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Molecular dosimetry of aflatoxin DNA adducts in humans and experimental rat models.

Primary hepatocellular carcinoma is one of the most common and lethal cancers in the world. It is particularly prevalent on the continents of Africa and Asia. A number of epidemiological studies have associated the exposure status of people to aflatoxin B1 as being important in the etiology of liver cancer. However, to date these studies have relied upon the criteria of presumptive intake data, rather than relying upon quantitative analyses of aflatoxin DNA adduct and metabolite content obtained by monitoring biological fluids from exposed people. Information obtained by monitoring exposed individuals for specific DNA adducts and metabolites will define the pharmacokinetics of aflatoxin B1 in people, thereby facilitating risk assessments. In combination with the human monitoring studies is the need to use animals models to help provide an interpretable data base for the human results. Animal models are also going to be critical to the development of chemoprotection strategies. The molecular dosimetry studies described in this chapter support the concept that measurement of the major, rapidly excised AFB-N7-guanine adduct in urine is an appropriate dosimeter for estimating exposure status and possibly risk in individuals consuming this mycotoxin.

Aflatoxins↗

Sequence specificity of aflatoxin B1-induced mutations in a plasmid replicated in xeroderma pigmentosum and DNA repair proficient human cells.

The mutagenic spectrum induced by aflatoxin-DNA lesions in DNA repair deficient and repair proficient human cells was investigated. The reactive metabolite aflatoxin B1-8,9-epoxide was synthesized and reacted in vitro with the shuttle vector plasmid pS189. Plasmids were transfected into human fibroblasts and allowed to replicate, and the recovered plasmids were screened in indicator bacteria for plasmid survival and mutations in the supF marker gene. Sequence data were obtained from 71 independently arising mutants recovered from DNA repair deficient xeroderma pigmentosum (XP) cells [XP12BE(SV40)] and 60 mutants recovered from a DNA repair proficient cell line (GM0637). Plasmid survival was lower and mutation frequency higher with the XP cells, and the mutation hotspots differed substantially for the 2 cell lines. Most mutations (> 90%) were base substitutions at G:C pairs, only about one-half of which were G:C-->T:A transversions, the expected predominant mutation. One-third of the mutations at GG sites and none of those at isolated Gs were G:C-->A:T transitions. Tandem base substitutions also occurred only at GG sites and were found only with XP cells. The location of mutation hotspots with either cell line did not correlate with the level of modification within the sequence as assessed by a DNA polymerase stop assay. These results suggest that the DNA repair deficiency associated with XP can influence not only the overall frequency of mutations but also the distribution of mutations within a gene. The finding of transition mutations exclusively at GG sites may be of predictive value in attempts to link dietary aflatoxin exposure to cancers associated with specific mutations in the c-ras oncogene and the p53 tumor suppressor gene.

Aflatoxin B1↗

[Efficacy and stability of ammoniation process as aflatoxin B1 decontamination technology in rice].

Rice, a cereal widely used in Venezuela for human and animal nutrition, is susceptible to aflatoxin contamination in the field and during storage. Therefore, the goal of this research was the evaluation of the efficacy and permanence of the ammoniation process through high pressure/high temperature (HP/HT) and atmospheric pressure/moderate temperature (AP/MT) conditions applied to rice samples artificially contaminated with aflatoxin B1. For this purpose a 2k design was drawn up considering the temperature, the rice moisture and the process time as variables. Under both sets of conditions, aflatoxin B1 concentration was reduced in a range of 90% to 100%. After in vitro stomach digestion simulation, toxin reversion ranged from 0% to 19%. In conclusion, the process efficacy and permanence were achieved through the use of high temperature and long process time for both sets of conditions (HP/HT and AP/MT), respectively.

Aflatoxin B1↗

Inhibition of aflatoxin production & growth of Aspergillus flavus by eugenol & onion & garlic extracts.

Efficacy of natural non-toxic materials including extracts of onion and garlic as well as eugenol was tested against aflatoxin production by A. flavus in liquid SMKY medium and in maize grains. Maximum inhibition in the mycelial growth occurred with garlic extract (61.94%), whereas inhibition of aflatoxin production was highest (60.44%) due to onion extract. Eugenol was most suitable for inhibiting aflatoxin production (60.35%) on maize grains.

Aflatoxins↗

Use of high-performance liquid chromatography to assess airborne mycotoxins. Aflatoxins and ochratoxin A.

An HPLC analytical method combining methanol-deionised water (80:20, v/v) extraction, methanol-acetonitrile (50:50, v/v) extraction and fluorescence detection was implanted to analyse ochratoxin A and aflatoxins B1, B2, G1 and G2 of air samples collected during the usual production process in a number of workplaces of a coffee factory to assess the occupational exposure of the engaged workers. The average levels of airborne ochratoxin A and aflatoxins were less than 1.2 and 0.4 ng/m3, respectively, using 50 L air samples. When 150 L air samples were used, levels lower than 0.04 ng/m3 ochratoxin A and 0.013 ng/m3 for aflatoxins B1, B2, G1 and G2, could be detected.

