Mycotoxins as human carcinogens—the <Emphasis Type="Italic">IARC Monographs</Emphasis> classification

Humans are constantly exposed to mycotoxins (e.g. aflatoxins, ochratoxins), mainly via food intake of plant and animal origin. The health risks stemming from mycotoxins may result from their toxicity, in particular their carcinogenicity. In order to prevent these risks, the International Agency for Research on Cancer (IARC) in Lyon (France)—through its IARC Monographs programme—has performed the carcinogenic hazard assessment of some mycotoxins in humans, on the basis of epidemiological data, studies of cancer in experimental animals and mechanistic studies. The present article summarizes the carcinogenic hazard assessments of those mycotoxins, especially aflatoxins (aflatoxin B₁, B₂, G₁, G₂ and M₁), fumonisins (fumonisin B₁ and B₂) and ochratoxin A (OTA). New information regarding the genotoxicity of OTA (formation of OTA-DNA adducts), the role of OTA in oxidative stress and the identification of epigenetic factors involved in OTA carcinogenesis–should they indeed provide strong evidence that OTA carcinogenicity is mediated by a mechanism that also operates in humans–could lead to the reclassification of OTA.     

In situ absorption of aflatoxins in rat small intestine

To evaluate the rate at which the four main aflatoxins (aflatoxins B₁, B₂, G₁ and G₂) are able to cross the luminal membrane of the rat small intestine, a study about intestinal absorption kinetics of these mycotoxins has been made. In situ results obtained showed that the absorption of aflatoxins in rat small intestine is a very fast process that follows first-order kinetics, with an absorption rate constant (k _a ) of 5.84±0.05 (aflatoxin B₁), 4.06±0.09 (aflatoxin B₂), 2.09±0.03 (aflatoxin G₁) and 1.58±0.04 (aflatoxin G₂) h^–1, respectively.     

Fungal and aflatoxin contamination of medicinal herbs

Since the consumption of aromatic and medicinal herbs has been increasing in the last years, the Argentinian Health Authorities are concerned to control the quality and security of them. Fungal and aflatoxin contamination are two parameters to be taken into account, to ensure the harmlessness of the phytomedicinal products. In 81 different samples, grouped in end products (EP), raw material (RM) and at harvest (SH), fungal flora (enumeration and identification) as well as naturalAspergillus flavus and aflatoxin occurrence were investigated. In all samples fungal counts fulfilled the international general recommendation limits (maximum 10⁵ cfu/g). Predominant flora was made up by xerophilic species ofAspergillus(100%), byPeniciIlium (< 50%) and in less percentage byFusarium (5.6%). Among the Aspergilli, A.flavus was present in all the three groups of samples. Using a TLC method, 47% of A. flavus isolates were toxinogenic, producing aflatoxin B₁ and B₂. In herbs, 4.7% of RM samples were naturally contaminated with aflatoxins B₁ and B₂. Considering the carcinogenic activity of aflatoxins it is essential to regulate them in the raw material (vegetal drug).     

Aflatoxin is degraded at different temperatures and pH values by mycella of Aspergillus parasiticus

Summary Blended 9-day-old mycelia of Aspergillus parasiticus NRRL 2999 were tested for their ability to degrade aflatoxins B₁ and G₁ at 7,19,28,36, and 45°C. Rates for degradation of aflatoxin B₁ and G₁ were maximum at 28°C. Intermediate rates of aflatoxin degradation were observed at 19 and 36°C while little aflatoxin was degraded at 7 and 45°C. Five different pH values (2.0, 3.0, 4.0, 5.0, and 6.5) were also tested to determine the effect of pH on ability of blended 9-day-old mycelia of A. parasiticus NRRL 2999 to degrade aflatoxins. The ability of mycelia to degrade aflatoxin was pH-dependent. Of the pH values tested, greatest rates of aflatoxin B₁ and G₁ degradation occurred when pH was in the range of 5 to 6.5. Little aflatoxin was degraded at pH 4.0 and essentially no aflatoxin was degraded by mycelia at pH 2.0 or 3.0 although some aflatoxin was degraded by acid conditions only at pH values of 4 or less.     

Aflatoxins B1 and G1 solubility in standard solutions and stability during cold storage

In the present work we study the use of different solvents to store aflatoxins B₁ and G₁ standard solutions. We have obtained significant differences between aflatoxin B₁ and G₁ In ethyl acetate, methanol and water, with aflatoxin G₁ being less stable. We recommend chloroform as the election solvent to store the aflatoxin solutions. The fact that aflatoxins are highly stable in water may have a potential use in experiments of biological activity.     

