Effect of hexavalent chromium on histone biotinylation in human bronchial epithelial cells

Toxicology Letters 228 (2014) 241–247

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Effect of hexavalent chromium on histone biotinylation in human bronchial epithelial cells Bo Xia a,b,c , Xiao-hu Ren a,b , Zhi-xiong Zhuang b , Lin-qing Yang b , Hai-yan Huang b , Li Pang c , De-sheng Wu b , Jia Luo a , You-li Tan a , Jian-jun Liu b,∗∗ , Fei Zou a,∗ a Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, China b Key Laboratory of Modern Toxicology of Shenzhen, Medical Key Laboratory of Health Toxicology, Laboratory of Modern Toxicology, Shenzhen Centre for Disease Control and Prevention, Shenzhen 518055, China c College of Food Science and Technology, Hunan Agricultural University, East Renmin Road, Changsha 410128, Hunan, China

h i g h l i g h t s • • • •

Cr(VI) (≥2.5 ␮M) induced histone deacetylation in 16HBE cells. Cr(VI) (≤0.6 ␮M) increased histone biotinylation in 16HBE cells. Cr(VI) (≥0.6 ␮M) affected the distribution of biotinidase in 16HBE cells. Cr(VI)-induced histone deacetylation takes part in adjusting histone biotinylation.

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Article history: Received 14 April 2014 Received in revised form 7 May 2014 Accepted 8 May 2014 Available online 21 May 2014 Keywords: Chromium(VI) Histone acetylation Histone biotinylation

a b s t r a c t Chromium is a potent human mutagen and carcinogen. The capability of chromium to cause cancers has been known for more than a century, and numerous epidemiological studies have been performed to determine its carcinogenicity. In the post-genome era, cancer has been found to relate to epigenetic mutations. However, very few researches have focused on hexavalent chromium (Cr(VI))-induced epigenetic alterations. The present study was designed to investigate whether Cr(VI) would affect the level of a newfound epigenetic modification: histone biotinylation. Histone acetylation and histone biotinylation were studied in detail using human bronchial epithelial (16HBE) cells as an in vitro model after Cr(VI) treatment. Our study showed that Cr(VI) treatment decreased histone acetylation level in 16HBE cells. In addition, low doses of Cr(VI) (≤0.6 ␮M) elevated the level of histone biotinylation. Furthermore, immunoblot analysis of biotinidase (BTD), a major protein which maintains homeostasis of histone biotinylation, showed that the distribution of BTD became less even and more concentrated at the nuclear periphery in cells exposed to Cr(VI). Moreover, Cr(VI)-induced histone deacetylation may take part in the regulation of histone biotinylation. Together, our study provides new insight into the mechanisms of Cr(VI)-induced epigenetic regulation that may contribute to the chemoprevention of Cr(VI)-induced cancers and may have important implications for epigenetic therapy. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Chromium, a commonly used industrial metal, is the most abundant heavy metal in the Earth’s crust and is widely used by humans in various manufacturing industries, such as leather tanning and

∗ Corresponding author. Tel.: +86 20 61648301. ∗∗ Corresponding author. Tel.: +86 13501580129. E-mail addresses: [email protected] (J.-j. Liu), public [email protected] (F. Zou). http://dx.doi.org/10.1016/j.toxlet.2014.05.010 0378-4274/© 2014 Elsevier Ireland Ltd. All rights reserved.

wood treatment, which caused environmental pollution and health concern worldwide (Vutukuru, 2005). For more than 100 years, numerous epidemiological studies have been performed on workers exposed to hexavalent chromium (Cr(VI)) to determine its carcinogenicity (Holmes et al., 2008). The epidemiology study on chromate production workers in 1948 showed that, compared to 1.4% in the reference population, 21.8% of the chromate workers deaths were due to respiratory cancer (Machle and Gregorius, 1948). However, the potential molecular mechanisms of carcinogenicity of Cr(VI) remain unclear.

