Aflatoxin B1 Molecules Research Articles

Transformation of Adsorbed Aflatoxin B1 on Smectite at Elevated Temperatures

AbstractAflatoxins cause liver damage and suppress immunity. Through adsorption, smectites can be used to reduce the bioavailability of aflatoxins. To further reduce the toxicity of aflatoxins and to eliminate the treatments of aflatoxin-loaded smectites, the ability to degrade the aflatoxin adsorbed to non-toxic or less toxic compounds is desirable. The objective of the present study was to investigate the effects of temperature and the exchange cation on the transformation of adsorbed aflatoxin B1 on smectite. An AfB1-Ca-smectite (sm) complex was synthesized. To enhance the Lewis acidity of the complexes, the exchanged calcium in the complex was replaced with Mn and Cu to obtain AfB1-Mn-sm and AfB1-Cu-sm complexes, respectively. The aflatoxin-sm complexes and pure aflatoxin B1 were dried at 60°C in aluminum cups, and heated from 100 to 200°C in 25°C steps. Aflatoxin B1 and its transformation products were extracted with methanol after the heat treatment. The extracts were analyzed using UV spectroscopy, high performance liquid chromatography (HPLC)-fluorescence/UV, ultra-performance liquid chromatography (UPLC)-photodiode array (PDA), and electrospray ionization-tandem quadrupole-mass spectrometry (ESI-TQDMS). The solid residues were analyzed using Fourier-transform infrared spectroscopy (FTIR). The UV and FTIR spectra of the AfB1-sm clay residue extracts obtained after heating had decreased AfB1 peak intensities and shifted peak positions with increased heating temperature. Significant shifts in band positions and changes in the shape of the UV spectra were observed in the extracts from the AfB1-Ca-sm complex heated at 175°C, the AfB1-Cu-sm complex heated at 150°C, and the AfB1-Mn-sm complex heated at 125°C. The HPLC and UPLCMS analyses of AfB1-sm complex extracts indicated a gradual decrease in AfB1 concentration with increased heating temperature and the formation of aflatoxins B2, B2a, M1, M2, and other unidentified compounds. No new compound was observed in the extracts of pure aflatoxin B1 after a comparable heating experiment. These results suggest that smectite can effectively convert aflatoxin to other less toxic forms at elevated temperatures. Smectite ion exchange with Cu or Mn transition-metal cations and heat treatment induced more efficient conversion of the adsorbed aflatoxin B1 molecules to other compounds.

Low cost quantitative digital imaging as an alternative to qualitative in vivo bioassays for analysis of active aflatoxin B1

Aflatoxin B1 (AFB1) producing fungi contaminate food and feed and are a major health concern. To minimize the sources and incidence of AFB1 illness there is a need to develop affordable, sensitive mobile devices for detection of active AFB1. In the present study we used a low cost fluorescence detector and describe two quantitative assays for detection of detoxified and active AFB1 demonstrating that AFB1 concentration can be measured as intensity of fluorescence. When the assay plate containing increasing concentrations of AFB1 is illuminated with a 366nm ultraviolet lamp, AFB1 molecules absorb photons and emit blue light with peak wavelength of 432nm. The fluorescence intensity increased in dose dependent manner. However, this method cannot distinguish between active AFB1 which poses a threat to health, and the detoxified AFB1 which exhibits no toxicity. To measure the toxin activity, we used a cell based assay that makes quantification more robust and is capable of detecting multiple samples simultaneously. It is an alternative to the qualitative duckling bioassay which is the “gold-standard” assay currently being used for quantitative analysis of active AFB1. AFB1 was incubated with transduced Vero cells expressing the green fluorescence protein (GFP) gene. After excitation with blue light at 475nm, cells emitted green light with emission peak at 509nm. The result shows that AFB1 inhibits protein expression in a concentration dependent manner resulting in proportionately less GFP fluorescence in cells exposed to AFB1. The result also indicates strong positive linear relationship with R2=0.90 between the low cost CCD camera and a fluorometer, which costs 100 times more than a CCD camera. This new analytical method for measuring active AFB1 is low in cost and combined with in vitro assay, is quantitative. It also does not require the use of animals and may be useful especially for laboratories in regions with limited resources.

