Toxigenicity of enniatins from Western Australian Fusarium species to brine shrimp (Artemia franciscana)

The high prevalence (14 of 24 isolates) of enniatin-producing isolates from Western Australian Fusarium species isolated from pasture legumes associated with sheep feed refusal and rat deaths, and the high toxicity of their crude extracts to brine shrimp (Artemia franciscana) from a previous study warranted further investigation of this class of mycotoxin. Crude extracts from Fusarium acuminatum, Fusarium avenaceum, Fusarium tricinctum and Fusarium sambucinum, along with enniatins A, A1, B and B1 purified from a Western Australian strain of F. acuminatum using semi-preparative HPLC, were bioassayed using brine shrimp. All Fusarium isolates produced both enniatins B and B1, except for F. tricinctum WAC 8019, and 11 of the 17 isolates produced enniatin A1. Overall, all of the F. avenaceum isolates produced high amounts of enniatins, in particular enniatin B. One isolate of F. acuminatum (WAC 5715) and of F. tricinctum (WAC 11486) also produced high amounts of both enniatins B and B1. Only F. acuminatum WAC 5715 produced enniatin A among the tested isolates. All four purified enniatins A, A1, B, B1, individually and in combination, caused brine shrimp toxicity after 6 h of exposure, implicating that this emerging class of mycotoxin as a cause of the acute toxicity to brine shrimp observed. The mixture of all four enniatins was the most toxic to brine shrimp compared to purified individual enniatins, where the relative toxicity order was B > B1 > A1 > A. Enniatin B was the individual most toxic enniatin with some bioactivity at 5 μg/mL and almost 100% brine shrimp death at 50 μg/mL after 24 h of exposure. This study is the first report to confirm the acute toxicity of enniatins A, A1, B and B1 to brine shrimp, and also highlights the need for further investigation of the potential toxicity of these cyclic hexadepsipeptides to animals and humans.     

Multiple regression analysis as a tool for the identification of relations between semi-quantitative LC-MS data and cytotoxicity of extracts of the fungus Fusarium avenaceum (syn. F. arthrosporioides).

The cytotoxicity of methanolic extracts from rice cultures of 53 Fusarium avenaceum strains, which had been isolated from different host organisms in Northern Europe, Canada and Australia/New Zealand, was investigated in a rat hepatoma (H4IIE-W), porcine epithelial kidney (PK-15), foetal feline lung fibroblast, dog lymphoblast (D3447), and a human hepatocarcinoma (Hep G2) cell line using the Alamar Bluetrade mark assay. All extracts were screened for known fungal metabolites using high-performance liquid chromatography with photodiode array and mass spectrometric detection, and both known and unknown metabolites were semi-quantified. Known metabolites that were determined in the cultures include acuminatopyrone, 2-amino-14,16-dimethyloctadecan-3-ol (2-AOD-3-ol), antibiotic Y, aurofusarin, chlamydosporol, chlamydospordiol, enniatins, fusarin A and C, and moniliformin. Multiple regression analysis was used in order to relate fungal metabolites to the cytotoxicity of the extracts. Separate linear regression models were constructed for each cell line. Eleven different fungal metabolites were related to the cytotoxicity (P<0.05). Out of these, nine metabolites were siginificantly related to the cytotoxicity in only one of the five models, while two, namely enniatins and 2-AOD-3-ol, were significant contributors in three or four regression models, respectively. This paper describes how multiple regression analysis may be applied for the assignment of bioactivity/toxicity to the constituents of a multi-component mixture.     

Influence of Carbohydrates on Secondary Metabolism in Fusarium avenaceum

Fusarium avenaceum is a widespread pathogen of important crops in the temperate climate zones that can produce many bioactive secondary metabolites, including moniliformin, fusarin C, antibiotic Y, 2-amino-14,16-dimethyloctadecan-3-ol (2-AOD-3-ol), chlamydosporol, aurofusarin and enniatins. Here, we examine the production of these secondary metabolites in response to cultivation on different carbon sources in order to gain insight into the regulation and production of secondary metabolites in F. avenaceum. Seven monosaccharides (arabinose, xylose, fructose, sorbose, galactose, mannose, glucose), five disaccharides (cellobiose, lactose, maltose, sucrose and trehalose) and three polysaccharides (dextrin, inulin and xylan) were used as substrates. Three F. avenaceum strains were used in the experiments. These were all able to grow and produce aurofusarin on the tested carbon sources. Moniliformin and enniatins were produced on all carbon types, except on lactose, which suggest a common conserved regulation mechanism. Differences in the strains was observed for production of fusarin C, 2-AOD-3-ol, chlamydosporol and antibiotic Y, which suggests that carbon source plays a role in the regulation of their biosynthesis.     

