DNA-damaging effects of genotoxins in mixture: modulation of covalent binding to DNA.

Modulation of DNA adduct formation by pre-existing adducts was examined in synthetic oligonucleotides and genomic DNA (calf thymus); genotoxins studied were N-acetoxy-acetylaminofluorene (N-AcO-AAF), aminofluorene (AF), aflatoxin B1-8,9-epoxide (AFB1-8,9-epoxide), and dimethylsulfate (DMS). Oligodeoxynucleotides containing either guanine-C8-AAF (Gua-C8-AAF) or Gua-C8-AF adducts and a neighboring unadducted guanine (G) (target G), located 1, 2, or 4 nucleotides from the adduct, were reacted, as single- (ss) or double-stranded (ds) substrates, with dimethylsulfate (DMS) or AFB1-8,9-epoxide. A modified Maxam-Gilbert technique showed that the presence of the AAF adduct lowered the extent to which AFB1-8,9-epoxide, but not DMS, reacted with target G. Binding of AFB1-8,9-epoxide to the target G was attenuated (> or =5-fold) when the target was located immediately adjacent to an AAF, but not AF, adduct in ds-DNA. Reaction with AFB1-8,9-epoxide increased when the target G was located 2 or 4 nucleotides from the AAF adduct. Pretreatment of calf thymus DNA with AAF (0-1.8% nucleotides modified) reduced levels of Gua-N7-AFB1 adducts formed after subsequent treatment with AFB1-8,9-epoxide. Pretreatment of calf thymus DNA with AFB1 did not alter levels of adducts formed after subsequent treatment with N-AcO-AAF. The supposition that aflatoxin B1-binding to DNA may be altered by conformational changes in the helix, due to the presence of a pre-existing AAF adduct, is supported by the absence of an effect by AF and confirmation of local denaturation of the oligomer helix by use of chemical probes hydroxylamine and diethylpyrocarbonate. Nonetheless, the importance of changes in the nucleophilicity of neighboring nucleotides and local steric effects cannot be ruled out.     

Aetiological differences in demographical,clinical and pathological characteristics of hepatocellular carcinoma in The Gambia

Background: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide, with a high burden in West Africa. Data evaluating aetiological differences in HCC presentation from this region are limited. Aims: The aim of this study was to describe the demographical, clinical and pathological characteristics of HCC by aetiology (hepatitis B or C infection, aflatoxin associated). Methods: One hundred and ninty‐three cases of HCC diagnosed between 1997 and 2001 in The Gambia were analysed. Characteristics were compared by aetiology using χ²‐tests, student t‐test and Wilcoxon's rank sum tests as appropriate. Results: The prevalence of hepatitis B surface antigen, hepatitis C antibody and aflatoxin‐associated 249^serTP53 mutations among HCC patients was 60, 20 and 38% respectively. The typical HCC patient was a 49‐year‐old male positive for hepatitis B surface antigen presenting with hepatomegaly (93%), abdominal pain (94%) and weight loss (95%) 8 weeks after symptom onset. Most patients had multifocal lesions with background cirrhosis. The median largest tumour was 10.3 cm and the median α‐fetoprotein level was 500 ng/ml. Eighty‐four per cent of patients had advanced HCC (patients not meeting the Milan criteria) at presentation. Conclusions: Irrespective of aetiological agent, HCC among West Africans presents at very advanced stages. Few clinical or pathological differences exist by aetiology. More studies are needed to understand the mechanisms of hepatocarcinogenesis among these patients as well as identify high‐risk populations in which early detection through screening will be beneficial.     

Aflatoxin M1 Levels in Commonly Consumed Cheese and Yogurt Samples in Ankara, Turkey

The potential hazardous human exposure to aflatoxin M₁ via consumption of milk and milk products has been demonstrated by several workers. Considering its risk to human health, determination of aflatoxin M₁ levels in dairy products is important. Since there are limited data available on the occurrence of aflatoxin M₁ levels in dairy products in Turkey, the aim of the present study was to investigate the presence of this toxin in various types of commonly consumed cheese and yogurt samples in the capital city of Turkey—Ankara. For this purpose, 39 samples of cheese and 40 samples of yogurt were randomly collected from supermarkets in Ankara. Aflatoxin M₁ levels were determined by a competitive enzyme-linked immunosorbent assay (ELISA) kit. Aflatoxin M₁ was detected in 11 cheese samples ranging from 78.20 to 188.44 ng/kg. Thirty-two of the 40 yogurt samples had aflatoxin M₁ levels between 61.61 and 365.64 ng/kg. The results of this study indicated the importance of continuous surveillance of commonly consumed cheese and yogurt samples for aflatoxin M₁ contamination in Turkey.     

