Mycotoxin Aflatoxin B1 Research Articles

Modulation of the spleen transcriptome in domestic turkey (Meleagris gallopavo) in response to aflatoxin B1 and probiotics.

Poultry are highly susceptible to the immunotoxic effects of the food-borne mycotoxin aflatoxin B1 (AFB1). Exposure impairs cell-mediated and humoral immunity, limits vaccine efficacy, and increases the incidence of costly secondary infections. We investigated the molecular mechanisms of AFB1 immunotoxicity and the ability of a Lactobacillus-based probiotic to protect against aflatoxicosis in the domestic turkey (Meleagris gallopavo). The spleen transcriptome was examined by RNA sequencing (RNA-seq) of 12 individuals representing four treatment groups. Sequences (6.9Gb) were de novo assembled to produce over 270,000 predicted transcripts and transcript fragments. Differential expression analysis identified 982 transcripts with statistical significance in at least one comparison between treatment groups. Transcripts with known immune functions comprised 27.6% of significant expression changes in the AFB1-exposed group. Short exposure to AFB1 suppressed innate immune transcripts, especially from antimicrobial genes, but increased the expression of transcripts from E3 ubiquitin-protein ligase CBL-B and multiple interleukin-2 response genes. Up-regulation of transcripts from lymphotactin, granzyme A, and perforin 1 could indicate either increased cytotoxic potential or activation-induced cell death in the spleen during aflatoxicosis. Supplementation with probiotics was found to ameliorate AFB1-induced expression changes for multiple transcripts from antimicrobial and IL-2-response genes. However, probiotics had an overall suppressive effect on immune-related transcripts.

Effect of 8-oxoguanine glycosylase deficiency on aflatoxin B1 tumourigenicity in mice.

The mycotoxin aflatoxin B1 (AFB1) may initiate cancer by causing oxidatively damaged DNA, specifically by causing 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) lesions. Base excision repair removes these lesions, with 8-oxoguanine glycosylase (OGG1) being the rate-limiting enzyme. The aim of this study was to determine the effect of ogg1 deficiency on AFB1-induced oxidatively damaged DNA and tumourigenesis. Female wild-type, heterozygous and homozygous ogg1 null mice were given a single dose of 50mg/kg AFB1 or 40 µl dimethyl sulfoxide (DMSO) ip. Neither ogg1 genotype nor AFB1 treatment affected levels of oxidised guanine in lung or liver 2h post-treatment. AFB1-treated ogg1 null mice showed exacerbated weight loss and mortality relative to DMSO-treated ogg1 null mice, but AFB1 treatment did not significantly increase lung or liver tumour incidence compared with controls, regardless of ogg1 genotype. Suspect lung masses from three of the AFB1-treated mice were adenomas, and masses from two of the mice were osteosarcomas. No osteosarcomas were observed in DMSO-treated mice. All liver masses from AFB1-treated mice were adenomas, and one also contained a hepatocellular carcinoma. In DNA from the lung tumours, the K-ras mutation pattern was inconsistent with initiation by AFB1. In conclusion, ogg1 status did not have a significant effect on AFB1-induced oxidatively damaged DNA or tumourigenesis, but deletion of one or both alleles of ogg1 did increase susceptibility to other aspects of AFB1 toxicity.

Influence of the Compounds Used in Immunochromatographic Test-system on its Analytical Characteristics: Multi-factor Optimization of the System for Aflatoxin B1

Analytical parameters of immunochromatographic test-systems, such as the detection limit and working range, depend on many factors. However, the process of choosing the concentration and composition of reagents applied to test strip membranes and choosing the membranes themselves has not been systematically described for commercially produced tests or for scientific studies. Here, we present the results of a study to determine how the properties of the test system components, using mycotoxin aflatoxin B1 as an example, affect its analytical performance. A comparison of different membranes used for immunochromatography was performed as well as reagent concentrations and composition of intermolecular conjugates. Optimization of the immunochromatographic system indicated a limit of aflatoxin B1 detection equal to 0.2 ng/mL. The effectiveness of the system in testing plant extracts with minimal (twice) dilution prior to analysis was determined and the final content of methanol in the course of immunoassay was 35%.