In vitro cytotoxicity of fungi spoiling maize silage

Penicillium roqueforti, Penicillium paneum, Monascus ruber, Alternaria tenuissima, Fusarium graminearum, Fusarium avenaceum, Byssochlamys nivea and Aspergillus fumigatus have previously been identified as major fungal contaminants of Danish maize silage. In the present study their metabolite production and in vitro cytotoxicity have been determined for fungal agar and silage extracts. All 8 fungal species significantly affected Caco-2 cell viability in the resazurin assay, with large variations for each species and growth medium. The 50% inhibition concentrations (IC₅₀) of the major P. roqueforti metabolites roquefortine C (48 μg/mL), andrastin A (>50 μg/mL), mycophenolic acid (>100 μg/mL) and 1-hydroxyeremophil-7(11),9(10)-dien-8-one (>280 μg/mL) were high. Fractionating of agar extracts identified PR-toxin as an important cytotoxic P. roqueforti metabolite, also detectable in maize silage. The strongly cytotoxic B. nivea and P. paneum agar extracts contained patulin above the IC₅₀ of 0.6 μg/mL, however inoculated onto maize silage B. nivea and P. paneum did not produce patulin (>371 μg/kg). Still B. nivea infected maize silage containing mycophenolic acid (∼50 mg/kg), byssochlamic acid and other metabolites, was cytotoxic. In contrast hot-spots of P. roqueforti, P. paneum, M. ruber and A. fumigatus were not more cytotoxic than uninoculated silage.     

Cytotoxicity of enniatins A, A1, B, B1, B2 and B3 from Fusarium avenaceum

The enniatins A, A1, B, B1, B2 and B3 were purified from hexane-extracts of Fusarium avenaceum rice cultures, using semi-preparative HPLC, after precipitation of lipids. Their toxicity, as well as the toxicity of the related fungal metabolite beauvericin (Bea) and the trichothecenes deoxynivalenol (DON) and T-2 toxin, was tested in two cell lines of human origin (hepatocellular carcinoma-line Hep G2 and fibroblast-like foetal lung cell line MRC-5) by using the BrdU and Alamar Blue™ assays. All the compounds evoked toxicity in the in vitro assays at the concentrations tested. The MRC-5 cell line in combination with the BrdU assay resulted in the lowest inhibitory concentrations (IC₅₀) for exposure with enniatins in the range 0.8 μM (enniatin A) to 3.6 μM (enniatin B). The cytotoxicity of DON in the BrdU assay was comparable to the cytotoxicity of enniatins A, B and Bea in a multiple regression model, while DON was significantly more cytotoxic than the enniatins in the Alamar Blue™ assay. This study indicates that enniatins, fungal metabolites that are commonly found in grain in Northern Europe, may have an underestimated toxic potential.     

Phomenins and fatty acids from Alternaria infectoria

Taxa of the Alternaria infectoria species group are the predominant Alternaria spp. found in cereals in Northern Europe. While several pyrones have been isolated from A. infectoria and described as taxonomical markers for species identification, information about the bioactivity of metabolites from the fungus is missing. Bioassay-guided fractionation of rice culture extracts from several strains of A. infectoria linked the observed toxicity of the extracts in MRC-5 cells to free fatty acids, i.e. linoleic acid and α-linolenic acid. The fungus also produced a cytotoxic pyrone, which upon isolation and NMR spectroscopic analysis was identified as a mixture of phomenins A and B (approximately 10:1), which have not previously been isolated from an Alternaria species.     

A Novel Metabolite from Aspergillus ochraceus JGI 25 Showing Cytotoxicity to Hela Cells

This study aims at the isolation of filamentous fungi, extraction of metabolites, and evaluation of the cytotoxic properties on HeLa cells and normal human lymphocytes. We isolated fungi from the soil by serial dilution method. One of the isolates was chosen and identified as Aspergillus ochraceus Wilhelm (Trichocomaceae) by standard techniques. The metabolites were extracted using methanol. Different concentrations of the extract were evaluated for their potential anticancer activity on HeLa cells by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assay and the safety of the extract was checked on normal human lymphocytes. The extract was purified by chromatographic techniques like thin-layer chromatography and high-performance liquid chromatography, and subjected to mass spectrometric analysis. The extract showed significant cytotoxic potential on HeLa cells at low concentrations with a half maximal inhibitory concentration value of <50 μg/ml. The extract gave 10 fractions by thin layer chromatography, and fraction B had higher toxicity than the rest. This fraction gave a single peak by high-performance liquid chromatography and had a mass-to-charge ratio of 905.65, which did not match any of the earlier known fungal metabolites or metabolites from other strains of A. ochraceus. The metabolite from A. ochraceus is alkaloid in nature, cytotoxic to HeLa cells, and appears to be a novel with anticancer potentials, which could be explored further for characterization of the active component.     

