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At least 19 recordsLinked to original sources
PubMed2005

Analysis of duplicate 24-hour diet samples for aflatoxin B1, aflatoxin M1 and ochratoxin A.

In the spring and autumn of 1994, a total diet study, in which 123 participants collected duplicates of their 24-hour diets, was carried out. The goal of this study was to determine the mass fractions of a number of analytes in these duplicate diets, so as to be able to establish oral daily intake values. After measurements were carried out for pesticides, PCBs, elements, sterols, nitrate and nitrite, and fatty acids, the duplicate diet study was concluded with analyses for aflatoxin M1, aflatoxin B1 and ochratoxin A. For this purpose a method of analysis was developed, that could simultaneously determine these mycotoxins at very low levels. The method involved chloroform extraction, liquid-liquid extraction, immunoaffinity cleanup and liquid chromatography. The method was supplemented with a procedure to confirm the identity of chromatographic peaks, assumed to represent aflatoxin M1, aflatoxin B1 and ochratoxin A. The method was in-house validated. Recoveries ranged from 68-74% for aflatoxin M1 (at spiking levels from 30-120 ng/kg, c.v. 7.6%), from 95-97% for aflatoxin B1 (at spiking levels from 50-200 ng/kg, c.v. 2.8%), and from 75-84% for ochratoxin A (at spiking levels from 150-600 ng/kg, c.v. 4.3%). Limits of quantitation (defined as signal/noise = 10) were estimated to be 24, 5 and 16 ng/kg lyophilised material for aflatoxin M1, aflatoxin B1 and ochratoxin A respectively. The newly developed method was used to analyse 123 samples of 24-hour diets. Aflatoxin M1 was detectable in 48% of the samples; the toxin contents remained below the limit of quantitation in all samples. Aflatoxin B1 could be detected in 42% of the samples; in 25% of the samples the levels were above the limit of quantitation. Ochratoxin A could be quantified in all samples. The analytical results were further processed to estimate levels of intake. Intake levels for the aflatoxins were very low, and could not reliably be established. The mean ochratoxin A intake was estimated to be 1.2 ng/kg body weight per day. This is well below the tolerable daily intake established by JECFA at 14 ng/kg body weight per day. The current dietary intake of ochratoxin A in the Netherlands is concluded to pose no appreciable health risk.

Adolescent↗
PubMedDate not supplied

Comparison of rates of enzymatic oxidation of aflatoxin B1, aflatoxin G1, and sterigmatocystin and activities of the epoxides in forming guanyl-N7 adducts and inducing different genetic responses.

The genotoxicity of the dihydrofurans aflatoxin B1 (AFB1), aflatoxin G1 (AFG1), and sterigmatocystin (STG) was examined in a bacterial system in which the induction of SOS repair is monitored with the umuC gene linked to a lacZ reporter gene in plasmid pSK1002. Human liver microsomal cytochrome P-450NF oxidized the dihydrofurans (in the presence of calf thymus DNA) to give guanyl-N7 adducts in the order AFB1 greater than STG greater than AFG1. The order of the umu response seen was STG greater than AFB1 greater than AFG1, when either the dihydrofurans were activated enzymatically or the synthetic epoxides of the dihydrofurans were added directly to the bacteria. Thus, the umu response per molecule of guanyl-N7 DNA adduct follows the order STG greater than AFB1 greater than AFG1. A similar pattern has been reported in the literature for Salmonella typhimurium base substitution dependent his reversions, but the pattern AFB1 greater than STG greater than AFG1 has been found for bacterial frame-shift-dependent mutagenesis and hepatocarcinogenesis. The guanyl-N7 adduct derived from AFG1 has considerably less of all of these biological activities per molecule. Neither guanine imidazole ring opening nor apurinic site formation appears to be a factor involved in the differential biological responses seen with the three guanyl-N7 adducts. These findings indicate that these structurally related guanyl-N7 DNA adducts have intrinsic differences which give rise to divergent biological responses.

Aflatoxin B1↗
PubMedDate not supplied

Simultaneous screening of fumonisin B1, aflatoxin B1, and zearalenone by line immunoblot: a computer-assisted multianalyte assay system.

