A novel structural biological assessment for reproductive and genetic toxicity in farm animals using nuclear magnetic resonance technique

在本研究中，我们利用结构生物学技术，即三维和功能体内核磁共振（NMR）观察技术，开发了一种新的评估农场动物环境内分泌干扰物（EDs）生殖和遗传毒性和致畸性的方法。(1)体内评估：采用新颖的体内^<31> p -和^1H-NMR波谱技术和^<31> p -和^1H-NMR成像技术，准确、无创地评估EDs致致畸性。以极低剂量（0.001 ~ 1,000 ng/kg体重）给药妊娠小鼠（天然雌激素用雌二醇17β、阳性对照剂用己烯雌酚、合成雌激素用双酚A、植物雌激素等），获得胚胎体内^<31>P-NMR谱，无创定量评价能量代谢（^<31>P-ATP）。胚胎ATP水平明显降低，但常规组织学和生化方法未见明显毒理学变化。因此，体内核磁共振技术是高度敏感的（至少比传统方法灵敏1000倍），可用于EDs和许多环境污染物的毒理学评估。(2)体外评价：采用体外培养的卵母细胞、精子、受精卵和胚胎评价EDs和环境污染物的生殖和遗传毒性。使用共聚焦激光显微镜，通过无创实时成像技术观察DNA芳基化、类固醇激素受体mRNA表达异常。

In the present investigation, we developed a novel assessment method for reproductive and genetic toxicity and teratogenicity of environmental endocrine disrupters (EDs) in farm animals using structural biological techniques, i.e. three dimensional and functional in vivo nuclear magnetic resonance (NMR) observation techniques.(1) In vivo assessment : the novel in vivo ^<31>P-and ^1H-NMR spectoroscopy and ^<31>P-and ^1H-NMR imaging techniques for accurate and noninvasive assessment of teratogenicity induced by EDs were applied. Mice with pregnancy were administered EDs (estradiol 17beta as natural estrogen, diethylstilbestrol, synthetic estrogen, used as positive control reagent, bisphenol A, phyto estrogens, and so on) at extremely low dose (0.001-1,000 ng/kg of body weight), and then in vivo ^<31>P-NMR spectra of embryos were acquired noninvasively and quantitatively to evaluate the energy metabolism (^<31>P-ATP). A significant decrease in embryo ATP level was seen, but no significant toxicological changes were detected by conventional histological and biochemical methods. Thus, in vivo NMR techniques are highly sensitive (at least 1,000-fold more sensitive than conventional methods) and are useful for toxicological assessment of EDs and many environmental pollutants.(2) In vitro assessment : Cultured oocytes, sperm, fertilized eggs and embryos were used to assess the reproductive and genetic toxicity of EDs and environmental pollutants. DNA arkylation, abnormalities of steroid hormone receptor mRNA expression were visualized by noninvasive-real time imaging techniques using a confocal laser microscopy.