Comparative metabolism of aflatoxin B1 in mouse, rat and human primary hepatocytes using HPLC-MS/MS.

Aflatoxin B1 (AFB1) is a highly hepatotoxic and carcinogenic mycotoxin produced by Aspergillus species. The compound is mainly metabolized in the liver and its metabolism varies between species. The present study quantified relevant AFB1- metabolites formed by mouse, rat, and human primary hepatocytes after treatment with 1 µM and 10 µM AFB1. The use of liquid chromatographic separation coupled with tandem mass spectrometric detection enabled the selective and sensitive determination of phase I and phase II metabolites of AFB1 over incubation times of up to 24 h. The binding of AFB1 to macromolecules was also considered. The fastest metabolism of AFB1 was observed in mouse hepatocytes which formed aflatoxin P1 as a major metabolite and also its glucuronidated form, while AFP1 occurred only in traces in the other species. Aflatoxin M1 was formed in all species and was, together with aflatoxin Q1 and aflatoxicol, the main metabolite in human cells. Effective epoxidation led to high amounts of DNA adducts already 30 min post-treatment, especially in rat hepatocytes. Lower levels of DNA adducts and fast DNA repair were found in mouse hepatocytes. Also, protein adducts arising from reactive intermediates were formed rapidly in all three species. Detoxification via glutathione conjugation and subsequent formation of the N-acetylcysteine derivative appeared to be similar in mice and in rats and strongly differed from human hepatocytes which did not form these metabolites at all. The use of qualitative reference material of a multitude of metabolites and the comparison of hepatocyte metabolism in three species using advanced methods enabled considerations on toxification and detoxification mechanisms of AFB1. In addition to glutathione conjugation, phase I metabolism is strongly involved in the detoxification of AFB1.

Research progress in toxicological effects and mechanism of aflatoxin B1 toxin.

Fungal contamination of animal feed can severely affect the health of farm animals, and result in considerable economic losses. Certain filamentous fungi or molds produce toxic secondary metabolites known as mycotoxins, of which aflatoxins (AFTs) are considered the most critical dietary risk factor for both humans and animals. AFTs are ubiquitous in the environment, soil, and food crops, and aflatoxin B1(AFB1) has been identified by the World Health Organization (WHO) as one of the most potent natural group 1A carcinogen. We reviewed the literature on the toxic effects of AFB1 in humans and animals along with its toxicokinetic properties. The damage induced by AFB1 in cells and tissues is mainly achieved through cell cycle arrest and inhibition of cell proliferation, and the induction of apoptosis, oxidative stress, endoplasmic reticulum (ER) stress and autophagy. In addition, numerous coding genes and non-coding RNAs have been identified that regulate AFB1 toxicity. This review is a summary of the current research on the complexity of AFB1 toxicity, and provides insights into the molecular mechanisms as well as the phenotypic characteristics.

Efficient Prevention of Aspergillus flavus Spores Spread in Air Using Plasmonic Ag-AgCl/α-Fe2O3 under Visible Light Irradiation.

Aspergillus flavus is a kind of widespread fungi that can produce carcinogenic, teratogenic, and mutagenic secondary metabolites known as aflatoxins. Aspergillus flavus mainly spread through the means of fungal spores in air, thus preventing the spores spread is an effective strategy to control aflatoxins contamination from source. Herein, a rapid and efficient control way to prevent the spread of Aspergillus flavus spores in air was demonstrated. Ag-AgCl nanoparticles were combined with tetrahedral α-Fe2O3 to form plasmonic composites that presented 93.65 ± 1.53% prevention rate of Aspergillus flavus spores under 50 min visible light irradiation. The efficient activity was attributed to the synergy effect of Ag including intrinsic disinfection, electron sink, and localized surface plasmon resonance effect, which were proven by photoelectric characterization, density functional theory, and finite difference time domain methods. The calculated work functions of α-Fe2O3, Ag, and AgCl were 3.71, 4.52, and 5.38 eV, respectively, which could accelerate photoinduced carrier transfer through Ag during photoreaction. Moreover, it was found that the intrinsic disinfection of Ag and hydroxyl radical from photocatalytic reaction were the main factors to the prevention of Aspergillus flavus spores, which resulted in the destruction of spore structure and the leakage of intracellular protein with 62.15 ± 2.63 µg mL-1. Most important, it was proven that the composites also showed high activity (90.52 ± 1.26%) to prevent Aspergillus flavus spore spread in the storage process of peanuts. These findings not only provided useful information for an efficient and potential strategy to prevent Aspergillus flavus contamination but also could be as a reference in toxic fungi control.

