Production and characterization of monoclonal antibodies against sterigmatocystin o‐methyltransferase

Monoclonal antibodies (MAbs) against sterigmatocystin (ST) O‐methyltransferase (OMTase), an enzyme responsible for the conversion of ST to O‐methylsterigmatocystin (OMST) in the late stage of aflatoxin biosynthesis, were produced by fusion of P3/NS‐1/1‐AG4–1 myeloma cells with spleen cells isolated from Balb/c mice that had been immunized with a purified ST‐OMTase preparation. Two clones, 1D9 and 8F11, which produced antibodies showing highest affinity toward ST‐OMTase, were chosen for antibody production and characterization. Enzyme‐linked immunosorbent assay (ELISA) analysis of various fungal extracts showed that the MAbs obtained from either ID9 or 8F11 were highly specific for the ST‐OMTase. Results of ELISA analysis using MAb obtained from clone 8F11 correlated well with those of ST‐OMTase activity in fungal extracts determined by spectrofluorometry, and only MAb 8F11 was capable of neutralizing part of the ST‐OMTase activity. Immunochemical analysis of various fungal extracts with MAb 1D9 revealed that the antibodies primarily reacted with the 40‐kDa ST‐OMTase purified with DEAE‐Sephadex chromatography, but reacted with the 46‐kDa ST‐OMTase species in the crude protein extracts of Aspergillus parasiticus.     

Mechanisms of butylated hydroxytoluene chemoprevention of aflatoxicosis--inhibition of aflatoxin B1 metabolism

Chemoprevention of toxicoses and/or cancer through the use of nutrients or pharmacologic compounds is the subject of intense study. Among the many compounds examined, food additives such as antioxidants are being considered due to their ability to reduce disease formation by either induction or inhibition of key enzyme systems. One such compound, butylated hydroxytoluene (BHT), has been found to protect against cancer formation caused by exposure to aflatoxin B₁ (AFB₁) in rodents. We have shown that dietary BHT protects against clinical signs of aflatoxicosis in turkeys, a species that is very susceptible to this mycotoxin. In this study, the effect of BHT on AFB₁ metabolism and other cytochrome P450 (CYP)-related enzyme activities in turkey liver microsomes was examined to discern possible mechanisms of BHT-mediated protection against aflatoxicosis. Ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), prototype activities for CYP1A1 and 1A2, respectively, were decreased in the BHT fed (4000 ppm) animals, while oxidation of nifedipine, a prototype activity for CYP3A4, was increased. However, BHT added to microsomal incubations inhibited these CYP activities in a concentration-related manner. Importantly, BHT inhibited conversion of AFB₁ to the reactive intermediate AFB₁-8,-9-epoxide (AFBO), exhibiting Michaelis-Menton competitive inhibition kinetics (Ki = 0.81 μM). Likewise, microsomes prepared from turkeys fed BHT were significantly less active in AFBO formation compared to those from control birds. When turkeys were fed BHT for up to 40 days, residual BHT was present in liver, breast meat, thigh meat and abdominal fat in concentrations substantially below U.S. FDA guidelines for this antioxidant, but in concentrations greater than the Ki, likely sufficient to inhibit bioactivation of AFB₁in vivo. BHT-induced hydropic degeneration in the livers of BHT fed animals was significantly greater in birds that remained on BHT treatment for up to 30 days, but this lesion diminished in animals fed for 40 days or when returned to a control diet. The data indicate that the observed chemopreventive properties of BHT in turkeys may be due, at least in part, to its ability to inhibit hepatic AFB₁ epoxidation and also that the BHT-induced hydropic degeneration is reversible and does not appear to cause long-term effects.     

aflN Is Involved in the Biosynthesis of Aflatoxin and Conidiation in Aspergillus flavus

Aspergillus flavus poses a threat to society economy and public health due to aflatoxin production. aflN is a gene located in the aflatoxin gene cluster, but the function of AflN is undefined in Aspergillus flavus. In this study, aflN is knocked out and overexpressed to study the function of AflN. The results indicated that the loss of AflN leads to the defect of aflatoxin biosynthesis. AflN is also found to play a role in conidiation but not hyphal growth and sclerotia development. Moreover, AlfN is related to the response to environmental oxidative stress and intracellular levels of reactive oxygen species. At last, AflN is involved in the pathogenicity of Aspergillus flavus to host. These results suggested that AflN played important roles in aflatoxin biosynthesis, conidiation and reactive oxygen species generation in Aspergillus flavus, which will be helpful for the understanding of aflN function, and will be beneficial to the prevention and control of Aspergillus flavus and aflatoxins contamination.     

Metabolism of aflatoxin B1 in turkey liver microsomes: the relative roles of cytochromes P450 1A5 and 3A37

