The process of preserving crops by fermentation in silos is under the control of the farmer to a much lesser degree compared to the level of control by the manufacturer over the production of other fermented foods, such as cheese and yoghurt. Additives designed to direct the extent and pattern of the fermentation are relatively unpopular in most countries, and their use is not guaranteed to remove the risk of undesirable components in silage. Hazards to animal health associated with silage fall into three categories: (1) undesirable micro-organisms e.g. Listeria, enterobacteria, clostridia and moulds; (2) undesirable chemicals, e.g. mycotoxins, and (3) excess acidity and other metabolic disorders. In some regions of Europe, the production of silage is discouraged or prohibited because of the risk of undesirable microbes. The principal risk in these areas is that of the secondary fermentation of cheese made from milk contaminated by bacterial spores, rather than a direct hazard of contaminated silage to animal health. With the possible exception of high dry matter silage conserved in large bales, respiratory hazards to animals from moulds and their spores generally are less from silage than hay. Mycotoxins and phytoestrogens may survive the ensiling period and constitute risks to animal health. Relatively little is known about the epidemiology of diseases that may be linked to undesirable chemicals and excess acidity in silage. Therefore, research is needed to define epidemiologically and mechanistically the risks to animal health and to the human food chain from silages contaminated with pathogenic bacteria and mycotoxins, and to understand more completely the relationships between the physical and chemical compositions of silage and metabolic disorders in animals. Copyright © 1999 John Wiley & Sons, Ltd.

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 C18 column and UV detection at 254 nm. The calibration curves for indospicine in horsemeat extract were linear over the concentration range 0.4 µg ml⁻¹ to 20 µg ml⁻¹, while for indospicine in serum, the linear range was from 0.17 µg ml⁻¹ to 16.67 µg 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. Copyright © 1999 John Wiley & Sons, Ltd.

A rapid HPLC method with fluorescence detection of pectenotoxin-2 (PTX2), a polyether macrolide toxin, in microalgae is presented. A dienophile reagent, DMEQ-TAD, was used for precolumn fluorescence labeling. PTX2 could be quantitatively detected in the range 1–200 ng. This method confirmed the occurrence of PTX2 in net haul samples mostly composed of dinoflagellates Dinophysis spp. collected in the Adriatic Sea, Italy and Mutsu Bay, Japan. Copyright © 1999 John Wiley & Sons, Ltd.

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 × 10⁴ 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. Copyright © 1999 John Wiley & Sons, Ltd.

Fumonisins are mycotoxins produced by the maize pathogen Gibberella fujikuroi mating population A and frequently contaminate maize. Wild-type G. fujikuroi produces four B-series fumonisins, FB₁, FB₂, FB₃ and FB₄. These toxins are identical in structure except for the number and positions of hydroxyls along their linear carbon backbone. To elucidate the genetic and biosynthetic relationships among these fumonisins, we conducted meiotic and biochemical analyses of G. fujikuroi mutants with altered fumonisin production that resulted from defective alleles at three loci, Fum1, Fum2 and Fum3. These mutants produced either no fumonisins, only FB₂ and FB₄, or only FB₃ and FB₄. Genetic analyses revealed the orientation of the Fum loci along linkage group 1 of the fungus. The mutants were grown together in pair-wise combinations to determine if their fumonisin production phenotypes could be complemented. When FB₃- and FB₂-producing mutants were grown together, complementation occurred. However, when a nonproducing mutant was grown with a FB₂- or FB₃-producing mutant, complementation did not occur or was incomplete. When purified FB₂, FB₃, or FB₄ was fed to mutant cultures, FB₄ was converted primarily to FB₂, FB₃ was converted to FB₁ and FB₂ was not converted. The results from these assays suggest a previously unrecognized branch in the fumonisin biosynthetic pathway. Published in 1999 by John Wiley & Sons, Ltd.

