Ochratoxin A induces abnormal tryptophan metabolism in the intestine and liver to activate AMPK signaling pathway

Background Ochratoxin A(OTA)is a mycotoxin widely present in raw food and feed materials and is mainly pro-duced by Aspergillus ochraceus and Penicillium verrucosum.Our previous study showed that OTA principally induces liver inflammation by causing intestinal flora disorder,especially Bacteroides plebeius(B.plebeius)overgrowth.However,whether OTA or B.plebeius alteration leads to abnormal tryptophan-related metabolism in the intestine and liver is largely unknown.This study aimed to elucidate the metabolic changes in the intestine and liver induced by OTA and the tryptophan-related metabolic pathway in the liver.Materials and methods A total of 30 healthy 1-day-old male Cherry Valley ducks were randomly divided into 2 groups.The control group was given 0.1 mol/L NaHCO3 solution,and the OTA group was given 235μg/kg body weight OTA for 14 consecutive days.Tryptophan metabolites were determined by intestinal chyme metabolomics and liver tryptophan-targeted metabolomics.AMPK-related signaling pathway factors were analyzed by Western blot-ting and mRNA expression.Results Metabolomic analysis of the intestinal chyme showed that OTA treatment resulted in a decrease in intesti-nal nicotinuric acid levels,the downstream product of tryptophan metabolism,which were significantly negatively correlated with B.plebeius abundance.In contrast,OTA induced a significant increase in indole-3-acetamide levels,which were positively correlated with B.plebeius abundance.Simultaneously,OTA decreased the levels of ATP,NAD+and dipeptidase in the liver.Liver tryptophan metabolomics analysis showed that OTA inhibited the kynurenine metabolic pathway and reduced the levels of kynurenine,anthranilic acid and nicotinic acid.Moreover,OTA increased the phosphorylation of AMPK protein and decreased the phosphorylation of mTOR protein.Conclusion OTA decreased the level of nicotinuric acid in the intestinal tract,which was negatively correlated with B.plebeius abundance.The abnormal metabolism of tryptophan led to a deficiency of NAD+and ATP in the liver,which in turn activated the AMPK signaling pathway.Our results provide new insights into the toxic mechanism of OTA,and tryptophan metabolism might be a target for prevention and treatment.

REVIEW Risk Assessment of the Mycotoxin Ochratoxin A

Ochratoxin A (OA) is a mycotoxin which has been found to occur in foods of plant origin, in edible animal tissues, as well as in human blood sera and tissues. The ability of OA to move up the food chain is aided by its long half-life in certain edible animal species. In this report, an evaluation of the health risks to Canadians due to the presence of OA in food products is presented. The first part of the report deals with the physicochemical aspects, mycology, laboratory production, analytical methods, and natural occurrence in plant products, animal products, and human tissues. The stability of OA in foods and feeds, the effects of food processing, and the removal from foods and feeds by physicochemical means are also discussed. From these data, the worst case estimate for the daily exposure of Canadians to OA, from the consumption of pork-based food products and cereal foods, is approximately 5 ng OA/kg body wt (mean of eaters) for young children, the highest consumption group on a body weight basis. The second part of the report deals with the metabolic disposition as well as the available toxicity database for OA in laboratory animals, farm animals, and humans. The major target for OA toxicity in all mammalian species tested is the kidney, and endemic nephropathies affecting livestock as well as humans have been attributed to OA. OA is also teratogenic, and in the fetus the major target is the developing central nervous system. Recent studies have provided 'clear evidence' of the carcinogenicity of OA in two rodent species. OA was found to be nonmutagenic in various microbial and mammalian gene mutation assays, but weak genotoxic activity to mammalian cells was noted. In addition, OA was found to suppress immune function. Based on the NTP carcinogenicity study with OA in rats, the estimated tolerable daily intake in humans ranges from 0.2 to 4.2 ng OA/kg body wt, depending on the method of extrapolation used. In view of the toxic properties of OA, it is recommended that exposure to OA be kept to a minimum. In Canada, further monitoring programs are required to better define the overall residue profile of OA in cereal grains, animal feeds, animal food products, and human blood. Such data are required to better assess dietary exposure and to ascertain the need for regulatory controls or other control mechanisms. (c)1989 Academic Press, Inc.

