Immunodetection and immunopurification of aflatoxins using a high affinity monoclonal antibody to aflatoxin B1

A monoclonal antibody was obtained from BALB/c mice immunized with aflatoxin Bl (AFB1) conjugated to bovine serum albumin. This IgG2a antibody, ASCI, with K light chain has a high specificity for AFB1. In an indirect enzyme-linked immunosorbent assay the antibody litre in ascites fluid was 1: 6000 for 50% binding to plates coated with aflatoxin-poly-L-lysine. The assay is sensitive to 2.5 pg aflatoxin/assay. ASCI cross-reacts with closely related aflatoxin metabolites such as AFB2, AFM1 and AFG1. However, ASCI displays negligible cross-reactivity with other related aflatoxin analogues such as AFM2, AFP1, AFQ1 and aflatoxicol. An immunoabsorbent was prepared by coupling ASCI antibody to Ultrogel AcA 22. This immunomatrix was used to purify aflatoxins at 0–1 ng/ml levels from contaminated body fluids such as bovine milk. The antibody affinity column was regenerated and re-used several times. Owing to its high specificity for AFB1 and AFM1, ASCI will be of value in immunodetection and immunopurification of these toxins in various foodstuffs.     

Aflatoxin Binders II: Reduction of aflatoxin M1 in milk by sequestering agents of cows consuming aflatoxin in feed

Sequestering agents bind dietary aflatoxin B1 (AFB1) and reduce absorption from an animal's gastrointestinal tract. As a result, they protect an animal from the toxic effects of AFB1 and reduce transfer of the metabolite, aflatoxin M1 (AFM1), into milk. Three experiments, using late-lactation Holstein cows fed AFB1-contaminated feed, were conducted to evaluate several potential sequestering agents for their abilities to prevent or reduce the transmission of AFM1 into milk. Six agents previously tested in our laboratory for AFB1 binding in vitro were evaluated in these experiments. These were: SA-20, an activated carbon (AC-A); Astra-Ben-20, a sodium bentonite (AB-20); MTB-100, an esterified glucomannan (MTB-100); Red Crown, a calcium bentonite (RC); Flow Guard, a sodium bentonite (FG); and Mycrosorb, a sodium bentonite (MS). Five of the six sequestering agents significantly (P < 0.01) reduced AFM1 contamination of milk (AB-20, 61%; FG, 65%; MS, 50%; MTB-100, 59%; and RC, 31%); whereas, AC-A, activated carbon, had no effect on AFM1 transmission at 0.25% of feed. By the first milking (1 day after cows consumed contaminated feed), AFM1 appeared in milk, then reached maximum levels after three days, and was absent from milk within four days after AFB1 was removed from the feed. Sodium bentonites at 1.2% of feed showed good potential as AFB1 binders; MTB-100, a yeast cell wall product, was equally effective at 0.05% in feed. Potential AFB1 binding agents should be evaluated experimentally to demonstrate efficacy. Our data show that sequestering agents can reduce AFM1 in milk of cows fed AFB1-contaminated feed.     

Carryover of aflatoxin from feed to milk in dairy cows with low or high somatic cell counts

Aflatoxin M1 (AFM1) residues in milk are regulated in many parts of the world and can cost dairy farmers significantly due to lost milk sales. Additionally, due to the carcinogenicity of this compound contaminated milk can be a major public health concern. Thirty-four lactating dairy cows were utilised to investigate the relationship between somatic cell counts (SCC), milk yield and conversion of dietary aflatoxin B1 (AFB1) into milk AFM1 (carryover (CO)). The AFM1 in milk increased as soon as the first milking after animal ingestion with a pattern of increment up to the observed plateau (between 7th and 12th days of AFB1 ingestion). There was a significant (P < 0.01) effect of the milk yield whereas no effect could be attributed to the SCC levels or to the milk yield × SCC interaction. Similarly, the main effect of milk yield was also observed (P < 0.01) on the total amount of AFM1 excreted during the ingestion period. Although the plasma concentration of gamma-glutamyl transferase was significantly affected by aflatoxin administration, levels of this liver enzyme were within the normal range for lactating dairy cows. The current data suggest that milk yield is the major factor affecting the total excretion of AFM1 and that SCC as an indicator of mammary gland permeability was not related to an increase in AFM1 CO.     