Aflatoxins from the endophytic fungus Aspergillus sp. Y-2 isolated from the critically endangered conifer Abies beshanzuensis.

Two new [asperxins A (1) and B (2)] and four known (3-6) aflatoxins were isolated and identified from the endophytic fungus Aspergillus sp. Y-2, which was derived from the needles of the critically endangered conifer Abies beshanzuensis. Their structures were elucidated by extensive spectroscopic methods. Among the isolates, compounds 1 and 5 showed considerable cytotoxicities against the A549 and Hela human cancer cell lines, with IC50 values in the range of 7.5-16.8 µM.

Encapsulation in chitosan-based nanomatrix as an efficient green technology to boost the antimicrobial, antioxidant and in situ efficacy of Coriandrum sativum essential oil.

The present investigation deals with first time report on encapsulation of Coriandrum sativum essential oil (CSEO) in chitosan nanomatrix as a green nanotechnology for enhancing its antimicrobial, aflatoxin inhibitory and antioxidant efficacy. Chitosan nano biopolymer entrapped CSEO as prepared through ionic gelation process showed broad spectrum fungitoxicity against molds infesting stored rice and also exhibited enhanced bioefficacy than unencapsulated CSEO. The CSEO entrapped in chitosan nanomatrix lead to decrement in important fungal membrane biomolecule i.e. ergosterol and leakage of UV-absorbing substances along with vital cellular ions. The CSEO encapsulation in selected biopolymer nanomatrix effectively checked methylglyoxal (the aflatoxin inducer) biosynthesis, confirming antiaflatoxigenic mode of action. The physico-chemical properties, considerable decrease in lipid peroxidation and improved in situ AFB1 suppressive as well as antifungal potential of CSEO nanocapsules suggested the deployment of chitosan based nano biopolymer for encapsulation of essential oils as an ecofriendly technology for application in food industries in order to enhance the shelf life and control the fungal and aflatoxin contamination of stored rice.

Aflatoxin-Guanine DNA Adducts and Oxidatively Induced DNA Damage in Aflatoxin-Treated Mice in Vivo as Measured by Liquid Chromatography-Tandem Mass Spectrometry with Isotope Dilution.

Dietary exposure to aflatoxin B1 (AFB1) is a significant contributor to the incidence of hepatocellular carcinomas globally. AFB1 exposure leads to the formation of AFB1-N7-guanine (AFB1-N7-Gua) and two diastereomers of the imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B1 (AFB1-FapyGua) in DNA. These adducts lead to G → T transversion mutations with the ring-opened adduct being more mutagenic than the cationic species. Accurate measurement of these three adducts as biomarkers in DNA and urine will help identify dietary exposure to AFB1 as a risk factor in the development of hepatocellular carcinoma worldwide. Herein, we report an improved methodology for the measurement of AFB1-N7-Gua and the two diastereomers of AFB1-FapyGua using liquid chromatography-tandem mass spectrometry with isotope dilution. We measured the levels of these compounds in liver DNA of six control mice and six AFB1-treated mice. Levels varying from 1.5 to 45 lesions/106 DNA bases in AFB1-treated mice were detected depending on the compound and animal. No background levels of these adducts were detected in control mice. We also tested whether the AFB1 treatment caused oxidatively induced DNA base damage using gas chromatography-tandem mass spectrometry with isotope dilution. Although background levels of several pyrimidine- and purine-derived lesions were detected, no increases in these levels were found upon AFB1 treatment of mice. On the other hand, significantly increased levels of (5' R)- and (5' S)-8,5'-cyclo-2'-deoxyadenosines were observed in liver DNA of AFB1-treated mice. The impact of this work is expected to achieve the accurate measurement of three AFB1-DNA adducts and oxidatively induced DNA lesions as biomarkers of AFB1 exposure as germane to investigations designed for the prevention of aflatoxin-related hepatocellular carcinomas and for determining the effects of genetic deficiencies in human populations.

