Effects of flavonoids on CYP1 expression in RL95-2 endometrial carcinoma cells

RL95-2 endometrial cancer cells were used to study cytochrome P450-mediated chemopreventative mechanisms of four flavonoids found in foods. To investigate enzymatic CYP1 inhibition, intact cells were induced with benzo(a)pyrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Quercetin, kaempferol and myricetin inhibited CYP1 activity dose-dependently with IC₅₀s ranging from 2.2 to 4 μM; while amentoflavone was inactive. Further experiments were designed to determine if flavonoids also interacted with the AhR or caused a decrease in CYP1 protein or mRNA expression. CYP1A1 protein expression was inhibited in cells co-treated with TCDD and quercetin, kaempferol or myricetin compared with TCDD alone, but amentoflavone was ineffective. Relatively higher (∼7-fold) basal levels of CYP1B1 protein were not significantly affected by flavonoid treatments. In general, at the message level significant inhibition of induced CYP1A1 or CYP1B1 was not detected following flavonoid cotreatment. Despite the common inhibitory effects of quercetin, kaempferol, and myricetin on induced CYP1A1-dependent activity and protein expression, the mechanisms of CYP1 inhibition in this cell line are complex and dependent on the CYP gene, AhR inducer and the flavonoid.     

超高效液相色谱-串联质谱法测定食品中黄曲霉毒素B1和M1的方法研究

应用超高效液相色谱-串联质谱联用技术（UPLC-MS/MS）,在多反应离子检测（MRM）方式下对食品中的黄曲霉毒素B1（AFB1）和M1（AFM1）污染量进行检测.样品经乙腈-水混合试剂提取,中性氧化铝柱净化,经Agilem ZORBAX Eclipse plus C18色谱柱（100 mm×4.6 mm,1.8 μm）,以甲醇和10 mmol/L NH4Ac溶液（含0.1％的甲酸）为流动相洗脱分离,以MRM方式进行定量分析.AFB1和AFM1分别在0.12～6.12 μg/L和0.11～2.28 μg/L质量浓度范围内线性关系良好,相对标准偏差（RSD）＜5％,加标回收率为81.3％～97.2％.该方法具有低成本、准确、快速、简便、灵敏度高等优点,可满足我国对AFB1和AFM1检测限要求.     

比对实验中黄曲霉毒素B1的检验与分析

本文综合国标法,色谱法及试剂盒法三种方法进行修正,把1：1的甲醇水改为7：3的甲醇水直接加进样品中,删除国标法及试剂盒自带方法中采用石油醚转移步骤,把手动振摇、蒸发等步骤改为漩涡振荡及离心方法,改良了样品的提取方法,操作方便,减少交叉污染,使结果更稳定。     

Co-occurrence of aflatoxins B 1 , B 2 , G 1 , G 2 , zearalenone and fumonisin B 1 in Brazilian corn

Two hundred and fourteen unprocessed corn samples (1997-98 harvest), collected at wholesale markets in different regions in Brazil, were surveyed for the occurrence of mycotoxins. The samples were analysed for aflatoxins B 1 , B 2 , G 1 , G 2 , zearalenone and fumoni1sin B 1 using in-house validated methods. The occurrence of aflatoxin B 1 , zearalenone and fumonisin B 1 was found in 38.3, 30.4 and 99.1% of the samples, respectively. Aflatoxin B 1 , zearalenone and fumonisin B 1 contamination levels varied from 0.2 to 129, 36.8 to 719, and 200 to 6100 μg/kg, respectively. The cooccurrence of the two carcinogenic mycotoxins aflatoxin B 1 and fumonisin B 1 was observed in 100% of the aflatoxin-contaminated samples (82 samples). Cooccurrences of aflatoxin B 1 : zearalenone: fumonisin B 1 and aflatoxin B 1 : aflatoxin B 2 : fumonisin B 1 were found in 18 and 43 samples, respectively.     