Aflatoxin is degraded by heated and unheated mycelia,filtrates of homogenized mycelia and filtrates of broth cultures of Aspergillus parasiticus

Steaming one-half of a lot of 9-day-old mycelia of Aspergillus parasiticus NRRL 2999 for 6 min resulted in little or no subsequent degradation of aflatoxin B₁ or G₁ by these mycelia. The other half of these mycelia was not heat-treated and degraded aflatoxins B₁ and G₁ Filtrates of the growth substrate which remained after the mycelium was removed from 8- to 15-day old cultures of A. parasiticus NRRL 2999 did not degrade substantial amounts of aflatoxin B₁ or G₁, whereas mycelia originally produced on these filtrates degraded substantial amounts of both aflatoxins. The supernatant fluid from homogenates of 9-day-old mycelia of A. parasiticus NRRL 2999 degraded aflatoxins B₁ and G₁ when 0.1 M or 1.0 M phosphate buffer, pH 6.5, was used to suspend the homogenate. These data support the hypothesis that the aflatoxin degrading factor(s) present in the mycelium of A. purasiticus is/are enzyme(s) or at least influenced by enzyme(s).     

The effects of aflatoxin B1 and palmotoxins B0 and G0 on the germination and leaf colour of the cowpea (vigna sinensis)

Aflatoxin B₁, crude aflatoxins and palmotoxins B₀ and G₀ were tested for seed germination and chlorophyll formation using the cowpea. It was found that aflatoxin B and crude aflatoxins inhibited both chlorophyll formation and seed germination and palmotoxins inhibited these processes to a lesser extent. 3 — Indolylacetic acid reduced the inhibitory effects of the aflatoxins in seed germination and chlorophyll formation in the cowpea.     

Production of aflatoxin byAspergillus parasiticus and its control

The aim of the present work was to investigate the production of aflatoxin byAspergillus parasiticus and to find out the possible ways to control it. Of 40 food samples collected from Abha region, Saudi Arabia, only 25% were contaminated with aflatoxins. Oil-rich commodities had the highly contaminated commodities by fungi and aflatoxins while spices were free from aflatoxins.Bacillus megatertum andB cereus were suitable for microbiological assay of aflatoxins. Czapek’s-Dox medium was found a suitable medium for isolation of fungi from food samples. The optimal pH for the growth ofA. parasiticus and its productivity of aflatoxin B₁ was found at 6.0, while the best incubation conditions were found at 30°C for 10 days. D-glucose was the best carbon source for fungal growth, as well as aflatoxin production. Corn steep liquor, yeast extract and peptone were the best nitrogen sources for both fungal growth and toxin production (NH₄)₂HPO₄ (1.55 gL^-1) and NaNO₂ (1.6 gL^-1) reduced fungal growth and toxin production with 37.7% and 85%, respectively. Of ten amino acids tested, asparagine was the best for aflatoxin B₁ production. Zn²⁺ and Co²⁺ supported significantly both fungal growth, as well as, aflatoxin B₁ production at the different tested concentrations. Zn²⁺ was effective when added toA. parasiticus growth medium at the first two days of the culture age. The other tested metal ions expressed variable effects depending on the type of ion and its concentration. Water activity (a_w) was an important factor controlling the growth ofA. parasiticus and toxin production. The minimum a_w for the fungal growth was 0.8 on both coffee beans and rice grains, while a_w of 0.70 caused complete inhibition for the growth and aflatoxin B₁ production. H₂O₂ is a potent inhibitor for growth ofA. parasiticus and its productivity of toxins. NaHCO₃ and C₆H₅COONa converted aflatoxin B₁ to water-soluble form which returned to aflatoxin B₁ by acidity. Black pepper, ciliated heath, cuminum and curcuma were the most inhibitory spices on toxin production. Glutathione, quinine, EDTA, sodium azide, indole acetic acid, 2,4-dichlorophenoxy acetic acid, phenol and catechol were inhibitory for both growth, as well as, aflatoxin B₁ production. Stearic acid supported the fungal growth and decreased the productivity of AFB₁ gradually. Lauric acid is the most suppressive fatty acid for both fungal growth and aflatoxin production, but oleic acid was the most potent supporter. Vitamin A supported the growth but inhibited aflatoxin B₁ production. Vitamins C and D₂ were also repressive particularly for aflatoxin production The present study included studying the activities of some enzymes in relation to aflatoxin production during 20-days ofA. parasiticus age in 2-days intervals. Glycolytic enzymes and pyruvate-generating enzymes seems to be linked with aflatoxin B₁ production. Also, pentose-phosphate pathway enzymes may provide NADPH for aflatoxin B₁ synthesis. The decreased activities of TCA cycle enzymes particularly from 4th day of growth up to 10th day were associated with the increase of aflatoxin B₁ production. All the tested enzymes as well as aflatoxin B₁ production were inhibited by either catechol or phenol.     

