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

本研究利用结构生物学技术，即三维和功能性活体核磁共振（NMR）观察技术，建立了一种评价环境内分泌干扰物（EDs）对家畜生殖、遗传毒性和致畸性的新方法。(1)体内评估：应用新型体内13 P<31>和131 H-NMR光谱学以及13 <31>P和131 H-NMR成像技术，准确和无创地评估ED诱导的致畸性。以极低剂量（0.001- 1，000 ng/kg体重）对妊娠小鼠施用ED（作为天然雌激素的雌二醇17 β、作为阳性对照试剂的己烯雌酚、合成雌激素、双酚A、植物雌激素等），然后<31>无创地和定量地获取胚胎的体内13 P-NMR光谱以评估能量代谢（13 <31>P-ATP）。胚胎ATP水平显着下降，但没有显着的毒理学变化检测到常规的组织学和生化方法。因此，体内NMR技术是高度敏感的（比常规方法敏感至少1,000倍），并且可用于ED和许多环境污染物的毒理学评估。(2)体外评估：采用体外培养的卵母细胞、精子、受精卵和胚胎评价了EDs和环境污染物的生殖和遗传毒性。采用激光共聚焦显微镜观察DNA烷基化、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.