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 fluorescent aptamer sensor demonstrates a weak fluorescence emission of Cy3 and a high fluorescence emission of Cy5 due to the quenching effect of BHQ2. The double-stranded DNA structure will be disintegrated in the presence of AFB1, resulting the removal of Cy3 and the close of Cy5 with BHQ2. 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, indicating it had good application potential in practical samples.

In Vivo Kinetics and Biotransformation of Aflatoxin B1 in Dairy Cows Based on the Establishment of a Reliable UHPLC-MS/MS Method

The in vivo kinetics of aflatoxin B1 (AFB1) and its carry-over as aflatoxin M1 (AFM1) in milk as well as the toxin loads in the tissue of dairy cows were assessed through a repetitive feeding trial of an AFB1-contaminated diet of 4 μg kg−1 body weight (b.w.) for 13 days. This was followed by a clearance period that ended with a single dose trial of an AFB1-contaminated diet of 40 μg kg−1 b.w. An ultra-high performance liquid chromatography tandem mass spectrometry method was developed and successfully validated by the determination of linearity (R2 ≥ 0.990), sensitivity (lower limit of quantification, 0.1–0.2 ng ml−1), recovery (79.5–111.2%), and precision relative standard deviation (RSD) ≤14.7%) in plasma, milk, and various tissues. The repetitive ingestion of AFB1 indicated that the biotransformation of AFB1 to AFM1 occurred within 48 h, and the clearance period of AFM1 in milk was not more than 2 days. The carry-over rate of AFM1 in milk during the continuous ingestion experiment was in the range of 1.15–2.30% at a steady state. The in vivo kinetic results indicated that AFB1 reached a maximum concentration of 3.8 ± 0.9 ng ml−1 within 35.0 ± 10.2 min and was slowly eliminated from the plasma, with a half-life time (T1/2) of 931.1 ± 30.8 min. Meanwhile, AFM1 reached a plateau in plasma (0.5 ± 0.1 ng ml−1) at 4 h after the ingestion. AFB1 was found in the heart, spleen, lungs, and kidneys at concentrations of 1.6 ± 0.3, 4.1 ± 1.2, 3.3 ± 0.9 and 5.6 ± 1.4 μg kg−1, respectively. AFM1 was observed in the spleen and kidneys at concentrations of only 0.7 ± 0.2 and 0.8 ± 0.1 μg kg−1, respectively. In conclusion, the in vivo kinetics and biotransformation of AFB1 in dairy cows were determined using the developed UHPLC-MS/MS method, and the present findings could be helpful in assessing the health risks to consumers.

Opposite Direction for Seasonal Variation of Aflatoxin M1 in Bulk Milk and Aflatoxin B1 in Rations: Results From a Prospective Study in Selected Dairy Farms of Qazvin Province, Iran

The presence of aflatoxin M1 (AFM1) in 24h bulk milk and aflatoxin B1 (AFB1) in concurrent total mixed rations (TMR) and feed ingredients were assessed in 12 large dairy operations. The bulk milk was sampled on days 1, 15 and 30 during winter and summer (n=72). Total mixed rations (n=48) and feed ingredients (n=230) were sampled two times with a 30-day interval. Aflatoxin was measured using direct competitive ELISA kits with detection limits of 1-81 ngkg−1 for milk and 1.25-101.25 ngkg−1 for feeds. Aflatoxin M1 was identified in all milk samples (100%), ranging from 2.03 to >81 ngkg−1, with a median of 70 ngkg−1 and averaging 61.25±28.91 ngkg−1 in winter and 54.20±25.51 ngkg−1 in summer (P=0.279). Contaminations <81 ngkg−1 (below the Iranian standard of 100 ngkg-1) were detected in 76% (n=55/72) of samples. Contaminations >81 ngkg−1 were detected in 24% (n=17/72) of samples and were more frequent in winter than in summer (42% vs. 6%). Sixty-nine percent of the winter milk samples (n=25/36) had contaminations above the median (70 ngkg−1). A reverse result was detected in summer. The chance of contaminations above median was higher in winter than in summer (OR=5.33, P=0.007). All TMR and ingredient samples had higher AFB1 contaminations in summer (P<0.05). Six TMR samples had non-detectable (<1.25 ngkg-1) values (5 in winter) and 7 samples had levels >101.25 ngkg-1 (all in summer). The chance of TMR contamination above the median (716 ngkg-1) was 5.57 times higher in summer than in winter (P=0.002). Seventy percent of the TMR samples had contaminations above the median in summer. Elevated levels of AFB1 of rations in summer (1375.50±905.02 vs. 537.05±558.79; P<0.002) did not result in elevation of AFM1 in milk, probably due to reduced AFB1 metabolism in the liver and lower dry matter intakes caused by heat stress. The AFB1 content of grain mix succeeded by corn silage, wet beet pulp, dry beet pulp and alfalfa hay were correlated with TMR contamination. Ration AFB1 and milk AFM1 were not correlated. Based on the results, a great majority of milk produced in the studied farms could have AFM1 contaminations below the Iranian standard limit (100 ngkg-1). Contaminations below 50 ngkg-1 appear to be achievable and affordable. Intensifying the controlling measures in summer, when the feed contaminations are elevated, may reduce the overall milk contamination.

