Suzanne Froscio

Cylindrospermopsin Genotoxicity and Cytotoxicity: Role Of Cytochrome P-450 and Oxidative Stress

Cylindrospermopsin (CYN) is a cyanobacterial toxin found in drinking-water sources world wide. It was the likely cause of human poisonings in Australia and possibly Brazil. Although CYN itself is a potent protein synthesis inhibitor, its acute toxicity appears to be mediated by cytochrome p-450 (CYP450)-generated metabolites. CYN also induces genotoxic effects both in vitro and in vivo, and preliminary evidence suggests that tumors are generated by oral exposure to CYN. To understand the role of CYP450-activated CYN metabolites on in vitro genotoxicity, this study quantified the process in primary mouse hepatocytes using the COMET assay in both the presence and absence of CYP450 inhibitors known to block acute CYN cytotoxicity. CYN was cytotoxic at concentrations above 0.1 μM(EC50 = 0.5 μM) but produced significant increases in Comet tail length, area, and tail moment at 0.05 μM and above; hence genotoxicity is unlikely to be secondary to metabolic disruption due to toxicity. The CYP450 inhibitors omeprazole (100 μM) and SKF525A (50 μM) completely inhibited the genotoxicity induced by CYN. The toxin also inhibits production of glutathione (GSH), a finding confirmed in this study. This could potentiate cytotoxicity, and by implication genotoxicity, via reduced reactive oxygen species (ROS) quenching. The lipid peroxidation marker, malondialdehyde (MDA) was quantified in CYN-treated cells, and the effect of the reduced glutathione (GSSG) reductase (GSSG-rd.) inhibitor 1,3-bis(chloroethyl)-l-nitrosourea (BCNU) on both MDA production and lactate dehydrogenase (LDH) leakage was examined. MDA levels were not elevated by CYN treatment, and block of GSH regeneration by BCNU did not affect lipid peroxidation or cytotoxicity. It therefore seems likely that CYP450-derived metabolites are responsible for both the acute cytotoxicity and genotoxicity induced by CYN. This work was funded by the Cooperative Research Centre for Water Quality and Treatment. The authors thank Yvette de Graaf for help with the COMET assay, and Dr. Tanya Lewanowitsch for useful and timely advice.

MICROCYSTINS (CYANOBACTERIAL TOXINS) IN DRINKING WATER ENHANCE THE GROWTH OF ABERRANT CRYPT FOCI IN THE MOUSE COLON

Microcystis aeruginosa produces toxic cyclic peptides called microcystins, potent hepatotoxins that have been implicated in tumor promotion in skin and liver. The model used in this investigation was the azoxymethane (AOM)-induced aberrant crypt focus (ACF) in the male C57Bl/6J mouse colon. Three intraperitoneal (i.p.) injections of 5 mg/kg AOM were administered at 7-d intervals to mice; 19 d after the last AOM injection, drinking water containing Microcystis extract was commenced and continued for a further 212 d. The content of microcystins in the drinking water was determined by mouse bioassay, high-performance liquid chromatography (HPLC), capillary eletrophoresis, and protein phosphatase inhibition. The doses employed were 0, 382, and 693 micrograms/kg bodyweight/d at the midpoint of the trial. Following postmortem examination blood cells, serum enzymes and organ pathology were investigated. A significant microcystin dose-dependent increase in the area of aberrant crypt foci was observed. There was no marked increase in the number of crypts/colon. Two overt colonic tumors (approximately 30 mm3) were seen in microcystin-treated mice, and one microscopic colonic tumor in an AOM-alone-treated mouse. This investigation provides the first evidence for the stimulation of preneoplastic colon tumor growth by microcystin.Microcystis aeruginosa produces toxic cyclic peptides called microcystins, potent hepatotoxins that have been implicated in tumor promotion in skin and liver. The model used in this investigation was the azoxymethane (AOM)-induced aberrant crypt focus (ACF) in the male C57Bl/6J mouse colon. Three intraperitoneal (ip) injections of 5 mg/ kg AOM were administered at 7-d intervals to mice; 19 d after the last AOM injection, drinking water containing Microcystis extract was commenced and continued for a further 212 d. The content of microcystins in the drinking water was determined by mouse bioassay, high-performance liquid chromatography (HPLC), capillary eletrophoresis, and protein phosphatase inhibition. The doses employed were 0, 382, and 693 µg/kg bodyweight/d at the midpoint of the trial. Following postmortem examination blood cells, serum enzymes and organ pathology were investigated. A significant microcystin dose-dependent increase in the area of aberrant crypt foci was observed. There was no marked increase in the number of crypts/colon. Two overt colonic tumors (~30 mm3) were seen in microcystin-treated mice, and one microscopic colonic tumor in an AOMalone-treated mouse. This investigation provides the first evidence for the stimulation of preneoplastic colon tumor growth by microcystin.

Limited uptake of the cyanobacterial toxin cylindrospermopsin by Vero cells

Cylindrospermopsin (CYN) is a cyanobacterial toxin increasingly found in drinking water sources worldwide. Toxicity studies have shown CYN can induce effects in a range of different cell types with primary hepatocytes consistently shown to be the most sensitive cellular model. How CYN enters the intracellular environment is not clear, although the size and hydrophilic nature of the toxin suggest it would not readily cross a lipid bilayer. In this study, a Vero cell line expressing green fluorescent protein (GFP) was used to monitor for CYN uptake based on the toxins potent effects on protein synthesis. Effects on the GFP signal were compared with inhibitors cycloheximide (CHEX) and emetine. While CYN potency was demonstrated in a cell-free system (CYN>CHEX>emetine) it was considerably reduced in the Vero-GFP cell model (CHEX, emetine>>CYN). In contrast to other inhibitors, CYN effects on GFP signal increased 6 fold over 4-24 h incubation indicating slow, progressive uptake of the toxin. Confirming that the uptake process is not energy dependent CYN entry also occurred at 4 degrees C, while competition experiments excluded the uracil nucleobase transporter system as potential mechanism for CYN uptake. Dilution of media enhanced CYN uptake by Vero-GFP cells although mechanism by which this occurred is unknown.

