Aerosolizable Marine Phycotoxins and Human Health Effects: In Vitro Support for the Biogenics Hypothesis.
ABSTRACT: Respiratory exposure to marine phycotoxins is of increasing concern. Inhalation of sea spray aerosols (SSAs), during harmful Karenia brevis and Ostreopsis ovata blooms induces respiratory distress among others. The biogenics hypothesis, however, suggests that regular airborne exposure to natural products is health promoting via a downregulation of the mechanistic target of rapamycin (mTOR) pathway. Until now, little scientific evidence supported this hypothesis. The current explorative in vitro study investigated both health-affecting and potential health-promoting mechanisms of airborne phycotoxin exposure, by analyzing cell viability effects via cytotoxicity assays and effects on the mTOR pathway via western blotting. To that end, A549 and BEAS-2B lung cells were exposed to increasing concentrations (ng·L-1 - mg·L-1) of (1) pure phycotoxins and (2) an extract of experimental aerosolized homoyessotoxin (hYTX). The lowest cell viability effect concentrations were found for the examined yessotoxins (YTXs). Contradictory to the other phycotoxins, these YTXs only induced a partial cell viability decrease at the highest test concentrations. Growth inhibition and apoptosis, both linked to mTOR pathway activity, may explain these effects, as both YTXs were shown to downregulate this pathway. This proof-of-principle study supports the biogenics hypothesis, as specific aerosolizable marine products (e.g., YTXs) can downregulate the mTOR pathway.
Project description:Lipophilic phycotoxins are secondary metabolites produced by phytoplanktonic species. They accumulate in filter-feeding shellfish and can cause human intoxication. Regulatory limits have been set for individual toxins, and the toxicological features are well characterized for some of them. However, phycotoxin contamination is often a co-exposure phenomenon, and toxicological data regarding mixtures effects are very scarce. Moreover, the type and occurrence of phycotoxins can greatly vary from one region to another. This review aims at summarizing the knowledge on (i) multi-toxin occurrence by a comprehensive literature review and (ii) the toxicological assessment of mixture effects. A total of 79 publications was selected for co-exposure evaluation, and 44 of them were suitable for toxin ratio calculations. The main toxin mixtures featured okadaic acid in combination with pectenotoxin-2 or yessotoxin. Only a few toxicity studies dealing with co-exposure were published. In vivo studies did not report particular mixture effects, whereas in vitro studies showed synergistic or antagonistic effects. Based on the combinations that are the most reported, further investigations on mixture effects must be carried out.
Project description:In vitro and in vivo studies have shown that phycotoxins can impact intestinal epithelial cells and can cross the intestinal barrier to some extent. Therefore, phycotoxins can reach cells underlying the epithelium, such as enteric glial cells (EGCs), which are involved in gut homeostasis, motility, and barrier integrity. This study compared the toxicological effects of pectenotoxin-2 (PTX2), yessotoxin (YTX), okadaic acid (OA), azaspiracid-1 (AZA1), 13-desmethyl-spirolide C (SPX), and palytoxin (PlTX) on the rat EGC cell line CRL2690. Cell viability, morphology, oxidative stress, inflammation, cell cycle, and specific glial markers were evaluated using RT-qPCR and high content analysis (HCA) approaches. PTX2, YTX, OA, AZA1, and PlTX induced neurite alterations, oxidative stress, cell cycle disturbance, and increase of specific EGC markers. An inflammatory response for YTX, OA, and AZA1 was suggested by the nuclear translocation of NF-κB. Caspase-3-dependent apoptosis and induction of DNA double strand breaks (γH2AX) were also observed with PTX2, YTX, OA, and AZA1. These findings suggest that PTX2, YTX, OA, AZA1, and PlTX may affect intestinal barrier integrity through alterations of the human enteric glial system. Our results provide novel insight into the toxicological effects of phycotoxins on the gut.
