Abstract

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 have 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 muscles, but liver damage is considered as the most significant one since cirrhosis or acute yellow atrophy of the liver can occur and death may result from these 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 established 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 ultrastructural changes of the phosphorus-intoxicated rat liver are extremely variable in result and not reproducible(Jézéquel, 1958; Ghoshal et at., 1969; Ganote and Otis, 1969). The mechanism of fatty degeneration is still controversial. Recently, however, a very interesting speculation was postulated by Choshal 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 rat on the early stage and 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 200 gm. 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-Steams Co.) was injected intramuscularly in a dosage of 10 mg. per 100 gm. 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 or 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 light 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 mm3 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 400-500Å 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 cell 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 occupying large areas of cytoplasmic spaces. The studded 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 these patterns until 24 hours after phosphorus injection but markedly 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 absence 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 hepatic lobule but involved in almost the entire lobules even in the early 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 toxic effect of yellow phosphorus completely and the lipoperoxidation theory seems not to be convincing in cases of fellow phosphorus poisoning.