G. Eaglesham

FIRST REPORT OF THE CYANOTOXINS CYLINDROSPERMOPSIN AND DEOXYCYLINDROSPERMOPSIN FROM RAPHIDIOPSIS CURVATA (CYANOBACTERIA)

A strain of Raphidiopsis (Cyanobacteria) isolated from a fish pond in Wuhan, P. R. China was examined for its taxonomy and production of the alkaloidal hepatotoxins cylindrospermopsin (CYN) and deoxy‐cylindrospermopsin (deoxy‐CYN). Strain HB1 was identified as R. curvata Fritsch et Rich based on morphological examination of the laboratory culture. HB1 produced mainly deoxy‐CYN at a concentration of 1.3 mg·g−1 (dry wt cells) by HPLC and HPLC‐MS/MS. CYN was also detected in trace amounts (0.56 μg·g−1). A mouse bioassay did not show lethal toxicity when tested at doses up to 1500 mg dry weight cells·kg−1 body weight within 96 h, demonstrating that production of primarily deoxy‐CYN does not lead to significant mouse toxicity by strain HB1. The presence of deoxy‐CYN and CYN in R. curvata suggests that Raphidiopsis belongs to the Nostocaceae, but this requires confirmation by molecular systematic studies. Production of these cyanotoxins by Raphidiopsis adds another genus, in addition to Cylindrospermopsis, Aphanizomenon, and Umezakia, now known to produce this group of hepatotoxic cyanotoxins. This is also the first report from China of a CYN and deoxy‐CYN producing cyanobacterium.

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.

Isolation and identification of the cyanotoxin cylindrospermopsin and deoxy-cylindrospermopsin from a Thailand strain of Cylindrospermopsis raciborskii (Cyanobacteria).

A strain of Cylindrospermopsis (Cyanobacteria) isolated from a fishpond in Thailand was examined for its taxonomy based upon morphology and 16S rRNA gene sequence. It was also examined for production of the hepatotoxic cyanotoxin called cylindrospermopsin (CYN) and deoxycylindrospermopsin (deoxy-CYN). The strain (CY-Thai) was identified as C. raciborskii (Woloszynska) Seenaya and Subba Raju based upon morphological examination which was confirmed by 16S rRNA gene sequences and phylogenetic comparisons based upon its 16S rRNA gene. The alkaloid heptatotoxin CYN was confirmed using mouse bioassay, HPLC and HPLC-MS/MS while deoxy-CYN was confirmed using HPLC-MS/MS. The mouse bioassay gave a minimum lethal dose at 250mg dry weight cells/kg body weight within 24h and 125mg/kg at 72h, with signs of poisoning the same as in literature reports for CYN. HPLC chromatographic comparison of the CY-Thai toxin with standard CYN gave the same retention time and an absorbance maximum at 262nm. HPLC-MS/MS confirmed the presence of CYN (M+H 416) and deoxy-CYN (M+H 400). The CYN content in strain CY-Thai was estimated at 1.02mg/g and approximately 1/10 of this amount for deoxy-CYN. This is the first report from Asia of a CYN, deoxy-CYN producing Cylindrospermopsis raciborskii.

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.u2003©1999 John Wiley & Sons, Inc.u2003Environ 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.u2003©1999 John Wiley & Sons, Inc.u2003Environ Toxicol 14: 163–165, 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.

Acute Human Self-Poisoning with the N-Phenylpyrazole Insecticide Fipronil –A GABAA-Gated Chloride Channel Blocker

Objective: Fipronil, a broad spectrum N‐phenylpyrazole insecticide that inhibits GABAA‐gated chloride channels, has been in use since the mid‐1990s. A high affinity for insect compared to mammalian GABA receptors results in lower animal toxicity than other insecticides blocking this channel. To date, only two accidental cases of fipronil poisoning in humans have been published. Case Series: We report seven patients with fipronil self‐poisoning seen prospectively in Sri Lanka together with pharmacokinetics for four patients. Non‐sustained generalized tonic‐clonic seizures were seen in two patients (peak measured plasma fipronil concentrations 1600 and 3744 µg/L); both were managed with diazepam without complications. A patient with a peak measured plasma concentration of 1040 µg/L was asymptomatic throughout his stay. Plasma concentration was still high at discharge 3–4 days post‐ingestion when the patients were well. Retrospective review of > 1000 pesticide poisoning deaths since 1995 found only one death from fipronil‐based products. In contrast to the good outcome of the above cases, this patient required intubation and ventilation and had continuous fits despite therapy with barbiturates and benzodiazepines. Conclusions: Our experience with prospectively observed patients suggests that fipronil poisoning is characterized by vomiting, agitation, and seizures, and normally has a favorable outcome. Management should concentrate on supportive care and early treatment of seizures. However, further experience is needed to determine whether increased susceptibility to fipronil or larger doses can produce status epilepticus.

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.