Eighteen maize samples were assayed for fumonisin B₁ (FB₁) and B₂ content by immunoaffinity column coupled with high performance liquid chromatography (HPLC). The FumoniTest columns were used once for the isolation of fumonisins (single-use column method). In the second part of the assay the columns were regenerated. After elution with methanol, PBS solution was left on the column for one day at room temperature to regenerate the columns (regenerated column method). The efficiency of columns regenerated twice was tested by determining FB₁ recovery and the reproducibility of the determinations. The recovery rate of FB₁ proved to be 82 % by the single-use column method (RSD: 5.7 %) and 82.6 % (RSD: 5.6 %) by the regenerated column method; 500–8000 ng FB₁ loaded onto the columns did not affect column performances. Nearly identical values were obtained when the FB₁ content of fumonisin-containing maize samples was determined by both methods. The results indicate that the FumoniTest columns can be regenerated by the method applied at least twice without decrease in column performance. The fumonisin affinity, capacity and specificity of the regenerated columns were not changed. Thus, columns regenerated in this way can be used for determining the fumonisin content of maize samples at least three times. Copyright © 1999 John Wiley & Sons, Ltd.

Trichothecenes are potent inhibitors of cytoplasmic protein synthesis which can affect the severity of plant diseases such as wheat head scab. While many trichothecene-producing fungi share the initial biosynthetic intermediates, Fusarium sp. are unique in the production of trichothecenes containing an oxygen function at C-3. Although the initial trichothecene and the final products have a C-3 hydroxyl group, the intermediate steps are acetylated at C-3. By using Chlamydomonas reinhardtii, a unicellular plant with a well-defined genetic system, we were able to test the proposal that trichothecenes with a C-3 hydroxyl are more toxic to plants, as well as demonstrate that C. reinhardtii is a promising plant trichothecene bioassay system. Seven pairs of trichothecenes with either a C-3 hydroxyl or C-3 acetyl group were assayed. Our results confirm that trichothecenes acetylated at C-3 were far less toxic to Chlamydomonas than those with a C-3 hydroxyl group. Published in 1999 by John Wiley & Sons, Ltd.

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. Copyright © 1999 John Wiley & Sons, Ltd.

Using monoclonal anti-fumonisin B₁ antibody (anti-FB₁) and avidin–biotin–peroxidase system, liver and kidneys of broiler chicks were evaluated for the detection and distribution of fumonisins (FBs). One hundred and fifty micrograms of FB₁ or culture extract of Fusarium moniliforme str. 113F containing 150 µg of FB₁ and 4 µg of FB₂ were administered into the vitelline sac of 1-day old, specific pathogen-free chicks. The animals were killed 24 h after injection, and renal and hepatic tissues submitted for immunohistochemical analysis. FBs were detected in the epithelial cells of convoluted distal and proximal tubules of the kidneys, as well as in the cytoplasm of hepatocytes. This novel immunohistochemical method developed is expected to be an efficient way for monitoring the target of the FB toxins in tissues. Copyright © 1999 John Wiley & Sons, Ltd.

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. Copyright ­© 1999 John Wiley & Sons, Ltd.

This paper reviews the toxicology of culmorins, a family of compounds found in grains contaminated by Fusarium graminearum and related fungi. We include the results of an Ames test and studies based on Quantitative Structure-Activity Relationships. Culmorin has low toxicity in several in vitro assays and in one study in swine and is Ames test negative. Culmorin is moderately antifungal. QSAR analysis suggested that the plant compound longifolene was similar. Longifolene is a GRAS compound used in cosmetics and is also moderately antifungal. Copyright © 1999 John Wiley & Sons, Ltd.

Monk seals in Cape Blanc (Western Sahara coast) suffered a mass mortality during May–July 1997 which was attributed to a morbillivirus. High performance liquid chromatography (HPLC) analysis on tissues of seals killed during the outbreak and on related fauna showed peaks with retention times coincident with those of some saxitoxin derivatives but their identity was not proved. Here we present results of further HPLC analyses that unambiguously prove the identity of these toxins by mass spectrometry (MS), supporting the hypothesis that this mortality of monk seals was caused by biotoxins rather than by a morbillivirus. Copyright © 1999 John Wiley & Sons, Ltd.

