Cytoskeletal toxicity of pectenotoxins in hepatic cells.

BACKGROUND AND PURPOSE: Pectenotoxins are macrocyclic lactones found in dinoflagellates of the genus Dinophysis, which induce severe liver damage in mice after i.p. injection. Here, we have looked for the mechanism(s) underlying this hepatotoxicity. EXPERIMENTAL APPROACH: Effects of pectenotoxin (PTX)-1, PTX-2, PTX-2 seco acid (PTX-2SA) and PTX-11 were measured in a hepatocyte cell line with cancer cell characteristics (Clone 9) and in primary cultures of rat hepatocytes. Cell morphology was assessed by confocal microscopy; F- and G-actin were selectively stained and cell viability measured by Alamar Blue fluorescence. KEY RESULTS: Clone 9 cells and primary hepatocytes showed a marked depolymerization of F-actin with PTX-1, PTX-2 and PTX-11 (1-1000 nM) associated with an increase in G-actin level. However, morphology was only clearly altered in Clone 9 cells. PTX-2SA had no effect on the actin cytoskeleton. Despite the potent F-actin depolymerizing effect, PTX-1, PTX-2 or PTX-11 did not decrease the viability of Clone 9 cells after 24-h treatment. Only prolonged incubation (> 48 h) with PTXs induced a fall in viability, and under these conditions, morphology of both Clone 9 and primary hepatocytes was drastically changed. CONCLUSIONS AND IMPLICATIONS: Although the actin cytoskeleton was clearly altered by PTX-1, PTX-2 and PTX-11 in the hepatocyte cell line and primary hepatocytes, morphological assessments indicated a higher sensitivity of the cancer-like cell line to these toxins. However, viability of both cell types was not altered.

Presumptive tremorgenic mycotoxicosis in a dog in New Zealand, after eating mouldy walnuts.

CASE HISTORY: A 1-year-old, intact male Labrador-cross dog vomited after eating walnuts that had been on the ground for 5 months. The dog then developed tremors, ataxia, increased salivation, and hyperaesthesia. CLINICAL FINDINGS: The dog had marked generalised tremors, ataxia and a temperature of 39.9 degrees C. Both pupils were of normal size and normally responsive to light. Vomiting was induced, and walnut shell was visible in the vomitus. DIAGNOSIS: Due to the sudden onset of tremors, lack of exposure to other convulsive toxins, and the evidence of ingestion of walnuts, the provisional diagnosis was tremorgenic mycotoxicosis. The dog was treated symptomatically, and made a full recovery over 18 hours. Tremorgenic mycotoxins were detected within walnuts collected from the dog's environment. CLINICAL RELEVANCE: Fungi that produce tremorgenic mycotoxins are present in New Zealand. Intoxication should be suspected in dogs that suddenly develop muscle tremors, especially if there is a history of ingestion of mouldy food 2-3 hours prior to the development of tremors.

Use of ELISA to identify Protoceratium reticulatum as a source of yessotoxin in Norway.

Picked cells of Protoceratium reticulatum collected from five locations in Norway were shown by ELISA analysis to contain yessotoxins (YTXs). The production of yessotoxin (YTX) was verified by culturing followed by LC-MS analysis of one of the Norwegian isolates. This is the first report of the biogenic origin of YTXs in Norway. The sensitivity of the ELISA method made it possible to quantitate YTXs in algal cultures, net-hauls, and in single cells of P. reticulatum. The cells picked from cultures and net-hauls contained 18-79 pg YTXs per cell. Dilution series and analyses of cells from non-YTX-producing algal species demonstrated the presence of only minimal matrix effects on the ELISA, probably attributable to the presence of salts. The sensitivity of this method makes it possible to search for other possible producers of YTXs, and might also make it possible to follow the YTXs through the food chain. This method allows, for the first time, measurement of the variability in toxin content within a population of dinoflagellate cells--rather than just the average amount of toxin per cell.

Molecular analysis of two cytochrome P450 monooxygenase genes required for paxilline biosynthesis in Penicillium paxilli, and effects of paxilline intermediates on mammalian maxi-K ion channels.

