Mechanism Of Liver Injury In Aflatoxin B₁ Subacute Posioning Ducklings

Contamination of Aflatoxin B₁(AFB₁)is ubiquitous in feeds.Livestock consumption of the feed contaminated with AFB₁ can cause serious problems to their health and growth performance,and may pose great potential risks for livestock products.China is the world's largest duck production countries,accounting for about 80%of world production.Duckling is the most sensitive species to the toxic effects of AFB₁ among poultry.Elucidation of the toxicity mechanism of AFB₁ to duck is prerequisite to reduce the incidence of AFB₁ poisoning of ducks.Liver is both the main organ for AFB₁metabolism and biotransformation,and the target organ of AFB₁,and the liver damage of ducklings has typical pathological changes.Therefore,systematically screening the potential candidate genes that could be involved in the hepatotoxic mechanism of AFB₁and revealing the molecular mechanism of the hepatotoxicity of AFB₁ in ducklings can provide the scientific basis for development of methods used to prevent and/or reduce aflatoxicosis in ducklings.The present study intends to investigate the hepatotoxic effects of AFB₁ in ducklings.Then,RNA-seq method was adopted to systematically analyze the response of the hepatic transcriptome to AFB₁ exposure to ducklings.Furthermore,the differentially expressed genes,as well as the KEGG pathway and GO functions were also analyzed.According to the analysis,the crucial candidate genes that could be involved in the hepatotoxic mechanism of AFB₁ have been screened.Finally,gene overexpression and RNAi technologies were used to explore the functional and mechanism properties of the candidate genes involved aflatoxicosis using duckling primary hepatocytes.Part 1:We have established the AFB₁-induced liver injury model in ducklings according to the contaminated levels in duck production practice.24 8-day-old ducklings were divided into 4 treatment groups.Each group received an oral dose of AFB₁ at 0,10,20,40 mg/kg·BW per day for 2 weeks.At the end of the study,all birds of each treatment group were slaughtered to collect blood and for serologic and the hepatic antioxidant parameters determination,as well as the liver histological examinations.The main results are as follows:1.Compared to the control,the final BW of the ducklings was decreased(P<0.05)8.6 and 18.9%when AFB₁ was administrated at the doses of 20 and 40?g/kg·BW,respectively.The same trend was also found for 20 and 40 mg/kg·BW AFB₁treatment group with a decrease of 6.6 and 17.8%,respectively.Growth performance was not significantly affected by oral administration of AFB₁ at the dose of 10?g/kg·BW for 2weeks.2.Compared to the control,AFB₁ administration led to significantly increased(P<0.05)activities of ALT(35.3%and 73.9%)and AST(28.8 and 137.3%),along with decreased(P<0.05)contents of TP(20.8 and 55.6%)and ALB(27.8 and 67.5%)in the serum of ducklings at the dose of 20 and 40?g/kg·BW,respectively.Serum biochemistry was not significantly affected by oral administration of AFB₁ at the dose of 10mg/kg·BW.3.Compared to the control,AFB₁ administration led to decreased(P<0.05)activities of GPX(17.5%and 34.5%),SOD(12.7 and 18.1%),CAT(17.1 and 31.4%),and concentration of GSH(30.9%and 43.4%),along with increased(P<0.05)contents of MDA(160.0%and 251.4%)in the serum of ducklings at the dose of 20 and 40?g/kg·BW,respectively.AFB₁ administration at the dose of 10?g/kg·BW significantly increased(P<0.05)the hepatic content of MDA(48.6%),while no significant effect on other hepatic antioxidant indexes was observed.4.Different concentrations of AFB₁ treatment led to pathological changes in the liver of ducklings.Compared with the control group,10 mg/kg.BW administration led to slight bile duct epithelial hyperplasia,larger cytoplasmic vacuoles in hepatocytes.20 mg/kg.BW administration caused partial necrosis of hepatocytes,bile duct epithelial cell obviously proliferation in the portal area,and with increased and concentrated intracellular lipid droplets in hepatocytes.40 mg/kg.BW administration led to massive hepatocytes necrosis,bile duct epithelial cell hyperplasia which occupied about half of the liver tissue,along with decreased lipid content in hepatocytes.Part 2:Based on the duckling toxicological experiment,the liver samples from control(0?g/kg·BW)and AFB₁(40?g/kg·BW)were chosen and RNA-seq technology was used to systematically analyze response of the hepatic transcriptome to AFB₁exposure to ducklings.The main results are as follows:1.Two RNA-Seq libraries were created from pooled samples and a total of 14.9 Gb data of sequence were produced.After filtered adapters,trimmed ambiguous and low-quality reads,60,723,216 and 73,435,290 high-quality and clean reads were obtained,accounting for 90.4%and 89.7%of total raw reads respectively.After the assembled transcripts were functionally annotated by searching against the NCBI NR and Swiss-Prot protein database,38,110 transcripts were aligned to the database,which accounting for39.3%of