发育毒性体外筛选模型的研究现状和进展

利用发育毒性体外评价模型开展相关安全性评价,可避免人体或动物实验耗时、费力等缺点,适用于大规模体外高通量筛选。目前常用的体外评价模型中,胚胎干细胞实验、大鼠全胚胎培养以及体外胚胎肢芽培养实验因与体内实验结果一致性较强,已得到广泛的认可和推荐。但与一般毒理学体外试验发展相比,由于发育毒性受试物在体内消化代谢路线路径的差异,使得发育毒性试验的筛选实验的发展和完善受到阻碍,因此合理的利用新技术、新方法,提高筛选体系的合理性、系统性成为未来体外评价模型的方向。     

新型烟草制品毒理学评价研究进展

为了解新型烟草制品的健康风险评估进展,对加热非燃烧型烟草制品、无烟气烟草制品和电子烟三类新型烟草制品的毒理学评价研究进行了综述。临床前毒理学评价是新型烟草制品健康风险评估程序中一个重要组成部分。1碳加热型和电加热型卷烟的毒理学评价方法包括细菌致突变试验、染色体畸变分析、姊妹染色单体交换试验、细胞毒性试验、DNA损伤分析、胞内相关酶分析等体外试验,以及皮肤涂抹致肿瘤、烟气吸入动物模型实验。加热非燃烧型卷烟的毒性低于传统卷烟。2无烟气烟草制品的毒理学评价方法包括Ames试验、细胞毒性试验、细胞增殖试验、细胞凋亡试验、染色体畸变分析、姊妹染色体交换试验、微核分析等体外测试,以及动物模型实验。无烟气烟草制品对人体健康有不利影响。3电子烟临床前毒理学评价研究较少。因此,发展一系列适合不同类型烟草制品的体外、体内毒理学测试方法,科学、客观、全面地评价新型烟草制品的健康影响,将成为我国烟草相关风险评估工作的重要内容之一。     

卷烟安全性毒理学评价体系的建立

针对卷烟烟气复杂体系安全性毒理学评价的难题,在国内首次研制了卷烟烟气动物染毒装置和卷烟体外毒理学主流烟气暴露装置,建立了包括动物吸烟急性毒性实验、鼠伤寒沙门氏菌诱变性试验(Ames试验)、细胞毒性试验、微核试验、细胞恶性转化试验、细胞氧化损伤试验等1项体内毒理学试验方法和5项体外毒理学试验方法的卷烟安全性毒理学评价体系,为定量评价卷烟烟气对机体损伤程度提供了完善的研究平台.     

呕吐毒素的食品污染、吸收代谢及肠道毒性研究进展

呕吐毒素又名脱氧雪腐镰刀菌烯醇（deoxynivalenol,DON）,是食品中污染最广泛的单端孢霉烯族化合物,且这类化合物是致吐和厌食毒性最强的真菌毒素。DON由镰刀菌产生,对动物和人体的肠道系统、免疫系统、神经系统、肝、肾、脾等器官和组织产生毒性,具有较强的细胞毒性,其中肠道为其毒性作用的第一道屏障。本文综述DON的食品污染特性、吸收代谢及不同研究模型的肠道毒性,并针对其易转化的特点,总结DON衍生物的肠道毒性,为今后DON及其衍生物的毒理学研究提供参考。     

食品包装材料中有害物质双酚A二缩水甘油醚 体外代谢研究

目的通过模拟体内代谢,对双酚A二缩水甘油醚(BADGE)的体外基本代谢情况进行研究。方法采用肝微粒体、肝S9 2种体外代谢试剂,通过模拟体内肝脏代谢,对BADGE的代谢行为及其代谢产物进行研究。通过代谢试剂浓度及代谢时间条件的优化,采用高效液相色谱-串联质谱(HPLC-MS/MS)作为检测手段对BADGE的体外代谢产物进行分析确证。结果体外代谢最佳孵化时间为60 min,最佳体外代谢试剂浓度为0.5mg/m L,在肝S9及肝微粒体2种体外代谢试剂的作用下,BADGE发生显著的代谢反应。结论本研究与传统的动物试验相比,节约了时间、精力,对食品包装材料的毒理学研究和安全性评价有重要的推动作用。     

