Methotrexate가 백서 간에 미치는 영향에 관한 조직화학적 연구

Abstract

[한글]

Methotrexate투여 및 phenobarbital전처치 후 methotrexate투여가 간실질세포에 미치는 영향을 추구하기 위하여 glucose-6-phosphatase활성의 변화와 형태학적 변화를 연관시켜 관찰하였다.

체중 200gm 내외의 백서 70마리를 사용하여 생리식염수만을 투여한 정상대조군, phenobarbital투여군, methotrexate단독투여군, phenobarbital전처치 후 methotrexate투여군의 네군으로 나눠 phenobarbital투여군은 체중 kg당 50mg의 phenobarbital을 24시간 간격으로 2회 투여하였고 methotrexate 단독투여군은 methotrexate를 체중 kg당 3mg투여하였다.

Phenobarbital전처치 후 methotrexate투여군은 phenobarbital을 24시간 간격으로 2회 투여한 후 24시간 후 methotrexate를 체중 kg당 3mg 투여하였다. 각 약제는 복강내 주사로 투여하였고 약제의 투여 후 4시간, 8시간, 16시간, 24시간, 72시간 후에 동물을 도살하여 일반적 조직변화의 양상을 보기 위해 hematoxylin-eosin염색을, g1ycogen의 소장을 보기 위해 periodic acid Schiff반응염색을 하였으며 glucose-6-phosphatase활성의 변화를 보기 위해 Wachstein-Meisel변법 (Wachstein & Meisel, 1956)에 따라 효소조직화학 염색을 하였으며 부수적으로 지방의 변화를 보기위해 oil redO염색을 시행하여 관찰하였다.

1. Hematoxylin-eosin염색소견은 methotrexate단독투여한 동물 모두 16시간 이후 약간의세포종창이 나타났으나 괴사 및 염증은 나타나지 않았다.

2. Periodic acid-Schiff염색반응소견은 16시간 이후 희생시킨 동물에서 약한 반응을 나타내었으며 phenobarbital전처치에는 유의할만한 차이가 없었다.

3. Glucose-6-phosphatase 효소조직화학표본 소견은 methotrexate투여 8시간 후 희생시킨 예부터 뚜렷한 glucose-6-phosphatase활성변화가 나타나기 시작하여 점차 감소되다가

72시간 후 희생시킨 예에서는 효소의 활성이 약간 회복되는 양상을 나타내었으며 phenobarbital전처치한 후 methotrexate투여한 동물에서 8시간 후부터 효소의 활성이 감소되어 phenobarbital단독투여군보다 더욱 심한 감소를 보였으며 72시간 후 희생시킨 동물에서도 효소활성이 회복되지 않았다.

4. Oil red O염색소견은 모든 실험동물에서 세포내에 염색되는 지방과립을 관찰할 수 없었다.

이상의 결과로 보아 methotrexate에 의한 간독성은 glucose-6-phosphatase를 이용한 효소조직화학방법으로 더 조기에 검출할 수 있을 것으로 사료되며 phenobarbital로 전처치한 경우 효소조직화학적 방법으로는 methotrexate투여군에 비해 더 심한 변화를 나타냄을

관찰할 수 있었다.

[영문]

This experiment was attempted to observe the toxic effects of administrations of methotrexate alone and phenobarbital prior to methotrexate treatment on liver by demonstrating the histological changes and glucose-6-phosphatase activity.

70 male and female albino rats (Sprague-Dawley strain) weighing around 200 gram were used for this study and divided into the following four groups.

Group Ⅰ; treated with saline

Group Ⅱ: treated with phenobarbital alone

Group Ⅲ: treated with methotrexate alone

The saline was injected intraperitoneally in single dose of 1.0 ml per kg, phenobarbital in two doses of 50 mg per kg at intervals of 24 hours, and methotrexate in single dose of 3.Omg per kg.

Group Ⅳ; pretreated with phenobarbital prior to methotrexate

Phenobarbital was given intraperitoneally in two doses of 50 mg per kg at intervals of 24 hours and methotrexate in single dose of 3 mg per kg at 24 hours after second injection of phenobarbital.

The animals of each group were killed at 4, 8, 16, 24, and 72 hours after last injection of drug. Overall histological changes of liver were observed by routine hematoxlin-eosine staining technique, the degree of glycogen accumulation by PAS staining, the patter of fatty change by oil red O staining. A modified method of Wachstein-Meisel (Wachstein and Meisel, 1956) was applied for the demonstration of glucose-6-phosphatase activity.

From this study, the following results were obtained.

1. In both groups treated with methotrexate alone and pretreated with phenobarbital prior to methotrexate, some cell swellings appeared after 16 hours but cell necrosis or infilteration of inflammatory cell could not be found.

2. Accumulation of glycogen in cytoplasm have become to be reduced weakly after 16 hours in both groups treated with methotrexate alone and pretreated with phenobarbital.

3. Glucose-6-phosphatase activity in animals treated with methotrexate alone started to decrease markedly after 8 hours and recovered at 72 hours. The progressive decrease of enzymatic activity in phenobarbital pretreated group was more significant than that in methotrexate alone treated group.

4. No fatty granule was found in both control and experimental groups. Above results showed that treatment with phenobarbital prior to methotrexate treatment induced histochemical changes in liver more severly than treatment of methotrexate alone. In addition, it could be suggested that early detection of hepatotoxicity induced by methotrexate can be accomplished more by using the histochemical method for glucose-6-phosphatase than general histological method.