A line immunoblot assay was developed for the simultaneous screening of fumonisin B1 (FB1), aflatoxin B1 (AFB1), and zearalenone (ZEA). Monoclonal antibodies for each of these toxins were immobilized as multiple lines on nitrocellulose membrane strips and sectored into hydrophobic compartments to minimize use of reagents. A modified enzyme-linked immunosorbent assay was conducted whereby free mycotoxins and horseradish peroxidase-labeled mycotoxins competed for binding to the nitrocellulose-bound antibodies. Color intensity of lines formed by a precipitating substrate was inversely related to mycotoxin concentration. Detection limits for the individual mycotoxins, as determined by visually comparing the color intensity of positive strips to negative controls, were 500, 0.5, and 3 ng/mL for FB1, AB1, and ZEA, respectively. Line density was quantitatively assessed also by using a camera, video monitor, and microcomputer equipped with a video-digitizing board. The assay could be used to determine range values for the various mycotoxins in extracts of spiked corn in less than 30 min, and values could be recorded on the microcomputer hard disk. This combination of line immunoblot assay and image analysis, termed computer-assisted multianalyte assay system, should be applicable to the simultaneous screening of multiple mycotoxins and other agricultural residues in food.

Aflatoxin B1↗
PubMed1988

Comparative metabolism and DNA binding of aflatoxin B1, aflatoxin M1, aflatoxicol and aflatoxicol-M1 in hepatocytes from rainbow trout (Salmo gairdneri).

DNA binding and metabolism patterns of 3H-labeled aflatoxin B1 (AFB1) and its phase I metabolites, aflatoxicol (AFL), aflatoxin M1 (AFM1), and aflatoxicol-M1 (AFL-M1), were compared in freshly prepared rainbow trout (Salmo gairdneri) hepatocytes. Aflatoxins were incubated with hepatocytes for periods up to 1 h, cellular DNA was isolated and specific activities determined by scintillation counting and Burton analysis. Data for (pmol bound aflatoxin/micrograms DNA)/(mumol dose) versus time fit a linear function (P less than 0.002) passing nearly through the origin for each aflatoxin. DNA binding at 1 h relative to AFB1 was: AFL, 0.53 +/- 0.07; AFM1, 0.81 +/- 0.20 AFL-M1, 0.83 +/- 0.24. Statistical analysis indicated that binding of AFL, AFM1 and AFL-M1 were significantly less than that of AFB1. HPLC analysis of the cellular supernatants indicated that the major metabolites were AFL, AFB1, AFL-M1, and AFM1 from AFB1, AFL, AFM1 and AFL-M1 substrates, respectively. Small quantities of hydroxylated metabolites and glucuronides also were detected in some of the incubations. The time-course data suggested that initial formation of major metabolites was rapid and that, by 20-30 min, net changes in metabolite levels decreased or approached zero. Because the four compounds possess a 8,9-double bond, DNA binding could be due to activation of the parent substrates as well as of their phase I metabolites. Based on current mutagenicity data and limited carcinogenicity studies, AFM1 and AFL-M1 have binding levels which are higher than expected compared to AFB1 and AFL.

Aflatoxin B1↗
PubMed1983

Purified form of cytochrome P-450 from rainbow trout with high activity toward conversion of aflatoxin B1 to aflatoxin B1-2,3-epoxide.

Aflatoxin B1, the most potent hepatic chemical carcinogen known, is activated to the putative product aflatoxin B1-2,3-epoxide via a cytochrome P-450-dependent reaction. Mt. Shasta rainbow trout is the most sensitive species known to the hepatocarcinogenic effects of aflatoxin B1. We have previously isolated and purified a minor form of cytochrome P-450 from this strain of rainbow trout, with a lambda max in the carbon monoxide-reduced difference spectrum of 449.5 nm and a molecular weight of 54,000. In this study, we have compared in a reconstituted system this trout P-450 to trout cytochrome P-448 and rat cytochrome P-450 and P-448 for metabolism and activation of aflatoxin B1. Trout cytochrome P-450 had much higher activity towards aflatoxin B1 and a greater degree of regioselectivity in the formation of aflatoxin B1-2,3-dihydroxy-2,3-dihydrodiol and was much more efficient in producing aflatoxin B1 covalent adducts with DNA. The existence of such a form of cytochrome P-450 in Mt. Shasta rainbow trout may be responsible for the acute sensitivity of this strain to the carcinogenic effects of aflatoxin B1.

