APOPTOSIS INDUCED IN CULTURED RAT EMBRYOS BY INTRA-AMNIOTICALLY MICROINJECTED SODIUM-NITROPRUSSIDE

Previously, we reported that massive cell death was induced in the mes encephalic area of cultured rat embryos after embryos of gestational d ay 10.5 were intra-amniotically microinjected with sodium nitroprussid e (SNP, 800 mu M) and cultured for 24 hr at 37 degrees C. The massive cell death apparently was the result of NO-mediated embryotoxicity. Da mage was concentration dependent and tissue specific. In follow-up stu dies, we now report evidence that NO generated from SNP induces apopto sis in organogenesis stage cultured rat embryos. Nile blue sulfate (NB S) staining suggested that microinjections of 400 mu M SNP induced apo ptosis in the mesencephalic area. Since we observed no massive cell de ath (''white caps'') at this concentration, it appeared that early sta ges of apoptosis preceded ''white cap'' formation. At 800 mu M SNP, to tal disintegration of cell bodies was evident and may have resulted fr om later stages of aoptosis or necrosis, or both. The ''white caps'' p er se, an accumulation of disintegrated cell bodies, did not stain wit h NBS, probably due to total loss of cell integrity and resultant coag ulation. 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 c an damage DNA directly and that in situ 3' end-labeling by terminal tr ansferase detects not only apoptosis but also random DNA breakage. Inc reased 3' end-labeling and a ''DNA ladder'' were detectable within 5-1 0 hr after exposure of day 10.5 embryos to 400 or 800 mu M of microinj ected SNP. Some smear background was also observed in the ''ladder.'' Rostral aspects of embryos exhibited more prominent indices of apoptos is than caudal regions. The results suggested that microinjections of SNP into the amniotic fluid of day 10.5 cultured rat embryos induces N O-mediated cell death in the mesencephalic and rhombencephalic regions by the process of apoptosis or of both apoptosis and necrosis, depend ing on the timing, concentration, and stage of gestation. (C) 1996 Wil ey-Liss, Inc.