Article pubs.acs.org/crt
Trichloroethylene-Induced DNA Methylation Changes in Male F344 Rat Liver Yan Jiang,†,‡ Jiahong Chen,‡,§ Cong Yue,‡,§ Hang Zhang,‡,§ and Tao Chen*,‡,§ †
Department of Physiology, School of Biology and Basic Medical Sciences, ‡Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, §Department of Toxicology, School of Public Health, Soochow University, Suzhou 215123, China S Supporting Information *
ABSTRACT: Trichloroethylene (TCE), a common environmental contaminant, causes hepatocellular carcinoma in mice but not in rats. To understand the mechanisms of the species-specific hepatocarcinogenecity of TCE, we examined the methylation status of DNA in the liver of rats exposed to TCE at 0 or 1000 mg/kg b.w. for 5 days using MeDIP-chip, bisulfite sequencing, COBRA, and LC-MS/MS. The related mRNA expression levels were measured by qPCR. Although no global DNA methylation change was detected, 806 genes were hypermethylated and 186 genes were hypomethylated. The genes with hypermethylated DNA were enriched in endocytosis, MAPK, and cAMP signaling pathways. We further confirmed the hypermethylation of Uhrf2 DNA and the hypomethylation of Hadhb DNA, which were negatively correlated with their mRNA expression levels. The transcriptional levels of Jun, Ihh, and Tet2 were significantly downregulated, whereas Cdkn1a was overexpressed. No mRNA expression change was found for Mki67, Myc, Uhrf1, and Dnmt1. In conclusion, TCE-induced DNA methylation changes in rats appear to suppress instead of promote hepatocarcinogenesis, which might play a role in the species-specific hepatocarcinogenecity of TCE.
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INTRODUCTION Trichloroethylene (TCE), a widely used industrial organic solvent, is a common environmental contaminant that has been found in air, soil, groundwater, and even food. Liver damage caused by TCE has been frequently reported in occupational users, but epidemiological studies of the association between TCE exposure and human liver cancer are still contradictory.1−4 Animal experiments showed that TCE exposure causes hepatocellular carcinoma (HCC) in mice but not in rats.5 The species-specific carcinogenicity of TCE makes it difficult to assess the risk of TCE to human liver. In 2012, the International Agency for Research on Cancer categorized TCE as a human carcinogen (class I) based on epidemiological studies indicating a strong association between occupational exposure and kidney cancer.6,7 It has been suggested that S-(1,2-dichlorovinyl)-L-cysteine, a mutagenic metabolite of TCE, is responsible for TCE-induced kidney cancer.8 However, neither TCE nor trichloroacetic acid, the major TCE metabolite in liver, has been proved to be mutagenic.9 Thus, nongenotoxic mechanisms, especially © 2016 American Chemical Society
epigenetic mechanisms, may play an important role in the hepatocarcinogenecity of TCE. DNA methylation, a well-studied epigenetic mechanism, is susceptible to environmental chemicals.10−12 The level of promoter DNA methylation is usually inversely related to gene expression, and alterations in DNA methylation are one of the earliest events in cancer development.13−15 TCE-induced DNA methylation changes have been reported in mouse liver, which was thought to be important for tumor development.16,17 However, whether TCE can induce similar DNA methylation alterations in rat liver is unclear. Species-specific effects are a characteristic of nongenotoxic carcinogens, which may be due to differences in physiological and biological responses to these agents.18 Understanding the modes of action by which chemicals induce liver tumors in different animals is important for an accurate prediction of their risk to humans. Accumulating evidence has shown that Received: July 26, 2016 Published: September 12, 2016 1773