Project description:Spirolide and gymnodimine macrocyclic imine phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. Analysis of 13-desmethyl spirolide C and gymnodimine A by binding and voltage-clamp recordings on muscle-type alpha1(2)betagammadelta and neuronal alpha3beta2 and alpha4beta2 nicotinic acetylcholine receptors reveals subnanomolar affinities, potent antagonism, and limited subtype selectivity. Their binding to acetylcholine-binding proteins (AChBP), as soluble receptor surrogates, exhibits picomolar affinities governed by diffusion-limited association and slow dissociation, accounting for apparent irreversibility. Crystal structures of the phycotoxins bound to Aplysia-AChBP ( approximately 2.4A) show toxins neatly imbedded within the nest of ar-omatic side chains contributed by loops C and F on opposing faces of the subunit interface, and which in physiological conditions accommodates acetylcholine. The structures also point to three major features: (i) the sequence-conserved loop C envelops the bound toxins to maximize surface complementarity; (ii) hydrogen bonding of the protonated imine nitrogen in the toxins with the carbonyl oxygen of loop C Trp147 tethers the toxin core centered within the pocket; and (iii) the spirolide bis-spiroacetal or gymnodimine tetrahydrofuran and their common cyclohexene-butyrolactone further anchor the toxins in apical and membrane directions, along the subunit interface. In contrast, the se-quence-variable loop F only sparingly contributes contact points to preserve the broad receptor subtype recognition unique to phycotoxins compared with other nicotinic antagonists. These data offer unique means for detecting spiroimine toxins in shellfish and identify distinctive ligands, functional determinants and binding regions for the design of new drugs able to target several receptor subtypes with high affinity.
Project description:Many detection methods for phycotoxins, bioactive compounds produced by harmful algae, focus on one compound or a class of related compounds. Multiple harmful algal species often co-occur in the environment, however, emphasizing the need to analyze for the presence of multiple groups of marine and freshwater phycotoxins in environmental samples, e.g., extracts from solid phase adsorption toxin tracking (SPATT). Two methods were developed to screen for 13 phycotoxins (microcystin-RR, -LR, -YR, azaspiracid-1, -2, karlotoxin 3, goniodomin A, brevetoxin-2, yessotoxin, pectenotoxin-2, dinophysistoxin-1, -2, and okadaic acid) in organic SPATT extracts using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) equipped with a trapping dimension (trap) and at-column dilution (ACD). The performance of each compound under 36 combinations of chromatographic conditions was characterized, and two final methods, acidic and basic, were selected based on peak shapes, signal intensities, resolution, and the separation in time of positive and negative MS ionization modes. Injection volumes of up to 1 mL were possible through trap/ACD technology, resulting in limits of detection between 0.001 and 0.05 µg/L across the analytes. Benefits highlighted in this study, beyond the improved detection limits and co-detection of multiple toxin groups, include the ability to inject samples of 100% organic solvent, ensuring analyte stability and streamlining workflow through the elimination of laborious sample preparation steps.
Project description:White matter degeneration and demyelination are nonnegligible pathological manifestations of Alzheimer's disease (AD). The damage of myelin sheath consisting of oligodendrocytes is the basis of AD's unique early lesions. Shenzhiling oral liquid (SZL) was the effective Chinese herbal compound approved by the Food and Drug Administration (FDA) for the treatment of AD in China, which plays the exact therapeutic role in clinical AD patients. However, its molecular mechanism remains unclear to date. For this purpose, an in vitro mode of streptozotocin- (STZ-) induced rat oligodendrocyte OLN-93 cell injury was established to mimic the pathological changes of myelin sheath of AD and investigate the mechanism of SZL protecting injured OLN-93 cell. The results showed that STZ can decrease cell viability and downregulate the activity of PI3K/Akt-mTOR signalling pathway and the expression of myelin sheath-related proteins (MBP, MOG, and PLP) in OLN-93 cells. Both SZL-medicated serum and donepezil (positive control) can protect cells from STZ-caused damage. SZL-medicated serum increased OLN-93 cell viability in a dose- and time-dependent manner and enhanced the activity of PI3K/Akt-mTOR signalling pathway. The inhibitor of PI3K (LY294002) inhibited the protective effect of SZL-medicated serum on the STZ-injured OLN-93 cells. Furthermore, rapamycin, the inhibitor of mTOR, inhibited the promotion of cell viability and upregulation of p-mTOR and MBP caused by SZL-medicated serum. In conclusion, our data indicate that SZL plays its therapeutic role on AD by promoting PI3K/Akt-mTOR signalling pathway of oligodendrocytes. Thus, the present study may facilitate the therapeutic research of AD.
