Natural toxins最新文献

We have modified the cell-based directed cytotoxicity assay for sodium channel and calcium channel active phycotoxins using a c-fos-luciferase reporter gene construct. In this report we describe the conceptual basis to the development of reporter gene assays for algal-derived toxins and summarize both published and unpublished data using this method. N2A mouse neuroblastoma cells, which express voltage-dependent sodium channels, were stably transfected with the reporter gene c-fos-luc, which contains the firefly luciferase gene under the transcriptional regulation of the human c-fos response element. The characteristics of the N2A reporter gene assay were determined by dose response with brevetoxin and ciguatoxin. Brevetoxin-1 and ciguatoxin-1 induced c-fos-luc with an EC50 of 4.6 and 3.0 ng ml(-1), respectively. Saxitoxin caused a concentration-dependent inhibition of brevetoxin-1 induction of c-fos-luc with an EC50 of 3.5 ng ml(-1). GH4C1 rat pituitary cells, which lack voltage-dependent sodium channels but express voltage-dependent calcium channels, were also stably transfected with the c-fos-luc. GH4C1 cells expressing c-fos-luciferase were responsive to maitotoxin (1 ng ml(-1)) and a putative toxin produced by Pfiesteria piscicida. Although reporter gene assays are not designed to replace existing detection methods used to measure toxin activity in seafood, they do provide a valuable means to screen algal cultures for toxin activity, to conduct assay-guided fractionation and to characterize pharmacologic properties of algal toxins.

Cyanotoxins produced by cyanobacteria (blue-green algae) include potent neurotoxins and hepatotoxins. The hepatotoxins include cyclic peptide microcystins and nodularins plus the alkaloid cylindrospermopsins. Among the cyanotoxins the microcystins have proven to be the most widespread, and are most often implicated in animal and human poisonings. This paper presents a practical guide to two widely used methods for detecting and quantifying microcystins and nodularins in environmental samples-the enzyme linked immunosorbant assay (ELISA) and the protein phosphatase inhibition assay (PPIA).

A novel micro-extraction technique was applied to the extraction of biologically active macrocyclic imines known as spirolides from pooled individual cells isolated from spirolide-rich plankton material. For comparison, this method was also applied to pooled individual cells isolated from a unialgal culture of the marine dinoflagellate Alexandrium ostenfeldii (Paulsen) Balech & Tangen, a species known to produce spirolides. Both athecate cells and motile forms of gonyaulacoid dinoflagellates derived from size-fractionated plankton material from Nova Scotia, Canada were sorted and pooled by the glass micropipette isolation technique and by flow cytometry. The development of a highly sensitive analytical method for spirolides (detection limit 2 ng ml(-1) for spirolide B) using liquid chromatography-mass spectrometry (LC-MS) and application to micro-extracted samples allowed the accurate determination of spirolide composition in as few as 50 cells. Total spirolide concentrations (fmol cell(-1)) calculated from pooled micropipette isolated cells were very consistent with those based upon bulk- or micro-extractions of A. ostenfeldii cells from unialgal batch cultures in exponential growth phase. The results of the pooled cell selection from field material from two sites in Nova Scotia confirmed the association of spirolides with vegetative cells of A. ostenfeldii and related athecate forms. Combining these techniques represents a highly sensitive method for the analysis of marine toxins within complex plankton matrices, even when the toxigenic species is in low abundance, by enrichment of the target organism.

Fusarium fungi are widely found in agricultural products, worldwide and can produce a great variety of mycotoxins. Fumonisins, produced by F. moniliforme, and deoxynivalenol, produced by F. graminearum, are two such mycotoxins that have received considerable attention as food safety concerns by regulatory agencies. High Performance Liquid Chromatography/Mass Spectrometry (HPLC/MS) was found to be a convenient analytical method to detect and quantify the naturally occurring fumonisin homologs and deoxynivalenol in extracts from grains and food products. The fumonisins are detected primarily as protonated molecules in the positive ion electrospray ionization (ESI) mode as they elute from a C-18 reverse phase column during a methanol water gradient containing acetic acid to facilitate chromatography. Deoxynivalenol can be detected as positive or negative ions in the atmospheric pressure chemical ionization (APCI) mode or in the negative ion ESI mode. One nanogram amounts of fumonisins or deoxynivalenol injected into the HPLC system are easily detected with signal to noise allowing detection limits of 1 microg g(-1) or better to easily be achieved with minimal clean-up of grain extracts.

An enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody was used to determine microcystin (MC) concentrations in water supplies and water plant samples collected between November 1995 and October 1996, from five regions of Paraná, Brazil. In addition, the presence of Microcystis sp. was monitored. Of the 50 samples obtained, 12 were from an urban lake, 8 from human water supplies, 10 from recreational lakes, 13 from farm waters used for animal pasture and 7 from aquaculture facilities. M. aeruginosa was positive in all locations. MCs were positive (>50 pg ml(-1)) in 9 samples (2 samples from human water supplies, 5 from recreational lakes and 2 from animal pasture). Heavy contamination with MCs was observed in water samples collected in May 1996 from 2 recreation (swimming-fishing sites at Itaipu dam, 6380 and 10,000 pg ml(-1)) and human supplies (6627 pg ml(-1)) samples. At these sites, a large bloom of Microcystis sp. was detected. Treatment with 1 ppm Cl- reduced MCs levels, although 267 pg ml(-1) remained in the water plant samples. Our data showed frequent occurrence of Microcystis sp., which may be a hazard to humans and animals in the state of Paraná. More detailed investigations are required to evaluate the risk of natural MC contamination in the water supplied in this region.

