Specific histone modifications regulate the expression of AhR in 16HBE cells exposed to benzo(a)pyrene

Abstract

An aryl hydrocarbon receptor (AhR) is a transcription factor mediating the responses to polycyclic aromatic hydrocarbon (PAH) compounds. To investigate the epigenetic mechanism involved in the regulation of AhR, we treated human bronchial epithelial cells (16HBE) with benzo(a)pyrene (BaP) and found a transcriptional suppression of AhR in a dose- and time-dependent manner. Suppression of AhR significantly attenuated the extent of BaP-induced CYP1A1 expression and the cell growth arrest, and conferred 16HBE cells insensitive to DNA damage. In addition, we found that the mRNA level of AhR was elevated more than twice in 16HBE cells treated with histone deacetylase inhibitor trichostatin A (TSA), indicating that AhR expression might be regulated via histone modification. Moreover, we showed that BaP or 3-MC treatment led to a reduction of acetylation at residues H3K9, H3K18 and H3K27, suggesting that histone modifications are associated with chemical exposure. Using chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR), we further demonstrate that the reduction of histones H3K18ac and H3K27ac is correlated with the decreased binding affinity with the AhR promoter in HBE cells treated with BaP or 3-MC. In addition, we identified that specific regions located at the transcriptional start site (TSS) of the AhR gene were responsible for H3K18ac- and H3K27ac-related transcriptional activity of the AhR promoter. Taken together, we identified that two specific histone modifications, H3K18ac and H3K27ac, were involved in regulation of the transcriptional activation of AhR, which might contribute to BaP-induced toxicity and the response to DNA damage.

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Introduction

Benzo(a)pyrene (BaP) is a model polycyclic aromatic hydrocarbon (PAH) and its metabolites are mutagenic and highly carcinogenic, listed as a Group 1 carcinogen by the IARC.¹ Metabolic activation of BaP by cytochrome P450s is critical for its toxicity and carcinogenicity.^2,3 BaP treatment induces cytochrome P4501A1 (CYP1A1) by binding to the aryl hydrocarbon receptor (AhR) in the cytosol.⁴ Studies on the animal model with AhR gene knockout revealed that lack of AhR diminished 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced teratogenesis and BaP-induced carcinogenesis in mice.^5,6In vitro studies have revealed that AhR is a critical factor in activation of chemical carcinogens by regulating the formation of metabolites and the effects of DNA breaks and anti-apoptosis.^7,8 Moreover, dysregulation of AhR expression was associated with many kinds of human diseases, such as hepatocellular carcinoma,⁹ leukemia,¹⁰ and asthma.¹¹

Activation of AhR induced by ligands is a multi-step process. When binding to the exogenous ligands, AhR releases, translocates into the nucleus and triggers the transcription of the target genes by binding to the xenobiotic response element (XRE) in their promoters and/or enhancer regions.^12,13 The agonists could directly bind to AhR in the cytoplasm, leading to a dissociation of AhR from the protein complex. Consequently, AhR translocates to the nucleus and forms heterodimers with an aryl hydrocarbon receptor nuclear translocator (ARNT), resulting in the transcriptional activation of a number of genes including cytochrome P450 1A1 (CYP1A1), NADPH dehydrogenase 1 (NQO1), aryl hydrocarbon receptor repressor (AhRR), and cyclooxygenase-2 (COX-2).¹⁴ Although the importance of AhR notably in mediating chemical toxicity and metabolism has been well characterized, the mechanism regulating the expression of AhR in response to AhR ligands remains elusive.

To address this issue, we sought specific regulatory pathways that play critical roles in AhR regulation. Our recent study identified miR-203 specifically suppressing the AhR expression in human cells in response to TCDD or BaP treatment via binding to the 3′-UTR region of AhR mRNA, indicating that epigenetic regulation was involved in the response to AhR ligands.¹⁵ Previous studies implied that histone modifications contributed to AhR regulation. HeLa cells treated with histone deacetylase or a DNA methyltransferase inhibitor resulted in an increase of AhR expression,¹⁶ while the histone deacetylase inhibitor restored the AhR-mediated response in mouse hepatoma cells expressing a low level of AhR.¹⁷ However, the key histone modifications which are responsible for regulation of AhR have remained unknown. In this study, we investigated the role of AhR in cellular response to BaP or 3-MC treatment and revealed that two specific histone modifications, H3K18ac and H3K27ac, were involved in regulation of the transcriptional activation of AhR, which might mediate BaP-induced toxicity.

