A number of fungal cultures were screened to select an organism suitable to be used in the detoxification of aflatoxin B1. They were co-cultured in Czapek-Dox-Casamino acid medium with aflatoxin B1 producing Aspergillus flavus. Several fungal cultures were found to prevent synthesis of aflatoxin B1 in liquid culture medium. Among these Phoma sp., Mucor sp., Trichoderma harzianum, Trichoderma sp. 639, Rhizopus sp. 663, Rhizopus sp. 710, Rhizopus sp. 668, Alternaria sp. and some strains belonging to the Sporotrichum group (ADA IV B14(a), ADA SF VI BF (9), strain 720) could inhibit aflatoxin synthesis by > or =90%. A few fungi, namely ADA IV B1, ADA F1, ADA F8, also belonging to the Sporotrichum group, were less efficient than the Phoma sp. The Cladosporium sp. and A. terreus sp. were by far the least efficient, registering <10% inhibition. The cultures which prevent aflatoxin biosynthesis are also capable of degrading the preformed toxin. Among these, Phoma sp. was the most efficient destroying about 99% of aflatoxin B1. The cell free extract of Phoma sp. destroyed nearly 50 microg aflatoxin B1 100 ml(-1) culture medium (90% of the added toxin), and this was more effective than its own culture filtrate over 5 days incubation at 28+/-2 degrees C. The degradation was gradual: 35% at 24 h, 58% at 48 h, 65% at 72 h, 85% at 96 h and 90% at 120 h. The possibility of a heat stable enzymatic activity in the cell free extract of Phoma is proposed.
A simplified procedure for the enzyme inhibition assay to measure okadaic acid and DTX-1 in mussels, based on the use of a commercially available enzyme preparation, is presented. The detection limit is 10 ng of toxin per g of digestive glands. Using Certified Reference Material (MUS-2), high accuracy and good precision is demonstrated for contamination levels higher than 32 ng g(-1). Twenty samples can be processed in about 9 h by one operator, at the cost of US$ 10 per sample. Some possibilities for further enhancing the sensitivity and reducing the processing time are discussed and a monitoring example is presented.
A liquid chromatography/mass spectrometry (LC/MS) method was developed for the sensitive and specific determination of azaspiracid and its two analogs, the causative toxins of azaspiracid poisoning that occurred in the Netherlands and Ireland. The LC/MS method provided a detection limit of 50 pg for azaspiracid. The sensitivity was approximately 8 x 10(4) times greater than the mouse bioassay. The method was used to confirm the presence of azaspiracids in toxic mussels collected at Arranmore Island, Ireland in 1997.
A negative mode liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS) method was developed to detect low concentrations of the diarrhetic shellfish poisoning (DSP) toxins okadaic acid and dinophysistoxin-1 (DTX-1). Detection relies upon monitoring the transition of negative precursor ions [M - H]- to a common fragment ion of m/z 255. Our limit of detection for okadaic acid with this method is 0.5 pg on column. LC-SRM MS has allowed us to detect persistent, low concentrations of DSP toxins from Singapore shellfish.
We recently described a high throughput receptor binding assay for paralytic shellfish poisoning (PSP) toxins, the use of the assay for detecting toxic activity in shellfish and algal extracts, and the validation of 11-[3H]-tetrodotoxin as an alternative radioligand to the [3H]-saxitoxin conventionally employed in the assay. Here, we report a dramatic increase in assay efficiency through application of microplate scintillation technology, resulting in an assay turn around time of 4 h. Efforts are now focused on demonstrating the range of applications for which this receptor assay can provide data comparable to the more time consuming, technically demanding HPLC analysis of PSP toxins, currently the method of choice for researchers. To date, we have compared the results of both methods for a variety of sample types, including different genera of PSP toxin producing dinoflagellates (e.g. Alexandrium lusitanicum, r2 = 0.9834, n = 12), size-fractioned field samples of Alexandrium spp. (20-64 microm; r2 = 0.9997, n = 10) as well as its associated zooplankton grazer community (200-500 microm: r2 = 0.6169, n = 10; >500 microm: r2 = 0.5063, n = 10), and contaminated human fluids (r2 = 0.9661, n = 7) from a PSP outbreak. Receptor-based STX equivalent values for all but the zooplankton samples were highly correlated and exhibited close quantitative agreement with those produced by HPLC. While the PSP receptor binding assay does not provide information on toxin composition obtainable by HPLC, it does represent a robust and reliable means of rapidly assessing PSP-like toxicity in laboratory and field samples. Moreover, this assay should be effective as a screening tool for use by public health officials in responding to suspected cases of PSP intoxication.
