DNA methyltransferase expression and DNA hypomethylation status in human hepatocytes following trichloroacetic acid exposure

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Highlights

  • TCA and 5-aza-dC could cause cell proliferation arrest in human normal hepatic L-02 cells.

  • TCA could induce the global genomic DNA hypomethylation in human normal hepatic L-02 cells.

  • Both DNMT1 and DNMT3a decreased with a time-dose-dependent manner following TCA exposure.

  • HepG2 cells have the high DNA methyltransferase expression level following TCA exposure.

  • DNMT1 and DNMT3a provide the therapeutic targets and biomarkers for TCA related diseases.

Abstract

Trichloroacetic acid (TCA) is one of the major metabolites of trichloroethylene (TCE) as the significant factor of environmental and occupational pollution. TCA has been shown to induce a series of epigenetic mutation in mouse liver. However, the epigenetic cytotoxicity of TCA is still in infancy. In this study, we explored the cellular biological characteristics, the genome DNA methylation status and the expression profile of DNA methyltransferases in human hepatic L-02 cells treated with TCA with certain time and dose effects. The cell cycle measured by flow cytometry revealed an increasing S + G2 (M) phase of TCA (0.9 mM 24 h, 48 h and 72 h) treated cells after a recovery day, and sub-G1 phase was not appeared. The levels of 5 -mC were decreased in TCA (0.9 mM 24 h and 72 h) treated cells by 5-mC immunolocalization process and HPCE (decreased from 27.2% to 50.1% respectively). Meanwhile, the mCpG% in normal L-02 cells and TCA (0.9 mM 48 h) treated cells was 79.6% ± 6.5% and 50.8% ± 3.8%, respectively (P < 0.05). It also revealed that treatment of L-02 cells with TCA induced decreased in DNMT1 and DNMT3a mRNA and protein levels with a time-dependent manner and a dose-response relationship, while DNMT3b had no obvious change. These results establish a link between DNA methyltransferases and Genome DNA hypomethylation, which is associated with TCA exposure.

Introduction

Trichloroacetic acid (TCA) might enhance the activation of T cells and disrupt various activities of peripheral T cells [1], which is related to human immune function and reduces human immunity. Trichloroethylene (TCE) has been widely used in many manufacturing industries [2], and it is a widespread water pollutant [3]. Although TCE is classified as carcinogenic to humans and is associated with kidney-specific toxicity [4,5], the toxicity study of TCA, a metabolite and a breakdown product of TCE, remains uncertain. In addition, TCA is a common organic contaminant of drinking water formed as by-products during chlorine disinfection. TCA could promote N-methyl-N-nitrosourea (MNU)-initiated foci of altered hepatocytes and induce the related liver tumors in B6C3F1 mice [5]. Moreover, it has reported that TCA could induce acute pulmonary injury and medicamentosa-like dermatitis due to the high concentration occupational exposure in Asia, especially in China [6,7].

Early-life environmental factors influences later-life susceptibility to cancer [8]. TCA has been testified to be nongenotoxic hepatocarcinogens since there is little evidence in vivo and vitro studies to indicate either genotoxic or mutagenic activity [9,10]. So there may exist a nongenotoxic mechanism in TCA induced tumors. TCA has been shown to increase cell proliferation and induced global DNA hypomethylation in mouse liver [11]. TCA has also been shown to up-regulate many protooncogenes, such as, H-ras, K-ras, Raf, C-fos, C-jun, IGF-II and C-myc, and lead to hypomethylation around the promoters of above genes [12]. Thus, epigenetic mechanisms play an important role in the toxic effect of TCA. However, the mechanism responsible for this chemical's hypomethylation and related biological effects remain unclear.

In this study, we analyzed the impact of TCA on L-02 Cells. Previous studies have indicated that the decreased methylation status of DNA in TCA induced may be associated with the abnormal expression of DNA methyltransferases. This abnormal expression may play an important role in the process of TCA induced tumors, for malignant cells often exhibit abnormal expression of DNA methyltransferases. This epigenetic process acts as an alternative to mutations to disrupt the function of tumor suppressor gene and can predispose genetic alterations which often lead to malignant transformation. Epigenetic changes are heritable, metastable, and often reversible, which alters the way DNA-encoded information, but does not involve changes in DNA sequences [13]. The mechanism of DNA methylation has not been clearly defined. DNA methyltransferases catalyze the transfer of methyl groups to CpG dinucleotides residing in many possible sequence contexts to generate hemimethylated or fully methylated DNA. There are several different kinds of DNA methyltransferases, such as DNMT1, 2, 3a, 3b and DNMT3L. DNMT1 is believed to be the “maintenance” enzyme, maintains the methylation pattern during DNA replication [14], and involves in de novo DNA methylation [15]. The catalytic activity of the purified DNMT2 with DNA substrates is very low and almost undetectable in direct biochemical assays [16]. DNMT3a and DNMT3b are known as de novo methyltransferases and have preferred target sites, which are different from DNMT1 [17]. DNMT3L does not have similar catalytic domain of other DNA methyltransferases nor any methyltransferase activity [18]. DNMT1, DNMT3a and DNMT3b were selected to our study.

The objective of this study was to examined the toxic effect of TCA on cell growth, cell cycle, apoptosis, global methylation status, and the expression of the methyltransferases DNMT1, DNMT3a and DNMT3b in the human hepatic cell lines, the data showed that down-regulated DNMT1 and DNMT3a caused the global hypomethylation in TCA treated L-02 cells, which provides new directions to interfere with TCA-related diseases, and health risks associated with TCA exposure are needed to be better understanding.

Section snippets

Reagents

TCA, 5-aza-dC, five standard bases (C, mC, G, T, A) and rabbit monoclonal antibodies of DNMT1 were from Sigma. Mouse monoclonal antibodies of DNMT3a and DNMT3b were from Santa Cruz Biotechnology. Anti-5-mC antibody (Mouse mAb) was from Calbiochem.

Cell lines

Human normal hepatic L-02 cell lines and human hepatic cellular cancer HepG2 cell lines were obtained from the Cell Bank of Chinese Academic of Science. L-02 cells were mainly young and middle-aged cells between the 10 generations and 30 generations

Effect of TCA on general biological characteristics

The L-02 cells proliferation was decreased when exposed to different concentration (0.1, 0.3, 0.9 mM) of TCA, and the 5-aza-dC also induced a down-regulation in cell proliferation, the difference had a statistical significance (P < 0.05), compared with normal L-02 cells (Fig. 1A–D).

Compared with normal L-02 cells, the G1 phase of TCA-re cells was significantly reduced by 10.5%–21.4% (p < 0.05), similar to HepG2 (Fig. 2A). S phase of the TCA (0.9 mM) treated cells showed decline of different

Discussion

Data showed that the treatment with TCA (≤0.9 mM) hadn't induced the L-02 cell proliferation arrest. However, the TCA treated cells with 24 h recover course can significantly increase the S + G2 (M) phase, and the exposure to TCA (≤0.9 mM) did not result in apoptosis of L-02 cells even for 72 h, but TCA (0.9 mM) had induced L-02 cells obvious hypomethylation, and the hypomethylation became even lower with the treated time prolonged. Meantime, TCA induced a marked down-regulation of DNMT1 and

Conflicts of interest

No potential conflicts of interest.

Acknowledgments

National Natural Science Foundation of China (81473014); Top Young Talents of Guangdong Hundreds of Millions of Projects (87316004); Jinan University High-level University Construction Public Health and Preventive Medicine Fund (JNUPHPM2016003)

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    These authors contributed equally to this work.

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