Study On The In Vitro Bioavailability Of Cadmium In Rice And Its Toxicity Mechanism To HL-7702 Cells

Backgrounds:Cadmium is a strong toxic heavy metal pollutant.In recent years,cultivated land in main regions of China has been polluted by cadmium,most of which are used for rice cultivation,leading to an increasingly serious problem of cadmium-contaminated rice.The traditional health risk assessment of cadmium in rice is usually based on the total amount of cadmium in rice and the toxicity of ionic cadmium.However,taking the bioavailability of cadmium and different toxicity in different forms of cadmium in account,the current evaluation methods often cannot truly reflect the health risk caused by cadmium exposure in rice.Therefore,in this study,rice with different degrees of cadmium contamination in a relatively large area of the country were collected firstly.Then the in vitro bioavailability of cadmium in rice was studied,and the HL-7702 cells were taken as the poisoning object to study the toxic effect and mechanism of cadmium in rice on liver cells finally.Through m RNA sequencing,the toxicity and mechanism of Cd-liver poisoning in rice were determined more accurately,so as to provide a reference for providing a theoretical basis for the formulation of Cd limit standard in grain.Methods:1.In vitro digestion simulation model-RIVM method combined with Caco-2 cell monolayer model was used.The apparent structure,the tightness,polarity and permeability of the cell monolayer was verified by performing the immunohistochemistry on the tight junction protein ZO-1 between Caco-2 cells,and measuring functional indicators of cells.After the establishment of the model,in vitro absorption and transport experiments were carried out to study the in vitro bioavailability of cadmium in rice with different pollution levels.2.Lower chamber fluid after transport in the monolayer model of Caco-2 cells was collected to simulate cadmium from rice and entering human blood circulation.HL-7702cells were used as the object of study and infected,untreated HL-7702 cells were used as blank control and cadmium chloride treated HL-7702 cells were used as positive control.Then m RNA sequencing technology was used to analyze the overall gene regulation level of HL-7702 cells.The key pathways and DEGs were screened for subsequent analysis eventually.3.According to the m RNA sequencing results,the key DEGs were screened under these conditions:related to cytotoxicity,with good enrichment and great difference in expression levels.RNA of infected HL-7702 cells was extracted and analyzed by real-time fluorescent quantitative PCR(RT-PCR)at m RNA level to study the cytotoxic mechanism of cadmium-contaminated rice on liver.Results:1.In vitro bioavailability of cadmium in riceA total of 102 kinds of rice with different cadmium contents were collected in this experiment.On the whole,samples containing less than 0.2mg/kg of cadmium accounted for 54.9%,those containing 0.2?0.4mg/kg of cadmium accounted for 27.5%,and those containing more than 0.4mg/kg of cadmium accounted for 17.6%.The cadmium content in rice ranged from 0.0123-2.4710mg/kg,with an average sample content of 0.2840mg/kg and a median of 0.1771mg/kg.The in vitro bioavailability of 96 rice samples was analyzed and tested,the median bioavailability of cadmium in rice samples of three ranges were 21.1%,25.5%and 28.74%,while the mean values were 23.22%,24.41%and 25.58%,respectively.The in vitro bioavailability of cadmium in rice ranged from 7.47-39.73%,with an average level of23.94%±6.91%and a median of 22.67%.The in vitro bioavailability of rice in the highest Cd concentration group was significantly higher than that in the lowest group(P<0.05).It was found that within a certain range(cadmium content was 0.05-0.4549mg/kg),there was a significant positive correlation between the cadmium content in rice and its bioavailability(correlation coefficient r=0.4289(P<0.0001).At the same time,the health risk assessment results of rice showed that the THQ value(target risk quotient value)of rice ingested without considering in vitro bioavailability was much higher than the THQ value after considering that,indicating that the traditional risk assessment methods did overestimate the potential harm of pollutants to a certain extent.2.m RNA sequencing results of Cd-Rice on HL7702 cellsAfter analysis,m RNA sequencing results showed that,compared with the Control group at different doses,with the increase of cadmium concentration,the number of DEGs in Rice-Cd group and Cd Cl₂ group increased significantly,too.At the same dose,the number of DEGs in the Rice-Cd group was higher than that in the Cd Cl₂ group,and the DEGs were mainly enriched in the signaling pathways related to the changes of cell cycle and intracellular matrix,such as the Hippo pathway and the p53 pathway,while in the Cd Cl₂group,the DEGs were mainly enriched in the signaling pathways related to the ability of cells to absorb metal ions,such as the MAPK pathway.3.Study on cytotoxic injury mechanism of HL-7702 induced by Cd in riceThe RT-PCR results showed that compared with the Control group,CCND3,BBC3,TP73,WNT1,and AXIN2 gene expressions were significantly down-regulated in the low-dose rice-Cd group(p<0.05),CCND3,WNT1 and AXIN2 genes were significantly down-regulated in the high-dose rice-Cd group(p<0.05),while there was no significant change in the cadmium chloride group.Combined with the functional mapping of DEGs,the toxicity of cadmium in rice to liver cells at the concentration of 0.5?M was related to the decrease in the signal feedback of p53 signaling pathway in HL-7702.When the concentration of Cd was 0.5?M and 1?M,Cd in rice affected the expression of the WNT1 gene in p53 and Hippo signaling pathway at the same time,and led to the down-regulation of the expression of anti-apoptosis genes AXIN2 and CCND3 eventually.There was no corresponding change in the cadmium chloride group,indicating that the ability of cadmium in rice to regulate apoptosis related genes was higher than that of cadmium chloride at the two doses selected in the experiment.Conclusion:Within a certain content range,the bioavailability of cadmium in rice was positively correlated with its content,and the THQ values of rice without bioavailability was significantly higher than that with bioavailability considered.The results of m RNA sequencing showed that cadmium in rice and cadmium chloride had the same characteristics in the process of liver cytotoxic injury:both changed the expression of genes related to inflammatory factors,and both affected some pathways related to cell metabolism.The difference is that the toxicity caused by cadmium in rice tends to change the cell cycle or induce cell apoptosis,while cadmium chloride tends to change the cell absorption of metal ions.After verification by RT-PCR,it was found that one of the mechanisms by which cadmium in rice exerts toxic effects on human liver cells is through changing the expression of growth factor WNT1 gene,which eventually leads to down-regulation of the expressions of anti-apoptosis genes AXIN2 and CCND3,and finally leads to cell apoptosis.