Establishment and characterization of cell lines (Kagura-1 and Kagura-2) from aflatoxin B1-induced rat hepatoma

Two hepatoma cell lines designated Kagura-1 and Kagura-2 were established from rat hepatocellular carcinomas induced by aflatoxin B1, and have been propagated for over two years. Both cell lines grew as monolayered sheets with a population doubling time of about 20 h. Chromosome counts of Kagura-1 cells ranged from 34 to 45 with a modal number of 40, while that of Kagura-2 cells ranged widely from 40 to 130 with a modal number of 65. Subcutaneous inoculation of cultured cells of both these lines into nude mice resulted in tumor formation. The histopathological appearances of the induced tumors were similar to those of the original tumors. Kagura-1 and Kagura-2 cell lines express at least two tumor markers, glutathione-S-transferase P and gamma-glutamyl transpeptidase; the level of c-myc messenger ribonucleic acid was also highly elevated.     

Isolation of a metastasizing cancer cell line from an aflatoxin B1-induced rat liver tumor.

An attempt was made to isolate cancer cell lines from liver tumors that had been induced by aflatoxin B1 (AFB1) in rats. A clonal cell line named AFB-1 was isolated from a liver tumor that was histologically diagnosed as hepatocellular carcinoma. When AFB-1 cells were inoculated into the subcutaneous tissue at the dorsal region of syngenic animals, they metastasized from the site of inoculation into the abdominal cavity to form many tumor nodules throughout the serous membrane and metastatic foci in the kidney and pancreas. They also metastasized into the thoracic cavity to form metastatic foci in the lung. This is the first instance where a metastasizing AFB1-induced cancer cell line has been isolated.     

Effect of organic solvent concentration on radioimmunoassay of aflatoxin B1

The effect of acetone and methanol contents in an aqueous casein-buffer solution pipetted with aflatoxin B1 into radioimmunoassay procedure, on some parameters of radioimmunochemical determination of aflatoxin B₁ has been studied. These organic solvents lower an antiserum titer and value of the zero specific binding, and at higher concentrations make worse the detection limit and the accuracy of radioimmunoassay. However, in radioimmunoassay of food extracts containing subresidual levels of aflatoxin, it could be advantageous to add the extract volume to the organic solvent concentration of 60%.     

Large-scale purification of the mycotoxins aflatoxin B1, B2 and G1

The isolation and purification of gram quantities of the important mycotoxins aflatoxin B1, B2 and G1 are described. The method involves final purification on a Waters Prep LC-500 instrument, loaded with silica cartridges, and elution with chloroform.     

Formation of S-[2-carboxy-1-(1H-imidazol-4-yl) ethyl]glutathione, a new metabolite of L-histidine, from cis-urocanic acid and glutathione by the action of glutathione S-transferase

Exposure of the skin to sunlight results in an increase of the content of epidermal trans-urocanic acid, a key metabolite of L-histidine, and also in occurrence of the isomerization of trans-urocanic acid to the cis isomer. S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]glutathione (GS(CIE)), an adduct of urocanic acid and glutathione, is a presumed origin of a urinary compound S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-L-cysteine (Cys(CIE)). The formation of GS(CIE) is stimulated by exposing the skin to sunlight irradiation. In this study we investigated an enzymatic formation of GS(CIE) from glutathione and cis-urocanic acid by incubation with rat liver extract that contained glutathione S-transferase (GST) at high activity. The formation of GS(CIE) was suppressed significantly when a liver extract depleted of GST activity was used. Enzymatic degradation of GS(CIE) with gamma -glutamyl transpeptidase resulted in the formation of N-[S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-L-cysteinyl]glycine, a metabolic intermediate between the glutathione adduct and Cys(CIE). A hydrolyzed product of GS(CIE) by HCl was identical with the urinary Cys(CIE). Compounds were analyzed by high-voltage paper electrophoresis, capillary electrophoresis, and fast atom bombardment mass spectrometry. From these results, we suggest that GS(CIE) formed from cis-urocanic acid and glutathione is an origin of the urinary compound Cys(CIE) and that the formation reaction is catalyzed mostly by the action of GST.     

Overpressured layer chromatographic determination of aflatoxin B1, B2, G1 and G2 in red paprika

Determination of aflatoxin B1 and total aflatoxin (B1 + B2 + G1 + G2) in red paprika powder is described using column chromatographic sample clean-up, overpressured layer chromatography (OPLC) separation and fluorescence densitometric evaluation. Two OPLC methods were developed for separation of the four aflatoxins. The detection limit and quantification limit of aflatoxins in red paprika were 0.5 and 1 μg/kg in both methods, respectively. Recovery experiment was carried out with sample containing 1.74 μg/kg aflatoxin B1 and 3.56 μg/kg total aflatoxins measured by European standard HPLC method. Mean recovery amounted to 78.5% (SD 16.1%, n = 5) for aflatoxin B1 and 81.8% (SD 17.1%, n = 5) for total aflatoxins in the case of method 1. It was 105.3% (SD 10.7%, n = 5) for aflatoxin B1 and 97.4% (SD 18.6%, n = 5) for total aflatoxins using the method 2. Despite of that the Hungarian climate is not proper for the toxin production of moulds high aflatoxin B1 contaminated red paprika purchased from the market was found, which may originate from mixing of imported paprika containing very high level toxin with Hungarian one.     

