偶氮染料的致突变分子机制及其毒代动力学研究

发布时间：2019-05-27 15:02

【摘要】：目的：通过制备偶氮染料毒性物质阳性暴露SD大鼠实验动物模型，利用组织病理学切片诊断技术和免疫组织化学法观察分析，进而探讨偶氮染料对实验动物机体的致突变损害和分子机制；采用液相色谱-串联质谱定量分析技术，开展偶氮染料在动物体内的毒性蓄积、组织分布及毒代动力学研究，对大鼠阳性暴露的损害进行定量评估，为临床上偶氮染料物质暴露污染提供理论基础和实验依据。 方法：(1)、通过制备偶氮染料阳性暴露的SD大鼠动物模型，结合病理学石蜡切片HE染色，对染料物质引起的损害进行病理学诊断分析。(2)、采用EnVision免疫组化法检查肝脏和肾脏器官中CYP1A1基因的蛋白表达水平，从分子生物学水平讨论其毒害程度和分子机制。(3)、建立了生物基质中4种染料物质的超快速液相色谱-串联质谱（UFLC-MS/MS）的定量分析方法，并实际应用于实验动物的组织分布、蓄积及毒代动力学的研究分析和定量评估。 结果：(1)、偶氮染料阳性处理组的大鼠肝组织均出现了不同程度的空泡症状，细胞间隔变厚，肝细胞脂肪变性，出现了大小不等的脂肪滴；肾脏组织出现程度不同的肾小管上皮细胞变性坏死，细胞界限模糊，对大鼠肝脏、肾脏造成了损伤危害。(2)、偶氮染料对大鼠肝脏CYP1A1表达分析结果显示空白对照组和苏丹染料处理组的平均阳性指数（IHC index）分别为138.89、1651.9、1220.35、490.16、1347.22，肾脏表达的阳性指数分别为17.69、295.68、128.31、196.73、400.33。说明偶氮染料处理组的肝细胞CYP1A1基因的蛋白表达水平明显高于对照组，且具有统计学意义（P 0.01），表明上调了该基因的蛋白表达水平，引起毒性效应和致损伤作用。(3)、针对不同基质的生物样品（血液、器官、组织等）成功建立了液相色谱-串联质谱联用检测法（LC-MS/MS），，该方法快速、稳定、低定量限（sub-ppb），适用于偶氮染料物质残留定量测定的实际工作，是生物样品中偶氮染料测定的稳健的定量方法。(4)、在50mg/kg4种苏丹染料的灌胃剂量下，组织分布实验中残留蓄积浓度值最高的是脂肪组织，含量最低的是心脏。(5)、4种偶氮染料分别于灌胃给药0.6046、1.362、4.196、4.341h后，大鼠的血药浓度达到最大值（Cmax分别为630.91、696.26、349.39、1304.61μg/L），在实验大鼠体内的平均半衰期（t1/2）分别为1.208h、2.611h、3.449h、3.411h，药-时曲线下面积值(AUC0-∞)分别为1150.03、2966.66、2706.37、10013.91μg·h/L，表明各种物质在动物机体内的生物利用度高、吸收速度快。 结论：偶氮染料阳性暴露对SD大鼠动物机体肝脏和肾脏产生了病理学损害作用，通过上调肝脏和肾脏内的CYP4501A1基因的蛋白表达水平，引起毒性效应和致损伤作用；偶氮染料在动物机体中易在组织器官内产生蓄积，脂肪组织中含量最高，毒代动力学数据表明4种染料物质的半衰期较短，生物利用度高，较易被吸收。
[Abstract]:Objective: to establish an experimental animal model of SD rats exposed to azo dye toxic substances, and to observe and analyze them by histochemical section diagnostic technique and immunohistochemistry. Furthermore, the mutagenic damage and molecular mechanism of azo dyes on experimental animals were discussed. The toxic accumulation, tissue distribution and toxicokinetics of azo dyes in animals were studied by liquid chromatography-tandem mass spectrometry (LC-MS), and the damage of positive exposure in rats was quantitatively evaluated. It provides theoretical and experimental basis for azo dye exposure to pollution in clinic. Methods: (1) the pathological diagnosis and analysis of the damage caused by dye substances were carried out by establishing the animal model of SD rats exposed to azo dye and combining with HE staining of pathological paraffin sections. (2), The protein expression of CYP1A1 gene in liver and kidney organs was detected by EnVision Immunohistochemical method, and its toxicity and molecular mechanism were discussed from the level of molecular biology. (3), A method for quantitative analysis of four dye substances in biological matrix by ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) was established and applied to the study, analysis and quantitative evaluation of tissue distribution, accumulation and toxicokinetics of experimental animals. Results: (1) in the azo dye positive group, the liver tissue of the rats showed vacuole symptoms in different degrees, the cell septum became thicker, the hepatocytes steatosis, and fat droplets of different sizes appeared. Renal tissue showed degeneration and necrosis of renal tubular epithelial cells and blurred cell boundaries, which caused damage to rat liver and kidney. (2), The results of analysis of CYP1A1 expression in rat liver by azo dye showed that the average positive index (IHC index) of blank control group and Sudan dye treatment group was 138.89, 1651.9, 1220.35490.16, 1347.22, respectively. The positive index of renal expression was 17.69295.68128.31196.73400.33. The results showed that the protein expression level of CYP1A1 gene in the group treated with azo dye was significantly higher than that in the control group (P < 0.01), indicating that the protein expression level of the gene was up-regulated. (3) liquid chromatography-tandem mass spectrometry (LC-MS/MS) was successfully established for biological samples of different matrices (blood, organs, tissues, etc.). The method is rapid and stable. Low fixed limit (sub-ppb), which is suitable for the quantitative determination of azo dye residues, is a robust quantitative method for the determination of azo dye in biological samples. (4) at the gastric perfusion dose of 50mg/kg4 Sudan dye, In the tissue distribution experiment, the residual accumulation concentration of adipose tissue was the highest, and the lowest content was the heart. (5) the four azo dyes were given 0.6046, 1.366, 4.196, 4.341 h, respectively, after intragastric administration of 0.6046, 1.362, 4.196, 4.341 h after intragastrically administration of 0.6046, 1.366, 4.196, 4.341 h, respectively. The blood concentration of rats reached the maximum (Cmax = 630.91696.26349.39, 1304.61 渭 g / L), respectively), the average half-life (T1 鈮

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