Carcinogen Aflatoxin B1 Research Articles

Aflatoxicosis: Lessons from Toxicity and Responses to Aflatoxin B1 in Poultry

This review is a comprehensive introduction to the effects of poultry exposure to the toxic and carcinogenic mycotoxin aflatoxin B1 (AFB1). The relationship between AFB1 sensitivity and metabolism, major direct and indirect effects of AFB1, recent studies of gene expression and transcriptome responses to exposure, and mitigation strategies to reduce toxicity are discussed. Exposure to AFB1 primarily occurs by consumption of contaminated corn, grain or other feed components. Low levels of residual AFB1 in poultry feeds can cause reduction in growth, feed conversion, egg production, and compromised immune functions, resulting in significant economic costs to producers. Thus, AFB1 acts as a “force multiplier” synergizing the adverse effects of microbial pathogens and other agents, and factors detrimental to poultry health. Domestic turkeys (Meleagris gallopavo) are one of the most sensitive animals known to AFB1 due, in large part, to a combination of efficient hepatic bioactivation by cytochromes P450 1A5 and 3A37, and deficient hepatic glutathione-S-transferase (GST)-mediated detoxification. Because of their sensitivity, turkeys are a good model to investigate chemopreventive treatments and feed additives for their ability to reduce AFB1 toxicity. Transcriptome analysis (RNA-seq) of turkey poults (liver and spleen) has identified AFB1-induced gene expression changes in pathways of apoptosis, carcinogenesis, lipid regulation, antimicrobial activity, cytotoxicity and antigen presentation. Current research focuses on further identifying the molecular mechanisms underlying AFB1 toxicity with the goal of reducing aflatoxicosis and improving poultry health.

Abstract 3745: Comprehensive integrative characterization of hepatocellular carcinoma: The TCGA HCC project

Abstract Background: Hepatocellular carcinomas (HCCs) are heterogenous primary liver cancers with pleomorphic histologic features. We present a comprehensive genetic, genomic, transcriptomic and proteomic analysis of 203 HCCs in The Cancer Genome Atlas Program (TCGA) on behalf of the TCGA Liver analysis working group to provide a deeper understanding of the molecular features of HCCs, to classify them in a clinically-relevant manner, and to provide a public resource that identifies potential targets for emerging therapies.Results: Mutation analyses confirmed the presence of TERT promoter mutations in 40% of HCCs. The analysis also revealed the first reported germline mutation in the TERT promoter in a 30 year old male, possibly reflecting an inherited predisposition to HCC. TERT promoter mutations were enriched in hepatitis C virus infected subjects. Analyses of hepatitis B virus (HBV)-induced HCCs demonstrated recurrent integrations of HBV in the TERT locus. Thus, HBV integration may substitute for TERT promoter mutations as an alternate mechanism of telomerase activation. Other tumor suppressor genes inactivated by mutation included the cell cycle genes TP53 (32% of cases), RB1 (8%), and PTEN (3%), the Wnt/beta catenin pathway gene AXIN1 (4%), and the chromatin remodeling genes ARID1A (9%), BAP1 (6%), ARID1B (4%), ARID2 (4%), and PBRM1 (3%). The oncogene CTNNB1 was mutated in 27% of HCCs. Mutations were also found in the MTOR pathway genes, MTOR (4%), TSC2 (4%) and TSC1 (3%). The patterns of mutational signatures observed included signatures characteristic of the known carcinogens aristolochic acid and aflatoxin B1. Clustering of mRNA, miRNA, or protein expression, DNA hypermethylation and DNA copy number profiles identified five, four, two, five and three clusters, respectively. Analysis of mRNA expression data identified five groups. In particular, Group 1 was associated with Asian race, higher histologic grade, and micro- and macrovascular invasion. Integrated clustering showed a grouping of HCCs into three superclusters: iCluster 1 had a low frequency of CDKN2A silencing, lack of CTNNB1 mutation, a normal-like methylation pattern, and overexpression of proliferation marker genes; iClusters 2 and 3 had a high frequency of CDKN2A silencing, CTNNB1 mutation, and overexpression of oxidative response genes; iCluster 3 tumors had a higher degree of chromosomal instability, high frequency of 17p loss, and a highly altered methylation profile. Copy number aberrations included gains in the MYC, VEGFA, CCND1, TERT, and MET loci and losses in the TP53, RB1, ERFFI1, CDKN2A, ARID1A and PTEN loci. Discussion: Based on integrated analysis of genome, transcriptome, methylome and proteome we showed that HCCs naturally separate into three to five distinct groups with associated molecular and clinical characteristics. Importantly, we identified a novel germline mutation in the TERT promoter that may be associated with an inherited risk for HCC. Note: This abstract was not presented at the meeting. Citation Format: Lewis R. Roberts, David A. Wheeler. Comprehensive integrative characterization of hepatocellular carcinoma: The TCGA HCC project. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3745. doi:10.1158/1538-7445.AM2015-3745