Aflatoxin B1↗
PubMed1997

Metabolic activation of aflatoxin B1 to aflatoxin B1-8,9-epoxide in woodchucks undergoing chronic active hepatitis.

Chronic hepatitis B virus infection as well as consumption of food contaminated with the mycotoxin aflatoxin B1 are considered to be 2 major risk factors for the development of primary liver cancer in humans. Furthermore, epidemiological surveys indicate that hepatitis B virus and aflatoxin B1 might act synergistically to induce primary liver cancer. In the present study, we have tested the hypothesis that the metabolic activation of aflatoxin B1 to aflatoxin B1-8,9-epoxide, the ultimate mutagenic and carcinogenic mycotoxin metabolite, is enhanced in an experimental model of chronic hepatitis using woodchucks, chronically infected with the woodchuck hepatitis virus. Woodchuck liver microsomes were incubated with radiolabeled aflatoxin B1, the resulting aflatoxin B1-8,9-epoxide was trapped as a glutathione conjugate and its formation rate was determined by a reversed-phase HPLC analysis. In woodchuck hepatitis virus-positive woodchucks, activation of aflatoxin B1 to aflatoxin B1-8,9-epoxide was reduced when compared to woodchuck hepatitis virus-free animals, and the extent of the reduction was dependent on the severity of the hepatitis. Hence, at least in woodchucks, a chronic hepadnaviral infection does not lead to an enhanced activation of aflatoxin B1.

Aflatoxin B1↗
PubMed1980

Quantitation of aflatoxin B1 and aflatoxin B1 antibody by an enzyme-linked immunosorbent microassay.

A specific microtest plate enzyme immunoassay has been developed for the rapid quantitation of aflatoxin B1 at levels as low as 25 pg per assay. Multiple-site injection of rabbits with an aflatoxin B1 carboxymethyloxime-bovine serum albumin conjugate was used for the production of hyperimmune sera. Dilutions of the purified antibody were air dried onto microplates previously treated with bovine serum albumin and glutaraldehyde and then incubated with an aflatoxin B1 carboxymethyloxime-horseradish peroxidase conjugate. The amount of enzyme bound to antibody was determined by monitoring the change in absorbance at 414 nm after the addition of a substrate solution consisting of hydrogen peroxide and 2,2'-azino-di-3-ethyl-benzthiazoline-6-sulfonate. Antibody titers determined in this manner closely correlated with those determined by radioimmunoassay. Competition assays as performed by incubation of different aflatoxin analogs with the peroxidase conjugate showed that aflatoxins B1 and B2 and aflatoxicol caused the most inhibition of conjugate binding to antibody. Aflatoxins G1 and G2 inhibited the conjugate binding to a lesser degree, whereas aflatoxins M1 and B2a had no effect of the assay.

Aflatoxins↗
PubMed1984

Effects of chronic exposure to aflatoxin B1 and aflatoxin M1 on the in vivo covalent binding of aflatoxin B1 to hepatic macromolecules.

Induction of resistance to aflatoxin B1 (AFB1) binding to cellular macromolecules in the rat by chronic exposure to AFB1 and aflatoxin M1 (AFM1) was investigated. The binding of [14C]AFB1 to liver macromolecules was measured in F-344 rats fed 0.5 ppb or 50 ppb AFM1 or 50 ppb AFB1 for 41 wk. The animals then received an intragastric dose of [14C]AFB1 at 5 micrograms/kg and were sacrificed 6 h later. Hepatic DNA, RNA, and protein were isolated by chloroform-phenol extraction and hydroxylapatite chromatography. In animals preexposed to 50 ppb AFB1, labeled AFB1 binding to DNA, RNA, and protein was decreased by 72%, 74%, and 61%, respectively. Preexposure to AFM1 resulted in a small reduction in binding to nucleic acids. Glutathione transferase activity was increased by 133% in animals fed 50 ppb AFB1, by 48% in those preexposed to 50 ppb AFM1, and remained at control values in rats fed 0.5 ppb AFM1. These results suggest that the induction of detoxification enzymes following chronic exposure to aflatoxin might contribute to the reduction in covalent binding of AFB1 to macromolecules.

