Study Of Aflatoxin B₁ Invoked Oxidative Stress, And Apoptosis Via Death Receptor Pathway In Hepatocytes

The fungal metabolites produced by Aspergillus flavus and Aspergillus parasiticus cause detrimental health effects on humans and animals.Particularly aflatoxin B₁（AFB₁）is the most widely studied and a well-known global carcinogen,producing hepatotoxic,genotoxic and immunotoxic effects in multiple species.AFB₁ is shown to provoke liver dysfunction by causing hepatocytes apoptosis and disturbing cellular enzymatic activities.In liver,AFB₁ causes apoptosis via extrinsic mechanism because of high expression of death receptor pathway.The detailed mechanism of AFB₁ induced hepatocytes apoptosis,via death receptor pathway still remains elusive.Considering this fact,the present study was designed to explore the mechanisms of hepatocytes apoptosis initiated by death receptors by investigating anatomical,histological,ultrastructural,biochemical,and flow cytometrical changes along with mRNA relative expression analysis of relevant genes.One hundred and fifty-six one-day-old healthy Cobb broilers were purchased from Chia Tai Group（Wenjiang,Sichuan,China）,and were randomly divided into two equal groups of three replicates,each replicate contained 26 birds.The Group were named as control group,which was fed on basal diet（AFB₁<0.001 mg/kg）,and AFB₁ group,which was fed on AFB₁ contaminated diet（AFB₁ =0.601 mg/kg）.The experiments were conducted for 21 days and results from the present study were as follows:Histopathologically,degenerative reversible lesions,such as slight to moderate hydropic degeneration and fatty vacuolar degeneration were observed,and proliferation of bile duct in portal area was seen in AFB₁ group[proliferation is often a kind of irreversible lesions].Moreover,irregular,fragmented and condensed nucleus,swollen mitochondria and swollen endoplasmic reticulum in the hepatocytes of chicken in AFB₁ group were also found when observed with electron microscope.The activities of CAT（Catalase）,GSH-Px（Glutathione Peroxidase）,SOD（Superoxide Dismutase）and Hydroxyl free radical scavenging were observed significantly low（p<0.05 or p<0.01）,while the contents of MDA（Malondialdehyde）were observed significantly high（p<0.05 or p<0.01）and GSH（glutathione）contents were seen low（p<0.05 or p<0.01）in AFB₁ group than those in control group.Flow cytometry analysis revealed a significantly higher percentage of apoptotic cells（p<0.05 or p<0.01）,while qPCR analysis showed significant up-regulation of death receptors Fas,TNFR1 and associated genes such as FADD,TRADD,TRAF2,Caspase 10,Caspase 8,Caspase 9 and Caspase 3 and down regulation of inhibitory apoptotic proteins XIAP and Bcl-2 in AFB₁ intoxicated birds.In summary,the results obtained from this novel and comprehensive study showed that dietary exposure to AFB₁ could increase apoptosis in chicken hepatocytes by inducing histopathological,ultrastructural,biochemical changes,and by initiating over expression of death receptor and associated genes.Moreover present study will facilitate better understanding of mechanisms and involvement of death receptor pathway in hepatocytes apoptosis induced by AFB₁ and ultimately may be helpful in bringing down the toxigenic potential of AFB₁.