Antimicrobial and enzymatic activity of anemophilous fungi of a public university in Brazil

ABSTRACT To the fungal microbiota the UFPE and biotechnological potential enzymatic and antimicrobial production. Air conditioned environments were sampled using a passive sedimentation technique, the air I ratio and the presence of aflatoxigenic strains evaluated for ANVISA. Icelles were to determine the enzymatic activity of lipase, amylase and protease metabolic liquids to determine antimicrobial activity. Diversity was observed in all CAV environments, CFU/m3 ranged from 14 to 290 and I/E ratio from 0.1 to 1.5. The of the fungal genera were: Aspergillus (50%), Penicillium (21%), Talaromyces (14%), Curvularia and Paecilomyces (7% each). Aspergillus sydowii (Bainier & Sartory) Thom & Church presented enzymatic activity and the Talaromyces purpureogenus Samson, Yilmaz, Houbraken, Spierenb., Seifert, Peterson, Varga & Frisvad presented antibacterial activity against all bacteria that all environments present fungal species biodiversity no toxigenic or pathogenic fungi were found, according to ANVISA legislation for conditioned environments and airborne filamentous fungi present potential for enzymatic and antimicrobial activity.

Correlating chemical sensitivity and basal gene expression reveals mechanism of action.

Changes in cellular gene expression in response to small-molecule or genetic perturbations have yielded signatures that can connect unknown mechanisms of action (MoA) to ones previously established. We hypothesized that differential basal gene expression could be correlated with patterns of small-molecule sensitivity across many cell lines to illuminate the actions of compounds whose MoA are unknown. To test this idea, we correlated the sensitivity patterns of 481 compounds with ∼19,000 basal transcript levels across 823 different human cancer cell lines and identified selective outlier transcripts. This process yielded many novel mechanistic insights, including the identification of activation mechanisms, cellular transporters and direct protein targets. We found that ML239, originally identified in a phenotypic screen for selective cytotoxicity in breast cancer stem-like cells, most likely acts through activation of fatty acid desaturase 2 (FADS2). These data and analytical tools are available to the research community through the Cancer Therapeutics Response Portal.

Isolation and identification of toxigenic and non-toxigenic fungi in samples of medicinal plants from the market / Isolamento e identificação de fungos toxigênicos e não toxigênicos em amostras plantas medicinais do comércio

ABSTRACT: The consumption of preparations of medicinal plants has been increasing during the last decades in occidental societies. The presence of toxigenic fungi in a plant product may represent a potential risk of contamination, because of aflatoxins and ochratoxins. In this study, 12 samples of medicinal plants were analyzed in relation to the level of fungal contamination, and the presence of producers of ochratoxin A and aflatoxins was assessed by visualization of fungi using a cromatovisor in coconut milk. Most of the species found belong to the genus Cladosporium, Fusarium, Aspergillus and Penicillium. Species producing ochratoxin A were present in 2 samples (16.7%), Melissa and Hibiscus. Species producing aflatoxin were found in samples of Jacaranda decurrens (8.33%). This study suggests that herbs, if stored improperly, can provide the growth of fungi and should be examined before consumption.