Incidence of aflatoxin producing strains and aflatoxin contamination in dry fruit slices of quinces (Cydonia oblonga Mill.) from the Indian State of Jammu and Kashmir

An investigation was undertaken to obtain data on the occurrence of aflatoxins and the aflatoxin producing potential of Aspergillus flavus strains isolated from dry fruit slices of quinces produced in jammu and Kashmir, India. A total of 147 A. flavus isolates recovered from dr fruit slices were grown in liquid rice flour medium and screened for the production of various aflatoxins by thin layer chromatography. The results showed that 23.14% of the tested isolates were aflatoxigenic, producing aflatoxins B₁and B₂ in varying amounts. Aflatoxins G₁ and G₂ were not detected. All 25 of the investigated market samples were also found to be aflatoxin B₁ positive and the level of contamination ranged from 96 to 8164 g/kg of the dry fruit which is quite high in comparison to the permissible level of 30 ppb. As per these results biochemical composition of dry fruit slices of quinces, along with climatic conditions seem to be very favourable for aflatoxin production by the toxigenic A. flavus strains. Therefore,monitoring of aflatoxins in dry fruit slices of quincesis recommended for this region.This revised version was published online in October 2005 with corrections to the Cover Date.     

Evaluation of different genotypes of nontoxigenic Aspergillus flavus for their ability to reduce aflatoxin contamination in peanuts

Aflatoxins produced by the fungus Aspergillus flavus are potent carcinogens and account for large monetary losses worldwide in peanuts, maize, and cottonseed. Biological control in which a nontoxigenic strain of A. flavus is applied to crops at high concentrations effectively reduces aflatoxins through competition with native aflatoxigenic populations. In this study, eight nontoxigenic strains of A. flavus belonging to different vegetative compatibility groups and differing in deletion patterns within the aflatoxin gene cluster were evaluated for their ability to reduce aflatoxin B₁ when paired with eight aflatoxigenic strains on individual peanut seeds. Inoculation of wounded viable peanut seeds with conidia demonstrated that nontoxigenic strains differed in their ability to reduce aflatoxin B₁. Reductions in aflatoxin B₁ often exceeded expected reductions based on a 50:50 mixture of the two A. flavus strains, although one nontoxigenic strain significantly increased aflatoxin B₁ when paired with an aflatoxigenic strain. Therefore, nontoxigenicity alone is insufficient for selecting a biocontrol agent and it is also necessary to test the effectiveness of a nontoxigenic strain against a variety of aflatoxigenic strains.     

Destruction of aflatoxins B1 and G1 in bread making

The effect of processing steps as well preservatives used in French bread making namely propionic acid and/or potassium sorbate (0.2%) on the destruction of aflatoxins B₁ and G₁ was studied. Mixing and baking processes showed marked destruction of aflatoxins B₁ and G₁; being 71.2% and 52.5% for aflatoxin B₁ after mixing and baking steps, while reaching 73.9% and 54.5% for aflatoxin G₁. Fermentation step caused additional 15.3% and 15.0% destruction of aflatoxins B₁ and G₁. On the other hand, aflatoxin B₁ destruction was 79.2% and 50.7% when propionic acid was used and 75.3 and 56.7% in the presence of potassium sorbate and after mixing and baking steps respectively. Concerning aflatoxins G₁ it was found that mixing and baking steps showed destruction of 81.9% and 53.4% in the presence of propionic acid and 75.1 and 49.4% in the presence of potassium sorbate in this respective order. Generally, it can be concluded that using propionic acid as preservative appeared to be more effective on the destruction of aflatoxins B₁ and G₁ than potassium sorbate in French bread making.     

A review of the dose-response induction of DNA adducts by aflatoxin B1 and its implications to quantitative cancer-risk assessment

The induction of DNA adducts by aflatoxin B₁ in the liver has been extensively reviewed in a quantitative cancer-risk assessment of aflatoxins (CDHS, 1990). Rat is the most sensitive species for aflatoxin tumorigenesis and liver is the most sensitive site. In vitro DNA-adduct studies were mostly on adduct identification and specificity of binding. In vivo studies provided dose-response relationship of aflatoxin B₁, binding to DNA and DNA-adduct formation. Most in vivo studies were conducted in rats. The dose-response curves of DNA-adduct induction after ingestion or injection treatments in this species were reviewed. A linear dose-response relationship was observed in both injection and ingestion studies at low doses. For cancer-risk assessment, this observation is consistent with the assumption of the linear dose-response risk-assessment model for genotoxic agents, and justifies the use of this model for quantitative cancer-risk assessment for aflatoxins.     