Aflatoxin B1 adsorption by the natural aluminosilicates - concentrate of montmorillonite and zeolite

Aflatoxin B1 adsorption by the concentrate of bentonite clay - montmorillonite and the natural zeolite - clinoptilolite and was investigated at the initial toxin concentration 4 ppm, with different amonunts of solid phase in suspension (10, 5, 2 and 1 mg/10 mL) and different pH values - 3, 7 and 9. Results indicated that for both minerals, decreasing the amount of solid phase in suspension, decrease the amount of active sites relevant for adsorption of aflatoxin B1. Thus, for concentrate of montnorillonite, at the lowest level of solid phase in suspension (1 mg/10 mL), aflatoxin B1 adsorption indexes were 97% at pH 3, 88% at pH 7 and 82% at pH 9, while for the natural zeolite, adsorption of toxin was 9% at pH 3 and 7% at pH 7 and 9. Since inorganic cations in minerals are mainly responsible for aflatoxin B1 adsorption, even the natural zeolite - clinoptilite has much higher cation exchange capacity (the content of inorganic exchangeable cations) compared to the concentrate of montmorillonite, adsorption of aflatoxin B1 by this mineral is much lower. Comparing the molecular dimensions of aflatoxin B1 molecule with the dimension of channels of clinoptilolite and interlamellar space of montmorillonite it is obvious that this toxin is adsorbed only at the external surface of clinoptilolite while in the montmorillonite all active sites are equally available for its adsorption. Thus, the concentrate of montmorillonite posess by higher adsorption capacity for aflatoxin B1. Results presented in this paper confirmed the fact the differences in the structure of minerals led to their different efficiency for adsorption of aflatoxin B1. Mineralogical and chemical composition, determination of cation exchange capacity, etc., are very important parameters influencing the effectiveness of minerals as aflatoxin B1 adsorbents. [Projekat Ministarstva nauke Republike Srbije, br. 451-03-2802-IP Tip1/142, br. 172018 i br. 34013] <br><br><font color="red"><b> This article has been corrected. Link to the correction <u><a href="http://dx.doi.org/10.2298/HEMIND170208003E">10.2298/HEMIND170208003E</a><u></b></font>

Aflatoxin B1 Detection Using a Highly-Sensitive Molecularly-Imprinted Electrochemical Sensor Based on an Electropolymerized Metal Organic Framework.

A sensitive electrochemical molecularly-imprinted sensor was developed for the detection of aflatoxin B1 (AFB1), by electropolymerization of p-aminothiophenol-functionalized gold nanoparticles in the presence of AFB1 as a template molecule. The extraction of the template leads to the formation of cavities that are able to specifically recognize and bind AFB1 through π-π interactions between AFB1 molecules and aniline moities. The performance of the developed sensor for the detection of AFB1 was investigated by linear sweep voltammetry using a hexacyanoferrate/hexacyanoferrite solution as a redox probe, the electron transfer rate increasing when the concentration of AFB1 increases, due to a p-doping effect. The molecularly-imprinted sensor exhibits a broad linear range, between 3.2 fM and 3.2 µM, and a quantification limit of 3 fM. Compared to the non-imprinted sensor, the imprinting factor was found to be 10. Selectivity studies were also performed towards the binding of other aflatoxins and ochratoxin A, proving good selectivity.