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As a well known potent oxidant, the formation of stable adducts caused by Cr(VI) is thought to contribute to its cytotoxic and genotoxic effects (Zhitkovich, 2005). Chemical and toxicological characteristics of chromium differ markedly on the basis of its valence state. Cr(VI) enters the cell through non-specific anion channels, and is subjected to a series of metabolic reductions to form the final stable metabolite Cr(III) (Stearns and Wetterhahn, 1994). These reactive intermediates and final products of Cr(VI) are able to induce the formation of stable DNA single or double-strand breaks, which may lead to a broad spectrum of DNA damage (Shi et al., 2004). The damage induced by Cr(VI) can lead to DNA replication inhibition, aberrant cell cycle checkpoints, dysregulated DNA repair mechanisms, which may all play an important role in Cr(VI) carcinogenesis (Nickens et al., 2010). Recently, many data are interesting and shed light on the role of Cr(VI) in epigenetic modifications, which are changes in cellular functions that occur without altering the genetic material resulting in tumorigenesis. The earliest report showed that Cr(VI) was able to induce DNA methylation and silence the expression of the gpt transgene in a cell line expressing the bacterial gpt reporter gene (Klein et al., 2002). Furthermore, in chromate induced human lung cancers, DNA methylation was found to be increased in the promoter region of the DNA mismatch repair (hMLH1) gene (Labra et al., 2004) and the tumor suppressor gene p16 (Kondo et al., 2006). Interestingly, Cr(VI) could cross link the histone deacetylase 1–DNA methyltransferase 1 complexes to the chromatin of the Cyp1a1 promoter and inhibit histone marks induced by AHR-mediated gene transactivation, including trimethylation of H3 Lys-4, phosphorylation of histone H3 Ser-10 and various acetylation marks in histones H3 and H4 (Schnekenburger et al., 2007). These data suggested that, other than genotoxic effects, epigenetics may contribute to the carcinogenicity of Cr(VI). Biotinylation of lysine (K) residues in histones is a novel, enigmatic histone modification. The discovery that biotinidase (BTD) has biotinyl-transferase activity, in addition to biotinidase hydrolase activity, presents possibility that biotin may participate directly in epigenetic changes, and the specific transfer of biotin to histones by BTD provides a possible explanation for why biotin is found in the nucleus of eukaryotic cells and the nature of its role in the regulation of protein transcription (Hymes and Wolf, 1999). Although biotin content in the nucleus is relatively small, it has been shown to regulate the transcription of glucokinase synthesis in early studies (Chauhan and Dakshinamurti, 1991). The fact that biotinylation increased for all classes of histones in G1, S, G2 and M phase of the cell cycle compared to G0 phase suggests that biotinylation of histones is a part of a general mechanism in nucleus (Stanley et al., 2001). The aim of the current study was to investigate whether Cr(VI) would affect the level of histone biotinylation, if it is so then what is the underlying mechanism? In our previous study, we found that Cr(VI) caused down regulation of BTD in human bronchial epithelial (16HBE) cells by modifications of histone acetylation (Xia et al., 2011). In the current study, we further investigated the effect of Cr(VI) on histone biotinylation. In addition, we also performed analyses of the distribution of the two histone biotinylation-related proteins, BTD and holocarboxylase synthetase (HCS), in Cr(VI)induced cells. Furthermore, we examined the effect of Cr(VI) on histone acetylation and tested the hypothesis that Cr(VI)-induced modifications of histone acetylation contribute to the regulation of histone biotinylation. Our study demonstrated that treatment of 16HBE cells with low doses of Cr(VI) (≤0.6 ␮M) increased the level of histone biotinylation. In addition, Cr(VI)-induced histone deacetylation caused down regulation of BTD, and that at least partially resulted in the level of histone biotinylation revert to normal. These results suggest that Cr(VI) can modulate the level of histone biotinylation processes involving histone acetylation.