Conversion of 11-hydroxy-O-methylsterigmatocystin to aflatoxin G1 in Aspergillus parasiticus

In aflatoxin biosynthesis, aflatoxins G(1) (AFG(1)) and B(1) (AFB(1)) are independently produced from a common precursor, O-methylsterigmatocystin (OMST). Recently, 11-hydroxy-O-methylsterigmatocystin (HOMST) was suggested to be a later precursor involved in the conversion of OMST to AFB(1), and conversion of HOMST to AFB(1) was catalyzed by OrdA enzyme. However, the involvement of HOMST in AFG(1) formation has not been determined. In this work, HOMST was prepared by incubating OrdA-expressing yeast with OMST. Feeding Aspergillus parasiticus with HOMST allowed production of AFG(1) as well as AFB(1). In cell-free systems, HOMST was converted to AFG(1) when the microsomal fraction, the cytosolic fraction from A. parasiticus, and yeast expressing A. parasiticus OrdA were added. These results demonstrated (1) HOMST is produced from OMST by OrdA, (2) HOMST is a precursor of AFG(1) as well as AFB(1), and (3) three enzymes, OrdA, CypA, and NadA, and possibly other unknown enzymes are involved in conversion of HOMST to AFG(1).     

Interaction of aflatoxin with L-ascorbic acid: a kinetic and mechanistic approach.

Aflatoxins containing B(1), B(2), G(1) and G(2) obtained by growing Aspergillus parasiticus on SMKY liquid medium were tested for cytotoxicity (hemolysis) on RBC suspension in the presence and absence of L-ascorbic acid (AA). The results revealed that hemolysis was significantly increased on increasing the concentration of aflatoxin (0.5-3 microg ml(-1)). It was also found that pretreatment with AA (5-100 microg ml(-1)) significantly decreased aflatoxin-induced hemolysis. The solution chemistry of the interaction of aflatoxin with AA in aqueous solutions showed enhanced conversion of AFB(1) and AFG(1) to AFB(2) and AFG(2), respectively. Hemolytic, kinetic and mechanistic aspects of the interactions of aflatoxins and AA are discussed.     

Production of M-/GM-group aflatoxins catalyzed by the OrdA enzyme in aflatoxin biosynthesis

Aspergillus parasiticus produces the minor aflatoxins M(1) (AFM(1)), M(2) (AFM(2)), GM(1) (AFGM(1)), and GM(2) (AFGM(2)), as well as the major aflatoxins B(1) (AFB(1)), B(2) (AFB(2)), G(1) (AFG(1)), and G(2) (AFG(2)). Feeding of A. parasiticus with aspertoxin (12c-hydroxyOMST) caused AFM(1) and AFGM(1), and cell-free experiments using the microsomal fraction of A. parasiticus and aspertoxin caused production of AFM(1), indicating that aspertoxin is a precursor of AFM(1) and AFGM(1). Feeding of the same fungus with O-methylsterigmatocystin (OMST) caused AFM(1) and AFGM(1) together with AFB(1) and AFG(1); feeding with dihydroOMST (DHOMST) caused AFM(2) and AFGM(2) together with AFB(2) and AFG(2). Incubation of either the microsomal fraction or OrdA enzyme-expressing yeast with OMST caused production of aspertoxin together with AFM(1) and AFB(1). These results demonstrated that the OrdA enzyme catalyzes both 12c-hydroxylation reaction from OMST to aspertoxin and the successive reaction from aspertoxin to AFM(1). In contrast, feeding of the fungus with AFB(1) did not produce any AFM(1), demonstrating that M-/GM-aflatoxins are not produced from B-/G-aflatoxins. Furthermore, AFM(1) together with AFB(1) and AFG(1) was also produced from 11-hydroxyOMST (HOMST) in feeding experiment of A. parasiticus, whereas no aflatoxins were produced when used the ordA deletion mutant. These results demonstrated that OrdA enzyme can also catalyze 12c-hydroxylation of HOMST to produce 11-hydroxyaspertoxin, which serves as a precursor for the production of AFM(1) and AFGM(1). The same pathway may work for the production of AFM(2) and AFGM(2) from DHOMST and dihydroHOMST through the formation of dihydroaspertoxin and dihydro-11-hydroxyaspertoxin, respectively.     