Aflatoxins↗

[Characterization of immobilized aflatoxin-detoxizyme].

Aflatoxins, found in contaminated food, are potent hepatocarcinogen. The aflatoxin-detoxiczyme (ADTZ) isolated from the edible fungus Armillariella sp., detoxifies aflatoxin B1 (AFB1). This paper reports on the characterization of immobilized ADTZ using a hydrophobic adsorption method. The ADTZ was isolated from cryo-homogenated fungus, previously cultivated at 24 - 28 degrees C for 20 - 30 days, using n-alkyl amino-agar beads. Various adsorption conditions of the enzyme to n-alkyl or n-octyl amino-agar beads were carried out. The effects of enzyme immobilization on different alkyl amino-agar beads, at different pH values (5.5 - 7.5), at different temperature (20 - 40 degrees C) and at different salt concentrations were investigated. The enzyme activity was measured at OD360 by reacting 133.3 ng/mL of AFB1 at 30 degrees C for 30 min with the immobilized ADTZ. The Km value of the immobilized enzyme, determined using Schematic Linewearver-Burk plot, is 3.308 x 10(-3) mol/L, lower than that of free enzyme, which is 2.16 x 10(-6) mol/L. This indicated the affinity of the detoxiczyme to AFB1 decreased after immobilization. The immobilized enzyme activity in oil-phase (n-hexane) was also studied with different concentration of water. After the treatment of the immobilized ADTZ, the toxin no longer causes liver toxicity in the rat toxicity test, no longer causes mutagenicity in Ames test and is no longer toxic in the chicken embryo test. Results also indicated that the pH stability, the thermostability and the freezing stability of ADTZ were improved after the immobilization.

Absorption↗

[Search for lesions caused by aflatoxin B1 poisoning in 3 industrial chicken farms on the Ivory Coast].

A systematic search for feed contamination by aflatoxin-B1 as well as for lesions indicating an acute or chronic aflatoxicosis was made in three groups of industrially raised hens in Côte-d'Ivoire. The contamination rates were low although they were 6 to 10 times higher than the official standards. No lesion due to an acute or chronic intoxication by aflatoxins was observed. However, the repeated intake of small amounts of aflatoxins might eventually have repercussions on the technics-economical performance either by a direct action of the toxin or via the development of an immune depression in the animals.

Aflatoxin B1↗

Determination of aflatoxin B1 in medical herbs: interlaboratory study.

A method was developed for the determination of aflatoxin B1 in medical herbs (senna pods, botanical name Cassia angustifolia; devil's claw, botanical name Harpagophytum procumbens; and ginger roots, botanical name Zingiber officinale). The method, which was tested in a mini-collaborative study by 4 laboratories, is based on an immunoaffinity cleanup followed by reversed-phase high-performance liquid chromatography separation and fluorescence detection after post-column derivatization. It allows the quantitation of aflatoxin B1 at levels lower than 2 ng/g. A second extractant (acetone-water) was tested and compared to the proposed methanol-water extractant. Several post-column derivatization options (electrochemically generated bromine, photochemical reaction, and chemical bromination) as well as different integration modes (height versus area) were also investigated. No differences were found depending on the choice of derivatization system or the signal integration mode used. The method was tested for 3 different matrixes: senna pods, ginger root, and devil's claw. Performance characteristics were established from the results of the study and resulted in HorRat values ranging from 0.12 to 0.75 with mean recoveries from 78 to 91% for the extraction with methanol-water and HorRat values ranging from 0.10-1.03 with mean recoveries from 98 to 103% for the extraction with acetone-water. As a result, the method, with all tested variations, was found to be fit-for-purpose for the determination of aflatoxin B1 in medical herbs at levels of 1 microg/kg and above.

Aflatoxin B1↗

Interlaboratory study of the Charm ROSA Safe Level Aflatoxin M1 Quantitative lateral flow test for raw bovine milk.