2. Materials and methods 2.1. Chemicals, materials and cell lines Potassium chromate (K2 CrO4 ) was purchased from Sigma–Aldrich. It was dissolved in sterile deionized water to 50 mM as the stock solution and stored at 4 ◦ C. The stock solution was diluted with minimum essential medium (MEM) (without FBS) to experimental concentration before the chemical treatment. MEM, heatinactivated fetal bovine serum (FBS), antibiotics (streptomycin and penicillin) and trypsin-EDTA solution were all purchased from Gibco BRL-Life Technologies (Grand Island, NY, USA). Dimethyl sulfoxide (DMSO) was purchased from BIO BASIC Inc. The human bronchial epithelial (16HBE) cell line was a kind gift from Prof. Gruenert D.C. (California University, CA, USA). Trichostatin A (TSA) was from Sigma and stored at −20 ◦ C. 2.2. Cell culture and chemical treatment Cells were cultured in MEM medium supplemented with 10% (v/v) heatinactivated FBS and antibiotic supplement (penicillin 100 U/ml and streptomycin 100 ␮g/ml) at 37 ◦ C in a humidified incubator with 5% CO2 . When the cultured cells had grown to about 80% confluence, they were treated with different concentrations of Cr(VI) generally ranged from 0.3 to 5 ␮M for 24 h, and 0.1% sterile deionized water was used as the solvent control. In previous publications on the toxicology of chromate, concentrations of chromate ranged from 0.2 to 800 ␮M, and incubation times ranged from 1 min to several days (Holmes et al., 2008). Therefore, we chose 24 h as the incubation time in our study, and 5 ␮M (<1/2 LC50 ) was chosen as the highest concentration in this study. TSA was added to the medium after treatment with Cr(VI) to the termination of the experiments. Detailed treatment procedures are outlined in the following sections or described in the figure legends. 2.3. Confocal microscopy analysis Fluorescence staining experiments were conducted as described before (Narang et al., 2004). After treatment with Cr(VI) for 24 h, 16HBE cells, grown on coverslips, were fixed in freshly prepared 4% paraformaldehyde in PBS for 5 min followed by washing 3 times with PBS prior to permeabilization by 0.3% Triton-X in PBS for 5 min. Fixed cells were then incubated with primary antibodies diluted 1:1000 in 1% BSAPBST for about 16 h at 4◦ C, washed with PBS and incubated with fluorescein-labeled secondary antibody diluted 1:400 in PBST at 37◦ C for 30 min in the dark and counterstained with DAPI in PBS for 5 min. All the immunofluorescence staining were observed using Confocal Laser Scanning Microscopy (Leica Tcs sp5, Leica Microsystems) under dark field and the images were analyzed by Image-Pro Plus software (IPP). The following antibodies were used in this study: BTD (K-17) Antibody (sc48432, santa cruz), HCS (N-19) Antibody (sc-23732, santa cruz), IgG-FITC Secondary Antibodies (santa cruz). 2.4. Histone extraction Histone extraction was performed as described previously (Shechter et al., 2007). Briefly, cells were collected by trypsinization and centrifugation, and washed twice with PBS, then the cell pellet was resuspended in hypotonic lysis buffer (10 mM Tris pH 8.0, 1.5 mM MgCl2 , 1 mM KCl, 1 mM dithiothreitol, 20 mM N-ethylmaleimide, 10 mM Na-butyrate, phosphatase inhibitors and protease inhibitor cocktail) and incubated for 30 min at 4◦ C. The cells were then centrifuged and resuspended in 0.4 M HCl and incubated overnight at 4◦ C. Soluble chromatinized histones were precipitated by trichloroacetic acid (TCA) and the histone pellet was then dissolved in 100 ␮l ddH2 O. Because there were no housekeeping proteins left after histone extraction, we quantified the content of histones in each sample using Micro BCA Protein Assay Kit (23235, Pierce), which was used for normalization of the levels of histone modifications. The histone solution was separated on 15% SDS/PAGE gel. The following antibodies were used in this study: anti-acetyl-histone H3 Antibody (06-599, Upstate), anti-acetyl-histone H4 Antibody (06-598, Upstate), Streptavidin HRP Conjugate (21130, Pierce). 2.5. Western blot analysis After the indicated treatments, total protein of 16HBE cells was extracted with lysis buffer (7 M Urea, 2 M Thiourea, 4%CHAPS, 10 mM Tris). The lysates were then centrifuged and the insoluble debris was discarded. After boiling at 99◦ C for 5 min, proteins were then separated on 10% polyacrylamide gels, and transferred to polyvinylidene fluoride membranes. Membranes were blocked in Tris-buffered saline (TBS) containing 0.1% (vol/vol) Tween 20 and 5% fat-free milk for 1 h at room temperature, and then incubated with primary antibodies overnight at 4◦ C and with secondary antibodies for 1 h at room temperature. GAPDH was used for normalization of protein levels. Antibody signals were detected using Image J quantification software. The following antibodies were used in this study: HDAC2 (C-8) Antibody (sc-9959, santa cruz), HDAC3 (N-19) Antibody (sc-8138, santa cruz), BTD (K-17) Antibody (sc-48432, santa cruz), HCS (N-19) Antibody (sc-23732, santa cruz).

