HepG2 liver cells treated with fumonisin B1 in galactose supplemented media have altered expression of genes and proteins known to regulate cholesterol flux

Fumonisin B1 (FB1) contributes to mycotoxicosis in animals and has been associated with the incidence of some cancers in humans. The effect of FB1 on lipidomic profiles, sphingolipids and cholesterol levels have been demonstrated in experimental models, however, the events leading to altered cholesterol levels are unclear. This study investigates the molecular mechanisms that regulate the effect of FB1 on cholesterol homeostasis in galactose supplemented HepG2 liver cells. Galactose supplementation is a proven method utilised to circumvent the Crabtree effect exhibited by cancer cells, which forces cancer cells to activate the mitochondria. HepG2 cells were cultured in galactose supplemented media and treated with FB1 (IC50 = 25 μM) for 6 h. Cell viability was determined using the MTT assay. Metabolic status was evaluated using ATP luciferase assay, and cholesterol regulatory transcription factors (SIRT1, SREBP-1C, LXR, LDLR, PCSK9, and ABCA1) were investigated using western blotting and qPCR. FB1 in galactose supplemented HepG2 cells increased gene expression of SIRT1 (P<0.05), SREBP-1C, LXR, and LDLR; however, PCSK9 (P<0.05) was decreased. Furthermore, protein expression of SIRT1, LXR, and LDLR was elevated upon FB1 treatment, while SREBP-1C and PCSK9 were reduced. The data provides evidence that SIRT1 reduced the expression of PCSK9 and deacetylated LXR to prevent degradation of LDLR. This could result in a dysregulated cholesterol flux, which may contribute to FB1 mediated toxicity.

Evaluation of the impact of pre- and post-harvest maize handling practices on mycotoxin contamination on smallholder farms in Guatemala

Mycotoxins are secondary metabolites produced by fungi including Aspergillus and Fusarium that commonly contaminate crops, such as maize, resulting in economic losses and food insecurity. Mycotoxins can contaminate crops during pre- and post-harvest stages. Consumption of mycotoxin-contaminated foods has been linked to a variety of negative health outcomes including liver cancer, stunting, and neural tube defects. In countries such as Guatemala where maize constitutes a major portion of the diet, mycotoxins can be a significant contributor to disease burden. This review describes maize pre- and post-harvest practices in Guatemala that can lead to the development of mycotoxins and subsequent exposure to humans and animals, current information gaps, and opportunities for future research. There are specific challenges to minimising fungal growth and subsequent mycotoxin production during storage of maize in Guatemala, including reducing moisture content, minimising pest damage, and controlling temperature. Research on maize-handling practices that are associated with the greatest mycotoxin exposure in Guatemala is needed to prioritise allocation of resources and reduce exposure.

A novel chemical marker of tremorgenic mycotoxicosis detected by gas-chromatography/mass-spectrometry

Tremorgenic mycotoxicosis can arise from dietary exposure to secondary metabolite products of various fungal species, particularly those from the Penicillium genus. Although general toxin screens often rely on gas chromatography-mass spectrometry (GC/MS) and well-developed mass spectral library databases, two principal representative Penicillium mycotoxins, roquefortine and penitrem A, are unamenable to GC/MS owing to high molecular weights, low volatilities and/or high thermal instabilities. Reliance on GC/MS screens alone could therefore inadvertently result in failure to collect evidence of exposure to such tremorgenic mycotoxins. In this report we describe a newly discovered tremorgenic marker compound (TMC), the presence of which correlates highly with conclusive exposure to Penicillium toxins in explanation of clinical manifestations of tremorgenic mycotoxicosis. According to detailed mass spectral deconvolution, the compound is 210.0892 molecular weight, and amenable to GC/MS whether chemically underivatized or derivatized by methylation or trimethylsilylation. 1D and 2D NMR (nuclear magnetic resonance) studies on the isolated compound determined the TMC to be the Penicillium product terrestric acid, C11H14O4, which matches the molecular formula determined by high resolution mass spectrometry and thus provides an excellent target for assessment of mycotoxicosis by GC/MS.