The gene cluster required for paxilline biosynthesis in Penicillium paxilli contains two cytochrome P450 monooxygenase genes, paxP and paxQ. The primary sequences of both proteins are very similar to those of proposed cytochrome P450 monooxygenases from other filamentous fungi, and contain several conserved motifs, including that for a haem-binding site. Alignment of these sequences with mammalian and bacterial P450 enzymes of known 3-D structure predicts that there is also considerable conservation at the level of secondary structure. Deletion of paxP and paxQ results in mutant strains that accumulate paspaline and 13-desoxypaxilline, respectively. These results confirm that paxP and paxQ are essential for paxilline biosynthesis and that paspaline and 13-desoxypaxilline are the most likely substrates for the corresponding enzymes. Chemical complementation of paxilline biosynthesis in paxG (geranygeranyl diphosphate synthase) and paxP, but not paxQ, mutants by the external addition of 13-desoxypaxilline confirms that PaxG and PaxP precede PaxQ, and are functionally part of the same biosynthetic pathway. A pathway for the biosynthesis of paxilline is proposed on the basis of these and earlier results. Electrophysiological experiments demonstrated that 13-desoxypaxilline is a weak inhibitor of mammalian maxi-K channels (Ki=730 nM) compared to paxilline (Ki=30 nM), indicating that the C-13 OH group of paxilline is crucial for the biological activity of this tremorgenic mycotoxin. Paspaline is essentially inactive as a channel blocker, causing only slight inhibition at concentrations up to 1 microM.

Integrated enzyme-linked immunosorbent assay screening system for amnesic, neurotoxic, diarrhetic, and paralytic shellfish poisoning toxins found in New Zealand.

Enzyme-linked immunosorbent assays (ELISAs) were developed for amnesic, neurotoxic, and diarrhetic shellfish poisoning (ASP, NSP, and DSP) toxins and for yessotoxin. These assays, along with a commercially available paralytic shellfish poisoning (PSP) ELISA, were used to test the feasibility of an ELISA-based screening system. It was concluded that such a system to identify suspect shellfish samples, for subsequent analysis by methods approved by international regulatory authorities, is feasible. The assays had sufficient sensitivity and can be used on simple shellfish extracts. Alcohol extraction gave good recovery of all toxin groups. The ease of ELISAs permits the ready expansion of the system to screen for other toxins, as new ELISAs become available.

Metal complexes of the mycotoxins sporidesmin A and gliotoxin, investigated by electrospray ionisation mass spectrometry.

The mycotoxin sporidesmin A (spdA), responsible for the intoxication of animals, causing facial eczema, has been investigated by electrospray ionisation mass spectrometry. Protonated [spdA+H](+) and deprotonated [spdA-H](-) ions are observed in positive and negative ion modes respectively. Reduced spdA, formed by cleavage of the disulfide bond by Na[BH(4)] gives an ion [spdA+H](-), and forms ions of the type [2spdA+M](2-) with a range of divalent metal ions M(2+)=Zn(2+), Cd(2+), Hg(2+), Sn(2+) and Fe(2+). Sodium-containing analogues [2spdA+M+Na](-) are observed, particularly at high cone voltages, where they are stable towards cone voltage-induced fragmentation, indicating appreciable stability of the (spdA)(2)M system. A competition experiment between Cd(2+) and Zn(2+) demonstrates that reduced spdA has a higher affinity for Cd(2+) ions. The related gliotoxin (gtx) forms analogous [2gtx+M](2-) and [2gtx+M+Na](-) ions. The reduction and metal complexation of spdA can be monitored by (1)H NMR spectroscopy, and results in chemical shift changes for those protons adjacent to the sulfur atoms. The isolation of a polymeric cadmium-spdA complex is also reported.

Metal complexes of sporidesmin D and dimethylgliotoxin, investigated by electrospray ionisation mass spectrometry.

