Lysosomal cysteine proteinases and their role in visceral yolk sac mediated nutrition and embryotoxicity.

The embryotoxicity of certain chemicals has been associated with an inhibition of protein degradation in the visceral yolk sac (VYS), an extraembryonic membrane responsible for embryonic amino acid supply prior to the formation of the functional placenta. This thesis tests the hypothesis that chemicals which inhibit a unique compliment of proteolytic enzymes present in the organogenesis-stage VYS can produce embryotoxicity and dysmorphogenesis through distinct mechanisms related to the disruption of VYS nutritional function. Conclusive evidence for the presence of lysosomal cysteine proteinases (LCPs) in the organogenesis-stage VYS is provided by experiments demonstrating that VYS tissue extracts hydrolyze selective substrates in a manner characteristic of this enzyme class. Further experiments indicate that only one member of this class (cathepsin L) is highly active in the VYS during early organogenesis and is therefore primarily responsible for VYS proteolysis. The first direct evaluation of two model toxicants for their embryotoxicity and effects on VYS function in vitro was performed. Rat conceptuses exposed to peptidyl diazomethanes (specific, irreversible inhibitors of the LCPs) in vitro showed selective embryonic cell death and dysmorphogenesis, and characteristic biochemical and histological changes indicative of inhibition of VYS proteolysis. The antimalarial drug chloroquine was also evaluated because of its known ability to inhibit lysosomal proteolysis by a different mechanism (increasing lysosomal pH). Chloroquine exposure was also embryotoxic and produced many of the same effects as the peptidyl diazomethanes. Clear differences between the biochemical and histological manifestations of embryotoxicity of the two agents were also noted, however. It is concluded that the embryotoxicity of chloroquine and the peptidyl diazomethanes is effected, in part, through inhibition of lysosomal cysteine proteinases, but that other direct embryonic effects also contribute to the final outcome. These findings provide valuable information for improving the mechanistic understanding of teratological data obtained in rodents and should be of value for use in human risk assessment.PhDToxicologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/104077/1/9500877.pdfDescription of 9500877.pdf : Restricted to UM users only