Controlled growth of immunogold for amplified optical detection of aflatoxin B1.

A simple, sensitive and cost-effective method for the analysis of the mycotoxin aflatoxin B1 (AFB1) has been established based on controlled growth of immunogold. AFB1-BSA conjugate modified magnetic beads were employed as capture probe and anti-AFB1 antibody-coated gold colloids were used as detection probe for the immunological recognition of AFB1, as well as for signal transduction. The immune recognition event is converted into the gold enlargement signal which can be quantitatively measured by UV-vis spectroscopy. The autocatalytic enlargement of immunogold was conducted in aqueous solution containing chloroauric acid, hexadecyltrimethylammonium bromide and ascorbic acid. The reaction could be stopped by the addition of sodium thiosulfate. The final absorbance and resonance light scattering intensity were highly dependent on immunogold concentration. After gold enhancement, the sensitivity of the immunoassay was improved and total assay time reduced to 1 h. Under optimized conditions, the linear range and lower detection limit was 0.01-1 ng mL(-1) and 7 pg mL(-1), respectively. The proposed method offers great promise for sensitive detection of other mycotoxins and organic pollutants.

Silica-coated liposomes loaded with quantum dots as labels for multiplex fluorescent immunoassay

This manuscript describes synthesis and followed application of silica-coated liposomes loaded with quantum dots as a perspective label for immunoaasay. The hollow spherical structure of liposomes makes them an attractive package material for encapsulation of multiple water-insoluble quantum dots and amplifying the analytical signal. Silica coverage ensures the stability of the loaded liposomes against fusion and internal leakage during storage, transporting, application and also provides groups for bioconugation. For the first time these nanostructures were employed for the sensitive multiplex immunochemical determination of two analytes. As a model system mycotoxins zearalenone and aflatoxin B1 were detected in cereals. For simplification of multiassay results’ evaluation the silanized liposomed loaded with QDs of different colors were used. The IC50 values for the simultaneous determination of zearalenone and aflatoxin B1 were 16.2 and 18µgkg−1 for zearalenone and 2.2 and 2.6µgkg−1 for aflatoxin B1 in wheat and maize, respectively. As confirmatory method, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used.

Cytotoxic assessment of the regulated, co-existing mycotoxins aflatoxin B1, fumonisin B1 and ochratoxin, in single, binary and tertiary mixtures

Aflatoxin B1 (AFB1), ochratoxin A (OTA) and fumonisin B1 (FB1) are contaminants which have been shown to regularly co-occur in a range of foods. However, only a small number of studies have evaluated the interactive effect of binary and tertiary mycotoxins. The present study evaluated the effects of low levels of each mycotoxin in combination at their EU regulatory limits. Toxic effect with respect to cell viability was measured by MTT and neutral red assays, assessing mitochondria and lysosome integrities respectively. Individual toxicity showed that OTA (10 μg/ml) was the most cytotoxic mycotoxin in all three cell lines studied (caco-2, MDBK and raw 264.7). Binary combinations were cytotoxic to the MDBK cell line in the order [OTA/FB1] > [AFB1/FB1] > [AFB1/OTA], whilst all effects observed were classified as being additive. Tertiary combinations of AFB1, FB1 and OTA at the EU regulatory limits were tested and not found to exhibit measurable cytotoxicity in MDBK, caco-2 or raw 264.7 cells. However by increasing these concentrations above the legal limits to OTA (3 μg/ml), FB1 (8 μg/ml) and AFB1 (1.28 μg/ml), cytotoxicity was observed with up to 26% reduction in cell viability and synergistic effects were evident with regard to mitochondrial integrity.

Hydrophilic, bright CuInS2 quantum dots as Cd-free fluorescent labels in quantitative immunoassay.