Biosynthetic origin of aflatoxin G1: confirmation of sterigmatocystin and lack of confirmation of aflatoxin B1 as precursors

The origin of aflatoxin G1 was studied using mutant strains of Aspergillus parasiticus blocked early in the pathway and by tracing 14C-labelled aflatoxin B1 (AFB1) in wild-type A. flavus and A. parasiticus strains. Sterigmatocystin (ST) was a precursor of AFB1, AFG1 and AFG2 in the four mutants examined. The identity of AFG1 was confirmed by mass spectrometry. No evidence for conversion of AFB1 to AFG1 was found. A rigorously controlled study of conversions of radioactivity based on preparative thin-layer chromatography of aflatoxins demonstrated that low levels of aflatoxin interconversions previously reported in the literature might actually be artifacts.     

Conversion of a new metabolite to aflatoxin B2 by Aspergillus parasiticus

A new metabolite which could be converted to aflatoxin (AF) B2 was detected during cofermentation analysis of two nonaflatoxigenic strains (SRRC 2043 and SRRC 163) of Aspergillus parasiticus. SRRC 2043, which accumulates the xanthone O-methylsterigmatocystin (OMST), a late precursor in the AFB1 pathway, was observed to accumulate another chemically related compound (HOMST; molecular weight, 356); SRRC 163 is blocked early in the pathway and accumulates averantin. During cofermentation of the two strains, levels of OMST and HOMST were observed to be greatly reduced in the culture, with simultaneous production of AFB1, AFB2, and AFG1. Intact cells of SRRC 163 were able to convert pure OMST or its precursor, sterigmatocystin, to AFB1 and AFG1 without AFB2 accumulation; the same cells converted isolated HOMST to AFB2 with no AFB1 or AFG1 production. The results indicate that AFB2 is produced from a separate branch in the AF biosynthetic pathway than are AFB1 and AFG1; AFB2 arises from HOMST, and AFB1 and AFG1 arise from sterigmatocystin and OMST.     

Conversion of a new metabolite to aflatoxin B2 by Aspergillus parasiticus.

A new metabolite which could be converted to aflatoxin (AF) B2 was detected during cofermentation analysis of two nonaflatoxigenic strains (SRRC 2043 and SRRC 163) of Aspergillus parasiticus. SRRC 2043, which accumulates the xanthone O-methylsterigmatocystin (OMST), a late precursor in the AFB1 pathway, was observed to accumulate another chemically related compound (HOMST; molecular weight, 356); SRRC 163 is blocked early in the pathway and accumulates averantin. During cofermentation of the two strains, levels of OMST and HOMST were observed to be greatly reduced in the culture, with simultaneous production of AFB1, AFB2, and AFG1. Intact cells of SRRC 163 were able to convert pure OMST or its precursor, sterigmatocystin, to AFB1 and AFG1 without AFB2 accumulation; the same cells converted isolated HOMST to AFB2 with no AFB1 or AFG1 production. The results indicate that AFB2 is produced from a separate branch in the AF biosynthetic pathway than are AFB1 and AFG1; AFB2 arises from HOMST, and AFB1 and AFG1 arise from sterigmatocystin and OMST.     

Automated column-switching high-performance liquid chromatography for the determination of aflatoxin M1

An extractionless method for determining aflatoxin M1 (AFM1), a major metabolite of aflatoxin B1 (AFB1), in human urine was developed. The biological fluid is injected directly into the chromatographic system after simple dilution and centrifugation. A pre-column, packed with a cation-exchange phase and coupled on-line to a column-switching liquid chromatography (LC) system, is used for sample pre-treatment and concentration. The analytes are non-selectively desorbed with the LC eluent and cleaned by means of a column-switching procedure. Pre-treatment and analysis were performed within 40 min. Average AFM1 recovery reached 97% in the 10–100 ng/l range of urine. The detection limit of AFM1 in urine and milk was 2.5 ng/l for 1 ml of injected sample. A comparison with an immunoaffinity column clean-up and LC method was performed. The method was applied to determine AFM1 in the urine of AFB1 gavaged rats, and in the urine of both potentially exposed and supposedly unexposed workers. The method was also extended to milk.     

