Mold flora and aflatoxin contamination of stored and cooked samples of pearl millet in the Paharia tribal belt of Santhal paragana, Bihar, India.

Stored and cooked samples of pearl millet (Pennesetum typhoides), which is regularly consumed as food by the Paharia tribe in the hilly regions of Santhal Pargana, Bihar State, India, that were harvested in January 1989 were analyzed for mold flora, natural occurrence of Aspergillus flavus and A. parasiticus, and incidence and levels of aflatoxin B1. Of the 22 fungal species isolated, A. flavus and A. parasiticus were the predominant species (63.8%) during the rainy season, followed by other species of Aspergillus, Penicillium, Fusarium, Rhizopus, Helminthosporium, and Curvularia. Screening of 169 A. flavus and A. parasiticus strains showed that 59 of them were toxigenic, producing various combinations of aflatoxins B1, B2, G1, and G2. The amounts of aflatoxin B1 ranged between 4 and 30 mg/100 ml of liquid medium. Analysis of stored and cooked samples also revealed a high incidence and alarming levels of naturally produced aflatoxin B1. Forty-nine of 75 stored and 16 of 38 cooked samples contained various combinations of aflatoxins. The levels of aflatoxin B1 ranged between 17 and 2,110 ppb in stored samples and 18 and 549 ppb in cooked samples. The correlation of insect damage with A. flavus and A. parasiticus incidence and quantity of aflatoxin B1 was found to be insignificant.     

Mold flora and aflatoxin contamination of stored and cooked samples of pearl millet in the Paharia tribal belt of Santhal paragana, Bihar, India

Stored and cooked samples of pearl millet (Pennesetum typhoides), which is regularly consumed as food by the Paharia tribe in the hilly regions of Santhal Pargana, Bihar State, India, that were harvested in January 1989 were analyzed for mold flora, natural occurrence of Aspergillus flavus and A. parasiticus, and incidence and levels of aflatoxin B1. Of the 22 fungal species isolated, A. flavus and A. parasiticus were the predominant species (63.8%) during the rainy season, followed by other species of Aspergillus, Penicillium, Fusarium, Rhizopus, Helminthosporium, and Curvularia. Screening of 169 A. flavus and A. parasiticus strains showed that 59 of them were toxigenic, producing various combinations of aflatoxins B1, B2, G1, and G2. The amounts of aflatoxin B1 ranged between 4 and 30 mg/100 ml of liquid medium. Analysis of stored and cooked samples also revealed a high incidence and alarming levels of naturally produced aflatoxin B1. Forty-nine of 75 stored and 16 of 38 cooked samples contained various combinations of aflatoxins. The levels of aflatoxin B1 ranged between 17 and 2,110 ppb in stored samples and 18 and 549 ppb in cooked samples. The correlation of insect damage with A. flavus and A. parasiticus incidence and quantity of aflatoxin B1 was found to be insignificant.     

Exposure measurement of aflatoxins and aflatoxin metabolites in human body fluids. A short review

Aflatoxins are highly toxic secondary fungal metabolites mainly produced by Aspergillus flavus and A. parasiticus. Human exposure to aflatoxins may result directly from ingestion of contaminated foods, or indirectly from consumption of foods from animals previously exposed to aflatoxins in feeds. This paper focuses on exposure measurement of aflatoxins and aflatoxin metabolites in various human body fluids. Research on different metabolites present in blood, urine, breast milk, and other human fluids or tissues including their detection techniques is reviewed. The association between dietary intake of aflatoxins and biomarker measurement is also highlighted. Finally, aspects related to the differences between aflatoxin determination in food versus the biomarker approach are discussed.     

Bioremediation of aflatoxins by some reference fungal strains.

Aspergillus parasiticus RCMB 002001 (2) producing four types of aflatoxins B1, B2, G1, and G2 was used in this study as an aflatoxin-producer. Penicillium griseofulvum, P. urticae, Paecilomyces lilacinus, Trichoderma viride, Candida utilis, Saccharomyces cerevisiae as well as a non-toxigenic strain of Aspergillus flavus were found to be able to exhibit growth on aflatoxin B1-containing medium up to a concentration of 500 ppb. It was also found that several fungal strains exhibited the growth in co-culture with A. parasiticus, natural aflatoxins producer, and were able to decreased the total aflatoxin concentration, resulting in the highest inhibition percentage of 67.2% by T viride, followed by P. lilacinus, P. griseofulvum, S. cerevisiae, C. utilis, P. urticae, Rhizopus nigricans and Mucor rouxii with total aflatoxin inhibition percentage of 53.9, 52.4, 52, 51.7, 44, 38.2 and 35.4%, respectively. The separation of bioremediation products using GC/MS revealed that the toxins were degraded into furan moieties.     