Detection of epigenetic aberrations in the development of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer death worldwide. Hepatocarcinogenesis is a complex, multistep process. It is now recognized that HCC is a both genetic and epigenetic disease; genetic and epigenetic components cooperate at all stages of hepatocarcinogenesis. Epigenetic changes involve aberrant DNA methylation, posttranslational histone modifications and aberrant expression of microRNAs all of which can affect the expression of oncogenes, tumor suppressor genes and other tumor-related genes and alter the pathways in cancer development. Several risk factors for HCC, including hepatitis B and C virus infections and exposure to the chemical carcinogen aflatoxin B1 have been found to influence epigenetic changes. Their interactions could play an important role in the initiation and progression of HCC. Discovery and detection of biomarkers for epigenetic changes is a promising area for early diagnosis and risk prediction of HCC.

Prenatal exposure of mice to the human liver carcinogen aflatoxin B1 reveals a critical window of susceptibility to genetic change.

It has become axiomatic that critical windows of susceptibility to genotoxins exist and that genetic damage in utero may be a trigger for later life cancers. Data supporting this critical window hypothesis are remarkably few. This study provides a quantitative bridge between DNA damage by the liver carcinogen aflatoxin B1 (AFB1 ) during prenatal development and the risk of later life genetic disease. AFB1 was given to pregnant C57BL/6J mice, carrying F1 gestation day 14 (GD14) embryos of the B6C3F1 genotype. Ultra-high performance liquid chromatography and mass spectrometry (UPLC-MS) using aflatoxin-(15) N5 -guanine adduct standards afforded measurement of the AFB1 -N(7) -Gua and AFB1 -FAPY adducts 6-hr post dosing in liver DNA of mothers and embryos. A parallel cohort gave birth and the livers of the F1 were analyzed for mutations in the gpt gene at 3 and 10 weeks of age. The data revealed mutational spectra dominated by G:C to T:A mutations in both the mother and offspring that are characteristic of AFB1 and distinct from background. It was shown that adducts in GD14 embryos were 20-fold more potent inducers of mutagenesis than adducts in parallel-dosed adults. This sensitivity enhancement correlated with Ki67 staining of the liver, reflecting the proliferative potential of the tissue. Taken together, these data provide insight into the relative genetic risks of prenatal and adult exposures to AFB1 . Early life exposure, especially during the embryonic period, is strikingly more mutagenic than treatment later in life. Moreover the data provide a baseline against which risk prevention strategies can be evaluated.

Carcinogenic effects of aflatoxin B1 among wheat handlers.