EGCG和PC-B2 联合用药对AFB1 致肝细胞损伤的保护作用及其机制

目的:探讨多酚类植物化学物表没食子儿茶素没食子酸酯(EGCG)与原花青素B2(PC-B2),对黄曲霉素B1(AFB1)起的肝细胞损伤保护作用及机制。方法:选用人胚肝细胞(L-02细胞)进行体外实验研究,实验分为6组,分别为空白组,溶剂对照组,AFB_1染毒组,AFB_1染毒+EGCG组,AFB_1染毒+PC-B_2组和AFB_1染毒+EGCG+PC-B_2组。采用MTT检测细胞存活度,通过单细胞凝胶电泳试验(SCGE)检测细胞DNA损伤,通过流式细胞法检测细胞凋亡,采用Western blot法检测凋亡信号通路相关蛋白B细胞淋巴瘤/白血病-2(Bcl-2),Bcl-2相关X蛋白(Bax),半胱氨酸天冬氨酸蛋白酶-3(Caspase-3),Caspase-9,p53蛋白表达的水平。结果:EGCG与PC-B_2均能降低AFB_1所致L-02细胞的生长抑制,使AFB_1对肝细胞的生长抑制率从61.12%降至42.18%和46.72%;EGCG与PC-B_2联用则效果更佳。SCGE实验结果表明EGCG与PC-B_2单用与联用均能减小AFB_1引起的L-02细胞DNA损伤(P0.05)。EGCG与PC-B_2单用与联用均能显著降低AFB_1所致L-02细胞的早期凋亡率(P0.05)。Western blot实验结果表明,AFB_1染毒后,EGCG和/或PC-B_2处理均能显著降低Caspase-3,Caspase-9的表达(P0.01),提高Bcl-2/Bax比值,而对p53表达影响不明显。结论:EGCG与PC-B_2通过降低L-02细胞DNA损伤与细胞凋亡率,从而降低AFB_1对人肝细胞的损伤作用,其作用机制可能与下调Caspase-3,Caspase-9的表达,升高Bcl-2/Bax有关。     

联苯双酯对大鼠黄曲霉毒素B1代谢及肝毒性的影响

目的研究抗肝炎药联苯双酯对大鼠黄曲霉毒素B₁代谢和肝毒性的影响。方法大鼠po联苯双酯300 mg·kg^-1·d^-1, 连服3 d后ip黄曲霉毒素B₁1.5 mg·kg^-1。给黄曲霉毒素B₁16 h后测定血清ALT和AST水平,观察联苯双酯对黄曲霉毒素B₁引起肝损伤的保护作用以及对体外代谢的影响。结果联苯双酯(300 mg·kg^-1·d^-1,连服3 d)可明显降低黄曲霉毒素B₁引起的大鼠血清转氨酶升高，增加低毒代谢产物AFM₁的生成。联苯双酯还可增加大鼠肝脏细胞色素P450总量和胞浆GSH含量，诱导P450 2B1介导的PROD和GST的活性。此外，联苯双酯对P450 3A介导的红霉素脱甲基酶和P450 1A介导的EROD也有一定的诱导作用。结论联苯双酯可通过增加大鼠肝脏对AFB₁代谢的解毒功能起到肝保护作用。     

Identification and Quantification of a Toxigenic and Non-Toxigenic Aspergillus flavus Strain in Contaminated Maize Using Quantitative Real-Time PCR

Aflatoxins, which are produced by Aspergillus flavus, are toxic to humans, livestock, and pets. The value of maize (Zea mays) grain is markedly reduced when contaminated with aflatoxin. Plant resistance and biological control using non-toxin producing strains are considered effective strategies for reducing aflatoxin accumulation in maize grain. Distinguishing between the toxin and non-toxin producing strains is important in determining the effectiveness of bio-control strategies and understanding inter-strain interactions. Using polymorphisms found in the fungal rRNA intergenic spacer region (IGS) between a toxigenic strain of A. flavus (NRRL 3357) and the non-toxigenic strain used in the biological control agent Afla-Guard^® (NRRL 21882), we developed a set of primers that allows for the identification and quantification of the two strains using quantitative PCR. This primer set has been used to screen maize grain that was inoculated with the two strains individually and co-inoculated with both strains, and it has been shown to be effective in both the identification and quantification of both strains. Screening of co-inoculated ears from multiple resistant and susceptible genotypic crosses revealed no significant differences in fungal biomass accumulation of either strain in the field tests from 2010 and 2011 when compared across the means of all genotypes. Only one genotype/year combination showed significant differences in strain accumulation. Aflatoxin accumulation analysis showed that, as expected, genotypes inoculated with the toxigenic strain accumulated more aflatoxin than when co-inoculated with both strains or inoculated with only the non-toxigenic strain. Furthermore, accumulation of toxigenic fungal mass was significantly correlated with aflatoxin accumulation while non-toxigenic fungal accumulation was not. This primer set will allow researchers to better determine how the two fungal strains compete on the maize ear and investigate the interaction between different maize lines and these A. flavus strains.     

