Undifferentiated murine embryonic stem cells used to model the effects of the blue-green algal toxin cylindrospermopsin on preimplantation embryonic cell proliferation.

Undifferentiated mouse embryonic stem cell (mES) proliferation in vitro resembles aspects of in vivo pre-implantation embryonic development. mES were used to assess the embryo-toxicity of cylindrospermopsin (CYN), a water contaminant with an Australian Drinking Water Guideline (ADWG) of 1 µg/L. mES exposed to 0-1 µg/mL CYN for 24-168 h were subjected to an optimised crystal violet viability assay. mES exposed to retinoic acid ± 1 µg/L CYN differentiated into neural-like cells confirmed by morphological examination and RT-PCR for Oct4, Brachyury and Nestin. The CYN No Observed Effect Concentration (OEC) was 0.5 µg/mL, the Lowest OEC was 1 µg/mL (p < 0.001, n = 3), and the IC50 was 0.86 µg/mL after 24 h. The ADWG 1 µg/L CYN did not affect differentiation or proliferation after 72 h, but decreased proliferation after 168 h (p < 0.05). We conclude that higher algal bloom-associated CYN concentrations have the potential to impair in vivo pre-implantation development, and the mES crystal violet assay has broad application to screening environmental toxins.

Nodularin exposure induces SOD1 phosphorylation and disrupts SOD1 co-localization with actin filaments.

Apoptotic cell death is induced in primary hepatocytes by the Ser/Thr protein phosphatase inhibiting cyanobacterial toxin nodularin after only minutes of exposure. Nodularin-induced apoptosis involves a rapid development of reactive oxygen species (ROS), which can be delayed by the Ca2+/calmodulin protein kinase II inhibitor KN93. This apoptosis model provides us with a unique population of highly synchronized dying cells, making it possible to identify low abundant phosphoproteins participating in apoptosis signaling. Here, we show that nodularin induces phosphorylation and possibly also cysteine oxidation of the antioxidant Cu,Zn superoxide dismutase (SOD1), without altering enzymatic SOD1 activity. The observed post-translational modifications of SOD1 could be regulated by Ca2+/calmodulin protein kinase II. In untreated hepatocytes, a high concentration of SOD1 was found in the sub-membranous area, co-localized with the cortical actin cytoskeleton. In the early phase of nodularin exposure, SOD1 was found in high concentration in evenly distributed apoptotic buds. Nodularin induced a rapid reorganization of the actin cytoskeleton and, at the time of polarized budding, SOD1 and actin filaments no longer co-localized.

Cylindrospermopsin, a blue-green algal toxin, inhibited human luteinised granulosa cell protein synthesis in vitro.

The blue-green algal toxin cylindrospermopsin (CYN) inhibits protein synthesis, and CYP450 enzymes metabolise CYN to cytotoxic endproducts. Human chorionic gonadotrophin (hCG) stimulates the de novo synthesis of StAR and CYP450 aromatase. Human IVF-derived granulosa cells (GC) (n=7) were exposed to 0-5µM CYN±1IU/ml hCG for 2-24h. After 24h pre-culture GC responded to hCG by increasing estradiol 17ß (E(2)) and progesterone (P(4)) synthesis. Three micromolar of CYN±1IU/ml hCG for 24h was not cytotoxic and did not affect basal or hCG-stimulated E(2) or P(4) production, but did inhibit protein synthesis (p<0.05, n=4). hCG-stimulated steroidogenesis was not reduced by CYN, suggesting a lack of effect on StAR or CYP450 aromatase protein synthesis. hCG enhanced the effects of CYN on GC protein synthesis. Twenty four hours exposure to 0.1µM CYN did not affect GC, supporting the establishment of a 0.0024µM Guideline level for CYN in public water supplies.

A method for acetonitrile-free microcystin analysis and purification by high-performance liquid chromatography, using methanol as mobile phase.

Cyanobacteria produce a wide range of potent toxins, including hepatotoxic microcystins. HPLC methods for microcystin analysis and purification almost invariably include acetonitrile in the elution gradient mobile phase. The recent, acute, global acetonitrile shortage requires that adequate methods are available for microcystin analysis and purification without the need for acetonitrile. Here we present a convenient methanol-based method for effective HPLC analysis and purification of the toxins, with full separation of a range of microcystin variants.

