that an intestinal inflammation was induced by OA exposure in marine medaka. This study showed that the environmental concentrations of OA could harm to the intestinal microbiota thus threatening the health of marine medaka, which hints that the chemical ecology of microalgal toxins should be paid attention to in future studies.

Apoptosis and oxidative stress of mouse breast carcinoma 4T1 and human intestinal epithelial Caco-2 cell lines caused by the phycotoxin gymnodimine-A.

Gymnodimine-A (GYM-A) is a cyclic imine phycotoxin produced by some marine dinoflagellates. It can cause rapid death of mice via intraperitoneal administration and frequently accumulate in shellfish potentially threatening human health. In this study, four different cell lines were exposed to GYM-A for the viability assessment. Results showed that GYM-A was cytotoxic with concentration-dependent pattern to each cell type, with mean IC50 values ranging from 1.39 to 2.79 µmol L-1. Results suggested that the loss of cell viability of 4T1 and Caco-2 cells was attributed to apoptosis. Furthermore, the collapse of mitochondrial membrane potential and caspases activation were observed in the GYM-A-treated cells. Reactive oxygen species (ROS) and lipid peroxides (LPO) levels were markedly increased in 4T1 and Caco-2 cells exposed to GYM-A at 2 µmol L-1, and the oxidative stress in 4T1 cells was more obvious than that in Caco-2 cells. Additionally, unusual ultrastructure impairment on mitochondria and mitophagosomes occurred in the GYM-A-treated cells. These results suggested that an ROS-mediated mitochondrial pathway for apoptosis and mitophagy was implicated in the cytotoxic effects induced by GYM-A. This is the first report to explore the cytotoxic mechanisms of GYM-A through apoptosis and oxidative stress, and it will provide theoretical foundations for the potential therapeutic applications of GYM-A.

Adverse effects of plastic leachate and its component 2,4-DTBP on the early development of zebrafish embryos.

Plastic waste has become a global environmental problem threatening the health of aquatic organisms especially via leachate. In this study, the test of zebrafish embryo showed adverse effects of leachate from some agricultural mulching films after UV light aging for 60 h. A typical phenolic antioxidant 2,4-di-tert-butylphenol (2,4-DTBP) was detected in the leachate and tested further for the zebrafish embryo biotoxicity. The microplastic leachate (6, 8 g/L, mass concentration measured by weight of plastic) increased the death and malformation rates, and reduced the hatching rate, heart rate, and body length of zebrafish larvae in the 96-hour early development period. Similar adverse effects were also caused by the 2,4-DTBP (0.01, 0.1, 1.0 mg/L, corresponding to 0.049, 0.49, and 4.85 µM) to some degree but could not completely explain the significant influences caused by the plastic leachate. Transcriptome analysis of zebrafish embryos exposed to the 2,4-DTBP for 96 h showed that the protein, fat, and carbohydrate digestion and absorption pathways, pancreatic secretion, PPAR signaling pathway, tryptophan metabolism, and adipocytokine signaling pathway were considerably down-regulated, but the cholesterol metabolism pathway was up-regulated in larval zebrafish. The altered transcriptional expression of mRNA at early development stage (96 h post fertilization) of zebrafish suggested that the 2,4-DTBP caused reduction of digestive capacity and pancreatic secretory function, and adversely affected processes associated with energy metabolism and glycolipid metabolism of larval zebrafish. This study helps us further understanding the effects of plastic leachate on the early development of fishes.

Toxicity and potential underlying mechanism of Karenia selliformis to the fish Oryzias melastigma.

