Aflatoxin B₁투여가 흰쥐 간조직의 LDH-Isozyme에 미치는 영향에 관한 연구
(A) study on tissue-specific alteration of lactic dehydrogenase isozymes of the rat liver in aflatoxin B₁intoxication
The discovery of aflatoxins in animal food-stuffs and the rapid development of knowledge of their potency as biologically active agents have given rise to be obvious and important questions concerning the risks to human health posed these or other food borne mold produced toxins.
Aflatoxicosis, in particular, has been demonstrated as hepatomas and hepatomal injuries in a small dose. For most species, the LD^^50 values of aflatoxin B^^1, the most potent group, is the ranger 0.5-10mg/kg body weight (Allcroft, 1965).
The precise mechanism of aflatoxicosis has not yet been determined, but acute experiment in several species have revealed the compounds are primarily hepatotoxins, with inhibitory effects similar to the action of actinomycin D.
(Garren, et al. 1963, Cs'anyi, et al. 1967, Trakatellis et al, 1964, Clifford, et al. 1966).
Studies on biochemical responses to aflatoxins by many investigators have resulted in inhibitions of DNA dependent RNA polymerase activities(Frieduman, et al. 1966 , Gelboin, et al. 1966, Clifford, et al. 1967, Moul, et al. 1968) and consequencetly, protein synthesis.
Based on present knowledge of the mechanisms of cellular biochemical action, these alterations would be expected to result also in changes in the electron transport system, such as cytochrom system, of carbohydrate metabolism.
And it might be anticipated that various dehydrogenases in the oxidation reduction system would be affected as well.
In particular, lactic dehydrogenase enzymes acting between aerobic (TCA cycle) and anaerobic carbohydrate metabolisms (EM path way) would be expected to be altered following aflatoxicosis.
It has reported the LDH isozyme patterns were changed in Hela cells.(Kine, et al. 1964, Richterich, and Burger, 1963).
The present investigation was therefore undertaken in order to know the alteration of lactic dehydrogenase isozymes in the liver of male rats by aflatoxin B^^1 and to determine the possibility to specify these alterations on cancer as a method of diagnosis.
Materials and Methods
Sixty healthy male albino rats weighing from 85 to 105 grams were used in the experiments and the rats were divided in 4 groups containing 15 animals.
Each experimental group was given the aflatoxin B^^1 in 1.0mg/kg weight, intraperitoneally once a days for 1 to 3 days.
After 1,2 and 3 days, each grop animals were killed by decapitation and livers were removed and preparated tissue homogenate 0.25M cold sucrose solution by all glass homogenizer in 4℃ cold room.
It was centrifuged at 600 xg for 15 min. to be free from nuclear fraction. The supernatant was again centrifuged at 12,500 xg for 30 min. to remove the mitochondrial fraction and was used for enzyme assays.
Each LDH isozyme of the tissue was separated by electrophoresis in cellulose acetate strip(Sepraphore Ⅲ, Gelman Co.) following method of Preston, et al. (1965).
H-and M-isozymes of LDH were differentiated with diethylaminoethyl-cellulose by the method of Bergmeyer, et al. (1963), and the activity was measured by Neiland method (1955), as the rate of reduction of NAD**+ when lactate to pyruvate in
content of protein.
The protein content was analyzed by the micro-Kjeldahl technique(Koch, et al. 1924).
The contents of DNA and RNA in the liver tissue were measured by Schmidt-Thanhauser method (1955) and Fiske SubbaRow method(1955).
Liver tissue from all experimental animals were observed by
electronmicroscophy(Hitachi Hu-11E, 75KV. 50μ objective aperture).
Results and Discussion
The experiment results on acute intoxication of rats by aflatoxins were as follows:
1. The M-type of LDH activity was dominant in the normal liver tissue.
2. A significant decrease of LDH activity induced by the animals receiving aflatoxin B^^1.
3. The LDH-H type was more inhibited than M-type in liver tissue following acute intoxication.
4. The protein content in the treated tissue was significantly decreased.
5. The content of DNA was markedly increased while the RNA was decreased in liver tissue of rats aflatoxin treated.
6. Histological patterns of the liver were markedly changed to destroy cristae in mitochondria by the treatment of aflatoxin B^^1.
As is apparent from the present data, the liver tissue examined showed liver LDH activities decreased and M type isozyme decreased also before the indication of clinical syndroms in the tissue.
It was suggested the aflatoxin interferes with glycolysis, first suppressing the aerobic glycoysis and gradually the more general carbohydrate metabolism pathways.
Based on present data and knowledge of mechanisms of gene transcription and translation, alteration in DNA dependent RNA synthesis would be expected to result in changes in protein metabolism and to inhibit the DNA dependent RNA polymerase in
vivo experiment. In the histological reviews, it would be shown that all dehydrogenase containing LDH in eneergy metabolism are inhibited by aflatoxins.
These alterations would be based in search of clinical findings of aflatoxicosis producing liver cancer.