Effects Of Cadmium Exposure On Liver Metabolism And Epigenetic Patterns

Objective:Cadmium is a heavy metal that is ubiquitous in the environment.It can enter the human body through smoke and dust inhalation,dietary intake,and skin contact.Due to its long half-life and low excretion rate,it can accumulate in various organs such as the liver,kidney and reproductive system for a long time,which will have various adverse effects on health.The liver is a human detoxification organ,and it plays a great role in removing the toxicity of heavy metals in the body.Epidemiologists pointed out that urine cadmium,hair cadmium or blood cadmium levels are closely related to abnormal liver function.Although there is sufficient evidence that cadmium is toxic to liver,its biological mechanism is still unclear.At present,cadmium has been proven to interfere with gene expression and signal transduction,inhibit DNA damage repair,change cell apoptosis,and induce oxidative stress autophagy and carcinogenesis.Since cadmium has a weak binding force on DNA,the epigenetic pathway may be one of the main mechanisms of its toxicity.Therefore,this project conducts an in-depth discussion on the epigenetic modification effects of liver disease induced by cadmium,and analyzes the important role of DNA methylation in the occurrence of cadmium-induced liver disease.The whole-genome methylation detection and transcriptome sequencing was used to clarify the potential mechanism of chronic cadmium stress-induced liver disease.Methods:1.Rat subchronic cadmium exposure.48 healthy male SD rats were randomly divided into 6 groups,8 rats in each group,defined as different doses of cadmium exposure groups,respectively.0 mg/kg bw/day,2.5 mg/kg bw/day,5 mg/kg bw/day,10 mg/kg bw/day,20 mg/kg bw/day and 40 mg/kg bw/day were administered to rats daily by gavage After exposure to cadmium for 12 weeks,the body weight was recorded every week,and then the rats were anesthetized to death,the liver tissue and blood were obtained.ICP-MS was used to detect blood cadmium levels,automatic biochemical analyzer was used to detect serum liver function indicators,and HE staining was used to observe liver pathology.2.Whole-genome methylation analysis.In order to study the effects of low and high-dose cadmium exposure on rat liver,we selected the control group,low-dose group(5 mg/kg)and high-dose group(40 mg/kg)for Whole Genome Bisulfite Sequencing.After the DNA was extracted and purified,high-throughput sequencing was performed.Through identification of differentially methylated sites,annotation of differentially methylated regions and enrichment of differentially methylated genes,the role of methylation in cadmium hepatotoxicity could be explored.3.Transcription level analysis of DNA methylation events related to cadmium exposure.Tissue RNA was extracted for RNA-seq sequencing,and the differences in gene expression under two or more conditions were compared,and specific genes related to cadmium exposure were identified,and then comparative enrichment analysis was used to further analyze these specificities genetic biological significance.The rat normal liver cell line BRL-3A was cultured in DMEM medium with Cd Cl₂at final concentrations of 0,0.5?M,2?M,and 5?M.Cell RNA was extracted after 12weeks,and fluorescence real-time quantitative PCR was used to detect specific DNA methylation events.Results:1.Compared with the control group,low-dose cadmium intervention rat have no significant changes in body weight.When the dose exceeds 10 mg/kg bw/d,the growth of the rat was inhibited.At the same time,compared with the control group,AST began to appear statistically significant in the 5 mg/kg dose group,and ALT began to appear statistically significant in the 20 mg/kg dose group.After 12 weeks of intragastric administration of cadmium chloride,only hepatocyte eosinophilia and inflammatory cell infiltration occurred in the low-dose group.With the increase of the dose,liver lobule structure disorder and connective tissue hyperplasia in the portal area gradually appeared.2.After 12 weeks of cadmium exposure,there was no significant difference in the overall methylation level of the genome among the groups.The effect of low-dose cadmium intervention on DNA methylation was less than that of the high-dose group,and there was a difference of 400 differential methylation regions between them.At the same time,after high-dose cadmium treatment,the differentially methylated regions are more concentrated in the promoter region.The enrichment analysis of GO and KEGG showed that differentially methylated genes were most enriched in the"metabolic pathway".3.Perform enrichment analysis was conducted on the differential genes of the transcriptome.Among the top 10 enriched metabolic pathways,6 pathways of them are related to lipid metabolism:"steroid hormone biosynthesis","retinol metabolism","steroid biosynthesis","linoleic acid metabolism","fatty acids degradation","arachidonic acid metabolism".The Protein-protein Interaction analysis of differential genes shows that Cyp1a1 was in the central region,which is also closely related to lipid metabolism.Verifications were made for Adh4,Adh6,Acat2 in the fatty acid degradation metabolic pathway,and Cyp1a1,Cyp2b2,Pla2g2d,Cyp2j4,Cyp2c6v1and Cyp2c22 in the arachidonic acid metabolic pathway.The expression patterns are consistent with the transcript data.Conclusions:1.The toxic effects of different doses of Cd exposure on the liver are heterogeneous.The exposure to cadmium slows down the growth and development of the body,and this effect was more pronounced in groups with high dose exposure.Through the detection of biochemical indicators to detect the effect of different concentrations of Cd Cl₂on the liver of rats,we found that AST and ALT changed significantly and had a concentration-dependent effect when the cadmium intervention concentration reached a certain threshold.Histopathology showed that low-dose cadmium exposure only manifested as inflammatory cell infiltration.With the increase of the dose,liver lobule structure disorder and connective tissue hyperplasia in the portal area gradually appeared.2.Cadmium exerts its toxic effects by changing the metabolic state of the liver,and the metabolic changes occur before the toxic phenotype.Through Whole Genome Bisulfite Sequencing,the most differentially methylated genes were concentrated in the"metabolic pathway".Through the liver tissue transcriptome sequencing analysis,"sterol hormone synthesis","cholesterol metabolism"and other lipid metabolism pathways were enriched.Which proved that changes in liver metabolism,especially lipid metabolism pathways,play an important role in cadmium toxicity.In intervention group with low exposure,the difference in lipid metabolism appeared to be particularly prominent,and there was no obvious organic liver disease,suggesting that lipid metabolism disorders may be a precursor to liver toxicity after cadmium intervention.