Comparison of aflatoxin B1 and aflatoxin G1 binding to cellular macromolecules in vitro, in vivo and after peracid oxidation; characterisation of the major nucleic acid adducts.

A comparison between [14C]aflatoxin B1 (AFB1) and [14C]aflatoxin G1 (AFG1) binding to rat liver and kidney cellular macromolecules has shown AFG1-DNA and-ribosomal RNA binding to be lower in both organs. For both mycotoxins more was bound to nucleic acids than to protein. Two hours after intraperitoneal injection (60 microgram/100 g) of [14C] AFB1, 40 ng, 151 ng/mg. Loss of radioactivity bound to liver DNA for both [14C]AFB1 and protein respectively and for [14C]AFG1 the respective figures were 10, 7 and 1 ng/mg. Loss of liver bound radioactivity to DNA for both [14C]AFG1 and [14C]AFG1 appeared to be biphasic indicating that an enzymic DNA repair process may be operating. In vitro binding studies also showed less AFG1 was bound to exogenous DNA after microsomal activation than AFB1. This difference was not a result of differences in the chemical reactivity of the "ultimate" electrophilic species, the respective expoxides, since chemical activation studies using 3-chloroperbenzoic acid showed similar amounts of AFG1 and AFB1 to be converted to the epoxides and to bind to DNA. Studies on the distribution coefficients of the two mycotoxins showed AFB1 to be more lipophilic than AFG1 and this may be an important factor in determining the weaker carcinogenicity of the latter compound. Characterisation of the major AFG1-DNA adduct formed in vitro, in vivo and after peracid oxidation showed it to have the structure trans-9,10-dihydro-9-(7-guanyl)-10-hydroxy-aflatoxin G1. This adduct is similar to that obtained from AFB1 by activation in vivo, in vitro and after peracid oxidation.     

In vitro metabolism of aflatoxin B1 by larvae of navel orangeworm, Amyelois transitella (Walker) (Insecta, Lepidoptera, Pyralidae) and codling moth, Cydia pomonella (L.) (Insecta, Lepidoptera, Tortricidae)

Larvae of the navel orangeworm (NOW), Amyelois transitella (Walker), a major pest of almonds and pistachios, and the codling moth (CM), Cydia pomonella (L.), the principal pest of walnuts and pome fruits, are commonly found in tree nut kernels that can be contaminated with aflatoxin, a potent carcinogen. The ability of larvae of these insects to metabolize aflatoxin B1 (AFB1) was examined. A field strain of NOW produced three AFB1 biotransformation products, chiefly aflatoxicol (AFL), and minor amounts of aflatoxin B2a (AFB2a) and aflatoxin M1 (AFM1). With AFL as a substrate, NOW larvae produced AFB1 and aflatoxicol M1 (AFLM1). A lab strain of CM larvae produced no detectable levels of AFB1 biotransformation products in comparison to a field strain which produced trace amounts of only AFL. Neither NOW nor CM produced AFB1-8,9-epoxide (AFBO), the principal carcinogenic metabolite of AFB1. In comparison, metabolism of AFB1 by chicken liver yielded mainly AFL, whereas mouse liver produced mostly AFM1 at a rate eightfold greater than AFL. Mouse liver also produced AFBO. The relatively high production of AFL by NOW compared to CM may reflect an adaptation to detoxify AFB1. NOW larvae frequently inhabit environments highly contaminated with fungi and, hence, aflatoxin. Only low amounts, if any, of this mycotoxin occur in the chief CM hosts, walnuts, and pome fruits. Characterizations of enzymes and co-factors involved in biotransformation of AFB1 are discussed.     