Sensitive detection of aflatoxin B1 molecules on gold SPR chip surface using functionalized gold nanoparticles

Due to the warm and favourably humid climate of Southern Hungary, the maize is one of the most important crops. The protection against crop damage caused by fusarium and Aspergillus species is essential. Detection of aflatoxin B1 (AFB1) molecules in cereal crops by selective sensors is important, while they can cause serious diseases in humans and animals if they enter the food chain. Our main objective was to develop selective AFB1 sensor with increased sensitivity applying βCD-functionalized gold nanoparticles (AuβCD NPs) in surface plasmon resonance (SPR) measuring apparatus. The nanoparticles ca. 10 nm in diameter were prepared in the presence of thiol-modified cyclodextrin. The adsorption isotherms of AFB1 on bare, thiol-modified cyclodextrin and AuβCD NPs covered Au film surface were calculated using SPR platform. The AFB1 concentration can be quantitatively determined in the 0.001–23.68 ng/mL range. The AuβCD NPs were found to be highly sensitive and exhibited a remarkably low limit of detection (LOD; 1 pg/mL) without using other analytical reagents.

Surface-enhanced Raman Scattering of Aflatoxin B1 on Silver by DFT Method

The structure, electrostatic properties, and Raman spectra of aflatoxin B1 (AFB1) and AFB1-Ag complex are studied by density functional theory with B3LYP/6-311G(d,p)/Lanl2dz basis set. The results show that the surface-enhanced Raman scattering (SERS) and pre-resonance Raman spectra of AFB1-Ag complex strongly depend on the adsorption site and the excitation wavelength of the incident light. The SERS factors are found to enhance 102−103 order compared to normal Raman spectrum of AFB1 molecule due to the larger static polarizabilities of the AFB1-Ag complex, which directly results in the stronger chemical enhancement in SERS spectra. The pre-resonance Raman spectra of AFB1-Ag complex are explored at 266, 482, 785, and 1064 nm incident light wavelength, in which the enhancement factors are about 102−104, mainly caused by the charge-transfer excitation resonance. The vibrational modes are analyzed to explain the relationship between the vibrational direction and the enhanced Raman intensities.

Degradation and detoxification of aflatoxin B1 by Pseudomonas putida

Aflatoxin is a mycotoxin produced by Aspergillus flavus and a common contaminant of food and feed, posing health hazards to humans and animals alike. The aim of this study is to explore the ability of Pseudomonas putida to degrade aflatoxin B1 (AFB1). The toxigenic strain of A. flavus was isolated from sugarcane and used to produce AFB1 in yeast extract sucrose medium. Two P. putida strains, MTCC 1274 and 2445, were cultured in mineral salt glucose medium (MSG) containing AFB1. The AFB1 was analyzed qualitatively and quantitatively by TLC and HPLC. It was found that Pseudomonas sp. can tolerate AFB1 in the medium (0.2 μg ml−1), and degrades it very efficiently. Within 24 h of incubation, AFB1 was reduced to an undetectable level in MSG medium. Analysis with TLC, HPLC, UV spectrophotometer, gas chromatography mass spectrometry (GC–MS), and Fourier transform infra-red spectroscopy (FT-IR) showed that AFB1 was bio-transformed to structurally different compounds (AFD1, AFD2, and AFD3), with the modified furan and lactone ring on the AFB1 molecule. A toxicity study on the HeLa cells showed that the new compounds formed are less toxic when compared with AFB1.

A simple and rapid optical biosensor for detection of aflatoxin B1 based on competitive dispersion of gold nanorods

This report illustrates a promising one-step and label-free optical biosensor for determination of aflatoxin B1 (AFB1) that is most commonly found in foods and highly dangerous even at very low concentrations. In this research, gold nanorods (GNRs) were employed as a sensing platform, which showed high stability under high ionic strength conditions without addition of any stabilizing agent. GNR-AFB1–BSA (bovine serum albumin) conjugates aggregated after mixing with free antibodies, resulting in significant changes in absorption intensity. At the same time the existence of AFB1 molecules in samples caused dispersion of nanorods, as a result of competitive immune-reaction with antibodies. By taking advantages of the competitive dispersion of GNRs, the developed method could effectively reduce false results caused by undesirable aggregation, which is a big problem for spherical gold nanoparticles. Absorption intensity of UV–vis spectra served as the sensing indicator, with dynamic light scattering (DLS) measurement as another sensing tool. The designed biosensing system could detect AFB1 in a linear range from 0.5 to 20ngmL−1, with a good correlation coefficient of 0.99. And the limit of detection (LOD) was 0.16ngmL−1, indicating an excellent sensitivity with absorbance result. The recoveries of the spiked AFB1 in real peanut samples ranged from 94.2% to 117.3%. Therefore the proposed nano-biosensor was demonstrated to be sensitive, selective, and simple, providing a viable alternative for rapid screening of toxins in agriculture products and foods.

