Abstract

Lysosomes are the main component of an intracellular digestive system which has been recognized in a wide variety of cells, both in plants and animals. They were first discovered in rat livers, by do Duve et al. (1955), through biochemical studies that revealed the existence of a distinct group of cytoplasmic particles, surrounded by a membrane, and containing acid hydrolases. Lysosomes are important in cellular pathology because they are almost invariably involved in the response of the organism to the challenging agents and because the lysosomal reaction often becomes an important contributing pathogenic factor as well as a component of the symptomatology. Lysosomes may also mediate tissue damage by releasing their enzymes directly into the cell sap or surrounding tissues. Such an effect was observed following the addition of excess vitamin A to fetal bone rudiments in organ culture(Fell and Thomas, 1960; Thomas et al., 1960; Dingle, 1963). The exact mechanism of the effect of excess vitamin A on the membranes of lysosomes is still controversial. The mechanism of intracytoplasmic release of lysosomal hydrolases facilitated by the action of vitamin A is thought to be either rupture of the lysosomal membrane(Dingle and Fell, 1961 ; Weissmann et al., 1963; do Duve, 1963; Roels et al., 1969), or leakage across the lysosomal membrane (Schin and Cleven, 1965; Trump et al., 1965; Weissmann, 1965; Comolli, 1967; Friedman et al., 1969). The purpose of the present study is to determine the effect of excess vitamin A to the lysosomal membrane and the stabilize effect of cortisone. Materials and Methods: Sixty five adult albino rats weighting around 200 gm. were used regardless of their sex. The mental animals were divided into three groups as follow: Group Ⅰ : control animals (7 rats) Group Ⅱ : animals treated with large doses of vitamin A(29 rats) Group Ⅲ : animals treated with large doses of vitamin A and cortisone (29 rats) Vitamin A was administered orally in the amount of 500 international units per 1 gm. of body weight daily. Cortisone was injected intramuscularly in a dosage of 0.05 mg. per 1gm. of body weight daily. The control animals received salada oil only with the same volume as vitamin A treated animals. The rats from each experimental group and one from control were killed respectively on the first, third, 5th, 7th, 10th, 15th and 20th days after adminitration of vitamin A and vitamin A with cortisone. The liver tissues for the light microscopic examination were obtained immediately. For the light microscopic examination, tissues were fixed in 10% neutral formalin and embedded in paraffin. Six μ thick sections were made and stained with hematoxylin-eosin, and periodic acid Schiff reaction for glycogen. Frozen sections were also made to be stained for fat with oil red O. Blocks of liver for routine electron microscopic analysis were fixed in 1% osmium teraoxide with veronal buffer (PH 7.4) at 0-4°C. these were dehydrated, embedded in Epon, and examined in Hitachi HU-IIE electron microscope after staining with uranyl acetate and lead hydroxide. Electron microscopic histochemical demonstration of acid phosphatase was accomplished as follows: Thin slices of liver were fixed for 2-4 hours in cold 4% sodium cacodylate-buffered glutaraldehyde and washed overnight in cacodylate buffer containing 7.5% sucrose. Small cubes of tissue were then incubated according to Gomori method for the demonstration of acid phosphatase. The incubated tissues were then postfixed in osmium teraoxide, dehydrated and embedded in Epon. these sections were examined unstained in the electron microscope.
Result
and Discussion: In the light microscopic examination in group Ⅱ, vacuoles in the liver cells were limited to the peripheral zone and extended to midzone by 7th day and were persistent throughout the remaining periods of the experiment. These vacuoles were found to be fat droplets by oil red O stain. Necrosis or ballooning of liver cells were not found. In groups Ⅲ, ballooning of liver cells was noted on the first day after treatment and extended and persisted throughout the remaining periods of the experiment. At the 10th day, fat vacuoles were also found with ballooning of liver cells. Slight dilatation of rough endoplasmic reticulum and normal appearance of lysosomes were observed on the third day in group Ⅱ by electron microscopic examination. But the activity of acid phosphatase was aggregated outside the lysosomes and intracytoplasm. It is suggested that the excess vitamin A facilitated the leakage of enzymes across the lysosomal membranes rather than actual rupture. On the 5th day in group Ⅱ, the rough endoplasmic reticulum was made dilated and noted the part of desruction of cytoplasm and acid phosphasases were distributed intracytoplasm and along the rough endoplasmic reticulum. It is suggested that thease change were caused by released and newly formed enzymes. In the more deranged cytoplasm, presence of lipid droplets and myelin figures were noted on the 7th day in group Ⅱ. In group Ⅲ, the cytoplasmic organelles were more preserved than in group Ⅱ. The release of acid phosphatase was delayed. In summary, severe injury of liver cells was not produced by the administration of large doses of citamin A. However, mild fatty changes were observed. The intracytoplasmic release of acid phosphatases of lysosomes is considered to be due to the increase of permeability of the lysosomal membrane rather than the rupture of it. Cortisone appears to delay both formation of lipids and release of lysosomal enzymes.