Study On Toxic Effect And Mechanism Of Cadmium In Rice On ICR Mice

Backgrounds:Cd is a persistent pollutant which is highly toxic and widely exists in environment and food.The highest total exposure in China is due to the high proportion of Cd in food from 90%.It is worth noting that the average contribution rate of rice is 58.6%.At present,the international research on Cd mainly focuses on the Cd pollution situation and its harm,and mainly focuses on the Cd of ionic states as the main research object.Cd in rice mainly binds to proteins in the form of complexes and has different toxicity from ionic Cd.Therefore,the difference between the toxicity of CdCl₂ and three different levels of Cd contaminated rice is the main purpose of the study.It provides more scientific basis for the determination of Cd limit index in food safety standards.Method:1.84 SPF grade male ICR mice were randomly divided into 7 groups,including Control group,Normal Rice group(Rice-N),physical mixing of normal Rice andCdCl₂solution(CdCl₂+Rice-N),CdCl₂ group,and experimental group(Rice with Cd concentration 0.1mg/kg,Rice-L group;Rice Cd concentration 0.4mg/kg,Rice-M group;Rice with Cd concentration 0.7mg/kg,Rice-H group).Mice were weighted,observed and recorded regularly,and they were passive euthanasia at the end of the 8^th week.The blood and various organs were collected,Cd accumulation in organs and blood was determined.Tissues were cut and stained with Hematoxylin and Eosin(H&E).The kit was used to mearsure the concentration of AST and ALT contents in the serum and liver were determined,determination of serum biochemical indicators(ALB,AST,ALT,CR,BUN,CK,GLU),and the concentration of MDA,T-SOD,CAT,GSH-PX and GSH in the liver tissues.2.mRNA sequencing was used to analyze the global gene regulation in the liver of infected mice.The differentially expressed genes(DEGs)in each group were compared,followed by GO(Gene Ontology)enrichment and KEGG pathway enrichment analysis,and some DEGs were screened for further study.3.According to the DEGs screened by mRNA sequencing results,real-time fluorescence quantitative PCR(RT-PCR)was used to detect AKT1,EGFR,MAP2K6,MAPK13,CD19,PIK3R6,PRKAA1 and e IF4E genes in MAPK and PI3K-Akt/mTOR signaling pathways in the liver of model mice from mRNA levels.The effects of CdCl₂and cadmium in rice on MAPK and pi3k-akt/mTOR signaling pathways in mouse liver were studied.Results:1.The influence on physiological and oxidation in mice by cadmium-contaminated riceThe BW(Body Weight)of the experimental mice showed an overall increasing trend,and BW of the mice gradually increased slowly as the time of infection increased.The high-dose group(CdCl₂ group,CdCl₂+Rice-N group,Rice-H group)showed slower action than the Control group and Rcie-N group.The long-term low dose of CdCl₂ had little effect on the viscera coefficient of mice.When rice matrix was present,the effect of CdCl₂ on the liver and kidney of mice was more significant than that of cadmium-contaminated rice.Cd was mainly distributed in the liver and kidney in mice,but less in the heart,spleen,lungs and blood.For Cd-Rice,When the concentration of Cd in rice increased,the accumulation of Cd in mouse tissue also increased.For CdCl₂+Rice-N group,Cd accumulation in liver tissues was significantly higher than that of Rice-H group(same Cd concentration with CdCl₂)(P<0.05),and the Cd accumulation in the kidney tissues of the CdCl₂+Rice-N group was significantly lower than that of the Rice-H group(P<0.05).when the Cd concentration in Cd-contaminated Rice was 0.4mg/kg(Rice-M),there were significant effects on the liver and kidney compared with mice in the Rice-N group(P<0.05),with the increase of Cd concentration in rice,the effect on liver and kidney of mice increased.At the same cadmium concentration,the serum ALT content in the CdCl₂+Rice-N group was significantly higher than Rice-H group(P<0.05),there was no significant difference in the contents of CR and BUN in the serum of mice in the Rice-H group and the CdCl₂+Rice-N group(P>0.05).Cd-Rice of 0.4mg/kg and 0.7mg/kg(Rice-M,Rice-H)could cause oxidative damage to the liver of mice,and the degree of damage was increased with the increase of Cd concentration in Cd-Rice.CAT activity in the liver of mice in the CdCl₂+Rice-N group was significantly lower than Rice-H group(same Cd concentration)(P<0.01).2.mRNA sequencing results of Cd-Rice on mice liverAt the same concentration of Cd in CdCl₂ and Rice,the number of DEGs between CdCl₂ group andControl group,the number of DEGs between CdCl₂+Rice-N group and Rice-N group was greater than that between Rice-H