Study of interactions between DNA and aflatoxin B1 using electrochemical and fluorescence methods

In this study, a carbon paste electrode modified with N-butylpyridinium hexafluorophosphate (BPPF6) ionic liquid and DNA was introduced as an electrochemical biosensor to study the interaction between DNA and aflatoxin B1 molecules. For this purpose, variations in oxidation peak current of guanine in various concentrations of aflatoxin B1 were measured by using the differential pulse voltammetry (DPV) method. According to this study, the binding constant of DNA–aflatoxin B1 was found to be 3.5×106M−1. This modified electrode was also used for determination of low concentrations of aflatoxin B1 by using differential pulse voltammetry. A linear dynamic range from 8.00×10−8 to 5.91×10−7M and a limit of detection of 2.00×10−8M resulted from DPV measurements. To confirm our results, a fluorescence study was also performed. It resulted in a binding constant of 2.8×106M−1, which is in good agreement with that obtained from electrochemical study.

Effect of water molecules on the fluorescence enhancement of Aflatoxin B1 mediated by Aflatoxin B1:β-cyclodextrin complexes. A theoretical study

In order to explain the observed fluorescence enhancement of Aflatoxin B1 (AFB1) when forming AFB1:beta-cyclodextrin (AFB1:beta-CD) inclusion complexes, we have performed a theoretical (quantum chemistry calculations) study of AFB1 and AFB1:beta-CD in vacuum and in the presence of aqueous solvent. The AM1 method was used to calculate the absorption and emission wavelengths of these molecules. With the help of density functional theory (DFT) and time-dependent DFT (TDDFT) vibrational frequencies and related excitation energies of AFB1 and AFB1.(H2O)m = 4,5,6,11 were calculated. On the basis of these calculations we propose a plausible mechanism for the fluorescence enhancement of AFB1 in the presence of beta-CD: (1) before photoexcitation of AFB1 to its S1 excited state, there is a vibrational coupling between the vibrational modes involving the AFB1 carbonyl groups and the bending modes of the nearby water molecules (CG + WM); (2) these interactions allow a thermal relaxation of the excited AFB1 molecules that results in fluorescence quenching; (3) when the AFB1 molecules form inclusion complexes with beta-CD the CG + WM interaction decreases; and (4) this gives rise to a fluorescence enhancement.

Adsorption of aflatoxin B1 on montmorillonite

The isothermal adsorption and the adsorptive mechanism of aflatoxin B1 (AFB1) on calcium montmorillonite (Mont) were studied in vitro trials. The maximum amounts of AFB1 on Mont in aqueous solution at 2 and pH 8 were 613.5 and 628.9 microg of AFB1 /g of Mont, respectively. The structure of AFB1-Mont was measured by x-ray diffraction and infrared absorption spectrum. The results suggested that the mechanism of AFB1 on Mont was AFB1 sorbed onto the edge of Mont by a double hydrogen bond, and AFB1 molecules did not penetrate into the interlayer area of Mont. The Mont, when added to the diet of broiler chicken at 0.5%, significantly diminished the adverse effects of feeding 200 microg of AFB1 /kg of feed. And the concentrations of Ca, P, Cu, Fe, Zn in the broiler bones were not affected by AFB1 and Mont, but the concentrations of Mn, Pb, and F were decreased by Mont.