Development of an Enzyme-Linked Immunosorbent Assay Method Specific for the Detection of G-Group Aflatoxins

To detect and monitor G-group aflatoxins in agricultural products, we generated class-specific monoclonal antibodies that specifically recognized aflatoxins G₁ and G₂. Of the final three positive and stable hybridomas obtained, clone 2G6 produced a monoclonal antibody that had equal sensitivity to aflatoxins G₁ and G₂, and did not cross-react with aflatoxins B₁, B₂, or M₁. Its IC₅₀ values for aflatoxins G₁ and G₂ were 17.18 ng·mL⁻¹ and 19.75 ng·mL⁻¹, respectively. Using this new monoclonal antibody, we developed a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA); the method had a limit of detection of 0.06 ng·mL⁻¹. To validate this CI-ELISA, we spiked uncontaminated peanut samples with various amounts of aflatoxins G₁ and G₂ and compared recovery rates with those determined by a standard HPLC method. The recovery rates of the CI-ELISA ranging from 94% to 103% were comparable to those of the HPLC (92% to 102%). We also used both methods to determine the amounts of G-group aflatoxins in five peanut samples contaminated by aflatoxin B₁-positive, and their relative standard deviations ranged from 8.4% to 17.7% (under 20%), which demonstrates a good correlation between the two methods. We further used this CI-ELISA to assess the ability of 126 fungal strains isolated from peanuts or field soils to produce G-group aflatoxins. Among these, seven stains producing different amounts of G-group aflatoxins were identified. Our results showed that the monoclonal antibody 2 G6-based CI-ELISA was suitable for the detection of G-group aflatoxins present in peanuts and also those produced by fungi.     

Biomonitoring of concurrent mycotoxin exposure among adults in Sweden through urinary multi-biomarker analysis

Mycotoxin producing moulds may contaminate numerous agricultural commodities either before harvest or during storage. A varied diet consisting of different foods may therefore be contaminated with a range of mycotoxins. The aim of the present study was to study concurrent exposure to mycotoxins through urinary multi-biomarker analysis, as well as its possible associations with the diet.Urinary samples from 252 adults, participating in the Swedish national dietary survey Riksmaten 2010–11, were collected together with a 4-day diet record. Concurrent mycotoxin exposure was studied using a multi-biomarker LC-MS/MS method. The results revealed that exposure to mycotoxins is common and concurrent exposure to more than one toxin was found in 69% of the study population. However, when comparing the number of toxins detected with the reported consumption data it was difficult to distinguish food patterns which would indicate an increased risk of exposure to many mycotoxins simultaneously.This is the first study to investigate concurrent mycotoxin exposure and urinary levels of fumonisin B₁ (FB₁), fumonisin B₂ (FB₂), nivalenol (NIV), ochratoxin A (OTA), zearalenone (ZEA), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL) and de-epoxydeoxynivalenol (DOM-1) among adults in Sweden.     

Effects of a Calcium Bentonite Clay in Diets Containing Aflatoxin when Measuring Liver Residues of Aflatoxin B1 in Starter Broiler Chicks

Research has shown success using clay-based binders to adsorb aflatoxin in animal feeds; however, no adsorbent has been approved for the prevention or treatment of aflatoxicosis. In this study, growth and relative organ weights were evaluated along with a residue analysis for aflatoxin B₁ in liver tissue collected from broiler chickens consuming dietary aflatoxin (0, 600, 1200, and 1800 µg/kg) both with and without 0.2% of a calcium bentonite clay additive (TX4). After one week, only the combined measure of a broiler productivity index was significantly affected by 1800 µg/kg aflatoxin. However, once birds had consumed treatment diets for two weeks, body weights and relative kidney weights were affected by the lowest concentration. Then, during the third week, body weights, feed conversion, and the productivity index were affected by the 600 µg/kg level. Results also showed that 0.2% TX4 was effective at reducing the accumulation of aflatoxin B₁ residues in the liver and improving livability in birds fed aflatoxin. The time required to clear all residues from the liver was less than one week. With evidence that the liver’s ability to process aflatoxin becomes relatively efficient within three weeks, this would imply that an alternative strategy for handling aflatoxin contamination in feed could be to allow a short, punctuated exposure to a higher level, so long as that exposure is followed by at least a week of a withdrawal period on a clean diet free of aflatoxin.