We report on the synthesis of core-shell CuInS2/ZnS quantum dots (QDs) in organic solution, their encapsulation with a PEG-containing amphiphilic polymer, and the application of the resulting water-soluble QDs as fluorescent label in quantitative immunoassay. By optimizing the methods for core synthesis and shell growth, CuInS2/ZnS QDs were obtained with a quantum yield of 50% on average after hydrophilization. After conjugation with an aflatoxin B1-protein derivative, the obtained QDs were used as fluorescent labels in microplate immunoassay for the quantitative determination of the mycotoxin aflatoxin B1. QDs-based immunoassay showed higher sensitivity compared to enzyme-based immunoassay.

In vivo treatment with aflatoxin B1 increases DNA oxidation, base excision repair activity and 8-oxoguanine DNA glycosylase 1 levels in mouse lung

Carcinogenicity of the mycotoxin aflatoxin B1 (AFB1), which is produced by Aspergillus fungi, is associated with bioactivation of AFB1 to AFB1-8,9-exo-epoxide and formation of DNA adducts. However, AFB1 also causes 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation in mouse lung DNA, suggesting that oxidative DNA damage may also contribute to AFB1 carcinogenicity. The oxidative DNA damage 5-hydroxy-2′-deoxycytidine (5-OHdC) may also contribute to AFB1 carcinogenicity. The objective of the present study was to determine the effect of treatment of mice with AFB1 on pulmonary and hepatic: 8-OHdG and 5-OHdC levels; base excision repair (BER, which repairs oxidative DNA damage) activities; and on levels of 8-oxoguanine DNA glycosylase (OGG1, the rate-limiting enzyme in the BER of 8-OHdG). Female A/J mice were treated with vehicle (dimethyl sulfoxide) or 50mg/kg AFB1 ip. Oxidative DNA damage was measured using HPLC with electrochemical detection, BER activity was assessed using an in vitro assay that employs a substrate plasmid DNA with 8-OHdG lesions, and OGG1 protein levels were determined by immunoblotting. Two hours post treatment, AFB1 increased 8-OHdG levels in mouse lung DNA by approximately 69% relative to control (p<0.05), but did not alter 8-OHdG levels in liver or 5-OHdC levels in lung or liver (p>0.05). AFB1 treatment also increased BER activity in mouse lung by approximately 87% (p<0.05) but did not affect hepatic BER activity (p>0.05). Levels of OGG1 immunoreactive protein were increased in both lung (20%) and liver (60%) (p<0.05). These results are consistent with oxidative DNA damage contributing to the carcinogenicity of AFB1 in this model.

Natural mycobiota and aflatoxin B1 presence in bee pollen collected in Serbia

Total fungal count, incidence of fungi and aflatoxin B1 (AFB1) concentration were studied in 33 samples of bee pollen randomly collected from beekeepers in Serbia. The total number of fungi was determined by dilution method whereas AFB1 was detected using the Enzyme-Linked Immuno-Sorbent Assay (ELISA). The mycological estimation showed the presence of nine genera of fungi as followed: Acremonium, Alternaria, Aspergillus, Cladosporium, Epiccocum, Fusarium, Mucor, Penicillium and Rhizopus, with total number ranging from 1 x 103 to 1 x 105 CFU g-1. The results have shown the predominance of the fungi from the genera Aspergillus and Alternaria. Among Aspergillus species it was observed that the most frequent species was A. flavus with incidence of 27.27 %. Mycotoxin AFB1 was detected as 100% positive in all samples (100%) with an average concentration of 8.61 ?g kg-1. The obtained results indicated that honey bee pollen must be strictly controlled during its manipulation in the harvesting and manufacturing. Therefore, the implementation of good manufacturing (beekeeping) practice to define procedures for honeybee products could be crucial to reduce the risk of possible contamination and provide natural and safety product without risk on the human health.