Novel toxic effects associated with a tropical Limnothrix/Geitlerinema-like cyanobacterium

The presence of a toxic strain of a fine filamentous cyanobacterium belonging to the Oscillatorialean family Pseudanabaenacea was detected during a survey of cyanobacterial taxa associated with the presence of cylindrospermopsin in dams in Central Queensland (Australia). The strain, AC0243, was isolated and cultured, its genomic DNA extracted and 16S RNA gene sequenced. Phylogenetic analysis placed AC0243 with Limnothrix species, although this genus appears polyphyletic. Moreover, not all morphological characters are consistent with this genus but more closely fit the description of Geitlerinema unigranulatum (R.N. Singh) Komárek and Azevedo. The potential toxic effects of AC0243 extract were assessed chemically and biologically. Cell free protein synthesis was inhibited by the extract. Exposure of Vero cells to the extract resulted in a significant reduction in cellular ATP levels following 24–72 h incubation. The presence of cylindrospermopsin was excluded based on the nature of responses obtained in cell and cell‐free assays; in addition, (i) it could not be detected by HPLC, LC‐MS, or immunological assay, and (ii) no genes currently associated with the production of cylindrospermopsin were found in the genome. Other known cyanobacterial toxins were not detected. The apparent novelty of this toxin is discussed.

Cyanotoxins are not implicated in the etiology of coral black band disease outbreaks on Pelorus Island, Great Barrier Reef

Cyanobacterial toxins (i.e. microcystins) produced within the microbial mat of coral black band disease (BBD) have been implicated in disease pathogenicity. This study investigated the presence of toxins within BBD lesions and other cyanobacterial patch (CP) lesions, which, in some instances ( approximately 19%), facilitated the onset of BBD, from an outbreak site at Pelorus Island on the inshore, central Great Barrier Reef (GBR). Cyanobacterial species that dominated the biomass of CP and BBD lesions were cultivated and identified, based on morphology and 16S rRNA gene sequences, as Blennothrix- and Oscillatoria-affiliated species, respectively, and identical to cyanobacterial sequences retrieved from previous molecular studies from this site. The presence of the cyanotoxins microcystin, cylindrospermopsin, saxitoxin, nodularin and anatoxin and their respective gene operons in field samples of CP and BBD lesions and their respective culture isolations was tested using genetic (PCR-based screenings), chemical (HPLC-UV, FTICR-MS and LC/MS(n)) and biochemical (PP2A) methods. Cyanotoxins and cyanotoxin synthetase genes were not detected in any of the samples. Cyanobacterial species dominant within CP and BBD lesions were phylogenetically distinct from species previously shown to produce cyanotoxins and isolated from BBD lesions. The results from this study demonstrate that cyanobacterial toxins appear to play no role in the pathogenicity of CP and BBD at this site on the GBR.

Evaluation of the Abraxis Strip Test for Microcystins™ for use with wastewater effluent and reservoir water.

Rapid tests for the microcystin-type cyanobacterial toxins that are designed to be able to be used in the field have recently become available. The tests provide a semi-quantitative result over a relatively narrow concentration range (10-fold) and are available with detection limits relevant for drinking water and recreational water compliance testing (1 μg/L and 10 μg/L, respectively). The aim of this research was to assess the applicability of these tests for the determination of microcystin-related toxicity in treated effluent from the Western Treatment Plant and potable source water from Tarago Reservoir, both near Melbourne, Australia. Accuracy, precision, cross-reactivity, matrix effects and inter-operator variability were assessed. The claimed mLR concentration response range of the tests was confirmed within reasonable limits, although the false negative and false positive rates were significant for spike concentrations below 2.5 μg/L (Recreational Strip Test). Inter-operator variability was reasonably high (CV=23%) and this was exacerbated by the use of untrained scorers. Contributing to this was significant inter-assay variability in test band intensity (CV=28%). The strip tests responded to all 8 microcystin analogues tested and also to a mixture of another 7 analogues contained in a Certified Bloom Material. Cross-reactivity was always greater than 50%. Matrix effects due to the test waters or to cyanobacterial cell material were also relatively minor, being of the order of 2-fold at the maximum. Overall, these Strip Tests were found to be reliable for relatively rapid detection of microcystins around the upper limits of their response ranges, as recommended by the manufacturer. While the Recreational Water Strip test was less reliable in the lower ranges, it can be used in conjunction with the Drinking Water Strip test to reduce uncertainty around the 1 μg/L concentration. Despite limitations, both strip tests provide near real-time information which can assist with day to day operational decisions. When results indicate microcystin concentrations near compliance limits it is recommended that use of the test kits should be supported by accurate quantitative toxin testing together with traditional algal cell counts, and possibly emerging qPCR methods for species and toxin gene detection.

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 17β (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.

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.

Novel cytotoxicity associated with Anabaena circinalis 131C.

The production of secondary metabolites by cyanobacteria is extensively varied. Anabaena spp. have been shown to produce the toxins saxitoxin, anatoxin-a, anatoxin-a(s), cylindrospermopsin and microcystin. In this study we show cytotoxicity associated with Anabaena circinalis 131C that could not be attributed to its high saxitoxin content. Analytical, ELISA and enzymatic methods excluded the presence of other known cyanobacterial toxins leading to the suggestion of novel toxicity.