Rat hepatoma cells show extreme sensitivity to aflatoxin B1

Aflatoxin B₁ (Afl B₁) is a potent hepatotoxin and hepatocarcinogen that requires metabolic activation to exert its cytotoxic and genotoxic effects. FU-5 cells are a rat hepatoma cell line originally derived from Reuber hepatoma cells. We found that the FU-5 cell line and an FAO-1 subclone showed extreme sensitivity to Afl B₁. However, the FAO-1 line was about 50 times more sensitive to Afl B₁ cytotoxicity compared with the parental FU-5 line. Upon exposure to Afl B₁, the FAO-1 line produced one major metabolite, which we identified as Aflatoxicol (AFl R₀). In constrast, the FU-5 line produced Afl R₀ and many polar metabolites that may be detoxification products. The FAO-1 cell bound slightly more Afl B₁ to its DNA than did the FU-5 line. The differences in metabolism of Afl B₁ may account for the differential cytotoxicity that this mycotoxin exerted on FAO-1 and FU-5 cells. Although both hepatoma cell lines were efficient at converting Afl B₁ into cytotoxic metabolites, they bound small amounts of Afl B₁ to their DNA and were inefficient in converting Afl B₁ into mutagenic metabolites. In addition, both lines were incapable of converting 2-acetylaminofluorene, benzo(a)pyrene, and dimethylnitrosamine into cytotoxic metabolites.     

Interactions between two carnobacteriocins Cbn BM1 and Cbn B2 from Carnobacterium maltaromaticum CP5 on target bacteria and Caco-2 cells

Two purified class IIa carnobacteriocins Cbn BM1 and Cbn B2, from Carnobacteriummaltaromaticum CP5, were evaluated for antimicrobial activity against pathogenic, spoilage and lactic acid bacteria. Then, the presence of a synergistic mode of action of these two carnobacteriocins on Listeria sp., Enterococcus sp. and Carnobacterium sp. was investigated. A synergistic mode of action between Cbn BM1 and Cbn B2 on sensitive target bacteria was demonstrated using the FIC index method. Combinations of carnobacteriocins enhanced their antibacterial activities and MICs were significantly reduced, between 2- and 15-fold, by the addition of the second bacteriocin. To improve the safety of the bacteriocins as biopreservative agents, the cytotoxicity of the combination of theses two bacteriocins was determined on Caco-2 cell line. However, these two peptides used alone or in combination, at concentration 100-fold higher than those required for antimicrobial activity, were not cytotoxic. This suggests that the two carnobacteriocins produced by C. maltaromaticum CP5 could be potential natural agents for food preservation.     

2-Amino-14,16-dimethyloctadecan-3-ol, a new sphingosine analogue toxin in the fungal genus Fusarium

2-Amino-14,16-dimethyloctadecan-3-ol (2-AOD-3-ol) was isolated from the cytotoxic rice culture extract of a strain of Fusarium avenaceum, which had previously been isolated from Norwegian grain. The structural information was obtained from LC-MS/MS, GC-MS, NMR spectroscopy and high-resolution MS data. The metabolite has a striking similarity to sphinganine, an intermediate in the biosynthesis of the sphingolipids. This similarity is a major feature of the so-called sphingosine analogue toxins; the most studied being the AAL toxins and the fumonisins. 2-AOD-3-ol was found to be cytotoxic to the rat hepatoma cell line H4IIE-W and to the porcine epithelial kidney cell line PK(15) at concentrations (EC₅₀) of 16 and 24 μM, respectively. The metabolite has been found in F. avenaceum inoculated wheat that was treated to support ideal conditions for Fusarium growth, demonstrating that the fungus has the potential to produce the metabolite under field conditions, which may occur in Northern Europe.     

