Association Between Ribosomal DNA Copy Number Variation And Hexavalent Chromium Exposure

ObjectiveTo investigate the rDNA copy number of hexavalent-chromium-exposed workers and its influencing factors,and to further verifies the rDNA copy number and its variation mechanism in vitro studies.MethodsIn the population study,one hundred and twelve workers exposed to hexavalent chromium in an electroplating plant in Zhejiang Province were selected as the exposure group.Sixty-eight people without hexavalent chromium exposure,and have not exposure to toxic or harmful chemicals or ionizing radiation in recent three months were selected as the control group.The general demographic information about the subjects was collected by self-made health questionnaire.The concentrations of heavy metals such as chromium were measured by inductively coupled plasma mass spectrometry（ICP-MS）after collected peripheral blood of the two groups.After extracting whole blood DNA,real-time fluorescence quantitative PCR（RT-qPCR）was used to detect the rDNA copy number（RCN）.Compare the difference of rDNA copy number between exposed and control population,and to analyze the correlation between rDNA copy number and work duration,blood chromium,etc.Multiple linear regression was used to analyze the influencing factors of the RCN.In vitro experiments,human lymphoblastic cell line（HMy2.CIR）and human normal pulmonary epithelial cells（BEAS-2B）were treated with 0-20 or 0-4μM K₂Cr₂O₇ for 24hours respectively.Cells in the timepoints of exposure for 24 hours,and repair for 3 days were collected,the RCN was detected after extraction of DNA.HMy2.CIR and human peripheral blood mononuclear cell（PBMC）were treated with 5μM K₂Cr₂O₇ for 24 hours.Cells in the timepoints of exposure for 24 hours,repair for 3 days,and 7 days were collected,and RCN was detected after extraction of DNA.HMy2.CIR were treated with 5μM K₂Cr₂O₇ for 24 hours.Cells in the timepoints of exposed for 24 hours,repair for 3 days,and 7 days were collected.After extracting the cells’total RNA,transcriptome sequencing was performed.The possible biological functions of differentially expressed mRNA were revealed by GO and KEGG enrichment analysis.Nucleolar proteins were screened by Genecard and NPD databases,and then the expression of nucleolar proteins was verified by RT-qPCR.The siRNA corresponding to nucleolar protein was transfected into HMy2.CIR by liposome transfection Treated the cell by 5μM K₂Cr₂O₇ at the same time,and the cells was detoxified after 24 hours exposure.Cells in the timepoints of exposure for 24 hours,and repair for 3 days were collected,and the RCN and the mRNA expression of nucleolar protein were detected.ResultsThe blood chromium median level of the exposure group was 1.26（0.04-4.53）ppb,which was higher than the control group（0.04-0.08）ppb（P<0.05）.The normalized copy numbers（NCNs）of 28S,18S,5.8S and 5S rDNA of the exposure group were 1.14（0.85-10.69）,1.49（1.13-15.51）,1.37（0.91-14.21）and 1.22（0.94-2.69）respectively,and were higher than controls（P<0.001）.Age（β=0.440,95%CI=0.170-0.711）,blood chromium level（β=1.385,95%CI=0.771-2.000）,and blood zinc level（β=-0.003,95%CI=-0.005--0.001）were included in the multiregression model for 45S rDNA copy number.The influencing factors of 5S rDNA copy number were age（β=0.075,95%CI=0.026-0.124）and blood chromium level（β=0.143,95%CI=0.037-0.249）.RCNs of 0-20μM Cr（Ⅵ）-exposed HMy2.CIR showed a dose-dependent increasing（P<0.05）.When BEAS-2B was exposed to 0-20μM Cr（Ⅵ）,the RCN did not change at 24hours,but increased in a dose-dependent model at 3 days after detoxication.RCNs of 5μM Cr（Ⅵ）-exposed PBMC showed a gradual upward trend（P<0.05）,while the RCNs of 5μM Cr（Ⅵ）-exposed HMy2.CIR increased firstly and then declined to normal level.The results of transcriptome sequencing showed that there were 135 differentially expressed genes at 24 hours after exposure,117 differentially expressed genes at 3 days after detoxification,and 113differentially expressed genes at 7 days after detoxification.The potential biological functions of these differentially expressed genes are G1/S phase transition of mitotic cell cycle,translation initiation,DNA damage response signal transduction by p53 class mediator,ribosome biosynthesis and so on.Fourty nucleolar proteins were selected from differentially expressed genes for validation.Compare with controls,except TRIM35,the mRNA expressions of selected nucleolar proteins were significant different in the exposure group more than one time point.We selected HRAS protein with high mRNA expression at 3 d after detoxification for further study.In HMy2.CIR cell line,mRNA expression of this nucleolar protein showed no significant different between exposure and control groups.At 3 d and 7 d after detoxification,the relative expression levels were 2.17±0.10 and 1.45±0.08,respectively,both were higher than the controls（P<0.01）.After the transfection of siRNA,the mRNA expression level of HRAS was 0.76±0.03,and was lower than controls（P<0.05）.The NCN values of 28S,18S,5.8S and 5S rDNA in the siHRAS group were 1.29±0.04,1.53±0.05,1.39±0.04 and 1.32±0.01,respectively,all higher than Mock group（P<0.05）.ConclusionsRCNs of blood DNA of Cr（Ⅵ）-exposed workers were higher than controls,and RCNs were positively correlated with blood chromium level.RCNs of Cr（Ⅵ）-exposed HMy2.CIR showed a dose-dependent increasing,but the variation of RCN of Cr（Ⅵ）-exposed BEAS-2B was not obvious.RCNs of Cr（Ⅵ）-exposed HMy2.CIR increased firstly and then declined to normal level.Cr（Ⅵ）exposure can induce differentially expressed genes in HMy2.CIR.The possible biological functions of these genes include DNA damage response mediated by P53 signal transduction and ribosome biosynthesis.Inhibition of mRNA expression of HRAS can induce rDNA copy number increase.