Intrafungal distribution of aflatoxins among conidia and sclerotia of Aspergillus flavus and Aspergillus parasiticus

This research examines the distribution of aflatoxins among conidia and sclerotia of toxigenic strains of Aspergillus flavus Link and Aspergillus parasiticus Speare cultured on Czapek agar (21 days, 28 degrees C). Total aflatoxin levels in conidia and sclerotia varied considerably both within (intrafungal) and among strains. Aspergillus flavus NRRL 6554 accumulated the highest levels of aflatoxin (conidia: B1, 84000 ppb; G1, 566000 ppb; sclerotia: B1, 135000 ppb; G1, 968000 ppb). Substantial aflatoxin levels in conidia could place at risk those agricultural workers exposed to dust containing large numbers of A. flavus conidia. Cellular ratios of aflatoxin B1 to aflatoxin G1 were nearly identical in conidia and sclerotia even though levels of total aflatoxins in these propagule types may have differed greatly. Aflatoxin G1 was detected in sclerotia of all A. flavus strains but in the conidia of only one strain. Each of the A. parasiticus strains examined accumulated aflatoxin G1 in both sclerotia and conidia. These results are examined in the context of current evolutionary theory predicting an increase in the chemical defense systems of fungal sclerotia, propagules critical to the survival of these organisms.     

Role of mycoferritin from Aspergillus parasiticus (255) in secondary metabolism (aflatoxin production)

Aspergillus parasiticus (255), a non-toxigenic isolate showed the presence of secondary metabolites-aflatoxins (B1, B2, G1, G2) when grown in yeast extract sucrose media but not in basal media, thus demonstrating its toxigenic potential. Native PAGE of the crude protein isolated at different growth periods of A. parasiticus in yeast extract sucrose media containing iron showed prominent expression of mycoferritin from day four onwards. The production of aflatoxins was also maximal on day four, both in the presence and absence of iron. Indicators of oxidative stress metabolites such as reactive oxygen species, thiobarbituric acid reactive species, reduced and oxidized glutathione and antioxidant enzymes like superoxide dismutase and glutathione peroxidase were analyzed both in the presence and absence of iron and the experimental data suggest oxidative stress as a pre-requisite for aflatoxin production. The pro-oxidant role of iron was minimized by induction of mycoferritin and the concomitant alterations in oxidative stress parameters imply an antioxidant role to mycoferritin in secondary metabolism, a finding of significance that has not been reported previously in fungal systems.     

Aflatoxin biosynthesis cluster gene cypA is required for G aflatoxin formation

Aspergillus flavus isolates produce only aflatoxins B1 and B2, while Aspergillus parasiticus and Aspergillus nomius produce aflatoxins B1, B2, G1, and G2. Sequence comparison of the aflatoxin biosynthesis pathway gene cluster upstream from the polyketide synthase gene, pksA, revealed that A. flavus isolates are missing portions of genes (cypA and norB) predicted to encode, respectively, a cytochrome P450 monooxygenase and an aryl alcohol dehydrogenase. Insertional disruption of cypA in A. parasiticus yielded transformants that lack the ability to produce G aflatoxins but not B aflatoxins. The enzyme encoded by cypA has highest amino acid identity to Gibberella zeae Tri4 (38%), a P450 monooxygenase previously shown to be involved in trichodiene epoxidation. The substrate for CypA may be an intermediate formed by oxidative cleavage of the A ring of O-methylsterigmatocystin by OrdA, the P450 monooxygenase required for formation of aflatoxins B1 and B2.     

Aflatoxin production by entomopathogenic isolates of Aspergillus parasiticus and Aspergillus flavus

Fourteen isolates of Aspergillus parasiticus and 2 isolates of Aspergillus flavus isolated from the mealybug Saccharicoccus sacchari were analyzed for production of aflatoxins B1, B2, G1, and G2 in liquid culture over a 20-day period. Twelve Aspergillus isolates including 11 A. parasiticus and 1 A. flavus produced aflatoxins which were extracted from both the mycelium and culture filtrate. Aflatoxin production was detected at day 3 and was detected continually for up to day 20. Aflatoxin B1 production was greatest between 7 and 10 days and significantly higher quantities were produced by A. flavus compared to A. parasiticus. Aflatoxin production was not a stable trait in 1 A. parasiticus isolate passaged 50 times on agar. In addition to loss of aflatoxin production, an associated loss in sporulation ability was also observed in this passaged isolate, although it did maintain pathogenicity against S. sacchari. An aflatoxin B1 concentration of 0.16 micrograms/mealybug (14.2 micrograms/g wet wt) was detected within the tissues of infected mealybugs 7 days after inoculation. In conclusion, the ability of Aspergillus isolates to produce aflatoxins was not essential to the entomopathogenic activity of this fungus against its host S. sacchari.     