卷烟烟气体外毒理学测定方法研究进展

综述了卷烟烟气体外毒理学测定方法的研究现状及发展趋势,并对采用体外毒理学方法评定卷烟烟气的基因毒性和细胞毒性的原理进行了简要介绍。      

邻苯二甲酸酯毒理学研究进展

邻苯二甲酸酯(PAEs)作为重要的聚氯乙烯塑料增塑剂在工业上应用广泛。目前,PAEs在海洋、大气、饮用水及动植物体内均被不同程度检出,对生态环境造成日益严重的污染。结合国内外毒理学研究成果,文章概述PAEs的人体暴露,并从肝脏、生殖发育系统、细胞、肾脏、神经毒性及对水生生物的毒性等方面详细探讨PAEs对动物健康的危害及其可能的毒性机制,以及对人体健康潜在的影响;在此基础上,对目前存在的问题及进一步研究的方向进行了探讨和展望。     

赭曲霉素A污染及毒性研究进展

赭曲霉素A(Ochratoxin A)是曲霉属和青霉属一些产毒菌株次级代谢产物,是一种重要食品污染物,具有强烈肾毒性和一定肝毒性、神经毒性及免疫毒性,并具有致癌、致畸、致突变性。该文介绍赭曲霉素常见产生菌及其对食品污染,毒理学特性及检测方法。     

电子烟烟液体外毒理学评价用细胞筛选研究

  目的  目前, 国内外尚无电子烟制品体外毒理学评价的统一方法, 本研究旨在确定适合于电子烟制品体外毒理学评价用细胞。  方法  以人口腔角质细胞(HOK)、人鼻腔上皮细胞(RPMI 2650)、人支气管上皮细胞(Beas-2b)、人肺成纤维上皮细胞(HPF)、人肺泡上皮细胞(HPAEpiC)为候选细胞, 采用细胞毒性法和Annexin V-FITC/PI双染法细胞凋亡试验作为评价方法, 综合细胞的耐受性、细胞增殖特性及试验操作性选取适合于电子烟体外毒理学评价用细胞。  结果  细胞毒性和细胞凋亡试验结果均显示, 不同的受试细胞对相同的样品测试结果定性结果一致, 样品间相对定量趋势一致。候选检测用细胞的细胞倍增时间显示, 在24 h的测试周期内, RPMI 2650细胞和HPF细胞的增殖较快。  结论  综合细胞的耐受性、细胞增殖特性及试验操作性, 建议选取上呼吸道细胞RPMI 2650和下呼吸道细胞HPF为电子烟体外毒理学测试用细胞。      

胚胎干细胞试验及其在毒理学发育毒性安全性评价中的应用

为解决胚胎毒性体内筛选方法需要大量的动物及人力、耗时、耗力的问题,介绍了胚胎干细胞试验及其在毒理学发育毒性安全性评价中的应用.胚胎干细胞试验可同时检测不同受试物对细胞增殖和分化的影响,通过受试物对细胞及个体的发育毒性和致畸能力的判断,将受试物分成3类, 即无胚胎毒性、弱胚胎毒性和强胚胎毒性,该试验方法大大提高了体外替代实验的预测符合率.干细胞试验方法有望成为研究受试物胚胎毒性的体外替代实验模型。     

水晶鹅肝冻制备方法研究

以普通鹅肝和鸭肝为主要原料,研制了新型水晶鹅肝冻的工艺流程和最佳工艺配方.研究了单甘脂、蛋黄粉等乳化剂对肝冻的乳化作用,以及鸭肝、β-环状糊精、糯米粉的添加量对肝冻感官品质的影响,卡拉胶和海藻酸钠添加量对肝冻热稳定性的影响.通过正交实验分析,最终得到肝冻的最佳工艺配方为:糯米粉7％、鸭肝:鹅肝为1∶2(w/w)、β-环状糊精3％ (w/w)、单甘脂:蛋黄为1∶2(w/w);海藻酸钠:卡拉胶为1∶10.     

