황린(黃燐)투여가 백서간세포에 미치는 영향에 관한 전자현미경적 연구

Abstract

[한글]

Ultrastructura1 Changes of Rat Liver Cells Induced by Yellow Phosphorus

Sung Ho Kim, M.D.

Department of Medical Science The Graduate School, Yonsei University

(Directed by Professors: Dong Sik Kim and Yoo Bock Lee)

Yellow Phosphorus, one of the allotropic forms of elemental phosphorus, is highly

toxic to human being but yellow phosphorus-containing roach poisons and other

chemical substances are still available. When sufficient quantities haute been

ingested, acute hemorrhagic inflammation of the esophagus and stomach is induced

and followed by fatty degeneration of the liver, kidneys, heart, and the voluntary

muacles, but liver damage is considered as the mast significant one since cirrhosis

or acute yellow atrophy of the liver can occur and death may result from those

causes (LaDue et al., 1944; Goodman and Gilman, 1965; Harrison et al., 1966). The

morphologic changes of the liver induced by yellow phosphorus had been rather well

establiahed with light microscopic studies and fatty degeneration and necrosis at

the peripheries of lobules of the liver have been accepted as a characteristic

findings of phosphorus intoxication (Popper and Schaffner, 1957; Robbins, 1967).

The ultrastructural changes at the early stage of intoxication, however, not fully

established yet because previous studies on the ultrastruotural changes of the

phosphorus-intoxicated rat liver are extremely variable in result and not

reproducible(Jezequel, 1958; Ghoshal et al., 1969; Ganote and Otis, 1969). The

mechanism of fatty degeneration is still controversial. Recently, however, a very

interesting speculation was postulated by Ghoshal et al. (1969,1971) explaining

that lipoperoxidation theory, which was very attractive in case of carbon

tetrachloride poisoning, was consistent with mechanism of yellow phosphorus

intoxication in morphological aspects.

The purpose of this paper is to describe the ultrastructural changes in the liver

cells of the yellow phosphorus intoxicated fat on the earthy stage find to discuss

the possibility of lipoperoxidation mechanism by studying the effect of DL-

alpha-tocopherol, a very powerful antioxidant.

Materials and Methods

Thirty two adult albino rats weighing around 200gm. each were used regardless of

their sex. The animals were divided into three groups as follows :

Group Ⅰ: control animals

Group Ⅱ : yellow phosphorus treated animals

Group Ⅲ : vitamin E and phosphorus treated animals

Phosphorus was suspended in olive oil (0.5% mixture w/v) and administered into

peritoneal cavities in the amount of 0.75mg. per 100gm. of body weight.

DL-alpha-tocopherol (Tofaxin, made by Wintrop- Stearns Co.) was injected

intramuscularly in a debase of 10mg. per 100gm. of body Weight daily for 3 days

prior to yellow phosphorus treatment and at time Of yellow phosphorus injection.

The control animals received olive oil only with the same volume with animals of

group Ⅱ of Ⅲ. At intervals of 6, 12, 24 and 48 hours after the administration of

yellow phosphorus, the animals were killed. The liver and other organs were

examined grossly and the specimens for the light and electron microscopic

examination were obtained immediately from the liver.

Specimens for the fight microscopic examinations were fixed in 10% neutral

formalin and embedded in paraffin. Sections of 6 micron thick were made and stained

with hematoxylin-eosin for studies of overall histologic change and periodic acid

Schiff reaction for glycogen. Frozen sections were also made and stained with oil

red O for demonstration of fat.