Epidemiological studies have demonstrated that serum aflatoxin B1 (AFB1) is a hepatocarcinogenic mycotoxin and contributor to the high rate of hepatocellular carcinoma (HCC). The prevalence of liver cancer in Egypt is particularly worrisome. In a registry-based analysis of occupational risk for HCC, significant excesses were observed especially for grain mill workers. The aim of this study was to assess the hepatic carcinogenicity of AFB1 in wheat handlers. Serum AFB1/albumin (AFB1/Alb), alpha-fetoprotein (AFP), alpha-l-fucosidase (AFU), and arginase were estimated in exposed wheat handlers including millers and bakers. The control group was composed of non-occupationally exposed workers. AFB1/Alb and AFU were significantly higher among workers employed as bakers compared to mill workers and controls. Mill workers had higher levels of AFB1/Alb than the controls. AFB1/Alb, AFP, and AFU were all significantly higher and arginase was significantly lower among HCC cases compared to the other groups. There was a significant correlation between AFU and AFB1/Alb in bakers and between AFP and AFB1/Alb in HCC cases. Arginase was inversely correlated with AFB1/Alb in HCC cases. AFB1/Alb was significantly correlated with the duration of exposure in bakers. Wheat handlers exposed to Aspergillus flavus have a high risk of elevated serum AFB1/Alb levels and AFU.

Occurrence of aflatoxin M₁ in UHT milk and infant formula samples consumed in Burdur, Turkey.

Aflatoxins are toxic fungal metabolites found in foods and feeds. Aflatoxin M₁ (AFM₁), a metabolite of the potent carcinogen aflatoxin B₁(AFB₁), occurs in milk from animals consuming feed contaminated with AFB1. The aim of this study was to investigate the occurrence and levels of AFM₁ in ultra-high temperature (UHT) milk and infant formula samples consumed in Burdur City. During 2011, 41 samples of UHT milk and 33 samples of infant formula were randomly collected from different supermarkets and drug stores. The occurrence and contamination levels of AFM₁ in the samples were investigated using the competitive enzyme-linked immunoabsorbent assay (ELISA) method. AFM₁ was detected in 30 samples (73.2%) in concentrations ranging from 6.42 to 71.33 ng/L (mean level, 17.76 ng/L). Altogether, only three (7.3%) samples of UHT milk were contaminated at levels above the Turkish legal limit (50 ng/L). However, AFM₁ was not detected in all infant formula samples. It is concluded that despite the widespread occurrence of AFM₁ in UHT milk samples, the contamination levels were not serious for public health.

In vivo treatment with aflatoxin B1 increases DNA oxidation, base excision repair activity and 8-oxoguanine DNA glycosylase 1 levels in mouse lung

Carcinogenicity of the mycotoxin aflatoxin B1 (AFB1), which is produced by Aspergillus fungi, is associated with bioactivation of AFB1 to AFB1-8,9-exo-epoxide and formation of DNA adducts. However, AFB1 also causes 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation in mouse lung DNA, suggesting that oxidative DNA damage may also contribute to AFB1 carcinogenicity. The oxidative DNA damage 5-hydroxy-2′-deoxycytidine (5-OHdC) may also contribute to AFB1 carcinogenicity. The objective of the present study was to determine the effect of treatment of mice with AFB1 on pulmonary and hepatic: 8-OHdG and 5-OHdC levels; base excision repair (BER, which repairs oxidative DNA damage) activities; and on levels of 8-oxoguanine DNA glycosylase (OGG1, the rate-limiting enzyme in the BER of 8-OHdG). Female A/J mice were treated with vehicle (dimethyl sulfoxide) or 50mg/kg AFB1 ip. Oxidative DNA damage was measured using HPLC with electrochemical detection, BER activity was assessed using an in vitro assay that employs a substrate plasmid DNA with 8-OHdG lesions, and OGG1 protein levels were determined by immunoblotting. Two hours post treatment, AFB1 increased 8-OHdG levels in mouse lung DNA by approximately 69% relative to control (p<0.05), but did not alter 8-OHdG levels in liver or 5-OHdC levels in lung or liver (p>0.05). AFB1 treatment also increased BER activity in mouse lung by approximately 87% (p<0.05) but did not affect hepatic BER activity (p>0.05). Levels of OGG1 immunoreactive protein were increased in both lung (20%) and liver (60%) (p<0.05). These results are consistent with oxidative DNA damage contributing to the carcinogenicity of AFB1 in this model.