Ochratoxin A in dried figs, raisings, apricots, dates on Iranian retail market

Ochratoxin A (OTA) is among the most important mycotoxins, and the International Agency for Research on Cancer (IARC) classifies it as possibly carcinogenic to humans (group 2B). A total of 121 samples of dried fruits from the central zone of Iran were analyzed for OTA by enzyme linked immunosorbent assay (ELISA) technique. The recovery percentages of OTA in spiked dried fruit samples at concentrations of 5, 10 and 20 ng/g were found to be 84.9%, 89.3% and 90.4% as mean, respectively. OTA was found in 20.7% of the analyzed samples by average concentration of 6.7 ± 3.9 ng/g. The incidence rates of OTA contamination in dried fig, raising, apricots, and date samples were 10.4%, 44.7%, 6.7% and 10.0%, respectively. The concentrations of OTA in 7.9% of contaminated dried raising samples and 2.1% of dried fig samples were higher than maximum tolerance limit accepted by European Union (10 ng/g). This value reflects that the analyzed samples have a minimal contribution to toxicological risk. To our best knowledge, the present study is the first report of the presence of OTA by ELISA in dried fruit samples in Iran.

Characterization and Comparison of Ochratoxin A-Ovalbumin(OTA-OVA) Conjugation by Three Methods

In order to generate an antibody against a small hapten molecule, the hapten is cross-linked with carrier protein to make it immunogenic. In this study, the hapten （ochratoxin A, OTA） was coupled to ovalbumin （OVA） by an active ester reaction. To develop a technique for detecting the conjugation, the hapten-protein conjugate （OTA-OVA） was characterized thoroughly by immunoarray technology, ultraviolet （UV） spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry （MALDI-TOF-MS）, respectively. The molecular weight of OTA-OVA was 50 350.141 Da, and the molecular weight of OVA was 44 887.506 Da, which were determined by MALDI-TOF-MS, respectively. In OTA-OVA, the molecular coupling ratio was 13：1 by MALDI-TOF-MS while the molecular coupling ratio was 10：1 by UV. In this experiment, UV and MALDI-TOF-MS were selected as the efficient methods to evaluate the cross-linking effect and calculate the molecular coupling ratio.

Aptamer based detection and separation platforms for ochratoxin A:A systematic review

Ochratoxin A(OTA),one of the most dangerous mycotoxins for human health,has been subjected to numerous studies for separation and detection in minimal amounts.Aptamers as novel recognition elements have been employed to fabricate ultrasensitive biosensors for the detection of OTA and designing delicate analytical tools.This review attempted to comprehensively examine all reported aptamer-based detection and separation platforms for ochratoxin.The most relevant databases were considered to discover all specific aptamers for dealing with OTA.Aptamer-based detection and separation devices specified for OTA were searched for,analyzed,discussed,and classified based on their specifications.The optical aptasensors have gathered a higher interest than electrochemical aptasensors,which can achieve a lower limit of detections.Moreover,some extraction platforms based on these aptamers were also found.However,aptamer-based devices seem to have some challenges in their application.

Recent Advances in Detection of Ochratoxin-A

Ochratoxin-A[7-(L-β-phenylalanylcarbonyl)-carboxyl-5-chloro-8-hydroxy-3,4-dihydro-3R-methyl-isocumarin, OTA] is a common food contaminant mycotoxin that enters the human body through the consumption of improperly stored food products. Upon ingestion, it leads to immuno-suppression and immuno-toxicity. OTA has been known to produce nephrotoxic, teratogenic, and carcinogenic activity (via oxidative DNA damage) in several species. This review introduces potentials of electrochemical biosensor to provide breakthroughs in OTA detection through improved selectivity and sensitivity and also the current approaches for detecting OTA in food products.