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Fig. 1. Treatment of 16HBE cells with various concentrations of Cr(VI) for 24 h decreased the levels of acetylation of histone H3 and H4. Histones were extracted and quantified as input control, and the levels of histone H3 and H4 acetylation were determined using western blot analysis after treating the cells with Cr(VI) for 24 h. Western blot data are presented in terms of percentage versus the results using non-Cr(VI) treated controls, which was assigned a value of 100%, and as means ± standard deviations. Significant difference versus non-Cr(VI)-treated control, *P < 0.05.

2.6. Statistical analysis Statistical analysis was conducted using an one-way ANOVA with post hoc Bonferroni correction. The data were presented as mean ± SD. All experiments were repeated at least three times independently. Results were considered to be statistically significant at P ≤ 0.05. SPSS 18.0 was used for the statistical analysis.

3. Results 3.1. Cr(VI) reduced histone H3 acetylation levels in 16HBE cells To examine whether Cr(VI) has epigenetic effects on histone acetylation, 16HBE cells were treated with Cr(VI) for 24 h. At the termination of the experiments, cells were harvested and histones were extracted for western blotting using antibody specific to H3Ac and H4Ac. The result revealed that high doses of Cr(VI) decreased global levels of histone H3 acetylation and histone H4 acetylation (Fig. 1). However, only H3 acetylation was significantly reduced. As histone acetylation is in part regulated by HDACs, we determined further whether Cr(VI) treatment altered the protein expression levels of HDAC2 and HDAC3 in 16HBE cells. Western

blot analysis showed that Cr(VI) increased both HDAC2 and HDAC3 levels significantly, as shown in Fig. 2.

3.2. Effects of Cr(VI) on histone biotinylation in 16HBE cells There is increasing evidence that histones are also covalently modified by the vitamin biotin (Camporeale et al., 2004; Hymes et al., 1995; Stanley et al., 2001), mediated by HCS (Narang et al., 2004) and BTD (Hymes et al., 1995). To further verify the effect of Cr(VI) on histone biotinylation, 16HBE cells were treated with Cr(VI) for 24 h, cells were then harvested and histones were extracted for western blotting using Streptavidin HRP Conjugate, an exclusive peroxidase-conjugated streptavidin biotin-binding protein. At doses lower than 0.6 ␮M, Cr(VI) increased the levels of histone biotinylation significantly, whereas this effect was absent at higher doses (Fig. 3). Subsequently, the protein expression levels of HCS and BTD in 16HBE cells were investigated by performing western blotting. As shown in Fig. 4, Cr(VI) increased the protein expression levels of HCS and reached significance at 5 ␮M. Different from HCS, 0.3 ␮M Cr(VI) significantly increased the protein

Fig. 2. Treatment of 16HBE cells with various concentrations of Cr(VI) for 24 h increased the protein expression levels of HDAC2 and HDAC3. The levels of HDAC2 and HDAC3 in cell lysates were determined using western blot analysis after treating the cells with Cr(VI) for 24 h. Data are presented in terms of percentage versus the results using non-Cr(VI) treated controls, which was assigned a value of 100%, and as means ± standard deviations. Significant difference versus non-Cr(VI)-treated control, *P < 0.05.

