Archives of Toxicology

, Volume 91, Issue 5, pp 2135–2150 | Cite as

Butyrate alters expression of cytochrome P450 1A1 and metabolism of benzo[a]pyrene via its histone deacetylase activity in colon epithelial cell models

  • Ondřej Zapletal
  • Zuzana Tylichová
  • Jiří Neča
  • Jiří Kohoutek
  • Miroslav Machala
  • Alena Milcová
  • Michaela Pokorná
  • Jan Topinka
  • Mary Pat Moyer
  • Jiřina Hofmanová
  • Alois Kozubík
  • Jan Vondráček
Toxicokinetics and Metabolism

Abstract

Butyrate, a short-chain fatty acid produced by fermentation of dietary fiber, is an important regulator of colonic epithelium homeostasis. In this study, we investigated the impact of this histone deacetylase (HDAC) inhibitor on expression/activity of cytochrome P450 family 1 (CYP1) and on metabolism of carcinogenic polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP), in colon epithelial cells. Sodium butyrate (NaBt) strongly potentiated the BaP-induced expression of CYP1A1 in human colon carcinoma HCT116 cells. It also co-stimulated the 7-ethoxyresorufin-O-deethylase (EROD) activity induced by the 2,3,7,8-tetrachlorodibenzo-p-dioxin, a prototypical ligand of the aryl hydrocarbon receptor. Up-regulation of CYP1A1 expression/activity corresponded with an enhanced metabolism of BaP and formation of covalent DNA adducts. NaBt significantly potentiated CYP1A1 induction and/or metabolic activation of BaP also in other human colon cell models, colon adenoma AA/C1 cells, colon carcinoma HT-29 cells, or in NCM460D cell line derived from normal colon mucosa. Our results suggest that the effects of NaBt were due to its impact on histone acetylation, because additional HDAC inhibitors (trichostatin A and suberanilohydroxamic acid) likewise increased both the induction of EROD activity and formation of covalent DNA adducts. NaBt-induced acetylation of histone H3 (at Lys14) and histone H4 (at Lys16), two histone modifications modulated during activation of CYP1A1 transcription, and it reduced binding of HDAC1 to the enhancer region of CYP1A1 gene. This in vitro study suggests that butyrate, through modulation of histone acetylation, may potentiate induction of CYP1A1 expression, which might in turn alter the metabolism of BaP within colon epithelial cells.

Keywords

CYP1A1 Butyrate Polycyclic aromatic hydrocarbons DNA adducts Histone deacetylases Colon epithelial cells 

Introduction

Colorectal cancer (CRC) is a major cause of cancer mortality and morbidity in Western world (Markowitz and Bertagnolli 2009). Most of CRC are sporadic, and both dietary and lifestyle factors have been strongly implicated in the CRC; nevertheless, the molecular mechanisms underlying the pathogenesis of CRC are still not fully understood. Polycyclic aromatic hydrocarbons (PAHs) belong among the dietary carcinogens that are formed during high temperature cooking, which are classified as either possibly carcinogenic or carcinogenic to humans (IARC 2010). The epithelial intestinal cells can come to a direct contact with these contaminants in digested food (Cavret and Feidt 2005); however, these compounds, such as benzo[a]pyrene (BaP), must undergo metabolic activation in mammalian cells via the action of cytochrome P450 family 1 enzymes (CYP1), in order to become mutagenic/carcinogenic (Phillips 1999). CYP1 family members play a key role in the production of ultimate genotoxic (+)-anti-7α,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydro-BaP (BPDE) metabolite of BaP, which forms covalent DNA adducts and is a prerequisite for induction of further forms of DNA damage (Xue and Warshawsky 2005).

Butyrate is a short-chain fatty acid produced by bacterial fermentation of dietary fiber within colon (primarily by clostridial strains within clusters IV and XIVa) that is a major energy source for colon epithelial cells (Bultman 2014; Irrazábal et al. 2014). It plays a key role in colonic epithelium homeostasis, and both butyrate and related molecules have been extensively studied as potential chemopreventive agents in CRC, because butyrate supports growth of normal colonocytes, while it simultaneously decreases proliferation and/or induces apoptosis/differentiation of neoplastic CRC cells (Bultman 2014). Butyrate has been shown to improve intestinal barrier function and to inhibit inflammation, factors contributing both to tumor initiation and promotion in colon (Irrazábal et al. 2014; Schilderink et al. 2013; Schwabe and Jobin 2013). Multiple mechanisms have been proposed to contribute to the effects of butyrate in CRC, including e.g.: (1) its role as an energy source (for normal colonocytes); (2) its action as both histone deacetylase (HDAC) inhibitor and modulator of activity of histone acetyltransferases (HATs) (Donohoe et al. 2012); or (3) butyrate-mediated activation of specific G-protein coupled receptors (Bultman and Jobin 2014; Singh et al. 2014). Although butyrate is generally considered to provide protection against CRC, this view has also been challenged, since additional genetic or nutritional factors may significantly modulate the action of butyrate in colon epithelium, and butyrate has been even shown to support experimental carcinogenesis in mouse models of CRC (Belcheva et al. 2014). The epidemiology studies also do not provide a clear support for the protective role of butyrate in CRC (Bultman and Jobin 2014). The effects of butyrate may depend on further dietary factors (in particular on lipids), the sources of butyrate or a timing of exposure toward this short-chain fatty acid (Lupton 2004). Nevertheless, the protective effects of butyrate on CRC development have also been proposed to include protection against DNA damage elicited by carcinogens, which could be mediated via increased activation of xenobiotic-metabolizing enzymes, such as glutathione-S-transferases (Scharlau et al. 2009).