Materials and methods

Chemical and reagents

Benzo(a)pyrene (BaP), 3-methylcholanthrene (3-MC), trichostatin A (TSA), and actinomycin D (ActD) were purchased from Sigma-Aldrich (St Louis, MO, USA). Primers used for real-time quantitative PCR (qPCR) and plasmid constructs were synthesized by Generay Biotechnology (Shanghai, China) and the sequences are listed in ESI Tables S1 and S2.† The following primary antibodies were used: rabbit anti-H3K9ac (Cell signaling, 9649), H3K14ac (Active motif technology, 39599), H3K18ac (Abcam, ab1191), and H3K27ac (Active motif, 39133), H3 (Abcam, ab10799). Rabbit anti-AhR and mouse anti-β-actin were purchased from Proteintech Group (Chicago, USA).

Cell culture

The human bronchial epithelial cell line (16HBE14σ, 16HBE) was a gift from Dr D.C. Gruenert (University of California, San Francisco, CA, USA). 16HBE cells were cultured in Minimum Essential Media (MEM) (Gibco) supplemented with 10% fetal bovine serum (Gibco) under a humid atmosphere at 37 °C with 5% CO₂.

RT-qPCR

Total RNA was extracted with the Trizol reagent (Invitrogen, Life technologies, Branford, USA) and reverse transcription was performed with a RT-qPCR Kit (Toyobo, Tokyo, Japan). The mRNA expression of target genes was detected using the Vii7 Real-time PCR system (Applied Biosystems, Branford, USA) and the SYBR Green Real time PCR Master Mix-Plus (Toyobo, Tokyo, Japan) with the standard program. Three independent experiments were performed.

Immunoblotting

Cells were lysed with RIPA buffer (50 mM Tris, 1% SDS, 10 mM EDTA, 1 mM PMSF) containing a protease inhibitor tablet (Roche, Indianapolis, IN) and followed by centrifugation to discard the insoluble substance. 50 μg of total protein was loaded and resolved by 7–14% gradient SDS-PAGE and followed by immunoblotting with specific antibodies.

Chromatin immunoprecipitation (ChIP)

16HBE cells were treated with 10 μM BaP for 24 h. 1 × 10⁷ cells were cross-linked with 1% formaldehyde and stopped by adding glycine solution. After washing with ice-cold PBS, the cells were harvested and lysed with the lysis buffer for 30 min and homogenized in a dounce homogenizer to release the nuclei. The pellet was collected and digested with micrococcal nuclease for 10 min. The supernatant that contains the fragment of sheared chromatin was incubated with specific antibodies for ChIP with protein G magnetic beads in 4 °C, overnight. The next day, the chromatin antibody complexes were eluted and incubated with reverse crosslink buffer to reverse the cross-links followed by protein K treatment. Finally, the DNA was purified and precipitated with phenol-chloroform and glycogen. Each primer was designed for the specific region. The result of the percent input was calculated by 100 × 2^{(Ct-adjusted Input-Ct-enriched)}. Input DNA Ct was adjusted from 10% to 100% equivalent by log₂10.

Establishment of stable cell lines

The short hairpin sequences for suppressing AhR expression were synthesized and cloned into a lentiviral plasmid pLKO.1, generating the vectors pLKO-SHGFP, pLKO-SHAhR-1 and pLKO-SHAhR-2. Each vector was transfected into 293 T cells together with vectors VSV-G and pCMV-ΔR8.91 via calcium phosphate precipitation. The lentiviral supernatants were collected to infect 16HBE cells followed by selection with puromycin (2 μg ml⁻¹). After culture for three passages, the cell lines were confirmed by immunoblotting.

Cell proliferation

For cell proliferation studies, 2 × 10⁴ cells were seeded in a 12-well plate for 24 h before treatment with 10 μM BaP or DMSO (0.1%, v/v). The media were changed every 48 h during BaP treatment. The cell number was counted at 0 h, 48 h, 72 h, 96 h and 120 h after BaP treatment using a Z2 Particle Counter and Size Analyzer (Beckman-Coulter, Miami, FL, USA). Three independent experiments were performed.

Cytokinesis-blocked micronucleus (CBMN) assay

For CBMN assay, 5 × 10⁴ 16HBE cells were seeded and treated with 10 μM BaP for 48 h. Vehicle DMSO was used as a control. Next, cells were blocked in cytokinesis by adding cytochalasin B (Sigma-Aldrich, St Louis, MO, USA) at a final concentration of 4 μg ml⁻¹. After 36 h incubation, cells were harvested sequentially for hypotonic treatment, fixation, coating and staining. 1000 cytokinesis-blocked cells were examined according to the criteria described by Fenech et al.¹⁸ The numbers of micronuclei per 1000 binucleated cells were recorded.

