Maree J. Smith

Stability of cylindrospermopsin, the toxin from the cyanobacterium, Cylindrospermopsis raciborskii: Effect of pH, temperature, and sunlight on decomposition

Cylindrospermopsin is a powerful hepatotoxin produced by the cyanobacterium Cylindrospermopsis raciborskii. It is considered a potential threat to livestock, wildlife, and humans, and is the suspected cause of an outbreak of hepatoenteritis on Palm Island, Queensland, Australia, and various stock poisoning incidents around Australia. In this study, the stability of cylindrospermopsin was investigated using different parameters, including visible and UV light, sunlight, temperature and pH. Cylindrospermopsin decomposes rapidly (half‐life of 1.5 h) when exposed to sunlight in an algal extract solution; however, no decomposition was recorded in pure cylindrospermopsin and Milli‐Q water solutions. Cylindrospermopsin decomposes slowly in temperatures ranging from 4 to 50°C at pH 7. After 10 weeks at 50°C, cylindrospermopsin had degraded to 57% of the original concentration. This degradation was accompanied by an increase in another compound which is believed to be structurally related to cylindrospermopsin. Boiling does not cause a significant degradation of cylindrospermopsin within 15 min. Initial investigations indicate that cylindrospermopsin is degraded slowly under artificial light ranging from 42, 29, and 9 μE m−1 s−1 and in darkness. Degradation of cylindrospermopsin was not affected by changes in pH. Experiments were performed in sterile conditions. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 155–161, 1999

Blooms of the cylindrospermopsin containing cyanobacterium, Aphanizomenon ovalisporum (Forti), in newly constructed lakes, Queensland, Australia

The cyanobacterium, Aphanizomenon ovalisporum (Forti) is reported herein for the first time in Australia. Its distribution appears to be restricted to an isolated subtropical region which has distinctive water quality parameters including ready availability of nutrients and relatively high chloride and hardness levels. Blooms of A. ovalisporum in Queensland, Australia, formed a thick brown surface scum from spring to autumn in newly constructed shallow lakes. During such blooms, the water and cellular material were both found to contain cylindrospermopsin, a water soluble toxin that produced fatty livers with hepatocyte necrosis in mice similar to the toxicity produced by Cylindrospermopsis raciborskii (Wolosz.). Toxin levels in freeze‐dried A. ovalisporum are approximately 25% of those present in freeze‐dried C. raciborskii. However, A. ovalisporum appears to release more of the produced toxin into the water body than does C. raciborskii.

Use of HPLC-MS/MS to monitor cylindrospermopsin, a blue–green algal toxin, for public health purposes

Increasing reports of blooms of the blue–green alga Cylindrospermopsis raciborskii (C. raciborskii), which contains the hepatotoxic alkaloid cylindrospermopsin (CYN), have led to public health concerns in Australia. The toxicology of CYN appears complex and is still being elucidated. We have utilized the combination of sensitivity and specificity afforded by coupling high performance liquid chromatography (HPLC) to a tandem mass spectrometer (MS/MS) to produce an assay which is suitable for monitoring low CYN concentrations in water samples. Intact algal cells in the water sample are lysed by a freeze–thaw cycle. After filtration (0.45 μm filter), 110 μL is injected. The HPLC uses an Altima C18 (250×4.6 mm, 5 μm) column at 40°C. Chromatography utilizes a linear gradient from 1 to 60% methanol over 5 min, with a final isocratic stage holding at 60% methanol for 1 min. The mobile phase is buffered to 5 mM with ammonium acetate. The transition from the M+H ion (416 m/z) to the 194 m/z fragment is monitored. Linearity of this assay is 1–600 μg/L [peak area=304×CYN (μg/L)−569; r2=1.000 (n=7)]. Using a single point standard curve, total coefficients of variation were 26.4, 10.5, 12.6, and 10.7% at 0.78, 5.2, 104, and 1040 μg/L. This assay is utilized in conjunction with algal cell counts and mouse bioassays to monitor water bodies for public health purposes. The rationale used in employing these methods is discussed. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 151–154, 1999

Deoxycylindrospermopsin, an analog of cylindrospermopsin from Cylindrospermopsis raciborskii

Cylindrospermopsin (CYN) is a hepatotoxic alkaloid found in the blue–green alga Cylindrospermopsis raciborskii (C. raciborskii). Data indicating CYN alone does not account for the toxicity of freeze dried cultures of C. raciborskii have been presented recently. In an attempt to explain these data, we have purified and characterized the structure of an analog of CYN, deoxycylindrospermopsin (deoxy‐CYN). Three mice dosed intraperitoneally (IP) with 0.8 mg/kg of deoxy‐CYN showed no toxicity after 5 days. Comparison with the toxicity of CYN (5 day median lethal dose approximately 0.2 mg/kg IP) and its relative abundance in C. raciborskii suggest deoxy‐CYN does not contribute significantly to the toxicity of C. raciborskii. The additional toxicity of freeze dried C. raciborskii over pure CYN, therefore, remains unexplained. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 163–165, 1999