Effects of sex, weight, diet and hCG administration on levels of skatole and indole in the liver and hepatic activities of cytochromes P4502E1 and P4502A6 in pigs

Cytochromes P4502E1 (CYP2E1) and P4502A6 (CYP2A6) catalyse metabolic reactions of skatole and indole metabolism. The objectives of this study were as follows: to evaluate whether activities of CYP2E1 and CYP2A6 in pigs of two live weights (LW) differ between males and females; to investigate whether activities of CYP2E1 and CYP2A6 are affected by hCG stimulation; and to investigate whether the levels of skatole and indole in the liver and the activities of CYP2E1 and CYP2A6 are affected by raw potato starch (RPS). Female pigs expressed higher CYP2A6 activity at 90kg LW, and higher CYP2E1 activity at 115kg LW compared to male pigs. Skatole levels in the liver were higher in male pigs than in female pigs at both LW, whereas indole levels were higher in males only at 115 kg LW. Neither levels of indolic compounds in the liver nor enzyme activities were affected by hCG stimulation. The inclusion of RPS in the diet reduced skatole levels in the liver in both sexes and increased CYP2A6 activity in female pigs. It was concluded that the incidence of boar taint may depend on both skatole amount, which reach the liver, and the activities of enzymes involved in skatole metabolism, which may vary depending on sex, live weight, and diet.     

2011-2012年食用植物油中黄曲霉毒素B1的调查

为调查我国市场食用植物油油中黄曲礞毒素B1是螽符合国家标准规定,本论义膈两年的时间,对988个传统烹调油样品、342个新兴功放类油样品中的黄曲霉毒素B1进行了检测。结果显示：2011年传统烹调油合格率为99．22％,新必功效类油样,铺及凋味汕合格宰为95．03％;2012年传统烹调油合格率为99．37％,新必功效类油样品及调味油合格半为96．90％。由此可见,食用油产品的合格牢征逐年提高。     

Occurrence of fumonisins B1 and B2 in Portuguese maize and maize-based foods intended for human consumption

Fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) are mycotoxins mainly produced by Fusarium verticillioides and Fusarium proliferatum, which are field pathogens of maize. A survey was conducted on the incidences of FB₁ and FB₂ in both maize and derived products purchased in Portugal. The analytical method involved extraction with methanol-water, clean-up by immunoaffinity column and derivatization with naphthalene-2,3-dicarboxaldehyde. Determination was carried out by high-performance liquid chromatography (HPLC) with spectrofluorimetric detection, with liquid chromatography/mass spectrometry (LC/MS) confirmation. The presence of FB₁ and FB₂ was determined in 67 samples of maize and maize-based foods, such as flour, semolina, starch, sweet maize, cornflakes and other breakfast cereals, and snacks collected in 2005. FBs were found in 15 samples at concentrations ranging from 113 to 2026 µg kg^-1. Two of the samples showed higher contamination levels than the limits established by the European Commission Regulation. None of the samples contained levels of fumonisins that would lead to an exposure exceeding the tolerable daily intake (TDI).     

Fumonisin B1, fumonisin B2, zearalenone and ochratoxin A contamination of maize in Croatia

Mycotoxins are products of moulds that frequently contaminate maize. In this study the presence of mycotoxins fumonisin B₁ (FB₁), fumonisin B₂ (FB₂), zearalenone (ZEA) and ochratoxin A (OTA) was determined in 49 maize grain samples collected in autumn 2002. The most frequent finding was that of FB₁(100%), followed by ZEA (84%) and OTA (39%), while FB₂ was found only in three samples. The co-occurrence of two and three mycotoxins was found in 55 and 37% of samples, respectively. The concentrations (mean ± SD) of FB₁, ZEA and OTA in positive samples were 459.8 ± 310.7, 3.84 ± 6.68 and 1.47 ± 0.38 µg kg^-1, respectively, and the concentrations of FB₂ in three positive samples were 68.4, 109.2 and 3084.0 µg kg^-1. Although such low concentrations of mycotoxins are not a significant source of exposure in countries with a European diet, a few samples with extreme values indicate that thorough control is needed.     