CYP1A1 protein is xenobiotic-metabolizing enzyme that has been detected in both normal and tumor colon mucosa, and which is inducible by the aryl hydrocarbon receptor (AhR) ligands in colon cells (for recent reviews, see Beyerle et al. 2015; Gundert-Remy et al. 2014). Presently, the data on CYP1A1 regulation and/or activity in human colon are limited. Nevertheless, both we and others have observed that CYP1A1 expression/activity can be induced by the AhR ligands in human colon cancer cell models and that it plays a major role in formation of genotoxic BaP metabolites in human colon cells in vitro (Hockley et al. 2008; Kabátková et al. 2015). The AhR is a principle regulator of CYP1A1 expression; however, transcriptional regulation of CYP1A1 also depends on a number of additional regulatory mechanisms, including the activity of transcriptional co-regulators modifying chromatin (Androutsopoulos et al. 2009). Importantly, factors modifying histone acetylation, such as HDAC inhibitors, could have a major impact on CYP1A1 regulation. Since enterocytes are directly exposed to millimolar concentrations of butyrate, which is a natural HDAC inhibitor, it can be hypothesized that butyrate might affect both the expression/activity of CYP1A1 and metabolism of compounds such as BaP.

The impact of HDAC inhibition on CYP1A1 regulation remains in part controversial, since conflicting results have been reported, indicating that HDAC role could be both species- and tissue-dependent. Most of the previous studies evaluating the impact of HDAC inhibitors on induction of CYP1A1 or other CYP1 enzymes have relied on application of trichostatin A (TSA). Several studies have suggested that inhibition of HDAC by TSA leads to a significant up-regulation of CYP1A1 in fibroblasts, hepatoma cells or other cell types (Beedanagari et al. 2010b; Gradin et al. 1999; Tigges et al. 2013; Xu et al. 1997), while others have observed this effect only in murine, but not in human cells (Suzuki and Nohara 2007). In contrast, yet additional studies have reported that TSA inhibits CYP1A1 or CYP1B1 induction in human cell lines, which has been tentatively linked to inhibition of cytoplasmic HDAC6 (Hooven et al. 2005; Kekatpure et al. 2009). The data on the effects of butyrate on CYP1A1 induction are less comprehensive, and again, conflicting results have been reported concerning its effects. Butyrate has been found to co-stimulate the AhR ligand-mediated CYP1A1 induction in human skin fibroblasts or in human cervical cancer cells (Haarmann-Stemmann et al. 2007), while it did not modulate CYP1A1 or Cyp1a1 transcription in human and mouse hepatoma cell lines, respectively (Gradin et al. 1999; Wei et al. 2004). Nevertheless, butyrate has been shown to significantly modify both presence of HDAC1 within mouse Cyp1a1 promoter/enhancer and acetylation of core histones within the same regulatory regions (Schnekenburger et al. 2007). The reports summarized above seem to indicate that the impact of HDAC inhibition by butyrate on CYP1A1 regulation could be cell type-specific and that butyrate can be expected to modulate chromatin properties within regulatory regions of human CYP1A1 gene. However, there is presently a lack of data based on human colon cell models, where the effects of butyrate could be of relevance, because of its high levels within intestinal lumen.

Taken together, unlike the impact of butyrate on energy metabolism in colon cells, the effects of butyrate on metabolism of xenobiotics have received far less attention during recent years. Several lines of evidence suggest that butyrate might alter the inducibility of the AhR target genes, such as CYP1A1 within colon epithelial cells. In the present in vitro study, we therefore investigated the effects of butyrate on induction of CYP1A1 and BaP bioactivation in various human colon cell models. Our results suggest that butyrate, through its HDAC-inhibitory activity, may significantly potentiate the AhR-dependent induction of a key enzyme involved in metabolism of BaP, CYP1A1, which may, in turn, modulate its genotoxic action within colon epithelial cells.

Materials and methods

Chemicals

BaP (CAS no. 50-32-8, purity 99.9%) was provided by Ehrenstorfer (Augsburg, Germany), stock solutions were prepared in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored in the dark. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was purchased from Cambridge Isotope Laboratories (Andover, MA), dissolved in DMSO and stored in the dark. Sodium butyrate (NaBt; purity 98.5%) was provided by Sigma-Aldrich (Prague, Czech Republic), stock solutions were prepared in ultrapure water, obtained from a Milli-Q UF Plus water system (Millipore, Molsheim, France). TSA (purity 98%) was provided by Sigma-Aldrich, and stock solutions were prepared in DMSO. Suberanilohydroxamic acid/vorinostat (SAHA, purity 98%) was provided by Cayman Chemical (Ann Arbor, Michigan). BaP metabolite standards (BaP-r-7,t-8,t-9,c-10-tetrahydrotetrol(±) (BaP-tetrol I-1), BaP-r-7,t-8,t-9,t-10-tetrahydrotetrol(±) (BaP-tetrol I-2), BaP-r-7,t-8,c-9,t-10-tetrahydrotetrol(±) (BaP-tetrol II-1), BaP-trans-7,8-dihydrodiol(±) (BaP-7,8-DHD), BaP-trans-9,10-dihydrodiol (BaP-9,10-DHD), BaP-trans-4,5-dihydrodiol(±) (BaP-4,5-DHD), BaP-3,6-dione, BaP-1,6-dione, BaP-6,12-dione, 1-OH-BaP, 3-OH-BaP and 9-OH-BaP) were all obtained from the National Cancer Institute’s Chemical Carcinogen Standard Reference Repository (Midwest Research Institute, Kansas City, MO). Ethyl acetate (p.a. ACS), methanol (p.a. ACS), and methanol (HPLC gradient grade) were all purchased from Merck.