Synthesis and Characterization of Different Crystalline Calcium Silicate Hydrate: Application for the Removal of Aflatoxin B1 from Aqueous Solution

Different crystalline calcium silicate hydrates (CSH) were synthesized under specific hydrothermal conditions and several methods were used to analyze samples. Amorphous calcium silicate hydrates (ACSH) mainly consists of disordered calcium silicate hydrate gel (C‐S‐H gel) and crystalline calcium silicate hydrates (CCSH) consists of crystallized tobermorite. The adsorption of carcinogenic aflatoxin B1 (AFB1) onto ACSH and CCSH was investigated. The adsorption kinetics was studied using pseudo‐first‐order and pseudo‐second‐order kinetic models and intraparticle diffusion model. The pseudo‐second‐order model provided the best correlation and the intraparticle diffusion controlled the adsorption process of AFB1 onto CCSH. Adsorption isotherm parameters were obtained from Langmuir and Freundlich and the adsorption data fitted to Freundlich much better. Based on the results of N2 adsorption/desorption, adsorption kinetics, and adsorption isotherms, the adsorption mechanism of AFB1 onto CCSH was developed. All results indicate that CCSH has a great potential to be a safe, easy‐made, and cost‐effective material for the control of AFB1 contamination.

Electrogenerated chemiluminescence of magnesium chlorophyllin a aqueous solution and its sensitive response to the carcinogen aflatoxin B1

The chlorophylls, crucial participants of photosynthesis, are large conjugated molecules with special electron donor–acceptor properties. Based on this, many investigations on the electrogenerated chemiluminescence (ECL) of chlorophyll a (Chl a) were performed in organic solvents, but no efficient signals were detected. Herein, ECL research of magnesium chlorophyllins a (Chlorins a) from simple saponification of the natural Chl a was carried out, and highly efficient and stable ECL signal was obtained for the first time. The mechanism study indicated that the ECL resulted from radical ion annihilation. Under the optimal conditions, the effect of the key gas O2 on the ECL of Chlorins a aqueous solution was investigated, and the recoverable inhibition of O2 was obviously observed. What is more, owing to the strong non-covalent interaction between Chlorins a and the carcinogen aflatoxin B1 (AFB1), ECL intensity of Chlorins a aqueous solution exhibited fast, sensitive and selective response to AFB1 with a low detection limit of 0.027ppb at the signal-to-noise ratio of 3. The costless and environmentally friendly ECL method opens a new potential way of the rapid detection of AFB1 in practical application.

Hepatitis B virus X protein co-activates pregnane X receptor to induce the cytochrome P450 3A4 enzyme, a potential implication in hepatocarcinogenesis

Hepatitis B virus X protein is a key regulator of hepatocarcinogenesis. The pregnane X receptor is a xenobiotic nuclear receptor that plays a role in the regulation of drug-metabolizing enzymes including the cytochrome P450 3A4, an enzyme important for the bioactivation of the liver carcinogen aflatoxin B1. To identify novel host factor that interacts with hepatitis B virus X protein and the functional interaction between hepatitis B virus X protein and pregnane X receptor in hepatocarcinogenesis. Co-immunoprecipitation, glutathione S-transferase pull-down, and chromatin immunoprecipitation were utilized to assess the interaction between hepatitis B virus X protein and pregnane X receptor. The functional relevance of hepatitis B virus X protein-pregnane X receptor interaction was investigated in cell cultures and hepatocellular carcinoma samples. We observed that hepatitis B virus X protein and pregnane X receptor co-localize in hepatic cells. Pregnane X receptor interacted with hepatitis B virus X protein via the ligand-binding domain of pregnane X receptor. Functionally, hepatitis B virus X protein increased the transcriptional activity of pregnane X receptor. Pregnane X receptor was able to recruit hepatitis B virus X protein to the CYP3A4 gene promoter. In clinic samples, the expression of pregnane X receptor was high in hepatitis B virus-associated liver cirrhosis and stage I hepatocellular carcinoma, but low in state II and stage III hepatocellular carcinoma. We revealed a novel function of hepatitis B virus X protein in co-activating pregnane X receptor. The increased expression of pregnane X receptor and its target gene CYP3A4 are potential biomarkers for the early stage of hepatitis B virus-associated hepatocarcinogenesis.