Fluorometric analysis of iodinated aflatoxin in minicultures ofAspergillus flavus andAspergillus parasiticus

Summary A convenient miniassay for aflatoxin has been developed for cultures ofAspergillus flavus andA. parasiticus grown for 3–10 days in 10 ml of a coconut extract medium. The sensitivity of the assay, as measured by photofluorometry (365 nm maximum excitation; 445 nm maximum emission), is of the order of 0.01 M (3.12 ng/ml) for aflatoxin B₁ dissolved in aqueous iodine (0.26 mM). High performance liquid chromatography, monitored by fluorometric analysis of both an aflatoxin B₁ standard and selected culture filtrates, confirmed the sensitivity of the assay and indicated specificity for iodine-enhanced fluorescence of aflatoxin in the coconut extract medium. Thin layer chromatography further confirmed the aflatoxin titers and the specificity for enhancement of aflatoxins B₁ and G₁ in culture filtrates.Alabama Agricultural Experiment Station Journal No. 6-871297.     

Association between vegetative compatibility and aflatoxin production by <Emphasis Type="Italic">Aspergillus</Emphasis> species during intraspecific competition

Intraspecific competition is the basis for biological control of aflatoxins, but there is little understanding of the mechanism(s) by which competing strains inhibit toxin production. Evidence is presented that demonstrates a relationship between strength of the vegetative compatibility reaction and aflatoxin production in Aspergillus flavus and A. parasiticus using the suspended disk culture method. Combining wild-type aflatoxin-producing isolates belonging to different vegetative compatibility groups (VCGs) resulted in a substantial reduction in aflatoxin yield. Pairs of aflatoxin-producing isolates within the same VCG, but showing weak compatibility reactions using complementary nitrate-nonutilizing mutants, also were associated with reduced levels of aflatoxin B₁. In contrast, pairings of isolates displaying a strong compatibility reaction typically produced high levels of aflatoxins. These results suggest that interactions between vegetatively compatible wild-type isolates of A. flavus and A. parasiticus are cooperative and result in more aflatoxin B₁ than pairings between isolates that are incompatible. Successful hyphal fusions among spore germlings produce a common mycelial network with a larger resource base to support aflatoxin biosynthesis. By comparison, vegetative incompatibility reactions might result in the death of those heterokaryotic cells composed of incompatible nuclei and thereby disrupt the formation of mycelial networks at the expense of aflatoxin biosynthesis. The content of this paper was presented at the 50th Anniversary Meeting of the Mycological Society of Japan, June 3–4, 2006, Chiba, Japan     

Aflatoxins: Detection,toxicity, and biosynthesis

Aflatoxins are toxic and carcinogenic secondary metabolites produced mainly byAspergillus flavus andAspergillus parasiticus. The aflatoxins present in food and feed are hazardous to both human and animal health. A number of studies have been conducted on the detection, toxicity, biosynthesis, and regulation of aflatoxins due to the discovery of serious aflatoxicosis in farm animals, and the presence of aflatoxins in many food products. There are many reviews that focus on the biosynthesis of aflatoxin, yet there are few examinations of the overall aspects of aflatoxins, including detection, toxicity, and the regulation on biosynthesis. Thus, the goal of this article is to give an overview of the overall aspects of aflatoxins. This review consists of four parts; i) detection methods for aflatoxins, ii) the toxicity mechanism of aflatoxin B1, iii) gene cluster for aflatoxin biosynthesis, and iv) the regulation of aflatoxin biosynthesis.     