Specimens for the electron microscopic examinations were cut in 1 ㎣ size and

fixed in 6% glutaraldehyde in phosphate buffer pH 7.4 for 2 hours at 4℃ and post

fixation was followed in 1% osmium tetraoxide in phosphate buffer at pH 7.4 for 2

hours. They were embedded in Epon 812 and cut into 470-5OOÅ thickness with glass

knife. After staining with uranyl acetate and lead hydroxide, observation was made

with Hitachi model HU 11-E electron microscope,

Result and Discussion

In the liver of control rats, no stainable fat was demonstrated by oil red O

method, but in group Ⅱ, fatty metamorphosis was demonstrable at 6 hours after

administration of yellow phosphorus and involved almost the entire lobule except

some hepatic parenchymal cells adjacent to central vein. It advanced in degree

through 12th hour and reached its peak at 24th hour and maintained this until the

48th hour, Scattered parenchymal sell necrosis and round cell infiltrations were

also noted but did not reveal any zonal character. In group Ⅲ, the fatty

metamorphosis did not occur at the 6th hour but at 12th hour mild changes were

noted which also advanced with time. Vitamin E failed to prevent inflammation and

necrosis. PAS Positive material was mildly decreased in the liver throughout the

experimental periods as compared with the control liver.

The most significant ultrastructural changes were found in rough endoplasmic

reticulum and were very distinctive. The rough endoplasmic reticulum was markedly

hyperplastic and arranged in parallel fashion ocoupying large areas of cytoplasmic

spaces. The studied ribosome was intact and free ribosome was not increased.

Polysome formation, however, was not demonstrable. These changes were noted at the

6th hour and maintained those patterns until 24 hours after phosphorus injection

but maredly decreased in degree at 48th hour. The fat globules were identified at

6th hour not in the cisternae but in cytoplasm and these findings were considered

as evidences of difference between carbon tetrachloride and yellow phosphorus

intoxication.

In group Ⅲ, the same morphologic changes took place except the at sence of fat

globule at 6th hour.

The smooth endoplasmic reticulum, lysosome, mitochondria, microbody, Golgi

complex and nucleus were not significantly altered in their morphologic characters

in both groups Ⅱ and Ⅲ.

In summary, the fatty changes induced by yellow phosphorus were not confined to

the periphery of the hepntic lobule but involved in almost the entire lobules even

in the earthy stage. The most significant and quite distinctive ultrastructural

changes were early hyperplasia of the rough endoplasmic reticulum and their

parallel arrangement. Vitamin E failed to prevent the tonic effect of yellow

phogphorus completely and the lipoperoxidation theory seams not to be convincing in

cases of yellow phosphorus poisoning.

[영문]

Yellow Phosphorus, one of the allotropic forms of elemental phosphorus, is highly toxic to human being but yellow phosphorus-containing roach poisons and other chemical substances are still available. When sufficient quantities haute been ingested, acute hemorrhagic inflammation of the esophagus and stomach is induced

and followed by fatty degeneration of the liver, kidneys, heart, and the voluntary muacles, but liver damage is considered as the mast significant one since cirrhosis or acute yellow atrophy of the liver can occur and death may result from those causes (LaDue et al., 1944; Goodman and Gilman, 1965; Harrison et al., 1966). The morphologic changes of the liver induced by yellow phosphorus had been rather well establiahed with light microscopic studies and fatty degeneration and necrosis at the peripheries of lobules of the liver have been accepted as a characteristic findings of phosphorus intoxication (Popper and Schaffner, 1957; Robbins, 1967).

The ultrastructural changes at the early stage of intoxication, however, not fully established yet because previous studies on the ultrastruotural changes of the phosphorus-intoxicated rat liver are extremely variable in result and not reproducible(Jezequel, 1958; Ghoshal et al., 1969; Ganote and Otis, 1969). The mechanism of fatty degeneration is still controversial. Recently, however, a very interesting speculation was postulated by Ghoshal et al. (1969,1971) explaining

that lipoperoxidation theory, which was very attractive in case of carbon tetrachloride poisoning, was consistent with mechanism of yellow phosphorus intoxication in morphological aspects.

The purpose of this paper is to describe the ultrastructural changes in the liver cells of the yellow phosphorus intoxicated fat on the earthy stage find to discuss the possibility of lipoperoxidation mechanism by studying the effect of DL-

alpha-tocopherol, a very powerful antioxidant.

