Accumulation and depuration of the cyanobacterial toxin cylindrospermopsin in the freshwater mussel Anodonta cygnea.

Cylindrospermopsin (CYN) is a toxic alkaloid produced by several genera of freshwater cyanobacteria. This compound has been implicated in outbreaks of human sickness and the death of domestic and wild animals. Given that several of the cyanobacterial genera known to produce CYN are common components of the phytoplankton of freshwaters including aquaculture facilities, we studied the accumulation of CYN in the freshwater mussel (swan mussel) Anodonta cygnea. Anodonta were exposed to CYN-producing cultures of the cyanobacterium Cylindrospermopsis raciborskii for 16 days and were found to accumulate the toxin to concentrations up to 2.52 microg g tissue dry weight(-1). There was considerable variation in the concentrations of CYN detected in different parts of the body. At the end of a 2-week accumulation period the distribution of CYN in the body of Anodonta was as follows: haemolymph (68.1%), viscera (23.3%), foot and gonad (7.7%) and mantle (0.9%). No CYN was detected in the gills or adductor muscle of any animals. Following a 2-week depuration period, approximately 50% of the toxin remained in the tissues. Based on the recently derived guideline value for CYN in human drinking water (1 microg l(-1)) and the concentrations of this compound in animal tissues reported here, there is a clear need for the increased monitoring of this compound in organisms grown for human and animal consumption.     

Context-dependent sensitization to ethanol in zebrafish (Danio rerio)

One of the most robust and readily measurable effects of moderate doses of ethanol on zebrafish behavior is locomotor hyperactivity. Two experiments were designed to examine the effects of repeated exposures to ethanol on ethanol-induced locomotor hyperactivity, and to determine whether these effects are context-dependent. Adult, wild-type zebrafish were given repeated exposure to ethanol in the presence of one contextual stimulus (A), while exposed to water in the presence of a second contextual stimulus (B). Exposure to ethanol consistently induced locomotor hyperactivity. After repeated exposures, animals tested with ethanol in the ethanol-paired context (A) showed sensitization of locomotor activity. When tested with ethanol in the unpaired context (B), however, sensitization was not observed. When tested in the absence of ethanol, there were no differences in responding to the paired and unpaired stimuli. This is the first demonstration of ethanol-induced locomotor sensitization in zebrafish. Moreover, this sensitization was context-specific, indicating that learning can modify drug-induced behaviors in zebrafish.     

First report on cylindrospermopsin producing Aphanizomenon flos-aquae (Cyanobacteria) isolated from two German lakes.

Three single-filament isolates of Aphanizomenon flos-aquae from two German lakes were found to produce remarkable amounts of the cyanobacterial hepatotoxin cylindrospermopsin (CYN). CYN-synthesis of the strains were evidenced both by LC-MS/MS analysis and detection of PCR products of gene fragments which are implicated in the biosynthesis of the toxin. The strains contain CYN in the range of 2.3-6.6 mg g(-1) of cellular dry weight. To our knowledge this is the first report of CYN in A. flos-aquae.     

Acute and long-term effects after single loading of functionalized multi-walled carbon nanotubes into zebrafish (Danio rerio)

Carbon nanotubes (CNTs) are widely explored for biomedical applications, but there is very limited information regarding their in vivo biodistribution and biocompatibility. Here, we report the in vivo biodistribution and long-term effects of functionalized multi-walled carbon nanotubes (MWCNTs) in developing zebrafish. The fluorescent-labeled MWCNTs were introduced into zebrafish embryos at 1-cell stage and at 72 h post fertilization through microinjection. After single injection, both acute and long-term interactions between zebrafish and functionalized MWCNTs were studied. The injected FITC-BSA-MWCNTs (at 1-cell stage) were allocated to all blastoderm cells of the embryos through proliferation, and were distinctively excluded from the yolk cell. When introduced into the circulation system, FITC-BSA-MWCNTs moved easily in the compartments and finally were cleaned out by the body at 96 h after the loading. At early stages, the treated zebrafish embryos generated immune response by accumulating circulating white blood cells at the trunk region. Under transmission electron microscope, many lysosome-like vesicles were observed in the blastoderm cells of the treated embryos. The zebrafish loaded with MWCNTs had normal primordial germ cells at early stage and produced second generation later on. However, the larvae of the second generation had obviously lower survival rates as compared to the untreated groups, suggesting a negative effect on the reproduction potential. These results suggest that extensive purification and functionalization processes can help improve the biocompatibility of CNTs. This study also indicates that purified CNTs may have long-term toxicity effects when they were delivered into the body.     