all the assembled transcripts.2.We found 749 transcripts showing 2-fold or greater differentially expressions between the control and AFB₁treatment.Specifically,compared to the control,512transcripts were up-regulated while 237 transcripts were down-regulated in AFB₁treatment.3.The GO analysis results showed that 391 differentially expressed genes were associated with biological process,cellular compound and molecular functions.Within the biological process category,the most abundant groups included metabolic process,oxidation-reduction process,and immune response,as well as other appealing groups,which included negative regulation of apoptotic process and fatty acid metabolic process.Under the cellular components category,the mitochondrion,nucleus,and extracellular region were the most highly represented GO terms.In the category of molecular function,catalytic activity,oxidoreductase activity and heme binding were the most enriched.4.The KEGG pathway analysis results showed that 293 differentially expressed genes were fell into the pathways.The most abundant groups were metabolic pathways,biosynthesis of secondary metabolites,and glutathione metabolism,along with other appealing groups included fatty acid metabolism,chemical carcinogenesis and metabolism of xenobiotics by cytochrome P450 pathways.5.Compared to the control,40?g/kg·BW AFB₁ administration enhanced(P<0.05)mRNA levels of phase I metabolizing enzymes CYP46A1,CYP3A9-like and CYP4B1-like,along with decreased(P<0.05)CYP2H1,CYP1A5,CYP27A1,CYP2K1-like and CYP2F3-like.Meanwhile,AFB₁ administration also upregulated(P<0.05)mRNA levels of phase II detoxification enzymes GSTA1,GSTA3 and GSTK1.6.The mRNA expression levels of 6 differential genes(CYP1A5,CYP2H1,GST3,Fasn,MT and MDM2)were measured by q-PCR to verify the accuracy of the RNA-seq dtermination.The results showed that hepatic mRNA levels of CYP1A5,CYP2H1,Fasn and MT were decreased(P<0.05),along with increased(P<0.05)GST3 and MDM2 by AFB₁ treatment,respectively,which were in accordance with the RNA-Seq results and indicated that the RNA-Seq results were reliable.MT and MDM2 possibly play important role in the liver injury of ducklings induced by AFB₁.Part 3:Based on the sequencing results and reference,we hypothesize that metallothionein(MT)and ubiquitin protein ligase E3(MDM2)might play crucial roles in AFB₁-induced hepatotoxity.Gene overexpression and Si RNA technologies were used in duckling primary hepatocytes to explore the functional and mechanism properties of MT and MDM2 involved in sub-acute aflatoxicosis of ducklings.The main results are as follows:1.The primary hepatocytes of ducklings treated with 25,50,75,100 and 125 ng/m L AFB₁ for 24 h,resulted in its viabilities decreased(P<0.05)14.2%,29.8%,47.9%,67.3%and 70.8%,respectively,by MTT assay.The IC₃₀of AFB₁(50 ng/m L)was used to perform gene function validation analysis.2.Overexpression of MT and knockdown of MDM2 genes expression in primary hepatocytes of ducklings was established.Compared to the control,the expression of mRNA and protein of MT in ducklings primary hepatocytes were increased(P<0.05)by56-fold and 1.5-fold,respectively.The expression of mRNA and protein of MDM2 in primary hepatocytes of ducklings were decreased(P<0.05)by 42.7%and 50.0%,respectively.3.The effect of MT and MDM2 in AFB₁-induced hepatotoxicity was explored.Compared to the control,the cell viability of primary hepatocytes was decreased(P<0.05)30%after AFB₁treatment for 24 h.AFB₁ treatment also increased(P<0.05)the apoptotic rate of primary hepatocytes.Interestingly,overexpression of MT in hepatocytes prevented and/or alleviated these alteration induced by AFB₁ treatment.Knockdown of MDM2 expression has no significant effect(P>0.05)on cell viability and apoptotic rate of primary hepatocytes compared to AFB₁ treatment group.4.Compared to the control,AFB₁ treatment led to decreased(P<0.05)activities of GPX(85.2%)and SOD(43.6%),as well as increased(P<0.05)concentrations of MDA(94.1%)and AFBO-DNA(43.5-folds).Notably,overexpression of MT in hepatocytes prevented and/or alleviated these changes induced by AFB₁ treatment.No significant differences(P>0.05)were found in GST activity among the 3 groups.5.Compared to the control,AFB₁ treatment led to decreased(P<0.05)mRNA levels of oxidation-protective genes PTGR,AR,Trx R,tumor suppressor p53,and antiapoptotic inner mitochondrial membrane protein Bcl-2,along with increased(P<0.05)mRNA level of apoptotic genes Caspase3.Strikingly,overexpression of MT in hepatocytes prevented and/or alleviated these changes induced by AFB₁ treatment.In summary,the study come to the following conclusions:Ducklings administrated with 20?g/kg·BW AFB₁ can cause liver subacute poisoning damage.AFB₁ induced abnormal expression of genes involved in I-phase enzyme metabolism,?-phase enzyme binding,redox process,carcinogenesis,apoptosis,cell cycle and fatty acid metabolism in the liver.The results also revealed that liver injury induced by AFB₁ was relevant to down-regulation of MT.