Aflatoxin B 1 -induced hepatic steatosis: role of carbonyl compounds and active diols on steatogenesis

Aflatoxin B1 (AFB1) exerts a chronic carcinogenic and an acute toxic effect on animals. Whereas the mechanism for carcinogenicity is known, no mechanism has been proposed for the toxic action. Among the most prominent signs of aflatoxicosis in several species, including birds and mammals, are hypolipidaemia, hypocholesterolaemia, and hypocarotenaemia, associated with severe hepatic steatosis and weight loss. We suggest that these signs of acute imbalance of lipid metabolism can be the result of the chemical modification (blocking) of key lysyl residues on the LDL protein B-100 by the activated AFB1 molecule. Modified LDLs are not recognised by their specific receptors and thus are rejected by peripheral cells. Upon return to the liver, the modified particles bind to the sinusoidal lining cells. Lipid starvation of peripheral tissues takes place while fat accumulates in the liver. This abnormal state is maintained and reinforced by further modification of nascent apoproteins, which in turn become unable to receive a lipid load for as long as aflatoxin continues to be available in the liver.

Carcinogen-nucleic acid interactions: equilibrium binding studies of aflatoxin B1 with the oligodeoxynucleotide d(ATGCAT)2 and with plasmid pBR322 support intercalative association with the B-DNA helix

Equilibrium binding of aflatoxin B1 (AFB1) to the oligodeoxynucleotide d(ATGCAT)2 was examined by using 1H NMR. AFB1 binds to double-stranded d(ATGCAT)2 with an apparent binding constant of 3.7 x 10(3) M-1. The equilibrium is rapid on the NMR time scale; the observed 1H NMR spectrum represents the population-weighted average of the chemical shifts arising from the free and bound states of the oligodeoxynucleotide and the AFB1. The spectrum of d(ATGCAT)2 exhibits exchange broadening in the presence of AFB1, manifested as decreases in apparent T2 relaxation times for the d(ATGCAT)2 base protons. Upon binding to d(ATGCAT)2, the AFB1 signals are shifted upfield, indicative of increased shielding. The adenine H2 protons are also shifted upfield in the presence of the carcinogen. Small changes in chemical shift are observed for other d(ATGCAT)2 protons. A substantial decrease in the nonselective T1 relaxation time is observed for the adenine H2 protons in the presence of AFB1. Competition binding experiments in which the competing ligands actinomycin D, ethidium bromide, and spermidine were individually added to an AFB1-d(ATGCAT)2 equilibrium mixture showed that addition of 1 equiv of actinomycin D or 4 equiv of ethidium bromide was sufficient to displace bound AFB1 from d(ATGCAT)2. In contrast, the addition of spermidine did not result in the displacement of bound AFB1 molecules and may have slightly enhanced binding, presumably due to stabilization of the DNA duplex. 1H NOESY experiments confirmed that the overall conformation for the d(ATGCAT)2 duplex was right-handed both in the absence and in the presence of AFB1. Equilibrium binding of AFB1 to d(ATGCAT)2 is greatly diminished at higher temperatures at which the oligodeoxynucleotide is single-stranded.(ABSTRACT TRUNCATED AT 250 WORDS)

Destruction of Aflatoxin B1 with Sodium Bisulfite: Isolation of the Major Product Aflatoxin B1S