Karenia selliformis can produce toxins such as gymnodimines, and form microalgal blooms causing massive mortality of marine life such as fish and shellfish, and resulting in serious economic losses. However, there are a few of studies on the toxic effects of K. selliformis on marine organisms and the underlying mechanisms, and it is not clear whether the toxins produced by K. selliformis affect fish survival through the food chain. In this study, a food chain was simulated and composed by K. selliformis-brine shrimp-marine medaka to investigate the possibility of K. selliformis toxicity transmission through the food chain, in which fish behavior, histopathology and transcriptomics changes were observed after direct or indirect exposure (through the food chain) of K. selliformis. We found that both direct and indirect exposure of K. selliformis could affect the swimming behavior of medaka, manifested as decreased swimming performance and increased "frozen events". Meanwhile, exposure to K. selliformis caused pathological damage to the intestine and liver tissues of medaka to different degree. The effect of direct exposure to K. selliformis on swimming behavior and damage to fish tissues was more severe. In addition, K. selliformis exposure induced significant changes in the expression of genes related to energy metabolism, metabolic detoxification and immune system in medaka. These results suggest that toxins produced by K. selliformis can be transferred through the food chain, and that K. selliformis can destroy the intestinal integrity of medaka and increase the absorption of toxins, leading to energy metabolism disorders in fish, affecting the metabolic detoxification capacity of the liver. Our finding provides novel insight into the toxicity of K. selliformis to marine fish.

Reaction mechanism and detecting properties of a novel molecularly imprinted electrochemical sensor for microcystin based on three-dimensional AuNPs@MWCNTs/GQDs.

Microcystins with leucine arginine (MC-LR) is a virulent hepatotoxin, which is commonly present in polluted water with its demethylated derivatives [Dha7] MC-LR. This study reported a low-cost molecularly imprinted polymer network-based electrochemical sensor for detecting MC-LR. The sensor was based on a three-dimensional conductive network composed of multi-walled carbon nanotubes (MWCNTs), graphene quantum dots (GQDs), and gold nanoparticles (AuNPs). The molecularly imprinted polymer was engineered by quantum chemical computation utilizing p-aminothiophenol (p-ATP) and methacrylic acid (MAA) as dual functional monomers and L-arginine as a segment template. The electrochemical reaction mechanism of MC-LR on the sensor was studied for the first time, which is an irreversible electrochemical oxidation reaction involving an electron and two protons, and is controlled by a mixed adsorption-diffusion mechanism. The sensor exhibited a great detection response to MC-LR in the linear range of 0.08-2 µg/L, and the limit of detection (LOD) is 0.0027 µg/L (S/N = 3). In addition, the recoveries of the total amount of MC-LR and [Dha7] MC-LR in the actual sample by the obtained sensor were in the range from 91.4 to 116.7%, which indicated its great potential for environmental detection.

Effects of marine phycotoxin dinophysistoxin-1 on the growth and cell cycle of Isochrysis galbana.

The phycotoxin dinophysistoxins are widely distributed in the global marine environments and potentially threaten marine organisms and human health. The mechanism of the dinophysistoxin toxicity in inhibiting the growth of microalgae is less well understood. In this study, effects of the dissolved dinophysistoxin-1 (DTX1) on the growth, pigment contents, PSII photosynthetic efficiency, oxidative stress response and cell cycle of the marine microalga Isochrysis galbana were investigated. Growth of I. galbana was significantly inhibited by DTX1 with 0.6-1.5 µmol L-1 in a 96-h batch culture, corresponding the 96 h-EC50 of DTX1 at 0.835 µmol L-1. The maximum quantum yield of PSII (Fv/Fm), and light utilization efficiency (α) were obviously reduced by DTX1 at 1.5 µmol L-1 during 96-h exposure. Contents of most of pigments were generally reduced by DTX1 with a dose-depend pattern in microalgal cells except for diatoxanthin. The ROS levels were increased by DTX1 with 0.6-1.5 µmol L-1 after 72-h exposure, while the contents or activities of MDA, GSH, SOD and CAT were significantly increased by DTX1 at 1.5 µmol L-1 at 96 h. The inhibitory effect of DTX1 on the growth of I. galbana was mainly caused by the production of ROS in the cells. Cell cycle analysis showed that the I. galbana cell cycle was arrested by DTX1 at G2/M phase. This study enhances the understanding of the chemical ecology effects of DTX1 on marine microalgae and also provides fundamental data for deriving water quality criteria of DSTs for marine organisms.