Multicolor immunochromatographic strip test based on gold nanoparticles for the determination of aflatoxin B1 and fumonisins

The authors describe the preparation of desert rose-like gold nanoparticles (DR-GNPs) with a plasmon resonance band at 620 nm which gives them a blue color. They have a hydrodynamic diameter of ～72 nm and were prepared by a seeding growth approach. The DR-GNPs were characterized by UV-vis spectroscopy, transmission electron microscopy and dynamic light scattering. These nonspherical GNPs were used as a label for the antibody in an immunochromatographic strip test (ICST). Despite their particular shape and the higher surface area compared to spherical gold nanoparticles, the DR-GNPs are useful blue labels for the GNP-based strip test. A multicolor ICST for aflatoxin B1 and fumonisins is described that employs both blue DR-GNPs and red spherical GNPs. It allows for simultaneous rapid determination of the two mycotoxins in maize flour, with visual cut-off levels of 2 μg?kg^-1 for aflatoxin B1 and of 1000 μg?kg^-1 for fumonisins.

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Graphical abstract Blue desert rose-like gold nanoparticles (DR-GNPs) were synthesized, characterized and applied as label for the ImmunoChromatographic Strip Test (ICST) technique, in which red spherical GNPs (s-GNPs) are usually employed. The combined use of the blue DR-GNP and red s-GNPs allowed developing of an intuitive multicolor ICST for the simultaneous detection of aflatoxin B1 and fumonisins in maize flour.

     

Therapeutic ability of some plant extracts on aflatoxin B1 induced renal and cardiac damage

The aim of the study was to investigate the therapeutic effect of aqueous extracts of some traditional medicinal plants (including Camellia sinensis leaves, Carum carvi seeds, Alpinia galangal rhizomes, Boswellia serrata resins and Cenchona officinalis bark) compared to the anticancer drug, methotrexate (MTX) against aflatoxicosis induced by aflatoxin-B1 (AFB1) in both kidneys and hearts of rats. Administration of AFB1 induces oxidative stress in kidneys of AFB1-treated rats through elevating the level of malondialdehyde (MDA) and depleting the levels of tissue antioxidants, glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G-6-PDH) and vitamin C. Also the result revealed that aflatoxicosis interfere with the cellular energy supply of rat hearts through its inhibitory action on some markers of energy metabolism indicated by a decrease in glucose and glycogen contents of heart and a reduction in the activities of some glycolytic enzymes, phosphogluco-isomerase (PGI), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) compared to normal healthy animals.Supplementation of the aqueous extracts of the different plant used, effectively ameliorated the deviation induced in both kidneys and hearts of animals in response to AFB1 administration. This effect was evident through reducing MDA level and up-regulating the inhibitory effect of AFB1 on the levels of antioxidants in kidneys as well as the energetic biomarkers in hearts. However, administration of MTX to AFB1-treated rats dramatically amplified the toxic effect of aflatoxicosis, indicated by marked increment in MDA level and decrease in the levels of antioxidants in kidneys of AFB1-MTX group in relation to AFB1 group. Also the same response was found in the bioenergetic markers of hearts. From the current investigation, it can be suggested that supplementation of the extracts of the different plants presented in this study was beneficial in modulating the alterations induced in kidney and heart under the toxic effects of AFB1.     

Simultaneous enzyme immunoassay for the screening of aflatoxin B1 and ochratoxin A in chili samples

Membrane-based immunoassay has been developed for simultaneous estimation of aflatoxin B₁ (AFB₁) and ochratoxin A (OA) in chili samples. The combined estimation of both the mycotoxins is more economical in respect of time, work and materials than two separate assays. The method uses a low cost test device consisting of a membrane with immobilized anti-AFB₁ and anti-OA antibodies and a filter paper attached to a polyethylene card below the membrane. It allows direct analysis of sample extracts containing substantial amount (40%) of methanol. This permits the use of two-fold diluted sample extracts resulting in minimum dilution error. The limit of quantitation obtained was 2 and 10 μg kg⁻¹ for AFB₁ and OA, respectively. The tolerance of 40% methanol was found to be due to the application of small size (0.8 mm diameter) spots on membranes, as the tolerance decreases to 20% with gradual increase in spot size. The combined method is capable of producing acceptable results to analyze AFB₁ and OA in chili with accuracy and precision. The AFB₁ and OA values obtained for spiked and naturally contaminated chili samples by the simultaneous method were in good correlation with those measured by individual ELISA. The method offers a simple, rapid and cost-effective screening tool to meet the requirements of the rapidly evolving EU legislation.     