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Chemical Research in Toxicology Table 1. Top Five Enriched Signaling Pathways by KEGG Analysisa count
P value
genes involved
endocytosis
21
0.023
MAPK signaling pathway
19
0.031
cytokine−cytokine receptor interaction cAMP signaling pathway Salmonella infection
17
0.024
15 10
0.042 0.0094
Arf5, Arfgap2, Acap2, Met, Rab11f ip4, Rab22a, Rab5c, Sh3glb1, Sh3glb2, Arpc5, Ap2a1, Chmp1b, Eea1, Egf r, Egf, Epn2, Fgfr2, Folr1, Ntrk1, Pip5k1b, Vps37b Elk1, Faslg, Rap1a, Rasgrf 2, Taok3, Cacng2, Cacng8, Dusp1, Dusp10, Egf r, Egf, Fgf 22, Fgf r2, Flnb, Hspb1, Il1a, Ntrk1, Ppm1a, Akt3 Faslg, Met, Acvr2a, Bmpr1a, Bmpr1b, Clcf1, Ccl12, Egf r, Egf, If nk, Il1a, Il5, Prl, Tnfrsf11b, Tnf rsf12a, Tnf rsf 21, Tnf rsf 25 Htr6, Abcc4, Atp1a4, Atp1b2, Atp2b3, Gpr81, Rap1a, Rock2, Calm2, Ednra, Npr1, Pde4d, Plce1, Akt3, Vav2 Mk1, Rilp, Rock2, Wasf1, Arpc5, Flnb, Il1a, Pkn2, Rhog, Tlr5
term
a
Bold represents hypomethylated genes. system using SYBR select master mix (Thermo Fisher Scientific). The amplification conditions were as follows: one cycle at 95 °C for 2 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. GAPDH and Ldha were used as internal controls. Designed primers are listed in Table S1. Fold changes were calculated by the comparative cycle threshold method (2−ΔΔCt) as described previously.24 Only genes with a fold change above 1.5 were taken into consideration. Bisulfite Sequencing and Combined Bisulfite Restriction Analysis (COBRA). Genomic DNA (500 ng) from each rat liver tissue was bisulfite-modified with the BisulFlash DNA methylation kit (Epigentek Group Inc., cat. no. P-1026-050), followed by PCR amplification. Primers are listed in Table S1. For bisulfite sequencing, the PCR products were separated by 2% agarose gel electrophoresis, purified with an E.Z.N.A. gel extraction kit (Omega), and cloned into a pGM-T vector (Tiangen, Beijing, China). Positive clones were selected by blue-white screening and sequenced using an ABI737 machine. For COBRA, the PCR products were digested with the corresponding restriction enzymes (Thermo Fisher Scientific) and analyzed on a 2.5% agarose gel. Liquid Chromatography Tandem Mass Spectrometry (LCMS/MS). LC-MS/MS were performed as described previously.23 Briefly, 3−5 μg of genomic DNA was hydrolyzed by nuclease P1 for 1 h at 37 °C, followed by treatment with alkaline phosphatase for 30 min. The hydrolyzed DNA was loaded into an Agilent 1100 series LC coupled to an API 3000 triple quadrupole mass spectrometer equipped with a turbo ion spray source (Thermo Fisher Scientific). Separation was accomplished on a Thermo 150 mm × 2.1 mm, 5 μm column with a mobile phase consisting of 0.1% formic acid and methanol. Quantitation of 2′-deoxycytosine and 5-methyl-2′-deoxycytidine was conducted using external standards. Statistics Analysis. Results are expressed as the mean ± SD. Differences between different groups were analyzed using Student’s t test at a significance level of p < 0.05.
