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.

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.

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.

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

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.

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.

Effect of the aflatoxins B1 and M1 on bovine oocyte developmental competence and embryo morphokinetics.

Aflatoxins are considered as reproductive toxins for mammalian species. Here, we studied the effect of aflatoxin B1 (AFB1) and its metabolite aflatoxin M1 (AFM1) on the development and morphokinetics of bovine embryos. Cumulus oocyte complexes (COCs) were matured with AFB1 (0.032, 0.32, 3.2, 32 µM) or AFM1 (0.015, 0.15, 1.5, 15, 60 nM), then fertilized and the putative zygotes were cultured in an incubator equipped with a time-lapse system. Exposing COCs to 32 µM AFB1 or 60 nM AFM1 reduced the cleavage rate, whereas exposing them to 3.2 or 32 µM AFB1 further reduced the blastocyst formation. A delay was recorded for the first and second cleavages in a dose-dependent manner for both AFB1- and AFM1-treated oocytes. A delay was recorded in the third cleavage in the AFM1-treated group. To explore potential mechanisms, subgroups of COCs were examined for nuclear and cytoplasmic maturation (n = 225; DAPI and FITC-PNA, respectively), and mitochondrial function was examined in a stage-dependent manner. COCs were examined for their oxygen consumption rates (n = 875; Seahorse XFp analyzer) at the end of maturation, MII-stage oocytes were examined for their mitochondrial membrane potential (n = 407; JC1), and putative zygotes were examined using a fluorescent time-lapse system (n = 279; IncuCyte). Exposing COCs to AFB1 (3.2 or 32 µM) impaired oocyte nuclear and cytoplasmic maturation and increased mitochondrial membrane potential in the putative zygotes. These alterations were associated with changes in the expression of mt-ND2 (32 µM AFB1) and STAT3 (all AFM1 concentrations) genes in the blastocyst stage, suggesting a carryover effect from the oocyte to the developing embryos.

Mycobiota and mycotoxins (aflatoxins and ochratoxin) associated with some Saudi date palm fruits.

This study aimed to determine the mycological profile of the retail date fruits distributed in different markets at Taif, Saudi Arabia. The presence of aflatoxins and ochratoxin A was also measured. Twenty-two fungal species belonging to 12 genera were isolated from 50 different date samples. Aspergillus flavus, A. niger, Penicillium chrysogenum, and Rhizopus stolonifer were the most prevalent species among isolated fungi. Eighty isolates of A. flavus and 36 of A. niger were detected for their aflatoxins and ochratoxin production potentials using thin layer chromatography. Toxicity test using Artimia larvae indicated that seven out of 18 A. flavus isolates had aflatoxins potentials, while nine out of 36 isolates of A. niger were ochratoxigenic. The quadruplex polymerase chain reaction using specific primers demonstrated the presence of four genes: nor A, ver 1, omt A, and avf A in seven A. flavus toxigenic isolates. Nine A. niger toxigenic isolates showed positive results for the presence of the PKS gene. In conclusion, the present study highlighted the potential hazards of mycotoxins on human health from consuming raw dates. Rapid molecular detection methods described here might help the food authorities to assure the safety of raw dates distributed in local markets.

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.

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.

Volatiles of antagonistic soil yeasts inhibit growth and aflatoxin production of Aspergillus flavus.

Minimizing Aspergillus flavus growth is an effective strategy to mitigate aflatoxin contamination in food and agricultural products. In the present investigation, we attempted to utilize soil-associated yeasts from the Western and Eastern Ghats of India against A. flavus to reduce aflatoxin contamination. Forty-five yeast isolates were screened against A. flavus using overlay and dual plate assays. Among them, 12 isolates effectively inhibited the growth of A. flavus. The 18S rDNA gene sequence analysis identified the twelve antagonistic isolates as belonging to Saccharomyces cerevisiae, Suhomyces xylopsoci, Pichia kudriavzevii, and Candida tropicalis. From the isolated yeasts, S. cerevisiae strains were selected for further evaluation based on the potential antagonistic activity. Volatiles of S. cerevisiae effectively suppressed the mycelial growth of A. flavus (P < 0.05) up to 92.1 % at 7 DAI. Scanning electron microscopic images of the fungus exposed to volatiles showed hyphal deformity and mycelial damage. Aflatoxin B1 (AFB1) production was drastically reduced up to 99.0 % in the volatile-exposed fungus compared to the control. The yeast strain YKK1 showed consistent Aspergillus flavus growth inhibition (80.7 %) and AFB1 production (98.1 %) for 14 days. Gas chromatography-mass spectrophotometry analysis of the yeast volatiles revealed the presence of antimicrobial compounds, including 1-pentanol, 1-propanol, ethyl hexanol, ethanol, 2-methyl-1-butanol, ethyl acetate, dimethyl trisulfide, p-xylene, styrene, and 1,4-pentadiene. The evaluated compounds of yeast volatiles, including ethyl acetate, hexanal, 1-propanol, 1-heptanol, 1-butanol, and benzothiazole, inhibited the fungal growth and AFB1 production of Aspergillus flavus when applied as pure chemicals. Benzothiazole at 5 mM was responsible for a high level of growth inhibition (23.6 %) and reduction of AFB1 synthesis (93.5 %). Hence, volatile compounds produced by soil yeast strains could be a potential biocontrol mechanism against aflatoxin contamination.