Effects of adrenergic agents on the expression of zebrafish (Danio rerio) vitellogenin Ao1

Teleost vitellogenins (VTGs) are large multidomain apolipoproteins, traditionally considered to be estrogen-responsive precursors of the major egg yolk proteins, expressed and synthesized mainly in hepatic tissue. The inducibility of VTGs has made them one of the most frequently used in vivo and in vitro biomarkers of exposure to estrogen-active substances. A significant level of zebrafish vtgAo1, a major estrogen responsive form, has been unexpectedly found in heart tissue in our present studies. Our studies on zebrafish cardiomyopathy, caused by adrenergic agonist treatment, suggest a similar protective function of the cardiac expressed vtgAo1. We hypothesize that its function is to unload surplus intracellular lipids in cardiomyocytes for “reverse triglyceride transportation” similar to that found in lipid transport proteins in mammals. Our results also demonstrated that zebrafish vtgAo1 mRNA expression in heart can be suppressed by both α-adrenergic agonist, phenylephrine (PE) and β-adrenergic agonist, isoproterenol (ISO). Furthermore, the strong stimulation of zebrafish vtgAo1 expression in plasma induced by the β-adrenergic antagonist, MOXIsylyl, was detected by Enzyme-Linked ImmunoSorbent Assay (ELISA). Such stimulation cannot be suppressed by taMOXIfen, an antagonist to estrogen receptors. Thus, our present data indicate that the production of teleost VTG in vivo can be regulated not only by estrogenic agents, but by adrenergic signals as well.     

Embryonic developmental toxicity of selenite in zebrafish (Danio rerio) and prevention with folic acid

Selenium (Se) is an essential micronutrient, but also a potential toxin, which may be absorbed in excess. Relatively little is known about selenium embryotoxicity in zebrafish. In this study, we evaluated the effect of selenite exposure in zebrafish embryos. Selenite treatment decreased survival and resulted in abnormal development in a dose- and time-dependent manner. We observed irregular growth of neurons in selenite treated embryos, characterized by the absence of neurons in the brain, trunk and tail. Selenite exposure also induced defects in heart function, such as bradycardia and cardiac dysplasia with irregular and smaller chamber shape. In addition, selenite exposure caused ectopic cell proliferation, apoptosis, and a change in the pattern of DNA methylation. Our results suggested that supplementation with folic acid (FA) ameliorated the cardiac and neural defects in selenite-treated embryos. In conclusion, we demonstrated that selenite exposure caused cardiac and neural defects in zebrafish embryos and that folic acid protected against this embryotoxicity. It will give insight into the risk assessment and prevention of Se-mediated embryotoxicity.     

A comparative study of Florida strains of Cylindrospermopsis and Aphanizomenon for cylindrospermopsin production.

The toxin cylindrospermopsin (CYN) is produced by a variety of cyanobacterial genera. One of these, Cylindrospermopsis raciborskii, is generally assumed to be the source of CYN in lakes and rivers in Florida, USA. However, in this study, none of the eight Florida isolates of this species tested contained the genetic determinants involved in toxin production nor did they produce CYN. We show for the first time that Aphanizomenon ovalisporum isolated from a pond in this state has the genes putatively associated with CYN production. Analysis by liquid chromatography with mass spectrometric detection (LC/MS) revealed that it produced CYN in the range of 7.39-9.33 microg mg(-1) freeze-dried cells. 16S rDNA sequences of this strain showed 99.6% and 99.9% identity to published A. ovalisporum and Anabaena bergii 16S sequences, respectively. These results help to explain the general lack of a defined relationship between the abundance of C. raciborskii in freshwater ecosystems of Florida and observed concentrations of CYN. The latter observation raises the potential that previous reports of CYN may be coincidental with unrecorded presence of another CYN-producing species.     

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.     

Development of a flow-through system for the fish embryo toxicity test (FET) with the zebrafish (Danio rerio)

The acute fish test is still a mandatory component in chemical hazard and risk assessment. However, one of the objectives of the new European chemicals policy (REACH – Registration, Evaluation, Authorization and Restriction of Chemicals) is to promote non-animal testing. For whole effluent testing in Germany, the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) has been an accepted and mandatory replacement of the fish test since January 2005. For chemical testing, however, further optimization of the FET is required to improve the correlation between the acute fish test and the alternative FET. Since adsorption of the test chemical to surfaces may reduce available exposure concentrations, a flow-through system for the FET using modified commercially available polystyrene 24-well microtiter plates was developed, thus combining the advantages of the standard FET with those of continuous delivery of test substances. The advantages of the design presented include: small test footprint, availability of adequate volumes of test solution for subsequent chemical analysis, and sufficient flow to compensate for effects of non-specific adsorption within 24 h. The flow-through test system can also be utilized to conduct longer-term embryo larval fish tests, thus offering the possibility for teratogenicity testing.     