Two different strains of the dinoflagellate Protoceratium reticulatum collected at Harima Nada and Yamada Bay in Japan were cultured and analyzed by fluorometric HPLC for yessotoxin production. Only the Yamada Bay strain produced yessotoxin. The toxin together with its analog, 45,46,47-trinoryessotoxin, were isolated from larger scale culture and unambiguously confirmed by (1)H NMR and MS measurements. This is the first confirmation of the biogenetic origin of yessotoxin in Japan, where the toxin was first reported. The results also indicate that the production of yessotoxins by P. reticulatum differs from strain to strain, in a similar way to that observed in many other toxigenic dinoflagellates such as Dinophysis spp. and Alexandrium spp.

From an ethyl acetate fraction of the brown alga Cystophora retroflexa several halogenated phlorotannins were isolated. Most of the compounds are derivatives of diphlorethol penta-acetate and triphlorethol-A hepta-acetate. The majority turned out to be chlorinated and/or brominated. Only one iodinated substance, 2-iodophloroglucinol triacetate, was isolated. The structure of this derivative and the following compounds have been characterized previously: 2([D])-bromodiphlorethol penta-acetate, 3([A1])-bromodiphlorethol penta-acetate, 4([D])-bromo-diphlorethol penta-acetate, 4([D])-chlorodiphlorethol penta-acetate, 3([A1])-chlorotriphlorethol-A hepta-acetate, 4([D])-bromotriphlorethol-A hepta-acetate and 4([D])-chlorobisfucopentaphlorethol-A nonadeca-acetate. Ten halogenated phlorethols and two chlorinated fucophlorethols are described for the first time and characterized as their acetates: 2([B])-bromotriphlorethol-A hepta-acetate, 2([D])-bromotriphlorethol-A hepta-acetate, 2([B]), 2([D])-dibromotriphlorethol-A hepta-acetate, 3([A1]), 5([A1])-dichlorotriphlorethol-A hepta-acetate, 3([A1]), 4([D])-dichlorotriphlorethol-A hepta-acetate, 3([A1])-chloro-4([D])-bromotriphlorethol-A hepta-acetate. 2([B]), 4([D])-dichlorotriphlorethol-A hepta-acetate, 2([D]), 3([A1])-dibromotriphlorethol-A hepta-acetate, 3([A1])-bromo-2([D])-chlorotriphlorethol-A hepta-acetate, 2([D])-bromotetraphlorethol-C nona-acetate, 4([D])-chlorofucotriphlorethol-B dodeca-acetate and 4([D])-chlorobisfucotetraphlorethol-A heptadeca-acetate.

During the summer of 1995, about 20 spot-billed ducks died unnaturally in a pond (Shin-ike) in Nishinomiya, Hyogo Prefecture, Japan. The suspected cause was the sudden appearance of toxic freshwater bloom of cyanobacteria. However, no birds died in a nearby pond (Oo-ike) in which the cyanobacteria was also present. Morphological observation of these cyanobacteria by microscope revealed that they were almost unialgal and were both Microcystis aeruginosa. The lyophilized algal cell powder from Shin-ike contained large amounts of microcystins which showed acute toxicity for mouse, while that from Oo-ike had only a very small amount of microcystin-RR which did not show acute toxicity. Autopsy of one of the birds revealed that the liver was necrotic and severely jaundiced with a dark green color, suggesting the toxicity of the microcystins. These results point to the cause of the unnatural death of spot-billed ducks in Shin-ike as being the sudden appearance of toxic Microcystis aeruginosa. This was due to eutrophication of the pond, following the influx of untreated sewage related to damage from the Great Hanshinn Earthquake of January 1995. This is the first experimental report of toxic cyanobacteria being implicated in the mass death of wild birds in Japan.

Two new analogs of azaspiracid, azaspiracid-2 and azaspiracid-3, were isolated from mussels collected at Arranmore Island, Ireland in 1997 as additional causes of human intoxication. Their structures were determined to be 8-methylazaspiracid and 22-demethylazaspiracid, respectively by NMR and negative ion FAB CID MS/MS experiments.

Since January 1993, neurological symptoms and rapid deaths (5 to 10 min) were typically observed in the mouse bioassay of acetone extracts of digestive glands from Arcachon and Toulon (France) during the winter season. It was assumed initially that a new lipophilic toxin was present because tests using the AOAC mouse bioassay for paralytic shellfish toxins on acid extracts of whole shellfish meat were negative, no known lipophilic toxins were detected and no toxic phytoplankton species were observed in the area during the poisoning events. In this study, however, preparative isolation of the toxic factor from toxic mussel digestive glands has revealed the presence of paralytic shellfish toxins, the principal ones being gonyautoxins-2 and -3 at Arcachon and gonyautoxins-1, -4, -2 and -3 at Toulon. The toxin concentrations recorded were below levels harmful to consumers and therefore represent a false positive in the mouse bioassay for lipophilic toxins based upon acetone extraction. The origin of the toxins remains to be determined.