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Abstract
Summary
Aflatoxins are one of the most dangerous mycotoxins known, owing to their high toxicity to both animals and humans. It is the most potent hepatic mitogen and carcinogen.
The present study on aflatoxicosis in mice consisted of two main experiments, acute and chronic experimental modules.
In the acute experimenal module, the mechanism of hepatocellular injury and the histopathological alterations of the liver induced by single oral administration of AFB1 (3 mg/ kg b.wt) were studied. Fourty-two mice were used and sacrificed 1 hr, 6 hrs, 12 hrs, 24 hrs, 1 week, 2 weeks and 3 weeks post toxin administration.
In the chronic experimental module, the efficiency of a cocktail of antioxidants to improve or prevent the toxic effect of AFB1 when adminstred to mice in a low dose (50 ug/kg b.wt) and longer duration was tested. The hepatocacinogenic effects of AFB1 were also studied. Sixty-eight mice were used and sacrificed 1 month, 2 months, 3 months and four months post toxin administration.
The effect of AFB1 on animals in the acute experimental module was studied by histopathological, biochemical and molecular techniques. Liver from each mice in acute group and its control counterpart was sampled and preserved in neutral buffered formalin solution for histopathological examination and in glutraldehyde solution (5%) for semithin sections. Blood samples were cellected from each animal then plasma was used for biochemical analysis of plasma levels of LP and TAO. Frozen liver samples were used for molecular studies that included detection of DNA fragmentation analysis and expression of caspases by SDS-PAGE.
The histopathological results of liver from mice treated with AFB1 were expressed by vascular and hepatocelluler changes. The vascular changes were in the form of congestion of hepatic vasculature, thrombosis, haemorrhage and perivascular mononuclear cellular infiltration. The hepatocellular changes included degenerative and necrobiotic changes of liver cells that progressed to liver cell death either by necrosis and apoptosis. Proliferation of Kupffer cells, oval cells and bile duct epithelium were also seen.
Biochemical analysis revealed significant increase in the plasma LP and significant decrease in plasma TAO in AFB1 treated group. DNA fragmentation analysis showed characteristic laddering pattern of apoptosis. SDS-PAGE revealed expression of caspase-1 of molecular weight 45 kd and caspase-3 of molecular weight 32 kd.
The observed vascular changes may be attributed to the direct angiopathic effects of AFB1 on the hepatic vasculature. While, the toxic effects of AFB1 were related principally to binding of its metabolites to the macromolecules of the cells. AFB1 also blocked the formation of proteins essential for the integrity of cell membranes which played a role in the development of the observed necrobiotic and necrotic changes. The observed apoptosis may be due to direct binding AFB1 metabolite (8,9-epoxide) to the guanine bond to produce DNA adducts causing DNA strand breaks or indirect through oxidative stress and development of reactive oxygen species (ROS) and release of free radicals.
In the chronic experimental module, the effects of AFB1 and antioxidants on mice were studied by histopathological studies and biochemical analysis of plasma levels of LP and TAO. The histopathological examination of liver samples from animals treated with AFB1 only revealed vascular changes and hepatocelluler changes. The vascular changes were in the form of congestion, thrombosis, perivascular infiltration and sinusoidal macrophagal reaction.
The hepatocellular changes were expressed by necrobiotic changes, necrosis, apoptosis and preneaplastic changes of the liver. The preneoplastic changes of the liver included megelocytosis with dysplastic nuclei, preneoplastic liver nodules, Kupffer cell hyperplasia that progressed to Kuppfer cell sarcoma and bile duct hyperplasia and dysplasia.
The hepatocarcinogenic effect is due to formation of AFB-8,9 epoxide (AFBO). AFBO react with DNA to form adducts. These adducts formation are regarded as the primary and critical event in the AFB1 carcinogenesis.
Supplementation with antioxidants improved the histopathological picture of liver. Although, the vascular changes were persistent, the necrobiotic and necrotic changes of the liver were less prominent and had low incidence. The biochemical findings of the chronic experimental module revealed a significant increase of plasma LP products in chronic AFB1 treated group when compared with AFB1 and antioxidant treated group. In addition, the plasma level of TAO in the antioxidant group was significantly higher compared with other groups.
Antioxidants protected the mice against the development of preneoplastic changes of any cellular elements in the liver. This protective effect may be attributed to the inhibition of the binding effect of AFB1 to DNA in liver. Antioxidants also had antiapoptotic action that protected the cells from actions of reactive oxygen species by scavenging the free radicals and breaking the process of lipid peroxidation.
Histopathological changes of other organs (kidneys, lungs and heart) revealed vascular and cellular changes. The vascular changes were expressed by congestion of the blood vessels, perivascular mononuclear infiltration and haemorrhage. The cellular changes expressed by degenerative changes in kidney tissues.