Heterologous expression and functional characterization of avian mu-class glutathione S-transferases

Hepatic glutathione S-transferases (GSTs: EC2.5.1.1.8) catalyze the detoxification of reactive electrophilic compounds, many of which are toxic and carcinogenic intermediates, via conjugation with the endogenous tripeptide glutathione (GSH). Glutathione S-transferase (GST)-mediated detoxification is a critical determinant of species susceptibility to the toxic and carcinogenic mycotoxin aflatoxin B1 (AFB1), which in resistant animals efficiently detoxifies the toxic intermediate produced by hepatic cytochrome P450 bioactivation, the exo-AFB1-8,9-epoxide (AFBO). Domestic turkeys (Meleagris gallopavo) are one of the most sensitive animals known to AFB1, a condition associated with a deficiency of hepatic GST-mediated detoxification of AFBO. We have recently shown that unlike their domestic counterparts, wild turkeys (Meleagris gallopavo silvestris), which are relatively resistant, express hepatic GST-mediated detoxification activity toward AFBO. Because of the importance of GSTs in species susceptibility, and to explore possible GST classes involved in AFB1 detoxification, we amplified, cloned, expressed and functionally characterized the hepatic mu-class GSTs tGSTM3 (GenBank accession no. JF340152), tGSTM4 (JF340153) from domestic turkeys, and a GSTM4 variant (ewGSTM4, JF340154) from Eastern wild turkeys. Predicted molecular masses of tGSTM3 and two tGSTM4 variants were 25.6 and 25.8kDa, respectively. Multiple sequence comparisons revealed four GSTM motifs and the mu-loop in both proteins. tGSTM4 has 89% amino acid sequence identity to chicken GSTM2, while tGSTM3 has 73% sequence identity to human GSTM3 (hGSTM3). Specific activities of Escherichia coli-expressed tGSTM3 toward 1-chloro-2,4-dinitrobenzene (CDNB) and peroxidase activity toward cumene hydroperoxide were five-fold greater than tGSTM4 while tGSTM4 possessed more than three-fold greater activity toward 1,2-dichloro-4-nitrobenzene (DCNB). The two enzymes displayed equal activity toward ethacrynic acid (ECA). However, none of the GSTM proteins had AFBO detoxification capability, in contrast to recombinant alpha-class GSTs shown in our recent study to possess this important activity. In total, our data indicate that although turkey hepatic GSTMs may contribute to xenobiotic detoxification, they probably play no role in detoxification of AFBO in the liver.

Alpha-Class Glutathione S-Transferases in Wild Turkeys (Meleagris gallopavo): Characterization and Role in Resistance to the Carcinogenic Mycotoxin Aflatoxin B1

Domestic turkeys (Meleagris gallopavo) are one of the most susceptible animals known to the toxic effects of the mycotoxin aflatoxin B1 (AFB1), a potent human hepatocarcinogen, and universal maize contaminant. We have demonstrated that such susceptibility is associated with the inability of hepatic glutathione S-transferases (GSTs) to detoxify the reactive electrophilic metabolite exo-AFB1-8,9-epoxide (AFBO). Unlike their domestic counterparts, wild turkeys, which are relatively AFB1-resistant, possess hepatic GST-mediated AFBO conjugating activity. Here, we characterized the molecular and functional properties of hepatic alpha-class GSTs (GSTAs) from wild and domestic turkeys to shed light on the differences in resistance between these closely related strains. Six alpha-class GST genes (GSTA) amplified from wild turkeys (Eastern and Rio Grande subspecies), heritage breed turkeys (Royal Palm) and modern domestic (Nicholas strain) turkeys were sequenced, and catalytic activities of heterologously-expressed recombinant enzymes determined. Alpha-class identity was affirmed by conserved GST domains and four signature motifs. All GSTAs contained single nucleotide polymorphisms (SNPs) in their coding regions: GSTA1.1 (5 SNPs), GSTA1.2 (7), GSTA1.3 (3), GSTA2 (3), GSTA3 (1) and GSTA4 (2). E. coli-expressed GSTAs possessed varying activities toward GST substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA), cumene hydroperoxide (CHP). As predicted by their relative resistance, livers from domestic turkeys lacked detectable GST-mediated AFBO detoxification activity, whereas those from wild and heritage birds possessed this critical activity, suggesting that intensive breeding and selection resulted in loss of AFB1-protective alleles during domestication. Our observation that recombinant tGSTAs detoxify AFBO, whereas their hepatic forms do not, implies that the hepatic forms of these enzymes are down-regulated, silenced, or otherwise modified by one or more mechanisms. These data may inform of possible molecular mechanisms of resistance to AFB1, and may also have the benefit of identifying genetic markers which could be used to enhance AFB1 resistance in modern domestic strains.