Cell culture

HCT116 and HT-29 cell lines were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). HCT116 and HT-29 cells were cultured in McCoy’s 5A medium supplemented with 1× penicillin/streptomycin mix (PAA Laboratories GmbH, Pasching, Austria). Both cell lines were grown with 10% fetal bovine serum (FBS; Gibco, Invitrogen, Carlsbad, CA, USA). Human colon adenoma cell line AA/C1 was kindly provided by prof. C. Paraskeva (Department of Pathology and Microbiology, University of Bristol, Bristol, UK). AA/C1 cells were cultured in high-glucose DMEM supplemented with hydrocortisone (1 µg/ml), insulin (8 µg/ml) and 20% FBS. The NCM460D™ cells, derived originally from normal human colon mucosal epithelium (Moyer et al. 1996), were provided by INCELL Corporation (San Antonio, TX, USA). The cells were routinely propagated under standard conditions in M3:10™ medium provided by the supplier of the cell line (INCELL Corporation), supplemented with 10% FBS. Human colon adenocarcinoma Caco-2 cells (ATCC) were cultured in DMEM supplemented with 1× penicillin/streptomycin mix and 10% FBS. In order to elicit their differentiation, Caco-2 cells were seeded at initial density of 40,000 cells/cm2 into 35-mm cell culture dishes and grown for 21 days in DMEM supplemented with 10% FBS and antibiotics, with the medium exchange every two days. At this time point, Caco-2 cell reportedly fully cease to proliferate, reach an optimum degree of morphological and functional differentiation, and they are sensitive to various AhR ligands (Pandrea et al. 2000; Le Ferrec et al. 2002). All cell lines were cultured at 37 °C in 5% CO2 and 95% humidity.

Western blotting

Cells were washed twice with cold PBS and lysed in SDS sample buffer (1% SDS, 10% glycerol, 0.1 M Tris pH 7.4, protease inhibitor cocktail (Sigma-Aldrich), 1 mM Na3VO4, 1 mM NaF), heated for 10 min at 90 °C and sonicated. Protein concentrations were estimated using Bio-Rad DC Protein Assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of proteins were separated on SDS–polyacrylamide gels and transferred onto a PVDF membrane (Millipore, Prague, Czech Republic). The following primary antibodies were used: anti-CYP1A1 (cat. no. H00001543-B01P, Abnova, Taipei, Taiwan); anti-acetyl-histone H3 (Lys14, cat. no. 07-353, Millipore, Temecula, CA); anti-acetyl-histone H4 (Lys16, cat. no.07-329, Millipore); and anti-β-actin (cat. no. A5441, Sigma-Aldrich). Anti-rabbit and anti-mouse secondary antibodies conjugated with horseradish peroxidase, and ECL-Plus reagents were purchased from GE Healthcare (Little Chalfont, UK), and they were used according to the manufacturer’s instructions.

Real-time quantitative RT-PCR (qRT-PCR)

After treatment cells were washed twice with cold PBS and lysed with cell lysis buffer. Total RNA was isolated from cells using the NucleoSpin®RNA II purification kit (Macherey-Nagel, Düren, Germany). The amplification of the samples was carried out with Superscript III Platinum One-Step qRT-PCR kit (Invitrogen, Carlsbad, CA, USA). Primers and the respective probes were provided by Generi Biotech (Hradec Králové, Czech Republic) or Roche Diagnostics (Mannheim, Germany). qRT-PCRs were performed using RotorGene 6000 (Corbett Life Science, Qiagen) thermocycler. The sequences of primers and probes have been reported previously (Kabátková et al. 2015).

TOP/FOP luciferase reporter assay of the transcriptional activity of β-catenin

HCT116 cells were plated in 24-well plates in complete cultivation medium without antibiotics. After 24-h cultivation, cells were transiently transfected with pRL-TK vector (constitutively active vector encoding Renilla luciferase) and either Super 8× TOPFlash (β-catenin-responsive firefly luciferase reporter plasmid) construct or Super 8× FOPFlash construct (the negative control) (Veeman et al. 2003), as previously described (Kabátková et al. 2015). The transfections were carried out in a total volume 500 μl containing 50 ng pRL-TK vector and either 200 ng Super 8× TOPFlash construct or 200 ng Super 8× FOPFlash construct, and 1 μl of Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA). Transfection mix was removed 6 h later, and cells were cultivated for another 24 h in McCoy’s medium with antibiotics, followed by exposure to indicated compounds for 24 h. For luciferase assay Dual-Luciferase Reporter Assay System (Promega, Madison, WI) was used, according to the manufacturer’s instructions. The firefly luciferase activity was normalized against the Renilla luciferase activity, in order to control for transfection efficiency, and a fold increase in TOPFlash activity compared to FOPFlash was calculated.