The extreme sensitivity of turkeys to aflatoxin B₁ (AFB₁) is associated with efficient epoxidation by hepatic cytochromes P450 (P450) 1A5 and 3A37 to exo-aflatoxin B₁-8,9-epoxide (exo-AFBO). The combined presence of 1A5 and 3A37, which obey different kinetic models, both of which metabolize AFB₁ to the exo-AFBO and to detoxification products aflatoxin M₁ (AFM₁) and aflatoxin Q₁ (AFQ₁), respectively, complicates the kinetic analysis of AFB₁ in turkey liver microsomes (TLMs). Antisera directed against 1A5 and 3A37, thereby individually removing the catalytic contribution of these enzymes, were used to identify the P450 responsible for epoxidating AFB₁ in TLMs. In control TLMs, AFB₁ was converted to exo-AFBO in addition to AFM₁ and AFQ₁ confirming the presence of functional 1A5 and 3A37. Pretreatment with anti-1A5 inhibited exo-AFBO formation, especially at low, submicromolar (~ 0.1 μM), while anti-3A37, resulted in inhibition of exo-AFBO formation, but at higher (> 50 μM) AFB₁ concentrations. Metabolism in immunoinhibited TLMs resembled that of individual enzymes: 1A5 produced exo-AFBO and AFM₁, conforming to Michaelis-Menten, while 3A37 produced exo-AFBO and AFQ₁ following the kinetic Hill equation. At 0.1 μM AFB₁, close to concentrations in livers of exposed animals, 1A5 contributed to 98% of the total exo-AFBO formation. At this concentration, 1A5 accounted for a higher activation:detoxification (50:1, exo-AFBO: AFM₁) compared to 3A37 (0.15: 1, exo-AFBO: AFQ₁), suggesting that 1A5 is high, while 3A4 is the low affinity enzyme in turkey liver. The data support the conclusion that P450 1A5 is the dominant enzyme responsible for AFB₁ bioactivation and metabolism at environmentally-relevant AFB₁ concentrations in turkey liver.     

Higher susceptibility to aflatoxin B1‐related hepatocellular carcinoma in glycine N‐methyltransferase knockout mice

In both humans and rodents, males are known to be more susceptible than females to hepatocarcinogenesis. We have previously reported that glycine N‐methyltransferase (GNMT) interacts with aflatoxin B₁ (AFB₁) and reduces both AFB₁‐DNA adduct formation and hepatocellular carcinoma (HCC) in mice. We also reported that 50% of the males and 100% of the females in a small group of Gnmt null (Gnmt?/?) mice developed HCC, with first dysplastic hepatocellular nodules detected at mean ages of 17 and 16.5 months, respectively. In our study, we tested our hypothesis that male and female Gnmt?/? mice are susceptible to AFB₁ carcinogenesis, and that the absence of Gnmt expression may accelerate AFB₁‐induced liver tumorigenesis. We inoculated Gnmt?/? and wild‐type mice intraperitoneally with AFB₁ at 7 days and 9 weeks of age and periodically examined them using ultrasound. Dysplastic hepatocellular nodules were detected in six of eight males and five of five females at 12.7 and 12 months of ages, respectively. Dysplastic hepatocellular nodules from 5/8 (62.5%) male and 4/5 (80%) female Gnmt?/? mice were diagnosed as having HCC, ～6 months earlier than AFB₁‐treated wild‐type mice. Results from microarray and real‐time PCR analyses indicate that five detoxification pathway‐related genes were downregulated in AFB₁‐treated Gnmt?/? mice: Cyp1a2, Cyp3a44, Cyp2d22, Gsta4 and Abca8a. In summary, we observed overall higher susceptibility to AFB₁‐related HCC in Gnmt?/? mice, further evidence that GNMT overexpression is an important contributing factor to liver cancer resistance.     

酸枣仁中黄曲霉毒素B1残留测定的不确定度评定

 目的 对高效液相色谱法测定酸枣仁中黄曲霉毒素B1残留量的不确定度进行评定。方法 对HPLC测定酸枣仁中黄曲霉毒素B1残留量的全过程进行分析,同时结合酸枣仁中黄曲霉毒素B1的HPLC方法验证数据,对分析过程中称量、供试品溶液及对照品溶液制备等影响因素引起的不确定度进行评价,最后根据各分量计算出合成不确定度并进行了扩展。结果 酸枣仁中黄曲霉毒素残留量B1为6.218 μg·kg^-1,置信概率（P）为95%时,其扩展不确定度为0.496 μg·kg^-1。 结论 该方法适用于HPLC测定酸枣仁中黄曲霉毒素B1残留量的不确定度评定,为黄曲霉毒素分析过程的不确定度评定提供了重要参考。     

植物乳杆菌F22对肝脏和肠道微生物菌群的影响研究

目的研究植物乳杆菌F22(Lactobacillus.plantarum F22)对肝脏和肠道微生物菌群的影响。方法 5 w龄雄性昆明小鼠60只,随机分为:低剂量植物乳杆菌F22[1×109 CFU/0.5ml磷酸缓冲液(PBS)]+黄曲霉毒素B1(AFB1)(250μg/kg bw)、高剂量植物乳杆菌F22(1×1010 CFU/0.5 ml PBS)+AFB1(250μg/kg bw)、AFB1(250μg/kg bw)、和无菌PBS液4组,Ⅰ、Ⅱ、Ⅲ、Ⅳ分别灌胃对应试物共10 d,之后,测定血清指标、肝组织结构及肠道菌群数量。结果 AFB1攻毒使小鼠血清白蛋白(ALB)、球蛋白(GLOB)和总蛋白(TP)含量均降低(P0.05);谷草转氨酶(GOT)、谷丙转氨酶(GPT)和碱性磷酸酶(AKP)活性极显著升高(P0.01);肝脏组织切片可见肝细胞广泛性变性和坏死,肠道菌群失调,乳酸菌、双歧杆菌和总厌氧菌数量下降,肠杆菌、肠球菌和总需氧菌数量升高。在灌胃植物乳杆菌F22后,以上现象均得到改善,并随剂量增加恢复效果更好。结论植物乳杆菌F22作为肠道益生菌对AFB1中毒小鼠肝脏和肠道微生态失调具有缓解作用。