Enniatin B and beauvericin are common in Danish cereals and show high hepatotoxicity on a high‐content imaging platform

Mycotoxins are fungi‐born metabolites that can contaminate foods through mould‐infected crops. They are a significant food/feed‐safety issue across the globe and represent a substantial financial burden for the world economy. Moreover, with a changing climate and fungal biota, there is now much discussion about emerging mycotoxins that are measurable at significant levels in crops world‐wide. Unfortunately, we still know very little about the bioavailability and toxic potentials of many of these less characterized mycotoxins, including the large family of enniatins. In this study, we present new occurrence data for enniatin A, A1, B, B1 and beauvericin in four Danish crops: oat, wheat, and barley from the 2010 harvest, and rye from 2011 harvest. The occurrence of the four enniatins were B > B1 > A1 > A. Enniatin B was detected in 100% of tested samples regardless of crop type. In addition to occurrence data, we report a proof‐of‐concept study using a human‐relevant high‐content hepatotoxicity, or “quadroprobe,” assay to screen mycotoxins for their cytotoxic potential. The assay was sensitive for most cytotoxic compounds in the 0.009–100 µM range. Among eight tested mycotoxins (enniatin B, beauvericin, altenariol, deoxynivalenol, aflatoxin B1, andrastin A, citrinin, and penicillic acid), enniatin B and beauvericin showed significant cytotoxicity at a concentration lower than that for aflatoxin B1, which is the archetypal acute hepatotoxic and liver‐carcinogenic mycotoxin. Hence, the quadroprobe hepatotoxicity assay may become a valuable assessment tool for toxicity assessment of mycotoxins in the future. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1658–1664, 2017.     

Enniatin B and Deoxynivalenol Activity on Bread Wheat and on Fusarium Species Development

Fusarium head blight (FHB) is a devastating wheat disease, mainly caused by Fusarium graminearum (FG)—a deoxynivalenol (DON)-producing species. However, Fusarium avenaceum (FA), able to biosynthesize enniatins (ENNs), has recently increased its relevance worldwide, often in co-occurrence with FG. While DON is a well-known mycotoxin, ENN activity, also in association with DON, is poorly understood. This study aims to explore enniatin B (ENB) activity, alone or combined with DON, on bread wheat and on Fusarium development. Pure ENB, DON, and ENB+DON (10 mg kg⁻¹) were used to assess the impacts on seed germination, seedling growth, cell death induction (trypan blue staining), chlorophyll content, and oxidative stress induction (malondialdehyde quantification). The effect on FG and FA growth was tested using ENB, DON, and ENB+DON (10, 50, and 100 mg kg⁻¹). Synergistic activity in the reduction of seed germination, growth, and chlorophyll degradation was observed. Conversely, antagonistic interaction in cell death and oxidative stress induction was found, with DON counteracting cellular stress produced by ENB. Fusarium species responded to mycotoxins in opposite directions. ENB inhibited FG development, while DON promoted FA growth. These results highlight the potential role of ENB in cell death control, as well as in fungal competition.     

A Strategy for Quality Control of Menispermum dauricum DC Based on Cytotoxic Activity and HPLC Fingerprint Analysis

The rhizome of Menispermum dauricum DC known as a traditional Chinese medicine, with high content of alkaloids, has been found to possess antitumor activity. In this research, an attempt to correlate fingerprinting with bioactivity was made for quality control of M. dauricum. Firstly, the cytotoxicity of extracts from ten batches of samples against human breast MCF-7 cancer cells was estimated by [3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazoliumbromide] assay. Then, cytotoxic activity-integrated fingerprints were established by high performance liquid chromatography. Eight peaks were selected as the common peaks to evaluate the similarities of samples and hierarchical clustering analysis was used to identify and classify different samples into groups. Assays for determinations of total alkaloids and dauricine contents enabled cytotoxicity coefficient of each extract. The potential usefulness of employing cytotoxicity coefficient was investigated by a combination of Pearson correlation coefficients and multiple linear regression analysis as being the reliable parameter to evaluate the herbal extracts. The results indicated that the level of dauricine (peak 8 in the fingerprint) correlated closely with cytotoxicity and played a significant role in the cytotoxicity of Bei Dou-Gen and could be related to its antitumor properties. It is proposed that the cytotoxicity coefficient value with a cytotoxic activity-integrated fingerprint of key biomarkers (dauricine) may be useful indicators to adopt for the quality control of M. dauricum. The analysis of cytotoxic-activity-integrated fingerprint could correlate fingerprinting with bioactivities and would provide a reasonable strategy for quality control of complex mixture of herbal medicines.     