Hepatocellular carcinoma and food contamination: Ochratoxin A as a great prompter

Ochratoxin A (OTA) is a secondary metabolite of Asper-gillus and Penicillium, microorganisms that can be haz-ardous to health when present as food contaminants. OTA is a potent member of a group of mycotoxins. Prolonged exposure to mycotoxins in the diet is related to cancer, among other diseases. Hepatocellular car-cinoma (HCC) accounts for 70%-90% of primary liver cancers and is the third leading cause of cancer-related deaths worldwide. In a recent study, Ibrahim et al proposed a correlation between the incidence of HCC and contamination with OTA. Analysis of OTA in serum samples showed that HCC patients had the highest incidence of OTA of the subjects examined (5-fold higher than that of the control group). OTA levels were significantly increased in HCC patients. This study demonstrates that chronic exposure to high levels of OTA may be associated with a high risk of liver cancer development. Future epidemiologic studies of HCC should focus on good practices in food preparation, food storage and the consumption of OTA-containing foods.

Determnation of ochratoxin A in grain by monoclonal antibody－based enzyme－linked immunosorbent assay

The simple rapid and sensitive enzyme-linked immunosorbent assay (ELISA) methods, di-rect and indirect ELISA, for quantitation of ochratoxin A in cereal had been developed by theutilization of monoclonal antibody on immunomicroplate. Direct FLIAS was found to be less timeconsuming than indirect ELISA. For direct FLISA, recovery of 1 -500 ppb OA added to wheat was78.9-100.0% and rice was 88.9- 120.0%. For indirect EI.IAS, recovery of 1-500 ppb OA addedto wheat was 79.0- 110.0% and rice was 82.0 120.0%. The minimal detection level for OA was Ippb. Analyses of 31 samples that caused humanintoxicant for OA showed that the ELISA resultsagreed wtll with those obtained by thin-layer chromatogrdphy.

Intercalation of Phenylalanine, Isocoumarin and Ochratoxin A (OTA) into LDH’s

Phenylalanine, isocoumarin and Ochratoxin A (OTA) have been intercalated within the interlayer space of layered double hydroxides. Synthesis of these nanocompounds was achieved via co-precipitation. Their physicochemical properties were studied by element chemical analysis, powder X-ray diffraction, infrared spectroscopy and thermal analyses. The presence of OTA in the interlayer is demonstrated by the study of LC-FD Analysis. On the other hand, these studies allow to check how some of the toxin is on the surface of the nanocomposite.

Peculiarities of feed contamination with citrinin and ochratoxin A

Occurrence of citrinin and ochratoxin A in different feed ingredients and compound feeds was screened by accredited methods based on the indirect competitive enzyme-linked immunosorbent assay. High frequency co-occurrence of both toxins was found in wheat grain and processed sunflower seeds. Citrinin levels exceeded those of ochratoxin A in the majority of co-contaminated feed samples, and the ratio of (1.1 - 10):1 proved to be the most frequent. A possible role of Aspergillus and Penicillium fungi in separate and simultaneous OTA and CIT occurrence in feeds is also discussed.

Efficacy of Organo-Modified Nano Montmorillonite to Protect against the Cumulative Health Risk of Aflatoxin B₁and Ochratoxin A in Rats