RESUMO: O consumo das plantas medicinais vem aumentando nas últimas décadas nas sociedades ocidentais, porém, a presença de fungos toxigênicos nestas plantas pode representar um risco em potencial de contaminação devido à produção de aflatoxinas e ocratoxinas. Neste trabalho, 12 amostras de plantas medicinais foram analisadas em relação ao nível de contaminação por fungos, enquanto a presença de produtores de ocratoxina A e aflatoxinas foi avaliada pela visualização em cromatovisor dos fungos em meio de leite de coco. A maioria das espécies encontradas pertence aos gêneros Cladosporium, Fusarium, Aspergillus e Penicillium. Espécies produtoras de ocratoxina A estavam presentes em 2 amostras (16,7%), Melissa e Hibisco. Espécies produtoras de aflatoxina foram encontradas na amostra de Carobinha (8,33%). Este trabalho sugere que as ervas, sendo armazenadas inadequadamente, proporcionam o crescimento de fungos e, por isso, estes devem ser examinados antes do consumo.

Enhanced phenotypic alterations of alveolar type II cells in response to Aflatoxin G1 -induced lung inflammation.

Recently, we discovered that Aflatoxin G1 (AFG1 ) induces chronic lung inflammatory responses, which may contribute to lung tumorigenesis in Balb/C mice. The cancer cells originate from alveolar type II cells (AT-II cells). The activated AT-II cells express high levels of MHC-II and COX-2, may exhibit altered phenotypes, and likely inhibit antitumor immunity by triggering regulatory T cells (Tregs). However, the mechanism underlying phenotypic alterations of AT-II cells caused by AFG1 -induced inflammation remains unknown. In this study, increased MHC-II expression in alveolar epithelium was observed and associated with enhanced Treg infiltration in mouse lung tissues with AFG1 -induced inflammation. This provides a link between phenotypically altered AT-II cells and Treg activity in the AFG1 -induced inflammatory microenvironment. AFG1 -activated AT-II cells underwent phenotypic maturation since AFG1 upregulated MHC-II expression on A549 cells and primary human AT-II cells in vitro. However, mature AT-II cells may exhibit insufficient antigen presentation, which is necessary to activate effector T cells, due to the absence of CD80 and CD86. Furthermore, we treated A549 cells with AFG1 and TNF-α together to mimic an AFG1 -induced inflammatory response in vitro, and we found that TNF-α and AFG1 coordinately enhanced MHC-II, CD54, COX-2, IL-10, and TGF-ß expression levels in A549 cells compared to AFG1 alone. The phenotypic alterations of A549 cells in response to the combination of TNF-α and AFG1 were mainly regulated by TNF-α-mediated induction of the NF-κB pathway. Thus, enhanced phenotypic alterations of AT-II cells were induced in response to AFG1 -induced inflammation. Thus, AT-II cells are likely to suppress anti-tumor immunity by triggering Treg activity.

Aflatoxins of type B and G affect porcine dendritic cell maturation in vitro.

The toxic effects of highly carcinogenic mycotoxins, especially aflatoxins (AF), on key antigen-presenting cells, such as dendritic cells (DC), are largely unknown. To elucidate the effect of AF on DC function, porcine monocyte-derived DC (MoDC) were treated with a mixture of several AF (i.e., AFB1, AFB2, AFG1, and AFG2) and the phagocytic capacity, the membrane expression level of several DC activation markers, the T-cell proliferation-inducing capacity, and the cytokine secretion pattern were assessed. As compared to untreated MoDC, AF significantly up-regulated the expression of the co-stimulatory molecules CD25 and CD80/86. However, the phagocytic activity of MoDC was not affected by AF treatment. While the cytokine secretion pattern of AF-treated MoDC was similar to control MoDC, the T-cell proliferation-inducing capacity of MoDC was increased upon aflatoxin treatment. The results indicate that a mixture of naturally occurring AF enhances the antigen-presenting capacity of DC, which could explain the observed immunotoxicity of AF by breaking down tolerance and further emphasizes the need to reduce the admissible level of AF in agricultural commodities.

Antimicrobial aflatoxins from the marine-derived fungus Aspergillus flavus 092008.