The Fusarium mycotoxins enniatins and beauvericin cause mitochondrial dysfunction by affecting the mitochondrial volume regulation,oxidative phosphorylation and ion homeostasis

The mechanisms of cell toxicity of mycotoxins of the enniatin family produced by Fusarium sp. enniatin B, a mixture of enniatin homologues (3% A, 20% A₁, 19% B, 54% B1) and beauvericin, were investigated. In isolated rat liver mitochondria, exposure to submicromolar concentrations of the enniatin mycotoxins depleted the mitochondrial transmembrane potential, uncoupled oxidative phosphorylation, induced mitochondrial swelling and decreased calcium retention capacity of the mitochondria. The mitochondrial effects were strongly connected with the potassium (K⁺) ionophoric activity of the enniatins. The observed enniatins induced K⁺ uptake by mitochondria. This shows that the enniatins acted as ionophores highly selective for potassium ions. The effects were observed in potassium containing media whereas less or no effect remained to be observed when K⁺ was partially or totally replaced by isomolar concentrations of Na⁺. The rank order of enniatin induced mitochondrial impairment was beauvericin > enniatin mixture > enniatin B. Exposure to the enniatins depleted the mitochondrial membrane potential also in intact human neural (Paju), murine insulinoma (Min-6) cells as well as boar spermatozoa. Exposure to enniatin B in media with physiological (4 mM) or low (<1 mM) but not in high (60 mM) external concentration of K⁺ induced hyperpolarization of the spermatozoal plasma membrane indicating enniatin that catalysed efflux of the cytosolic K⁺ ions. These results indicate that the cellular toxicity targets of the enniatin mycotoxins are the mitochondrion and the homeostasis of potassium ions.     

Sequence Divergence of the Enniatin Synthase Gene in Relation to Production of Beauvericin and Enniatins in Fusarium Species

Beauvericin (BEA) and enniatins (ENNs) are cyclic peptide mycotoxins produced by a wide range of fungal species, including pathogenic Fusaria. Amounts of BEA and ENNs were quantified in individual rice cultures of 58 Fusarium strains belonging to 20 species, originating from different host plant species and different geographical localities. The species identification of all strains was done on the basis of the tef-1α gene sequence. The main aim of this study was to analyze the variability of the esyn1 gene encoding the enniatin synthase, the essential enzyme of this metabolic pathway, among the BEA- and ENNs-producing genotypes. The phylogenetic analysis based on the partial sequence of the esyn1 gene clearly discriminates species producing exclusively BEA from those synthesizing mainly enniatin analogues.     

Mycotoxins,Phytoestrogens and Other Secondary Metabolites in Austrian Pastures: Occurrences,Contamination Levels and Implications of Geo-Climatic Factors

Pastures are key feed sources for dairy production and can be contaminated with several secondary metabolites from fungi and plants with toxic or endocrine-disrupting activities, which possess a risk for the health, reproduction and performance of cattle. This exploratory study aimed to determine the co-occurrences and concentrations of a wide range of mycotoxins, phytoestrogens and other secondary metabolites in grazing pastures. Representative samples of pastures were collected from 18 Austrian dairy farms (one sample per farm) between April to October 2019. After sample preparation (drying and milling) the pastures were subjected to multi-metabolite analysis using LC-MS/MS. In total, 68 metabolites were detected, including regulated zearalenone and deoxynivalenol (range: 2.16–138 and 107–505 μg/kg on a dry matter (DM) basis, respectively), modified (3-deoxynivalenol-glucoside, HT-2-glucoside) and emerging Fusarium mycotoxins (e.g., enniatins), ergot alkaloids and Alternaria metabolites along with phytoestrogens and other metabolites. Aflatoxins, fumonisins, T-2 toxin, HT-2 toxin and ochratoxins were not detected. Of the geo-climatic factors and botanical diversity investigated, the environment temperature (average of 2 pre-sampling months and the sampling month) was the most influential factor. The number of fungal metabolites linearly increased with increasing temperatures and temperatures exceeding 15 °C triggered an exponential increment in the concentrations of Fusarium and Alternaria metabolites and ergot alkaloids. In conclusion, even though the levels of regulated mycotoxins detected were below the EU guidance levels, the long-term exposure along with co-occurrence with modified and emerging mycotoxins might be an underestimated risk for grazing and forage-fed livestock. The one-year preliminary data points out a dominant effect of environmental temperature in the diversity and contamination level of fungal metabolites in pastures.     