The present study was performed to investigate whether the lichen rock tripe (Lasallia pustulata) can be used as food during survival situations. The effects of 30% lichen supplementation given to female Balb/c mice were studied on growth rate, metabolism and immune functions. After 3 weeks on this diet, it was found that the lichen supplementation did not affect the growth rate or the well-being of the animals. The growth rate tended to be higher in the lichen group when compared to control mice. Food consumption was similar in both groups, but with a trend towards slightly higher intake (12%) in the lichen group. The heart, liver, kidney and lymphoid organ (spleen and thymus) weights were not affected by the lichen. Histological hematoxylin eosin staining showed that all these organs were normal. Plasma glucose levels were unchanged, but plasma urea levels decreased by 24% (p < 0.05) with the lichen diet. Red and white blood cells and the number of lymphoid cells in the thymus and spleen were normal. The activity of thymocytes and spleen T-lymphocytes were not affected by the lichen diet, but spontaneous cell-mediated cytotoxicity (NK cells) tended (n.s.) to increase and spleen B-lymphocyte activity increased by 40% (p < 0.05). This study shows that the lichen rock tripe has immune stimulating effects important for host defence reactions and can be used as food in survival situations without any adverse effects on the metabolism.
Two corn powder samples implicated in the human food poisoning that occurred in Guangxi province in 1989, and eight wheat and two barley samples linked to an episode that involved about 130,000 people in gastrointestinal disorders in Anhui province in 1991 were analyzed for trichothecenes including deoxynivalenol (DON), nivalenol (NIV) and their esters, zearalenone (ZEA) and fumonisins (FMs) by gas chromatography/mass spectroscopy and high performance liquid chromatography, and T-2 toxin by enzyme-linked immunosorbent assays. DON was detected in all samples as a major trichothecene (16-51,450 microg kg(-1)), and NIV was in one corn, one barley and all wheat at relatively low levels (10-6935 microg kg(-1)). ZEA was found in all corn and barley, and six wheat samples (46-3079 microg kg(-1)). In addition, 3-acetyl-DON (2544 microg kg(-1)) and 15-acetyl-DON (2537 microg kg(-1)) were detected separately in one corn and one wheat sample. The highest levels of these mycotoxins were found in one wheat sample associated with the human intoxication in Anhui province. FMs in corn were below 1000 microg kg(-1). Risks of DON and ZEA on the people who consumed the causative cereals were assessed.
The holdings of eight collections of fungi have been examined for organisms isolated from wood and/or trees. Further selection of these fungi has been made according to their reported ability to produce volatile, biologically active metabolites. It is emphasized that the isolates in the collections do not necessarily produce such metabolites. The list of fungi fulfilling these conditions is slightly augmented by reports we have found in the literature, where the fungi concerned have not yet been deposited. The biochemistry of these compounds is considered with particular emphasis on their biosynthesis including that by Homo sapiens. The physiological and toxicological activity of these metabolites is reviewed especially with reference to their potential role in the complex symbioses existent in, for example, a tree. The review concludes with a discussion of areas of botany deserving increased attention in the hope that this will stimulate further work. The statements in the review are based on 173 references.