Electrospray ionisation mass spectrometry (ES-MS) has been used to probe the coordination chemistry of metabolites such as sporidesmin D (spdD), found in the saprophytic fungus Pithomyces chartarum, and the related bisdethiobis(methylthio)gliotoxin (dimethylgliotoxin, Megtx). SpdD forms complexes of the type [spdD+M(MeCN)] and [2spdD+M]+ (M=Cu, Ag) and, at higher cone voltages, [spdD+M]+. The bis(ligand) ion [2spdD+M]+ was observed at very high cone voltages, indicating it has appreciable stability; the proposed structure of this species has a four-coordinate metal ion with two bidentate spdD ligands, coordinated through their SMe groups. 1H NMR titrations of spdD with K+, Ag+ and Cu+ provided additional evidence for complex formation with the soft metals. SpdD forms only relatively weak complexes with Zn2+, Cd2+, Co2+ and Mn2+, in keeping with the known reduced tendency of these metals to form stable thioether complexes. ES-MS studies of Megtx showed similar results to spdD, with stable adducts formed with Cu+ and Ag+ ions. The X-ray crystal structure of spdD is also reported.

Congener-independent immunoassay for microcystins and nodularins.

Cyanobacteria (blue-green algae) (e.g., Microcystis and Nodularia spp.) capable of producing toxic peptides are found in fresh and brackish water worldwide. These toxins include the microcystin (MC) heptapeptides (>60 congeners) and the nodularin pentapeptides (ca. 5 congeners). Cyanobacterial cyclic peptide toxins are harmful to man, other mammals, birds, and fish. Acute exposure to high concentrations of these toxins causes liver damage, while subchronic or chronic exposure may promote liver tumor formation. The detection of cyclic peptide cyanobacterial toxins in surface and drinking waters has been hampered by the low limits of detection required and that the present routine detection is restricted to a few of the congeners. The unusual beta-amino acid ADDA (4E,6E-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid) is present in most (>80%) of the known toxic penta- and heptapeptide toxin congeners. Here, we report the synthesis of two ADDA-haptens, the raising of antibodies to ADDA, and the development of a competitive indirect ELISA for the detection of microcystins and nodularins utilizing these antibodies. The assay has a limit of quantitation of 0.02-0.07 ng/mL (depending on which congeners are present), lower than the WHO-proposed guideline (1 ng/mL) for drinking water, irrespective of the sample matrix (raw water, drinking water, or pure toxin in PBS). This new ELISA is robust, can be performed without sample preconcentration, detects toxins in freshwater samples at lower concentrations than does the protein phosphatase inhibition assay, and shows very good cross-reactivity with all cyanobacterial cyclic peptide toxin congeners tested to date (MC-LR, -RR, -YR, -LW, -LF, 3-desmethyl-MC-LR, 3-desmethyl-MC-RR, and nodularin).

New analogue of gymnodimine from a Gymnodinium species.

A spiroimine, gymnodimine B (1), was isolated from cells recovered by filtration from cultures of a marine dinoflagellate, Gymnodinium sp. Its structure was identified by one- and two-dimensional NMR spectroscopy and mass spectrometry. Gymnodimine B is similar in structure to gymnodimine (2) but contains an exocyclic methylene at C-17 and an allylic hydroxyl group at C-18.

Terpendole M, a novel indole-diterpenoid isolated from Lolium perenne infected with the endophytic fungus Neotyphodium lolii.

Terpendole M (1), a novel indole-diterpenoid, was isolated from perennial ryegrass (Lolium perenne) infected with the endophytic fungus Neotyphodium lolii. It was identified as 14alpha-hydroxyterpendole C by NMR and mass spectral techniques. The known indole-diterpenoids paspaline (3) and 13-desoxypaxilline (4) were also isolated from perennial ryegrass for the first time. Terpendole M was less tremorgenic than terpendole C (2) in a standard mouse bioassay. These findings provide clues to the biogenesis of the lolitrem neurotoxins, and information on the structure-activity relationships within the indole-diterpenoids.

Paxilline-negative mutants of Penicillium paxilli generated by heterologous and homologous plasmid integration.

Using a monoclonal antibody based ELISA, 600 pAN7-1 plasmid-tagged mutants of Penicillium paxilli were screened for paxilline accumulation and one paxilline-negative mutant, YI-20, was identified. A molecular analysis of this mutant showed that pAN7-1 was inserted at a single site but was present as 4-6 copies arranged in a head-to-tail tandem array. Rescue of flanking sequences and analysis of the corresponding genomic region revealed that YI-20 has an extensive deletion at the site of pAN7-1 integration. Probing of a CHEF gel with the same sequences showed that associated with the deletion is a rearrangement of chromosome Va. Targeted gene disruption of wild-type sequences adjacent to the site where pAN7-1 inserted, resulted in the generation of two additional paxilline-negative mutants; both were single crossovers with deletions extending outside the region mapped. Neither of these new mutants had a rearrangement of chromosome Va, suggesting that deletion of genes on this chromosome is responsible for the paxilline-negative phenotype. Telomeric fingerprinting of genomic digests of P. paxilli, combined with pulsed-field gel electrophoresis of chromosomal DNA, established that there are a minimum of eight chromosomes in this fungus.