Evaluation of Cassia occidentalis for in vitro cytotoxicity against human cancer cell lines and antibacterial activity

Objective:

To evaluate the in vitro cytotoxicity and antibacterial properties of Cassia occidentalis (whole plant) via alcoholic, hydro-alcoholic, and aqueous extracts against eight human cancer cell lines from six different tissues and four bacterial strains.

Material and Methods:

in vitro cytotoxicity against the human cancer cells, cultured for 48h in presence of different concentrations C. occidentalis extracts and percentage of cell viability, was evaluated using the sulforhodamine-B (SRB) assay. The antibacterial activity was performed using the standard protocol against bacterial strains.

Results:

It was observed that aqueous extract of C. occidentalis (whole plant) had more potential than hydro-alcoholic and alcoholic extracts against HCT-15, SW-620, PC-3, MCF-7, SiHa, and OVCAR-5 human cancer cell lines at 100, 30, and 10 μg/ml in a dose-dependent manner. The hydro-alcoholic extract showed potential against Bacillus subtillis.

Conclusion:

The plant can be explored for the possible development of lead molecules for drug discovery.     

Antibacterial activity of the enniatin B,produced by Fusarium tricinctum in liquid culture,and cytotoxic effects on Caco-2 cells

The enniatins (ENs) are bioactive compounds of hexadepsipeptidic structure produced by several strains of Fusarium sp. The EN B was purified from extracts of Fusarium tricinctum growth on liquid culture of potato dextrose broth (PDB), using a semipreparative liquid chromatography (LC) followed by an analytical LC. The purity and the structure of the isolated compound were confirmed by the determination of the extinction coefficient and with electrospray ionization–mass spectrometry (ESI-MS) study.

The pure fraction of EN B was utilized to determine the antibiotic effects on several bacterial strains that are considered normally pathogens of the intestinal tract: Escherichia coli, Enterococcus faecium, Salmonella enterica, Shigella dysenteriae, Listeria monocytogenes, Yersinia enterocolitica, Clostridium perfringens, Pseudomonas aeruginosa, and Staphylococcus aureus, and to study the cytotoxic effects on Caco-2 differentiated and undifferentiated cells.

The results obtained demonstrated that in several antibiograms, EN B induced the inhibition of the grown microorganisms tested and no significant differences over control were detected when Caco-2 cells were exposed to EN B, at any of the concentrations used.     

Fusarium mycotoxin enniatin B: Cytotoxic effects and changes in gene expression profile

The mycotoxin enniatin B, a cyclic hexadepsipeptide produced by the plant pathogen Fusarium, is prevalent in grains and grain-based products in different geographical areas. Although enniatins have not been associated with toxic outbreaks, they have caused toxicity in vitro in several cell lines. In this study, the cytotoxic effects of enniatin B were assessed in relation to cellular energy metabolism, cell proliferation, and the induction of apoptosis in Balb 3T3 and HepG2 cells. The mechanism of toxicity was examined by means of whole genome expression profiling of exposed rat primary hepatocytes. Enniatin B altered cellular energy metabolism and reduced cell proliferation in Balb 3T3 and HepG2 cell lines. Furthermore, the proportion of apoptotic cell populations of Balb 3T3 cells slightly increased. On the other hand, enniatin B caused necrotic cell death in primary hepatocytes. Gene expression studies revealed the alteration of energy metabolism due to effects on mitochondrial organization and function and the assembly of complex I of the electron transport chain.     

Cane toad toxicity: An assessment of extracts from early developmental stages and adult tissues using MDCK cell culture

Extracts of the cane toad (Bufo [Chaunus] marinus) adversely affected the growth of Mardin-Darby canine kidney (MDCK) cells during culture. In a similar manner to ouabain treatment, application of toad extracts over a 24 h period resulted in high levels of cytotoxicity, as indicated by cell detachment, increased membrane permeability and loss of mitochondrial function. Cell viability and growth were unchanged for controls (PBS) and increased with the application of Limnodynastes peronii tadpole and adult frog extracts. We investigated the general cytotoxicity of cane toad developmental stages (e.g. eggs, embryonic hatchlings, tadpoles and post-metamorphic toadlets) as well as selected adult tissues (e.g. skin, gut, liver). Our results showed that pre-metamorphic cane toad aqueous extracts used at 1 mg/ml on MDCK cells generated cytotoxicity levels comparable to ouabain treatment (3 μM). After normalisation, extracts from 2-3-month-old toadlets appeared less toxic than pre- and early metamorphic stages. Adult tissues revealed a gradient of cytotoxicity levels ranging from non-toxic brain to highly toxic dorsal skin extracts.