LOCALIZATION OF MICROCYSTIN SYNTHETASE GENES IN COLONIES OF THE CYANOBACTERIUM MICROCYSTIS USING FLUORESCENCE IN SITU HYBRIDIZATION(1).

To better understand the production of microcystins (MCs) in Microcystis colonies, fluorescence in situ hybridization (FISH) methods were developed to detect DNA involved in the synthesis of these cyanobacterial hepatotoxins. Using colonies of Microcystis aeruginosa (Kütz.) Kütz. isolated from environmental blooms of cyanobacteria and from a colony-forming, MC-producing laboratory strain of Microcystis, amplified PCR products were observed, coincident with positive controls. The total MC content of individual colonies of Microcystis, determined by ELISA, showed a positive correlation with colony cross-sectional area. FISH analysis of Microcystis colonies gave high fluorescence in comparison to negative controls, indicating the presence of MC synthetase DNA (mcyA) in situ. FISH analysis for MC synthetase genes has the potential to be developed into an effective early warning tool for drinking and recreational water management.

Effects of blue-green algal toxin cylindrospermopsin (CYN) on human granulosa cells in vitro.

The blue-green algal toxin cylindrospermopsin (CYN) occurs in public water supplies. CYN was hepatotoxic when administered orally to mice, and cytotoxic and genotoxic to human cell lines. To determine the effects of CYN on primary human IVF-derived granulosa cells, 0-1 microg/ml CYN was added to cells for 2, 4 or 6h+/-hCG (n=6), or for 24, 48 and 72 h (n=6). Cytotoxicity was evaluated by MTT assay, and secreted progesterone or estrogen quantified by radioimmunoassay. 24h exposure to 1 microg/ml CYN was cytotoxic (p<0.05), whereas 0.0625 microg/ml CYN did not cause cytotoxicity or affect estrogen production, but did inhibit basal progesterone production (p<0.01). Similarly, 6h exposure to 1 microg/ml CYN did not affect cytotoxicity or hCG-stimulated estrogen production, but did inhibit hCG-stimulated progesterone production (p<0.01). In this in vitro assay, CYN inhibited progesterone production and therefore has the potential to be an endocrine disrupter by changing the progesterone:estrogen ratio in women.

Production of antibodies against microcystin-RR for the assessment of purified microcystins and cyanobacterial environmental samples.

Microcystins (MC) are cyanobacterial hepatotoxins responsible for animal-poisoning and human health incidents. Immunoassays provide a sensitive means to detect these toxins, although cross-reactivity characteristics of different antibodies are variable, and most antibodies have been produced against MC-LR. Here, we have produced the first polyclonal antibodies against the commonly occurring variant, MC-RR, and compared them with MC-LR antibodies for the analysis of purified MCs and cyanobacterial environmental samples. Both antisera cross-reacted with all MCs tested, and with the related cyanobacterial hepatotoxin nodularin-R, but not with non-toxic cyanobacterial peptides. In general, better cross-reactivity characteristics were observed with the MC-RR antisera and limits of quantification were lower for most variants, with all MCs tested and nodularin-R having limits of quantification of 0.31 nM or below. The antisera had different affinities to mixtures containing pooled MC-LR and MC-RR, with MC-LR antisera underestimating total MC concentration when MC-RR represented over 70% of the total MC pool. Both antisera correlated well with HPLC-UV data when incorporated into ELISAs to screen previously characterised environmental samples from Aland, Finland. MC-RR antisera are useful for screening samples containing multiple MCs, and particularly for samples primarily containing MC-RR variants.

Microcystin analysis in single filaments of Planktothrix spp. in laboratory cultures and environmental blooms.