group and Rice-N group.The number of DEGs in the three rice groups with different Cd concentrations showed that Cd-Rice had no significant effect on the dose of DEGs in the liver of mice.The DEGs between CdCl₂ group andControl group,and the DEGs between CdCl₂+Rice-N and Rice-N group were mainly enriched by GO,mainly enriched entries related to lipids,redox and enzyme activities.The DEGs between Rice-H group(same Cd concentration)with Rice-N group enriched the entries of molecular metabolism correlation,enzyme activity correlation and neuromodulation correlation.For three kinds of Cd-Rice,the DEGs between Rice-L with Rice-N group were mainly enriched with entries related to molecular metabolism,ion transport and lipid enzyme activity.The DEGs between Rice-M with Rice-N group mainly enriched the entries related to enzyme reaction.KEGG analysis showed that both CdCl₂ andCd-Rice with the same Cd concentration could regulate the MAPK signaling pathway to cause liver damage in mice,while Cd-Rice could also regulate other signaling pathways such as PI3K-Akt.3.Preliminary study on the mechanism of liver injury in mice by the regulation of MAPK and PI3K/Akt/mTOR pathway by Cd-Rice.The up-regulation and down-regulation degree of genes in MAPK and PI3K-Akt/mTOR signaling pathway were significantly correlated with the concentration of Cd in rice in a dose-effect manner,the higher the Cd concentration in rice,the greater the up-regulation or down-regulation degree.In MAPK signaling pathway,for CdCl₂ andCd-Rice(same Cd concentration),there was no significant difference in the expression of AKT1 in the liver of mice in the CdCl₂+Rice-N group and Rice-H group compared with the Rice-N group(P>0.05),CdCl₂ caused extremely significant down-regulated expression of AKT1 in mouse liver(P<0.001);EGFR in CdCl₂ group andCdCl₂+Rice-N was significantly down-regulated(P<0.01),while Rice-H group was significantly up-regulated on the contrary(P<0.001);MAP2K6 was significantly down-regulated in CdCl₂ group,CdCl₂+Rice-N group and Rice-H group,and the down-regulated multiple of MAP2K6 in CdCl₂+Rice-N group was significantly higher than that in Rice-H group(P<0.05);MAPK13 was significantly up-regulated in CdCl₂ group,CdCl₂+Rice-N group and Rice-H group,and the up-regulated multiple of MAPK13 in mouse liver of CdCl₂+Rice-N group was significantly higher than that of Rice-H group(P<0.05).In PI3K-Akt/mTOR signaling pathway,for CdCl₂ andCd-Rice(same Cd concentration),the down-regulation of CD19 in mouse liver in Rice-H group was higher than CdCl₂+Rice-N group(P<0.05).The expression of PIK3R6 in the liver of Rice-H group was significantly up-regulated(P<0.01),there was no significant PIK3R6 gene expression in mouse liver in the CdCl₂+Rice-N group compared with the Rice-N group and the CdCl₂ group compared with the Control group(P>0.05);The significantly down-regulated multiple of PRKAA1 in CdCl₂+Rice-N group was significantly higher than Rice-H group(P<0.05);EFI4E expression in the liver of mice in CdCl₂+Rice-N group and Rice-H group was significantly up-regulated(P<0.05),the upregulation multiple of e FI4E in the liver of Rice-H group was significantly higher than CdCl₂+Rice-N group(P<0.05),there was no significant difference in the gene expression between CdCl₂ group andControl group(P>0.05).Conclusion:When the concentration of Cd in rice increases,the accumulation of Cd in rice in mice increases,the damage to the liver and kidneys increases,and the degree of oxidative damage to the liver increases.CdCl₂ andCd-Rice with the same Cd concentration have different Cd accumulation of in vivo,Cd in Rice was more likely to accumulate in the kidney while CdCl₂ was more likely to accumulate in the liver.CdCl₂ caused more damage and oxidative damage to mouse liver tissue than Cd in rice.Compared with Cd-Rice with the same Cd concentration,the influence of Cd in CdCl₂ on mouse liver was more extensive than that in Cd-Rice.Both of them could affect the enzyme activity in the process of mouse liver toxicity injury,and they have their own regulatory mechanisms,but there was an association between the toxic mechanisms in the liver of mice.When CdCl₂ andCd-Rice of the same Cd concentration regulate MAPK signal transduction pathway,CdCl₂ inhibits the secretion of cytokines and promotes the apoptosis of liver cells to a higher degree than that of Cd-Rice,thus reducing the body's response to injury.Cd in rice is stronger than CdCl₂ in weakening the response to injury and promoting cell transformation in the PI3K-Akt/mTOR signal transduction pathway,thereby increasing the possibility of cell cancer.