Production of ultrasensitive antibodies against aflatoxin B1

AIMS: To produce specific antibodies against the haptenic fungal toxin aflatoxin B1 (AFB1) and apply these antibodies in immunochemical assays for aflatoxins. METHODS AND RESULTS: Rabbits were immunized using an AFB1-bovine serum albumin conjugate and serum titres determined by double-antibody enzyme immunoassay. High titres of antibodies with very high affinity for AFB1 were obtained 15 and 4 weeks after the initial immunization and the first booster immunization respectively. The antibodies were employed in enzyme immunoassay (EIA) and immunoaffinity chromatography (IAC) methods for aflatoxins. With a detection limit of 15.8 pg ml(-1) for AFB1, the EIA employing these antibodies is the most sensitive test for AFB1 described so far. In IAC columns, these antibodies provided high binding capacity for all major aflatoxins, including AFB1, AFB2, AFG1 and AFG2. CONCLUSION: The antibodies described here are useful for the analysis of trace levels of aflatoxins. SIGNIFICANCE AND IMPACT OF THE STUDY: Polyclonal antibody-based EIA and IAC methods for aflatoxin analysis offer a suitable alternative to the more expensive monoclonal antibody-based methods.     

Aflatoxin B1 dihydrodiol antibody: production and specificity.

A specific antibody for 2,3-dihydro-2,3-dihydroxyaflatoxin B1 (AFB1-diol) was prepared, and its reactivity was characterized for the major aflatoxin (AF) B1 (AFB1) metabolites. Reductive alkylation was used to conjugate AFB1-diol to ethylenediamine-modified bovine serum albumin (EDA-BSA) and horseradish peroxidase for use as an immunogen and an enzyme-linked immunosorbent assay (ELISA) marker, respectively. High reactant ratios, 1:5 and 1:10, for AFB1-diol-EDA-BSA (wt/wt) resulted in precipitated conjugates which were poorly immunogenic. However, a soluble conjugate obtained by using a 1:25 ratio of AFB1-diol to EDA-BSA could be used for obtaining high-titer AFB1-diol rabbit antibody within 10 weeks. Competitive ELISAs revealed that the AFB1-diol antibody detected as little as 1 pmol of AFB1-diol per assay. Cross-reactivity of AFB1-diol antibody in the competitive ELISA with AF analogs was as follows: AFB1-diol, 100%; AFB1, 200%; AFM1, 130%; AFB2a, 100%; AFG1, 6%; AFG2, 4%; aflatoxicol, 20%; AFQ1, 2%; AFB1-modified DNA, 32%; and 2,3-dihydro-2-(N7-guanyl)-3-hydroxy AFB1, 0.6%. These data indicated that the cyclopentanone and methoxy moieties of the AF molecule were the primary epitopes for the AFB1-diol antibody. The AFB1-diol competitive ELISA was subject to substantial interference by human, rat, and mouse serum albumins but not by BSA, Tris, human immunoglobulin G, or lysozyme. By using a noncompetitive, indirect ELISA with an AFB1-modified DNA solid phase, a modification level of one AFB1 residue for 200,000 nucleotides could be determined.     

Aflatoxin B1 dihydrodiol antibody: production and specificity

A specific antibody for 2,3-dihydro-2,3-dihydroxyaflatoxin B1 (AFB1-diol) was prepared, and its reactivity was characterized for the major aflatoxin (AF) B1 (AFB1) metabolites. Reductive alkylation was used to conjugate AFB1-diol to ethylenediamine-modified bovine serum albumin (EDA-BSA) and horseradish peroxidase for use as an immunogen and an enzyme-linked immunosorbent assay (ELISA) marker, respectively. High reactant ratios, 1:5 and 1:10, for AFB1-diol-EDA-BSA (wt/wt) resulted in precipitated conjugates which were poorly immunogenic. However, a soluble conjugate obtained by using a 1:25 ratio of AFB1-diol to EDA-BSA could be used for obtaining high-titer AFB1-diol rabbit antibody within 10 weeks. Competitive ELISAs revealed that the AFB1-diol antibody detected as little as 1 pmol of AFB1-diol per assay. Cross-reactivity of AFB1-diol antibody in the competitive ELISA with AF analogs was as follows: AFB1-diol, 100%; AFB1, 200%; AFM1, 130%; AFB2a, 100%; AFG1, 6%; AFG2, 4%; aflatoxicol, 20%; AFQ1, 2%; AFB1-modified DNA, 32%; and 2,3-dihydro-2-(N7-guanyl)-3-hydroxy AFB1, 0.6%. These data indicated that the cyclopentanone and methoxy moieties of the AF molecule were the primary epitopes for the AFB1-diol antibody. The AFB1-diol competitive ELISA was subject to substantial interference by human, rat, and mouse serum albumins but not by BSA, Tris, human immunoglobulin G, or lysozyme. By using a noncompetitive, indirect ELISA with an AFB1-modified DNA solid phase, a modification level of one AFB1 residue for 200,000 nucleotides could be determined.     

