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.

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.

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.

Human fatalities from cyanobacteria: chemical and biological evidence for cyanotoxins.

An outbreak of acute liver failure occurred at a dialysis center in Caruaru, Brazil (8 degrees 17' S, 35 degrees 58' W), 134 km from Recife, the state capital of Pernambuco. At the clinic, 116 (89%) of 131 patients experienced visual disturbances, nausea, and vomiting after routine hemodialysis treatment on 13-20 February 1996. Subsequently, 100 patients developed acute liver failure, and of these 76 died. As of December 1996, 52 of the deaths could be attributed to a common syndrome now called Caruaru syndrome. Examination of phytoplankton from the dialysis clinic's water source, analyses of the clinic's water treatment system, plus serum and liver tissue of clinic patients led to the identification of two groups of cyanobacterial toxins, the hepatotoxic cyclic peptide microcystins and the hepatotoxic alkaloid cylindrospermopsin. Comparison of victims' symptoms and pathology using animal studies of these two cyanotoxins leads us to conclude that the major contributing factor to death of the dialyses patients was intravenous exposure to microcystins, specifically microcystin-YR, -LR, and -AR. From liver concentrations and exposure volumes, it was estimated that 19.5 microg/L microcystin was in the water used for dialysis treatments. This is 19.5 times the level set as a guideline for safe drinking water supplies by the World Health Organization.

Mechanism of action of fotemustine, a new chloroethylnitrosourea anticancer agent: evidence for the formation of two DNA-reactive intermediates contributing to cytotoxicity.

Methyl excision repair deficient human tumor cells (Mer-) were found to be hypersusceptible to killing by the antimelanoma agent fotemustine (FM) implicating alkylation of O6 guanine as the major contributor to toxicity. Preincubation of the drug in aqueous solution for 5 min resulted in an immediate reduction in cytotoxicity (35-50%), in vitro DNA alkylation (31%), and DNA interstrand cross-linking (40%) followed by a second reaction with considerably slower kinetics. Electrospray ionisation mass spectrometry (ESI-MS) showed that in aqueous solution FM rearranged rapidly to form either a metastable tautomer or decomposed to form a highly reactive diazohydroxide (t1/2 < 2 min). These results suggest the presence of two DNA-reactive species relevant to biological activity. Coincubation of ellagic acid (an inhibitor of O6-guanine alkylation) with FM inhibited in vitro ISC, suggesting that the O6-chloroethyl lesion is the predominant cause of the cross-link. On the basis of these findings, we propose that FM breaks down to form a short-lived intermediate, 2-chloroethyldiazohydroxide, which rapidly generates O6-guanine lesions responsible for the drug's initial activity and a long lived iminol tautomer responsible for the remaining O6 guanine alkylation and cytotoxicity.