The oral toxicity for mice of the tropical cyanobacterium Cylindrospermopsis raciborskii (Woloszynska)

Exposure of humans and domestic animals to Cylindrospermopsis raciborskii and its associated toxin cylindrospermopsin in their drinking water will normally be by ingestion. Studies of the cyanobacterium to date have involved dosing mice by the intraperitoneal route, which excludes the possible influence on its toxicity of the alimentary tract barrier. In the present study, outbred MF1 male mice were fasted overnight and then given a single oral dose suspended in normal saline of freeze‐dried C. raciborskii culture containing 0.2% cylindrospermopsin. The median lethal dose was in the range 4.4–6.9 mg/kg alkaloid equivalent. Death occurred from 2 to 6 days after dosing, and pathological changes included marked fatty liver, often with periacinar coagulative necrosis, acute renal tubular necrosis, atrophy of the thymic cortex and the lymphoid follicles in the spleen, subepicardial and myocardial hemorrhages, and multiple ulcerations of the esophageal part of the gastric mucosa. The syndrome was consistent with that already reported for cylindrospermopsin dosed parenterally. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 135–142, 1999

Hepatic xenobiotic metabolism of cylindrospermopsin in vivo in the mouse

Cylindrospermopsin (CYN) is a hepatotoxin isolated from the blue-green alga Cylindrospermopsis raciborskii. The role of both glutathione (GSH) and the cytochrome P450 enzyme system (P450) in the mechanism of toxicity of CYN has been previously investigated in in vitro systems. We have investigated the role of GSH and P450 in vivo in mice. Mice pre-treated with buthionine sulphoximine and diethyl maleate to deplete hepatic GSH prior to dosing with 0.2mg/kg CYN showed a seven-day survival rate of 5/13 while the control group rate was 9/14. Dosing mice with 0.2mg/kg CYN produced a small decrease in hepatic GSH with a characteristic rebound effect at 24h. The magnitude of this effect is however small and combined with the non-significant difference in survival rates after GSH depletion suggest depletion of GSH by CYN could not be a primary mechanism for CYN toxicity. Conversely, pre-treatment with piperonyl butoxide, a P450 inhibitor, protected mice against CYN toxicity giving a survival rate of 10/10 compared with 4/10 in the control group (p < 0.05 Chi squared) and was protective at doses up to 0.8 mg/kg, suggesting activation of CYN by P450 is of primary importance in the mechanism of action.

Degradation of the cyanobacterial toxin, cylindrospermopsin, from Cylindrospermopsis raciborskii, by chlorination.

Cylindrospermopsin, a potent cyanobacterial toxin produced by Cylindrospermopsis raciborskii and other cyanobacteria, is regularly found in water supplies of Queensland, Australia. This study focussed on the effectiveness of chlorination as a water treatment procedure for cylindrospermopsin degradation. The results demonstrate that relatively low chlorine doses (<1 mg l(-1)) are sufficient for degradation of cylindrospermopsin, when the dissolved organic carbon content is low. However, if organic matter other than cylindrospermopsin is present in the solution, the effectiveness of chlorine for cylindrospermopsin degradation is reduced as other organic matter present consumes chlorine. Under the experimental conditions using samples with a solution pH of 6-9, a residual chlorine concentration of 0.5 mg l(-1)99% of cylindrospermopsin. Toxin degradation via chlorination occurs within the first minute and no difference was observable between degradation in an open system and in a closed system. With a decrease of the pH from 6 to 4 a reduction in the efficiency of chlorine for degradation of cylindrospermopsin was observable, a possible indication that cylindrospermopsin is more stable to chlorine degradation at lower pH. However, in normal water treatment this is not relevant since the pH is consistently higher than 6.

A sensitive and specific assay for glutathione with potential application to glutathione disulphide, using high-performance liquid chromatography-tandem mass spectrometry

We have utilised the combination of sensitivity and specificity afforded by coupling high-performance liquid chromatography (HPLC) to a tandem mass spectrometer (MS-MS) to produce an assay which is suitable for assaying glutathione (GSH) concentrations in liver tissue. The sensitivity suggests it may also be suitable for extrahepatic tissues. The method has been validated for GSH using mouse liver samples and also allows the assay of GSSG. The stability of GSH under conditions relevant to the assay has been determined. A 20-microl amount of a diluted methanol extract of tissue is injected with detection limits of 0.2 pmol for GSH and 2 pmol for GSSG. The HPLC uses an Altima C18 (150 x 4.6 mm, 5 microm) column at 35 degrees C. Chromatography utilises a linear gradient from 0 to 10% methanol in 0.1% formic acid over 5 min, with a final isocratic stage holding at 10% methanol for 5 min. Total flow rate is 0.8 ml/min. The transition from the M+H ion (308.1 m/z for GSH, and 613.3 m/z for GSSG) to the 162.0 m/z (GSH) and 355.3 m/z (GSSG) fragments are monitored.