Determination of aflatoxin B1 in alcoholic beverages: comparison of one- and two-photon-induced fluorescence

The qualitative and quantitative analysis of aflatoxin B₁ in a model system (water/ethanol), in different wines and in beer using one- and two-photon-induced fluorescence is discussed. The absorption and fluorescence properties of aflatoxin B₁ depend on the solvent and pH. The two-photon-absorption cross-section was calculated for aflatoxin B₁ in beer and wine (σ ₂ ∼ 25 GM) for excitation at 720 nm. A comparison of the one- and two-photon- induced fluorescence results showed that the disturbance due to background emission originating from matrix constituents is significantly reduced under two-photon-excitation conditions. The limit of detection for the one- and two-photon-induced fluorescence was determined.     

Colloidal gold based immunochromatographic strip for the simple and sensitive determination of aflatoxin B1 and B2 in corn and rice

We have developed a simple and fast immunochromatographic test strip for the simultaneous quantitation of aflatoxin B₁ and aflatoxin B₂ in corn and rice. The strip contains three pads (sample, conjugate, and absorbing pad) and uses the respective polyclonal antibodies immobilized on gold nanoparticles. Matrix interferences were minimized by application of fugacity theory. Clean-up of samples and pre-treatment of strip pads is not required. The visual detection limit is 0.1 ng mL^?1, and the process can be completed within 5 min. Out of 113 natural samples, 16 rice and 27 corn samples (38% in total) were aflatoxin positive and the test results were confirmed by HPLC. The strip shows, however, high cross reactivity to aflatoxins G₁, G₂, and M₁. We consider this strip to possess wide applicability because of its ease of use, sensitivity, stability, and low cost.

Mass spectral identification and positional mapping of aflatoxin B1—Guanine adducts in oligonucleotides

The biological consequences of a carcinogen—DNA adduct are defined by the structure of the lesion and its position within the genome. Electrospray ionization ion trap mass spectrometry (ESI-ITMS) is shown here to be a sensitive and rapid approach capable of defining both of these parameters. Three isomeric oligonucleotides of the sequence 5′-CCGGAGGCC modified by the potent human carcinogen aflatoxin B₁ (AFB₁) at different guanines were analyzed by ESI-ITMS. All three samples possessed the same molecular ion confirming the presence of an intact aflatoxin moiety in each oligonucleotide. In addition, each sample displayed a characteristic fragmentation pattern that permitted unambiguous identification of the site of modification within the sequence. Furthermore, an AFB₁-modified oligonucleotide was converted under alkaline conditions to its more stable formamidopyrimidine (FAPY) derivative. Analysis of this sample revealed the presence of a molecular ion corresponding to the presence of the FAPY adduct and a distinctive fragmentation pattern that paralleled the known chemical stability of the FAPY metabolite. This approach should be of general use in the determination of not only the nature and site of covalent modifications, but also the chemical stability of DNA adducts.     

Modelling of the impedimetric responses of an aflatoxin B1 immunosensor prepared on an electrosynthetic polyaniline platform

Aflatoxins are a group of mycotoxins that have deleterious effects on humans and are produced during fungal infection of plants or plant products. An electrochemical immunosensor for the determination of aflatoxin B₁ (AFB₁) was developed with AFB₁antibody (AFB₁-Ab) immobilized on Pt electrodes modified with polyaniline (PANi) and polystyrene sulphonic acid (PSSA). Impedimetric analysis shows that the electron transfer resistances of the Pt/PANi–PSSA electrode, the Pt/PANi–PSSA/AFB₁-Ab immunosensor and Pt/PANi–PSSA/AFB₁-Ab incubated in bovine serum albumin (BSA) were 0.458, 720 and 1,066 kΩ, respectively. These results indicate that electrochemical impedance spectroscopy (EIS) is a suitable method for monitoring the change in electron transfer resistance associated with the immobilization of the antibody. Modelling of EIS data gave equivalent circuits which showed that the electron transfer resistance increased from 0.458 kΩ for the Pt/PANi–PSSA electrode to 1,066 kΩ for the Pt/PANi–PSSA/AFB₁-Ab immunosensor, indicating that immobilization of the antibody and incubation in BSA introduced an electron transfer barrier. The AFB₁ immunosensor had a detection limit of 0.1 mg/L and a sensitivity of 869.6 kΩ L/mg.