Molds and mycotoxins in freshly harvested maize

Incidence of toxigenic fungi (molds) and concentration of mycotoxin aflatoxin B1 (AFB1), deoxynivalenol (DON), zearalenone (ZON) and fumonisin (FB1) were studied in the maize grains collected immediately after harvesting in 2012. A total of 29 maize samples were analyzed and the highest incidence was determined for fungal species of Rhizopus (56.41%), Aspergillus (43.66%) and Fusarium (14.97%) genera. Significantly lower incidence was obtained for species of genus Penicillium (3.31%), and especially for species of genera Acremonium (1.38%), Alternaria (0.75%) and Cladosporium (0.14%). Among toxigenic fungi Aspergillus flavus (36.69%) was the most common species of As?pergillus genus, whereas the Fusarium verticillioides with 14.69% of incidence was the predominant species of Fusarium genus. In all studied maize samples, the presence of AFB1, ZON and FB1 mycotoxins was established, except for DON which was established in 75.86% samples. AFB1 was detected in average concentration of 13.95 ?g kg-1 for 44.83% of samples, and average concentration higher than 40 ?g kg-1 for 55.17% of samples. The average concentrations of DON which was detected was 235 ?g kg-1, while it was 98.38 ?g kg-1 and 3590 ?g kg-1 for the presence of ZON and FB1, respectively. Moderate positive correla?tion was obtained between concentrations of AFB1 and FB1 (r=0.35), while weak positive correlation was established between concentrations ZON and DON (r=0.02).

Aminoacylase 1-catalysed deacetylation of bioactives epoxides mycotoxin-derived mercapturates; 3,4-epoxyprecocenes as models of cytotoxic epoxides

The mycotoxin aflatoxin B1 (AFB1) is a carcinogenic food contaminant which is metabolically activated by epoxydation. The metabolism of mycotoxins via the mercapturate metabolic pathway was shown, in general, to lead to their detoxication. Mercapturic acids thus formed (S-substitued-N-acetyl-l-cysteines) may be accumulated in the kidney and either excreted in the urine or desacetylated by Acylase 1 (ACY1) to yield cysteine S-conjugates. To be toxic, the N-acetyl-l-cysteine-S-conjugates first have to undergo deacetylation by ACY 1. The specificity and rate of mercapturic acid deacetylation may determine the toxicity, however the exact deacetylation processes involved are not well known. The aim of this study was to investigate the role of ACY1 in the toxicity of some bioactive epoxides from Aflatoxin B1. We characterized the kinetic parameters of porcine kidney and human recombinant aminoacylase-1 towards some aromatic and aliphatic-derived mercapturates analogue of mycotoxin-mercapturic acids and 3,4-epoxyprecocene, a bioactive epoxide derivated from aflatoxin. The deacetylation of mercapturated substrates was followed both by reverse phase HPLC and by TNBS method. Catalytic activity was discussed in a structure-function relationship. Ours results indicate for the first time that aminoacylase-1 could play an important role in deacetylating mercapturate metabolites of aflatoxin analogues and this process may be in relation with their cyto- and nephrotoxicity in human.