Endophytic fungi in indigenous Australasian grasses associated with toxicity to livestock.

Grazing of Echinopogon spp. by livestock in Australia has caused symptoms similar to those of perennial ryegrass staggers. We observed an endophytic fungus in the intercellular spaces of the leaves and seeds of New Zealand and Australian specimens of Echinopogon ovatus. Culture of surface-sterilized seeds from New Zealand specimens yielded a slow-growing fungus. An examination in which immunoblotting and an enzyme-linked immunosorbent assay (ELISA) were used indicated that E. ovatus plants from Australia and New Zealand were infected with fungi serologically related to Neotyphodium lolii (the endophyte of perennial ryegrass) and other Epichloe and Neotyphodium spp. endophytic in pooid grasses. No lolitrems (the indole-diterpenoids implicated as the causative agents of perennial ryegrass staggers), peramine analogs, or ergot alkaloids were detected in the infected specimens by high-performance liquid chromatography or ELISA. However, in endophyte-infected E. ovatus plants from New Zealand, analogs of the indole-diterpenoid paxilline (thought to be a biosynthetic precursor of the lolitrems and related tremorgens) were detected by ELISA, and N-formylloline was detected by gas chromatography. Endophyte-free specimens of New Zealand E. ovatus did not contain detectable paxilline analogs or lolines and were more palatable than infected specimens to adults of the pasture pest Listronotus bonariensis (Argentine stem weevil). Hyphae similar to those of the E. ovatus endophyte were also found in herbarium specimens of Echinopogon nutans var. major, Echinopogon intermedius, Echinopogon caespitosus, and Echinopogon cheeli. This appears to be the first time that an endophytic Neotyphodium species has been identified in grasses endemic to New Zealand or Australia.

Polyclonal antibodies to domoic acid, and their use in immunoassays for domoic acid in sea water and shellfish.

Ovine antibodies raised against conjugates linked through the secondary amino group of domoic acid (1) were used, together with activated-ester-derived conjugates of domoic acid (DA) as the plate coater, to develop a robust indirect competitive enzyme-linked immunosorbent assay (cELISA) for DA in shellfish and seawater. The ELISA was used to analyze shellfish samples for DA, and was compatible with several extraction procedures. The ELISA had a detection limit below 0.01 ng ml(-1), a limit of quantitation (LOQ) of 0.15 ng ml(-1) and a working range of 0.15-15 ng ml(-1) DA. The LOQ is equivalent to 38 ng g(-1) DA in shellfish flesh, assuming a 250-fold dilution during extraction. This is more than 500 times lower than the maximum permitted level (20 microg g(-1) flesh). The ELISA is designed for use alongside regulatory analyses, and, following formal validation, should be available for pre-screening of regulatory shellfish flesh samples. The ELISA was also shown to be appropriate for analysis of DA in algal cultures and in samples of seawater, and thus has the potential to provide early warning of developing algal blooms.

Photosensitivity in South Africa. IX. Structure elucidation of a beta-glucosidase-treated saponin from Tribulus terrestris, and the identification of saponin chemotypes of South African T. terrestris.

Saponin C, a beta-glucosidase-treated saponin isolated from ethanol-water extracts of a South African collection of Tribulus terrestris, was shown by one- and two-dimensional NMR spectroscopy to be ruscogenin 1-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-6)-acetylglucopyranoside++ +. GC-MS analysis of the hydrolysed ethanol-water (4:1) extracts of T.terrestris specimens from two of four sites, revealed high levels of ruscogenin and potentially lithogenic diosgenin saponins. Specimens from two other sites contained non-lithogenic saponins derived predominantly from tigogenin, neotigogenin, gitogenin and neo-gitogenin.

Hepatogenous photosensitization of ruminants by Brachiaria decumbens and panicum dichotomiflorum in the absence of sporidesmin: lithogenic saponins may be responsible.