Aflatoxin B1↗
PubMedDate not supplied

Oxidation of aflatoxins and sterigmatocystin by human liver microsomes: significance of aflatoxin Q1 as a detoxication product of aflatoxin B1.

Aflatoxin Q1 8,9-oxide was synthesized and found to yield lower levels of N7-guanyl adducts than obtained from aflatoxin B1 8,9-oxide when mixed with calf thymus DNA or Salmonella typhimurium TA 98 cells. However, when S. typhimurium TA 98 was treated with the (analogous) epoxides of aflatoxin B1, aflatoxin G1, aflatoxin Q1, or sterigmatocystin, the ratios of revertants to N7-guanyl DNA adducts were similar. Aflatoxin Q1 and aflatoxin B1 8,9-oxide (trapped here as the glutathione conjugate) are the major oxidative products formed from aflatoxin B1 at all substrate concentrations in human liver microsomes, and cytochrome P-450 (P-450) 3A4 appears to be the dominant enzyme involved in both oxidations, as judged by studies involving correlation of activities in different liver samples, chemical inhibition, immunoinhibition, and reconstitution with purified hepatic and yeast recombinant P-450 3A4. Aflatoxin Q1 is not appreciably oxidized in human liver microsomes and is not very genotoxic. The postulated formation of both aflatoxin Q1 and aflatoxin 8,9-oxide from aflatoxin B1 can be rationalized by a model in which P-450 3A4 binds the substrate in either of two different configurations. This is further demonstrated by the dichotomous effect of 7,8-benzoflavone--this flavone stimulates 8,9-epoxidation while inhibiting the 3 alpha-hydroxylation reaction to form aflatoxin Q1. Thus, the 3 alpha-hydroxylation of aflatoxin B1 to aflatoxin Q1 is viewed as a potentially significant detoxication pathway.

Aflatoxin B1↗
PubMed1997

Experimental induction of liver fibrosis in young guinea pigs by combined application of copper sulphate and aflatoxin B1.

Aflatoxin B1 alone (0.05 mg resp. 0.037 mg/kg/d), copper alone (6.6 mg/kg/d or 200 mg/l drinking water) or a combination of both was administered orally for 6 months to young guinea pigs from the first/second day of life. In the copper group there were no pathomorphological changes. For the aflatoxin B1 group, liver damage was established. In the combined group, liver injury was more frequent and more severe compared to the aflatoxin B1 group and biliary copper excretion was diminished compared with the copper group. Histologically, only the livers of this group exhibited degeneration, atrophy and steatosis of liver cells, inflammatory processes and a more or less prominent fibrosis. For childhood cirrhosis (ICC and ICT) a combined etiology--a liver damaging agent plus elevated alimentary copper--is a plausible hypothesis.

Aflatoxin B1↗
PubMed1982

Kinetics of transformation of aflatoxin B1 into aflatoxin M1 in lactating mouse: an ELISA analysis.

A new enzyme-linked immunosorbent assay (ELISA) was used to study the kinetics of transformation of aflatoxin B1 into aflatoxin M1 in lactating mice. Aflatoxin M1 concentration in the milk samples reached a maximum 30 min after injection of aflatoxin B1 and decreased thereafter. At the maximum time, the levels of aflatoxin M1 in the samples were proportional to the dosages administered. Aflatoxin B1 was also detected in the milk samples but at a lower concentration.

Aflatoxin B1↗
PubMed1981

Histochemical assessment of 3 beta-hydroxysteroid dehydrogenase activity in the testes of rats following acute administration of aflatoxin B1.

