Aflatoxin B1 및 G1이 백서 간에 미치는 영향에 관한 형태학적 및 자기방사법적 연구

Abstract

[한글]

[영문]

The aflatoxins are the metabolites of certain strains of aspergillus flavus that grows on ground nuts and on foodstuff (Sakai and Uraguchi, 1955; Borker, 1966; Hartley et al., 1963; Barnes, 1967; Smith and Mckernan, 1962), and are proved to be

toxic to several animals (Newberne et al., 1964; Allcroft and Carnaghan, 1962; Asao et al., 1963) have purified the crude aflatoxine B^^1, B^^2, G^^1 and G^^2 and found that their purified compounds are closely related each other in terms of

their structures and toxic effects on the cell metabolism of several animals.

It has been demonstrated that administration of aflatoxins to a variety of animals result in an inhibition of protein synthesis due to the effect on the RNA dependent polymerase, which took place rapidly in a matter of a few hours (clifford et al., 1967; Rogers and Newberne, 1967). Although they have structural similarity with each other, namely the only difference between B^^1 and G^^1 is that the dihydrofuran ring of the former is replaced by a lactone ring it was reported that LD 50 of G^^1 was three times higher than of B^^1 to 1 day old ducklings (Asao et

al., 1963). However, G^^1 is also carcinogenic when administered to the rats in tern(Wogan, 1968; Butler et al., 1969). It is also true as regard to a necrotizing dose of aflatoxin in case of human liver cell culture system (Zuckerman et al., 1967).

The purpose of present investigation is to determine the toxicity of aflatoxin G^^1 versus B^^1 by comparing the ultrastructural changes in the rat liver and also by comparing their degree of cellular incorporation of G^^1 and B^^1 in the liver cells as determined by autoradiographic technics.

Material and Methods

Male rats weighing about 200-220gm were used for the experiment. Aflatoxin B^^1 and G^^1 were dissolved in Dimethylformamide (DMF) and the tritium labelling was achieved by the method of Dr. W. Lizinsky (1966).

Experimental Schedule

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Group No. of animals Compounds given Dose

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Group Ⅰ 21 Dimethylformamide 0.15ml DMF containing H**3 650μc.

Group Ⅱ 21 Aflatoxin B^^1 0.87mg AFT-B^61 containing H**3

650μc dissolved in 0.15 ml DMF.

Group Ⅲ 21 Aflatoxin G^^1 0.87mg AFT-G^^1 containing H**3

650μc dissolved in 0.15 ml DMF.

Group Ⅳ 21 Aflatoxin G^^1 2.61mg AFT-G^^1 containing H**3

650μc dissolved in 0.15 ml DMF.

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Animals were sacrified at time interval of 1 hour, 6 hours, 18 hours, 24 hours, 7 days, 4 weeks and 8 weeks after a single administration of the aflatoxin by subcutaneous injection.

Histology and Electron Microscopy

The tissues were fixed in neutral buffered formalin and sections were prepared by standard paraffin embedding procedure and stained routinely with Hematoxylin and eosin, Periodic acd Schiff(PAS),, Methyl green pyronin and Feulgen reaction were

also applied.

For electron microscopy, the animals were killed by a blow on the head and bled immediately by severing the jugular vein. Specimen of the liver were taken from the median lobe and fixed for 2 hours at 4℃ in one percent osmium tetraoxide in veronal buffer with pH 7.4 (Palade, 1952). All tissues were dehydreated in graded alcohol and embedded in Epon 812 according to standard procedures (Luft, 1961).

Thin sections were cut with LKB Ultratome, then stained with uranyl acetate (Watson, 1958) and lead citrate (Millonig, 1961) and observed with the Hitach HU 11-E Electron Microscope.

Autoradiographic studies by light microscope

The tissue was embedded in paraffin by standard procedures, and cut in 2-4μ thickness. The sections were deparaffinized by immersing in xylene, and rinsed with distilled water. The slides were then coated with a nuclear emulsion Kodak NTB-2 and stored in a refrigerator at 4℃ for 30 days. The sectiones were developed in Kodak Dektol which diluted 1:2 with water at 20℃ fixd in Kodac acid fixer, and finally washed in running water. After drying the slides were stained by hematoxylin and eosin (Leblond, 1965), and examined under conventional light microscope.

Autoradiographic studies by electron microscope

Preparations were made by the method of Caro and Van Tubergen (1962). This involves direct application of a loop of L-4 nuclear sensitive emulsion to mount sections. The exposure period was for 100 days. It was developed by physical developer, followed by lead citrate staining. Routine observation was with Electron

Microscope.

Result and Discussions

The results showed that administration of aflatoxin B^^1 induced a marked nucleolar alteration from 6 hours after the injection. No significant nucleolar alteration were noted in animals treated with smaller amount of aflatoxin G^^1, but

injection of larger amount of aflatoxin G^^1 induced nucleolar alteration similar to aflaoxin B^^1 treatment. The nucleolar change was characterized by segregation of granular and fibrillar elements.

All three group showed cytoplasmic changes such as dilation of rough endoplasmic reticulum with detached ribosome, hyperplasia of smooth endoplasmic reticulum, increased number of lipid droplets and microbodies. But the mitochondrial alterationin aflatoxin G^^1 groups was more prominent than in aflatoxin B^^1 group.

Autoradiographic studies have shown the incorporation of both aflatoxin B^^1 and G^^1 on parenchymal cells, but afltoxin B^^1 incorporated more intensely than G^^1 groups, especially in the nucleus.

Autoradiographic findings on electron microscopy showed a marked difference of the size of grains between aflatoxin B^^1 and G^^1 groups, namely G^^1 being larger than B^^1. However, it was not certain that this difference in size of grains is related to the storage time of aflatoxin in liver cells.

In summary, the data obtained by present experiments indicate that both aflatoxin B^^1 and G^^1 exert toxic effect upon rat liver cells. However, the degree of toxic effect of aflatoxin B^^1 is much stronger than G^^1, as judged by nucleolar segregation, and capacity of nuclear incorporation.