In vitro embryotoxicity of the cysteine proteinase inhibitors benzyloxycarbonyl‐phenylalanine‐alanine‐diazomethane (Z‐Phe‐Ala‐CHN2) and benzyloxycarbony1‐phenylalanine‐phenylalanine‐diazomethane (Z‐Phe‐Phe‐CHN2)

Abstract

This study makes use of whole embryo culture to investigate the potential embryotoxicity of benzyloxycarbonyl-phenylalanine-alanine-diazomethane (Z-Phe-Ala-CHN2) and benzyloxycarbonyl-phenylalanine-phenylalanine-diazomethane (Z-Phe-Phe-CHN2), two low molecular weight, active site-directed and irreversible inhibitors of the lysosomal cysteine proteinases. Peptidyl diazomethanes are the most specific inhibitors available for lysosomal cysteine proteinases and can be hypothesized to interrupt visceral yolk sac (VYS)-mediated nutrition during early organogenesis. When added directly to the culture medium of gestational day 10-11 rat conceptuses, both compounds inhibited lysosomal cysteine proteinase activity in the VYS in a concentration-dependent fashion that correlated with the degree of embryotoxicity observed. Z-Phe-Ala-CHN2 and Z-Phe-Phe-CHN2 were also found to increase the protein content of the VYS, even though all other conceptual growth parameters decreased. This effect was dependent on the serum content of the culture medium and the exposure time. Histological examination of Z-Phe-Ala-CHN2-treated conceptuses revealed a dramatic increase in the size and number of vacuoles in the VYS endoderm epithelium, suggestive of inhibition of VYS proteolysis. At the same time, excessive cell death was observed throughout the neuroepithelium and in specific regions of the mesenchyme of the corresponding embryos. This cell death manifested morphological characteristics of apoptosis and could be detected by supravital staining with Nile Blue Sulphate. These findings provide additional evidence in support of the hypothesis that lysosomal cysteine proteinases play a critical role in VYS-mediated histiotrophic nutrition and suggest that peptidyl diazomethanes may be useful in further characterization of these enzymes. The possible direct effects of these inhibitors on embryonic cells and the relationships between interruption of VYS-mediated nutritional processes and embryonic cell death are discussed.