Elsevier

Toxicology Letters

Volume 249, 13 May 2016, Pages 15-21
Toxicology Letters

Methylation levels of P16 and TP53 that are involved in DNA strand breakage of 16HBE cells treated by hexavalent chromium

https://doi.org/10.1016/j.toxlet.2016.03.003Get rights and content

Highlights

  • Thehypermethylation of CpG1, CpG31 and CpG32 of p16 were observed in Cr(VI) treated groups.

  • The methylation level of CpG1 of p16 can enhance cell damage by regulating its expression or affecting some transcription factors to combine with their DNA strand sites.

  • The CpG1methylation level of p16 could be used as a biomarker of epigenetic effect caused by Cr(VI) treatment.

Abstract

The correlations between methylation levels of p16 and TP53 with DNA strand breakage treated by hexavalent chromium [Cr(VI)] remain unknown. In this research, Human bronchial epithelial cells (16HBE cells) in vitro and bioinformatics analysis were used to analyze the epigenetic role in DNA damage and potential biomarkers. CCK-8 and single cell gel electrophoresis assay were chosen to detect the cellular biological damage. MALDI-TOF-MS was used to detect the methylation levels of p16 and TP53. qRT-PCR was used to measure their expression levels in different Cr(VI) treatment groups. The transcription factors with target sequences of p16 and TP53 were predicted using various bioinformatics software. The findings showed that the cellular toxicity and DNA strand damage were Cr(VI) concentration dependent. The hypermethylation of CpG1, CpG31 and CpG32 of p16 was observed in Cr(VI) treated groups. There was significant positive correlation between the CpG1 methylation level of p16 and cell damage. In Cr(VI) treated groups, the expression level of p16 was lower than that in control group. The expression level of TP53 increased when the Cr(VI)concentration above 5 μM. About p16, there was significant negative correlation between the CpG1 methylation levels with its expression level. A lot of binding sites for transcription factors existed in our focused CpG islands of p16. All the results suggested that the CpG1 methylation level of p16 could be used as a biomarker of epigenetic effect caused by Cr(VI) treatment, which can enhance cell damage by regulating its expression or affecting some transcription factors to combine with their DNA strand sites.

Introduction

Chromium(Cr) and its compounds are basic chemical raw materials. They have been widely used in the industry and agriculture including chrome, dyes, paints, rubber and ceramics (Gao and Xia, 2011). While hexavalent chromium [Cr(VI)] is a strong oxidant which can cause multi-system disorders involving the skin and mucous membrane, liver and renal (Wang et al., 2011), immune system (Beaver et al., 2009), genetic damage and even lung cancer (Hara et al., 2010). It is widely accepted that DNA damages dominate the underlying mechanisms of Cr(VI)-induced carcinogenesis (Halasova et al., 2012, Wise and Wise, 2012).

Our previous studies had proved that occupational chromate exposure can increase apoptosis (Wang et al., 2012). Many studies have also showed that apoptosis was the main reason for many diseases even cancer (Cavallo et al., 2010). Many factors can regulate apoptosis process including cell cycle regulation such as TP53 and p16 (Qi et al., 2014). When cells were stimulated by genetic damage chemicals, it can cause DNA damage, and then induce accumulation and activation of p53 protein through the NF-κB pathway to influence other proteins’ expression such as p21 and p16 (Senba et al., 2010, Shibata-Kobayashi et al., 2013). p21 and p53 can induce the cells arrest at the junction of G1/S and G2/M phase of the cell cycle and control the progression of cell cycle to provide chance for DNA damage repairing (Sikdar et al., 2015, Zhu et al., 2015). When DNA damage failed to be repaired, p53 and p16 could be up-regulated to mediate apoptosis by various pathways. Therefore, it can be assumed that the epigenetic modifications and expression levels of TP53 and p16 could play an important role in the maintenance of genome integrity and cellular survival.

Recent studies suggest that DNA damage can modify DNA methylation patterns and lead to hypomethylation and, consequently, to genomic instability. DNA methylation is a regulated biological process in which the methyl unite was transferred to the specific bases by methyl transferase, and S-Adenosylmethionine (SAM) is the methyl donor. DNA methylation can regulate gene expression to cause changes of chromatin structure, maintain the stability of DNA and affect the interaction of DNA with protein, which can play an important role in pathological process of many diseases (Ali et al., 2011, Maunakea et al., 2010, Romanoski et al., 2015).

Previous studies showed that some environmental pollutants can affect TP53 and p16 expression and their aberrant CpG methylation. p16 gene methylation were significantly increased when exposed to arsenic and Polycyclic aromatic hydrocarbons (PAH) (Tyler and Allan, 2014, Zhang et al., 2015). In oral ingestion of Cr (VI) through drinking water could cause global DNA hypomethylation in blood cells from male rats (Wang et al., 2015). It is also indicated that the aberrant methylation of TP53 and p16 is involved in chromium carcinogenesis (Kondo et al., 1997, Kondo et al., 2006) and the expression and methylation level of p16(INK4a) are reduced in chromate lung cancer (Kondo et al., 2006). To explore the association between methylation and DNA damage will help to reveal whether epigenetic mechanism is involved in Cr(VI)-induced DNA damage. In this research, Human bronchial epithelial cells (16HBE cells) in vitro and bioinformatics analysis were used to analyze the methylation level of TP53 and p16 and understand whether DNA methylation can be a potential biomarker related to chromium carcinogenesis.

Section snippets

Cell line and chromium treatment

16HBE cells (tumor cell library of Chinese Academy of Medical Sciences, China) were cultured in DMEM supplemented with 10% fetal bovine serum, 100U/mL penicillin, and 100 μg/mL streptomycin, and maintained at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air.

Cells were treated with dichromate (Cr2O72−) (Sigma, USA) stoke solution diluted in cell culture medium at different concentrations. As the control group, cells were treated with the same volume of ddH2O (Sigma, USA) instead of

Cell survival rate

As was shown in Fig. 1, the cell survival rate was measured by the CCK-8. Comparing with the control group, the cell survival rate decreased significantly in a concentration dependent manner when the treatment concentrations above 12.5 μM during 24 and 48 h. And the cell survival rate decreased significantly at all studied concentrations when treated for 24 h.

The model of cell survival rate was analyzed by SPSS software to calculate the IC50 for Cr(VI) treatment concentration and time (Liu et al.,

Discussion

Many investigations had proved that Cr(VI) treatment can induce genetic damage and induction of cell apoptosis, which can contribute to its hazard on health (Fu et al., 2015, Xia et al., 2015). Our present research proved that the cell survival rate and the values of percentage of tail DNA(%), TL, TM and OTM were changed in a Cr concentration dependent manner.

The CpG islands methylation play an important role in the transcription regulation. The inverse relationship was shown between the

Conclusions

Cr(VI) treatment could up-regulate the methylation levels of CpG1, CpG31 and, CpG32 sites of p16, which inversely down-regulated its mRNA expression levels, and also can affect the combination of some transcription factors with corresponding sites of p16 gene strand to enhance cellular stress induced by Cr(VI) in HBE16 cells. It is suggested that the methylation level of these significant CpG sites could be used as a potential effective biomarker of epigenetic effect due to chromate exposure.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by the projects of National Natural Science Foundation of China (81273043) and by Doctor Fund of Ministry of Education of china (20120001110103).

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    These authors contributed equally to this work.

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