Study On The Relationship Between Hexavalent Chromium Exposure And DNA Methylation Status At The Promoter Of P16Gene

ObjectiveThe aim of our study was to investigate the effects of hexavalent chromium on DNA methylation at the promoter of p16gene with population investigation and in vitro study. Role of DNA methylation in cell cycle arrest induced by Cr (VI) was investigated in two cell lines. Our research will provide evidence to study the toxicity mechanisms of hexavalent chromium.MethodsIn the population investigation,192cases of workers occupationally exposed to hexavalent chromium [Cr (VI)] were selected from an electroplating factory, and the80controls selected from support or management crew in different electroplating factories were not exposed to toxic and harmful chemicals and ionizing radiation in recent three months. Those two groups were well matched in terms of age, sex, smoking, and drinking history. Peripheral blood from subjects was collected and inductively coupled plasma mass spectrometry (ICP-MS) was used to detect total blood chromium content. Genomic DNA was extracted from whole blood for determination of DNA methylation status of p16gene, using a methylation-specific PCR (MSP) method. The8-hydroxydeoxyguanosine (8-OHdG), malondialdehyde (MDA), glutathione (GSH), and p16protein in plasma were evaluated with Enzyme Linked Immunosorbent Assay (ELISA). The exposed population was divided into long exposed group (>36months) and short exposed group (≤36months) according to the duration of exposure to hexavalent chromium. The diference of DNA methylation of p16gene between long exposed group and short exposed group was evaluated by χ2test, and correlations between oxidative indicators and DNA methylation of p16gene were analyzed by Spearman rank correlation analysis.Human B lymphoblastoid cell line and A549human lung cell line were exposed to0,5,10,15mM potassium dichromate (K2Cr2O7) or0,1.25,2.5,5mg/cm2lead chromate (PbCrO4). The exposure time was2hours or24hours for potassium dichromate, and4 hours or24hours for lead chromate. Cells were collected after exposure to potassium dichromate or lead chromate, then cell cycle was detected by Fluorescence Activated Cell Sorter (FACS) with Propidium Iodide (PI) staining, and genomic DNA, total RNA, and protein were extracted from the cells. A methylated DNA quantification kit was used for detecting global methylation status of the DNA isolated from Cr (Ⅵ) treated samples. Both MSP and Pyrosequencing methods were used to examine methylation status at the promoter of the p16gene. Reverse transcription (RT) and real-time quantitative PCR (rt-qPCR) were performed for detection of the mRNA expression level of CDK4, CDK6, and p16gene. Western Blot was conducted to analyze the expression of p16protein.ResultsTotal Cr contents in blood from the population exposed to hexavalent chromium were significantly higher (p<0.01) than those from the controls. The percentage of DNA methylation of p16in exposed group was significantly higher than the controls (p<0.000). No statistical significance of the p16protein expression in plasma was observed between exposed group and control group (p>0.05). The duration of exposure to hexavalent chromium showed no significant influence on methylation of p16gene in the exposed populations (χ2=1.36, p>0.05). GSH was negatively correlated to methylation of p16gene (r=-0.156,p<0.05).The cell cycle arrest in human B lymphoblastoid cell line and A549cell line were not changed by short-term (2h and4h) exposure to K2rO7or PbCrO4. After treatment with K2Cr2CrO7or PbCrO4for24hours exposure, the percentages of G1-phase cells in human B lymphoblastoid cells increased significantly. Conversely, the percentages of S phase samples treated with Cr (Ⅵ) were significantly lower than those in control group (p<0.05, p<0.01), and no significant changes of the percentages of G2-phase cells were observed in both soluble and particulate chromate treatment groups. As to A549cells exposed to Cr (Ⅵ) for24h, cells were significantly (p<0.05, p<0.01) arrested at G1phase in medium and high dose groups and the percentages of G2/M-phase cells decreased significantly (p<0.05p<0.01). No significant changes in the percentages of S-phase cells were observed.Both K2Cr2O7and PbCrO4could decrease the percentages of5-mC in total DNA in a concentration dependent manner in these two cell lines. In human B lymphoblastoid cells, the decrease of global methylation levels was more obvious in short-term (2or4hours) exposure groups than those in long-term (24hours) exposure groups. Both the MSP and pyrosequencing asssy indicated that p16gene in both cell lines was hypermethylated and was not affected by hexavalent chromium.The expression of p16, CDK4, and CDK6gene in short-term (2or4hours) exposure groups remained unchanged in two cell lines. In A549cells, the expression of p16gene was significantly increased (p<0.01) while the expression of CDK4and CDK6gene were decreased (p<0.05, p<0.01) at moderate and high hexavalent chromium treated groups for24hours. The effects of Cr (Ⅵ) on expression of p16, CDK4, and CDK6gene in human B lymphoblastoid cells were similar to those of A549cells, but there were also some differences. No significant difference of CDK4gene expression was found between samples treated with PbCrO4for24hours in human B-lymphoblastoid cells, and significant increase of p16expression was only observed at1.25μg/cm group. The p16protein expression in both cell lines was significantly elevated after24h exposure. Our results showed that significant increase of p16protein expression was observed in human B lymphoblastoid cells exposed to15mM of K2Cr2O7and in A549cells exposed to5.0mg/cm2of PbCrO4.ConclusionOccupational exposure to hexavalent chromium can lead to higher DNA methylation rates of p16gene in exposed populations than those in control populations, but the expression of p16protein in plasma was not significantly changed. Our in vitro study indicated that soluble and particulate hexavalent chromium exposure can lead to cell cycle arrest at G1-phase in both human B lymphoblastoid cells and A549cells, which may be attributed to upregulation of p16gene, downregulation of CDK4/6gene, and hypomethylation of genomic DNA. However, the effects of hexavalent chromium on DNA methylation of p16gene were not obvious in two cell lines, suggesting that the upregulation of p16may be not through their DNA methylation.