A new aflatoxin, aflatoxin B(2b) (1), together with six known compounds, were isolated from the marine-derived fungus Aspergillus flavus 092008 endogenous with the mangrove plant Hibiscus tiliaceus (Malvaceae). The structure of 1 was determined by the spectroscopic and chemical methods. Compound 1 exhibited a moderate antimicrobial activity against Escherichia coli, Bacillus subtilis and Enterobacter aerogenes, with MIC values of 22.5, 1.7 and 1.1 M, respectively. Compound 1 also showed a weak cytotoxicity against A549, K562 and L-02 cell lines, with IC(50) values of 8.1, 2.0 and 4.2 M, respectively. The results showed that hydration and hydrogenation of (8)-double bond significantly reduces the cytotoxicity of aflatoxins, while the esterification at C-8 increases the cytotoxicity.

[Effect of aflatoxin G1 on the expression of HLA- I molecule in human esophageal epithelial cells].

OBJECTIVE: To explore the effect of AFG1 on the expression of human leukocyte antigen (HLA-I) molecule in human esophageal epithelial cells. METHOD: Western Blot and RT-PCT analysis was used to explore the effect of AFG1 on the antigen presenting function of primary cultured esophageal epithelial cells. RESULTS: The protein expression of HLA-ABC was significantly decreased in the groups treated with 100, 1000 and 2000 microg/L AFG1 as compared with the solvent control group (P < 0.05). Within the range of 100 - 2000 microg/L AFG1, the protein expression level of HLA-ABC of esophageal epithelial cells decreased gradually, and showed significant negative correlation (r = -0.921, n = 3, P < 0.01). The effect of AFG1 on the expression of HLA-ABC mRNA was further confirmed by RT-PCR analysis. The results showed that after treated with 100 microg/L, 1000 microg/L and 2000 microg/L AFG1, the expression of HLA-A mRNA was decreased significantly as compared with the solvent control (P < 0.05), and the expression of HLA-B mRNA was decreased in the 2000 microg/L AFG1 treated group (P < 0.05), while there was no effect on the expression of HLA-C mRNA. CONCLUSION: The expression of HLA- I molecule in human esophageal epithelial cells could be inhibited by AFG1 treatment.

The selectivity of austocystin D arises from cell-line-specific drug activation by cytochrome P450 enzymes.

The natural product austocystin D was identified as a potent cytotoxic agent with in vivo antitumor activity and selectivity for cells expressing the multidrug resistance transporter MDR1. We sought to elucidate the mechanism of austocystin D's selective cytotoxic activity. Here we show that the selective cytotoxic action of austocystin D arises from its selective activation by cytochrome P450 (CYP) enzymes in specific cancer cell lines, leading to induction of DNA damage in cells and in vitro. The potency and selectivity of austocystin D is lost upon inhibition of CYP activation and does not require MDR1 expression or activity. Furthermore, the pattern of cytotoxicity of austocystin D was distinct from doxorubicin and etoposide and unlike aflatoxin B(1), a compound that resembles austocystin D and is also activated by CYP enzymes to induce DNA damage. Theses results suggest that austocystin D may be of clinical benefit for targeting or overcoming chemoresistance.

Aflatoxin effetc on the oocysts morphometry and contribution on the morphology of Eimeria bateri Bhatia, Pandey and Pande, 1965 from the japanese quail Coturnix japonica in Brazil / Efeito da aflatoxina sobre a morfbmetria dos oocistos e contribuição para a morfologia de Eimeria bateri Bhatia, Pandey and Pande, 1965 da Codorna japonesa Coturnix japonica no Brasil

The purpose of this study was to characterize Eimeria bateri oocysts and to evaluate the aflatoxin effect in the morphometry of sporulated oocysts in Japanese quails infected naturally. Of a total of 50 quails naturally infected by E. bateri were randomly divided into two groups with 25 birds each. In one of them, quails were orally administered with aflatoxin in dose of 0.04 mg/kg body weight previously. Both experimental groups shed E. bateri oocysts. These oocysts were subspherical to ellipsoidal, 25.1 x 18.9 Lim, with bi-layered wall. Micropyle and residuum were absent, but one or more polar granules were present. Sporocysts elongate ovoid, 12.5 x 7.4 μm. Stieda and substieda bodies were present. Sporocyst residuum was dispersed and sporozoites presented a nucleus and a refractile body. Histograms confirmed the presence of a single species, E. bateri. Linear regression proved that E. bateri oocysts are polymorphic, due, basically, to shape of these oocysts. The comparative morphometry between two experimental groups demonstrated that the aflatoxin influenced significantly in the E. bateri oocysts.