Toxigenicity of enniatins from Western Australian Fusarium species to brine shrimp (Artemia franciscana)

The high prevalence (14 of 24 isolates) of enniatin-producing isolates from Western Australian Fusarium species isolated from pasture legumes associated with sheep feed refusal and rat deaths, and the high toxicity of their crude extracts to brine shrimp (Artemia franciscana) from a previous study warranted further investigation of this class of mycotoxin. Crude extracts from Fusarium acuminatum, Fusarium avenaceum, Fusarium tricinctum and Fusarium sambucinum, along with enniatins A, A1, B and B1 purified from a Western Australian strain of F. acuminatum using semi-preparative HPLC, were bioassayed using brine shrimp. All Fusarium isolates produced both enniatins B and B1, except for F. tricinctum WAC 8019, and 11 of the 17 isolates produced enniatin A1. Overall, all of the F. avenaceum isolates produced high amounts of enniatins, in particular enniatin B. One isolate of F. acuminatum (WAC 5715) and of F. tricinctum (WAC 11486) also produced high amounts of both enniatins B and B1. Only F. acuminatum WAC 5715 produced enniatin A among the tested isolates. All four purified enniatins A, A1, B, B1, individually and in combination, caused brine shrimp toxicity after 6 h of exposure, implicating that this emerging class of mycotoxin as a cause of the acute toxicity to brine shrimp observed. The mixture of all four enniatins was the most toxic to brine shrimp compared to purified individual enniatins, where the relative toxicity order was B > B1 > A1 > A. Enniatin B was the individual most toxic enniatin with some bioactivity at 5 μg/mL and almost 100% brine shrimp death at 50 μg/mL after 24 h of exposure. This study is the first report to confirm the acute toxicity of enniatins A, A1, B and B1 to brine shrimp, and also highlights the need for further investigation of the potential toxicity of these cyclic hexadepsipeptides to animals and humans.     

Cylindrospermopsin occurrence in two German lakes and preliminary assessment of toxicity and toxin production of Cylindrospermopsis raciborskii (Cyanobacteria) isolates.

Cylindrospermopsis raciborskii, a freshwater cyanobacterium of tropical origin, is not only increasingly found in (sub) tropical water bodies, but also in temperate regions. Since this species may produce potent toxins such as cylindrospermopsin (CYN) and paralytic shellfish poisons, its massive occurrence in water bodies used as drinking water sources or for recreation is of major concern. The proliferation of C. raciborskii in German water bodies has been documented for the past decade. We investigated the occurrence of CYN in field populations and isolates of C. raciborskii from two lakes, and assessed the toxicity of culture isolates using the mouse bioassay, primary rat hepatocytes and human derived cell lines. We show for the first time the occurrence of CYN in German water bodies. None of seven isolates of C. raciborskii contained CYN, however, all isolates were toxic to primary rat hepatocytes, human hepatoblastoma (HEP-G2) and human colon adenocarcinoma (CACO-2) cells. Methanolic extracts were more toxic than aqueous extracts. Three isolates tested in the mouse bioassay were toxic at a concentration of 800 mg kg(-1) showing liver and spleen damage and inflammation of the intestine. These results give strong evidence that the German isolates of C. raciborskii contain currently not identified or unknown toxins.     

Screening the toxic potential of Cylindrospermopsis raciborskii strains isolated from Lake Balaton, Hungary