Natural Occurrence of Aflatoxins from Maize in Iran

Fifty-one maize samples, intended for animal feed and human consumption, were collected from the four main maize production provinces in Iran and analyzed by high-performance liquid chromatography (HPLC) for contamination by four naturally occurring aflatoxin analogues (AFB1, AFB2, AFG1, and AFG2). AFB1 was detected in 58.3, and 80% of the maize samples obtained from Kermanshah and Mazandaran provinces, respectively. The maximum AFB1 (276.3 μg/kg) and highest level of total aflatoxins (AFT) (316.9 μg/kg) were detected in a maize sample collected from Kermanshah province. The mean aflatoxin level from contaminated samples (52.60 μg/kg) from Kermanshah was significantly higher (P < 0.0001) than those in maize from the other three provinces and exceeded all the maximum tolerated levels (MTLs) set for AFT in maize. The level of AFB1 in 15.68% of the total samples was above the MTL (5 μg/kg) for AFB1 in maize in Iran. The mean contamination level of AFT (23.86 μg/kg) in the positive samples was higher than MTL for maize in Iran (20 μg/kg) intended for animal feed. The levels of AFB1, AFB2, AFG1, and AFG2 ranged between not detected (<0.1 μg/kg) and 276.3, 30.4, 9.1, and 1.1 μg/kg in maize grain, respectively.     

2(3)-tert-butyl-4-hydroxyanisole inhibits oxidative metabolism of aflatoxin B1 in isolated rat hepatocytes

Previous studies indicate that dietary administration of phenolic antioxidants, 2(3)-tert-butyl-4-hydroxyanisole (BHA) and 3,5-di-tert-butyl-4-hydroxytoluene, inhibits the carcinogenic effect of a number of chemical carcinogens including aflatoxin B1 (AFB1). Induction of hepatic enzymes, such as glutathione S-transferase, UDP-glucuronyltransferase, and epoxide hydrolase, has been shown to be responsible for the reduction of AFB1 cytotoxic and carcinogenic effects. The effect of BHA on AFB1 activation was examined in vitro utilizing isolated rat hepatocytes and liver microsomes. In hepatocytes, the total AFB1 content and bound form of AFB1 were 3.4 and 1.4 pmol/10(6) cells, respectively. In the cell-free microsomal activating system, 2.2 pmol were activated per mg of microsomal protein during 60 min of incubation. BHA (0.1-0.5 mM) inhibited AFB1 activation and binding in both systems in a dose-dependent manner; in hepatocytes, 90% inhibition was observed at 0.5 mM. Analyzing various AFB1 adducts, BHA (0.25 mM)-treated hepatocytes contained a significantly reduced amount of AFB1 macromolecular adducts. The antioxidant neither stimulated nor inhibited the cytosolic glutathione S-transferase and microsomal UDP-glucuronyltransferase activities. Analysis of various hydroxylated (aflatoxins M1 and Q1 (AFM1 and AFQ1] and demethylated (aflatoxin P1 (AFP1] metabolites of AFB1 in both the conjugated and unconjugated form indicated that there was a 30-50% reduction of unconjugated AFP1, AFQ1, and AFM1, whereas AFB1 was increased 3-fold. There was no significant change of conjugated metabolites. The effect of BHA on AFB1 activation in hepatocytes was compared with that of other cytochrome P-450 inhibitors; the ED50 values of SKF 525A, BHA, and metyrapone were 9 microM, 40 microM, and 280 microM, respectively. In the cell-free microsomal system, biotransformation of AFB1 to AFP1, AFM1, and AFQ1 was also inhibited. Kinetic analysis of p-nitroanisole O-demethylase activity of rat liver microsomes demonstrated that BHA inhibited noncompetitively with an apparent Ki of 90 microM. In the absence of enzyme induction, the phenolic antioxidant, BHA, blocks the oxidative biotransformation of AFB1 in isolated hepatocytes.     