A method for determining fumonisin B1 in corn using immunoaffinity column clean-up and thin layer chromatography/densitometry

A method for determining fumonisin B₁(FB₁) in corn was developed and the clean-up optimized in order to give an extract suitable for one-dimensional thin layer chromatographic (TLC) analysis. FB₁ was extracted with a solution of methanol:water (80:20,v/v), purified through an immunoaffinity column and separated on a C₁₈ reversed phase TLC plate. The FB₁ was visualized with 0.1mol/l sodium tetraborate, 0.40mg/ml fluorescamine in acetonitrile and 0.01mol/l boric acid:acetonitrile (2:3,v/v) for fluorescence detection, and quantified by densitometric analysis. Water, acetonitrile:water (1:1v/v) and acetonitrile:water (4:1v/v) were evaluated as TLC solvents for running both standards and samples together with derivatization procedures aimed at improving separation, resolution, sensitivity and linearity. The mean recovery for FB₁ for spiked samples was found to be 85% and the linear equation of standard calibration curve by densitometric analysis gave an r2 value higher than 0.99. The maximum coefficient of variation for replicate analysis of spiked samples was 19%. The absolute amount of FB₁ standard detectable on a TLC plate was 2 ng, giving a detection limit for the method of 0.1mg/kg. The method has been shown to be robust in the application of FB₁ monitoring in corn (214 samples) collected in different regions of the country. FB₁ was detected in 99% of these samples in the range of 0.2 to 6 mg/kg.     

酶联免疫吸附结合免疫亲和柱－高效液相色谱法检测大米中黄曲霉毒素B1的研究

对大米中两种检测黄曲霉毒素B1方法的结合使用进行了研究,即采用酶联免疫吸附法对大米中黄曲霉毒素B1进行初筛,对可疑样品采用免疫亲和柱-高效液相色谱法进行验证。样品经甲醇水提取、免疫亲和柱净化、高效液相色谱分离、碘柱后衍生、荧光检测器测定。结果表明,黄曲霉毒素B1线性关系良好,相关系数为0.9999,检出限为0.50μg/kg,回收率为89.4%~95.2%,RSD值为1.88%~3.05%。两种方法的结合应用,适用于较大批量样品的检测。     

Comparison of two Analytical Methods (ELISA and LC-MS/MS) for Determination of Aflatoxin B1 in Corn and Aflatoxin M1 in Milk

The aim of this paper is to assess the closeness of agreement between results of ELISA and LC-MS/MS methods for determination of aflatoxin B₁ in corn and aflatoxin M₁ in milk. Samples of corn (n=100) and milk (n=250) were simultaneously analyzed using ELISA and LC-MS/MS methods, after the severe drought that affected Serbia in summer 2012 resulting in occurrence of aflatoxin B1 in corn and aflatoxin M₁ in milk. Regression analysis showed higher level of agreement between aflatoxin B₁ samples (R²=0.994), compared to aflatoxin M₁ samples (R²=0.920). However, both techniques were satisfactory in meeting the requirements for official control purposes.     

Simultaneous determination of aflatoxin B1 and ochratoxin A and their natural occurrence in Mediterranean virgin olive oil