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.

Advances in the Total Synthesis of Aflatoxins

Aflatoxins, which are produced by Aspergillus flavus, Aspergillus nomius, and Aspergillus parasiticus, are a group of pentacyclic natural products with difuran and coumarin skeletons. They mainly include aflatoxin B1, B2, G1, G2, M1, and M2. Biologically, aflatoxins are of concern to human health as they can be present as contaminants in food products. The unique skeletons of aflatoxins and their risk to human health have led to the publication of nine remarkable total syntheses (including three asymmetric syntheses) and ten formal total syntheses (including four asymmetric formal syntheses) of aflatoxins in the past 55 years. To better understand the mechanism of the biological activity of aflatoxins and their presence in samples from the food industry, this review summarizes progress in the total synthesis of aflatoxins.

An in Vitro Antifungal and Antiaflatoxigenic Properties of Commiphora myrrha and Prunus mahaleb

Aflatoxins and especially aflatoxin B, are the devastating contaminant of food and feed products with hazardous effects to mankind and his domestic animals. These investigations were set to evaluate the effect of various levels of Commiphora myrrha resin (1.0, 1.25, 2.25, and 3.25 g/100 ml) and Prunus mahaleb seed extract (0.75, 1.5, 2.5, and 3.5 g/100 ml) on the growth and aflatoxin secretion by two aflatoxigenic strains of Aspergillus flavus and A. parasiticus. The two plant extracts significantly (p&lt;0.05) decreased aflatoxin secretion, and inhibited the fungal growth. Resin of C. myrrha displayed 51.9-95.7% reduction in total aflatoxin secretion by A. flavus, and 46.9-92% for A. parasiticus, and Seed extract of P. mahaleb decreased aflatoxin up to 53.7-95.8% and 40-94.7%, respectively. The inhibition of aflatoxin B (B1 and B2) by myrrh resin and seed extract of mahaleb ranged between 51.7-93.5, 50-93.6% (A. flavus) and 39.5-89.7%, 37.9-93% (A. parasiticus). The mycelial dry weight of A. flavus and A. parasiticus ws decreased up to 46.1-58.7%, 28.9-51.3% (Myrrh resin), and between 45-56.9%, 33.3-55.9% (Mahaleb seed extract). Nonetheless, the two plant extracts did not detoxify aflatoxin B1. Therefore, it apparent that the resin of C. myrrha and seed extract of P. mahaleb affected the biosynthesis pathway of aflatoxins. Thus, they can be recommended as effective natural plant biopreservative against aflatoxin contamination of food and feed products.

Indoles Derived From Glucobrassicin: Cancer Chemoprevention by Indole-3-Carbinol and 3,3'-Diindolylmethane

Hydrolysis of glucobrassicin by plant or bacterial myrosinase produces multiple indoles predominantly indole-3-carbinol (I3C). I3C and its major in vivo product, 3,3'-diindolylmethane (DIM), are effective cancer chemopreventive agents in pre-clinical models and show promise in clinical trials. The pharmacokinetics/pharmacodynamics of DIM have been studied in both rodents and humans and urinary DIM is a proposed biomarker of dietary intake of cruciferous vegetables. Recent clinical studies at Oregon State University show surprisingly robust metabolism of DIM in vivo with mono- and di-hydroxylation followed by conjugation with sulfate or glucuronic acid. DIM has multiple mechanisms of action, the most well-characterized is modulation of aryl hydrocarbon receptor (AHR) signaling. In rainbow trout dose-dependent cancer chemoprevention by dietary I3C is achieved when given prior to or concurrent with aflatoxin B1, polycyclic aromatic hydrocarbons, nitrosamines or direct acting carcinogens such as N-methyl-N'-nitro-nitrosoguanidine. Feeding pregnant mice I3C inhibits transplacental carcinogenesis. In humans much of the focus has been on chemoprevention of breast and prostate cancer. Alteration of cytochrome P450-dependent estrogen metabolism is hypothesized to be an important driver of DIM-dependent breast cancer prevention. The few studies done to date comparing glucobrassicin-rich crucifers such as Brussels sprouts with I3C/DIM supplements have shown the greater impact of the latter is due to dose. Daily ingestion of kg quantities of Brussels sprouts is required to produce in vivo levels of DIM achievable by supplementation. In clinical trials these supplement doses have elicited few if any adverse effects. Sulforaphane from glucoraphanin can act synergistically with glucobrassicin-derived DIM and this may lead to opportunities for combinatorial approaches (supplement and food-based) in the clinic.