Alkaline comet assay

Cells were harvested after 48 h exposure. 90 μl of cell suspension was mixed with 10 μl 0.8% low-melting-point agarose and subjected to the 0.6% bottom agarose-coated slide. Then the slide was cooled down on ice and immersed in lysis buffer (2 M NaCl, 30 mM Na₂EDTA, 10 mM Tris, 10% DMSO, 1% Triton X-100, pH 10.0) for 2 h. Next, the slide was immersed in iced electrophoresis buffer (300 mM NaOH, 1 mM Na₂EDTA, pH 13.0) for 30 min following electrophoresis at 20 V and 300 mA for 20 min and neutralized with 200 mM Tris buffer (pH 7.5). After staining with 2 μg ml⁻¹ PI, the slide was photographed under a fluorescence microscope (Nikon, Eclipse Ti-E) and analyzed using the software CometScore™ Version 1.5 (Tritek Corporation).

Statistical analysis

Data were expressed as the mean ± SD of three independent experiments. The differences between two groups were assessed using an unpaired Student's test. And the differences among more than two groups were analyzed by a one-way ANOVA followed by a Mann–Whitney test using the software SPSS 13.0. Differences were considered statistically significant at P < 0.05.

Results

The effect of BaP or 3-MC on the expression of AhR

It has been demonstrated that AhR participated in the metabolism of environmental chemicals, such as PAHs and TCDD and mediated their toxic effects.^7,19 We first examined the effects of AhR agonists on AhR expression. High AhR levels were detected in HBE cells. We treated 16HBE cells with 10 μM BaP or 1 μM 3-MC for different time intervals. In the preliminary study, trypan blue staining was performed to calculate the IC₅₀, and the concentration of BaP or 3-MC used was approximately 10% of the IC₅₀. As shown in Fig. 1A, the BaP or 3-MC treatment decreased the mRNA levels of AhR by 28% or 19% after 12 h and this effect persisted through 48 h in 16HBE cells. In agreement with the changes in the mRNA level, we also observed the down-regulation of AhR protein in 16HBE cells (Fig. 1B). Meanwhile, we found that the expression of CYP1A1 increased in a time-dependent manner (Fig. 1C), indicating that the AhR-mediating pathway was activated by BaP or 3-MC. Previous studies have shown that the down-regulation of AhR protein was mediated via the 26S proteasome pathway following the nuclear export of AhR.²⁰ To eliminate the possibility that PAHs exposure promote the degradation of mRNA, we treated 16HBE cells with 10 μM BaP and 1 μg ml⁻¹ actinomycin D (ActD), a specific inhibitor of mRNA synthesis. As a result, we did not observe the effect of ActD on the reduction of AhR mRNA induced by BaP treatment (Fig. 1D), suggesting that BaP did not accelerate the decay of AhR mRNA when the transcription was inhibited. Taken together, these observations indicate that HBE cells exposed to AhR ligands, BaP or 3-MC lead to the transcriptional repression of AhR.

Fig. 1 The effect of BaP or 3-MC on the expression of AhR. 16HBE cells were treated with 10 μM BaP or 1 μM 3-MC, respectively, for different time points (12 h, 24 h, 36 h, and 48 h). RT-qPCR (A) and western blotting (B) were used to examine the levels of mRNA and protein of AhR (1 : 1500), respectively. (C) RT-qPCR was performed to examine the mRNA levels of CYP1A1. (D) 16HBE cells were treated with both BaP (10 μM) and ActD (1 μg ml⁻¹), BaP (10 μM) or ActD (1 μg ml⁻¹) respectively. The data of mRNA expression are expressed as mean ± SD for three independent experiments, *P < 0.05, compared with cells treated with DMSO. In a representative immunoblotting image, the value under each band indicates the fold change of protein expression relative to control cells.