As part of a study of plants involved in crystal-associated hepatogenous photosensitization diseases, samples of Brachiaria decumbens and Panicum dichotomiflorum on which cattle and goats had recently been photosensitized were analyzed. The level of saponins associated with these photosensitization outbreaks were determined by GC-MS. Only low levels of Pithomyces chartarum spores were present on the B decumbens, and all isolates obtained failed to produce sporidesmin.

First report of field outbreaks of ergot-alkaloid toxicity in South Africa.

Outbreaks of clinical disease caused by the ingestion of ergotized Lolium rigidum (annual ryegrass), which resulted in a substantial loss in production, have been reported. A number of outbreaks of a hyperthermia syndrome in cattle, characterized by severe loss in milk production, loss of body mass and reduced fertility, are described. In one major outbreak in March to April 1994, a milling company reported that 2,646 dairy cows on 29 farms had developed clinical signs. In this outbreak, significant levels of ergotamine, ergosine, ergocornine and ergocryptine were found in the milled dairy rations fed to the affected cows. Barley screenings containing ergotized annual-ryegrass seed was identified as the toxic component and probable source of the ergot alkaloids in the ration. The clinical syndrome was reproduced experimentally by feeding suspected feed to a group of nine high-producing Ayrshire cows. An outbreak of gangrenous necrosis of the extremities in young cattle in the winter of 1987 was also suspected of having been caused by ergot alkaloids in grain screenings.

Photosensitivity in South Africa. VIII. Ovine metabolism of Tribulus terrestris saponins during experimentally induced geeldikkop.

Geeldikkop was induced in a sheep by dosing it orally with a crude extract of the steroidal saponins from Tribulus terrestris. GC-MS analysis of the sheep's ruminal contents, bile, faeces and urine for free and conjugated sapogenins, revealed the general features of the metabolic pathway by which diosgenin and yamogenin glycosides were converted into the glucuronides of epismilagenin and episarsasapogenin, the major constituents of the biliary crystals that usually form during geeldikkop. Other steroidal saponins in the T. terrestris extract, including those derived from tigogenin, neotigogenin, gitogenin and neogitogenin appear to be non-lithogenic. The implications of these findings are discussed.

Photosensitivity in South Africa. VII. Chemical composition of biliary crystals from a sheep with experimentally induced geeldikkop.

Geeldikkop was induced in a sheep by oral administration of crude saponins from Tribulus terrestris. Centrifugation of the bile from this sheep gave a pale green sediment of crystalloid material which was insoluble in common organic solvents, but soluble in acetic acid. Analysis of the crystalloid material by 1H and 13C NMR, EDXA, TLC, LSIMS, and by acidic hydrolysis followed by TLC and GC-MS, revealed it to be composed principally of a 6:1 mixture of the calcium salts of the beta-D-glucuronides of the steroidal sapogenins epismilagenin and episarsasapogenin. The administered saponin was found to contain glycosides of the steroidal sapogenins diosgenin, yamogenin, epismilagenin, tigogenin, neotigogenin, gitogenin and neogitogenin in the ratio 10:7:1:11:7:35:25. A metabolic pathway for the conversion of diosgenin and yamogenin saponins to the biliary glucuronides is proposed.

An attempt to reproduce crystal-associated cholangitis in lambs in experimental dosing of sarsasapogenin or diosgenin alone and in combination with sporidesmin.

No liver damage occurred in a group of 21 lambs dosed intraruminally with up to 9 g of sarsasapogenin or diosgenin daily for 10 consecutive days. In contrast, seven out of 15 lambs dosed with 0.1 mg of sporidesmin/kg liveweight in combination with sarsasapogenin and three out of six lambs dosed with sporidesmin in combination with diosgenin developed liver lesions. These were typical of those induced by sporidesmin. One lamb dosed with sporidesmin in combination with 9 g of diosgenin developed a crystal-associated cholangitis typical of Panicurn intoxication and alveld. No sapogenins were detected in urine by gas chromatography-mass spectrometry. The results suggest that orally administered sarsasapogenin and diosgenin are either not hepatotoxic per se or are too poorly absorbed to elicit a toxic response. The results provide only weak evidence that sporidesmin may be involved in the aetiology of Panicurn intoxication.