Single filaments of Planktothrix spp. were isolated from laboratory cultures of P. agardhii (NIES 595) and P. rubescens (SL 03) and from four freshwater lakes in England and Turkey. Filament lengths were measured and microcystins were extracted by freeze-thawing and boiling. Microcystin analysis of the isolated single filaments was performed by ELISA using antibodies raised against microcystin-LR with a minimum detection limit (MDL) of 11 pg filament(-1). In some cases a high percentage of the filaments from the environmental samples and laboratory cultures were below the MDL of the assay. Based on the filaments with detectable microcystin contents, P. agardhii from Bassenthwaite Lake (England) had the lowest mean microcystin concentration (0.7 fg microm(-3)), and the highest microcystin concentration (2.9 fg microm(-3)) was measured in P. rubescens from Iznik Lake (Turkey). We investigated the relationship for filaments with microcystin contents above MDL and their biovolume. Relationships varied widely although P. agardhii from Bassenthwaite showed a better (positive) relationship between filament biovolume and microcystin content than P. rubescens from environmental samples. Under culture conditions, P. rubescens showed a good relationship between filament biovolume and toxin content.

Effects of the insecticide amitraz, an alpha2-adrenergic receptor agonist, on human luteinized granulosa cells.

BACKGROUND: Amitraz, an insecticide used to prevent tick and mite infestation of cattle, crops and dogs, is an alpha2-adrenergic receptor agonist that inhibits GnRH release and the ovulatory LH surge in rats. Noradrenalin, the physiological ligand for adrenergic receptors, inhibits progesterone production by IVF-derived granulosa cells, but the effects of amitraz are unknown. METHODS: Luteinized granulosa cells obtained from women undergoing ovarian stimulation were exposed to amitraz (1, 10, 50, 100 microg/ml) for 2-72 h, and to amitraz (50 microg/ml) +/- hCG or the specific alpha2-adrenergic receptor antagonist yohimbine, for 6 h. Cell numbers were determined by 3-(4, 5-dimethylthiazol-(2)-yl)-2, 5-diphenyl tetrazolium bromide(MTT) assay and hormone production by radioimmunoassay. RESULTS: Amitraz 10 microg/ml did not affect cell numbers or estrogen production, but reduced progesterone production to 58 +/- 8% (p < 0.01, 24 h, n = 6) of control values. Amitraz (100 microg/ml) was cytotoxic and caused a corresponding reduction in hormone production. Amitraz 50 microg/ml did not affect cell numbers or estrogen production, but reduced progesterone per cell production to 82 +/- 6% of control values after 6 h. This was prevented by 0.2 mmol/l yohimbine. Exposure to amitraz 50 microg/ml for 6 h exposure abolished hCG-stimulated progesterone production but not estrogen production. CONCLUSIONS: Amitraz inhibited basal and hCG-stimulated progesterone but not estrogen production. The inhibitory action of amitraz and its antagonism by yohimbine suggest that alpha2-adrenergic receptors are expressed by luteinized human granulosa cells.

Immunogold localisation of microcystins in cryosectioned cells of Microcystis.

Insights into the origins, function(s), and fates of cyanobacterial toxins may be obtained by an understanding of their location within cyanobacterial cells. Here, we have localised microcystins in laboratory cultures of Microcystis PCC 7806 and PCC 7820 by immunogold labelling. Cryosectioning was used for immunoelectron microscopy since microcystins were extracted during the ethanol-based dehydration steps routinely used for sample preparation. Microcystins were specifically localised in the nucleoplasm and were associated with all major inclusions of the microcystin-producing strains Microcystis PCC 7806 (MC(+)) and Microcystis PCC 7820, and labelling was preferentially associated with the thylakoids and around polyphosphate bodies. A mutant strain of Microcystis PCC 7806 (MC(-)) which does not produce microcystins was used as a control. Distribution of total gold label within each cell region or associated with inclusions indicated that most of the cells' microcystin pool was associated with the thylakoids (69%, PCC 7806 (MC(+)); 78%, PCC 7820), followed by the nucleoplasmic region (19%, PCC 7806 (MC(+)); 12%, PCC 7820). Cryosectioning is a useful technique since it reduces the extraction of microcystins during sample preparation for electron microscopy.

Modification of MTT assay conditions to examine the cytotoxic effects of amitraz on the human lymphoblastoid cell line, WIL2NS.

