长期低剂量经口摄入敌敌畏对大鼠毒性作用的代谢组学研究

发布时间：2018-02-25 21:22

本文关键词： 敌敌畏毒性代谢组学长期低剂量超高效液相色谱/飞行时间串联质谱　出处：《哈尔滨医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：用代谢组学方法研究长期低剂量经口摄入敌敌畏对大鼠的毒性作用及可能的作用机制，筛选敌敌畏早期暴露的敏感生物标志物，为评价敌敌畏长期低剂量暴露对人类机体的影响提供更为可靠的理论依据。方法：雄性Wistar大鼠（180g－220g）按体重随机分为四组：低剂量组（大鼠经口摄入敌敌畏的最大无作用剂量即NOAEL，2.4mg/kg bw/d），中剂量（7.2mg/kg bw/d,3×NOAEL），高剂量组（21.6mg/kg bw/d,9×NOAEL）和对照组。敌敌畏按照上述剂量通过饮水给予大鼠，连续染毒24周，对照组大鼠直接给予饮用水。在实验进行4周、8周、12周、16周、20周和24周时，采集大鼠的血液、尿液和组织样本，进行生化指标检测、组织病理学检测，同时应用超高效液相色谱－质谱联用仪对尿液和血浆进行代谢组学分析。结果：血清生化指标显示，与对照组比较，中、高剂量组的胆碱酯酶(ChE)在染毒4周后即明显升高（p＜0.05），谷氨酸氨基转移酶（ALT）、天门冬氨酸转氨酶（AST）、尿素氮（BUN）和肌酐（Cr）的含量在染毒12周后明显升高（p＜0.05）；高剂量组白蛋白(ALB)在染毒12周后含量亦明显降低（p＜0.05）。低剂量组的这几个指标在实验过程中均没有发现明显变化。组织病理学检测显示，实验进行12周时，中、高剂量组肝组织出现脂肪变性或是细胞坏死等异常改变，低剂量组和对照组在整个实验过程中无异常变化。尿液代谢组学研究在正、负离子模式下共发现12个代谢物的异常改变。敌敌畏的代谢产物邻苯二甲酸二甲酯（DMP），仅出现在染毒组，且随着染毒剂量的增高，强度增加。与对照组比较，除了DMP，低剂量组尿液中的这些代谢物与对照组相比没有显著差异（p＞0.05）。中剂量组中吲哚酚硫酸和硫酸雌酮在敌敌畏处理12周后强度增高（p＜0.05），乳糖醛酸在实验20周后强度升高（p＜0.05），尿酸、辛二酸、古洛糖酸、尿素和肌酐在实验8周后强度显著降低（p＜0.05），柠檬酸实验进行12周后强度降低（p＜0.05）；高剂量组中的吲哚酚硫酸实验8周后强度增高（p＜0.05），硫酸雌酮和乳糖醛酸实验12周后强度增强（p＜0.05），尿酸（负离子模式）、辛二酸和柠檬酸在染毒8周后强度降低（p＜0.05），尿酸（正离子模式）、尿素和肌酐在整个实验的24周强度均降低（p＜0.05）。血浆代谢组学分析显示，，与对照组相比，正、负离子模式下共发现10个代谢物的异常改变。低剂量组LysoPE(16:0/0:0)实验16周后强度升高（p＜0.05）；LysoPC(17:0/0:0)、脱氢鞘氨醇、C16脱氢鞘氨醇和C17脱氢鞘氨醇染毒16周后强度降低（p＜0.05）。中剂量组LysoPE(16:0/0:0)实验16周后强度升高（p＜0.05）；花生四烯酸、LysoPC(16:0/0:0)、 LysoPC(0:0/18:0)和C17脱氢鞘氨醇在实验12周后强度降低（p＜0.05）；LysoPC(15:0/0:0)和鞘氨醇染毒处理16周强度降低（p＜0.05）；LysoPC(17:0/0:0)、脱氢鞘氨醇、C16脱氢鞘氨醇整个实验过程全部降低（p＜0.05）。高剂量组中除代谢物LysoPE(16:0/0:0)实验12周后强度升高（p＜0.05）外，其余9个代谢物在实验过程中6个时间点强度全部降低（p＜0.05）。结论：DMP作为敌敌畏在尿液中的代谢产物，具有灵敏性和特异性，可以作为敌敌畏暴露的一个生物标志物；大鼠暴露于敌敌畏后，影响了糖类、脂类的代谢及机体的抗氧化系统；尿液代谢组学分析显示，在最大无作用剂量（NOAEL）水平上，没有发现敌敌畏对大鼠机体的异常影响；血浆代谢组学分析显示一些脂类的异常改变与脏器损伤有关；代谢组学技术在研究敌敌畏长期低剂量暴露毒性方面有独特的优势。
[Abstract]:Objective: toxicity using metabonomics method to study the long-term low dose oral intake of dichlorvos in rats and its possible mechanism, screening of sensitive biomarkers for early exposure to dichlorvos, evaluation of dichlorvos long-term low dose exposure effect on the human body to provide a more reliable theoretical basis.
Methods: male Wistar rats (180g 220g) were randomly divided into four groups: low dose group (maximum dichlorvos oral intake of rats had no effect dose NOAEL, 2.4mg/kg bw/d), middle dose (7.2mg/kg bw/d, 3 * NOAEL), high dose group (21.6mg/kg bw/d, 9 x NOAEL) and control group. According to the above dose by drinking dichlorvos was given to rats, after 24 weeks, the rats in the control group given direct drinking water. For 4 weeks, 8 weeks, 12 weeks, 16 weeks in the experiment, 20 weeks and 24 weeks, rats collected blood, urine and tissue samples for biochemical detection, histopathological analysis, instrument of urine and plasma metabonomics analysis combined with ultra performance liquid chromatography and by mass spectrometry.
Results: the serum biochemical index, compared with the control group, high dose group, cholinesterase (ChE) in rats after 4 weeks was significantly increased (P < 0.05), alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN) and creatinine (Cr) content increased significantly in after 12 weeks (P < 0.05); high dose group albumin (ALB) content in after 12 weeks were significantly lower (P < 0.05). The index of low dose group during the experiment were found no significant changes. By histopathology, the experiment for 12 weeks and in liver tissue of high dose group showed fatty degeneration or abnormal cell necrosis, low dose group and the control group had no abnormal changes during the whole experiment.
