Diatom-derived Polyunsaturated Aldehydes Research Articles

Diatom-Derived Polyunsaturated Aldehydes Activate Similar Cell Death Genes in Two Different Systems: Sea Urchin Embryos and Human Cells

Programmed cell death, such as apoptosis and autophagy, are key processes that are activated early on during development, leading to remodelling in embryos and homeostasis in adult organisms. Genomic conservation of death factors has been largely investigated in the animal and plant kingdoms. In this study, we analysed, for the first time, the expression profile of 11 genes involved in apoptosis (extrinsic and intrinsic pathways) and autophagy in sea urchin Paracentrotus lividus embryos exposed to antiproliferative polyunsaturated aldehydes (PUAs), and we compared these results with those obtained on the human cell line A549 treated with the same molecules. We found that sea urchins and human cells activated, at the gene level, a similar cell death response to these compounds. Despite the evolutionary distance between sea urchins and humans, we observed that the activation of apoptotic and autophagic genes in response to cytotoxic compounds is a conserved process. These results give first insight on death mechanisms of P. lividus death mechanisms, also providing additional information for the use of this marine organism as a useful in vitro model for the study of cell death signalling pathways activated in response to chemical compounds.

Diatom-Derived Polyunsaturated Aldehydes Are Unlikely to Influence the Microbiota Composition of Laboratory-Cultured Diatoms.

Diatom-derived oxylipins, including polyunsaturated aldehydes (PUA), are considered to have infochemical, allelochemical and bacteriostatic properties, with plausible roles as grazing deterrents and regulators of inter- and intraspecific competition. However, the extent and mechanisms of how PUA influence diatom–bacteria interactions remain unresolved. In this study, impacts on the diversity of the associated bacterial communities (microbiota) of two contrasting Skeletonema marinoi strains (a PUA and a non-PUA producer) were investigated under three nitrate conditions in batch culture. Further, the response of the culture microbiota was studied when spiked with PUA at ecologically relevant concentrations (86nM octadienal and 290nM heptadienal). Of the 741 identified OTUs, Proteobacteria was the most abundant phylum (62.10%), followed by Bacteroidetes (12.33%) and Firmicutes (6.11%). Escherichia/Shigella were the most abundant genera for all treatments. Similar communities were present in both spiked and non-spiked cultures suggesting they can tolerate PUA exposure at realistic concentrations. This study suggests that PUA are not major drivers of diatom–bacteria interactions in laboratory cultures.

Toxicity of diatom-derived polyunsaturated aldehyde mixtures on sea urchin Paracentrotus lividus development

Diatom-derived polyunsaturated aldehydes (PUAs), decadienal, heptadienal and octadienal, derive from the oxidation of fatty acids and have cytotoxic and anticancer effects. PUAs, tested separately, induce malformations in sea urchin Paracentrotus lividus embryos. Decadienal induces the worst malformations and lowest survival rates. Interestingly, decadienal, heptadienal and octadienal place in motion several genes to counteract their negative effects. To date, no studies are available reporting on the effects of PUA mixtures on marine invertebrates. Here we test binary and ternary mixtures on embryonic development of P. lividus. Our findings demonstrate that mixtures of PUAs act (i) at morphological level in synergistic way, being much more severe compared to individual PUAs; (ii) at molecular level also reveal an additive effect, affecting almost all fifty genes, previously tested using individual PUAs. This study is relevant from an ecological point of view since diatoms are a major food source for both pelagic and benthic organisms. This work opens new perspectives for understanding the molecular mechanisms that marine organisms use in reacting to environmental natural toxin mixtures such as diatom PUAs.

Assessment of sex-related variability of biomarkers in sea urchin (Paracentrotus lividus) from Bizerte lagoon, Tunisia

The marine environment is continually subjected to the action of stressors (including natural toxins), which represent a constant danger for benthic communities. In the present work using network analysis we identified ten genes on the basis of associated functions (FOXA, FoxG, GFI-1, nodal, JNK, OneCut/Hnf6, TAK1, tcf4, TCF7, VEGF) in the sea urchin Paracentrotus lividus, having key roles in different processes, such as embryonic development and asymmetry, cell fate specification, cell differentiation and morphogenesis, and skeletogenesis. These genes are correlated with three HUB genes, Foxo, Jun and HIF1A. Real Time qPCR revealed that during sea urchin embryonic development the expression levels of these genes were modulated by three diatom-derived polyunsaturated aldehydes (PUAs), decadienal, heptadienal and octadienal. Our findings show how changes in gene expression levels may be used as an early indicator of stressful conditions in the marine environment. The identification of key genes and the molecular pathways in which they are involved represents a fundamental tool in understanding how marine organisms try to afford protection against toxicants, to avoid deleterious consequences and irreversible damages. The genes identified in this work as targets for PUAs can be considered as possible biomarkers to detect exposure to different environmental pollutants.