An interlaboratory study of 21 public health, state agriculture, and industry laboratories in the United States tested raw commingled bovine milk containing aflatoxin M1 using the Charm Rapid One Step Assay (ROSA) Safe Level Aflatoxin M1 Quantitative lateral flow method. Blind coded sample pairs were fortified with 0, 300, 350, 400, 450, 500, and 550 parts per trillion (ppt) aflatoxin M1. A ROSA reader quantitatively interpreted test strips with ppt readings. Readings < or = 400 ppt were interpreted as negative, and readings >400 ppt were interpreted as positive. Initial positive samples were subsequently assayed 2 additional times. If both retest results were >400 ppt, the sample was called positive/ actionable relative to U.S. and Codex levels, 500 ppt. The concentration of 400 ppt was chosen for the positive/negative interpretation to provide 90% sensitivity with 95% confidence at the 500 ppt legislative level. The combined false negative rate was <5% (4 of 83) for samples at 500 and 550 ppt. The false violatives at 0, 300, 350, 400, and 450 ppt (n = 42 at each level) were 0, 0, 21, 14, and 93%, respectively. The 90% positive concentration with 95% confidence was 503 ppt by probit analysis. The average intralaboratory repeatability was 11% and average interlaboratory reproducibility was 13% for the fortified sample pairs. High-performance liquid chromatography analysis of the study samples by 5 laboratories showed 38% false negatives with the 500 and 550 ppt samples, and a 0% false-violative rate with samples less than the 500 ppt action level.

Aflatoxin M1↗

Transfer RNA methylase activity and capacity during aflatoxin B1-induced hepatocellular carcinogenesis.

Transfer RNA methylase (tRNA methylase) activity and capacity were monitored in whole-rat-liver preparations during the induction of hepatocellular carcinomas by an 8-week aflatoxin B1 dosing regimen that produced minimal toxic effects. Significant phases of elevated tRNA methylase capacity occurred at 6 to 9 weeks (20%) and 24 to 29 weeks (40%). No significant change in tRNA methylase activity was noted over the course of the 55-week experiment. Higher aflatoxin B1 doses, producing acute toxic liver damage, resulted in elevated tRNA methylase activity (50%) and capacity (30%) at least as early as 1 week after dosing. Experiments with individual nodular lesions excised from livers of rats continuously fed a diet containing 2 ppm aflatoxin B1 demonstrated similarly elevated tRNA methylase activities and capacities in hyperplastic (preneoplastic)nodules, with and without histological evidence of carcinoma.

Aflatoxins↗

Effect of long term feeding and withdrawal of aflatoxin B1 and ochratoxin A on kidney cell transformation in albino rats.

Subacute doses (1/20 LD50) of aflatoxin B1 and ochratoxin A were fed to weanling albino rats individually and in combination for 36 weeks and then rats were maintained on toxin free normal diet for a period of 24 weeks. Livers of rats were fatty, wherever aflatoxin was administered but the enzyme activity did not show significant differences among various groups. However, in a few individuals whose livers were severely affected, higher concentrations of urine creatinine, liver RNA and DNA, and ALT enzyme activity were recorded. Histopathological examination showed various stages of hepatoma and hepatocarcinoma including nodular hyperplasia, hypertrophy, vacuolisation, degeneration, pseudolobulation, cellular infiltration and fibrosis of liver of rats fed with aflatoxin individually and in combination. Few anaplastic cells in the corticomedullary region and nuclear enlargement of proximal tubular epithelium of kidney were found wherever combined toxin and ochratoxin alone were administered. Liver tumor expression was time dependent.

Administration, Oral↗

Aflatoxin carcinogenesis in Wistar rats enhanced by thyroideum POLFA.

Comparative studies of carcinogenesis were carried out in Wistar rats receiving Thyroideum Polfa and/or aflatoxin B1. Of decisive importance for the enhancement and acceleration of aflatoxin carcinogenesis, particularly in the kidneys, was the administration of Thyroideum before and during the treatment with aflatoxin B1.

Aflatoxin B1↗

[Comparison of three methods for screening aflatoxin-producing strains].

By using rice medium microcolumn method, aflatoxin-producing ability medium dish method (APA method), A and M medium thin-layer chromato scanner method (AM method), seventy-six strains of Aspergillus flavus from the foodstuffs in Zhejiang province were screened for aflatoxin-producing strains, and which were confirmed with thin-layer chromatography method. Using the above three methods, aflatoxin-producing strains rates were detected respectively in 11.8%, 9.2% and 18.4%. Twenty aflatoxigenic strains were determined positive by all three methods (26.3%). All three methods showed false negatives. The AM method and APA method both showed false positives. The causes of false negatives and positives were discussed and the accord-rate of AM method with other methods was the highest as compared with that of microcolumn or APA method. False negative rate was the highest with APA method and next in order was that of microcolumn method. False positives appeared in both APA method and AM method. The causes of false negative and positive were discussed. It was suggested that AM method be safe, simple, and reliable method screening for aflatoxigenic strains be it improved in some aspects.

Aflatoxins↗

Characterization of the toxicity of the mycotoxins aflatoxin, ochratoxin, and T-2 toxin in game birds. I. Chukar partridge.