Reported parameters of the MTT assay vary widely, and reflect a need to optimise the assay for different cell types. The MTT assay conditions for the human B-lymphocyte-derived cell line WIL2NS were optimised for MTT incubation and formazan development. The optimised MTT assay was validated by examining the effects of the acaride amitraz on WIL2NS. In pH-buffered media in the absence of cells, MTT formed formazan spontaneously, and absorbance was proportional to both the initial concentration of MTT and the time of incubation at 37 degrees C. One milligram per millilitre MTT was toxic to WIL2NS cells, but the accuracy of the standard curve was reduced when only 0.2 mg/ml MTT was used. Twenty percent SDS in 0.2 M HCl was preferable to DMSO as a solvent for formazan. Exposure to 0.035% amitraz resulted in a significant reduction in WIL2NS cell numbers after only 2 h of exposure. It was concluded that 0.035% of amitraz has the potential to adversely affect lymphocytes in the systemic blood system in humans, and that an optimised MTT assay was obtained by incubating WIL2NS cells with 0.45 mg/ml MTT for 17 h, followed by addition of acidified SDS for 1 h.

Streptococcus iniae: serological differences, presence of capsule and resistance to immune serum killing.

The biochemical profiles, presence of capsule, outer membrane protein profiles and serological interactions of isolates of Streptococcus iniae obtained from different geographical and fish host origins were examined. The isolates had very similar biochemical profiles using API 20 Strep but varied as to whether they were arginine dihydrolase-negative, -positive or -intermediate (AD-ve, AD+ve, AD+/-ve, respectively). Representatives of each AD type were compared in subsequent experiments. All types possessed a polysaccharide capsule. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of outer membrane proteins or whole cells revealed no difference in banding patterns between isolates. All isolates were resistant to trout normal and specific immune serum and grew well in the presence of added fresh normal serum. Serological analyses of the isolates revealed antigenic differences. Trout antiserum against the AD+ve isolate did not agglutinate the AD-ve or AD+/-ve isolates, while antisera against the latter 2 types showed low agglutinating activity with all 3 isolates. When whole live cells of AD-ve and AD+ve isolates were dot-blotted, antiserum to the AD+ve isolate did not stain the AD-ve isolate, but antiserum to the AD-ve isolate stained both AD types. However, if the cells were pre-treated with Proteinase K (to remove surface-exposed protein antigens), the AD+ve isolate was stained only by its homologous antiserum. These results suggest that while certain protein antigens of the different AD type strains are immunologically cross-reactive, the capsular antigens appear to be AD type-specific. Furthermore, the results suggest that the cross-reactive antigens on the AD-ve isolate are effectively hidden by the strain-specific capsule, while they are partially exposed on the AD+ve isolate.

Learning impairments in monkeys with combined but not separate excitotoxic lesions of the anterior and mediodorsal thalamic nuclei.

Clinical studies in humans and experiments in macaques suggest that damage to the anterior and the mediodorsal thalamus can induce a moderate amnesia, but a more dense impairment may result from substantial damage within the temporal lobes or their subcortical connections. Lesions of the anterior thalamus in macaques produce impairments which resemble those seen after lesions of the fornix-mamillary pathway, which carries projections from the hippocampus to the anterior thalamus, while lesions of the mediodorsal thalamus, which receives inputs from frontal and temporal cortex, produce moderate impairments on a wider range of memory tasks. In the present study, we have made bilateral excitotoxic lesions of either the anterior or the mediodorsal thalamus, or both, in marmoset monkeys. Monkeys with lesions of both thalamic nuclei were severely impaired on retention and new learning of examples of the visuospatial conditional task, a task which is specifically impaired by lesions of the fornix or hippocampus. They were not impaired on performance of a visuovisual conditional task on which monkeys with hippocampal lesions are impaired, nor were they impaired on any visual discrimination task, including the concurrent discrimination task on which monkeys with temporal neocortical ablations are impaired. Monkeys with separate lesions of either the anterior or the mediodorsal thalamus were not impaired on any of these tasks. These results suggest that the mediodorsal thalamus and the anterior thalamus are both involved in processing the output of the hippocampal-fornix-thalamic circuit. Dense amnesia may result from damage to circuits additional to the temporal lobe efferents to either the anterior or the mediodorsal nuclei.