Measurement of 7-ethoxyresorufin-O-deethylase (EROD) activity

After incubation with tested compounds or solvent control, medium was removed and the cells were washed twice with cold PBS. To each well 150 μl of ultrapure water was added to swell the cells and after 15 min incubation at room temperature, the plates were placed at −80 °C for at least 30 min to lyse the cells. To measure resorufin production, into 96-well plates, it was pipetted 20 µl of each samples in duplicates. To each well, they were added: 2 mM 3,3′-methylene-bis(4-hydroxycoumarin) (Sigma-Aldrich) in 50 μl of Tris-sucrose buffer (pH 8.0), and 25 μl 20 μM 7-ethoxyresorufin (Sigma-Aldrich) and the plates were pre-incubated for 20 min at 37 °C. After that, 25 μl of 1 mM NADPH solution were added per well, in order to start the reaction and plates were incubated for 1 h at 37 °C. The resorufin production was measured in Fluostar Galaxy (BMG Labtech GmbH, Ortenberg, Germany) with an excitation filter at 530 nm and an emission filter at 590 nm. Protein concentrations were estimated as described above.

Detection of DNA adducts

Detection of DNA adducts has been performed as described previously (Umannová et al. 2008). Briefly, cells were washed twice with cold PBS, scraped into Eppendorf tubes, centrifuged and the cell pellets were stored at −80 °C. DNA was isolated using RNAses A and T1 and proteinase K treatment followed by phenol/chloroform/isoamyl alcohol. DNA samples were kept at −80 °C until analysis. Briefly, DNA samples (6 μg) were digested with a mixture of micrococcal endonuclease (Sigma-Aldrich, St. Louis, MO, USA) and spleen phosphodiesterase (MP Biomedicals, Strasbourg, France) for 4 h at 37 °C. Nuclease P1 (Yamasa Corporation, Chiba-ken, Japan) was used for adduct enrichment. Labeled DNA adducts were resolved via multidirectional TLC on 10 cm × 10 cm PEI-cellulose plates. Autoradiography was carried out at −80 °C for 24 h. Radioactivity of distinct adduct spots was measured using liquid scintillation counting. To determine the exact amount of DNA in each sample, aliquots of the DNA enzymatic digest (1 μg of DNA hydrolysate) were analyzed for nucleotide content using reverse-phase high-performance liquid chromatography with UV detection, which simultaneously controlled for DNA purity. DNA adduct levels were expressed as relative DNA adduct levels per 108 nucleotides. A BPDE-DNA adduct standard was run in a parallel sample, in order to normalize the calculated DNA adduct levels.

Analysis of BaP metabolites

Cells were incubated with the test compounds or solvent control. After that, cells were washed twice with cold PBS, scraped into Eppendorf tubes, centrifuged and the cell pellets were stored at −80 °C. Determination of BaP metabolites was then performed as recently described (Kabátková et al. 2015). Briefly, cell pellets were extracted twice with 700 μl of ethyl acetate, combined extracts were dried under a stream of nitrogen, re-dissolved in 50 μl of methanol and aliquot of 10 µl was injected into the HPLC column. The Agilent 1200 chromatographic system (Agilent Technologies, Santa Clara, CA), consisting of a binary pump, vacuum degasser, autosampler and thermostated column compartment, was used for the LC–MS/MS analyses, using the conditions described previously (Kabátková et al. 2015). Following their determination, the levels of BaP metabolites were normalized to total protein concentrations, which were estimated as described above.

Chromatin immunoprecipitation (ChIP)

After incubation with tested compounds or solvent control, DNA–protein complexes were cross-linked with 1% formaldehyde and 125 mM glycine was added to stop the reaction. Samples were washed with PBS and sonicated in breaking buffer (0.5% SDS, 20 mM Tris pH 8.0, 2 mM EDTA, 0.5 mM EGTA, 0.5 mM PMSF and complete protease inhibitory tablet). Protein A-Sepharose 4B beads (Sigma-Aldrich, Prague, Czech Republic) in IP buffer (0.5% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0, 150 mM NaCl and 10% glycerol) were added for 2 h at 4 °C for non-specific binding. Pre-cleared supernatants were then incubated with beads and specific antibody anti-HDAC1 (cat. no. 05-100, Millipore) or no antibody overnight at 4 °C. Samples were then washed 1× with low salt wash (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0, 150 mM NaCl), 3× with high salt wash (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0, 500 mM NaCl), 1× by LiCl wash (2 mM EDTA, 20 mM Tris pH 8.0, 0.25 M LiCl, 1% NP40, 1% NaDeoxycholate) and 2× with TE wash (10 mM Tris pH 8.0, 1 mM EDTA). To isolate DNA, we incubated precipitates and inputs in elution buffer (1% SDS, 0.1 M NaHCO3) 2× for 15 min, after that 20 μl of 5 M NaCl was added per sample and they were heated to 65 °C for 3.5 h. After heating, 15 μl 1 M Tris pH 7.8, 2 μl glycoblue and 2 μl Proteinase K were added to each sample that were incubated at 37 °C for 30 min. Then we performed phenol/chloroform extractions and finally purified DNA with isopropanol, and DNA samples were then dried and dissolved in water. The amplification of the samples was carried out with SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich). Primers were provided by Generi Biotech (Hradec Králové, Czech Republic). RT-PCRs were performed using LightCycler system (Roche Diagnostics). For analysis of HDAC1 binding to the −0.8 kb enhancer region of human CYP1A1 gene, we used the following primer sequences: forward 5′-CAATCAAAGCACTAGCCACCCC-3′, reverse 5′-CGCCTCTTGATTGAAGGATCGG-3′.