Effects of aflatoxin B1 and monensin interaction on liver and intestine of poultry – influence of a biological additive (Pichia kudriavzevii RC001)

The aim of this study was to evaluate the effects of aflatoxin B1 (AFB1) and monensin (MONS) interaction on the liver and intestinal histological changes in poultry, and the influence of Pichia kudriavzevii RC001. One-day-old commercial line (Ross 308) broilers (n=120) were individually weighed and randomly assigned to 8 treatments (15 broilers/treatment, 5 broilers per cage and 3 replicates/treatment). The experimental diets were: Group 1: basal diet (BD); Group 2: BD + MONS (50 mg/kg); Group 3: BD + P. kudriavzevii RC001 (1 g/kg); Group 4: BD + AFB1 (100 μg/kg); Group 5: BD + MONS + P. kudriavzevii RC001; Group 6: BD + AFB1 + P. kudriavzevii RC001; Group 7: BD + AFB1 + MONS + P. kudriavzevii RC001; Group 8: BD + AFB1 + MONS. When MONS was added, the typical AFB1 macroscopic and microscopic alterations were intensified. The P. kudriavzevii RC001 cytotoxicity and genotoxicity assays with Vero cells and with broiler chicken’s erythrocytes, demonstrated that P. kudriavzevii RC001 neither were non-cytotoxic nor genotoxic. When MONS was added in the presence of P. kudriavzevii RC001, the toxic effect of AFB1 on liver was not prevented. When P. kudriavzevii was present alone, the same prevention of the pathological damage was observed in the intestine of poultry fed with AFB1. The smallest apparent absorption area was obtained when AFB1 and MONS were added in the feed (P<0.05). AFB1 and MONS interaction demonstrated important toxic effects. Although P. kudriavzevii was effective in ameliorating the adverse effects of AFB1 alone on liver pathology and gut morphology, it was not able to diminish the toxic effects of AFB1 in presence of MONS. It suggests that P. kudriavzevii could be used as feed additive or counteracting the toxic effects of AFB1 in poultry production in the absence of MONS.

Inhibition of Aspergillus flavus growth and aflatoxin B1 production by natamycin

Aspergillus flavus causes huge crop losses, reduces crop quality and has adverse effects on human and animal health. A large amount of food contaminated with aflatoxin can greatly increase the risk of liver cancer. Therefore, prevention and control of aflatoxin production have aroused attention of research in various countries. Natamycin extracted from Streptomyces spp. has been widely used in production practice due to its good specificity and safety. Here, we found that natamycin could significantly inhibit fungal growth, conidia germination, ergosterol and AFB1 production by A. flavus in a dose-dependent manner. Scanning electron microscope analysis indicated that the number of conidia was decreased, the outer wall of conidia was destroyed, and the mycelia were shrivelled and tangled by natamycin. RNA-Seq data indicated that natamycin inhibited fungal growth and conidia development of A. flavus by significantly down-regulating some genes involved in ergosterol biosynthesis, such as Erg13, HMG1 and HMG2. It inhibited conidia germination by significantly down-regulating some genes related to conidia development, such as FluG and VosA. After natamycin exposure, the decreased ratio of aflS/aflR caused by the down-regulation of all the structural genes, which subsequently resulted in the suppression of AFB1 production. In conclusion, this study served to reveal the inhibitory mechanisms of natamycin on fungal growth and AFB1 biosynthesis in A. flavus and to provide solid evidence for its application in controlling AFB1 contamination.

Abiotic factors affect growth and aflatoxin B1 production by Aspergillus flavus strains on chilli powder and red chillies

Chillies and chilli-based products are important spices on a global basis. The production, processing, transport and storage phases of chillies are prone to infection by Aspergillus Section Flavi and contamination with aflatoxins (AFs), especially aflatoxin B1 (AFB1) for which legislative limits exist in many countries. We have examined the effect of the interacting abiotic factors of water availability (water activity, aw; 0.995-0.850 aw) and temperature (15-37 °C) on (a) lag phases prior to growth, (b) growth, (c) AFB1 production and (d) contour maps of optimum and boundary conditions for colonisation and toxin production by three Aspergillus flavus strains on a 10% chilli-based medium. Additional studies with whole red chillies + A. flavus conidial inoculum on AFB1 contamination during storage for 10-20 days at 30 °C were also carried out. In vitro, the lag phases before growth were delayed by lower temperatures (15, 20 °C) and aw levels (0.928-0.901 aw). There was no statistical difference in growth between the three strains. Optimal growth was at 37 °C and 0.982 aw with no growth at 0.85 aw. Optimal temperature × aw conditions for AFB1 production were at 30 °C and 0.982 aw with no statistical difference in production between strains. No AFB1 was produced at 15-20 °C at 0.901 and 0.928 aw levels, respectively. In situ studies with A. flavus inoculated whole red chillies at 0.90 and 0.95 aw found that this species became the major component of the total fungal populations at 30 °C after 10-20 days storage. AFB1 contamination was above the European legislative limits (5 μg/kg) for spices at 0.90 aw after 20 days storage and at 0.95 aw after 10 and 20 days. This suggests that storage conditions of ≥0.90 aw, especially at ≥25-30 °C represents a significant risk of contamination with AFB1 at levels where rejection might occur, even after only 10-20 days storage.