Aflatoxin B1 (150 microgram) was administered i.p. to adult male rats on 3 occasions at intervals of 48 h. The testicular distribution and localisation of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was evaluated histochemically in rats killed 24 h after the last dose of aflatoxin, with or without human chorionic gonadotropin (HCG) stimulation. Diminished augmentation of seminal vesicle and prostate weights to HCG stimulation was recorded in male rats pretreated with aflatoxin. There were no convincing effects of aflatoxin pretreatment on the histochemical localisation and distribution of 3 beta-HSD in the testes of the rats used in this investigation.

3-Hydroxysteroid Dehydrogenases↗
PubMed1987

Carcinogenicity of dietary aflatoxin M1 in male Fischer rats compared to aflatoxin B1.

Aflatoxin M1 (AFM), an hydroxy metabolite of the potent carcinogenic mycotoxin aflatoxin B1 (AFB) is frequently found in milk and other dairy products. Sufficient amounts of AFM were produced to study the carcinogenicity of this compound. AFM was fed to male Fischer rats starting at 7 weeks up to 21 months of age. Agar-based semisynthetic diets contained 0.0, 0.5, 5.0, and 50.0 micrograms/kg of AFM or 50 micrograms/kg of AFB. Hepatocellular carcinomas were detected in two of 37 rats and neoplastic nodules were found in six of 37 rats fed 50 micrograms/kg AFM between 19 and 21 months. No nodules or carcinomas were observed in the lower AFM dose groups. Nineteen of 20 rats fed a diet containing 50 micrograms/kg of AFB developed hepatocellular carcinomas by 19 months of age. Carcinogenic potency of the aflatoxins was reflected by morphometric quantitation of foci detected in hematoxylin and eosin stained sections. Three rats fed the diet containing 50 micrograms/kg AFM developed intestinal carcinomas. None were observed in other groups. Under the conditions of this experiment AFM was found to be a weak hepatic carcinogen compared to AFB and to possess intestinal carcinogenicity.

Aflatoxin B1↗
PubMed1978

Reduced nicotinamide adenine dinucleotide phosphate-dependent formation of 2,3-dihydro-2,3-dihydroxyaflatoxin B1 from aflatoxin B1 by hepatic microsomes.

2,3-Dihydro-2,3-dihydroxyaflatoxin B1 (dihydrodiol) was formed as a major metabolite in the incubation of aflatoxin B1 with rat and hamster liver microsomes. The yield of the dihydrodiol was maximal at pH 6.5, was reduced nicotinmide adenine dinucleotide phosphate- and cytochrome P-450-dependent, and was increased 2- to 4-fold by pretreatment of the animals with phenobarbital; pretreatment with 3-methylcholanthrene did not alter the activity of rat hepatic microsomes. Inhibitors of epoxide hydrase did not lower the yield of the dihydrodiol in these systems. Negligible yields of the dihydrodiol were formed from aflatoxin B1 and rat liver microsomes in the presence of DNA. Little or no formation of the dihydrodiol was noted with microsomes from rat intestinal mucosa, kidney, or lung. These results further support the formation of aflatoxin B1 2,3-oxide as a major electrophilic metabolite of aflatoxin B1 in rat and hamster liver microsomal systems, since this highly reactive epoxide would be expected to hydrolyze readily to form the dihydrodiol.

Aflatoxins↗
PubMed1997

[Transformation of rat hepatocytes in an in vitro primary culture by aflatoxin B1].

Aflatoxin B1 (AFB1) is one of the major causative factors of hepatocellular carcinoma. In this study, the combined effects of AFB1 activated by human cytochrome p450 IA2 and c-myc in transformation of rat hepatocytes were investigated in an in vitro primary culture system. The expression vectors, Xm-6/c-myc was first constructed and their expression possibilities were examined in Alexander cells by immunocytochemistry. Then both c-myc and human cytochrome p450 IA2 expression vectors were sequentially transfected into newborn rat liver cells in serum-free primary culture. Results showed that p450 IA2 could activate AFB1 at concentrations as low as 5 ng/ml, and the activated AFB1 coupled with exogenous c-myc could induce rat hepatocytes to survive and grow beyond two-month limit in primary culture. During long-term in vitro culturing including four-month in crisis, one of the randomly selected transformed hepatocytes with the growth advantage became immortalized. Immunocytochemical assays for CK-18 and rat albumin plus observed electron microscopic features clearly confirmed these cells derived from epithelial hepatocytes. Further characterization showed that the process of immortalization was associated with chromosomal abnormalities and elevated expression of TGF alpha.