O objetivo deste estudo foi caracterizar os oocistos de Eimeria bateri e avaliar o efeito da aflatoxina na morfometria destes oocistos em codornas japonesas naturalmente infectadas. Cinqüenta codornas naturalmente parasitadas por E. bateri foram separadas aleatoriamente em dois grupos com 25 aves cada. Um dos grupos foi intoxicado experimentalmente com aflatoxina, por via oral, na dose de 0,04 mg/kg de peso vivo. Os dois grupos experimentais eliminaram oocistos de E. bateri nas fezes. Esses oocistos foram de subesféricos a elipsóides, 25,1 x 18,9 Lm, com parede dupla. A micrópila e o resíduo estavam ausentes, mas um ou vários grânulos polares estavam presentes. Esporocistos ovóides alongados, 12,5 x 7,4 L m. Os corpos de Stieda e substieda estavam presentes. O resíduo do esporocisto estava disperso e os esporozoítas apresentaram um núcleo e um corpo refráctil. Os histogramas confirmaram a presença de uma única espécie, E. bateri. A regressão linear comprovou que os oocistos de E. bateri são polimórficos, devido, basicamente, à forma desses oocistos. A morfometria comparativa entre os dois grupos experimentais, demonstrou que a aflatoxina influiu significativamente nos oocistos de E. bateri.

Modulation of aflatoxin biomarkers in human blood and urine by green tea polyphenols intervention.

To evaluate the efficacy of green tea polyphenols (GTPs) in modulating aflatoxin B(1) (AFB(1)) biomarkers, a total of 352 serum samples and 352 urine samples collected from a 3 month chemoprevention trial with 500 mg GTPs, 1000 mg GTPs and a placebo were measured for AFB(1)-albumin adducts (AFB-AA), aflatoxin M(1) (AFM(1)) and aflatoxin B(1)-mercapturic acid (AFB-NAC). Levels of AFB-AA at baseline were comparable for all three dose groups (P = 0.506). No significant differences were observed in AFB-AA levels in the placebo group over the 3 month period (P = 0.252). However, a significant reduction in AFB-AA levels was observed in the 500 mg group (P = 0.002). A marginally significant reduction in AFB-AA levels was also found in the 1000 mg group over the 3 month intervention period (P = 0.051). An analysis using a mixed-effects model indicated that the reduction in AFB-AA levels over time was dose and time dependent (dose-time interaction P = 0.049). There were no significant differences in median AFM(1) levels among the three study groups at the baseline (P = 0.832), 1 month (P = 0.188) and 3 months (P = 0.132) of the GTP intervention; however, reduction of 42 and 43% in median AFM(1) levels, as compared with the placebo, were found in 500 mg (P = 0.096) and 1000 mg (P = 0.072) groups at 3 months of the intervention. Significant elevations in median AFB-NAC levels and the ratio of AFB-NAC:AFM(1) were found in both 500 and 1000 mg groups compared with the placebo group at both 1 month (P < 0.001) and 3 months (P < 0.001) of GTPs intervention. These results demonstrate that GTPs effectively modulate AFB(1) metabolism and metabolic activation.

Binding of aflatoxins to the 20S proteasome: effects on enzyme functionality and implications for oxidative stress and apoptosis.