The correlation between changes in length of the different cell cycle stages and the toxicity of Alexandrium fundyense Balech was studied in semi-continuous cultures. Growth rates ranging from 0.031 d(-1) to 0.36 d(-1) were established at different temperatures or levels of phosphate limitation. In all treatments, G1 was the phase with the longest duration. Decrease in growth rate was associated with an increase in duration of the different cell cycle stages. Toxin content was always directly correlated to the duration of the G1 phase. In both the temperature treatments and the phosphate limitation experiments, toxin production rates remained constant for the respective range of conditions, implying that the variations in toxin content observed were a result of increasing periods of biosynthetic activity. Toxin accumulation was directly correlated to protein biosynthesis in all temperature treatments. In contrast, toxin content showed little correlation with protein content as phosphate limitation increased. Significant differences in toxin composition were observed between the temperature and phosphate treatments. Total concentrations of GTX II and III and C I and II were significantly higher in the phosphate-limited cultures, while the levels of STX, NEO and gonyautoxins I and IV remained virtually unchanged. We conclude that toxin biosynthesis in A. fundyense is coupled to the G1 phase of the cell cycle, that toxin synthesis is not down-regulated by phosphate deprivation and that interconversions among saxitoxin derivatives are influenced by the availability of phosphate.
The first discovered naturally occurring inhibitor of de novo sphingolipid biosynthesis was fumonisin B1. There are now 11 identified fungal inhibitors of ceramide synthase or 'fumonisin B1-like' compounds. With the exception of the australifungins, all other fungal ceramide synthase inhibitors are structurally sphingoid-like. There are several recently discovered fungal inhibitors of another enzyme in the de novo sphingolipid biosynthesis pathway: serine palmitoyltransferase (SPT). One of the SPT inhibitors is named ISP-I. While ceramide synthase inhibitors are toxic to animals, plants and fungi, the SPT inhibitors are not known to cause animal or plant disease, but are potent inhibitors of fungal growth. Very little is known about their toxicity in animals. There are at least 24 fungal SPT inhibitors produced by a variety of fungi. Given that the fungal inhibitors of sphingolipid biosynthesis are chemically and biologically diverse, two bioassays have been developed to screen for fumonisin-like or ISP-I-like activity in naturally contaminated products or fungal culture materials. These bioassays are based on the changes in free sphingoid base concentration that occur when the ceramide synthase or SPT are inhibited. The bioassays have the advantage that they are functionally rather than chemically specific and thus will detect ceramide synthase and SPT inhibitors regardless of their chemical structure.
Indospicine is a hepatotoxic amino acid that accumulates in the meat of horses that consume the legume Indigofera linnaei. A method to determine indospicine concentration in biological samples using an amino acid analyser has been reported, but the analysis time is long and therefore not suited to the analysis of large numbers of samples. A rapid and reliable method was developed for the analysis of indospicine in horsemeat and serum using High Performance Liquid Chromatography. Horsemeat and serum were extracted with either water or 0.01 N hydrochloric acid, respectively, and deproteinized by ultrafiltration. Precolumn derivatization of samples with phenylisothiocyanate was followed by separation of indospicine from other amino acids on a Pico-Tag C 18 column and UV detection at 254 nm. The calibration curves for indospicine in horsemeat extract were linear over the concentration range 0.4 microg ml(-1) to 20 microg ml(-1), while for indospicine in serum, the linear range was from 0.17 microg ml(-1) to 16.67 microg ml(-1). The mean recovery of indospicine in horsemeat extract was 87.2 +/- 6.8% and in serum was 97.3 +/- 9.9%. Analysis time for indospicine in horsemeat samples was 31 min and in serum samples was 36 min.
A new brevetoxin B analog, brevetoxin B4 (BTXB4), was isolated as the major toxin in greenshell mussels, Perna canaliculus, collected in New Zealand at the time of the neurotoxic shellfish poisoning incident. The new analog accounted for nearly two-thirds of the mouse lethality of the shellfish and was determined to be a mixture of N-myristoyl-BTXB2 and N-palmitoyl-BTXB2.