Synergism between aflatoxins in covalent binding to DNA and in mutagenesis in the photoactivation system

Aflatoxins (AFs) produce singlet oxygen upon their exposure to UV (365-nm) light. Singlet oxygen in turn activates them to mutagens and DNA-binding species. DNA binding and mutagenesis by AFs were enhanced in D2O as compared to reactions in H2O, and a singlet oxygen scavenger inhibited mutagenesis. DNA photobinding of 3H-AFB1 increased in the presence of unlabeled AFB2, and the addition of AFB2 enhanced mutagenesis by AFB1 in a synergistic manner. These results are compatible with the notion that singlet oxygen, formed by one aflatoxin molecule, can readily activate another aflatoxin molecule. This may bear an environmental implication in that the weakly carcinogenic AFB2, which is often produced in nature together with AFB1, may be important in enhancing the activation of AFB1 by sunlight.     

Aflatoxin M1 effects on Xenopus laevis development

BACKGROUND: The principal Aflatoxin B(1) (AFB(1)) hydroxylated metabolite excreted in milk is Aflatoxin M(1) (AFM(1)) classified in group 2B by the International Agency for Research on Cancer (IARC). Human exposure to AFM(1) is due to the consumption of contaminated dairy products and partly to endogenous production through AFB(1) liver metabolism. METHODS: Since no data are available on AFM(1) embryotoxicity, its lethal and teratogenic potential was investigated using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). Stage-8 blastulae were exposed to AFM(1) at 1, 4, 16, 64, and 256 microg/L concentrations until stage 47, free-swimming larva. RESULTS: A slight increase of mortality and malformed larva percents was found in AFM(1)-exposed groups but these differences were not statistically significant in comparison with the controls. CONCLUSIONS: Therefore, AFM(1) is a non-embryotoxic compound when evaluated with a FETAX model at concentrations under the conditions tested. However, AFM(1) merits further studies using mammals as experimental models to identify a possible risk during human pregnancy.     

The mercapturic acid pathway metabolites of a glutathione conjugate of aflatoxin B1

A glutathione conjugate of aflatoxin B1 (AFB1) which has previously been identified as 8,9-dihydro-8-(S-glutathionyl)-9-hydroxy aflatoxin B1 (AFB1-GSH) (E.J. Moss, D.J. Judah, M. Przybylski and G.E. Neal, Biochem. J., 210 (1983) 227-233) has been degraded in vitro to all of the intermediates of the mercapturic acid pathway (MAP) and the chromatographic and spectral characteristics of each of these compounds investigated. The cysteinylglycyl conjugate (AFB1-Cys.Gly) was prepared by incubating the AFB1-GSH conjugate with a rat hepatoma cell line rich in gamma-glutamyl-transpeptidase (GGT). Incubations of the AFB1-Cys.Gly conjugate with dipeptidase produced a metabolite, which was purified and characterized by 1H-NMR spectroscopy as 8,9-dihydro-8-(S-cysteinyl)-9-hydroxy aflatoxin B1 (AFB1-Cys). The N-acetyl derivative of the AFB1-Cys conjugate resulted from the incubation of the AFB1-GSH conjugate in vitro with isolated rat kidney cells. Mass spectral data were consistent with the compound being 8,9-dihydro-8-(S-cysteinyl-(N-acetyl))-9-hydroxy aflatoxin B1 (AFB1-Nac.Cys). A chromatographically identical compound was obtained by the chemical acetylation of AFB1-Cys.