Data analyses

Data were expressed as mean ± SD for at least three independent replications. Comparisons between treatments were made by one-way analysis of variance (ANOVA) with post hoc comparisons of the means made by Tukey’s range test. For comparisons of two groups, t test was used. If the variances were non-homogeneous, nonparametric alternative of ANOVA or Mann–Whitney U test were used. A P value of <0.05 was considered significant.

Results

NaBt co-stimulates induction of CYP1A1 mRNA, protein and activity in human colon HCT116 cells

Concentrations of butyrate vary between proximal and distal colon, as well as within colonic crypts gradient. Based on: (1) reported physiological concentrations of butyrate within mouse and human colon (Donohoe et al. 2012; Hamer et al. 2008); (2) distinct effects of both low (0.5 mM) and high (3 mM) NaBt concentrations on human colon carcinoma HCT116 cells (Donohoe et al. 2012); and (3) our own previous data on effects of high dose butyrate in the same cell line (Hofmanová et al. 2012), we selected the 0.5 and 3 mM concentrations for our experiments. As shown in supplementary Online Resource 1, NaBt at concentrations 1 mM and higher reduced numbers of adherent HCT116 cells, with 3 mM concentration inducing apoptosis, as indicated by the presence of cleaved poly(ADP-ribose) polymerase 1 (PARP) fragment in HCT116 cells (after 24-h treatment). The lower 0.5 mM NaBt had no effect on PARP cleavage and it did not reduce numbers of adherent HCT116 cells on its own.

Both concentrations of NaBt significantly up-regulated levels of the BaP-induced CYP1A1 and CYP1B1 mRNAs; this effect was more pronounced in case of CYP1A1 enzyme (Fig. 1a). NaBt (both 0.5 and 3 mM concentrations) also significantly increased levels of CYP1A1 protein, at both 24- and 48-h treatment times (Fig. 1b). We did not detect significant changes in CYP1B1 protein (data not shown). Based on this, we then selected the lower 0.5 mM concentration of NaBt, which did not alter HCT116 cell survival, for our further experiments. We next evaluated the impact of NaBt on induction of CYP1-dependent EROD activity. For these experiments, we selected TCDD as a strong AhR ligand, which does not show inhibitory effects on EROD activity, unlike some PAHs, including BaP (Shimada and Guengerich 2006). As shown in Fig. 1c, NaBt significantly potentiated EROD activity in HCT116 cells (almost fivefold as compared with TCDD).
Fig. 1

NaBt potentiates ligand-dependent induction of CYP1 enzymes. HCT116 cells were treated with NaBt (0.5 or 3 mM), BaP (10 µM), their combination or vehicle (DMSO 0.1%). a Levels of CYP1A1 and CYP1B1 mRNA transcripts after 24-h treatment were detected by qRT-PCR. The results are expressed as mean ± SD of three independent experiments. Double asterisk denotes significant difference between control (DMSO) and respective treatment (P < 0.01). Double hash denotes significant difference between BaP alone, and the samples treated with combination of BaP and NaBt (P < 0.01). b Cell lysates (obtained after 24- and 48-h treatments) were analyzed by Western blotting, using specific antibodies against CYP1A1 and β-actin (loading control). The results are representative of three independent experiments. c Cells were treated with NaBt (0.5 mM), TCDD (1 nM), their combination or vehicle (DMSO 0.1%) for 24 h. The results of detection of EROD activity were normalized to total protein levels and then expressed relative to the activity of sample with maximum EROD induction. The results are expressed as mean ± SD of three independent experiments. Double asterisk denotes significant difference between control (DMSO) and respective treatment (P < 0.01). Double hash denotes significant difference between TCDD alone, and the sample treated with combination of TCDD and NaBt (P < 0.01)

NaBt increases levels of major BaP metabolites and formation of DNA adducts in HCT116 cells

As the above results indicated that NaBt significantly up-regulates CYP1A1 expression and activity in HCT116 cells treated with BaP, we then examined its impact on BaP metabolism and formation of covalent DNA adducts by the major genotoxic BaP metabolite—BPDE. Using LC–MS/MS analysis, we determined intracellular levels of the following metabolites: (1) hydroxylated BaP (OH-BaP) metabolites (1-OH-BaP, 3-OH-BaP and 9-OH-BaP), BaP-dihydrodiol (BaP-DHD) metabolites (BaP-4,5-DHD, BaP-7,8-DHD and BaP-9,10-DHD) and BaP-tetrols (BaP-tetrol I-1, BaP-tetrol I-2 and BaP-tetrol II-1), which represent intermediate or final metabolites of the diol-epoxide pathway; and (2) BaP-diones representing final metabolites of the radical cation pathway (BaP-1,6-dione, BaP-3,6-dione and BaP-6,12-dione). As shown in Fig. 2a, NaBt significantly up-regulated formation of 3-OH-BaP, 9-OH-BaP, BaP-7,8-DHD, BaP-9,10-DHD, BaP-1,6-dione, BaP-3,6-dione, and three BaP-tetrol metabolites. This indicated that the enhanced CYP1A1 expression led to an overall increase of BaP metabolism. This is further supported by our observation that the levels of BaP metabolites were similarly increased in cell culture medium, when cells were co-treated with NaBt (data not shown). NaBt also significantly increased formation of covalent DNA adducts, as determined by the 32P-postlabeling at both 24- and 48-h treatment times (Fig. 2b).
Fig. 2