Ecological risk assessment for xylenes and propylbenzenes in aquatic environment using a species sensitivity distribution approach.

Xylenes and propylbenzenes (PBZs) are volatile aromatic hydrocarbons with high aquatic toxicity. Xylenes can be present in three isomers: o-xylene (OX), m-xylene (MX), and p-xylene (PX), while PBZs include two isomers: n-propylbenzene (n-PBZ) and isopropylbenzene (i-PBZ). Their accidental spills and improper discharges from petrochemical industries can cause severe contamination in water bodies posing potential ecological risks. In this study, the published acute toxicity data of these chemicals for aquatic species were collected to calculate hazardous concentrations protecting 95% species (HC5) using a species sensitivity distribution (SSD) approach. The acute HC5 values for OX, MX, PX, n-PBZ, and i-PBZ were estimated to be 1.73, 3.05, 1.23, 1.22, and 1.46 mg/L, respectively. The risk quotient (RQ) values calculated based on HC5 indicated their high risk (RQ: 1.23 ∼ 21.89) in groundwater, but low risk (RQ < 0.1) in natural seawater, river water, and lake water. When xylenes or PBZs leaked into the sea, they were expected to pose a high risk (RQ > 1) at the start and then a low risk (RQ < 0.1) after 10 days due to natural attenuation. These results may help to derive more reliable protection thresholds for xylenes and PBZs in aquatic environment and provide a basis for evaluating their ecological risks.

Effects of lipophilic phycotoxin okadaic acid on the early development and transcriptional expression of marine medaka Oryzias melastigma.

The lipophilic okadaic acid (OA)-group toxins produced by some species of Dinophysis spp. and Prorocentrum spp. marine dinoflagellates have been frequently and widely detected in natural seawater environments, e.g. 2.1∼1780 ng/L in Spanish sea and 5.63∼27.29 ng/L in the Yellow Sea of China. The toxicological effects of these toxins dissolved in seawater on marine fish is still unclear. Effects of OA on the embryonic development and 1-month old larvae of marine medaka (Oryzias melastigma) were explored and discussed in this study. Significantly increased mortality and decreased hatching rates occurred for the medaka embryos exposed to OA at 1.0 µg/mL. Diverse malformations including spinal curvature, dysplasia and tail curvature were also observed in the embryos exposed to OA and the heart rates significantly increased at 11 d post fertilization. The 96 h LC50 of OA for 1-month old larvae was calculated at 3.80 µg/mL. The reactive oxygen species (ROS) was significantly accumulated in medaka larvae. Catalase (CAT) enzyme activity was significantly increased in 1-month old larvae. Acetylcholinesterase (AChE) activity significantly increased with a dose-dependent pattern in 1-month old larvae. Differentially expressed genes (DEGs) were enriched in 11 KEGG pathways with Q value < 0.05 in 1-month old medaka larvae exposed to OA at 0.38 µg/mL for 96 h, which were mainly related to cell division and proliferation, and nervous system. Most of DEGs involved in DNA replication, cell cycle, nucleotide excision repair, oocyte meiosis, and mismatch repair pathways were significantly up-regulated, while most of DEGs involved in synaptic vesicle cycle, glutamatergic synapse, and long-term potentiation pathways were markedly down-regulated. This transcriptome analysis demonstrated that a risk of cancer developing was possibly caused by OA due to DNA damage in marine medaka larvae. In addition, the neurotoxicity of OA was also testified for marine fish, which potentially cause major depressive disorder (MDD) via the up-regulated expression of NOS1 gene. The genotoxicity and neurotoxicity of OA to marine fish should be paid attention to and explored further in the future.

Phase-contrast imaging with synchrotron hard X-ray reveals the effect of icariin on bone tissue morphology and microstructure in rabbits with early glucocorticoid-induced osteonecrosis of the femoral head.

