Lee Q P, Park H W, Thayer J, Mirkes P E, Juchau M R
Department of Pharmacology, University of Washington, Seattle 98195, USA.
Teratology. 1996 Jan;53(1):21-30. doi: 10.1002/(SICI)1096-9926(199601)53:1<21::AID-TERA3>3.0.CO;2-C.
Previously, we reported that massive cell death was induced in the mesencephalic area of cultured rat embryos after embryos of gestational day 10.5 were intra-amniotically microinjected with sodium nitroprusside (SNP, 800 microM) and cultured for 24 hr at 37 degrees C. The massive cell death apparently was the result of NO-mediated embryotoxicity. Damage was concentration dependent and tissue specific. In follow-up studies, we now report evidence that NO generated from SNP induces apoptosis in organogenesis stage cultured rat embryos. Nile blue sulfate (NBS) staining suggested that microinjections of 400 microM SNP induced apoptosis in the mesencephalic area. Since we observed no massive cell death ("white caps") at this concentration, it appeared that early stages of apoptosis preceded "white cap" formation. At 800 microM SNP, total disintegration of cell bodies was evident and may have resulted from later stages of aoptosis or necrosis, or both. The "white caps" per se, an accumulation of disintegrated cell bodies, did not stain with NBS, probably due to total loss of cell integrity and resultant coagulation. The majority of the coagulated dead cells in the "white caps" were heavily stained with 3,3'-diaminobenzidine via in situ 3' end-labeling with terminal transferase. However, it is now known that NO can damage DNA directly and that in situ 3' end-labeling by terminal transferase detects not only apoptosis but also random DNA breakage. Increased 3' end-labeling and a "DNA ladder" were detectable within 5-10 hr after exposure of day 10.5 embryos to 400 or 800 microM of microinjected SNP. Some smear background was also observed in the "ladder." Rostral aspects of embryos exhibited more prominent indices of apoptosis than caudal regions. The results suggested that microinjections of SNP into the amniotic fluid of day 10.5 cultured rat embryos induces NO-mediated cell death in the mesencephalic and rhombencephalic regions by the process of apoptosis or of both apoptosis and necrosis, depending on the timing, concentration, and stage of gestation.
此前，我们报道称，在妊娠第10.5天的大鼠胚胎羊膜腔内微量注射硝普钠（SNP，800微摩尔）并在37℃培养24小时后，培养的大鼠胚胎中脑区域会诱导大量细胞死亡。这种大量细胞死亡显然是一氧化氮介导的胚胎毒性的结果。损伤具有浓度依赖性和组织特异性。在后续研究中，我们现在报告证据表明，SNP产生的一氧化氮在器官发生阶段培养的大鼠胚胎中诱导细胞凋亡。硫酸尼罗蓝（NBS）染色表明，微量注射400微摩尔SNP会在中脑区域诱导细胞凋亡。由于在此浓度下我们未观察到大量细胞死亡（“白帽”），似乎细胞凋亡的早期阶段先于“白帽”形成。在800微摩尔SNP时，细胞体的完全解体很明显，这可能是凋亡后期或坏死阶段或两者共同作用的结果。“白帽”本身是解体细胞体的聚集物，不能被NBS染色，可能是由于细胞完整性完全丧失和随之而来的凝固。“白帽”中大多数凝固的死亡细胞通过末端转移酶原位3'末端标记被3,3'-二氨基联苯胺强烈染色。然而，现在已知一氧化氮可直接损伤DNA，并且末端转移酶原位3'末端标记不仅能检测细胞凋亡，还能检测随机DNA断裂。在第10.5天的胚胎暴露于400或800微摩尔微量注射的SNP后5至10小时内，可检测到3'末端标记增加和“DNA梯带”。在“梯带”中也观察到一些涂片背景。胚胎的头侧部分比尾侧区域表现出更明显的细胞凋亡指标。结果表明，向第10.5天培养的大鼠胚胎羊膜腔内微量注射SNP，根据妊娠时间、浓度和阶段，通过细胞凋亡过程或细胞凋亡与坏死两者共同作用，在中脑和后脑区域诱导一氧化氮介导的细胞死亡。
