Study On The Mechanisms Of Oxidative Damage And Potential Carcinogenicity Of BRL-3A Rat Hepatocytes Induced By Acrylamide Based On MRNA Sequencing

Acrylamide(AA)is a food contaminant that is certified by the International Agency for Cancer as a potential human carcinogen in Category 2A,which is detectable in both commercial processing and home cooking foods.AA can cause oxidative damage in experimental animals and eventually induce cancer.At present,the neurotoxicity,genotoxicity,reproductive toxicity and carcinogenicity of AA have been widely reported.However,the mechanism of liver cell injury remains unclear.In this paper,mRNA sequencing technology was used to study the molecular mechanism of oxidative damage and potential carcinogenicity of AA on BRL-3A rat hepatocytes,and the transcriptomic level regulation of glycidamide(GA),the epoxidation metabolite of AA,against BRL-3A rat hepatocytes was preliminary explored.The specific content of the thesis is as follows:(1)We evaluated the changes in the apparent damage indexes of rat liver cells induced by AA in BRL-3A.Oxidative damage factors SOD,GST and GSH-Px,DNA damage factors ROS and 8-OHdG,immune-related cytokines IL-6,TNF-? and IL-10 activities and levels in BRL-3A cells treated with AA(250-2000 ?M)were examined by enzyme-linked immunosorbent assay.The results showed that AA could cause significant oxidative damages by decreasing the activity of SOD,GST,and GSH-Px in BRL-3A hepatocytes.And it increased ROS,8-OHdG contents in BRL-3A cells to cause oxidative damage to DNA;at the same time,it promoted the secretion of hepatocyte pro-inflammatory cytokines TNF-? and IL-6 in BRL-3A rat liver cells and inhibit the secretion of IL-10,an anti-inflammatory cytokine,and caused inflammatory reactions in hepatocytes.(2)To further examine the mechanism of AA-induced oxidative damage and potential carcinogenicity of BRL-3A rat hepatocytes,we used mRNA sequencing technology to investigate the global gene regulation of BRL-3A rat hepatocytes after exposure to AA(2000 ?M).And preliminary investigations were conducted on the regulation of transcriptome levels of BRL-3A cells by GA.Compared with the control group,a total of 313 genes in the AA group were markedly differentially expressed(FC?2,Q<0.05),among which 115 genes were significantly upregulated and 198 genes were significantly downregulated.When compared with the control group,a total of 1202 genes were differentially expressed(FC?2,Q<0.05)in all genes with different expressions in GA group(875 ?M),among which 223 genes were significantly up-regulated and 979 genes were significantly down-regulated.At the same time,we conducted Gene Ontology(GO)enrichment analysis and KEGG enrichment analysis on genes that were significantly differently expressed.The results showed that in the AA-regulated differential genes,GO entries such as modified amino acid binding,regulation of synapse organization,and response to activity were enriched more,while in GA-regulated GO terms,many were enriched in GO items such as glutathione binding,cAMP binding and innervation.Furthermore,differential expression genes of BRL-3A rat hepatocytes regulated by AA and GA(p < 0.05,Q < 0.05,FC?2)were enriched most in global and overview maps and signal transduction KEGG pathways,including metabolism of xenobiotics by cytochrome P450,MAPK signaling pathway,NF-?B signaling pathway,TNF signaling pathway,ECM receptor interaction,etc.and oxidative stress,immune response,liver fibrosis related pathways.(3)Regulation of five genes related to important physiological functions such as cell proliferation,oxidative damage,DNA damage,and lipid metabolism in the Ras/MAPK signaling pathway induced by AA was analyzed via RT-PCR at the mRNA level.AA(250-2000 ?M)on BRL-3A rat liver cells resulted in the up-regulation of Fgf7,while the expression of pla2g4 a was significantly down-regulated.The same concentration of AA could cause the expression of Dusp5 and Gadd45 a upregulate significantly,and cause a high expression of Fos.Meanwhile,the up-regulation and down-regulation of the five target genes all showed a significant dose-effect relationship with AA.In summary,the evaluation of the apparent indicators confirmed the oxidative damage,DNA damage and inflammation induced by AA on BRL-3A rat hepatocytes.Moreover,the mechanism of oxidative damage and potential carcinogenic effects of AA on BRL-3A rat hepatocytes maybe closely related to the regulation of Ras/MAPK signaling pathway.AA may regulate the growth and proliferation of hepatocytes,affect its lipid metabolism pathway,trigger inflammatory reactions and cause organelle damage,DNA damage and excessive stress response,resulting in liver cell damage,and leading to liver cancer eventually.This thesis could provide a reliable theoretical basis for the use of natural botanic antioxidants to reduce the oxidative damage and carcinogenic effects of AA in food processing.