Background: Phase-contrast imaging (PCI) with synchrotron hard X-ray was used to observe the changes in bone tissue morphology and microstructure in rabbit models of early glucocorticoid-induced osteonecrosis of the femoral head (ONFH), and to evaluate the intervention effect of Icariin. Methods: Fifty mature New Zealand rabbits (weighing 2.5-3.0 kg) were randomly divided into a control group (n = 10), a glucocorticoid group (n = 20), and an Icariin group (n = 20). The glucocorticoid group and the Icariin group were sequentially injected with lipopolysaccharide (LPS) and methylprednisolone (MPS) to establish a glucocorticoid-induced ONFH animal model. The Icariin group was given Icariin solution when methylprednisolone was injected for the first time, and the control group and glucocorticoid group were given the same amount of normal saline. Animals were sacrificed after 6 weeks, and bilateral femoral head specimens were taken for research. The right femoral head was observed by PCI with synchrotron hard X-ray technology, and the left femoral head was verified by Micro-CT scanning and HE staining. Results: Forty-three animals (nine in the control group, sixteen in the glucocorticoid group, and eighteen in the Icariin group) were included in the study. PCI with synchrotron hard X-ray revealed that the trabecular bone in the glucocorticoid group was thinned, broken, and structurally damaged, whereas the trabecular bone in the Icariin group had normal volume, thickness, and a relatively intact structure. Micro-CT scan reconstruction and HE staining were used to verify the reliability of this technique in identifying osteonecrosis. Conclusion: The effects of Icariin were observed in an early glucocorticoid-induced ONFH rabbit model using PCI with synchrotron hard X-ray. Icariin weakens the destructive effect of glucocorticoids on bone tissue structure, improves bone tissue morphology, and stabilizes bone microstructure. This technique may provide a definitive, non-invasive alternative to histological examination for the diagnosis of early ONFH.

Cell cycle of microalga Isochrysis galbana arrested by neurotoxin ß-N-methylamino-l-alanine and corresponding molecular mechanisms.

The phycotoxin ß-N-methylamino-l-alanine (BMAA) has attracted attention due to its risks to marine organisms and human health. In this study, approximately 85 % of synchronized cells of the marine microalga Isochrysis galbana were arrested at the cell cycle G1 phase by BMAA at 6.5 µM for a 24-h exposure. The concentration of chlorophyll a (Chl a) gradually decreased, while the maximum quantum yield of PSII (Fv/Fm), the maximum relative electron transport rate (rETRmax), light utilization efficiency (α) and half-saturated light irradiance (Ik) reduced early and recovered gradually in I. galbana exposed to BMAA in 96-h batch cultures. Transcriptional expression of I. galbana analyzed at 10, 12, and 16 h disclosed multiple mechanisms of BMAA to suppress the microalgal growth. Production of ammonia and glutamate was limited by the down-regulation of nitrate transporters, glutamate synthase, glutamine synthetase, cyanate hydrolase, and formamidase. Diverse extrinsic proteins related to PSII, PSI, cytochrome b6f complex, and ATPase were influenced by BMAA at transcriptional level. Suppression of the DNA replication and mismatch repair pathways increased the accumulation of misfolded proteins, which was reflected by the up-regulated expression of proteasome to accelerate proteolysis. This study improves our understanding of the chemical ecology impacts of BMAA in marine ecosystems.

Occurrence and spatial distribution of paralytic shellfish toxins in seawater and marine organisms in the coastal waters of Qinhuangdao, China.