The reaction between aflatoxin B1 and sodium bisulfite yielded almost quantitatively a light yellow, highly fluorescent water-soluble product, aflatoxin B1S. Reverse-phase (C18) high pressure liquid chromatography (with and without the paired-ion technique), and normal- and reverse-phase thin layer chromatography revealed the ionic product at greater than 98% yield. The product's intense fluorescence, identical in color to that of aflatoxins B1 and B2, supported the conclusion that the lactone of aflatoxin B1 was not opened by bisulfite and that the reaction took place at some other location on the aflatoxin B1 molecule. The UV spectrum of aflatoxin B1S exhibited identical absorbance maxima to aflatoxin B1 and these maxima exhibited no bathochromic shift in alkaline solution. Aflatoxin B1S exhibited change to bright yellow fluorescence under 365nm UV light after being sprayed with 20% sulfuric acid in methanol. The IR spectrum of aflatoxin B1S indicated the vinylene group of the furofuran ring system of aflatoxin B1 was missing from aflatoxin B1S, whereas the coumarin-cyclopentenone ring system present in aflatoxin B1 was intact in aflatoxin B1S. Furthermore, the IR spectrum suggested the presence of a sulfonate moiety in B1S. Proton NMR spectra of aflatoxin B1S also indicated that the cyclopentenone region was intact and that the furofuran had undergone reaction with bisulfite. Integration of the NMR spectrum of aflatoxin B1S revealed 12.75 to 13.25 protons, consistent with a bisulfite addition across the double bond of aflatoxin B1 to form aflatoxin B1S. Aflatoxins B2 and G2 were not susceptible to the action of bisulfite, again supporting the conclusion that bisulfite acts at the unsaturation in the furofuran ring system of aflatoxin B1. There was no evidence that aflatoxin B2 was produced in the degradation of aflatoxin B1 by bisulfite under these conditions. The fact that previously reported chemical modifications of this furofuran ring system resulted in reduced toxicity support the potential utility of bisulfite as a means of detoxification of aflatoxin-contaminated commodities.

Polymyxin-B-Binding Capacities of Isolated Phospholipids from Salmonella typhimurium

5-hydroxycoumarin and aflatoxin B, were shorter than that of 5-methoxycoumarin by 75 nm and 40nm respectively. The relative fluorescent intensities of 5-methoxycoumarin, 5-hydroxycoumarin and aflatoxin B1 were estimated to be in the proportions 1 :2.1: 5.2 respectively. Although the coumarin molecule is non-fluorescent (Mattoo, 1956), these results indicate that the addition of a hydroxyl group in the 5-position causes a very pronounced fluorescence but that methylation of the phenolic group t o give 5-methoxycoumarin diminishes the fluorescence. The relatively high KQ value of aflatoxin B, makes it one of the most fluorescent coumarin derivatives. The KQ values of aflatoxin B1 in different solvents such as chloroform, methanol and ethanol have been determined by several workers (Carnaghan et al., 1963; Robertson et al., 1967). Our present results suggest that a n aqueous solution (pH2) is a superior medium for the quantitative determination of aflatoxin B1. The large enhancement of fluorescence of the aflatoxin B1 molecule over its central 5-methoxycoumarin moiety is undoubtedly due to the excitation of the rc (delocalized) electrons contained in the conjugated system of double bonds. The effect of pH on fluorescence can be associated with change in chemical species (Bridges & Williams, 1968). Examination of the variation of fluorescence intensity of aflatoxin B1 with pH will therefore be valuable in the attempt to determine the possible use of the furocoumarin compound as a p H indicator.

The crystal structures of aflatoxin B1. I. The structure of the chloroform solvate of aflatoxin B1 and the absolute configuration of aflatoxin B1

Aflatoxin B1. CHC13 forms orthorhombic crystals of space group P212121 with four molecules in the unit cell of dimensions a= 6.16, b= 8.04 and c= 36.25 A. The orientation of the aflatoxin B1 molecule was found by means of a minimum function, calculated for two intramolecular vectors between light atoms; its position was determined by calculating the value of the residual while the molecule was moved systematically through the unit cell. The presence of the chloroform molecule appeared after a Fourier synthesis. The structure consists of alternating layers parallel to the plane (001) of chloroform and aflatoxin B1 connected by CH. . . O hydrogen bonds. Within the aflatoxin B1 layer, strings of coplanar molecules are present in the directions [110] and [il0]). The molecules within a string have two short CH3... O contacts of 304 and 3.15 A, which points to a specific interaction. These contacts may be called hydrogen bonds. The absolute configuration of the aflatoxin Bx molecule is given.