The aim of the current study was to prepare organo-modified nano montmorillonite (OMNM) and to evaluate its chemopreventive effects against the hapatonephrotoxicity induced by aflatoxin B1 (AFB1) and ochratoxin A (OA) singly or in combination in rats. OMNM was prepared using Cetyltrimethylammoniumbromide (CTAB) as organic modifier. Eighty male Sprague Dawley were divided into 8 groups and treated for 8 weeks as follow: the control group;the group treated orally with AFB1 (80 μg/kg b.w.);the group treated with OA (100 μg/kg b.w.);the group treated with AFB1 plus OA, the group treated with OMNM (5 g/kg diet) and the groups treated with AFB1 and/or OA plus OMNM. At the end of treatment period, blood and tissue samples were collected from all animals for biochemical and histological analysis. The results revealed that the expansion in the basal spacing of the montmorillonite due to the intercalation of CTAB was 7.20?&#197 and the average particle size of OMNM was 120 nm. The in vivo results indicated that treatment with both AFB1 and OA singly or in combination resulted in a significant increase in liver and kidney function parameters, oxidative stress and tumor markers accompanied with a significant decrease in antioxidant enzyme activities and significant histological changes in liver and kidney tissues. These changes were severe in the group received the combined treatment of AFB1 and OA. OMNM alone did not show any toxic effect and it succeeded to prevent or at least diminish the toxic effects and the histological changes in liver and kidney. It can be concluded that treatment with AFB1 and OA has a synergistic toxic effects and OMNM is safe and it is a promise candidate as an additive to protect against the exposure to multi-mycotoxins in high risk population.

Unveiling the emerging role of curcumin to alleviate ochratoxin A-induced muscle toxicity in grass carp(Ctenopharyngodon idella):in vitro and in vivo studies

Background Ochratoxin A(OTA),a globally abundant and extremely hazardous pollutant,is a significant source of contamination in aquafeeds and is responsible for severe food pollution.The developmental toxicity of OTA and the potential relieving strategy of natural products remain unclear.This study screened the substance curcumin(Cur),which had the best effect in alleviating OTA inhibition of myoblast proliferation,from 96 natural products and investigated its effect and mechanism in reducing OTA myotoxicity in vivo and in vitro.Methods A total of 720 healthy juvenile grass carp,with an initial average body weight of 11.06±0.05 g,were randomly assigned into 4 groups:the control group(without OTA and Cur),1.2 mg/kg OTA group,400 mg/kg Cur group,and 1.2 mg/kg OTA+400 mg/kg Cur group.Each treatment consisted of 3 replicates(180 fish)for 60 d.Results Firstly,we cultured,purified,and identified myoblasts using the tissue block culture method.Through preliminary screening and re-screening of 96 substances,we examined cell proliferation-related indicators such as cell viability and ultimately found that Cur had the best effect.Secondly,Cur could alleviate OTA-inhibited myoblast differentiation and myofibrillar development-related proteins(Myo G and MYHC)in vivo and in vitro and improve the growth performance of grass carp.Then,Cur could also promote the expression of OTA-inhibited protein synthesis-related proteins(S6K1 and TOR),which was related to the activation of the AKT/TOR signaling pathway.Finally,Cur could downregulate the expression of OTA-enhanced protein degradation-related genes(murf1,foxo3a,and ub),which was related to the inhibition of the Fox O3a signaling pathway.Conclusions In summary,our data demonstrated the effectiveness of Cur in alleviating OTA myotoxicity in vivo and in vitro.This study confirms the rapidity,feasibility,and effectiveness of establishing a natural product screening method targeting myoblasts to alleviate fungal toxin toxicity.

免疫亲和层析技术协同酶联免疫吸附法检测赭曲霉毒素A

利用免疫亲和层析技术(immunoaffinity chromatography,IAC)对样品中赭曲霉毒素A(ochratoxin A,OTA)进行捕获与浓缩,利用酶联免疫吸附(enzyme-linked immunosorbent assay,ELISA)法对IAC捕获的目标物OTA进行测定。IAC与ELISA中的抗OTA单克隆抗体来自不同的克隆株,分属不同独特型,与OTA结合靶点的选择性存在差异,IAC与ELISA协同检测,可以有效过滤OTA结构类似物造成的干扰,提高免疫分析的特异性与灵敏度。该方法用于加标样品测定时,OTA的检出限为0.2 ng/g,定量限为0.4 ng/g,OTA的平均回收率为75.9%,与单一的ELISA法相比,该方法虽然回收率略低,但灵敏度可以显著提高,达到ELISA法的60倍。通过对49份样品的实际检测,该方法检出阳性样品与国标法的符合率达到100%,漏检率为0%,由此可见,该方法在准确性上表现出明显的优势。作为一种综合免疫分析技术,该方法不需要大型仪器设备,对操作环境没有严格要求,便于基层实验室对样品中OTA的分析。