In recent years, paralytic shellfish toxins (PSTs) have been prevalent in the coastal waters of Qinhuangdao, the west coast of the Bohai Sea, China. The content of PSTs in shellfish often exceeded the regulatory limit of 800 µg STX equivalent (eq.) kg-1, which poses a serious threat to human health. In this study, two surveys were conducted in May 2021 and May 2022 to investigate the distribution of PSTs in the coastal waters of Qinhuangdao. Seawater, surface sediment, phytoplankton, zooplankton, and other marine organism samples were collected, and the composition and concentration of PSTs were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Results showed that multiple PST components were detected in all seawater samples collected at different depths, mainly including GTX1/4, GTX2/3, dcGTX2, STX and C1/2, and the highest concentration of PSTs reached 244 ng STX eq. L-1. The sediment samples also contained low levels of C1/2 and GTX2/3. Trace amounts of C1/2 and GTX1-4 were detected in phytoplankton and zooplankton. Moreover, all bivalve shellfish samples were found to contain PSTs, and the scallop Azumapecten farreri and the ark clam Anadara kagoshimensis showed relatively high concentrations of 607 and 497 µg STX eq. kg-1, respectively. In addition, low levels of PSTs were also found in some non-traditional PST vectors, including whelk Rapana venosa, octopus Amphioctopus ovulum, goby Ctenotrypauchen chinensis, and greenling Hexagrammos agrammus. Results of this study improve the understanding of the distribution of PSTs in seawater and marine organisms and the potential risk of persistent PSTs in seawater to marine ecosystems and human health.

Possibility analysis of thyroid imaging parameters for dose adjustment in 131I treatment of hyperthyroidism.

BACKGROUND: To determine the thyroid uptake rate by correcting the background and analyze its clinical significance. METHODS: The study included 161 patients with hyperthyroidism. The thyroid uptake rate was calculated by drawing a 100 pixels ROI (region of interest) background, above and below the thyroid and correcting the thyroid ROI for background counting. At the same time, the clinical baseline characteristic parameters such as age and thyroid volume etc. of patients with hyperthyroidism were collected. The consistency of 99mTcO4uptake rate before treatment and 131I-uptake rate after treatment, and the correlation between uptake rate of thyroid and baseline characteristic parameters were also analyzed. RESULTS: The uptake rate of 99mTcO4 was found positively correlated with 3 h-radioactive iodine uptake (RAIU), 24 h-RAIU, 3 h/24 h conversion rate, thyroid volume, 131I activity free triiodothyronine (FT3) and free thyroxine (FT4), and showed negative correlation with age, effective half-life (P<0.05). The uptake rate of 131I was found positively correlated with 3 h-RAIU, 24 h-RAIU, 3 h/24 h conversion rate, thyroid volume, 131I activity, FT3, FT4 (P<0.05). In patients with positive thyrotrophin receptor antibody (TRAb), a significant positive correlation between uptake rate of 99mTcO4 and 131I (P<0.05) was observed. There was a high consistency between pretreatment uptake rate of 99mTcO4 and post-treatment uptake rate of 131I (P=0.009; W=0.7). CONCLUSIONS: The corrected thyroid uptake rate is remarkably correlated with clinical characteristic parameters of patients, which can be used to comprehensively evaluate the comprehensive condition of patients with hyperthyroidism.

Putative biosynthesis mechanism of the neurotoxin ß-N-methylamino-L-alanine in marine diatoms based on a transcriptomics approach.

The neurotoxin ß-N-methylamino-L-alanine (BMAA) has been presumed as an environmental cause of human neurodegenerative disorders, such as Alzheimer's disease. Marine diatoms Thalassiosira minima are demonstrated here to produce BMAA-containing proteins in axenic culture while the isomer diaminobutyric acid was bacterially produced. In the co-culture with Cyanobacterium aponinum, diatom growth was inhibited but the biosynthesis of BMAA-containing proteins was stimulated up to seven times higher than that of the control group by cell-cell interactions. The stimulation effect was not caused by the cyanobacterial filtrate. Nitrogen deprivation also doubled the BMAA content of T. minima cells. Transcriptome analysis of the diatom in mixed culture revealed that pathways involved in T. minima metabolism and cellular functions were mainly influenced, including KEGG pathways valine and leucine/isoleucine degradation, endocytosis, pantothenate and CoA biosynthesis, and SNARE interactions in vesicular transport. Based on the expression changes of genes related to protein biosynthesis, it was hypothesized that ubiquitination and autophagy suppression, and limited COPII vesicles transport accuracy and efficiency were responsible for biosynthesis of BMAA-containing proteins in T. minima. This study represents a first application of transcriptomics to investigate the biological processes associated with BMAA biosynthesis in diatoms.

