Simultaneous detection of AFB1 and aflD gene by "Y" shaped aptamer fluorescent biosensor based on double quantum dots.

The developed method for simultaneous detection of aflatoxin B1 (AFB1) and aflD genes can effectively monitor from the source and reduce the safety problems and economic losses caused by the production of aflatoxin, which can be of great significance for food safety regulations. In this paper, we constructed a sensitive and convenient fluorescent biosensor to detect AFB1 and aflD genes simultaneously based on fluorescence resonance energy transfer (FRET) between quantum dots (QDs) and a black hole quenching agent. A stable "Y" shaped aptasensor was employed as the detection platform and a double quantum dot labeled DNA fragment was utilized to be the sensing element in this work. When the targets of AFB1 and aflD genes were presented in the solution, the aptamer in the "Y" shaped probe is specifically recognized by the target. At this time, both Si-carbon quantum dots (Si-CDs) and CdTe QDs are far away from the BHQ1 and BHQ3 to recover the fluorescence. The linear range of the prepared fluorescence simultaneous detection method was as wide as 0.5-500 ng·mL-1 with detection lines of 0.64 ng·mL-1 for AFB1 and 0.5-500 nM with detection lines of 0.75 nM for aflD genes (3σ/k). This fabricated fluorescent biosensor was further validated in real rice flour and corn flour samples, which also achieved good results. The recoveries were calculated by comparing the known and found amounts of AFB1 which ranged from 88.4 to approximately 115.32% in the rice flour samples and 90.7 ~ 102.58% in the corn flour samples. The recoveries of aflD genes ranged from 84.32 to approximately 109.3% in the rice flour samples and 89.48 ~ 100.99% in the corn flour samples. Therefore, the proposed biosensor can significantly improve food safety and quality control through a simple, fast, and sensitive agricultural product monitoring and detection system.

In silico studies and development of a protein-based electrochemical sensor for selective and sensitive detection of aflatoxin B1.

Proteins from different species have been docked with aflatoxin B1 (AFB1) and identified 3 proteins (prostaglandin-E(2)9-reductase from Oryctolagus uniculus, proto-oncogene serine/threonine-protein kinase Pim-1 and human immunoglobulin G (hIgG)) as potential candidates to develop an electrochemical sensor. Fluorescence spectroscopy experiments have confirmed the interaction of hIgG with AFB1 with an affinity constant of 4.6 × 105 M-1. As a proof-of-concept, hIgG was immobilized on carbon nanocomposite (carbon nanotube-nanofiber, CNT-F)-coated glassy carbon electrode (GCE). FT-IR spectra, HR-TEM and BCA assay have confirmed successful immobilization of hIgG on the electrode (hIgG@CNT-F/GCE). The preparation of this protein electrochemical sensor requires only 1 h 36 min, which is fast as compared with preparing an electro immunosensor. hIgG@CNT-F/GCE has displayed an excellent AFB1 limit of detection (0.1 ng/mL), commendable selectivity in the presence of two other mycotoxins (ochratoxin A and patulin) and the detection of  AFB1 in spiked peanuts and corn samples.

A Highly Sensitive and Group-Specific Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of AFB1 in Agriculture and Aquiculture Products.