NaBt increases metabolism and bioactivation of BaP. a HCT116 cells were treated with BaP (10 µM) alone or in combination with NaBt (0.5 mM) for 24 h. Cell pellets were collected and BaP metabolites were analyzed by LC–MS/MS. The results were normalized to total protein levels and expressed as mean ± SD of three independent experiments. Single and double asterisk denote significant difference between BaP alone, and the samples treated with combination of BaP and NaBt (P < 0.05 and P < 0.01, respectively). b HCT116 cells were treated with NaBt (0.5 mM), BaP (10 µM), their combination or vehicle (DMSO 0.1%) for 24 or 48 h. The levels of covalent DNA adducts were determined by 32P-postlabeling. The results are expressed as mean ± SD of three independent experiments. Double asterisk denotes significant difference between BaP alone, and the samples treated with combination of BaP and NaBt (P < 0.01). Representative chromatograms of DNA adducts are shown at the top of the panel

NaBt co-stimulates EROD activity and formation of DNA adducts in additional models of colon cells

We then aimed to confirm that the observed effects of NaBt on induction of CYP1A1 and BaP metabolism are not limited to HCT116 cell line. We selected the AA/C1 non-tumorigenic cell line derived from adenoma from a patient with familial adenomatous polyposis (Williams et al. 1990), and the HT29 colon carcinoma cell line, as additional models of colon epithelial cells of tumor origin. As shown in Fig. 3a, NaBt (0.5 mM) significantly co-stimulated induction of EROD activity by TCDD in both cell lines. Again, NaBt significantly enhanced formation of covalent DNA adducts as compared to BaP alone, in both AA/C1 and HT-29 cells (Fig. 3b). This suggested that the effects of NaBt are not limited to one particular colon cell model and that this short-chain fatty acid may modulate BaP metabolism and bioactivation via CYP1A1 in various types of adenoma- or carcinoma-derived colon cancer cell models.
Fig. 3

NaBt increases CYP1 activity and formation of covalent DNA adducts in AA/C1 and HT-29 cells. a AA/C1 and HT-29 cells were treated with NaBt (0.5 mM), TCDD (1 nM), their combination or vehicle (DMSO 0.1%) for 24 h. The results of detection of EROD activity were normalized to total protein levels and then expressed relative to the activity of sample with maximum induction. The results are expressed as mean ± SD of three independent experiments. Double asterisk denotes significant difference between control (DMSO) and respective treatment (P < 0.01). Double hash denotes significant difference between TCDD alone, and the samples treated with combination of TCDD and NaBt (P < 0.01). b AA/C1 and HT-29 cells were treated with NaBt (0.5 mM), BaP (10 µM), their combination or vehicle (DMSO 0.1%) for 24 h. The levels of covalent DNA adducts were determined by 32P-postlabeling. The results are expressed as mean ± SD of three independent experiments. Double asterisk denotes significant difference between BaP alone and the sample treated with combination of BaP and NaBt (P < 0.01)

In order to evaluate the effects of butyrate also in cells derived from normal human colon mucosal epithelium, we then determined the effects of butyrate on induction of CYP1A1 mRNA/protein, EROD activity and formation of covalent DNA adducts in NCM460D cell line. As shown in Fig. 4a, NaBt significantly potentiated induction of both CYP1A1 mRNA and protein. This corresponded with enhanced induction of EROD activity, when TCDD was applied in combination with NaBt (Fig. 4b) and increased formation of covalent DNA adducts by NaBt and BaP, as compared to BaP alone (Fig. 4c).
Fig. 4

NaBt increases CYP1A1 expression EROD activity and formation of covalent DNA adducts in human colon epithelial NCM460D cells. a NCM460D cells were treated with NaBt (0.5 or 3 mM), BaP (10 µM), their combination or vehicle (DMSO 0.1%). a (left) Levels of CYP1A1 and CYP1B1 mRNA transcripts after 24-h treatment were detected by qRT-PCR. The results are expressed as mean ± SD of three independent experiments. Double asterisk denotes significant difference between control (DMSO) and respective treatment (P < 0.01). Single hash denotes significant difference between BaP alone, and the samples treated with combination of BaP and NaBt (P < 0.05) (right). Cell lysates (obtained after 24-h treatment) were analyzed by Western blotting, using specific antibodies against CYP1A1 and β-actin (loading control). The results are representative of three independent experiments. b NCM460D cells were treated with NaBt (0.5 mM or 3 mM), TCDD (1 nM), their combination or vehicle (DMSO 0.1%) for 24 h. The results of detection of EROD activity were normalized to total protein levels and then expressed relative to the activity of sample with maximum induction. The results are expressed as mean ± SD of three independent experiments. Double asterisk denotes significant difference between control (DMSO) and respective treatment (P < 0.01). Single hash denotes significant difference between TCDD alone, and the samples treated with the respective combination of TCDD and NaBt (P < 0.05). Double hash denotes significant difference between TCDD alone, and the samples treated with the respective combination of TCDD and NaBt (P < 0.01). c NCM460D cells were treated with NaBt (0.5 mM or 3 mM), BaP (10 μM), their combination or vehicle (DMSO 0.1%) for 24 h. The levels of covalent DNA adducts were determined by 32P-postlabeling. The results are expressed as mean ± SD of three independent experiments. Double asterisk denotes significant difference between BaP alone, and the samples treated with the respective combination of BaP and NaBt (P < 0.01). Representative chromatograms of DNA adducts are shown at the top of the panel

