Ultrastructural Study of Peroxisome Proliferation in Hepatocytes by Quick-Freezing and Deep-Etching Method

It had been emphasized that luminar continuities between sER and peroxisomes were detected by conventional electron microscopy. However, recent studies ruled out the luminar continuities between sER and peroxisomes. Lazarow et al. reported that peroxisomal proteins were synthesized on free ribosomes and postulated the existence of “peroxisomal reticulum” distinct from the ER. The object of this study is to clarify the proliteration mechanism of peroxisomes after administration of a peroxisome proliferator, DEHP (di-2- ethylhexyl phthalate).Mice treated with 2% DEHP for 1, 3, 5 and 7 days and normal mice were perfused with 2% paraformaldehyde in 0.1M phosphate buffered solution, pH 7.4, (PB) for 5 min. The livers were cut into small pieces, washed in PB to remove cytoplasmic soluble proteins and were fixed again with 2% paraformaldehyde-0.25% glutaraldehyde for 30 min. They were quickly frozen in isopentane-propane mixture (around -190 C) and fractured in liquid nitrogen to remove the damaged surface tissues. They were deeply etched in Eiko FD-3S machine (-95°C, 2-6xl0-7 Torr) and rotary shadowed with platinum.