Materials and Methods

Thirty two adult albino rats weighing around 200gm. each were used regardless of their sex. The animals were divided into three groups as follows :

Group Ⅰ: control animals

Group Ⅱ : yellow phosphorus treated animals

Group Ⅲ : vitamin E and phosphorus treated animals

Phosphorus was suspended in olive oil (0.5% mixture w/v) and administered into peritoneal cavities in the amount of 0.75mg. per 100gm. of body weight.

DL-alpha-tocopherol (Tofaxin, made by Wintrop- Stearns Co.) was injected intramuscularly in a debase of 10mg. per 100gm. of body Weight daily for 3 days prior to yellow phosphorus treatment and at time Of yellow phosphorus injection.

The control animals received olive oil only with the same volume with animals of group Ⅱ of Ⅲ. At intervals of 6, 12, 24 and 48 hours after the administration of yellow phosphorus, the animals were killed. The liver and other organs were examined grossly and the specimens for the light and electron microscopic

examination were obtained immediately from the liver.

Specimens for the fight microscopic examinations were fixed in 10% neutral formalin and embedded in paraffin. Sections of 6 micron thick were made and stained with hematoxylin-eosin for studies of overall histologic change and periodic acid Schiff reaction for glycogen. Frozen sections were also made and stained with oil red O for demonstration of fat.

Specimens for the electron microscopic examinations were cut in 1 ㎣ size and fixed in 6% glutaraldehyde in phosphate buffer pH 7.4 for 2 hours at 4℃ and post fixation was followed in 1% osmium tetraoxide in phosphate buffer at pH 7.4 for 2 hours. They were embedded in Epon 812 and cut into 470-5OOÅ thickness with glass knife. After staining with uranyl acetate and lead hydroxide, observation was made with Hitachi model HU 11-E electron microscope,

Result and Discussion

In the liver of control rats, no stainable fat was demonstrated by oil red O method, but in group Ⅱ, fatty metamorphosis was demonstrable at 6 hours after administration of yellow phosphorus and involved almost the entire lobule except some hepatic parenchymal cells adjacent to central vein. It advanced in degree

through 12th hour and reached its peak at 24th hour and maintained this until the 48th hour, Scattered parenchymal sell necrosis and round cell infiltrations were also noted but did not reveal any zonal character. In group Ⅲ, the fatty metamorphosis did not occur at the 6th hour but at 12th hour mild changes were

noted which also advanced with time. Vitamin E failed to prevent inflammation and necrosis. PAS Positive material was mildly decreased in the liver throughout the experimental periods as compared with the control liver.

The most significant ultrastructural changes were found in rough endoplasmic reticulum and were very distinctive. The rough endoplasmic reticulum was markedly hyperplastic and arranged in parallel fashion ocoupying large areas of cytoplasmic spaces. The studied ribosome was intact and free ribosome was not increased.

Polysome formation, however, was not demonstrable. These changes were noted at the 6th hour and maintained those patterns until 24 hours after phosphorus injection but maredly decreased in degree at 48th hour. The fat globules were identified at 6th hour not in the cisternae but in cytoplasm and these findings were considered

as evidences of difference between carbon tetrachloride and yellow phosphorus intoxication.

In group Ⅲ, the same morphologic changes took place except the at sence of fat globule at 6th hour.

The smooth endoplasmic reticulum, lysosome, mitochondria, microbody, Golgi complex and nucleus were not significantly altered in their morphologic characters in both groups Ⅱ and Ⅲ.

In summary, the fatty changes induced by yellow phosphorus were not confined to the periphery of the hepntic lobule but involved in almost the entire lobules even in the earthy stage. The most significant and quite distinctive ultrastructural changes were early hyperplasia of the rough endoplasmic reticulum and their

parallel arrangement. Vitamin E failed to prevent the tonic effect of yellow phogphorus completely and the lipoperoxidation theory seams not to be convincing in cases of yellow phosphorus poisoning.