epigenetic mechanisms, especially modifications in DNA methylation, can mediate gene−environment interactions and are key to cancer development.11,19,20 It has been reported that TCE induces distinct hepatic transcriptional differences between mice and rats.21 We further found that TCE-induced mRNA expression changes were correlated with DNA methylation alterations in the liver of mice.22 To assess if TCE can induce species-specific DNA methylation changes, in this study we examined the genome-wide liver DNA methylation profiles of rats exposed to TCE. The mRNA expression levels of genes involved in proliferation and DNA methylation regulation were also measured. On the basis of previous studies in mouse liver, a single dose of TCE at 1000 mg/kg b.w. for 5 days was chosen for this study.16,22
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EXPERIMENTAL PROCEDURES
Chemicals. TCE (CAS 79-01-6, ≥99.5% pure) was purchased from Adamas-Beta (Shanghai, China). Taq PCR mastermix (2×) and restriction digestion enzymes were obtained from Thermo Fisher Scientific (Beijing, China). All other chemicals and enzymes were from Sigma-Aldrich (Shanghai, China) unless otherwise stated. Animal Treatment. All animal procedures were approved by the Institutional Animal Care/Use Ethical Committee of Soochow University. Adult F344 male rats aged 6 weeks were obtained from Shanghai Lab Animal Research Center (Shanghai, China). The animals were housed (5 rats per cage) with a 12 h light and 12 h dark cycle at 22 ± 2 °C. The relative humidity was 55 ± 5%. After a 1 week adaption period, 10 rats were divided into 2 groups randomly with 5 rats in each group. The control rats were orally administered corn oil at 5 mL/kg/day for 5 days, and the treated rats, TCE at 1000 mg/kg/day in corn oil for 5 days. Approximately 90 min after the last dose, the rats were then euthanized. At necropsy, the liver was rapidly excised, weighed, frozen in liquid nitrogen, and stored at −80 °C. DNA and RNA Extraction. Genomic DNA and total RNA from rat liver tissues of each group were extracted by E.Z.N.A. DNA/RNA/ protein isolation kit (Omega Bio-tek, Inc., Norcross, USA) according to the manufacturer’s protocol. The concentration and quality of DNA and RNA were examined and quantified using a NanoDrop ND-2000 (Thermo Scientific). Methylated DNA Immunoprecipitation and Microarray (MeDIP-Chip) Analysis. DNA from 3 control samples was mixed, as was DNA from 3 TCE-exposed samples. Methylated DNA was immunoprecipitated using a magMeDIP kit (mc-magme-048; Diagenode, Liège, Belgium) as described previously.23 DNA labeling and hybridization were performed according to NimbleGen’s protocol. The immunoprecipitated CpG-methylated DNA from control and TCE-exposed samples was labeled with Cy5 and Cy3. The DNA samples were then hybridized onto the rat 3×720K CpG Island Plus RefSeq promoter array (05924545001; Roche NimbleGen, Madison, USA). Data scanning and collection were performed using NimbleScan, and only genes with a peak score above 3 were taken into consideration. Quantitative RT-PCR. First-strand cDNA was reverse transcribed using pre real-time cDNA synthesis master mix (Biomiga, Shanghai, China). Quantitative PCR was performed in an ABI Prism 7500
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RESULTS TCE-Induced DNA Methylation Changes. To compare our data with previously published data generated from mouse liver, we used the same TCE treatment conditions (a dose level of 1000 mg/kg b.w. for 5 days) for rats. Using MeDIP-chip, we identified 806 genes with DNA hypermethylation and 186 genes with DNA hypomethylation in TCE-treated rat liver compared with vehicle controls. KEGG analysis showed that genes showing DNA methylation changes, especially hypermethylated genes, were enriched in endocytosis, MAPK, and cAMP signaling pathways (Table 1 and Figure S1). In particular, Dnmt3a and Uhrf2, two genes involved in DNA methylation regulation, showed DNA hypermethylation in TCE-exposed rat liver samples compared with controls (Table S2). We further confirmed the DNA hypermethylation of the Uhrf2 promoter region and the hypomethylation of the Hadhb promoter region by bisulfite sequencing (Figure 1). No DNA methylation change was detected in the promoter regions of Ihh, Cdkn1a, Jun, Myc, Sfn, and Cdkn2a by bisulfite sequencing 1774
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Dnmt3a, Uhrf2, Hadhb, and Clock). Gata1 and Hadhb showed the largest DNA methylation changes (Table S2). Dnmt3a and Uhrf2 are involved in the regulation of DNA methylation, and the DNA methylation status of Clock is important for cancer development.26 In our results, all three DNA hypermethylated genes (Gata1, Dnmt3a, and Uhrf2) showed mRNA expression downregulation in TCE-exposed samples compared with vehicle controls (Figure 3). However, of the two DNA
Figure 1. DNA methylation status detected by bisulfite sequencing and LC-MS/MS in the liver of rats exposed to TCE or vehicle control. (A) Bisulfite sequencing of the Uhrf2 promoter region. (B) Bisulfite sequencing of the Hadhb promoter region. (C) Bisulfite sequencing of the Cdkn1a promoter region. (D) Global DNA methylation levels detected by LC-MS/MS. Open and closed circles indicate unmethylated and methylated CpG sites, respectively. MeC %, DNA methylation percent.