Olive oil, the most important dietary fat source of the Mediterranean diet, can be contaminated by mycotoxins. An efficient analytical method for the simultaneous determination of aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) in olive oil is reported. Thirty commercial samples of virgin olive oil, purchased in olive-press plants and supermarkets in southern Italy and North Africa, were analysed to verify the analytical procedure and monitor mycotoxin contamination. A simple, rapid and economic method was set up and tested for both the extractive step and the clean-up procedures for simultaneous AFB₁ and OTA determination in olive oil. Data obtained showed that OTA was detected with high frequency (80%) in samples from both geographical areas (up to 17.0 ng g^-1), while AFB₁ was found from three of four samples from North Africa (up to 2.4 ng g^-1). In addition, 'not labelled' oil samples proved to be more contaminated by OTA then 'labelled' samples (mean values of 2.47 and 0.66 ng g^-1, respectively). These findings indicate that olive oil can be significantly contaminated by mycotoxins and confirm that a scrupulous application of European Regulation 1019/2002 (European Commission 2002), which prohibits the sale of non-labelled olive oil, is strongly recommended. Conventional qualitative parameters such as peroxide number, spectrophotometric evaluation and acid values were not correlated with mycotoxin occurrence.     

QuEChERS萃取-UPLC-MS/MS测定花生酱中黄曲霉毒素B1方法的研究

建立了QuEChERS萃取-UPLC-MS/MS测定花生酱中黄曲霉毒素B1的快速检测方法。样品首先经过1%甲酸-乙腈溶液提取,提取液采用QuEChERS净化试剂(250mg MgSO4+100mg HC-C18+50mg PSA)净化后上机,UPLC-MS/MS正离子源多反应模式检测,外标法定量。黄曲霉毒素B1在1.0~5.0ng/mL范围内呈良好线性关系,相关系数(R^2)>0.999,4个水平的添加目标分析物黄曲霉毒素B1的回收率在81.7%~93.5%范围内,相对标准偏差(RSD)为2.4%~5.6%,检出限为0.03μg/kg;该试验方法具有简单、高效、经济、准确、回收率高、精密度好的优点,适用于花生酱样品中黄曲霉毒素B1的快速检测。     

Occurrence of aflatoxin B1, citrinin and ochratoxin A in rice in five provinces of the central region of Vietnam

The possible coexistence of three mycotoxins in rice, including aflatoxin B1 (AFB1), citrinin (CIT) and ochratoxin A (OTA), was investigated. The samples of rice were collected in large markets in five provinces of the central region of Vietnam. These toxins were extracted, purified and finally quantified by HPLC with fluorimetry detection. Contamination of AFB1 was found to be the most, followed by OTA, while contamination of CIT was insignificant. The coexistence of CIT with AFB1/OTA in rice was found in high percentage. Some samples overpassed the authorized limit by Europe in OTA and/or AFB1.     

Application of near infrared spectroscopy for rapid detection of aflatoxin B1 in maize and barley as analytical quality assessment

The establishment of fast and non-destructive methods for the evaluation of quality and safety of raw grains is being demanded nowadays to avoid toxic substance presence. Aflatoxin B1 (AFB1) has been recognised by the International Agency of Research on Cancer as a group 1 carcinogen for animals and humans and the EU Official Journal has established action levels for AFB1 presence in all feed materials between 5 and 20 ppb. Near infrared spectroscopy (NIRS) is an excellent candidate for a rapid and low-cost method for the detection of aflatoxins in cereals. This study assesses the utility of NIRS for rapid detection of mycotoxigenic fungi as AFB1. A total of 152 samples were involved and analysed for aflatoxin content. The results of spectroscopic models developed have demonstrated that NIRS technology is an excellent alternative for fast AFB1 detection in cereals. The best predictive model to detect AFB1 in maize was obtained using standard normal variate and detrending (SNVD) as scatter correction (r² = 0.80 and 0.82; SECV = 0.211 and 0.200 for grating and FT-NIRS instruments, respectively). In the case of barley, the best predictive model was developed using SNVD on the dispersive NIRS instrument (r² = 0.85 and SECV = 0.176) and using spectral data as log 1/R for FT-NIRS (r² = 0.84 and SECV = 0.183).     