The urine metabonomic study in positive and negative ion mode were found abnormal changes of 12 metabolites. Two metabolites of phthalic acid methyl ester (DMP), two had only appeared in the control group, and with the increased dose intensity increased. Compared with the control group, in addition to DMP, the low dose group in urine these metabolites compared with the control group had no significant difference (P > 0.05). The middle dose group in indoxyl sulfate and estrone sulfate in the treatment of dichlorvos intensity increased after 12 weeks (P < 0.05), lactobionic acid in the 20 week after strength increased (P < 0.05), uric acid, suberic acid, gulonic acid in the experiment, urea and creatinine after 8 weeks of strength decreased significantly (P < 0.05), citric acid experiment strength decreased after 12 weeks (P < 0.05); after 8 weeks of indoxyl sulfate experiment in the high dose group strength increased (P < 0.05), estrone sulfate and aldehyde lactobionic acid after 12 weeks of strength test enhanced (P < 0.05), urine Acid (negative ion mode), octanic acid and citric acid decreased after 8 weeks of exposure (P < 0.05), uric acid (positive ion mode), urea and creatinine decreased at 24 weeks of the whole experiment (P < 0.05).
Plasma metabonomics analysis showed that compared with the control group, positive and negative ion mode were found abnormal changes of 10 metabolites. Low dose of LysoPE group (16:0/0:0) after 16 weeks of experiment the strength increased (P < 0.05); LysoPC (17:0/0:0), C16 dehydrogenation of sphingosine, dehydrogenation of sphingosine and dehydrogenation of C17 sphingosine after 16 weeks after the intensity decreased (P < 0.05). The middle dose group LysoPE (16:0/0:0) after 16 weeks of experiment the strength increased (P < 0.05); four arachidonic acid, LysoPC (16:0/0:0), LysoPC (0:0/18:0) to reduce the strength and C17 in the 12 week after dehydrogenation of sphingosine (P < 0.05); LysoPC (15:0/0:0) and sphingosine after 16 week treatment intensity decreased (P < 0.05); LysoPC (17:0/0:0), C16 dehydrogenation of sphingosine, dehydrogenation of sphingosine throughout the experiment all decreased (P < 0.05). High dose group except metabolite LysoPE (16:0/0:0) after 12 weeks of experiment the strength increased (P < 0.05), the remaining 9 metabolites in experimental process in The intensity of the 6 time points was all decreased (P < 0.05).
Conclusion: DMP as a metabolite of dichlorvos in the urine, with high sensitivity and specificity, can be used as a biomarker of exposure to dichlorvos; rats were exposed to DDVP, affect carbohydrate metabolism and lipid, antioxidant system of the display; urine metabolic analysis, the maximum non effect dose (NOAEL) level, no abnormal dichlorvos effects on rat body; plasma metabonomics analysis showed some abnormal changes of lipid related organ damage; metabonomics technique in the study of dichlorvos exposure has the unique advantage of poison.

【学位授予单位】：哈尔滨医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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