Enzymatic Formation of G-Group Aflatoxins and Biosynthetic Relationship between G- and B-Group Aflatoxins

We detected biosynthetic activity for aflatoxins G₁ and G₂ in cell extracts of Aspergillus parasiticus NIAH-26. We found that in the presence of NADPH, aflatoxins G₁ and G₂ were produced from O-methylsterigmatocystin and dihydro-O-methylsterigmatocystin, respectively. No G-group aflatoxins were produced from aflatoxin B₁, aflatoxin B₂, 5-methoxysterigmatocystin, dimethoxysterigmatocystin, or sterigmatin, confirming that B-group aflatoxins are not the precursors of G-group aflatoxins and that G- and B-group aflatoxins are independently produced from the same substrates (O-methylsterigmatocystin and dihydro-O-methylsterigmatocystin). In competition experiments in which the cell-free system was used, formation of aflatoxin G₂ from dihydro-O-methylsterigmatocystin was suppressed when O-methylsterigmatocystin was added to the reaction mixture, whereas aflatoxin G₁ was newly formed. This result indicates that the same enzymes can catalyze the formation of aflatoxins G₁ and G₂. Inhibition of G-group aflatoxin formation by methyrapone, SKF-525A, or imidazole indicated that a cytochrome P-450 monooxygenase may be involved in the formation of G-group aflatoxins. Both the microsome fraction and a cytosol protein with a native mass of 220 kDa were necessary for the formation of G-group aflatoxins. Due to instability of the microsome fraction, G-group aflatoxin formation was less stable than B-group aflatoxin formation. The ordA gene product, which may catalyze the formation of B-group aflatoxins, also may be required for G-group aflatoxin biosynthesis. We concluded that at least three reactions, catalyzed by the ordA gene product, an unstable microsome enzyme, and a 220-kDa cytosol protein, are involved in the enzymatic formation of G-group aflatoxins from either O-methylsterigmatocystin or dihydro-O-methylsterigmatocystin.     

Rapid On-Site Sensing Aflatoxin B1 in Food and Feed via a Chromatographic Time-Resolved Fluoroimmunoassay

Aflatoxin B₁ poses grave threats to food and feed safety due to its strong carcinogenesis and toxicity, thus requiring ultrasensitive rapid on-site determination. Herein, a portable immunosensor based on chromatographic time-resolved fluoroimmunoassay was developed for sensitive and on-site determination of aflatoxin B₁ in food and feed samples. Chromatographic time-resolved fluoroimmunoassay offered a magnified positive signal and low signal-to-noise ratio in time-resolved mode due to the absence of noise interference caused by excitation light sources. Compared with the immunosensing performance in previous studies, this platform demonstrated a wider dynamic range of 0.2-60 μg/kg, lower limit of detection from 0.06 to 0.12 µg/kg, and considerable recovery from 80.5% to 116.7% for different food and feed sample matrices. It was found to be little cross-reactivity with other aflatoxins (B₂, G₁, G₂, and M₁). In the case of determination of aflatoxin B₁ in peanuts, corn, soy sauce, vegetable oil, and mouse feed, excellent agreement was found when compared with aflatoxin B₁ determination via the conversational high-performance liquid chromatography method. The chromatographic time-resolved fluoroimmunoassay affords a powerful alternative for rapid on-site determination of aflatoxin B₁ and holds a promise for food safety in consideration of practical food safety and environmental monitoring.     

Failure of plant tissues to metabolize aflatoxin B1?

After exposure of peas and wheat kernels to aflatoxin B₁ solutions the following aflatoxins could not be detected in seed extracts: aflatoxins M₁, B_2a, Q₁, aflatoxicol and tetrahydrodeoxyaflatoxin B₁.     

Production of aflatoxin on rice

A method has been developed for the production of aflatoxin by growing Aspergillus flavus strain NRRL 2999 on the solid substrate rice. Optimal yields, more than 1 mg of aflatoxin B₁ per g of starting material, were obtained in 5 days at 28 C. A crude product containing aflatoxins was isolated by chloroform extraction and precipitation with hexane from concentrated solutions. The crude product consisted of 50% aflatoxin in the following ratio: B₁-B₂-G₁-G₂, 100:0.15:0.22:0.02. Aflatoxin B₁ was separated from almost all the impurities and from the other aflatoxins by chromatography on silica gel with 1% ethyl alcohol in chloroform. Analytically pure aflatoxin B₁ was recrystallized from chloroform-hexane mixtures.     

Aflatoxins in mutants of Aspergillus flavus

Mutants ofAspergillus flavus were recovered following the irradiation of conidia with ultraviolet light. Analysis of the mutants for aflatoxins B₁, B₂, G₁, and G₂ indicated a wide range of variability in aflatoxin levels. None of the isolates produced the G toxins, and four produced little or no aflatoxin B₂. Production of B₁ and B₂ by the mutants ranged from 1.3 µ;g/ml to 967 µg/ml and zero to 30 µg/ml, respectively. The correlation between production of B₁ and B₂ was statistically significant. There was no apparent correlation between nutritional requirement or conidial color and aflatoxin production.