Project description:Marine sediments can reserve many environmental pollutants. Lipophilic marine phycotoxins (LMPs) are natural toxic substances widespread in the marine environment; however, evidence of their existence in sediment is scarce. In the present study, in order to explore the occurrence and distribution characteristics of LMPs in sediment, surface sediment samples collected from a tropical area of Daya Bay (DYB) at different seasons, were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). According to the results, up to six toxin compounds were detected in sediment samples from DYB, OA and DTX1 had the highest levels, followed by PTX2, homo-YTX, AZA2, and GYM. Although AZA2 and GYM were found in most of the sediment, OA, DTX1, homo-YTX, and PTX2 were the predominant toxin compounds, and PTX2 was the most ubiquitous toxin in sediment. The spatial distribution of LMP components in the sediment fluctuated with sampling times, partially according to the physical-chemical parameters of the sediment. There are likely several sources for LMPs existing in surface sediments, but it is difficult to determine contributions of a specific toxin-source in the sediment. Therefore, marine sediments may be a toxin reservoir for LMPs accumulation in benthic organisms via food chains.
Project description:Nasopharyngeal carcinoma (NPC) is a high morbidity and mortality cancer with an obvious racial and geographic bias, particularly endemic to Southeast China. Our previous studies demonstrated that Centipeda minima extract (CME) exhibited anti-cancer effects in human NPC cell lines. Arnicolide C and arnicolide D are sesquiterpene lactones isolated from Centipeda minima. In this study, for the first time, we investigated their anti-NPC effects and further explored the related molecular mechanisms. The effects of both arnicolide C and arnicolide D were tested in NPC cells CNE-1, CNE-2, SUNE-1, HONE1, and C666-1. The results showed that the two compounds inhibited NPC cell viability in a concentration- and time-dependent manner. As the inhibitory effect of arnicolide D was the more pronounced of the two, our following studies focused on this compound. Arnicolide D could induce cell cycle arrest at G2/M, and induce cell apoptosis. The molecular mechanism of cell cycle regulation and apoptosis induction was investigated, and the results showed that arnicolide D could downregulate cyclin D3, cdc2, p-PI3K, p-AKT, p-mTOR, and p-STAT3, and upregulate cleaved PARP, cleaved caspase 9, and Bax. Regulation of cyclin B1, cdk6, and Bcl-2 expression by arnicolide D showed dynamic changes according to dose and time. Taken together, arnicolide D modulated the cell cycle, activated the caspase signaling pathway, and inhibited the PI3K/AKT/mTOR and STAT3 signaling pathways. These findings provide a solid base of evidence for arnicolide D as a lead compound for further development, and act as proof for the viability of drug development from traditional Chinese medicines.