Review of good agricultural practices for smallholder maize farmers to minimise aflatoxin contamination

Maize is consumed world-wide as staple food, livestock feed, and industrial raw material. However, it is susceptible to fungal attack and at risk of aflatoxin contamination under certain conditions. Such contamination is a serious threat to human and animal health. Ensuring that the maize used by food industry meets standards for aflatoxin levels requires significant investment across the supply chain. Good Agricultural Practices (GAP) form a critical part of a broader, integrated strategy for reduction of aflatoxin contamination. We reviewed and summarised the GAP of maize that would be effective and practicable for aflatoxin control within high-risk regions for smallholder farmers. The suggested practicable GAP for smallholder farmers were: use of drought-tolerant varieties; timely harvesting before physiological maturity; sorting to remove damaged ears and those having poor husk covering; drying properly to 13% moisture content; storage in suitable conditions to keep the crop clean and under condition with minimally proper aeration, or ideally under hermetic conditions. This information is intended to provide guidance for maize growers that will help reduce aflatoxin in high-risk regions, with a specific focus on smallholder farmers. Following the proposed guidelines would contribute to the reduction of aflatoxin contamination during pre-harvest, harvest, and post-harvest stages of the maize value chain.

An update on genotoxic and epigenetic studies of fumonisin B1

Fumonisins (FBs), a widespread group of mycotoxins produced by Fusarium spp., are natural contaminants in cereals and foodstuffs. Fumonisin B1 (FB1) is the most toxic and prevalent mycotoxin of this group, and it has been reported that FB1 accounts for 70-80% of FBs produced by the mycotoxigenic strains. The mode of action of FB1 depends on the structural similarity with sphinganine/sphingosine N-acyltransferase. This fact causes an accumulation of sphingoid bases and blocks the sphingolipid biosynthesis or the function of sphingolipids. Diverse toxic effects and diseases such as hepatocarcinogenicity, hepatotoxicity, nephrotoxicity, and cytotoxicity have been reported, and diseases like leukoencephalomalacia in horses and pulmonary oedema in horses and swine have been described. In humans, FBs have been associated with oesophageal cancer, liver cancer, neural tube defects, and infantile growth delay. However, despite the International Agency for Research on Cancer designated FB1 as a possibly carcinogenic to humans, its genotoxicity and epigenetic properties have not been clearly elucidated. This review aims to summarise the progress in research about the genotoxic and epigenetics effects of FB1.

Association of exposure to deoxynivalenol with DNA methylation in white blood cells in children in China

Deoxynivalenol (DON) is a mycotoxin that commonly contaminates cereals worldwide. Dietary exposure to DON is a subject of great public health concern, but studies on the health effects of chronic exposure to DON are not available. In this study, we investigated the connection between DNA methylation levels and DON exposure in children. The DNA methylation status of white blood cells from 32 children aged 2~15 years old in Henan, China, was profiled. A total of 378 differentially methylated CpGs were identified between the high and low DON exposure groups, and 8 KEGG pathways were found to be significantly enriched among the differentially methylated genes. In addition, the quantitative methylation of EIF2AK4, EMID2 and GNASAS was analysed using the Sequenom MassARRAY platform. The results showed that the methylation level of EIF2AK4 was significantly different between the two groups, and the methylation levels were associated with exposure to DON. Conclusively, our study found that chronic exposure to DON during childhood could affect DNA methylation levels.

Interlaboratory collaboration to determine the performance of the Randox food diagnostics biochip array technology for the simultaneous quantitative detection of seven mycotoxins in feed

An inter-laboratory collaborative study was performed to evaluate the performance of the Biochip Array Technology (BAT) Myco 7 method. The Myco 7 Array is a method which simultaneously and quantitatively detects 20 mycotoxins including aflatoxins B1, B2, G1 and G2, ochratoxin A, deoxynivalenol, zearalenone, fumonisin B1, B2 and B3 and T-2 and HT-2 toxin. The BAT Myco 7 method was collaboratively evaluated by nine government and private Association of American Feed Control Officials (AAFCO) laboratories. Samples were analysed in a proficiency testing round format. Seventeen blind samples were analysed on the same equipment using Myco 7 kits. 99% of the results fell within an acceptable Z-score range of -2|<Z<|+2. Deoxynivalenol had a 100% Z-score pass rate, while a 99% pass was recorded for aflatoxins, zearalenone, ochratoxin A and fumonisins. T-2 toxin had a 97% Z-score pass rate. HorRat analysis for reproducibility used a range of 0.3<|HorRat|≤2. The target was met for deoxynivalenol, zearalenone, T-2 and HT-2 toxin, and aflatoxins B1, B2, G1 and G2 assays. Fumonisins and ochratoxin A assays had a 93% and 94% pass, respectively. The reproducibility co-efficiency of variation was between 16 and 20% meeting set criterion of <40% and is, therefore, fit-for-purpose for use in the AAFCO control programs for mycotoxins.