Are known cyanotoxins involved in the toxicity of picoplanktonic and filamentous north atlantic marine cyanobacteria?

Eight marine cyanobacteria strains of the genera Cyanobium, Leptolyngbya, Oscillatoria, Phormidium, and Synechococcus were isolated from rocky beaches along the Atlantic Portuguese central coast and tested for ecotoxicity. Strains were identified by morphological characteristics and by the amplification and sequentiation of the 16S rDNA. Bioactivity of dichloromethane, methanol and aqueous extracts was assessed by the Artemia salina bioassay. Peptide toxin production was screened by matrix assisted laser desorption/ionization time of flight mass spectrometry. Molecular analysis of the genes involved in the production of known cyanotoxins such as microcystins, nodularins and cylindrospermopsin was also performed. Strains were toxic to the brine shrimp A. salina nauplii with aqueous extracts being more toxic than the organic ones. Although mass spectrometry analysis did not reveal the production of microcystins or other known toxic peptides, a positive result for the presence of mcyE gene was found in one Leptolyngbya strain and one Oscillatoria strain. The extensive brine shrimp mortality points to the involvement of other unknown toxins, and the presence of a fragment of genes involved in the cyanotoxin production highlight the potential risk of cyanobacteria occurrence on the Atlantic coast.

Primary screening of the bioactivity of brackishwater cyanobacteria: toxicity of crude extracts to artemia salina larvae and paracentrotus lividus embryos

Cyanobacteria are a diverse group of Gram-negative bacteria that produce an array of secondary compounds with selective bioactivity against vertebrates, invertebrates, plants, microalgae, fungi, bacteria, viruses and cell lines. The aim of this study was to assess the toxic effects of aqueous, methanolic and hexane crude extracts of benthic and picoplanktonic cyanobacteria isolated from estuarine environments, towards the nauplii of the brine shrimp Artemia salina and embryos of the sea urchin Paracentrotus lividus. The A. salina lethality test was used as a frontline screen and then complemented by the more specific sea urchin embryo-larval assay. Eighteen cyanobacterial isolates, belonging to the genera Cyanobium, Leptolyngbya, Microcoleus, Phormidium, Nodularia, Nostoc and Synechocystis, were tested. Aqueous extracts of cyanobacteria strains showed potent toxicity against A. salina, whereas in P. lividus, methanolic and aqueous extracts showed embryo toxicity, with clear effects on development during early stages. The results suggest that the brackishwater cyanobacteria are producers of bioactive compounds with toxicological effects that may interfere with the dynamics of invertebrate populations.

Toxicity of chromated copper arsenate: a study in mice

Chromated copper arsenate (CCA) was widespread used as a chemical wood preservative with application in the construction of playground equipment, fences, jetties, and naval. Environmental protection agency (EPA) had limited the use of CCA-treated wood on 2002, due to probable implications on both human and environmental health. Although this fact, several industries pursue the use of this product within their manufactories. In addition, the durability of this wood for 60 years, makes these treated products an hazard to the public health. In the present work, studies were explored exposing mice to CCA, during 14, 24, 48, and 96 h for the assessment of acute toxicity of CCA. Kidney and liver were removed, prepared for histology and for metalloid, and copper content evaluation by high resolution inductively coupled plasma mass spectroscopy. The histological results evidenced apparently normal structures for control animals and group exposed to As2O5. On the contrary, the renal sections of the animals treated with CCA revealed epithelium cells desquamation, hyaline, and granular casts in renal tubules lumen. Furthermore, high levels of arsenic were detected in the kidney of animals treated with CCA over 14 and 48 h, being significantly greater than controls. Although this approach underlines the potential hazard of CCA on some vital organs, further testing may be required to establish the impacts on other functions.

Evaluation of the role of glutathione in the lead-induced toxicity in saccharomyces cerevisiae

The effect of intracellular reduced glutathione (GSH) in the lead stress response of Saccharomyces cerevisiae was investigated. Yeast cells exposed to Pb, for 3 h, lost the cell proliferation capacity (viability) and decreased intracellular GSH level. The Pb-induced loss of cell viability was compared among yeast cells deficient in GSH1 (∆gsh1) or GSH2 (∆gsh2) genes and wild-type (WT) cells. When exposed to Pb, ∆gsh1 and ∆gsh2 cells did not display an increased loss of viability, compared with WT cells. However, the depletion of cellular thiols, including GSH, by treatment of WT cells with iodoacetamide (an alkylating agent, which binds covalently to thiol group), increased the loss of viability in Pb-treated cells. In contrast, GSH enrichment, due to the incubation of WT cells with amino acids mixture constituting GSH (l-glutamic acid, l-cysteine and glycine), reduced the Pb-induced loss of proliferation capacity. The obtained results suggest that intracellular GSH is involved in the defence against the Pb-induced toxicity; however, at physiological concentration, GSH seems not to be sufficient to prevent the Pb-induced loss of cell viability.