Intracellular hydrolases of Aspergillus parasiticus and Aspergillus flavus

When the distribution profile of hydrolases in mycelial homogenates and culture filtrates of A. parasiticus and A. flavus was examined, six hydrolytic enzymes viz. N-acetyl-beta-glucosaminidase, aryl sulfatase, alkaline proteinase, cathepsin B, cathepsin D and aminopeptidase were detected in homogenate. The culture filtrates were devoid of any activity of these enzymes. The enzyme levels varied with the stage of incubation. The most abundant fungal exopeptidase showing preference for basic amino acid naphthylamides seems to be an aminopeptidase B. Incorporation of CEPA, an ethylene generating compound, stimulated the amino peptidase activity in the mycelium but inhibited the enzyme in vitro. The enzyme was also inhibited by different aflatoxins to varying degree. While aminopeptidase B was located intracellularly, a non-dialysable, heat-stable inhibitor of the enzyme was found to be secreted in the culture filtrate. This peptide inhibitor was however ineffective on the other enzymes.     

Inhibition of aflatoxin production by trifluoperazine in Aspergillus parasiticus NRRL 2999

Trifluoperazine, an anti-calmodulin agent, inhibited aflatoxin production by Aspergillus parasiticus NRRL 2999, without affecting the growth significantly. Culturing the organism for 3 days in the presence of 0.14mm trifluoperazine resulted in a generalized decrease in the production of all aflatoxins; the production of aflatoxin B1, a potent hepatocarcinogen, was inhibited to 88% under such conditions. Culturing 7-day-old preformed cultures in the presence of higher concentrations of trifluoperazine (>1mm) completely abolished production of all aflatoxins including AFB1. The inhibitory influence of trifluoperazine on aflatoxin production was accompanied by calmodulin-dependent phosphorylation of an 85kDa cytoplasmic calmodulin-binding protein. While the functions of calmodulin in mediating primary events of germination, growth and differentiation in fungi have earlier been reported, the present results indicate a possible role for calmodulin in the production of fungal toxins.     

Inhibition of aflatoxin production by trifluoperazine in Aspergillus parasiticus NRRL 2999

Trifluoperazine, an anti-calmodulin agent, inhibited aflatoxin production by Aspergillus parasiticus NRRL 2999, without affecting the growth significantly. Culturing the organism for 3 days in the presence of 0.14mm trifluoperazine resulted in a generalized decrease in the production of all aflatoxins; the production of aflatoxin B1, a potent hepatocarcinogen, was inhibited to 88% under such conditions. Culturing 7-day-old preformed cultures in the presence of higher concentrations of trifluoperazine (>1mm) completely abolished production of all aflatoxins including AFB1. The inhibitory influence of trifluoperazine on aflatoxin production was accompanied by calmodulin-dependent phosphorylation of an 85kDa cytoplasmic calmodulin-binding protein. While the functions of calmodulin in mediating primary events of germination, growth and differentiation in fungi have earlier been reported, the present results indicate a possible role for calmodulin in the production of fungal toxins.     

Precursor recognition by kinetic pulse-labeling in a toxigenic aflatoxin B1-producing strain of Aspergillus.

Kinetic pulse-labeling of aflatoxin pathway compounds was carried out in Aspergillus parasiticus, beginning with radioactive acetate. Norsolorinic acid, averufin, versicolorin A, and sterigmatocystin (all known as compounds which can be incorporated into the aflatoxin molecule) were radiotraced to follow their order of appearance. Aflatoxin species B1, B2, G1, and G2 were included. Norsolorinic acid and averufin appeared as early transient intermediates followed in order by versicolorin A, aflatoxins, and sterigmatocystin. To date, a mutually confirming array of results has been obtained with established precursors in wild-type strains of A. parasiticus and A. versicolor (as well as with an aflatoxin pathway mutant of A. parasiticus), which together establish a practical methodology for recognition of new pathway intermediates. The kinetic of pulse-labeling for sterigmatocystin in relation to aflatoxins suggests that duel branchlets may exist to flatoxins; i.e., sterigmatocystin may not be an obligatory aflatoxin precursor.