Aflatoxins (AFs) including AFB1, AFB2, AFG1 and AFG2 are widely found in agriculture products, and AFB1 is considered one of the most toxic and harmful mycotoxins. Herein, a highly sensitive (at the pg mL-1 level) and group-specific enzyme-linked immunosorbent assay (ELISA) for the detection of AFB1 in agricultural and aquiculture products was developed. The AFB1 derivative containing a carboxylic group was synthesized and covalently linked to bovine serum albumin (BSA). The AFB1-BSA conjugate was used as an immunogen to immunize mice. A high-quality monoclonal antibody (mAb) against AFB1 was produced by hybridoma technology, and the mAb-based ELISA for AFB1 was established. IC50 and limit of detection (LOD) of the ELISA for AFB1 were 90 pg mL-1 and 18 pg mL-1, respectively. The cross-reactivities (CRs) of the assay with AFB2, AFG1, and AFG2 were 23.6%, 42.5%, and 1.9%, respectively, revealing some degree of group specificity. Corn flour, wheat flour, and crab roe samples spiked with different contents of AFB1 were subjected to ELISA procedures. The recoveries and relative standard deviation (RSD) of the ELISA for AFB1 in spiked samples were 78.3-116.6% and 1.49-13.21% (n = 3), respectively. Wheat flour samples spiked with the mixed AF (AFB1, AFB2, AFG1, AFG2) standard solution were measured by ELISA and LC-MS/MS simultaneously. It was demonstrated that the proposed ELISA can be used as a screening method for evaluation of AFs (AFB1, AFB2, AFG1, AFG2) in wheat flour samples.

Proteomic analysis of ITPR2 as a new therapeutic target for curcumin protection against AFB1-induced pyroptosis.

Aflatoxin B1 (AFB1) is extremely carcinogenic and can cause liver cancer in humans and animals with continued ingestion. As a natural compound, curcumin (Cur) exhibits excellent anti-inflammatory, and anti-cancer properties with few side effects. In this study, a total of 60 male mice (6-week-olds, 15 per group). After one week of acclimatization feeding, the mice were divided into control group (Con), AFB1 group, curcumin group (Cur), and AF+Cur group. The mice were gavaged with curcumin (Cur, 100 mg/kg) and/or AFB1 (0.75 mg/kg). To identify a new therapeutic target for AFB1-induced pyroptosis, we performed proteomic profiling for curcumin alleviating liver injury caused by AFB1 to further validate the targets through volcano plot analysis, Venn analysis, heatmap analysis, correlation, cluster analysis, GO and KEGG enrichment. AFB1 exposure resulted in the loss of hepatocyte membrane, swelling of the endoplasmic reticulum, and a significant increase in transaminase (ALT and AST) contents, while curcumin greatly improved these changes. We found that differentially expressed proteins are enriched in the endoplasmic reticulum membrane and identified ITPR2 as a target of curcumin that alleviates AFB1-induced liver injury by proteomics. Furthermore, ITPR2 expression was detected by immunofluorescence, and qRT-PCR for mRNA expression of genes downstream of ITPR2 (calpain1, calpain2, caspase-12, caspase-3). ITPR2-activated endoplasmic reticulum stress-related proteins (calpain1, calpaini2, bcl-2, BAX, cl-caspase-12, cl-caspase-3), apoptosis (PARP) and pyroptosis (DFNA5) related proteins were examined by western blotting. The analysis showed that it effectively prevents AFB1-induced pyroptosis by lowering endoplasmic reticulum stress via interfering with ITPR2 and its downstream proteins (calpain1, calpain2, bcl-2, Bax) and inhibiting caspase-12/caspase-3 pathway. Conclusively, this study applied proteomic profiling to elucidate ITPR2 as a new target, which might give a new perspective on the mechanism of curcumin alleviating AFB1-induced pyroptosis.

Enhanced autophagy reversed aflatoxin B1-induced decrease in lactate secretion of dairy goat Sertoli cells.

The deleterious effects of aflatoxins, especially aflatoxin B1 (AFB1) which are widespread at all stages of food production, on the reproductive system have been widely reported in males. However, it is still far from fully understood about the toxic effect and molecular mechanism after exposure to AFB1 in various testicular cells, especially Sertoli cells (SCs) which provide various energy materials and support to the developing germ cells as nurse cells. In this work, we examined the effects of AFB1 in dairy goat SCs on lactate production and autophagy, and the role of autophagy on AFB1-induced reduction in lactate production. Mechanistically, AFB1 destroyed the energy balance and reduced the secretion of lactate in dairy goat SCs (P < 0.01), resulting in a reduced level of ATP (P < 0.01) and phosphorylation of AMPK (P < 0.01). Subsequently, activated AMPK triggers autophagy by directly phosphorylating ULK1 (P < 0.05). The enhancement of autophagy partially reversed the AFB1-induced decrease in lactate secretion by promoting glucose utilization (P < 0.01) and increasing the expression of proteins related to lactate secretion in dairy goat SCs (P < 0.05) such as GLUT1, GLUT3, LDHA, and MCT4. Collectively, our study suggests that AFB1 inhibits the secretion of lactate which supply for germ cell development by damaging the "Warburg-like" metabolism of dairy goat SCs. Moreover, autophagy contributes to the resistance of glucose metabolism damage induced by AFB1. DATA AVAILABILITY: All data generated or analyzed in this study are available from the corresponding authors upon request.