驴肝与猪肝、鸡肝和鹅肝之间的营养成分比较

目的探究驴肝与猪肝、鸡肝和鹅肝之间的营养成分差异。方法测定驴肝、猪肝、鸡肝和鹅肝的水分、灰分、蛋白质、粗脂肪、氨基酸、脂肪酸、矿物质元素、维生素的含量,并将驴肝与其他3种肝脏的营养成分作对比分析。结果驴肝蛋白质含量比猪肝、鸡肝和鹅肝高,不饱和脂肪酸含量和占总脂肪酸的比例均优于猪肝、鸡肝和鹅肝,矿物质元素中Zn含量明显高于其他3种肝脏。胆固醇含量、总脂肪酸含量均低于其他畜禽肝脏。结论从营养学角度看,驴肝是较优的动物性食品原料。     

Liver fibrosis is a common pathological change in the liver of dairy cows with fatty liver

Fatty liver (i.e., hepatic lipidosis) is a prevalent metabolic disorder in dairy cows during the transition period, characterized by excess hepatic accumulation of triglyceride (TG), tissue dysfunction, and cell death. Detailed pathological changes, particularly hepatic fibrosis, during fatty liver remain to be determined. Liver fibrosis occurs as a consequence of liver damage, resulting from the excessive accumulation of extracellular matrix, which distorts the architecture of the normal liver, compromising its normal synthetic and metabolic functions. Thus, we aimed to investigate liver fibrosis status and its potential causal factors including oxidative stress, hepatocyte apoptosis, and production of inflammatory cytokines in the liver of cows with fatty liver. Forty-five dairy cows (parity, 3–5) were selected, and liver biopsy and blood were collected on the second week postpartum (days in milk, 10–14 d). On the basis of the degree of lipid accumulation in liver, selected cows were categorized into normal (n = 25; TG <1% wet wt), mild fatty liver (n = 15; 1% ≤ TG <5% wet wt), and moderate fatty liver (n = 5; 5% ≤ TG <10% wet wt). Compared with normal cows, blood concentrations of nonesterified fatty acids and β-hydroxybutyrate, along with alanine aminotransferase and aspartate aminotransferase activities, were greater in the cows with fatty liver (mild and moderate). Hepatic extracellular matrix deposition, as indicated by Picrosirius red staining, was greater in cows with fatty liver than those with normal ones. In addition, we observed an increased proportion of collagen type I fiber in extracellular matrix with increased lipid accumulation in the liver. Compared with normal cows, the area of α-smooth muscle actin (α-SMA)-positive staining along with the mRNA abundance of collagen type I α 1 (COL1A1), ACTA2 (gene encoding α-SMA), and transforming growth factor-β (TGFB) were greater in cows with fatty liver. Compared with normal cows, hepatic contents of malondialdehyde, glutathione disulfide, and 8-isoprostane were greater, whereas total antioxidant capacity, the hepatic content of glutathione, and activities of antioxidant indicators, including superoxide dismutase, glutathione peroxidase, and catalase, were lower in cows with fatty liver. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells and abundance of apoptosis-related molecules BAX, CASP3, CASP8, and CASP9 were greater in cows with fatty liver. However, mRNA abundance of the anti-apoptotic gene BCL2 did not differ. The mRNA abundance of pro-inflammatory cytokines including tumor necrosis factor-α (TNFA), interleukin-1β (IL1B), and interleukin-6 (IL6) was greater in the liver of cows with fatty liver. Overall, the present study indicated that fibrosis is a common pathological response to liver damage and is associated with oxidative stress, hepatocyte death, and inflammation.     