Two samples of Narthecium asiaticum Maxim leaves collected in Japan were found to contain 103 µg g⁻¹ and 160 µg 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. Copyright © 1999 John Wiley & Sons, Ltd.

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. Copyright © 1999 John Wiley & Sons, Ltd.

Production of aflatoxin B₁ and fumonisin B₁ in pure and mixed cultures of Aspergillus flavus and Fusarium proliferatum were determined on irradiated maize seeds inoculated with different spore concentrations at 0.97 water activity (a_w) and a temperature of 25 °C. The highest levels of aflatoxin B₁ were produced by A. flavus at the lowest levels of inoculum (10³ spore ml⁻¹). There was no spore concentration influence on fumonisin B₁ production after 10, 20 and 35 days of incubation. When A. flavus was co-inoculated with F. proliferatum, aflatoxin B₁ production was inhibited. The higher the inocula levels of Fusarium produced, the higher the inhibition and this inhibition increased during the incubation period. Total inhibition was reached at 35 days of incubation. There was no interaction influence on fumonisin B₁ production at all inoculum levels assayed. These results suggest that under optimal environmental conditions of substrate, water activity and temperature, the interaction between A. flavus and F. proliferatum could produce inhibition of aflatoxin B₁ and stimulation of fumonisin B₁. Copyright © 1999 John Wiley & Sons, Ltd.

The fungal metabolite kojic acid, which is produced by Aspergillus and Penicillium species fungi that may be pathogens of both insects and plants, was a significant inhibitor of phenoloxidase of different representative beetle and caterpillar insect species. Fusaric acid and picolinic acid, produced by Fusarium spp., were also significant inhibitors of phenoloxidase, while dipicolinic acid and beauvericin were ineffective at concentrations tested. Previous reports of the ability of kojic and fusaric acid to inhibit defensive enzymes of plants suggest that these compounds may be important in allowing the producing fungi to be pathogens of both insects and plants. Published in 1999 by John Wiley & Sons, Ltd.

This article reviews current requirements for the analysis for mycotoxins in foods and identifies legislative as well as other factors that are driving development and validation of new methods. New regulatory limits for mycotoxins and analytical quality assurance requirements for laboratories to only use validated methods are seen as major factors driving developments. Three major classes of methods are identified which serve different purposes and can be categorized as screening, official and research. In each case the present status and future needs are assessed. In addition to an overview of trends in analytical methods, some other areas of analytical quality assurance such as participation in proficiency testing and reference materials are identified. © Crown copyright 1999. Reproduced with the permission of Her Majesty's Stationery Office. Published by John Wiley & Sons, Ltd.

High Performance Liquid Chromatography (HPLC) is an important tool for the study of PSP toxins. It provides an alternative to bioassays and gives the concentration of individual toxin isomers. The current HPLC protocol uses a post-column chemical reaction system (PCRS) to oxidize the saxitoxin ring system to form a fluorescent chromophore. This oxidation is sensitive to changes in the flow rate, temperature, pH and age of the reagents. We have previously shown that this oxidation can be accomplished using electrochemical techniques. Termed the electrochemical oxidation system (ECOS), this approach provides a simpler alternative to the traditional PCRS-based HPLC system. A detailed description of the construction and maintenance of an HPLC–ECOS system for the analysis of PSP toxins is presented. Comparisons of the mouse bioassay, HPLC–PCRS and HPLC–ECOS system are presented for three different sample matrices: toxic dinoflagellates (Alexandrium tamarense), geoduck (Panopea generosa) and scallops (Placopectin magellanicus). In all three cases, the correlation of the HPLC–ECOS system to the mouse bioassay is similar to that obtained using the HPLC–PCRS system for the analysis of PSP toxins. Copyright © 1999 John Wiley & Sons, Ltd.