Humic acids alleviate aflatoxin B1-induced hepatic injury by reprogramming gut microbiota and absorbing toxin.

Aflatoxin B1 (AFB1) is a hepatotoxic fungal metabolite that is widely present in food and can cause liver cancer. As a potential detoxifier, naturally occurring humic acids (HAs) may be able to reduce inflammation and restructure the gut microbiota composition; however, little is known about the mechanism of HAs detoxification as applied to liver cells. In this study, HAs treatment alleviated AFB1-induced liver cell swelling and the infiltration of inflammatory cells. HAs treatment also reinstated various enzyme levels in the liver disturbed by AFB1 and substantially alleviated AFB1-caused oxidative stress and inflammatory responses by enhancing immune functions in mice. Moreover, HAs increased the length of the small intestinal and villus height to restore intestinal permeability, which is impaired by AFB1. In addition, HAs reconstructed the gut microbiota, increasing the relative abundance of Desulfovibrio, Odoribacter, and Alistipes. In vitro and in vivo assays demonstrated that HAs could efficiently remove AFB1 by absorbing the toxin. Therefore, HAs treatment can ameliorate AFB1-induced hepatic injury by enhancing gut barrier function, regulating gut microbiota, and adsorbing toxin.

Hepatoprotective effects of a chemically-characterized extract from artichoke (Cynara scolymus L.) against AFB1-induced toxicity in rats.

This study was conducted to investigate the protective potential of a pharmaceutically formulated capsule of artichoke leaf powder (ArLP) against aflatoxin B1 (AFB1)-induced hepatotoxicity in male albino rats. In the 42-day experiment, rats were divided into five equal groups: (i) control, treated with sterile water, (ii) treated with 4% DMSO as AFB1 vehicle, (iii) ArLP of 100 mg kg-1 bw, (iv) AFB1 of 72 µg kg-1 bw, and (v) AFB1 plus ArLP. Exposure of rats to AFB1 resulted in hepatotoxicity as manifested by the intensification of oxidative stress, production of free radicals and significant increase in the activity levels of liver function enzymes relative to the control. Significant reductions in both the enzymatic and non-enzymatic antioxidant markers as well as histopathological abnormalities in liver tissues were also observed. Notably, the combined administration of ArLP with AFB1 clearly reduced AFB1-mediated adverse effects leading to the normalization of most of these parameters back to control levels. These findings clearly highlight the potential benefits of artichoke dietary supplements as a safe and natural solution in counteracting the adverse hepatotoxic effects conferred by AFB1 exposure. Further research is warranted to fully dissect the biochemical and molecular mechanism of action of the observed artichoke-mediated hepatoprotection.

Design of Pyrrole-Based Gate-Controlled Molecular Junctions Optimized for Single-Molecule Aflatoxin B1 Detection.