基于链置换扩增的电化学适配体生物传感器检测食品中的赭曲霉毒素A

为构建一种基于链置换扩增技术(SDA)和电化学适配体传感器技术检测食品中赭曲霉毒素A(OTA)的方法,根据OTA特异性适配体设计发卡结构,并在发卡结构的茎部设置SDA反应识别位点,进行SDA扩增。将扩增产物与修饰二茂铁(Fc)的电化学探针进行杂交,使电信号发生变化,从而建立电化学适配体传感器检测OTA的方法。通过对7组不同毒素的测定评价该方法的特异性,测定其灵敏度和检出限,并与赭曲霉毒素A酶联免疫分析方法(ELISA)国家标准(GB 5009.96-2016)进行对比试验。结果表明:最优条件下,电化学适配体传感器灵敏度线性范围为0.1 pg/mL~10 ng/mL,检出限(LOD)为0.05 pg/mL。当OTA存在时,检测结果为阳性,当OTA不存在时,检测为阴性,说明该方法特异性良好。在人工加标试验中,该电化学适配体传感器的加标回收率为96.60%~99.04%,ELISA(国家标准)的加标回收率为94.00%~98.50%,该方法的加标回收率优于国家标准。结论:该方法能够快速检测食品中的OTA,具有实用应用价值。

基于金纳米花的双信号适配体传感器检测红酒中真菌毒素

构建基于金纳米花(gold nanoflower,AuFL)和适配体的双信号荧光适配体传感器用于同时检测赭曲霉毒素A(ochratoxin A,OTA)和玉米赤霉烯酮(zearalenone,ZEN)。以AuFL为载体,将含OTA和ZEN适配体的单链DNA共价交联在AuFL表面,进一步将其与两种荧光染料(FAM和Cy3)标记的互补DNA杂交,制备DNA功能化纳米探针。荧光染料和AuFL之间发生荧光共振能量转移(fluorescence resonance energy transfer,FRET)导致纳米探针自身无荧光。加入OTA和ZEN后,FAM和Cy3从探针表面脱落会扰乱FRET过程,导致两者荧光的恢复。优化条件下,该双信号荧光适配体传感器对OTA检测的线性范围为0.05~500 ng/mL,检出限为0.017 ng/mL。对ZEN检测的线性范围为0.1~500ng/mL,检出限为0.033ng/mL。该双信号荧光适配体传感器具有良好的选择性和重现性,并能够成功应用于红酒实际样中OTA和ZEN的检测。

高效液相荧光色谱法同时测定六神曲中黄曲霉毒素、玉米赤霉烯酮和赭曲霉毒素A

目的建立一种用免疫亲和柱净化-柱后光化学衍生-高效液相色谱同时检测六神曲中黄曲霉毒素、玉米赤霉烯酮和赭曲霉毒素A的方法。方法样品采用60%乙腈超声提取,免疫亲和柱净化,采用XBridge^(®)Phenyl苯基色谱柱(4.6 mm×250 mm,5μm),以乙腈和0.1%磷酸溶液为流动相,梯度洗脱,光化学衍生仪衍生,通过切换荧光波长检测。结果黄曲霉毒素、玉米赤霉烯酮和赭曲霉毒素A标准曲线的线性范围分别为0.006~0.108、0.500~2.500和0.202~1.009 ng,6种毒素的线性关系在0.9994以上,检出限分别为1.2、100.0和40.3 ng/ml,平均加标回收率为73.2%~92.3%,相对偏差为2.1%~5.4%。结论该方法具有专属性强、操作方便等特点,能够有效用于六神曲中3种毒素的同时检测和安全质量控制。