Finally, in order to evaluate the effects of NaBt also in an alternative model of differentiated non-proliferating colonic cells, we used differentiated Caco-2 cells. Here, we observed a significant up-regulation of CYP1A1 and CYP1B1 mRNA (Fig. 5a), and CYP1A1 protein in cells treated with both BaP and 3 mM NaBt, as compared with BaP alone (Fig. 5b). BaP was a potent CYP1 inducer in non-proliferating Caco-2 cells and effects of NaBt were less pronounced under these cultivation conditions; they were significant only at the higher 3 mM concentration. This indicated that differentiated Caco-2 enterocytes could be more sensitive to AhR activation, which may perhaps limit the contribution of butyrate to the overall CYP1 induction. TCCD, a non-genotoxic AhR ligand, highly increased EROD activity in differentiated Caco-2 cells on its own (Fig. 5c) and the impact of NaBt on this endpoint was limited. Taken together, the above data obtained with NCM460D and differentiated Caco-2 cells suggested that butyrate may modulate CYP1 expression and/or activity also in cellular models better approximating normal colon epithelium.
Fig. 5

NaBt potentiates CYP1 induction in differentiated human colon carcinoma Caco-2 cells. a Differentiated Caco-2 cells were treated with NaBt (0.5 or 3 mM), BaP (10 μM), their combination or vehicle (DMSO 0.1%) for 24 h. Levels of CYP1A1 and CYP1B1 mRNA transcripts after 24-h treatment were detected by qRT-PCR. The results are expressed as mean ± SD of three independent experiments. Double asterisk denotes significant difference between control (DMSO) and respective treatment (P < 0.01). Double hash denotes significant difference between BaP alone, and the samples treated with the respective combination of BaP and NaBt (P < 0.01). b Whole cell lysates of differentiated Caco-2 cells, treated with indicated compounds for 24 h, were analyzed by Western blotting, using specific antibodies against CYP1A1 and β-actin (loading control). The results are representative of three independent experiments. c Differentiated Caco-2 cells were treated with NaBt (0.5 or 3 mM), TCDD (1 nM), their combination or vehicle (DMSO 0.1%) for 24 h. The results of detection of EROD activity were normalized to total protein levels and then expressed relative to the activity of sample with maximum induction. The results are expressed as mean ± SD of three independent experiments. Double asterisk denotes significant difference between control (DMSO) and respective treatment (P < 0.01)

The enhanced CYP1A1 expression is not linked to stimulation of Wnt/β-catenin signaling

Next, we explored potential mechanisms responsible for the effects of NaBt on CYP1A1 induction. First, we evaluated whether it could be linked to an increased Wnt/β-catenin signaling in HCT116 cells, because it has been reported that NaBt increases the activity of this pathway in colon cancer cells (Bordonaro et al. 2008), and this pathway has been shown to co-operate with the AhR in regulation of both rodent and human CYP1A1 (Braeuning et al. 2011; Kasai et al. 2013; Procházková et al. 2011; Vaas et al. 2014). Therefore, we performed the TOP/FOP luciferase reporter assay to evaluate the TCF/LEF transcriptional activity, which is dependent on β-catenin activity. We found that 3 mM NaBt, but not 0.5 mM NaBt, significantly increased the activity of Super 8× TOPFlash reporter construct (Fig. 6). As both concentrations similarly increased CYP1A1 expression/activity, this indicated that the observed effects on CYP1A1 induction were not related to the NaBt-induced activation of β-catenin signaling.
Fig. 6

Only high concentrations of NaBt activate Wnt/β-catenin signaling in HCT116 cells. Cells were co-transfected with two plasmids pRL-TK, and either Super 8× TOPflash or Super 8× FOPflash, and then treated with NaBt (0.5 or 3 mM), BaP (10 µM), their combination or vehicle (DMSO 0.1%) for 24 h. The results of TOP/FOP reporter gene assay are expressed as a ratio of between TOPflash versus FOPflash firefly luciferase activity, normalized to Renilla luciferase activity and then expressed relative to activity of control (DMSO). The results are expressed as mean ± SD of three independent experiments. Double asterisk denotes significant difference between control (DMSO) and respective treatment (P < 0.01)

HDAC inhibitors increase expression and activity of CYP1A1, as well as formation of covalent DNA adducts in HCT116 cells

We then investigated, whether the co-stimulatory effects of NaBt could be linked to inhibition of HDAC by NaBt. Here, we used two chemical HDAC inhibitors, TSA and SAHA (vorinostat), at 1 μM concentration (previously reported to significantly alter histone acetylation/proliferation of HCT116 cells); however, we also observed similar impact of lower TSA concentrations on CYP1A1 induction (data not shown). We found that both HDAC inhibitors, similar to NaBt, co-stimulated induction of CYP1A1 mRNA (Fig. 7a), EROD activity (Fig. 7b) and increased CYP1A1 protein levels (Fig. 7c). Finally, TSA significantly increased the formation of covalent DNA adducts in HCT116 cells, similar to NaBt (Fig. 7d). In addition, we observed similar effects of TSA and SAHA on CYP1A1 expression also in NCM460D cells derived from normal human colon mucosal epithelium (data not shown), suggesting that HDAC inhibitors may similarly regulate CYP1A1 levels also in cells that are not derived from tumor tissue. Together, these results indicated that NaBt may potentiate the effects of AhR ligands on induction of CYP1A1 via HDAC inhibition.
Fig. 7