Food contamination by aflatoxins is an urgent global issue due to its high level of toxicity and the difficulties in limiting the diffusion. Unfortunately, current detection techniques, which mainly use biosensing, prevent the pervasive monitoring of aflatoxins throughout the agri-food chain. In this work, we investigate, through ab initio atomistic calculations, a pyrrole-based Molecular Field Effect Transistor (MolFET) as a single-molecule sensor for the amperometric detection of aflatoxins. In particular, we theoretically explain the gate-tuned current modulation from a chemical-physical perspective, and we support our insights through simulations. In addition, this work demonstrates that, for the case under consideration, the use of a suitable gate voltage permits a considerable enhancement in the sensor performance. The gating effect raises the current modulation due to aflatoxin from 100% to more than 103÷104%. In particular, the current is diminished by two orders of magnitude from the µA range to the nA range due to the presence of aflatoxin B1. Our work motivates future research efforts in miniaturized FET electrical detection for future pervasive electrical measurement of aflatoxins.

Cloning and expression of the catalase gene (KatA) from Pseudomonas aeruginosa and the degradation of AFB1 by recombinant catalase.

BACKGROUND: Aflatoxin B1 (AFB1 ) poses a severe threat to human and animal health. Countries worldwide have invested considerable manpower and material resources in degrading aflatoxins. Enzyme degradation is the most efficient and environmentally friendly approach for modifying aflatoxin into less toxic molecules. Catalase is commonly used as a detoxification agent to decrease the contamination levels of aflatoxins in animal feeds. This study aimed to obtain recombinant catalase via gene engineering and determined whether a recombinant catalase could degrade AFB1 . RESULTS: The catalase gene (KatA) from Pseudomonas aeruginosa was cloned and expressed in Escherichia coli, and the expression conditions of this recombinant catalase were optimized. The recombinant catalase was isolated and purified using Ni-chelating affinity chromatography, and its ability to degrade AFB1 was evaluated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the expressed of catalase was approximately 55.6 kDa, which was subsequently purified using Ni-chelating affinity chromatography. The degradation rate of AFB1 by recombinant catalase in the presence of syringaldehyde was 38.79%. CONCLUSION: The degradation of AFB1 by a recombinant catalase has been reported for the first time. This study provides a new paradigm for the use of recombinant catalases in degrading AFB1 in food and feed. © 2022 Society of Chemical Industry.

A ratiometric fluorescent aptamer homogeneous biosensor based on hairpin structure aptamer for AFB1 detection.

On the basis of aptamer (Apt) with hairpin structure and fluorescence resonance energy transfer (FRET), a ratio fluorescent aptamer homogeneous sensor was prepared for the determination of Aflatoxin B1 (AFB1). Initially, the Apt labeled simultaneously with Cy5, BHQ2, and cDNA labeled with Cy3 were formed a double-stranded DNA through complementary base pairing. The fluorescence signal of Cy3 and Cy5 were restored and quenched respectively. Thus, the ratio change of FCy3 to FCy5 was used to realized the detection of AFB1 with wider detection range and lower limit of detection (LOD). The response of the optimized protocol for AFB1 detection was wider linear range from 0.05 ng/mL to 100 ng/mL and the LOD was 12.6 pg/mL. The sensor designed in this strategy has the advantages of simple preparation and fast signal response. It has been used for the detection of AFB1 in labeled corn and wine, and has good potential for application in real samples.

An efficient aggregation-induced electrochemiluminescent immunosensor by using TiO2 nanoparticles as coreaction accelerator and energy donor for aflatoxin B1 detection.

Herein, we fabricated a label-free ECL immunosensor for aflatoxin B1 (AFB1) detection. In this system, a small organic aggregation-induced electrochemiluminescence luminophore, 2,5-di-tetraphenylethylene-ylthiazolo [5,4-d] thiazole, was designed, named TPETTZ. Polyaniline-wrapped TiO2 nanoparticles (PANI/TiO2 NPs) complex was synthesized through one-step in situ oxidation polymerization of aniline, and performed excellent electrical conductivity and abundant amino groups. As an ECL accelerator, TiO2 nanoparticles (TiO2 NPs) promoted the oxidation of tri-n-propylamine (TPA) to generate more TPA•; in addition, it also acted as a donor to improve the ECL intensity of TPETTZ (acceptor) through electrochemiluminescence resonance energy transfer (ECL-RET). Encouraged by the above, under the existence of TPA, TPETTZ displayed a strong and continuously stable ECLanode signal due to the introduction of PANI/TiO2 NPs. Therefore, the immunosensor was constructed for AFB1 detection based on the quenching effect of target on the ECL signal, and a linearly decreasing ECL signal was obtained as the increasement of AFB1 in the range of 75 fg/mL to 100 ng/mL, with a lower detection limit of 27.5 fg/mL. Moreover, the as-prepared sensing platform performed a satisfactory anti-interference, stability, and reproducibility, and appeared a good accuracy in walnut sample analysis, presenting a promising application in the future.