Figure 3. Relative expression changes of mRNAs in TCE-treated rat livers compared with controls. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
hypomethylated genes (Handhb and Clock), only Handhb showed mRNA overexpression (Figure 3). The mRNA expression level of Clock did not change significantly (Figure 3). We also examined the mRNA expression levels of six genes involved in proliferation and DNA methylation regulation (Cdkn1a, Ihh, Jun, Mki67, Myc, Uhrf1, Dnmt1, and Tet2). Ihh, Jun, and Tet2 were downregulated, whereas Cdkn1a was upregulated; no significant mRNA expression change was found for other genes (Figure 3). The DNA methylation and mRNA
and COBRA (Figures 1 and 2). In addition, we found no significant DNA methylation change in the repetitive DNA elements of Line-1 (Figure 2B), which is usually used as an indicator of the global DNA methylation level.25 LC-MS/MS results further confirmed that TCE induced no global DNA methylation change in rat liver (Figure 1D). TCE-Induced mRNA Expression Changes. We first examined mRNA expression changes of the five genes with DNA methylation alteration according to MeDIP-chip (Gata1,
Figure 2. DNA methylation status detected by COBRA in the liver of rats exposed to TCE or vehicle control. (A) COBRA results of the promoter region of Ihh by Hinf I: 325 bp (140/108/77). (B) COBRA results of Line-1 by HpyCH4IV: 163 bp (115/43). (C, D) COBRA results of the promoter region of Jun by TaqI: 143 bp (74/44/25) and BstUI: 143 bp (47/37/35/24). (E, F) COBRA results of the promoter region of Myc by TaqI: 242 bp (125/77/39) and BstUI: 242 bp (101/100/41). (G) COBRA result of the promoter region of Sfn by TaqI: 229 bp (173/56). (H) COBRA result of the promoter region of Cdkn2a by TaqI: 505 bp (205/68/63/58/62). L, DNA ladder; P, positive control by treating rat genomic DNA with M.SssI; C, control samples; T, TCE-treated samples; M, methylation; UM, unmethylation. 1775
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Dnmt3a is essential for de novo methylation, and Tet2 catalyzes oxidative reactions of 5-methylcytosine and is important for the maintenance of DNA hypomethylation.19,34 Both Uhrf1 and Uhrf2 bind preferentially to hemimethylated DNA and can interact with Dnmts.35 Uhrf1 is required for DNA methylation maintenance, and Uhrf2 is important for cell differentiation.36 Therefore, dysregulation of Dnmt3a, Tet2, and Uhrf2 might be responsible for the abnormal DNA methylation changes in the liver of rats exposed to TCE. The drawback of this study is that a pooled sample of three replicates was used for the MeDIP array for economic reasons. This may introduce bias and make it impossible to do statistical analysis. However, we have validated the DNA hyper- and hypomethylation of Uhrf2 and Handhb. Future studies using individual samples will reveal more detailed information about TCE-induced DNA methylation alterations in rat liver. In conclusion, TCE induced DNA methylation changes in rat livers, which appears to suppress proliferation and might be beneficial for rat hepatocytes to resist carcinogenesis. The different DNA methylation patterns induced by TCE between rats and mice may play a possible role in the species-specific hepatotoxicity of TCE.