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. Copyright © 1999 John Wiley & Sons, Ltd.

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 µg g⁻¹ or better to easily be achieved with minimal clean-up of grain extracts. Published in 1999 by John Wiley & Sons, Ltd.

Evanescent wave-based fiber-optic immunosensors were studied for the detection of fumonisins and aflatoxins in maize. Two formats, competitive and non-competitive, were used. A competitive format was used to measure fumonisin B₁ (FB₁) in both spiked and naturally contaminated maize samples. Fumonisin monoclonal antibodies were covalently coupled to an optical fiber and the competition between FB₁ and FB₁ labeled with fluorescein (FB₁-FITC) for the limited number of binding sites on the fiber was assessed. The signal generated in the assay was inversely proportional to the FB₁ concentration. For samples, the concentration causing an inhibition of binding by 50 % (IC₅₀) was dependent upon the clean-up procedure used. Simple dilution of methanolic maize extracts yielded an assay with an IC₅₀ equivalent to 25 µg FB₁ g⁻¹ maize with a limit of detection of 3.2 µg g⁻¹ maize. Affinity column clean-up yielded an assay with an IC₅₀ equivalent to 5 µg FB₁ g⁻¹ maize (limit of detection 0.4 µg FB₁ g⁻¹). An HPLC method and the immunosensor method agreed well for naturally contaminated maize samples except when large amounts of other fumonisins that cross-react with the immunosensor were present. The second sensor format, for the mycotoxin aflatoxin B₁ (AFB₁), was a non-competitive assay using the native fluorescence of this mycotoxin. Because the fluorescence of AFB₁ itself was detected, the response of the sensor was directly proportional to the toxin concentration. The sensor, while capable of detecting as little as 2 ng ml⁻¹ of AFB₁ in solution was technically not an immunosensor, since the attachment of aflatoxin specific antibodies was not required. Sensors of the formats described have the potential to rapidly screen individual maize samples but require coupling with a clean-up technique to be truly effective. Published in 1999 by John Wiley & Sons, Ltd.

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). Copyright © 1999 John Wiley & Sons, Ltd.

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. Copyright © 1999 John Wiley & Sons, Ltd.

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 µm; r² = 0.9997, n = 10) as well as its associated zooplankton grazer community (200–500 µm: r² = 0.6169, n = 10; >500 µm: 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. Published in 1999 by John Wiley & Sons, Ltd.

Like all eucaryotic cells, yeasts are sensitive to trichothecenes, especially T-2 toxin and verrucarin A. Based on this sensitivity, a yeast bioassay was developed to evaluate the toxicity of corn samples. The bioassay was optimized using spiked maize extracts. The toxicity of samples was defined as toxicity equivalent to a certain concentration of T-2 toxin standards. The assay can be performed on crude extracts, but the results are more precise after column clean-up. The test can also be used for the screening of trichothecene toxicity in general. The relative standard deviation (RSD) at 85 % growth inhibition (EC₈₅) was 4.5 % for the T-2 toxin standards (n = 8). This corresponds to an initial T-2 toxin concentration of approximately 58 ppb in the corn sample. Samples containing 188 and 113 ppb T-2 toxin caused a growth inhibition higher than 85 %, whereas samples with toxin concentrations of 56 and 19 ppb had a growth inhibition less than 85 %. Therefore the test can be used for the qualitative evaluation of corn samples up to a level of 58 ppb ± 2.8 ppb. The bioassay is easy to perform with minimum requirements for equipment. Results can be obtained within 24 h and a large number of samples can be analysed daily. The costs are low and the results obtained are repeatable. With some modifications this test can be used for toxicity studies on trichothecene metabolites as well as for extracts with unknown compounds with properties similar to trichothecenes. Copyright © 1999 John Wiley & Sons, Ltd.

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. Published in 1999 by John Wiley & Sons, Ltd.

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. Published in 1999 by John Wiley & Sons, Ltd.