Heterologous expression and functional characterization of avian mu-class glutathione S-transferases

Hepatic glutathione S-transferases (GSTs: EC2.5.1.1.8) catalyze the detoxification of reactive electrophilic compounds, many of which are toxic and carcinogenic intermediates, via conjugation with the endogenous tripeptide glutathione (GSH). Glutathione S-transferase (GST)-mediated detoxification is a critical determinant of species susceptibility to the toxic and carcinogenic mycotoxin aflatoxin B1 (AFB1), which in resistant animals efficiently detoxifies the toxic intermediate produced by hepatic cytochrome P450 bioactivation, the exo-AFB1-8,9-epoxide (AFBO). Domestic turkeys (Meleagris gallopavo) are one of the most sensitive animals known to AFB1, a condition associated with a deficiency of hepatic GST-mediated detoxification of AFBO. We have recently shown that unlike their domestic counterparts, wild turkeys (Meleagris gallopavo silvestris), which are relatively resistant, express hepatic GST-mediated detoxification activity toward AFBO. Because of the importance of GSTs in species susceptibility, and to explore possible GST classes involved in AFB1 detoxification, we amplified, cloned, expressed and functionally characterized the hepatic mu-class GSTs tGSTM3 (GenBank accession no. JF340152), tGSTM4 (JF340153) from domestic turkeys, and a GSTM4 variant (ewGSTM4, JF340154) from Eastern wild turkeys. Predicted molecular masses of tGSTM3 and two tGSTM4 variants were 25.6 and 25.8kDa, respectively. Multiple sequence comparisons revealed four GSTM motifs and the mu-loop in both proteins. tGSTM4 has 89% amino acid sequence identity to chicken GSTM2, while tGSTM3 has 73% sequence identity to human GSTM3 (hGSTM3). Specific activities of Escherichia coli-expressed tGSTM3 toward 1-chloro-2,4-dinitrobenzene (CDNB) and peroxidase activity toward cumene hydroperoxide were five-fold greater than tGSTM4 while tGSTM4 possessed more than three-fold greater activity toward 1,2-dichloro-4-nitrobenzene (DCNB). The two enzymes displayed equal activity toward ethacrynic acid (ECA). However, none of the GSTM proteins had AFBO detoxification capability, in contrast to recombinant alpha-class GSTs shown in our recent study to possess this important activity. In total, our data indicate that although turkey hepatic GSTMs may contribute to xenobiotic detoxification, they probably play no role in detoxification of AFBO in the liver.

Alpha-Class Glutathione S-Transferases in Wild Turkeys (Meleagris gallopavo): Characterization and Role in Resistance to the Carcinogenic Mycotoxin Aflatoxin B1

Domestic turkeys (Meleagris gallopavo) are one of the most susceptible animals known to the toxic effects of the mycotoxin aflatoxin B1 (AFB1), a potent human hepatocarcinogen, and universal maize contaminant. We have demonstrated that such susceptibility is associated with the inability of hepatic glutathione S-transferases (GSTs) to detoxify the reactive electrophilic metabolite exo-AFB1-8,9-epoxide (AFBO). Unlike their domestic counterparts, wild turkeys, which are relatively AFB1-resistant, possess hepatic GST-mediated AFBO conjugating activity. Here, we characterized the molecular and functional properties of hepatic alpha-class GSTs (GSTAs) from wild and domestic turkeys to shed light on the differences in resistance between these closely related strains. Six alpha-class GST genes (GSTA) amplified from wild turkeys (Eastern and Rio Grande subspecies), heritage breed turkeys (Royal Palm) and modern domestic (Nicholas strain) turkeys were sequenced, and catalytic activities of heterologously-expressed recombinant enzymes determined. Alpha-class identity was affirmed by conserved GST domains and four signature motifs. All GSTAs contained single nucleotide polymorphisms (SNPs) in their coding regions: GSTA1.1 (5 SNPs), GSTA1.2 (7), GSTA1.3 (3), GSTA2 (3), GSTA3 (1) and GSTA4 (2). E. coli-expressed GSTAs possessed varying activities toward GST substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA), cumene hydroperoxide (CHP). As predicted by their relative resistance, livers from domestic turkeys lacked detectable GST-mediated AFBO detoxification activity, whereas those from wild and heritage birds possessed this critical activity, suggesting that intensive breeding and selection resulted in loss of AFB1-protective alleles during domestication. Our observation that recombinant tGSTAs detoxify AFBO, whereas their hepatic forms do not, implies that the hepatic forms of these enzymes are down-regulated, silenced, or otherwise modified by one or more mechanisms. These data may inform of possible molecular mechanisms of resistance to AFB1, and may also have the benefit of identifying genetic markers which could be used to enhance AFB1 resistance in modern domestic strains.