Potential of montmorillonite modified by an organosulfur surfactant for reducing aflatoxin B1 toxicity and ruminal methanogenesis in vitro.

BACKGROUND: Montmorillonite clay modified by organosulfur surfactants possesses high cation exchange capacity (CEC) and adsorption capacity than their unmodified form (UM), therefore they may elevate the adverse impact of aflatoxin B1 (AFB1) on ruminal fermentation and methanogenesis. Chemical and mechanical modifications were used to innovate the organically modified nano montmorillonite (MNM). The UM was modified using sodium dodecyl sulfate (SDS) and grounded to obtain the nanoscale particle size form. The dose-response effects of the MNM supplementation to a basal diet contaminated or not with AFB1 (20 ppb) were evaluated in vitro using the gas production (GP) system. The following treatments were tested: control (basal diet without supplementations), UM diet [UM supplemented at 5000 mg /kg dry matter (DM)], and MNM diets at low (500 mg/ kg DM) and high doses (1000 mg/ kg DM). RESULTS: Results of the Fourier Transform Infra-Red Spectroscopy analysis showed shifts of bands of the OH-group occurred from lower frequencies to higher frequencies in MNM, also an extra band at the lower frequency range only appeared in MNM compared to UM. Increasing the dose of the MNM resulted in linear and quadratic decreasing effects (P < 0.05) on GP and pH values. Diets supplemented with the low dose of MNM either with or without AFB1 supplementation resulted in lower (P = 0.015) methane (CH4) production, ruminal pH (P = 0.002), and ammonia concentration (P = 0.002) compared to the control with AFB1. Neither the treatments nor the AFB1 addition affected the organic matter or natural detergent fiber degradability. Contamination of AFB1 reduced (P = 0.032) CH4 production, while increased (P < 0.05) the ruminal pH and ammonia concentrations. Quadratic increases (P = 0.012) in total short-chain fatty acids and propionate by MNM supplementations were observed. CONCLUSION: These results highlighted the positive effects of MNM on reducing the adverse effects of AFB1 contaminated diets with a recommended dose of 500 mg/ kg DM under the conditions of this study.

SeMet attenuates AFB1-induced intestinal injury in rabbits by activating the Nrf2 pathway.

The aim of this study was to investigate the role of selenomethionine (SeMet) in alleviating AFB1 induced intestinal injury by inhibiting intestinal oxidative stress. Forty 35-day-old rabbits were divided randomly into 4 groups (control group, AFB1 group, 0.2 mg/kg Se + AFB1 group, 0.4 mg/kg Se + AFB1 group). From the first day of the experiment, the two treatment groups were fed 0.2 mg/kg SeMet or 0.4 mg/kg SeMet daily for 21 days. On the 17th day, all rabbits in the model group and the two treatment groups were given intragastric AFB1 daily for 5 days. The ADG, ADFI and FCR of the rabbits were examined. Rabbit jejunum tissue was collected for hematoxylin- eosin staining (HE), PCNA detection, immunofluorescence and WB. Intestinal tissue IL-1ß, IL-6 and TNF-α were examined by enzyme-linked immunosorbent assay (ELISA). The results showed that the production performance was decreased, the levels of ROS and MDA were increased in intestinal tissues, the activity of antioxidant enzymes was decreased and the expression levels of Nrf2 and HO-1 were decreased in AFB1-exposed rabbits. In addition, AFB1 induces an inflammatory response in the jejunum and promotes the expression of TNF-α, IL-6 and IL-1ß. SeMet pretreatment significantly improved the performance of the rabbits, alleviated intestinal oxidative stress and the inflammatory response. Therefore, we confirmed that SeMet protects against AFB1 induced oxidative damage and improves productivity in rabbits by activating the Nrf2/HO-1 signaling pathway.