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significant proportion of BTD protein is associated with the core nuclear matrix. Immunoblot analysis with HCS antibodies showed that HCS fractionates were primarily in cell nucleus, and Cr(VI) did not affect the distribution of HCS (Fig. 6). 3.4. Effect of Cr(VI) and TSA on histone biotinylation in 16HBE cells

Fig. 3. Effect of Cr(VI) on biotinylation of histones in 16HBE cells. Histones were extracted and quantified as input control, and the levels of histone biotinylation were determined using western blot analysis after treating the cells with Cr(VI) for 24 h. Western blot data are presented in terms of percentage versus the results using non-Cr(VI) treated controls, which was assigned a value of 100%, and as means ± standard deviations. Significant difference versus non-Cr(VI)-treated control, *P < 0.05.

expression levels of BTD, whereas higher doses decreased BTD levels dose dependently and approached significance at 5 ␮M. 3.3. Influence of Cr(VI) on the distribution of BTD and HCS in 16HBE cells We were also interested to examine the influence of Cr(VI) on the distribution of BTD and HCS in 16HBE cells. For this purpose, 16HBE cells were treated with various concentrations of Cr(VI) for 24 h. At the termination of the experiment, cells were fixed for fluorescence staining experiments. As shown in Fig. 5, BTD fractionates were found both in cell nucleus and cytoplasm in normal cells, and that treatment with Cr(VI) removed cytoplasmic BTD but left nuclear BTD in an evenly distributed, punctate pattern. Following treatment with Cr(VI), the distribution of BTD became less even and more concentrated at the nuclear periphery. BTD was still detected after high Cr(VI) treatment, indicating that a

To further verify the relationship between histone deacetylation and changes of histone biotinylation in 16HBE cells, we examined the effect of Cr(VI) and TSA on histone biotinylation. For this purpose, 16 HBE cells were treated with 5 ␮M Cr(VI) for 24 h, and then with/without 500 nM TSA for another 24 h. Cells were harvested, cell lysates prepared and subjected to the analysis of the protein expression levels of HCS and BTD using western blot analysis. While histones were extracted for western blotting using Streptavidin HRP Conjugate. As shown in Fig. 7A and B, treatment of cells with TSA increased the protein expression levels of BTD, but did not alter HCS. Furthermore, in Cr(VI)-induced cells, TSA significantly increased the protein expression levels of BTD. Analysis of histone biotinylation revealed that TSA increased the level of histone biotinylation both in cells treated with and without Cr(VI) (Fig. 7C and D). However, no significant changes have been observed. 4. Discussion Despite the well-recognized carcinogenic potential of chromium, the molecular mechanism underlying its celltransforming ability remains poorly understood (Salnikow and Zhitkovich, 2008). Epigenetic alterations have appeared as a key mechanism involved in carcinogenicity. Changes in normal epigenetic patterns necessary for transcriptional regulation may cause serious disruptions. Previous evidences suggested that Cr(VI) was able to alter epigenetic homeostasis which may play important roles in promoting carcinogenesis. In cultured mammalian cells and plants, Cr(VI) was found to be able to induce genomewide or gene-specific DNA methylation changes (Klein et al., 2002; Labra et al., 2004). However, in our study, we did not find significant changes in global DNA methylation in Cr(VI)-induced 16HBE cells (data not published). Several studies indicated that the level of H3K9me2 was increased after exposure to Cr(VI), which was known to be crucial for DNA methylation (Jackson et al., 2004; Tamaru et al., 2003). In addition, Cr(VI) was able to cross link the histone deacetylase

Fig. 4. Effect of Cr(VI) on the protein expression levels of HCS and BTD. The protein expression levels of HCS and BTD in cell lysates were determined using western blot analysis after treating the cells with Cr(VI) for 24 h. Western blot data are presented in terms of percentage versus the results using non-Cr(VI) treated controls, which was assigned a value of 100%, and as means ± standard deviations. Significant defference versus non-Cr(VI)-treated control, *P < 0.05.

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Fig. 5. Subcellular distribution of BTD in 16HBE cells. After treating the cells with Cr(VI) for 24 h, endogeneous BTD were detected by immunofluorescence using anti-BTD antibody and FITC-conjugated secondary antibody. The nucleus was revealed by DAPI.