Suppression of AhR attenuated BaP-induced cellular toxicity and DNA damage

To further explore the role of the down-regulation of AhR after BaP exposure, we generated stable 16HBE cells expressing two independent AhR-specific shRNAs (named 16HBESHAhR-1 and 16HBESHAhR-2 cells). Two shRNAs decreased AhR expression by approximately 70% compared to the control cells (16HBESHGFP) by immunoblotting analysis (Fig. 2A). Notably, suppression of AhR led to a decrease in CYP1A1 induction in 16HBE cells treated with 10 μM BaP or 3-MC for 48 h (Fig. 2B). Next, we performed cytokinesis-block micronucleus assay (CBMN), comet assay and detected the γH2AX level to examine whether the change of AhR expression had an impact on the DNA damage (Fig. 2C–E). As shown in Fig. 2C, we found that the frequencies of micronuclei in 1000 binucleated cells, tail moment of comet assay and γH2AX expression in response to 10 μM BaP treatment increased significantly in 16HBESHGFP cells. In contrast, the degree of DNA damage in 16HBESHAhR-1 and 16HBESHAhR-2 cells decreased over 40% compared to control cells, indicating that suppression of AhR conferred cells resistant to BaP-induced cytotoxicity and genome instability. Similar results were observed in 16HBESHAhR cells treated with 3-MC. Next, we examined the effect of AhR on cell proliferation. As expected, we observed an inhibitory effect on cell proliferation in HBE cells treated with 10 μM BaP. The suppression of AhR suppressed the proliferation of 16HBESHAhR-1 and 16HBESHAhR-2 cells by 21% and 25%, respectively, while a 35% inhibition in 16HBESHGFP cells was observed (Fig. 2D), indicating that an inhibitory effect of BaP on cell proliferation was greatly diminished when AhR was suppressed. Taken together, these results suggest that AhR acts as a regulator of CYP1A1 expression and attenuated the cytotoxicity and DNA damage induced by BaP.

Fig. 2 Suppression of AhR attenuated BaP-induced toxicity and DNA damage. (A) Lentiviral vectors encoding two independent shRNAs targeting AhR were introduced into 16HBE cells, generating stable cell lines, 16HBESHAhR-1 and HBESHAhR-2. The values under each band indicated the fold change of AhR levels normalized to β-actin expression relative to control cells. (B) RT-qPCR was performed to measure the expression of CYP1A1 in 16HBESHGFP, 16HBESHAhR-1, and HBESHAhR-2 cells exposed to 10 μM BaP or 1 μM 3-MC for 48 h. (C) Cytokinesis-blocked micronucleus (CBMN) assay in indicated cells treated with 10 μM BaP, 1 μM 3-MC or DMSO for 48 h. The frequency of micronuclei (MN) in 1000 binucleated (BN) cells is presented as mean ± SD for three independent experiments, *P < 0.05 compared to the SHGFP control, as determined by one-way ANOVA and followed by Dunnett's t test. (D) Tail moments of HBE cells indicated were determined by Comet assay. *P < 0.05 compared to the SHGFP control, as determined by one-way ANOVA and followed by Dunnett's t test. (E) γH2AX was detected with western blotting after BaP or 3-Mc for 48 h, with the H2A expression as normalization. (F) The indicated cells were treated with 10 μM BaP or DMSO for 48 h, 96 h, and 120 h, respectively. The cell number was counted at indicated time points. Data are reported as mean ± SD for three replicate wells, *P < 0.05, compared with control cells.

The effect of BaP or 3-MC on histone H3 acetylation

Previous studies have demonstrated that histone acetylation was involved in regulation of AhR expression.^16,21 It is clear that the acetylation of H3K9, H3K14, H3K18, and H3K27 is associated with open chromatin and active transcription.²² To explore the key histone acetylation involved in the transactivation of AhR in response to AhR agonists, we examined the effects of BaP or 3-MC on modifications of H3K9ac, H3K14ac, H3K18ac, and H3K27ac. Notably, we found that the levels of H3K9ac, H3K18ac, and H3K27ac significantly declined, accompanied with a remarkable decrease in AhR expression in response to BaP treatment (Fig. 3A and B). In contrast, we failed to detect a change in H3K14ac. Similar patterns were found when we treated 16HBE cells with 3-MC at different doses of 0, 0.1, 1.0 and 10.0 μM (Fig. 3A and B). These observations suggest that H3K9ac, H3K18ac, and H3K27ac may regulate AhR expression in response to AhR agonists.