Distribution of moulds and mycotoxins in maize grain silage in the trench silo

In this study the mycological and mycotoxicological contamination of maize silage samples collected from the bottom (BL), middle (ML) and top (TL) layer in the silage trench silo was studied. Mycological testing of the silage contamination showed the presence of five potential toxigenic fungi of the genera, Aspergillus, Fusarium, Mucor, Penicillium and Rhizopus with their total number ranging from 1 to 10 x 103 CFU g-1. Also, four mycotoxins were detected as followed, aflatoxin B1 (AFB1), zearalenone (ZON), deoxynivalenol (DON) and fumonisin B1 (FB1). Mycotoxins AFB1 and FB1 were detected as 100% positive in all the samples tested. An average concentration of AFB1 was 2.31 ?g kg-1, 2.51 ?g kg-1 and 2.81 ?g kg-1 in BL, ML and TL respectively, while the concentration of FB1 was 1292.00 ?g kg-1, 1298.00 ?g kg-1 and 1902.00 ?g kg-1 in BL, TL and ML respectively. Average concentration of toxin ZON in positive samples was 15.69 ?g kg-1 in BL and 30.68 ?g kg-1 in ML, while its presence in the samples taken from the top layer was not detected. An average concentration of DON toxin in positive samples were in the range of 41.71 ?g kg-1 for TL, 79.20 ?g kg-1 for ML, while it was 97.00 ?g kg-1 for BL. There was no statistically significant correlation between moisture content and total number of moulds and concentrations of certain mycotoxins. However, statistically highly significant correlations between the total number of moulds and AFB1 concentration (r = 1.0), ZON (r = 0.99) and FB1 (r = 0.91) were established, while weak correlation was obtained in the case of DON and total mould counts (r = 0.54).

Análises quali e quantitativas de micotoxinas em águas da cadeia produtiva do arroz por CCD e CCDAE

This study validated a simple and applied method for determining mycotoxins aflatoxin B1, aflatoxin B2, ochratoxin A, zearalenone and deoxynivalenol, in water from the rice production chain. Five solvent combinations for extraction were tested, with quantification performed by TLC/HPTLC and confirmation by LC-MS/MS. Mycotoxins in water from field and rice industries were evaluated. Mycotoxin recovery levels were around 90%. Two samples from rice parboiling waste were contaminated (deoxynivalenol/aflatoxin B1, 110/9 ng mL-1; and deoxynivalenol, 100 ng mL-1). Zearalenone, deoxynivalenol and ochratoxin A (36, 30 and 28%) were carried to soaking water during parboiling.

Functional Characterization of Alpha-Class Glutathione S-Transferases from the Turkey (Meleagris Gallopavo)

Six Alpha-class glutathione S-transferase (GST) subunits were cloned from domestic turkey livers, which are one of the most susceptible animals known to the carcinogenic mycotoxin aflatoxin B₁. In most animals, GST dysfunction is a risk factor for susceptibility toward AFB₁, and we have shown that turkeys lack GSTs with affinity toward the carcinogenic intermediate exo-aflatoxin B(1)-8-9-epoxide (AFBO). Conversely, mice are resistant to AFB₁ carcinogenesis, due to high constitutive expression of mGSTA3 that has high affinity toward AFBO. When expressed in Escherichia coli, all six tGSTA subunits possessed conjugating activities toward substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA), and cumene hydroperoxide (CHP) with tGSTA1.2 appearing most active. Interestingly, tGSTA1.1, which lacks one of the four Alpha-class signature motifs, possessed enzymatic activities toward all substrates. All had comparable activities toward AFBO conjugation, an activity absent in turkey liver cytosols. E. coli-expressed mGSTA3 conjugated AFBO with more than 3-fold greater activity than that of tGSTAs and had higher activity toward GST prototype substrates. Mouse hepatic cytosols had approximately 900-fold higher catalytic activity toward AFBO compared with those from turkey. There was no apparent amino acid profile in tGSTAs that might correspond to specificity toward AFBO, although tGSTA1.2, which had slightly higher AFBO-trapping ability, shared Tyr¹⁰⁸ with mGSTA3, a residue postulated to be critical for AFBO trapping activity in mammalian systems. The observation that recombinant tGSTAs detoxify AFBO, whereas their hepatic forms do not, implies that the hepatic forms of these enzymes are silenced by one or more regulatory mechanisms.