Aflatoxins (AF) are contaminants of improperly stored foods; they are potent genotoxic and carcinogenic compounds, exerting their effects through damage to DNA. They can also induce mutations that increase oxidative damage. The goal of this study was to evaluate the possibility that a third mechanism could be involved in the carcinogenic action of aflatoxins, namely, direct binding to key enzymes involved in the regulatory pathways of the cell cycle, thereby modulating enzyme functionality. The 20S constitutive and immunoproteasome peptidase and proteolytic activities were assayed in the presence of aflatoxins B1, G1 and M1. All three toxins activated multiple peptidase activities of the proteasome. Aflatoxin (AF) M1 was the most potent activator of proteasome activity, while the constitutive 20S proteasome was specifically stimulated by AFG1. Furthermore, the effects of AFB1 on cultured hepatoma cells were investigated and the various proteasomal activities determined with cell lysates were differently affected. Taking into account the key role of the proteasome in cellular defense against oxidative stress, the carbonyl group content and the activities of antioxidant enzymes in cell lysates were analyzed. The proapoptotic effect of AFB1 was also investigated by measuring caspase-3 activity and cellular levels of p27 and IkappaBalpha.

Dietary exposure to aflatoxin from maize and groundnut in young children from Benin and Togo, West Africa.

Aflatoxins are a family of fungal toxins that are carcinogenic to man and cause immunosuppression, cancer and growth reduction in animals. We conducted a cross-sectional study among 480 children (age 9 months to 5 years) across 4 agro-ecological zones (SS, NGS, SGS and CS) in Benin and Togo to identify the effect of aflatoxin exposure on child growth and assess the pattern of exposure. Prior reports on this study [Gong, Y.Y.,Cardwell, K., Hounsa, A., Egal, S., Turner, Hall, A.J., Wild, C.P., 2002. Dietary aflatoxin exposure and impaired growth in young children from Benin and Togo: cross sectional study. British Medical Journal 325, 20-21, Gong, Y.Y., Egal, S., Hounsa, A., Turner, P.C., Hall, A.J., Cardwell, K., Wild, C.P., 2003. Determinants of aflatoxin exposure in young children from Benin and Togo, West Africa: the critical role of weaning and weaning foods. International Journal of Epidemiology, 32, 556-562] showed that aflatoxin exposure among these children is widespread (99%) and that growth faltering is associated with high blood aflatoxin-albumin adducts (AF-alb adducts), a measure of recent past exposure. The present report demonstrates that consumption of maize is an important source of aflatoxin exposure for the survey population. Higher AF-alb adducts were correlated with higher A. flavus (CFU) infestation of maize (p=0.006), higher aflatoxin contamination (ppb) of maize (p<0.0001) and higher consumption frequencies of maize (p=0.053). The likelihood of aflatoxin exposure from maize was particularly high in agro-ecological zones where the frequency of maize consumption (SGS and CS), the presence of aflatoxin in maize (SGS) or the presence of A. flavus on maize (NGS and SGS) was relatively high. Socio-economic background did not affect the presence of A. flavus and aflatoxin in maize, but better maternal education was associated with lower frequencies of maize consumption among children from the northernmost agro-ecological zone (SS) (p=0.001). The impact of groundnut consumption on aflatoxin exposure was limited in this population. High AF-alb adduct levels were correlated with high prevalence of A. flavus and aflatoxin in groundnut, but significance was weak after adjustment for weaning status, agro-ecological zone and maternal socio-economic status (resp. p=0.091 and p=0.083). Ingestion of A. flavus and aflatoxin was high in certain agro-ecological zones (SS and SGS) and among the higher socio-economic strata due to higher frequencies of groundnut consumption. Contamination of groundnuts was similar across socio-economic and agro-ecological boundaries. In conclusion, dietary exposure to aflatoxin from groundnut was less than from maize in young children from Benin and Togo. Intervention strategies that aim to reduce dietary exposure in this population need to focus on maize consumption in particular, but they should not ignore consumption of groundnuts.

Interferon-alpha gene therapy prevents aflatoxin and carbon tetrachloride promoted hepatic carcinogenesis in rats.