Cylindrospermopsis raciborskii is becoming a major concern among cyanobacteria, due to its potential ability to produce toxic metabolites. We assessed the cytotoxic potential of four C. raciborskii strains (ACT 9502, ACT 9503, ACT 9504 and ACT 9505) isolated from Lake Balaton (Hungary), by lactate dehydrogenase (LDH) leakage measurements and by detecting morphological alterations in CHO-K1 (Chinese Hamster Ovary) cells. The Australian AQS (cylindrospermopsin producer) strain of C. raciborskii and purified cylindrospermopsin (CYN) were used as positive references in both the biochemical and morphological studies. Chemical analysis for known cyanotoxins was performed on aqueous extracts of ACT and AQS strains by the HPLC-MS technique.Comparing threshold values of LDH leakage data, different toxic potentials of cyanobacterial extracts are suggested in short term (3 h) and long (24 h) exposure regimes. In the acute (3 h) experiments the aqueous extract of the ACT 9505 strain proved to be most toxic (EC₅₀ = 7.4 mg mL⁻¹), while after 24 h the ACT 9504 extract was the most effective (EC₅₀ = 0.65 mg mL⁻¹). The extract of the AQS strain and the purified CYN exerted most of their toxic effects after 3 h exposure (EC₅₀ = 0.74 mg mL⁻¹, and 0.9 μg mL⁻¹ respectively).The morphological changes of CHO-K1 cells induced by the crude extracts of the ACT strains included fragmentation of the actin filaments then relocation of the depolymerized actin to the perinuclear region, resulting cell rounding and loss of adhesion. Exposure of CHO-K1 cells to the crude extract of the AQS strain, moreover, resulted cell shrinking and formation of filopodia, i.e. distinctly different cytological alterations from that induced by the ACT extracts and the purified CYN.Chemical analysis of the cyanobacterial crude extracts confirmed the presence of cylindrospermopsin in the extract of the AQS strain (8.5 mg CYN g⁻¹ dry weight), and none of the presently known cyanotoxins have been analytically confirmed in the extracts of the ACT strains isolated from the Lake Balaton.Although a significant toxicity of all four ACT C. raciborskii strains is confirmed by both biochemical and morphological studies, our results also pointed out the necessity of further studies to identify the toxic, but still unknown metabolic components produced by these cyanobacterial members of the phytoplankton communities.     

Cadmium sulfate and CdTe-quantum dots alter DNA repair in zebrafish (Danio rerio) liver cells

Increasing use of quantum dots (QDs) makes it necessary to evaluate their toxicological impacts on aquatic organisms, since their contamination of surface water is inevitable. This study compares the genotoxic effects of ionic Cd versus CdTe nanocrystals in zebrafish hepatocytes. After 24 h of CdSO₄ or CdTe QD exposure, zebrafish liver (ZFL) cells showed a decreased number of viable cells, an accumulation of Cd, an increased formation of reactive oxygen species (ROS), and an induction of DNA strand breaks. Measured levels of stress defense and DNA repair genes were elevated in both cases. However, removal of bulky DNA adducts by nucleotide excision repair (NER) was inhibited with CdSO₄ but not with CdTe QDs. The adverse effects caused by acute exposure of CdTe QDs might be mediated through differing mechanisms than those resulting from ionic cadmium toxicity, and studying the effects of metallic components may be not enough to explain QD toxicities in aquatic organisms.     

Protection against the toxicity of microcystin-LR and cylindrospermopsin in Artemia salina and Daphnia spp. by pre-treatment with cyanobacterial lipopolysaccharide (LPS)

Purified cyanobacterial lipopolysaccharide (LPS) was not acutely toxic to three aquatic invertebrates (Artemia salina, Daphnia magna and Daphnia galeata) in immersion trials. However, pre-exposure (24 h) to 2 ng mL⁻¹ LPS increased the LC₅₀ of microcystin-LR significantly in all 3 species. Similar results were observed with A. salina pre-treated with the same concentration of cyanobacterial LPS and subsequently exposed to cylindrospermopsin, increasing the LC₅₀ by 8. The findings indicate the need to include exposures to defined combinations of cyanotoxins, and in defined sequences, to understand the contributions of individual cyanotoxins in accounting for cyanobacterial toxicity to invertebrates in natural aquatic environments.     

Toxicity assessment of Amphidinium carterae, Coolia cfr. monotis and Ostreopsis cfr. ovata (Dinophyta) isolated from the northern Ionian Sea (Mediterranean Sea)

In many coastal areas the abundant proliferation of microalgae producing biotoxins determines the occurrence of Harmful Algal Blooms (HABs). Their presence in temperate waters is well documented and often associated with marine toxin-derived disease. The occurrence and toxicity of three harmful microalgae (Amphidinium carterae, Coolia cfr. monotis and Ostreopsis cfr. ovata) from the northern Ionian Sea (Mediterranean Sea) is hereby reported. The three dinoflagellates were sampled both on macroalgae and water and their morphology and occurrence were compared to those of other Mediterranean sites. The toxicity of the three cultured strains was tested by Artemia salina and hemolysis tests and their effects on the first stages of the sea urchin development was also evaluated. The contemporary presence of the three species inhibited the in vitro sea urchin embryonic development. But this action could be ascribed to the sole Ostreopsis as the addition of the single species to the sea urchins embryos evidenced no effects in presence of Amphidinium or Coolia cells, and an irregular segmentation in presence of Ostreopsis. In particular, this latter species exerted a cytotoxic effect in a dose-dependent manner, with a production of deformed embryos even at very low cell concentration (42 cells mL⁻¹). Nevertheless, when algal cell lysate was added, some effects on the sea urchin development was detected for each dinoflagellate, and also in this case Ostreopsis has proved to be the most toxic species. However, the lysate of Amphidinium and Ostreopsis strongly affects the A. salina nauplii vitality, while the hemolytic activity was very low for Amphidinium and Coolia lysate and very strong for Ostreopsis.Our results highlight the importance to monitoring the presence of these dinoflagellates whose effects may also be reflected on the early life stages of marine organisms, especially those species that are important from both an ecological and economic point of view, as the sea urchins are.     