Carry-over of aflatoxin B1 to aflatoxin M1 in high yielding Israeli cows in mid- and late-lactation.

The potent hepatotoxin and carcinogen aflatoxin B1 (AFB1) is a common mycotoxin contaminant of grains used in animal feeds. Aflatoxin M1 (AFM1) is the major metabolite of AFB1 in mammals, being partially excreted into milk, and is a possible human carcinogen. The maximum permitted concentration of AFM1 in cows’ milk is 0.05 μg/kg in Israel and the European Union. Since milk yield and the carry-over of AFB1 in the feed to AFM1 in the milk are highly correlated, it was considered important to determine the AFM1 carry-over in Israeli-Holstein dairy cows, distinguished by world record high milk production. Twelve such cows were used to determine AFM1 carry-over following daily oral administration of feed containing ~86 μg AFB1 for 7 days. The mean carry-over rate at steady-state (Days 3–7) was 5.8% and 2.5% in mid-lactation and late-lactation groups, respectively. The carry-over appears to increase exponentially with milk yield and could be described by the equation: carry-over% = 0.5154 e0.0521 × milk yield, with r2 = 0.6224. If these data truly reflect the carry-over in the national Israeli dairy herd, the maximum level of AFB1 in feed should not exceed 1.4 μg/kg, a value 3.6 times lower than the maximum residue level currently applied in Israel.

Stimulato-Inhibitory Response to Cumin Oil in Aflatoxin B1 Production of Aspergillus Species

Background: Aspergillus species produce the highly toxic and carcinogenic metabolite, Aflatoxin B1 (AFB1), on food and agricultural commodities. Some natural products are known to inhibit aflatoxin production. Objectives: With the aim of controlling aflatoxin production, the essential oils of Cuminum cyminum L. from the best known regions of Iran i.e. Alborz Mountain and Kerman region, were obtained by hydrodistillation. Materials and Methods: Antifungal activities of the oils to inhibit growth and aflatoxin productivity of A. flavus PICC-AF39, A. flavus PICC-AF24, and A. parasiticus NRRL-2999 were studied. Minimal inhibitory (MIC) and minimal fungicidal (MFC) concentrations of the oil were determined. Sub-MIC was selected for the measurement of aflatoxins B and G concentration. Samples were analyzed either using a high performance liquid chromatography (HPLC) method with some minor modifications. Aflatoxins (AFs) were determined by reverse-phase HPLC and fluorescence detector with post column derivatization (PCD) involving bromination. Results: A significant reduction in Aflatoxin production was noted which was not due to the inhibitory effect but because of antifungal property of the oil. Interestingly, the oil promoted toxin production for the reasons yet to be investigated. The extent of aflatoxin production was dependent on the concentration of essential oil used. All toxin-producing fungi in this study produced higher amount of aflatoxin at low concentrations of the oil. 400 ppm concentration of C. cyminum L. from Alborz Mountain increased aflatoxin production to over fourfold. Aflatoxin productivity was declined at high concentration of the oil. Conclusions: Antimicrobial and antitoxigenic properties of natural products need a firmly established criterion before they could be offered to application.