Retrovirus-mediated interferon alpha (IFN-alpha) gene transfer was evaluated with regard to its possible protective effects against aflatoxin B1 (AFB1)-initiated and carbon tetrachloride (CCl4)-promoted hepatic carcinogenesis in rats. To our knowledge, this is the first time an experimental in vivo gene therapy trial was conducted in Egypt. Two genes were examined in liver tissue by RT-PCR: the first was glutathione-S-transferase placental (GST-P) isoenzyme, as an early marker to detect hepatic malignancy; the second was IFN-alpha gene expression to detect the efficiency of gene uptake and its persistence after transduction. Forty male rats, divided equally into 4 groups, were included in the study: the first group was the control; the second group received CCl4 0.2 ml subcutaneously twice weekly for 12 weeks and AFB1 0.25 mg/kg body wt intraperitoneally twice weekly for 6 weeks; the third group received IFN-alpha (10(8) pfu) intravenously in the tail vein prior to the start of CCl4 and AFB1 injections; and the fourth group received IFN-alpha (10(8) pfu) by intrahepatic injection under ultrasonography guide after termination of the CCl4 and AFB1 injection schedule. The results showed that IFN-alpha has a marked and significant protective effect against hepatic fibrogenesis as well as hepatic carcinogenesis. Pathological examination of liver tissue proved that IFN-alpha minimized both fibrotic and cirrhotic processes. The amount of fibrosis was less in both groups receiving IFN-alpha, with more protection in the group that received IFN-alpha intravenously prior to CCl4 and AFB1. The results of RT-PCR showed that the IFN-alpha gene was significantly expressed in both groups receiving IFN-alpha, with a more intense expression in the group that received IFN-alpha by intrahepatic injection after termination of CCl4 and AFB1 injections. The IFN-alpha gene was detected after three months of gene transduction in rats receiving IFN-alpha intravenously prior to CCl4 and AFB1 and after one month of gene transduction in the post CCl4 and AFB1 rats. IFN-alpha gene was not expressed in the two groups that did not undergo gene transfer. Histopathological signs of premalignant macronodules were evident in the group receiving CCl4 and AFB1, but not IFN-alpha as well as in the group that received IFN-alpha at the end of the experiment. GST-P gene expression was also detected in these two groups, confirming early malignant transformation. In conclusion, IFN-alpha exerts significant protective effects, but more so when the gene is administered before fibrogenic and carcinogenic induction in hepatic tissues. IFN-alpha gene therapy may be justified in clinical trials for high-risk candidates with hepatic carcinogenesis.

Ser-249TP53 mutation in tumour and plasma DNA of hepatocellular carcinoma patients from a high incidence area in the Gambia, West Africa.

Hepatocellular carcinoma (HCC) is frequent in areas of high exposure to aflatoxin and high prevalence of HBV infection, such as western Africa and south-east China. A selective mutation in TP53 (AGG-->AGT at codon 249, Arg-->Ser) has been identified as a hotspot in HCCs from such areas, reflecting DNA damage caused by aflatoxin metabolites. Recent studies have shown that circulating free DNA can be retrieved from human plasma, and it is hypothesised that plasma DNA may serve as a source for biomarkers of tumorigenic processes. In our study, we have determined the prevalence of Ser-249 mutation, using a PCR-restriction digestion method, with selective use of short oligonucleotide mass spectrometry analysis (SOMA), in a series of 29 biopsy specimens of HCC from The Gambia in West Africa. Overall, we identified the Ser-249 mutation in 35% (10/29) of the tumours. In parallel, we tested 17 plasma samples from HCC patients with matching tumour tissue. The 249 status concordance between tumour tissues and matched plasma was 88.5%. These results indicate that the Ser-249 mutation is common in HCC in The Gambia (35%), although a higher prevalence has been reported in other regions with high population exposure to aflatoxin (e.g., eastern China: >50%). Moreover, our studies indicate that plasma is a convenient source of liver tumour-derived DNA, thus holding promise for earlier detection and diagnosis of cancer.