Nitric Oxide Mediates Nitrite-Sensing and Acclimation and Triggers a Remodeling of Lipids.

Nitric oxide (NO) is an intermediate of the nitrogen cycle, an industrial pollutant, and a marker of climate change. NO also acts as a gaseous transmitter in a variety of biological processes. The impact of environmental NO needs to be addressed. In diatoms, a dominant phylum in phytoplankton, NO was reported to mediate programmed cell death in response to diatom-derived polyunsaturated aldehydes. Here, using the Phaeodactylum Pt1 strain, 2E,4E-decadienal supplied in the micromolar concentration range led to a nonspecific cell toxicity. We reexamined NO biosynthesis and response in Phaeodactylum NO inhibits cell growth and triggers triacylglycerol (TAG) accumulation. Feeding experiments indicate that NO is not produced from Arg but via conversion of nitrite by the nitrate reductase. Genome-wide transcriptional analysis shows that NO up-regulates the expression of the plastid nitrite reductase and genes involved in the subsequent incorporation of ammonium into amino acids, via both Gln synthesis and Orn-urea pathway. The phosphoenolpyruvate dehydrogenase complex is also up-regulated, leading to the production of acetyl-CoA, which can feed TAG accumulation upon exposure to NO. Transcriptional reprogramming leading to higher TAG content is balanced with a decrease of monogalactosyldiacylglycerol (MGDG) in the plastid via posttranslational inhibition of MGDG synthase enzymatic activity by NO. Intracellular and transient NO emission acts therefore at the basis of a nitrite-sensing and acclimating system, whereas a long exposure to NO can additionally induce a redirection of carbon to neutral lipids and a stress response.

New inter-correlated genes targeted by diatom-derived polyunsaturated aldehydes in the sea urchin Paracentrotus lividus

The marine environment is continually subjected to the action of stressors (including natural toxins), which represent a constant danger for benthic communities. In the present work using network analysis we identified ten genes on the basis of associated functions (FOXA, FoxG, GFI-1, nodal, JNK, OneCut/Hnf6, TAK1, tcf4, TCF7, VEGF) in the sea urchin Paracentrotus lividus, having key roles in different processes, such as embryonic development and asymmetry, cell fate specification, cell differentiation and morphogenesis, and skeletogenesis. These genes are correlated with three HUB genes, Foxo, Jun and HIF1A. Real Time qPCR revealed that during sea urchin embryonic development the expression levels of these genes were modulated by three diatom-derived polyunsaturated aldehydes (PUAs), decadienal, heptadienal and octadienal. Our findings show how changes in gene expression levels may be used as an early indicator of stressful conditions in the marine environment. The identification of key genes and the molecular pathways in which they are involved represents a fundamental tool in understanding how marine organisms try to afford protection against toxicants, to avoid deleterious consequences and irreversible damages. The genes identified in this work as targets for PUAs can be considered as possible biomarkers to detect exposure to different environmental pollutants.

Diatom-derived polyunsaturated aldehydes activate cell death in human cancer cell lines but not normal cells.

Diatoms are an important class of unicellular algae that produce bioactive polyunsaturated aldehydes (PUAs) that induce abortions or malformations in the offspring of invertebrates exposed to them during gestation. Here we compare the effects of the PUAs 2-trans,4-trans-decadienal (DD), 2-trans,4-trans-octadienal (OD) and 2-trans,4-trans-heptadienal (HD) on the adenocarcinoma cell lines lung A549 and colon COLO 205, and the normal lung/brunch epithelial BEAS-2B cell line. Using the viability MTT/Trypan blue assays, we show that PUAs have a toxic effect on both A549 and COLO 205 tumor cells but not BEAS-2B normal cells. DD was the strongest of the three PUAs tested, at all time-intervals considered, but HD was as strong as DD after 48 h. OD was the least active of the three PUAs. The effect of the three PUAs was somewhat stronger for A549 cells. We therefore studied the death signaling pathway activated in A549 showing that cells treated with DD activated Tumor Necrosis Factor Receptor 1 (TNFR1) and Fas Associated Death Domain (FADD) leading to necroptosis via caspase-3 without activating the survival pathway Receptor-Interacting Protein (RIP). The TNFR1/FADD/caspase pathway was also observed with OD, but only after 48 h. This was the only PUA that activated RIP, consistent with the finding that OD causes less damage to the cell compared to DD and HD. In contrast, cells treated with HD activated the Fas/FADD/caspase pathway. This is the first report that PUAs activate an extrinsic apoptotic machinery in contrast to other anticancer drugs that promote an intrinsic death pathway, without affecting the viability of normal cells from the same tissue type. These findings have interesting implications also from the ecological viewpoint considering that HD is one of the most common PUAs produced by diatoms.