Human skin penetration of selected model mycotoxins

Dermal exposure data for mycotoxins are very scarce and fragmentary, despite their widespread skin contact and hazard toxicity. In this study, the transdermal kinetics of aflatoxin B1 (AFB1), ochratoxin A (OTA), fumonisin B1 (FB1), citrinin (CIT), zearalenone (ZEA) and T-2 toxin (T-2) were quantitatively evaluated, using human skin in an in vitro Franz diffusion cell set-up. All mycotoxins penetrated through the skin, except for FB1, which showed concentrations in the receptor fluid below the LoD, resulting in a Kp<3.24×10−6cm/h. OTA showed the highest permeation (Kp=8.20×10−4cm/h), followed by CIT (Kp=4.67×10−4cm/h). AFB1 and ZEA showed lower permeability rates (Kp=2.11 and 2.33×10−4cm/h, respectively). T-2 was found to have the lowest permeability (Kp=6.07×10−5cm/h). From literature-based mycotoxin-concentrations, dermal contact surface, exposure time and apparent Kp's obtained in this study, the daily dermal exposure (DDE) in two industrial and one residential scenario was estimated. Dermal exposure to the DNA-reactive genotoxic carcinogenic AFB1 can lead to a health risk for agricultural workers which are exposed to a mycotoxin contaminated solution in a worst case situation. For all the other investigated mycotoxins, no significant health risk is calculated after dermal contact in neither agricultural nor residential environments.

In Vitro - In Vivo Correlation of Gene Expression Alterations Induced by Liver Carcinogens

Although cultivated hepatocytes are widely used in the studies of drug metabolism, their application in toxicogenomics is considered as problematic, because previous studies have reported only little overlap between chemically induced gene expression alterations in liver in vivo and in cultivated hepatocytes. Here, we identified 22 genes that were altered in livers of rats after oral administration of the liver carcinogens aflatoxin B1 (AB1), 2-nitrofluorene (2-NF), methapyrilene (MP) or piperonyl-butoxide (PBO). The functions of the 22 genes have been classified into two groups. Genes related to stress response, DNA repair or metabolism and genes associated with cell proliferation, respectively. Next, rat hepatocyte sandwich cultures were exposed to AB1, 2-NF, MP or PBO for 24h and expression of the above mentioned genes was determined by RT-qPCR. Significant correlations between the degree of gene expression alterations in vivo and in vitro were obtained for the stress, DNA repair and metabolism associated genes at concentrations covering a range from cytotoxic concentrations to non-toxic/in vivo relevant concentrations. In contrast to the stress associated genes, no significant in vivo/in vitro correlation was obtained for the genes associated with cell proliferation. To understand the reason of this discrepancy, we compared replacement proliferation in vivo and in vitro. While hepatocytes in vivo, killed after administration of hepatotoxic compounds, are rapidly replaced by proliferating surviving cells, in vitro no replacement proliferation as evidenced by BrdU incorporation was observed after washing out hepatotoxic concentrations of MP. In conclusion, there is a good correlation between gene expression alterations induced by liver carcinogens in vivo and in cultivated hepatocytes. However, it should be considered that cultivated primary hepatocytes do not show replacement proliferation explaining the in vivo/in vitro discrepancy concerning proliferation associated genes.

PRESENCE OF AFLATOXIN M1 IN PASTEURIZED AND UHT MILK COMMERCIALIZED IN SHIRAZ, KHUZESTAN AND YAZD, IRAN

Aflatoxin M1 (AFM1) is a hydroxylated metabolite of the carcinogen aflatoxin B1 (AFB1), and may be found in the milk of lactating animals that have ingested feedstuffs contaminated with AFB1 Between Jun 2010 and March 2011, 149 samples of pasteurized (n=90) and UHT (n=59) milk were collected from supermarkets in the cities of Shiraz, Khuzestan and Yazed, Iran. All samples were analyzed for AFM1 contamination by ELISA and 142 (95.3%) were positive with mean concentrations 41 ng.l -1 . These concentrations are lower than the standads of Codex Alimentarius and FDA (500 ng.l -1 ), but 33 samples (22.1%) had higher concentrations than the maximum tolerance accepted by some European countries (50 ng.l -1 ). Mean concentrations of AFM1 in pasteurized and UHT milk were 39 and 46 ng.l -1 , respectively. Mean concentrations of AFM1 in fall and winter samples were significantly higher (P<0.05) than those of summer but differences between AFM1 concentrations of fall and winter samples were not significantly different. No significant differences in Concentrations of AFM1 in milk samples between Shiraz, Khuzestan and Yazd. Considering the results, this survey revealed a high frequency of AFM1 contamination in milk samples from central part of Iran.