1-DNA methyltransferase 1 complexes to the chromatin of the Cyp1a1 promoter and inhibit various histone marks, such as acetylation marks in histones H3 and H4 (Schnekenburger et al., 2007). Furthermore, global changes in various histone tail modifications were observed in human lung carcinoma A549 cells after exposure to Cr(VI) (Arita and Costa, 2009). In this study, we found that Cr(VI) decreased global levels of histone H3 acetylation significantly. In addition, Cr(VI) increased both HDAC2 and HDAC3 levels significantly. Interestingly, previous study showed that HDAC2 participated in the DNA-damage response and was rapidly recruited to DNA-damage sites to promote hypoacetylation (Miller et al., 2010). Furthermore, HDAC2-depleted cells were hypersensitive to

DNA-damaging agents and showed sustained DNA-damage signaling. Therefore, Cr(VI) may induce the expression of HDAC2 by causing DNA-damages, and then gave rise to histone H3 hypoacetylation. As a relatively new field of epigenetics, studies of biological roles for biotinylation of histones were scarce. However, it may participate in several biological processes. Firstly, biotinylation of histones increases in response to cell proliferation in human lymphocytes (Stanley et al., 2001). It increases early in the cell cycle (G1 phase) and remains increased during later phases (S, G2 and M phase). Then, studies in chicken erythrocytes showed that biotinylation of histones is enriched in transcriptionally silent chromatin

Fig. 6. Subcellular distribution of HCS in 16HBE cells. After treating the cells with Cr(VI) for 24 h, endogeneous HCS were detected by immunofluorescence using anti-HCS antibody and FITC-conjugated secondary antibody. The nucleus was revealed by DAPI.

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Fig. 7. Effect of Cr(VI) and TSA on histone biotinylation in 16HBE cells. Comparative effect of Cr(VI) and TSA on (A) the protein expression levels of HCS and BTD and (C) histone biotinylation in 16HBE cells were determined using western blot analysis after treating the cells with Cr(VI) for 24 h. (B) and (D) Western blot data are presented in terms of relative density of control as means ± standard deviations in different treatment groups. Significant difference versus control, *P < 0.05.

(Peters et al., 2002). Furthermore, biotinylation of histones might play important roles in the cellular response to DNA damage (Peters et al., 2002). Our results showed that Cr(VI) induced a significant increase of histone biotinylation in the 0.3 ␮M and 0.6 ␮M groups. Two major proteins, BTD and HCS, maintain homeostasis of histone biotinylation in cells. Changes in BTD and HCS directly impact the status of histone biotinylation and accordingly disturb the normal functions of biotinylation. In our previous work, we found that Cr(VI) caused down regulation of BTD in 16HBE cells (Xia et al., 2011). In this study, we found that BTD was distributed equally in cell nucleus and cytoplasm in normal cells, and treatment with Cr(VI) removed cytoplasmic BTD but left nuclear BTD. This observation highlights the function of BTD in maintaining histone biotinylation, and is consistent with the finding that BTD plays key roles in the preservation of histone biotinylation, which may also demonstrate the biological importance of histone biotinylation (Hassan and Zempleni, 2006). As another important histone biotinylation-correlated protein, the distribution of HCS was not affected by Cr(VI). It has recently become apparent that epigenetic modifications, such as DNA methylation and histone acetylation, can be dependent on one another, and that this crosstalk can be of physiological importance. Relationships among these epigenetic changes have implications for understanding normal development as well as somatic cell reprogramming and tumorigenesis (Cedar and Bergman, 2009). To investigate whether Cr(VI)-induced deacetylation of histone would affect the level of histone biotinylation, we

used TSA, a well-known HDAC inhibitor, as controls. Our results indicated that, TSA significantly increased the protein expression levels of BTD both in normal cells and Cr(VI)-induced cells, which is consistent with our previous work (Xia et al., 2011). Furthermore, Analysis of histone biotinylation showed that TSA increased the level of histone biotinylation both in cells treated with and without Cr(VI), though no significant changes have been found. These results indicate that the action of Cr(VI) is opposite to TSA. In summary, we are reporting for the first time that Cr(VI) can elevate the level of histone biotinylation in 16HBE cells, and Cr(VI)-induced histone deacetylation may take part in the regulation of histone biotinylation. These findings are of importance for understanding the relationship between histone biotinylation and histone acetylation in Cr(VI) induced cells. Further understanding the interplay between them could give important insights into the mechanisms of Cr(VI)-induced carcinogenesis.

Conflict of interest The authors declare that there are no conflicts of interest.

Transparency document The Transparency document associated with this article can be found, in the online version.

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