Abstract

The aflatoxins are the metabolites of certain strains of aspergillus flavus that grows on ground nuts and on foodstuff(Sakai and Uraguchi, 1955; Vorker, 1966; Hatley et al., 1963; Barnes, 1967; Smith and Makerana, 1962), and are proved to be toxic to seceral animals (Newberne et al., 1964; Allcroft and Canaghan, 1962; Asao et al., 1963) have purified the crude aflatoxine B₁, B₂, G₁ and G₂ 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 B₁, G₁ is that the dihydrofuran ring of the former is replaced by a lactone ring it was reported that LD 50 of G₁ was three times higher than of B₁ to 1 day old ducklings (Asao et al., 1963). However, G₁ 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 G₁ versus B₁ by comparing the ultrastructural changes in the rat liver and also by comparing their degree of cellular incorporation of B₁ and G₁ in the liver cells as determined by autoradiographic technics. Material and Methods: Male rats weighing about 200-220gm were used for the experiment. Aflatoxin B₁ and G₁ were dissolved in Dimethylformamide(DMF) and the tritium labelling was achieved by the method of Dr. W. Lisinsky(1966). Animals were sacrified at time interval of 1 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 and 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°C in one percent osmium tetraoxide in veronal buffer with pH 7.4(Palade, 1952). All tissues were dehydrated 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 (Milloning, 1961) and observed with the Hitach HU II-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°C for 30 days. The sectiones were developed in Kodak Dektol which diluted 1 : 2 with water at 20°C fixed 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 coventional 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 B₁ 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 G₁ but injection of larger amount of aflatoxin G₁ induced nucleolar alteration similar to afaoxin B₁ treatment. The nucleolar change was characterized by segregation of granular and fibrillar elements. All three group showed cytoplasmic changes such as dilation of rough endolpasmic reiculum with detached ribosome, hyperplasia of smooth endoplasmic reticulum, increased number of lipid droplets and microbodies. But the mitochondial alteration in aflatoxin G₁ group was more prominent than in aflatoxin B₁ group. Autoradiographic studies have shown the incorporation of both aflatoxin B₁ and G₁ on parenchymal cells, but aflatoxin B₁ incorporated more intensely than G₁ groups, especially in the nucleus. Autoradiographic findings on electron microscopy showed a marked difference of the size of grains between aflatoxin B₁ and G₁ group, namely G₁being larger than B₁. 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 B₁ and G₁ exert toxic effect upon rat liver cell. However, the degree of toxic effect of aflatoxin B₁ is much stronger than G₁as judged by nucleolar segregation, and capacity of nuclear incorporation.