不同动物肝脏中脂肪酸成分分析

采用气相色谱法对7种动物肝脏中脂肪酸组成进行分析。以猪、羊、鸡、鸭、鲫鱼、草鱼和鹅的肝脏为材料,采用KOH-乙醇皂化,HCl-甲醇酯化的方法进行样品处理。实验结果表明,猪肝中有16种脂肪酸,鸡肝中有12种脂肪酸,羊肝中有18种脂肪酸,鸭肝中有14种脂肪酸,鲫鱼肝中有20种脂肪酸,草鱼肝中有22种脂肪酸,鹅肝中有19种脂肪酸。能快速、准确、可靠的满足动物肝中脂肪酸检测分析要求。     

新型鹅肝酱的制备技术研究

以鹅肥肝为主要原料,研制了新型鹅肝酱的工艺流程和最佳工艺配方。研究了乳化剂对鹅肝的乳化作用,及搅拌时间、增稠剂、加水量对鹅肝酱品质的影响。通过感官评价及正交实验分析,最终得到的新型鹅肝酱的最佳工艺配方为:乳化搅拌时间15min、1%的单甘酯和酪蛋白、5%的CMC和β-环糊精、冰水120mL。对配制的鹅肝酱进行感官评价和低温贮藏试验,结果表明该鹅肝酱符合营养、美味和质量要求。     

桔甘片对急性酒精性肝损伤小鼠的保护作用

探讨桔甘片（Jie-Gan Tablets,JGT）对急性酒精肝损伤小鼠的保护作用。采取50%酒精一次性灌胃（i.g）的方法建立小鼠急性酒精性肝损伤模型。检测小鼠血清中谷草转氨酶（AST）、谷丙转氨酶（ALT）、甘油三酯（TG）水平,肝组织匀浆中丙二醛（MDA）的含量及谷胱甘肽过氧化酶（GSH-Px）的活性,采用H&E染色分析组织切片形态变化,评估JGT的肝保护作用。结果显示不同剂量JGT和联苯双酯预处理能够显著抑制小鼠体内两种血清转氨酶水平的升高（p<0.05）,抑制率最高达到61.81%。JGT高剂量组显著降低血清中TG水平（p<0.05）,降幅达到34.73%。与模型组相比,阳性对照组显著逆转了MDA水平上升这一现象（p<0.01）,JGT各给药组呈剂量依赖的方式降低MDA的含量（p<0.05）,且提高了抗氧化酶GSH-Px的活性（p<0.05）,其中高剂量组MDA含量降至1.74 nmol/mg,GSH-Px活力上升至828.81 U/mg pro。H&E染色结果表明,JGT预处理能够有效改善酒精诱导的肝损伤。由此可见,JGT对ALD具有一定的预防保护作用。     

大鲵肝肽酒制备及其对小鼠酒后肝脏保护作用研究

以大鲵肝脏和白酒为原料制备大鲵肝肽酒，通过酶解法制备不同分子质量肝肽，对其体外乙醇脱氢酶（ADH）激活活性、体外抗氧化活性和溶解度进行测定，并采用生理盐水、白酒、大鲵肝肽酒分别灌胃小鼠，对小鼠肝脏及血清中相关生化指标进行测定。结果表明，分子质量<3 000 Da肝肽（GSLP-Ⅲ）对ADH激活率最高（83.53%），其对1,1-二苯基-2-三硝基苯肼自由基（DPPH·）、羟自由基（·OH）和超氧阴离子自由基（O2-·）清除率均>83.50%，还原力最高（吸光度值0.44），在53%vol白酒中溶解度较高（94.35%）。与白酒组小鼠相比，大鲵肝肽酒组小鼠肝脏乙醇脱氢酶（ADH）、乙醛脱氢酶（ALDH）、超氧化物歧化酶（SOD）活力、还原型谷胱甘肽（GSH）含量分别提高66.58%、26.84%、15.84%、36.92%，丙二醛（MDA）、甘油三酯（TG）含量分别降低17.37%、26.39%，血清谷丙转氨酶（ALT）和谷草转氨酶（AST）活性分别降低21.82%、23.61%，说明大鲵肝肽酒对小鼠酒后肝脏具有一定的保护作用。     

Aspects of transition cow metabolomics—Part I: Effects of a metaphylactic butaphosphan and cyanocobalamin treatment on the metabolome in liver,blood, and urine in cows with different liver metabotypes