An electrochemical aptasensor based on target triggered multiple-channel DNAzymes cycling amplification strategy with PtFe@Co-MOF as signal amplifier.

A novel electrochemical aptasensor for the detection of Aflatoxin B1 (AFB1) was developed for the first time by using the target-triggered multiple-channel deoxyribozymes (DNAzymes) cycling amplified assay with Pt Fe doped NH2-Co-MOF (PtFe@Co-MOF) as a signal amplifier. In the presence of AFB1, a self-assembling cross-over nucleic structure could be triggered by AFB1 via two aptamers' structure switching for strand displacement, resulting in four channels of Mg2+-dependent DNAzyme recycling simultaneously to multiply the detection signals. These DNAzymes cyclically split the substrate sequence to release the PtFe@Co-MOF labeled detection probe (DP), which is subsequently hybridized with the capture probes on the Au-deposited glassy carbon electrode. The fabrication procedure was characterized by differential pulse voltammetry, and the results of the morphological and element composition characteristics methods were analyzed to determine the successful preparation of PtFe@Co-MOF. The limit of detection (LOD) for AFB1 detection was 2 pg mL-1 with a linear range from 5 pg mL-1 to 80 ng mL-1. By comparison, the enhanced detection sensitivity has been found to originate from the efficient shearing of DNAzymes, enhanced peroxidase-like capability, and multiple active sites of PtFe@Co-MOF. Besides, this aptasensor showed high specificity for AFB1 compared with similar mycotoxins and exhibited high accuracy with low experimental cost and easy operation. Furthermore, the unique design of electrochemical aptasensors could provide a promising platform for the onsite determination of AFB1, as well as other targets by replacing the aptamer and other core recognition sequences.

Fish feed mycobiota and aflatoxins in round fish tissues.

BACKGROUND: Round fish is one of the most consumed fish in Brazil. Farmed fish feed is based mainly on grains, which are susceptible to contamination by mold and mycotoxins. Aspergillus spp., Penicillium spp. and Fusarium spp. are the major mycotoxins producers. The presence of potentially toxigenic fungi in the diet is a concern due to the possibility of cumulative toxins in fish tissues, becoming a risk to food safety. This study aims to assess the mycobiota of fish feed and the occurrence of aflatoxin residues in round fish tissues. Feed and fish samples were collected from fish farming and fish pay properties. Feed was submitted to mold counting and mold identification. The round fish liver and muscle were submitted to the detection and quantification of aflatoxins B1 , B2 , G1 and G2 by high-performance liquid chromatography. RESULTS: In evaluated feed, mold counts in the samples ranged from 2.0 to 4.7 log colony forming units g-1 and the major genera found were Penicillium (61.5%) and Aspergillus (34.6). Aflatoxin B1 (AFB1 ) was detected in 70% liver samples and 43.3% muscle samples, at levels up to 5.70 and 1.13 µg kg-1 , respectively. CONCLUSION: It is concluded that, although the levels were lower than those recommended by Brazilian legislation, round fish are being exposed to diets naturally contaminated by aflatoxins and are susceptible to toxins accumulation in tissues. Therefore, regulations regarding feed should consider limits for mold and aflatoxin contamination in fish edible tissues should be monitored in order to ensure consumers' safety. © 2021 Society of Chemical Industry.

Lignin and organic free radicals in maize (Zea mays L.) seeds in response to aflatoxin B1 contamination: an optical and EPR spectroscopic study.