Evaluation and validation of two fluorometric HPLC methods for the determination of aflatoxin B1 in olive oil

Two methods for the determination of aflatoxin B₁(AFB₁) in olive oil were tested and compared. In method A the oil sample was mixed with methanol + water (60 + 40), extracted with hexane and then with chloroform. Chloroform was evaporated and the residue was dissolved with dichloromethane which was then transferred for clean-up onto a silica 'Sep-Pak' cartridge. The cartridge was pre-washed with hexane, ethyl ether and dichloromethane. AFB₁ was eluted with chloroform + acetone (9 + 1) and evaporated to dryness. In method B, the oil sample was mixed with methanol + water (80 + 20), shaken and centrifuged. The supernatant was diluted 1:10 with water and 10ml of the diluted mixture transferred to an 'Aflaprep' immunoaffinity column for the clean-up step. AFB₁ was eluted with acetonitrile and evaporated to dryness. AFB₁ from both methods was derivatized to its hemiacetal (AFB_2a ) and then quantitated by HPLC using a C₁₈ (60 A 4.6 x 250 mm) column with fluorescence detection. Both methods are simple, reliable and efficient, but method A showed a lower detection limit (2.8 ng/kg) than method B (56 ng/kg). With a 95% confidence level there was no significant difference in recovery between the two methods, which was 87.2% for method A and 84.8% for method B. In addition, application of a two-tailed F-test to the variances within spiked samples at concentrations 1, 2, 5 and 10 mu g/kg separately showed that there was no significant difference in the precisions of the two methods. Fifty samples of olive oil of Greek origin produced between 1995 and 1998 were examined with both methods for the presence of AFB₁. When analysing the samples with method B, the presence of AFB₁ was not detected. The use of method A revealed the presence of AFB₁ in 72% of the samples. The range of contamination was generally found to be very low (2.8-15.7 ng/kg), however one sample was contaminated with 46.3 ng/kg.     

Effects of ready to drink teas on AhR- and PXR-mediated expression of cytochromes P450 CYP1A1 and CYP3A4 in human cancer cell lines and primary human hepatocytes

A variety of xenobiotics are taken in the diet and they can interfere with regulatory pathways of drug metabolizing enzymes in humans. This can result in food-drug interactions, which is undesirable clinical situation where drug pharmacokinetics are influenced by dietary compounds. Xenobiotics-mediated food-drug interactions include the induction of drug metabolizing cytochromes P450. The expression of the most important inducible cytochromes CYP1A and CYP3A4 are regulated by xenoreceptors PXR and AhR.We examined extracts from 17 different flavoured ready to drink teas (RDTs) for their capabilities to activate PXR and AhR receptors and to induce CYP3A4 and CYP1A genes. Primary cultures of human hepatocytes and cancer cell lines HepG2 and LS174T were used as in vitro models. Gene reporter assays, RT-PCR and Western blots were performed.We identified three RDTs that induced CYP3A4 mRNA and protein, implying a potential for food-drug interactions. Several RDTs slightly elevated CYP1A1 expression or activated AhR.     

Natural occurrence of aflatoxins (B1 and M1) in feed,plasma and raw milk of lactating dairy cows in Beja,Tunisia, using ELISA

Beja is an agricultural area in northwest Tunisia. It contributes to national needs by offering cereals and milk to the market for human and animal consumption. A small number of studies on mycotoxin occurrence in feedstuffs and raw milk from lactating dairy cows in this region are available. Therefore, 226 samples were collected from farms and local markets during November 2008 until April 2010. Samples consisted of 112 raw cow milk, 56 blood from lactating cows and 58 feed destined for dairy cows. Plasma and feed were analysed for aflatoxin B₁ (AFB₁). Milk samples were analysed for aflatoxin M₁ (AFM₁). All samples were treated using a simultaneous methanolic-aqueous extraction, followed by immunoaffinity column clean-ups and were investigated by competitive enzyme-linked immunoabsorbent assay (ELISA). Recoveries were 80%–95% and 81%–92% for AFB₁ and AFM₁, respectively, while the limit of detection (LOD) was 0.01?µg/kg or µg/l for both mycotoxins. Results revealed the presence of AFB₁ in 84.4% of the feed samples (mean 18.7?±?1.4?µg/kg), and 39.2% of the plasma-examined samples (median 7.1?±?1.0?µg/l) were found to be contaminated at levels higher than the Tunisian and the European Union (EU) limit for dairy animals, which are 20 and 5?µg/kg in animal feed, respectively. AFM₁ was detected in 60.7% of the cow raw milk samples examined (median 13.6?±?1.4?µg/l). Contaminated levels were higher than the EU limit of 0.05?µg/l. It was concluded that more precaution should be taken on hygiene controls in order to prevent fungal contamination.     

