马度米星铵对水产动物的毒性、毒性机制及残留研究

Maduramicin ammonium is widely used as an anticoccidial drug for broilers due to its high anticoccidial activity and little drug-resistance. However, it has properties of narrow safety range and high toxicity, which frequently caused the target animals and human poisoning . Our preliminary data demonstrated that maduramicin ammonium could accumulate in crucian carp and crayfish, which are the main food for human beings and also one of the important ways for maduramicin ammonium to enter human body. Therefore, it is of important significance for rational use of veterinary drugs and food safety to investigate the toxicity and mechanism of maduramicin ammonium on crucian carp and crayfish as well as their metabolism and residue in vivo. This study intends to investigate the following scientific questions: 1) the toxic effect of maduramicin ammonium on crucian carp and crayfish; 2) the metabolism and residue of maduramicin ammonium and their effect on hepatic microsomal drug metabolizing enzyme P450; 3) the toxic molecular mechanism of aquatic animal cells caused by maduramicin ammonium. These data will be compared with the previous results of mammalian cells to reveal their differences in toxic mechanisms. This study will contribute to further investigating the effect of maduramicin ammonium on aquatic animals, ecological environment and human food safety.

马度米星铵抗球虫活性强，不易产生耐药性，是兽医临床上防治鸡球虫病的最常用药物之一。但其安全范围窄、毒性大，易引起靶动物和人的中毒。我们前期研究证实鲫鱼和克氏原鳌虾很可能是水体中马度米星铵的富集生物，二者作为人类重要的水产食品，是马度米星铵进入人体的重要途径，因此研究马度米星铵对鲫鱼和克氏原鳌虾的毒性、毒性机制、残留及代谢对兽医临床合理用药和人类食品安全具有重要意义。本项目拟研究马度米星铵对鲫鱼和克氏原鳌虾的毒性作用，阐明马度米星铵在水产动物的残留及对肝药酶P450活性的影响与毒性之间的关系；用体外细胞培养模型，评价马度米星铵对细胞增殖、凋亡和氧化应激的影响，探讨其细胞毒性的分子机制；比较马度米星铵对鸡、鲫鱼和克氏原鳌虾三种动物毒性机制的差异。为科学合理使用马度米星铵提供依据，也为深入研究马度米星铵对生态环境和人类食品安全的影响提供资料。

马度米星铵抗球虫活性强，不易产生耐药性，是兽医临床上防治鸡球虫病的最常用药物之一。但其安全范围窄、毒性大，易引起靶动物和人的中毒。我们前期研究证实克氏原鳌虾和鲫鱼很可能是水体中马度米星铵的富集生物，二者作为人类重要的水产食品，是马度米星铵进入人体的重要途径，因此研究马度米星铵对鲫鱼和克氏原鳌虾的毒性、毒性机制、残留及代谢对兽医临床合理用药和人类食品安全具有重要意义。本项目考察了马度米星铵对克氏原鳌虾和鲫鱼的急性毒性，获得了暴露96h的半数致死浓度（LC50）和安全浓度（SC）；同时考察了亚致死剂量浓度马度米星铵暴露28d对水产动物血液参数、组织抗氧化指标的影响及组织损伤作用，通过使用HE染色等手段观察组织变化，确定了毒性靶组织；建立了一种超高效液相色谱-串联质谱法（UPLC-MS/MS）用于检测水产动物血液和可食性组织中马度米星铵的残留量，获得了马度米星铵在水产动物体内的药动学参数和残留消除规律；明确了马度米星铵对鲫鱼和克氏原螯虾肝胰腺中药物代谢酶的影响，应用差速离心法提取肝微粒体，Lowry法测定肝微粒体蛋白浓度，紫外分光光度法测定CYP450酶系含量与活性，分析暴露28d后克氏原鳌虾和鲫鱼体内CYP450、CYP b5、ERND、APND、AH、GSTs、UGTs、NATs等代谢酶的活性变化，同时利用实时荧光定量PCR对CYP450、gst、CYP b5、CYP1A、CYP3A及CYP4T的mRNA表达水平进行检测，揭示了肝脏CYP450与毒性之间的关系；采用qRT-PCR检测了代谢和应激相关基因COX1、COX2、HSP70、MT、GST的mRNA表达水平，揭露了马度米星铵胁迫下，肝胰腺解毒和应激相关基因表达的变化。综上所述，本研究揭示了马度米星铵对水产动物的毒性效应与机制，阐明了马度米星铵在水产动物可食性组织中的残留消除规律，为保障生态环境健康和人类食品安全提供了可靠理论依据。