expression changes caused by TCE in the liver of mice and rats are summarized in Table 2. Table 2. Summary of mRNA and DNA Methylation Changes in the Liver Samples of Mice and Rats Exposed to TCEa mouse22 Cdkn1a Ihh Hadhb Uhrf 2 Uhrf1 Tet2 Dnmt1 Dnmt3a Jun Myc Mki67 Line-1
rat
mRNA
DNA methylation
mRNA
DNA methylation
↑ ↓ / / ↑ ↓ − ↓ ↑ − ↑ /
↓ ↑ / / / / / / − − / −
↑ ↓ ↑ ↓ − ↓ − ↓ ↓ − − /
− − ↓ ↑ − − − ↑ − − − −
a
Upward arrow, significantly upregulated; downward arrow, significantly downregulated; dash, not changed; slash, not examined.
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ASSOCIATED CONTENT
S Supporting Information *
DISCUSSION Previously, we found that the promoter regions of Cdkn1a and Ihh were hypo- and hypermethylated, respectively, in TCEexposed mouse liver, which was correlated negatively with their mRNA expression levels.22 In contrast, neither of these genes showed detectable DNA methylation changes in the liver of rats exposed to TCE, although TCE induced similar mRNA expression changes for these two genes in rats and mice. Using MeDIP-chip, we screened genome-wide DNA methylation profiles and identified 992 genes with DNA methylation changes in TCE-exposed rat livers compared with controls. Endocytosis, MAPK, and cAMP signaling pathways were among the top five affected pathways when most of the enriched genes were hypermethylated, indicating that the three pathways were suppressed instead of activated. It has been reported that TCE activates MAPK signaling in the liver of mice but not rats.21 Since endocytosis, MAPK, and cAMP signaling pathways are usually overactivated in cancer cells, with the capability of promoting cell proliferation, repression of the three pathways may be beneficial for rat hepatocytes to resist carcinogenesis.27−30 Consistently, the cell proliferation marker Mki67 and oncogene Jun, which were overexpressed in the liver of mice exposed to TCE, showed no mRNA expression change (Mki67) or even downregulation (Jun) in TCE-treated rat liver samples compared with controls. Furthermore, no detectable DNA methylation alteration was found in the promoter regions of Myc, Sfn, and Cdkn2a, which are cancer-related genes that have been reported to be transcriptionally regulated by DNA methylation.31−33 In mouse liver, TCE disturbed the mRNA expression levels of a number of genes involved in the regulation of DNA methylation, including Dnmt3a, Dnmt3b, Tet2, and Uhrf1. Here, as shown in Table 2, we found that the mRNA expression levels of Dnmt3a and Tet2 were significantly downregulated in the liver of rats treated with TCE, similar to that found in mouse livers. In addition, we detected a decrease in the mRNA expression level of Uhrf2. However, the mRNA expression level of Uhrf1, which was upregulation in mouse livers by TCE, did not change significantly in TCE-treated rat liver samples.
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.6b00257. Primer sequences used for qPCR and bisulfite PCR; list and pathway mapping of genes with altered DNA methylation alteration by MeDIP-chip in TCE-exposed rat liver compared with controls (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Telephone: +86-512-65882273. Fax: +86-512-65882233. Funding
This work was supported by projects sponsored by SRF for ROCS, SEM (grant number K513900115), the National Natural Science Foundation of China (grant number 81570284), and The Priority Academic Program Development of Jiangsu Higher Education Institutions. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS TCE, trichloroethylene; HCC, hepatocellular carcinoma; qPCR, quantitative polymerase chain reaction; COBRA, combined bisulfite restriction analysis; MeDIP-chip, methylated DNA immunoprecipitation and microarray; LC-MS/MS, liquid chromatography tandem mass spectrometry
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