BACKGROUND: Aflatoxin B1 (AFB1 ) is the most dangerous of the mycotoxins that contaminate cereal seeds naturally. A stress lignin formation is linked with the accumulation of reactive oxygen species causing a change in the redox status and formation of stable organic radicals, constituting the first layer of defense. The relationship between AFB1 and changes in lignin organic free radicals in seeds is not known, nor is the part of the seed that is more targeted. Using optical and electron paramagnetic resonance spectroscopy, we investigated AFB1 -induced changes in lignin and organic free radicals in seeds, and whether the inner and outer seed fractions differ in response to increasing AFB1 . RESULTS: Different changes in the content of lignin and free radicals with increasing AFB1 concentrations were observed in the two seed fractions. There was a significant positive linear correlation (R = 0.9923, P = 0.00005) between lignin content and AFB1 concentration in the outer fraction, and no correlation between the lignin content and the AFB1 concentration in the inner fraction. We found a positive correlation between the area of the green spectral emission component (C4) and the AFB1 concentration in the outer fraction. CONCLUSIONS: To the best of our knowledge, the results showed, for the first time, that maize seed fractions respond differently to aflatoxin with regard to their lignin and organic free radical content. Lignin content and (C4) area may be reliable indicators for the screening of lignin changes against AFB1 content in the seeds, and thus for seed protection capacity. © 2021 Society of Chemical Industry.

Efficacy of the antifungal metabolites of Streptomyces philanthi RL-1-178 on aflatoxin degradation with its application to prevent aflatoxigenic fungi in stored maize grains and identification of the bioactive compound.

Aflatoxin B1 is a potent carcinogen produced by Aspergillus flavus (A. flavus) and Aspergillus. parasiticus (A. parasiticus), mainly during grain storage. The efficacy of the freeze-dried culture filtrate of Streptomyces philanthi (S. philanthi) strain RL-1-178 (DCF) on degradation of aflatoxin B1 (AFB1) were evaluated and its bioactive compounds were identified. The DCF at a concentration of 9.0% (w/v) completely inhibited growth and AFB1 production of A. parasiticus TISTR 3276 and A. flavus PSRDC-4 after 7 days tested in yeast-extract sucrose (YES) medium and on stored maize grains after 28 and 14 days incubation, respectively. This indicated the more tolerance of A. parasiticus over A. flavus. The DCF and bacterial cells of S. philanthi were capable to degrade AFB1 by 85.0% and 100% for 72 h and 8 days, respectively. This confirmed the higher efficacy of the DCF over the cells. After separation of the DCF on thin-layer chromatography (TLC) plate by bioautography bioassay, each active band was identified by liquid chromatography-quadrupole time of flight mass spectrometer (LC-Q-TOF MS/MS). The results revealed two compounds which were identified as azithromycin and an unknown based on mass ions of both ESI+ and ESI- modes. The antifungal metabolites in the culture filtrate of S. philanthi were proved to degrade aflatoxin B1. It could be concluded that the DCF may be applied to prevent the growth of the two aflatoxin-producing fungi as well as the occurrence of aflatoxin in the stored maize grains.

A novel colorimetric biosensor for sensitive detection of aflatoxin mediated by bacterial enzymatic reaction in saffron samples.

Aflatoxin is regarded as the potent carcinogenic agent which is secreted from fungi and present in some food products. So far, many detection methods have been developed to determine the trace amounts of aflatoxin in foods. In the present study a colorimetric competitive assay for detection of aflatoxin B1 (AFB1) has been developed based on interaction of gelatin functionalized gold nanoparticles (AuNPs@gelatin) in specific enzymatic reaction. Bacterial supernatant containing gelatinase enzyme were used as the substrate that could digest the coated gelatin on the surface of AuNPs and following in the presence of NaCl medium ingredient resulted to color change of AuNPs colloidal solution from red to purple. It was observed that with addition of aflatoxin to the bacterial supernatant, aflatoxin could interfere in aggregation of AuNPs and inhibited the process which subsequently prevent the expected color change induced by AuNPs aggregation. The supernatant containing AuNPs were investigated to analyze their induced surface plasmon resonance spectra through UV-visible spectroscopy. The absorption values were directly proportional with the applied AFB1 concentration. The experiment conditions including incubation time, AuNPs concentration and pH were investigated. The obtained results showed that through this approach we could detect the AFB1 in a linear range from 10 to 140 pg ml-1, with detection limit of 4 pg ml-1. Real sample assay in saffron samples showed recoveries percentage of 92.4%-95.3%. The applied approach proposed simple, cost effective and specific method for detection of AFB1 toxin in food samples.