Dairy cows in modern production systems are at risk to develop metabolic disorders during the transition period. Reasons for individual differences in susceptibility, as well as the underlying pathomechanisms, are still only partially understood. The development of metaphylactic treatment protocols is needed. In this context, an on-farm prospective 3-fold blinded randomized study involving 80 German Holstein cows was performed throughout 1 yr. The trial involved a thorough recording of the production and clinical traits, clinical chemistry, and liver biopsies and blood and urine sampling at d 14 (mean: 12 d, range: 1–26 d) antepartum (AP), and d 7 (7, 4–13) and 28 (28, 23–34) postpartum (PP) for metabolomics analyses. Two groups received a treatment with butaphosphan and cyanocobalamin (BCC) at either the dosage recommended by the manufacturer or the double dosage (5 or 10 mL/100 kg of body weight 10% butaphosphan and 0.005% cyanocobalamin (Catosal, Bayer Animal Health), n = 20 in each group, parity: 4.2 ± 2.0 and 3.4 ± 1.3, respectively (mean ± SD)] and one group a placebo treatment (NaCl 0.9%, n = 40, parity: 4.0 ± 1.9). The animals were treated at 6 time points (7, 6, and 5 d AP, and 1, 2, and 3 d PP) via intravenous injection. Mass spectroscopy-based targeted metabolomics analysis of blood plasma and liver samples were performed using the AbsoluteIDQ p180 kit (Biocrates Life Sciences), whereas the urine samples were analyzed by nuclear magnetic resonance spectroscopy. Statistical analysis was performed using multivariate [partial least squares discriminant analysis (PLS-DA)] and univariate methods (linear mixed model). Multivariate data analysis (PLS-DA plots) of the liver metabolome revealed 3 different metabotypes (A = medium, B = minor, C = large alterations in liver metabolome profile between AP and PP status). Metabotype B animals were characterized by higher PP lipomobilization (stronger PP body condition decrease and higher blood bilirubin, fatty acids, gamma-glutamyltransferase, and triglyceride levels) and a higher occurrence of transition cow diseases, compared with the animals in metabotype C. Analysis of the feeding data showed that the period of metabotype B animals (calving in a distinct time frame) was characterized by a decreased grass silage quality. The PP liver metabolome of the metabotype C animals was characterized by higher concentrations of AA, acylcarnitines, lysoPC and sphingomyelins compared with metabotype B. For the metaphylactic treatment with BCC a dose-dependent effect was confirmed, differing between the metabotypes. In all matrices and metabotypes at various time points significant treatment effects were observed, with different profiles in clinical chemistry and as well in metabolomics data. The most clear-cut treatment effect was observed in metabotype B in the liver at 7 d PP, characterized by an increase in several acylcarnitines and phosphatidylcholines, indicating a more efficient influx and oxidation of fatty acids in mitochondria and thereby an increase in energy supply and more efficient triglyceride export in the liver. The results from the liver metabolomics analysis support the application of an indication-based metaphylactic treatment with BCC.     

Content of biologically active polyamines in livers of cattle, pigs and chickens after animal slaughter

Dietary polyamines putrescine (PUT), spermidine (SPD) and spermine (SPM) participate in an array of important human physiological roles, including tumour growth. Physicians and dieticians thus need reliable information on polyamine contents in foods. However, data for livers are lacking. We determined therefore the content of these polyamines 24 h after slaughter in livers of young bulls, cows, pigs and chicken in 58, 19, 36 and 38 samples, respectively. Polyamines were determined as N-benzamides by micellar electrokinetic capillary chromatography. Mean PUT contents about 25 mg kg⁻¹ were found in cattle livers, while very low or negligible contents were determined in livers of the other animals. Extremely high mean SPD contents of 122 and 161 mg kg⁻¹ were found in livers of bulls and cows, respectively and mean levels of 32 and 57 mg kg⁻¹ in livers of pigs and chicken. An opposite relation was observed for SPM. Its mean contents were 43, 35, 115 and 120 mg kg⁻¹ for bulls, cows, pigs and chicken livers, respectively. Thus, livers of the tested animal species belong among foods with the highest polyamine contents. However, the contents ranged very widely, that makes application of the results for the control of human nutrition rather difficult. Polyamine contents in bovine blood were found to be below the detection limits of 2.1, 1.0 and 1.4 mg kg⁻¹ for PUT, SPD and SPM, respectively. Thus, the blood content did not contribute to the substantial polyamine contents in bovine liver found in this study.