Teratogenic Effects of Diatom Metabolites on Sea Urchin Paracentrotus lividus Embryos

The diatom-derived polyunsaturated aldehydes (PUAs), 2-trans,4-trans-decadienal, 2-trans,4-trans-octadienal, 2-trans,4-trans,7-octatrienal, 2-trans,4-trans-heptadienal, as well as tridecanal were tested on early and later larval development in the sea urchin Paracentrotus lividus. We also tested the effect of some of the more abundant diatom polyunsaturated fatty acids (PUFAs) on development, in particular 5,8,11,14,17-eicosapentaenoic acid (EPA), one of the main precursors of diatom PUAs, as well as 4,7,10,13,16,19-docosahexaenoic acid (DHA), 6,9,12,15-octadecatetraenoic acid (stearidonic acid), 6,9,12-octadecatrienoic acid (γ-linolenic acid) and 9,12-octadecadienoic acid (linoleic acid). PUAs blocked sea urchin cell cleavage in a dose dependent manner and with increasing chain length from C7 to C10 PUAs, with arrest occurring at 27.27 μM with heptadienal, 16.13 μM with octadienal, 11.47 μM with octatrienal and 5.26 μM with decadienal. Of the PUFAs tested, only EPA and stearidonic acid blocked cleavage, but at much higher concentrations compared to PUAs (331 μM for EPA and 181 μM for stearidonic acid). Sub-lethal concentrations of decadienal (1.32–5.26 μM) delayed development of embryos and larvae which showed various degrees of malformations depending on the concentrations tested. Sub-lethal concentrations also increased the proportion of TUNEL-positive cells indicating imminent death in embryos and larvae. Using decadienal as a model PUA, we show that this aldehyde can be detected spectrophotometrically for up to 14 days in f/2 medium.

High Plasticity in the Production of Diatom-derived Polyunsaturated Aldehydes under Nutrient Limitation: Physiological and Ecological Implications

Diatoms have evolved a silicified cell wall that provides an efficient barrier against herbivores. These microalgae also produce chemical compounds such as polyunsaturated aldehydes (PUAs) that can potentially impair recruitment and cause malformations in the offspring of such grazers. We measured silica content as an indication of cell wall thickness, organic nutrient cell quotas, PUAs and polyunsaturated fatty acid cell content in Skeletonema marinoi grown under N-, P- and Si-limitation in continuous cultures. A 7.5 fold increase in PUA production was observed (27.5 fmol cell(-1)) in Si-limited cells with respect to the controls, while Si content decreased by 50%. PUA production decreased in nitrogen-limited cells to 0.14 fmol cell(-1) and increased 3 times in phosphorus-limited cells (7.53 fmol cell(-1)), while silica content increased by 20% in both cases. The substrate to product ratio suggested that production of the PUAs heptadienal and octadienal was limited by the amount of substrate under P- and Si-limitation, and by enzyme activity under N-limitation. Octatrienal production was likely limited by enzyme activity in all growth conditions. The high PUA levels produced under Si-limitation, when cells have a thinner cell wall, suggests a compensatory alternation of mechanical and chemical defense mechanisms.

Giant liposomes as delivery system for ecophysiological studies in copepods

Giant liposomes are proposed as a potential delivery system in marine copepods, the dominant constituent of the zooplankton. Liposomes were prepared in the same size range as the food ingested by copepods (mean diameter of about 7 microm). The encapsulation of a hydrophilic and high molecular mass fluorescent compound, fluorescein isothiocyanate-dextran (FitcDx), within the liposomes provided a means of verifying copepod ingestion when viewed with the confocal laser-scanning microscope. Females of the calanoid copepod Temora stylifera were fed with FitcDx-encapsulated liposomes alone or mixed with the dinoflagellate alga Prorocentrum minimum. Control copepods were incubated with the P. minimum diet alone. Egg production rates, percentage egg-hatching success and number of faecal pellets produced were evaluated after 24 h and 48 h of feeding. Epifluorescence of copepod gut and faecal pellets indicated that the liposomes were actively ingested by T. stylifera in both experimental food conditions, with or without the dinoflagellate diet. Ingestion rates calculated using 3H-labelled liposomes indicated that females ingested more liposomes when P. minimum was added to the solution (16% vs 7.6% of uptake). When liposomes were supplied together with the algal diet, egg production rate, egg-hatching success and faecal pellet production were as high as those observed for the control diet. By contrary, egg production and hatching success were very low with a diet of liposomes alone and faecal pellet production was similar to that recorded in starved females. This results suggest that liposomes alone did not add any nutritive value to the diet, making them a good candidate as inert carriers to study the nutrient requirements or biological activity of different compounds. In particular, such liposomes are proposed as carriers for diatom-derived polyunsaturated aldehydes, which are known to impair copepod embryo viability. Other potential applications of liposomes as a delivery system of drugs and nutrients in copepod mass cultivation, or as carriers of pollutants to study copepod physiology in ecotoxicological experiments, are also discussed.