Ferulic acid prevents aflatoxin B1-induced liver injury in rats via inhibiting cytochrome P450 enzyme, activating Nrf2/GST pathway and regulating mitochondrial pathway.

Aflatoxin B1 (AFB1) causes oxidative stress and hepatocyte apoptosis through its epoxidized metabolite AFBO, which is catalyzed by CYP450 enzymes. Ferulic acid (FA) is a phenolic acid commonly found in plants and is known for its antioxidant capacity. However, the role of FA in AFB1-induced liver injury is still elusive. In this study, rats were exposed to AFB1 and simultaneously treated with FA for 30 days. The results showed that I) FA alleviated the histopathological changes induced by AFB1, inhibited the elevation of serological indexes induced by AFB1, and reduced the production of AFBO in liver. II) AFB1-induced increase in CYP450 expression was significantly reduced by FA. The molecular docking results of FA and CYP2A6 showed high fitness score and interaction. III) FA obviously inhibited the production of MDA, and significantly activated the Nrf2/GST pathway and antioxidant enzymes (SOD and GST). IV) AFB1-induced hepatocyte apoptosis, the high expression of p53, bax, cyt-c, caspase-9, caspase-3, and the low expression of bcl-2 were all restored by FA. It has been suggested from these results that FA proved effective against AFB1-induced liver damage in rats via inhibiting CYP450 enzyme, promoting antioxidant pathway Nrf2/GST, activating antioxidant enzymes (SOD and GST), and regulating the mitochondrial pathway.

Toxicogenicity and mechanistic pathways of aflatoxin B1 induced renal injury.

The study investigated the toxicogenic effects, molecular mechanisms and proteomic assessment of aflatoxin B1 (AFB1 ) on human renal cells. Hek293 cells were exposed to AFB1 (0-100 µM) for 24 h. The effect on cell viability was assessed using the methylthiazol tetrazolium (MTT) assay, which also produced the half maximal inhibitory concentration (IC50 ) used in subsequent assays. Free radical production was evaluated by quantifying malondialdehyde (MDA) and nitrate concentration, while DNA fragmentation was determined using the single cell gel electrophoresis (SCGE) assay and DNA gel electrophoresis. Damage to cell membranes was ascertained using the lactate dehydrogenase (LDH) assay. The concentration of ATP, reduced glutathione (GSH), necrosis, annexin V and caspase activity was measured by luminometry. Western blotting and quantitative PCR was used to assess the expression of proteins and genes associated with apoptosis and oxidative stress. The MTT assay revealed a reduction in cell viability of Hek293 cells as the AFB1 concentration was increased, with a half maximum inhibitory concentration (IC50 ) of 32.60 µM. The decreased viability corresponded to decreased ATP concentration. The upregulation of Hsp70 indicated that oxidative stress was induced in the AFB1 -treated cells. While this implies an increased production of free radicals, the accompanying upregulation of the antioxidant system indicates the activation of defense mechanisms to prevent cellular damage. Thus, membrane damage associated with increased radical formation was prevented as indicated by the reduced LDH release and necrosis. In addition, cytotoxic effects were evident as AFB1 activated the intrinsic pathway of apoptosis with corresponding increased DNA fragmentation, p53 and Bax upregulation and increased caspase activity, but externalization of phosphatidylserine (PS), a major hallmark of apoptosis, did not occur in AFB1 treated renal cells. The results suggest that AFB1 induced oxidative stress leading to cell death by the intrinsic pathway of apoptosis in renal cells.