Aflatoxin B1↗
PubMed1998

Activation and detoxication of aflatoxin B1.

Aflatoxin B1 (AFB1) is a potent hepatocarcinogen in experimental animals and a hazard to human health in several parts of the world. Implementation of rational intervention plans requires understanding of aspects of the roles of individual chemical steps involved in its disposition. AFB1 is activated to AFB1 exo-8,9-epoxide primarily by cytochrome P450 (P450) enzymes, particularly P450 3A4. However, P450 3A4 and other P450s also oxidize AFB1 to less dangerous products. The exo-epoxide is unstable in H2O (t1/2 1 s at 25 degreesC, k=0.6 s-1) and the diol product undergoes base-catalyzed rearrangement to a dialdehyde that reacts with protein lysine residues. AFB1 exo-8, 9-epoxide reacts with DNA to give adducts in high yield (>98%). This interaction is characterized by a Kd of approximately 1.4 mM, intercalation between base pairs, and rapid reaction with the guanyl N7 atom (k approximately 40 s-1). A proton field on the periphery of DNA is postulated to catalyze hydrolysis and also conjugation. Rat and especially human epoxide hydrolase show very little rate acceleration of hydrolysis of AFB1 exo- or endo-8,9-epoxide. However, glutathione transferases (GSTs) can catalyze AFB1 exo-8,9-epoxide conjugation. Kinetic analysis indicates a range of ratios of kcat/Kd varying from 10 to 1700 s-1 M-1, with the polymorphic GST M1-1 having the highest activity of the human GSTs. Studies with human hepatocytes indicate a major role for GST M1-1 in AFB1 conjugation and that the model chemoprotective agent oltipraz can act by both inducing GSTs and inhibiting P450s.

Aflatoxin B1↗
PubMed2006

Unraveling the aflatoxin-FAPY conundrum: structural basis for differential replicative processing of isomeric forms of the formamidopyrimidine-type DNA adduct of aflatoxin B1.

Aflatoxin B1 (AFB) epoxide forms an unstable N7 guanine adduct in DNA. The adduct undergoes base-catalyzed ring opening to give a highly persistent formamidopyrimidine (FAPY) adduct which exists as a mixture of forms. Acid hydrolysis of the FAPY adduct gives the FAPY base which exists in two separable but interconvertible forms that have been assigned by various workers as functional, positional, or conformational isomers. Recently, this structural question became important when one of the two major FAPY species in DNA was found to be potently mutagenic and the other a block to replication [Smela, M. E.; Hamm, M. L.; Henderson, P. T.; Harris, C. M.; Harris, T. M.; Essigmann, J. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 6655-6660]. NMR studies carried out on the AFB-FAPY bases and deoxynucleoside 3',5'-dibutyrates now establish that the separable FAPY bases and nucleosides are diastereomeric N5 formyl derivatives involving axial asymmetry around the congested pyrimidine C5-N5 bond. Anomerization of the protected beta-deoxyriboside was not observed, but in the absence of acyl protection, both anomerization and furanosyl --> pyranosyl ring expansion occurred. In oligodeoxynucleotides, two equilibrating FAPY species, separable by HPLC, are assigned as anomers. The form normally present in duplex DNA is the mutagenic species. It has previously been assigned as the beta anomer by NMR (Mao, H.; Deng, Z. W.; Wang, F.; Harris, T. M.; Stone, M. P. Biochemistry 1998, 37, 4374-4387). In single-stranded environments the dominant species is the beta anomer; it is a block to replication.

Aflatoxin B1↗