Impact of excipients in the chronic toxicity of fluoxetine on the alga Chlorella vulgaris

Fluoxetine is a selective serotonin reuptake inhibitor (SSRI) widely used in the treatment of major depression. It has been detected in surface and wastewaters, being able to negatively affect aquatic organisms. Most of the ecotoxicity studies focused only in pharmaceuticals, though excipients can also pose a risk to non-target organisms. In this work the ecotoxicity of five medicines (three generic formulations and two brand labels) containing the same active substance (fluoxetine hydrochloride) was tested on the alga Chlorella vulgaris, in order to evaluate if excipients can influence their ecotoxicity. Effective concentrations that cause 50% of inhibition (EC50) ranging from 0.25 to 15 mg L−1 were obtained in the growth inhibition test performed for the different medicines. The corresponding values for fluoxetine concentration are 10 times lower. Higher EC50 values had been published for the same alga considering only the toxicity of fluoxetine. Therefore, this increase in toxicity may be attributed to the presence of excipients. Thus more studies on ecotoxicological effects of excipients are required in order to assess the environmental risk they may pose to aquatic organisms.

Saccharomyces cerevisiae Mutants Affected in Vacuole Assembly or Vacuolar H+-ATPase are Hypersensitive to Lead (Pb) Toxicity

Lead is an important environmental pollutant. The role of vacuole, in Pb detoxification, was studied using a vacuolar protein sorting mutant strain (vps16D), belonging to class C mutants. Cells disrupted in VPS16 gene, did not display a detectable vacuolar-like structure. Based on the loss of cell proliferation capacity, it was found that cells from vps16D mutant exhibited a hypersensitivity to Pb-induced toxicity, compared to wild type (WT) strain. The function of vacuolar H?-ATPase (VATPase), in Pb detoxification, was evaluated using mutants with structurally normal vacuoles but defective in subunits of catalytic (vma1D or vma2D) or membrane domain (vph1D or vma3D) of V-ATPase. All mutants tested, lacking a functional V-ATPase, displayed an increased susceptibility to Pb, comparatively to cells from WT strain. Modification of vacuolar morphology, in Pb-exposed cells, was visualized using a Vma2p-GFP strain. The treatment of yeast cells with Pb originated the fusion of the medium size vacuolar lobes into one enlarged vacuole. In conclusion, it was found that vacuole plays an important role in the detoxification of Pb in Saccharomyces cerevisiae; in addition, a functional V-ATPase was required for Pb compartmentalization.

Toxicity screening of Diclofenac, Propranolol, Sertraline and Simvastatin using Danio rerio and Paracentrotus lividus embryo bioassays

Early life-stage bioassays have been used as an alternative to short-term adult toxicity tests since they are cost-effective. A single couple can produce hundreds or thousands of embryos and hence can be used as a simple high-throughput approach in toxicity studies. In the present study, zebrafish and sea urchin embryo bioassays were used to test the toxicity of four pharmaceuticals belonging to different therapeutic classes: diclofenac, propranolol, simvastatin and sertraline. Simvastatin was the most toxic tested compound for zebrafish embryo, followed by diclofenac. Sertraline was the most toxic drug to sea urchin embryos, inducing development abnormalities at the ng/L range. Overall, our results highlight the potential of sea urchin embryo bioassay as a promising and sensitive approach for the high-throughput methods to test the toxicity of new chemicals, including pharmaceuticals, and identify several drugs that should go through more detailed toxicity assays.

Toxicity assessment of crude and partially purified extracts of marine Synechocystis and Synechococcus cyanobacterial strains in marine invertebrates

Among the Cyanoprokaryota, the genera Synechocystis and Synechococcus have rarely been studied with respect to potential toxicity. This is particularly true with marine environments where studies about the toxicity of cyanobacteria are restricted to filamentous forms at the warmer temperate and tropical regions and also to filamentous forms at cold seas such as the Baltic Sea. In this study, we describe the effects of cyanobacterial strains of the Synechocystis and Synechococcus genera isolated from the marine coast of Portugal, on marine invertebrates. Crude and partially purified extracts at a concentration of 100 mg/ml of freeze-dried material of the marine strains were tested for acute toxicity in nauplii of the brine shrimp Artemia salina, in the rotifer Brachionus plicatillis and in embryos of the sea urchin Paracentrotus lividus and the mussel Mytilus galloprovincialis. The cyanobacterial extracts, especially the crude extract, had an impact on A. salina nauplii. No significant toxic effects were registered against the rotifer. A negative impact of all strains was recorded on the embryonic development of the sea urchin, with toxic effects resulting in an inhibition of embryogenesis or development of smaller larvae. To the mussel embryos, the effects of cyanobacterial extracts resulted in a complete inhibition of embryogenesis. The results of all assays indicate that Synechocystis and Synechococcus marine strains contained toxic compounds to marine invertebrates.