Functional Characterization of Alpha-Class Glutathione S-Transferases from the Turkey (Meleagris Gallopavo)

Six Alpha-class glutathione S-transferase (GST) subunits were cloned from domestic turkey livers, which are one of the most susceptible animals known to the carcinogenic mycotoxin aflatoxin B₁. In most animals, GST dysfunction is a risk factor for susceptibility toward AFB₁, and we have shown that turkeys lack GSTs with affinity toward the carcinogenic intermediate exo-aflatoxin B(1)-8-9-epoxide (AFBO). Conversely, mice are resistant to AFB₁ carcinogenesis, due to high constitutive expression of mGSTA3 that has high affinity toward AFBO. When expressed in Escherichia coli, all six tGSTA subunits possessed conjugating activities toward substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA), and cumene hydroperoxide (CHP) with tGSTA1.2 appearing most active. Interestingly, tGSTA1.1, which lacks one of the four Alpha-class signature motifs, possessed enzymatic activities toward all substrates. All had comparable activities toward AFBO conjugation, an activity absent in turkey liver cytosols. E. coli-expressed mGSTA3 conjugated AFBO with more than 3-fold greater activity than that of tGSTAs and had higher activity toward GST prototype substrates. Mouse hepatic cytosols had approximately 900-fold higher catalytic activity toward AFBO compared with those from turkey. There was no apparent amino acid profile in tGSTAs that might correspond to specificity toward AFBO, although tGSTA1.2, which had slightly higher AFBO-trapping ability, shared Tyr¹⁰⁸ with mGSTA3, a residue postulated to be critical for AFBO trapping activity in mammalian systems. The observation that recombinant tGSTAs detoxify AFBO, whereas their hepatic forms do not, implies that the hepatic forms of these enzymes are silenced by one or more regulatory mechanisms.

Impact of fungal content of some Arabic nuts to induce kidney toxicity and agonistic action of natural resources

Human and animals are exposed to a great extent of the danger of mycotoxic infection and their toxins due to wide spread of fungal growth on grains and food. Aflatoxin B1 (AFB1) is one of the most dangerous toxins that are produced by fungi as a secondary metabolic products. This study aims to isolate and identify the fungi from some grains present in Arabian nuts. 30 samples were collected from commercial stores in Jeddah in the period from 2008 to 2009. Histological study was done on female rat kidney that was treated with isolated fungus Aspergillus flavus and the carcinogenic substance aflatoxin B1 (AFB1). Results showed the occurrence of several harmful effects of the experimental fungi and AFB1 on the kidney. Effects of “musk” and “sidr” extract as natural treating sources were confined in this study. They proved their effectiveness in treating renal mycotoxicity. Key words: Arabian nuts, Aspergillus flavus, aflatoxin B1, kidney, musk deer, sidr.

One-Step Detection of Aflatoxin-B1 Using scFv-Alkaline Phosphatase-Fusion Selected from Human Phage Display Antibody Library

A unique human phage display library was used to successfully generate a scFv to the highly carcinogenic toxin aflatoxin B1. Such an antibody has major potential applications in therapy and diagnostics. To further exploit its analytical capacity, the scFv was genetically fused to alkaline phosphatase, thereby generating a novel and highly sensitive self-indicating reagent. The performance of this reagent was further characterized, demonstrating its efficacy. The sensitivity of scFv-AP fusion was three-fold better than that of the scFv form. The ability of this human library to generate antibodies to a small hapten was clearly demonstrated and this is linked to its intrinsic diversity, which exceeds many existing conventional human libraries. Our results indicate that demography may influence the diversity of the repertoire of the library in terms of its capacity to generate antibodies to specific targets. Equally, the approach demonstrated should also be applicable for other haptens and larger antigens.