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.

[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.

[Influence of aflatoxin on Vibrio fischeri luminescence].

OBJECTIVE: In the present study, we aim to evaluate the inhibitory effect of aflatoxin on Vibrio fischeri luminescence. METHODS: V. fischeri culture is treated with aflatoxin or the culture broth of aflatoxin-producing strains, and the luminescence intensity of V. fischeri is detected to analyze the influence of aflatoxin on V. fischeri. RESULTS: The logarithmic value of aflatoxin concentration and the decrease ratio of V. fischeri luminescence is in a linear relationship. Based on the regression equation between aflatoxin concentration and luminescence decrease of V. fischeri, the toxin-producing status of different microbes can be detected quickly and exactly: all of six tested Aspergillus flavus strains show toxigenicity to V. fischeri, and their toxin yield reached 14.94 mg/L - 46.45 mg/L (represented by aflatoxin concentration), while the tested Aspergillus oryzae shows no toxigenicity. CONCLUSION: The above data showed that the luminescence change of V. fischeri could exactly reflect the capability of various microbes to produce toxin (especially aflatoxin), which provided a new clue for rapid detection of aflatoxin in industrial and agricultural production and could be developed as a potential method for aflatoxin assay.

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.

Genetic resistance to aflatoxin in Japanese quail.

Progress was rapid in attempts to develop lines of quail resistant to acute aflatoxicosis induced by oral dosing with aflatoxin. After five generations of selection, 8- and 11-fold differences were present in mortality between two selected lines and their respective control lines. These quail lines should be of value in investigating the physiological basis of resistance to aflatoxin.

Salt-induced conversion of B-DNA to Z-DNA inhibited by aflatoxin B1.

The carcinogen aflatoxin B1 was reacted with a polymer of alternating deoxyguanine and deoxycytosine residues to determine the effect that adduct formation has on the conversion of this polymer from the right-handed B-DNA form found at low salt concentrations to the left-handed Z-DNA form found at high salt concentrations. Reaction with aflatoxin strongly inhibited the salt-induced conversion of this polymer from B-DNA to Z-DNA. This inhibition could be detected even at relatively low binding levels.

Rapid and marked inhibition of rat-liver RNA polymerase by aflatoxin B-1.

From 15 minutes to 2 hours after the administration of aflatoxin B(1) invivo there is a 35-to 70-percent inhibition of DNA-directed RNA synthesis. The inhibition was reversed 12 and 24 hours later.

Aflatoxin B1: binding to DNA in vitro and alteration of RNA metabolism in vivo.

Aflatoxin B(1) binds to both native and denatured DNA, as shown by spectroscopy and equilibrium dialysis. It also strongly inhibits incorporation of cytidine into rat liver nuclear RNA and lowers the RNA content of the nucleus. The extremle toxicity and carcinogenicity of aflatoxin B(1) may be direct results of the affinity of this agent for DNA.

Morphologic, molecular and metabolic characterization of Aspergillus section Flavi in spices marketed in Lebanon.

Spices are used extensively in Lebanon not only to flavour foods but also for their medicinal properties. To date, no data are available regarding the nature of the toxigenic fungal species that may contaminate these products at the marketing stage in this country. Eighty samples corresponding to 14 different types of spices were collected throughout Lebanon to characterize the Aspergillus section Flavi contaminating spices marketed in Lebanon and the toxigenic potential of these fungal species. Most fungal genera and species were identified as belonging to Aspergillus section Flavi. Aspergillus flavus was the most frequent species, representing almost 80% of the isolates. Although identified as A. flavus by molecular analysis, some strains displayed atypical morphological features. Seven strains of A. tamarii and one A. minisclerotigenes were also isolated. Analyses of toxigenic potential demonstrated that almost 80% of strains were able to produce mycotoxins, 47% produced aflatoxins, and 72% produced cyclopiazonic acid, alone or in combination with aflatoxins.

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.