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观察技术，发展了一种新的环境内分泌干扰物(EDs)对家畜生殖和遗传毒性及致畸性的评估方法。(1)活体评估：应用新的活体内核磁共振波谱技术和^&lt；31&gt；P和^1H-核磁共振成像技术，准确和非侵入性地评估环境内分泌干扰物的致畸作用。以极低剂量(0.001~1,000 ng/kg体重)给妊娠小鼠注射雌二醇(天然雌激素、己烯雌酚、合成雌激素、双酚A、植物雌激素等)，在体内无创性、定量地获取胚胎的P-核磁共振谱，以评价能量代谢。胚胎ATP水平显著下降，但常规组织学和生化方法未检测到明显的毒理学变化。因此，体内核磁共振技术具有高度的敏感性(至少是常规方法的1000倍)，对于EDs和许多环境污染物的毒理学评估是有用的。(2)体外评估：使用培养的卵母细胞、精子、受精卵和胚胎来评估EDs和环境污染物的生殖和遗传毒性。用激光共聚焦显微镜非侵入性实时成像技术显示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.