Vitamin A의 대량투여가 백서간세포의 Lysosome에 미치는 영향에 관한 전자현미경적 연구

Abstract

[한글]

Ultrastructural Changes of Rat Liver Cells Induced by Large Doses of Vitamin A

Sang Hyok Nam, M.D.

Department of Medical Science The Graduate School, Yonsei University

(Directed by Professors: Dong Sik Kim, M.D. and Yoo Bock Lee, M.D.)

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 de Duve et al. (1955), through biochemical

studies that revealed the existence of a distinct group of cytoplasmic particles,

surrounding by a membrane, and containing acid hydrolases.

Lyaosomes 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 of ten 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; Din히e, 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 rupture of the

lysosomal membrane(Dingle and Fell, 1961; Weissmann et at., 1963; de Duve, 1963;

Roels et al., 1969), and 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 was 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 weighing around 200gm. were used regardless of their

sex. The experimental 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 rat7)

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

0.05mg. per 1 gm. of body weight daily. The control animals received salada oil

only with the same volume as vitamin A treated animals.

Three rats from each experimental group were killed respectively on the first,

third, 5th, 7th, 10th, 15th and 20th days after administration 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 fired in 1% osmium

tetraoxide with veronal buffer (PH 7.4) at 0-4℃. 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 tetraoxide, 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 group 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 phoshatase 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 destruction of cytoplasm and acid phosphatases were distributed

intracytoplasm and along the rough endoplasmic retioulum It is suggested that these

changes 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 vitamin 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.

[영문]

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 de Duve et al. (1955), through biochemical studies that revealed the existence of a distinct group of cytoplasmic particles, surrounding by a membrane, and containing acid hydrolases.

Lyaosomes 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 of ten 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; Din히e, 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 rupture of the lysosomal membrane(Dingle and Fell, 1961; Weissmann et at., 1963; de Duve, 1963; Roels et al., 1969), and 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 was 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 weighing around 200gm. were used regardless of their sex. The experimental 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 rat7)

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 0.05mg. per 1 gm. of body weight daily. The control animals received salada oil only with the same volume as vitamin A treated animals.

Three rats from each experimental group were killed respectively on the first, third, 5th, 7th, 10th, 15th and 20th days after administration 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 fired in 1% osmium tetraoxide with veronal buffer (PH 7.4) at 0-4℃. 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 tetraoxide, 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 group 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 phoshatase 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 destruction of cytoplasm and acid phosphatases were distributed intracytoplasm and along the rough endoplasmic retioulum It is suggested that these changes 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 vitamin 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.