Cytotoxicity and intestinal permeability of phycotoxins assessed by the human Caco-2 cell model.

Phycotoxins are a class of multiple natural metabolites produced by microalgae in marine and freshwater ecosystems that bioaccumulate in food webs, particularly in shellfish, having a great impact on human health. Phycotoxins are mainly leached and absorbed in the small intestine when human consumers accidentally ingest toxic aquatic products contaminated by them. To assess the intestinal uptake and damage of phycotoxins, a typical in vitro model was developed and widely applied using the human colorectal adenocarcinoma Caco-2 cell line. In this review, the application cases were summarized for multiple phycotoxins, including microcystins (MCs), cylindrospermopsins (CYNs), domoic acids (DAs), saxitoxins (STXs), palytoxins (PLTXs), okadaic acids (OAs), pectenotoxins (PTXs) and azaspiracids (AZAs). The results of the previous studies showed that each group of phycotoxins presented different cytotoxicity and mechanisms to Caco-2 cells, and significant discrepancies in the transport of phycotoxin across the Caco-2 cell monolayers. Therefore, this review describes the evaluation assays of the Caco-2 cell monolayer model, illustrates the principles of several primary cytotoxicity evaluation assays, and summarizes the cytotoxicity of each group of phycotoxins to Caco-2 cells line and their cellular transport, and finally proposes the development of multicellular intestinal models for future comprehensive studies on the toxicity and absorption of phycotoxins in the intestine. It will improve the understanding of Caco-2 cell monolayer models in the toxicology studies on phycotoxins and the potentially detrimental effects of microalgal toxins on the human intestine.

Stability and Chemical Conversion of the Purified Reference Material of Gymnodimine-A under Different Temperature and pH Conditions.

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.

Phytoplankton community and HAB species in the South China Sea detected by morphological and metabarcoding approaches.

The southern Chinese coast is one of the most developed regions in China and is an area where harmful algal blooms (HABs) have occurred frequently. In this study, differences in the phytoplankton community between microscopic observations and 18S rDNA metabarcoding were compared in 89 surface water samples collected from the southern Chinese coast and the western South China Sea (SCS). This is the first report investigating the phytoplankton community and HAB species using a combination of morphological and metabarcoding approaches in this sea area. There were substantial differences in phytoplankton community structure detected by the two methods. Microscopic observation revealed diatom predominance in the phytoplankton community, while metabarcoding indicated dinoflagellate dominance. The phytoplankton community structure obtained by microscopic observation better reflects the real situation in the water column. Metabarcoding annotated more species than morphospecies observed by microscopy. Haptophyta and Cryptophyta were the specific phyla detected in metabarcoding but were missed in microscopy due to their small size. Conversely, some taxa were found in microscopic analysis alone, such as species in Dinophysis, Prorocentrum, and Scrippsiella, suggesting some biases during metabarcoding and gaps in sequence databases. Metabarcoding is superior for detecting morphologically cryptic, small-sized and HAB taxa, such as unarmored dinoflagellates, nanosized hatophytes and chlorophytes, as well as multiple species in Alexandrium, Pseudonitzschia, and Chaetoceros in our study. A total of 62 HAB taxa were identified in this study, including blooming and potentially toxic species. Diatom abundances generally decreased southward, while those of dinoflagellates and haptophytes showed the opposite trend. Chlorophytes were mainly distributed in coastal waters, especially in the Pearl River Estuary. Phytoplankton community structures were shaped by nutrients and salinity, and phosphorus was the most limiting factor for phytoplankton growth. The phytoplankton community in the western SCS showed unique characteristics away from those in the coastal sea areas. The results suggest that the combination of morphological and metabarcoding approaches comprehensively reveals the phytoplankton community structure and diversity of HAB species.