Acute and repeated-doses (28 days) toxicity study of Hypericum polyanthemum Klotzsch ex Reichardt (Guttiferare) in mice

Hypericum polyanthemum, a South Brazilian species showed antidepressant-like and antinociceptive effects in rodents. Since limited information is available on the toxicity and safety profile of the Hypericum genus, we therefore investigated whether H. polyanthemum cyclo-hexane extract (POL) treatment could be associated with toxicity in preclinical setting using mice as an experimental model. These toxicity studies were based on the guidelines of the Organization for Economic Cooperation and Development (OECD-guidelines 423 and 407). Animals received POL single dose (2000 mg/kg, p.o.) or daily for 28-days (90, 450 and 900 mg/kg, p.o.). Acute toxicity study did not detect any clinical signs, changes in behavior or mortality. In repeated dose toxicity study, POL affected the body weight gain and induced biochemical, hematological and liver histological changes at 450 and 900 mg/kg. Mice treated with POL 90 mg/kg did not show any toxicity signs. In conclusion H. polyanthemum can be classified as safe (category 5) according to OECD acute toxicity parameters. However, the alterations observed after repeated treatment with high doses suggest that the liver could be the target organ on potential H. polyanthemum toxicity and point to the need of further toxicity studies.     

Toxicity of Bothrops neuwiedi complex (“yarará chica”) venom from different regions of Argentina (Serpentes, Viperidae)

We report a study of toxic and enzymatic activities of Bothrops neuwiedi complex venoms collected from specimens of different regions of Argentina and a pool of these same venoms. Were determined lethal, hemorrhagic and pro-coagulant (plasma and fibrinogen) doses and the neutralization of these activities by a bivalent antivenom. The electrophoretic pattern of different regions venom was studied by SDS-PAGE. All samples exhibited lethal potencies, hemorrhagic and coagulant (plasma and fibrinogen) activities with potencies concordant with previous studies. The only conspicuous difference in the toxicological pattern of Bothrops diporus venoms was the low-thrombin-like activity found in one sample. The antivenom used in this study could neutralize all the toxic activities tested and the neutralizing potency of the antivenom was comparable for all samples. Despite the wide distribution of B. neuwiedi complex throughout Argentina and the evident morphological variation between B. diporus (B. neuwiedi complex), this study establishes a remarkably similar toxicity profile throughout its range. This is the first systematic study on the regional variation of enzymatic and toxic activities of venom from species belonging to the B. neuwiedi complex, one of the snakes of highest sanitary importance in South America and their neutralization by the type of antivenom most commonly used in the South of South America.     

Safety assessment of methanol extract of red dragon fruit (Hylocereus polyrhizus): Acute and subchronic toxicity studies

Recently, the fruits of Hylocereus polyrhizus, known as red dragon fruit, have received much attention from growers worldwide. However, there is little toxicological information regarding the safety of repeated exposure to these fruits. The present study evaluated the potential toxicity of a methanol extract of H. polyrhizus fruit after acute and subchronic administration in rats. In the acute toxicity study, single doses of fruit extract (1250, 2500 and 5000 mg/kg) were administered to rats by oral gavage, and the rats were then monitored for 14 days. In the subchronic toxicity study, the fruit extract was administered orally to rats at doses of 1250, 2500 and 5000 mg/kg/day for 28 days. There was no mortality or signs of acute or subchronic toxicity. There was no significant difference in body weight, relative organ weight or hematological parameters in the subchronic toxicity study. Biochemical analysis showed some significant changes, including creatinine, globulin, total protein and urea levels. No abnormality of internal organs was observed between treatment and control groups. The lethal oral dose of the fruit extract is more than 5000 mg/kg and the no-observed-adverse-effect level (NOAEL) of the extract for both male and female rats is considered to be 5000 mg/kg per day for 28 days.