Fig. 3 The effect of BaP or 3-MC on histone acetylation. 16HBE cells were treated with BaP and 3-MC at concentrations of 0 μM, 0.1 μM, 1.0 μM, and 10.0 μM, respectively, for 48 h. (B) Immunoblotting analysis was performed to examine the levels of H3K9ac, H3K14ac, H3K18ac, and H3K27ac. Fold change of histone acetylation and AhR levels normalized to H3 expression are indicated. (B) Dose-dependent induction of AhR mRNA by BaP or 3-MC at indicated concentrations. (C) 16HBE cells were treated with TSA at concentrations of 0 nM, 100 nM, 200 nM, and 400 nM. The cell lysates isolated at 24 h were subjected to immunoblotting analysis using the antibodies indicated. (D) RT-qPCR was performed to examine the expression of AhR. The data of mRNA expression detection are reported as mean ± SD for three independent experiments, *P < 0.05, compared with cells treated with DMSO.

To determine whether the suppression of AhR expression was reversible, we treated 16HBE cells with trichostatin A (TSA), a histone deacetylase inhibitor for 24 h. As shown in Fig. 3C, H3K9ac, H3K14ac, H3K18ac, and H3K27ac levels dramatically increased in a dose-dependent manner. In concert with this observation, we revealed that mRNA expression of AhR significantly increased by 0.5–2.0 fold examined by qPCR (Fig. 3D), reinforcing the notion that histone acetylation modified AhR expression upon administration of AhR agonists.

Reduced H3K18ac and H3K27ac were associated with AhR down regulation

To further address specific histone acetylations which were responsible for the regulation of AhR expression, we performed ChIP-qPCR on 16HBE cells treated with 200 nM TSA. Specific antibodies against H3K9ac, H3K14ac, H3K18ac, or H3K27ac were applied for ChIP analysis followed by qPCR to quantitate the presence of different regions from F1 to F8 of the AhR promoter (Fig. 4A), where TSS is the transcription start site. To examine the effect under histone acetylation inhibitor treatment, we detected the status of histone acetylation on the AhR promoter. The regions of F3 and F6 were predicted to be the H3K27Ac-enriched peak in the promoter of AhR in the UCSC Genome Browser (https://genome.ucsc.edu/), therefore we choose these two regions as the representatives for the entire promoter (Fig. 4A). As a result, the intensity of signals representing F3 and F6 fragments showed a 4–15 fold increase in H3K9ac-, H3K14ac- or H3K27ac-IP fractions upon TSA treatment, while no obvious change was found in the H3K18ac-IP fraction compared with DMSO-treated control cells (Fig. 4B). Taken together, these results indicate that altered H3 lysine acetylation might be the key epigenetic patterns that regulate AhR expression.

Fig. 4 Reduced H3K18ac and H3K27ac were responsible for AhR regulation. (A) Schematic display of fragment region spanning ∼1000 bp of the TSS of the AhR promoter. (B) 16HBE cells were treated with 200 nM TSA for 24 h. The fragments amplified with primer 3 (F3) and primer 6 (F6) located at 300 bp upstream and downstream of TSS. (C–G) 16HBE cells were incubated with 10 μM BaP, 1 μM 3-MC or DMSO for 48 h. Chromatin immunoprecipitation (ChIP) was performed with antibodies against RNA pol II (a positive control), H3K9ac, H3K14ac, H3K18ac, and H3K27ac. The results are expressed as the percent of amplified signal intensity of chromatin precipitated by specific antibodies indicated relative to the total input of sheared chromatin.

We further define the extent of H3 acetylation localization over the AhR promoter in order to better understand its role in the chromatin structure and AhR regulation in response to BaP treatment. To this end, we mapped the AhR promoter by chromatin immunoprecipitation with antibodies against H3K9ac, H3K14ac, H3K18ac, H3K27ac using 8 sets of specific PCR primers, spanning from −800 bp to +200 bp of the AhR promoter, to compare the enrichment of DNA fragments in cell samples treated with BaP. The antibody against Pol II served as a positive control (Fig. 4C). As shown in Fig. 4D and E, no obvious changes were found in H3K9ac- and H3K14ac-corresponding signals between BaP treatment and controls (Fig. 4D and E). In contrast, 10 μM BaP treatment led to a remarkable reduction of H3K18ac- and H3K27ac-corresponding signals of the AhR promoter (Fig. 4F and G), which was in accord with the BaP-induced AhR suppression shown in Fig. 1. In particular, upon BaP exposure, H3K18ac-corresponding signals decreased by 50–60% in the region spanning from −800 bp to −300 bp. With respect to H3K27ac, BaP treatment led to a reduction of the signals by 35–45% at the region close to TSS (from −400 bp to 0 bp). Meanwhile, a similar trend of histone modifications was also observed in cells with 3-MC treatment (Fig. 4), where H3K18ac and H3K27ac were decreased significantly. Taken together, these results indicated that the reduced levels of H3K18ac and H3K27ac in response to BaP or 3-MC treatment might be the epigenetic signatures in regulating AhR.