Chukar partridges were fed diets containing 1.25, 2.5, or 5 ppm aflatoxin; 1, 2, or 4 ppm ochratoxin A (OA); or 4, 8, or 16 ppm T-2 toxin. Toxin-induced mortality was seen during the third week with 4 ppm OA (12.5%) and 16 ppm T-2 toxin (15%), compared with the mortality in control chukars fed no toxin (2.5%). Body weights were significantly decreased by the highest level of aflatoxin at 3 weeks of age, by the highest level of OA by 2 weeks of age, and by 8 and 16 ppm T-2 toxin by 1 week of age. Aflatoxin did not affect liver weight and OA did not increase kidney weight in 3-week-old chukars. There was a slight decrease in kidney weight in chukars fed 4 ppm OA; however, the decrease was related to the decrease in body weight produced by the toxin. Mouth lesions were seen at all levels of T-2 toxin fed.

Aflatoxins↗

In vivo effect of dietary factors on the molecular action of aflatoxin B1: role of copper on the catalytic activity of liver microsome.

Wealing rats were kept on a synthetic copper-free diet for 9-13 weeks and subsequently on the same diet but supplemented with copper at different levels for 2 weeks. The ability of liver microsomes to catalyze reactions of aflatoxin B1 leading to its activation and DNA adduct formation was measured after each period of experimental feeding. An increased molecular reactivity of aflatoxin B1 was evident during deficiency such that the degree of its conversion to its reactive metabolite and subsequent DNA adduct formation was higher with microsomes from copper deficient rats as compared to rats maintained on a normal supply of copper. These activities of microsomes returned to normal after supplementation of the diet with a small level of copper. The results emphasize the role of copper in the activation, and hence in modulating the carcinogenicity of aflatoxin B1.

Aflatoxin B1↗

Determination of aflatoxin concentrations in peanut butter by enzyme-linked immunosorbent assay (ELISA): study of three commercial ELISA kits.

Sixteen United Kingdom analytical laboratories participated in an evaluation of 3 commercially available enzyme-linked immunosorbent assay (ELISA) kits for analysis of aflatoxin in peanut butter. Each laboratory was sent 3 sets of 10 randomly numbered samples of peanut butter. Each set consisted of 5 pairs of undisclosed duplicates. Four of the sets of duplicates were naturally contaminated butters with "target" aflatoxin values (estimated by liquid chromatography) between 8 and 81 micrograms/kg. The fifth pair was a blank peanut butter containing approximately 3 micrograms/kg of total aflatoxins. A statistical treatment of the results of the study is presented, together with discussion of the relative merits of the different kits.

Aflatoxins↗

Evaluation of diets contaminated with aflatoxin and ochratoxin fed to growing pigs.

The effects of dietary aflatoxin and ochratoxin, fed singly and in combination, were evaluated in growing crossbred pigs. Five barrows (7 weeks old at beginning of study) per group were fed either control feed, 2.0 mg of aflatoxin (AF)/kg of feed, 2.0 mg of ochratoxin (OA/kg of feed, or 2.0 mg of AF and 2.0 mg of OA/kg of feed for 28 days. Production performance, serum biochemical, hematologic, and pathologic evaluations were made. Body weights were reduced by the combination treatment, whereas body weight gain was decreased by all toxin treatments. The effect of AF and OA in combination on body weight gain was additive. Liver weights were increased by the combination treatment, whereas kidney weights were increased only in the OA group. Aflatoxin caused decreases in serum calcium, sodium, phosphorus, urea nitrogen, cholesterol, and glucose concentrations, whereas OA alone caused decreases in serum phosphorus, cholesterol, and hematologic values. The AF-OA treatment induced decreases in mean corpuscular volume, packed cell volume, and in serum concentrations of phosphorus, cholesterol, and urea nitrogen. The AF-OA treatment increased serum alkaline phosphatase activities and triglycerides. It was concluded that AF and OA, singly or in combination, can affect clinical performance, serum biochemical and hematologic values, and organ weights of barrows. Although values of some measurements were affected more by the combination than by either toxin alone and suggested synergism or antagonism, the toxic interactions could best be described as additive.

Aflatoxins↗

Reduction of aflatoxin content of infected cowpea seeds during processing into food.

Cowpeas have been a major source of protein, especially in developing nations. Despite of the fact that aflatoxin contaminations have been found to be mainly common in substrates such as carbohydrates, detection of the toxins in raw cowpeas has also been reported [7]. It is therefore important and necessary to investigate fate of the aflatoxin when raw cowpeas are processed into various products. In Nigeria, cowpea seeds are usually consumed after boiling to softness and mixed with ingredients such as pepper, salt and palm-oil to form a porridge. Other processing methods involve wet milling the cowpea seeds and either steaming in aluminium cups or in leaves to form 'moinmoin', or frying in oil to form 'akara' balls. In this work therefore, the fate of the aflatoxins as a result of the processing of artificially contaminated cowpeas was investigated.

Aflatoxins↗