3种前处理-HPLC法测定婴幼儿配方食品中维生素B1

分别采用国标法(GB 5009.84-2016)、酸解法和等电点法处理试样,用高效液相色谱-荧光检测(HPLCFLD)法测定婴幼儿配方乳粉和婴幼儿谷类辅助食品中的维生素B1。结果表明,维生素B1在3.036~303.6 ng/mL范围内线性良好,相关系数R^2>0.999。3种方法的回收率和相对标准偏差(RSD,n=6)均符合要求。其中,国标法和酸解法的RSD值相对较小,等电点法的RSD值偏大。试样经国标法、酸解法处理测得维生素B1含量与标签一致,少数试样经等电点法处理测得维生素B1含量明显偏低。酸解法相较于国标法,省去了耗时最长的酶解步骤,仍获得良好回收率。可见,酸解法前处理简单,耗时短,结果准确度高,重现性好,适用于批量检测,总体优于国标法和等电点法。     

Effects of diet enriched with restructured meats,containing Himanthalia elongata, on hypercholesterolaemic induction,CYP7A1 expression and antioxidant enzyme activity and expression in growing rats

Meat and pork consumptions are very high in Spain. Seaweeds are rich in fibre, minerals, and bioactive substances. Due to the growing demand for healthier meats, this work studied the effect of diets containing restructured pork (RP) enriched with Himanthalia elongata (Sea Spaghetti) on: (1) cholesterolaemia; (2) liver cytochrome P450 7A1 (CYP7A1) expression; (3) liver antioxidant enzyme activities and gene expression; (4) the liver antioxidant substrate concentrations. Four groups of 10 Wistar rats each were fed a mix of 85% AIN-93 M rodent diet and 15% freeze-dried RM for 35 days. The control group (C) consumed control RP; the Sea Spaghetti (SS) group, RP with 5% Sea Spaghetti. Animals on added cholesterol diets (CholC and CholSS) consumed their basal C and SS diets enriched with cholesterol and cholic acid as hypercholesterolaemic agent. Food intake was significantly affected by the alga × cholesterol interaction and by dietary cholesterol (both p < 0.001). Plasma cholesterol was significantly affected by the cholesterol × alga interaction (p < 0.05). CholC rats showed significantly higher plasma cholesterol (p < 0.001) than did their C counterparts, whilst serum cholesterol of CholSS was significantly lower (p < 0.05) than that of CholC. The glutathione peroxide (GSSG) concentrations and all mRNA expressions were significantly affected by the cholesterol × alga interaction (at least p < 0.05). SS vs C group showed significant (at least p < 0.05) increases in superoxide dismutase (Mn-SOD and Cu,Zn-SOD), glutathione peroxidase (GPx) and decrease of glutathione reductase (GR) expressions, and increased GR activity, GSSG and the redox index. CholSS vs CholC showed significant (at least p < 0.05) increases of CYP7A1, GR and Cu,Zn-SOD expression but decreases in catalase, Mn-SOD and GPx expression, and increase of GR activity. In conclusion, Sea Spaghetti could be widely used in RP design. Its addition to non-cholesterol enriched RP diet reduced oxidation mechanisms. SS-RP partially blocked the effect of the hypercholesterolaemic agent, giving rise to a new balance of the antioxidant enzyme expression.