Discussion

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many responses to toxic environmental chemicals.²³ AhR linked the xenobiotic to the molecular events by triggering multiple intracellular signaling pathways, which lead us to explore the regulation of AhR and biological effects of dysregulated AhR in relation to cytotoxicity and DNA damage. In this study, we reveal that suppression of AhR transcription is associated with the reduced histone acetylation of H3K18ac and H3K27ac in response to the known AhR ligands, BaP and 3-MC. In addition, we clarify the correlation among suppression of AhR, CYP1A1 enzyme expression, and cellular response. The results of the present study provide additional insights into mechanisms regulating the transactivation of AhR.

Previous studies have demonstrated that the dysregulation of AhR play an important role in development of many kinds of diseases, such as smoking-induced lung cancer.²⁴ Activation of AhR results in an induction of enzyme CYP1A1/1B1, which may lead to increased formation of DNA adducts.²⁵ Given the evidence that AhR response to xenobiotic varied greatly,^26,27 we speculate that different mechanisms were involved in regulation of AhR and multiple levels at which AhR expression may be controlled. Epigenetic regulation on AhR activation has been reported previously. For example, AhR transcription was suppressed through promoter methylation that interrupted the accessibility in chromatin and inhibited the binding of transcription factor Sp1 to the promoter.²⁸ In addition, human cells treated with the histone deacetylase inhibitor (HDACi) and the DNA methyltransferase inhibitor resulted in a recovery of AhR transcription.^16,28 Prior studies from our laboratory have shown that miR-203 negatively regulates the expression of AhR, affecting the expression of its downstream genes and the resulting toxicity of the ligands.¹⁵ In this study, we sought to explore the correlation between BaP exposure and histone acetylation and the effect on regulation of AhR expression. We found that both H3K18ac and H3K27ac responded to ligands and regulated AhR transcription. Although ligand-induced histone modifications may not directly link to the activities in regulating specific genes, the recruitment of the AhR promoter in the ChIP fractions and a good correlation between the histone marks and ligand-induced AhR target gene transcription provide strong evidence and support the existence of this regulatory axis. Thus, histone marks might be useful parameters in the assignment of toxic equivalency.

Regulation of gene expression and chromatin remodeling can occur through post-translational modifications including acetylation, methylation, phosphorylation, glycosylation, sumoylation, and ADP-ribosylation.^29–31 Growing evidence has demonstrated that the aberrant histone modification plays an important role in cellular response to environmental chemicals and link to adverse health outcomes.^32–34 Thus, identification of specific epigenetic alterations, establishment of dose–response relationship and demonstrating human relevance are essential for the application of epigenetic biomarkers in risk assessment. It is increasingly recognized that altered histone methylation could be useful biomarkers in monitoring environmental chemical exposures and predicting the health endpoints in humans.^35,36 Indeed, in our unpublished data, we have identified several specific histone modifications that are associated with the extent of exposure and degree of DNA damage in peripheral lymphocytes of PAH-exposed workers. Here, we showed that histone acetylation of H3K18ac and H3K27ac was an important regulator for the AhR expression in response to ligands. However, the epigenetic mechanism composed by histone modification and other epigenetic factors, including DNA methylation and microRNAs (miRNAs), generate a complex network and dynamically regulate the transcriptional status of a specific gene. Thus, future studies are required to elucidate the comprehensive network of multiple epigenetic modes including DNA methylation, histone modifications, and miRNAs to clarify the function in induction of toxicity effects.

Conclusions

In summary, we identify that the decrease of two histone H3 acetylations, H3K18ac and H3K27ac, are correlated with down regulation of AhR transcription in response to BaP, which might function in avoiding the persistence of activated AhR target genes and confer cell resistance to cellular damage.

Acknowledgements

This work was supported by the key program of the NSFC (81430079, 81130050) and the NSFC (81072284, 81402715 and 81273098), the National “Twelfth Five-Year” Plan for Science & Technology (2014BAI12B02), the National Key Basic Research and Development Program (2010CB912803), the Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme GDUPS (2010), the Natural Science Foundation of Guangdong Province (S2012040007713), Research Fund for the Doctoral Program of Higher Education (20120171120067), and the Program of the Guangzhou City Pearl River New Star of Science and Technology (2013J2200020).

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4tx00088a

‡ These authors contributed equally to this work.

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