TP53 Mutations and HBX Status Analysis in Hepatocellular Carcinomas from Iran: Evidence for Lack of Association between HBV Genotype D and TP53 R249S Mutations

High incidence of HCC is mostly due to the combination of two major risk factors, chronic infection with hepatitis B (HBV) and/or C (HCV) viruses and exposure to the mycotoxin aflatoxin B1, which induces a particular mutation at codon 249 in TP53 (R249S). Eight genotypes of HBV are diversely found in high and low incidence areas. Regardless of documented strong associations between TP53 R249S mutation and HBV genotypes B, C, A or E, there is no report of such association for genotype D despite of the presence of aflatoxin in areas with high prevalence of HBV genotype D. In Iran, 3% of the population is chronically infected with HBV, predominantly genotype D. Twenty-one histologically confirmed HCC cases from Iran were analyzed for TP53 R249S and HBV double mutations 1762T/1764A, hallmarks of more pathogenic forms of HBV. We did not detect any of these mutations. In addition, we report the only case identified so far carrying both R249S mutation and chronic HBV genotype D, a patient from The Gambia in West Africa. This paper suggests that association between HBV genotype D and aflatoxin-induced TP53 mutation is uncommon, explaining the relatively lower incidence of HCC in areas where genotype D is highly prevalent.

Ochratoxin A reduces aflatoxin B1 induced DNA damage detected by the comet assay in Hep G2 cells

Mycotoxins aflatoxin B1 (AFB1) and ochratoxin A (OTA) can be present together in food commodities. These food contaminants are considered to be genotoxins, acting by different mechanisms. The aim of this work was to characterize combined genotoxic in vitro effects of both mycotoxins in Hep G2 cells. For this purpose, cytotoxicity was first determined in isolated and combined treatments in order to determine the dose range of genotoxicity studies. Co-exposure of cells to OTA+AFB1 for 24h resulted in additive effects. Genotoxicity was determined in Hep G2 cells by the modified comet assay with restriction enzymes (endo III and FPG). Significant reactive oxygen species formation was detected in both single and combined treatments. AFB1 was genotoxic after 3h with external metabolic activation (S9 mix) and after 24h without metabolic activation. Co-exposure to OTA significantly decreased DNA damage induced by AFB1, not only in breaks and apurinic sites but also in FPG-sensitive sites. The apparent contradiction between additive cytotoxic effects and antagonic genotoxic effects may be explained if AFB1 and OTA compete for the same CYPs, yielding more ROS but less AFB1 adducts.

Microbiological and safety evaluation of green table olives marketed in Italy

The microbiological and safety conditions of green table olives sold on the Italian market were evaluated on 40 samples, 20 loose and 20 packed in containers, purchased at street markets and supermarkets. The olives were analyzed for microflora and food safety indices, and for aflatoxin B1 and ochratoxin A occurrence, and the results showed acceptable security. There was wide heterogeneity in the microflora, the numerical values being in relation to the olive type. The microbial population was dominated by the yeasts and lactic bacteria responsible for the fermentation process, and their numbers, together with the metabolic activity, led to conditions unfavorable for the development of pathogenic microflora; hence, such microflora were always absent. However, fecal coliforms were occasionally found in cracked olives. Lactic Acid Bacteria and yeast classification was performed at the species level by molecular techniques. The mycotoxin aflatoxin B1 was detectable in 25% of the olive samples, in the range of 0.40–0.55 μg/kg, while ochratoxin A, present in 58% of the samples, was in the range of 0.20–3.90 μg/kg.

Development of the custom polymeric materials specific for aflatoxin B1 and ochratoxin A for application with the ToxiQuant T1 sensor tool

Two polymers were computationally designed with affinity to two of the most abundant mycotoxins aflatoxin B1 (AFB1) and ochratoxin A (OTA) for application in the ToxiQuant T1 System. The principle of quantification of AFB1 and OTA using the ToxiQuant T1 instrument comprised of a fluorimetric analysis of mycotoxins adsorbed on the polymer upon exposure to UV light. High affinity of the developed resins allowed the adsorption of both toxins as discrete bands on the top of the cartridge with detection limit as low as 1 ng quantity of mycotoxins.