基于磁性纳米粒子的比色适配体传感器检测赭曲霉毒素A

基于磁性纳米粒子(Fe_(3)O_(4)@Au)和纳米银(Ag NPs),通过DNA碱基互补配对,构建了一种新颖的比率比色传感器用于赭曲霉毒素A(Ochratoxin A,OTA)的检测。OTA适配体(aptamer)偶联的Fe_(3)O_(4)@Au作为参比信号,互补DNA(cDNA)偶联的Ag NPs作为响应信号,通过调控两者的比例以及DNA碱基互补,制备了具有两个紫外吸收峰的比率纳米探针(Fe_(3)O_(4)@Au-Ag)。没有OTA存在时,比率比色传感器的吸光度比值(A400/A580)保持恒定。对检测条件(时间、pH和温度)进行了优化,在优化条件下,该传感器对OTA检测呈现良好的线性关系,线性范围为0.01~1000 ng·mL^(-1),检出限为0.033 ng·mL^(-1)。该传感器具有良好的选择性、重现性和高灵敏度,并被成功应用于豌豆粉中OTA的检测,回收率为93.9%~96.0%。

纳米材料电化学生物传感器检测食品中赭曲霉毒素A的研究进展

赭曲霉毒素A(ochratoxin A,OTA)是一种污染食品的常见真菌毒素,长期食用OTA污染的食品会严重影响人体健康。随着生物传感器技术的发展,电化学生物传感器成为OTA检测的新方向。纳米材料在电化学传感器中的应用有效提高了其性能。该文综述使用纳米材料检测OTA的电化学生物传感器的原理和特点,并对其发展方向进行展望。

赭曲霉毒素A对动物的危害及脱毒研究进展

赭曲霉毒素A(OTA)是一种由曲霉或青霉产生的次级代谢产物,广泛污染谷物、水果和坚果等农产品和饲料,造成严重的经济损失。此外,越来越多的证据表明,OTA通过多种毒性作用(肾毒性、肝毒性、肠毒性、致癌性、致畸性和免疫毒性)对人类和动物的健康造成巨大威胁。因此,研究OTA的毒性机制,从多方面寻求解毒方法迫在眉睫。本文综述了OTA对动物的危害及其脱毒方法,重点讨论了各种OTA脱毒方法的优势和劣势,并对提高OTA脱毒效率的方法进行了深入探讨,以期降低饲料OTA的含量,有助于减少OTA对动物健康造成的危害。

A Label-free Fluorescent Aptasensor for Turn-on Monitoring Ochratoxin A Based on AIE-active Probe and Graphene Oxide

A label-free fluorescent aptasensor for specific and ultrasensitive monitoring ochratoxin A（OTA） was developed using the specific aptamer of OTA（OSA） as recognition dement, an aggregation-induced emission（AIE） molecule（a 9,10-distyrylanthracene with two ammonium groups, DSAI） as a fluorescent probe, and graphene oxide（GO） as a quencher. In the absence of OTA, the AIE probe DSAI and OSA complex（DSAI/OSA） is adsorbed on the GO surface, and the fluorescence of DSAI will be quenched efficiently via the fluorescence resonance energy transfer（FRET） from DSAI to GO. Upon the OTA addition, a more stable complex（OSA-OTA） is formed and released from GO. Meanwhile, DSAI and OSA-OTA can form a new complex（DSAI/OSA-OTA）, then the fluorescent signal of DSAI recovers gradually. Therefore, by introducing GO and DSAI, the fluorescence signal of DSAI can be easily turned from ＂off＂ to ＂on＂ after the addition of OTA, and the ultrasensitive detection of OTA by monitoring the change of the fluorescence signal of DSAI can be readily realized. The detection limit of the assay can reach 0.324 nmol/L with a linear detection range of 10-200 nmol/L. And the aptasensor exhibits high selectivity for OTA against other analogues. Moreover, it has been successfully applied for the detection of OTA in red wine samples.