Project description:BACKGROUND:Renal cell carcinomas (RCC) harboring a TFE3 gene fusion (TfRCC) represent an aggressive subset of kidney tumors. Key signaling pathways of TfRCC are unknown and preclinical in vivo data are lacking. We investigated Akt/mTOR pathway activation and the preclinical efficacy of dual mTORC1/2 versus selective mTORC1 inhibition in TfRCC. METHODS:Levels of phosphorylated Akt/mTOR pathway proteins were compared by immunoblot in TfRCC and clear cell RCC (ccRCC) cell lines. Effects of the mTORC1 inhibitor, sirolimus, and the dual mTORC1/2 inhibitor, AZD8055, on Akt/mTOR activation, cell cycle progression, cell viability and cytotoxicity were compared in TfRCC cells. TfRCC xenograft tumor growth in mice was evaluated after 3-week treatment with oral AZD8055, intraperitoneal sirolimus and respective vehicle controls. RESULTS:The Akt/mTOR pathway was activated to a similar or greater degree in TfRCC than ccRCC cell lines and persisted partly during growth factor starvation, suggesting constitutive activation. Dual mTORC1/2 inhibition with AZD8055 potently inhibited TfRCC viability (IC50 = 20-50 nM) due at least in part to cell cycle arrest, while benign renal epithelial cells were relatively resistant (IC50 = 400 nM). Maximal viability reduction was greater with AZD8055 than sirolimus (80-90% versus 30-50%), as was the extent of Akt/mTOR pathway inhibition, based on significantly greater suppression of P-Akt (Ser473), P-4EBP1, P-mTOR and HIF1α. In mouse xenograft models, AZD8055 achieved significantly better tumor growth inhibition and prolonged mouse survival compared to sirolimus or vehicle controls. CONCLUSIONS:Akt/mTOR activation is common in TfRCC and a promising therapeutic target. Dual mTORC1/2 inhibition suppresses Akt/mTOR signaling more effectively than selective mTORC1 inhibition and demonstrates in vivo preclinical efficacy against TFE3-fusion renal cell carcinoma.
Project description:Collapsin response mediator protein (CRMP)-2 and the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway are associated with common physiological functions such as neuronal polarity, axonal outgrowth and synaptic strength, as well as various brain disorders including epilepsy. But, their regulatory and functional links are unclear. Alterations in CRMP-2 expression that lead to its functional changes are implicated in brain disorders such as epilepsy. Here, we investigate whether changes in CRMP-2 expression, possibly regulated by mTOR-related signaling, correlates with neuronal growth and viability. Inhibition of mTOR and/or phosphoinositol-3-kinase (PI3K) led to deceased p-S6K, and p-S6 signals also reduced CRMP-2 expression. These changes corresponded to inhibition of neuronal viability and proliferation in cultured hippocampal HT-22 cells under both basal serum-free and serum- or insulin-induced mTOR pathway-activated conditions. CRMP-2 expression tended to be increased by mTOR activation, indicated by an increase in p-S6/S6 level, in pentylentetrazole (PTZ)-induced epileptic rat hippocampal tissues was also significantly reduced by mTOR inhibition. Knockdown of CRMP-2 by si-RNA reduced the neuronal viability without changes in mTOR signaling, and overexpression of CRMP-2 recovered the glutamate-induced neurotoxicity and decrease of mTOR signaling in HT-22 cells. In conclusion, CRMP-2 protein expression controlled by the PI3K-mTOR-S6K signaling axis exerts its important functional roles in neuronal growth and survival.
Project description:Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality globally. Because most patients are diagnosed at advanced stages of the disease, multi-targeted tyrosine kinase inhibitor sorafenib is the only available drug to show limited effectiveness. Novel and effective therapies are unmet medical need for advanced HCC patients. Given that the aberrant expression and activity of platelet-derived growth factor receptor ? (PDGFR?) are closely associated with the pathogenesis of HCC, here we present the discovery and identification of a novel PDGFR? inhibitor, N-(3-((4-(benzofuran-2-yl)pyrimidin-2-yl)oxy)-4-methylphenyl)-4-((4-methylpiperazin-1-yl)methyl)benzamide (E5) after comparison of different derivatives. We found that E5 inhibited proliferation and induced apoptosis in HCC cells. Since the pan-caspase inhibitor Z-VAD-FMK partially rescued HCC cells from E5-reduced cell viability, autophagic cell death triggered by E5 was subsequently investigated. E5 could induce the conversion of LC3-I to LC3-II, increase the expression of Atg5 and restore the autophagy flux blocked by chloroquine. Meanwhile, E5 was able to downregulate the PDGFR?/PI3K/AKT/mTOR pathway and to activate MAPK/ERK signaling pathway. Taken together, in addition to the possibility of E5 as a valuable drug candidate, the present study further supports the notion that targeted inhibition of PDGFR? is a promising therapeutic strategy for HCC.