Fumonisins are mycotoxins of world-wide distribution in maize infected by the fungus Fusarium verticillioides. They are highly toxic to certain livestock and are potential carcinogens. Exophiala spinifera, a black yeast fungus found on moldy maize kernels, was identified previously as capable of growing on fumonisin B1 as a sole carbon source and thus is a potential source for fumonisin detoxifying enzymes. Pure cultures of E. spinifera transform fumonisin B(1) to the amino polyol AP(1) plus free tricarballylic acid through the activity of a soluble extracellular esterase, and further transformation is evidenced by accumulation in culture supernatant of a less polar compound(s) lacking a fluorescamine-reactive amino group. A free amine is thought to be critical for biological activity of FB(1) or AP(1). As a first step towards characterizing this amine-modifying activity, we investigated the biotransformation of AP(1) by E. spinifera liquid cultures that had been previously grown in liquid medium containing AP(1) as a sole carbon source. Accumulation of AP(1)-derived metabolites was monitored by thin-layer chromatography of culture supernatants, and product metabolites were purified and evaluated by mass spectrometry and nuclear magnetic resonance. Two products of treatment of purified AP(1) with cultures of E. spinifera are shown to be N-acetyl AP(1) and a new compound, 2-oxo-12,16-dimethyl-3,5,10, 14,15-icosanepentol hemiketal (or 2-OP(1) hemiketal).
Two samples of Narthecium asiaticum Maxim leaves collected in Japan were found to contain 103 microg(-1) and 160 microg g(-1) dry matter of 3-methoxy-2(5H)-furanone respectively. 3-Methoxy-2(5h)-furanone was suggested to be the toxic principle of N. asiaticum causing nephrotoxicity in cattle in Japan. Two other furanones, which are thought to be non-toxic, were also isolated from the two samples. These were 4-methoxy-2(5H)-furanone and 5-hydroxy-4-methoxy-2(5H)-furanone.
A simple, very sensitive and rapid HPLC method was developed for the simultaneous quantitative analysis of both fusapyrone (FP) and deoxyfusapyrone (DFP), the two antifungal 3-substituted-4-hydroxy-6-alkyl-2-pyrones isolated from rice culture of Fusarium semitectum, in crude extracts. Such method was optimized on C-18 reverse phase column, using the isolated metabolites as standards, with a sequence of linear elution steps with a MeOH-H(2)O mixture and using an ultraviolet detector fixed at 285 nm, where both alpha-pyrones showed a characteristic absorption maximum. This method was used to quantify the bioactive metabolites in crude organic extracts from two F. semitectum strains. The recovery of FP and DFP was measured in a crude extract from a poor metabolite producer F. semitectum strain. The recovery values ranged from 84% to 99% for FP and from 99% to 101% for DFP, indicating that the method was close to quantitative recovery. Furthermore, an efficient medium pressure column chromatography and TLC combined method was developed for the isolation and purification of FP and DFP from fungal culture extracts.
Natural ToxinsVolume 7, Issue 6 p. 343-345 Editorial Advances in detection methods for fungal and algal toxins† Frances M. Van Dolah, Corresponding Author Frances M. Van Dolah Conference Organizer NOAA/National Ocean Service, Center for Coastal Environmental Health and Biomolecular Research, Charleston, South Carolina, USANOAA/National Ocean Service, Center for Coastal Environmental Health and Biomolecular Research, Charleston, South Carolina, USASearch for more papers by this authorJohn L. Richard, John L. Richard Conference Organizer Romer Laboratories, Inc., Union, Missouri, USASearch for more papers by this author Frances M. Van Dolah, Corresponding Author Frances M. Van Dolah Conference Organizer NOAA/National Ocean Service, Center for Coastal Environmental Health and Biomolecular Research, Charleston, South Carolina, USANOAA/National Ocean Service, Center for Coastal Environmental Health and Biomolecular Research, Charleston, South Carolina, USASearch for more papers by this authorJohn L. Richard, John L. Richard Conference Organizer Romer Laboratories, Inc., Union, Missouri, USASearch for more papers by this author First published: 13 December 2000 https://doi.org/10.1002/1522-7189(199911/12)7:6<343::AID-NT88>3.0.CO;2-%23Citations: 4 † This article is a US Government Work and is in the public domain in the USA. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume7, Issue6November/December 1999Pages 343-345 RelatedInformation
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.
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.
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.