HDAC inhibitors increase expression and activity of CYP1A1, as well as formation of covalent DNA adducts in HCT116 cells. a HCT116 cells were treated with TSA (1 µM), SAHA (1 µM), BaP (10 µM), their combination or vehicle (DMSO 0.1%) for 24 h. The levels of CYP1A1 mRNA transcripts were detected by qRT-PCR. The results are expressed as mean ± SD of three independent experiments. Double asterisk denotes significant difference between control (DMSO) and respective treatment (P < 0.01). Double hash denotes significant difference between BaP alone, and the samples treated with combination of BaP and TSA or SAHA (P < 0.01). b HCT116 cells were treated with TSA (1 µM), SAHA (1 µM), TCDD (1 nM), their combination or vehicle (DMSO 0.1%) for 24 h. The results of detection of EROD activity were normalized to total protein levels and then expressed relative to the activity of sample with maximum induction. The results are expressed as mean ± SD of three independent experiments. Double asterisk denotes significant difference between control (DMSO) and respective treatment (P < 0.01). Double hash denotes significant difference between TCDD alone, and the samples treated with combination of TCDD and TSA or SAHA (P < 0.01). c HCT116 cells were treated with NaBt (0.5 mM), TSA (1 µM), SAHA (1 µM), BaP (10 µM), their combination or vehicle (DMSO 0.1%) for 24 h. Cell lysates were analyzed by Western blotting, using specific antibodies against CYP1A1 and β-actin (loading control). The results are representative of three independent experiments. d HCT116 cells were treated with TSA (1 µM), BaP (10 µM), their combination or vehicle (DMSO 0.1%) for 24 h. The levels of covalent DNA adducts were determined by 32P-postlabeling. The results are expressed as mean ± SD of three independent experiments. Single asterisk denotes significant difference between BaP alone and the sample treated with combination of BaP and TSA (P < 0.05)

NaBt increases histone H3 and H4 acetylation and decreases HDAC1 binding to CYP1A1 enhancer

Since the above results pointed to the role of HDAC inhibition in effects of NaBt on CYP1A1 induction, we examined impact of NaBt on histone acetylation. We focused on two histone post-translational modifications, which have been shown to occur in response to NaBt treatment in mouse Cyp1a1 regulatory region—acetylation of histone H3 at Lys14 (K14) and acetylation of histone H4 at Lys16 (K16) (Schnekenburger et al. 2007). As shown in Fig. 8a, both H3 and H4 were acetylated at the respective Lys residues in response to NaBt treatment. Moreover, using ChIP analysis, we then confirmed that NaBt decreased the presence of HDAC1 within the enhancer region of human CYP1A1 gene (Fig. 8b). Together, these results indicated that NaBt, even at 0.5 mM concentration (which is well within a lower range of butyrate concentrations observed in the human colon), may significantly modulate HDAC1 action and histone acetylation within the context of CYP1A1 regulatory region.
Fig. 8

NaBt increases histone acetylation and displaces HDAC1 from CYP1A1 enhancer. a HCT116 cells were treated with NaBt (0.5 mM), BaP (10 µM), their combination or vehicle (DMSO 0.1%) for 24 h. Cell lysates were analyzed by Western blotting, using specific antibodies against acetyl-histone H3 (K14), acetyl-histone H4 (K16) and β-actin (loading control). The results are representative of three independent experiments. b HCT116 cells were pre-treated with NaBt (0.5 mM) for 16 h and then treated with BaP (10 μM) or vehicle (DMSO 0.1%) for 1.5 h. The presence of HDAC1 within the enhancer region of CYP1A1 was determined by ChIP assay. The results are expressed as mean ± SD of two independent experiments

Discussion

Short-chain fatty acids, such as butyrate, are important modulators of behavior and fate of colon epithelial cells. Apart from serving as an energy source, butyrate modulates a number of signaling pathways controlling cell proliferation, survival and differentiation, the essential processes involved in the constant renewal of colon epithelium (Bultman and Jobin 2014; Irrazábal et al. 2014; Sengupta et al. 2006). In contrast to this role of butyrate, its impact on xenobiotic-metabolizing enzymes within colon remains mostly unknown. Butyrate has been suggested to induce glutathione-S-transferases in colon cell models, and this has been proposed to potentially contribute to its beneficial effects via prevention of DNA damage (Scharlau et al. 2009). However, in contrast to this class of phase II enzymes, effects of butyrate on regulation on phase I enzymes participating in bioactivation of dietary carcinogens, such as CYPs, are not clear. Importantly, since butyrate is a HDAC inhibitor at the physiological levels found in the colon lumen, it might contribute to chromatin decondensation and modulate expression of enzymes contributing to metabolism of dietary carcinogens, such as BaP. In the present study, we therefore investigated potential impact of butyrate on induction of CYP1A1 expression/activity in various types colon epithelial cell models, its impact on metabolism of BaP (including formation of covalent DNA adducts), as well as potential mechanism(s) underlying the effects of butyrate on CYP1A1 induction.

Transcriptional regulation of CYP1A1 can be modulated by inhibition or stimulation of enzymes involved in the control of histone acetylation, HDACs and histone acetyltransferases (HATs), respectively. Butyrate has been suggested to contribute to histone modifications via both mechanisms (Donohoe et al. 2012). Although some previous studies reported co-stimulatory role of NaBt in the AhR ligand induced CYP1A1 transcription, other authors have not observed co-stimulatory effects of NaBt (Gradin et al. 1999; Haarmann-Stemmann et al. 2007; Schnekenburger et al. 2007; Wei et al. 2004). Here, we found that NaBt, at relatively low concentration, strongly co-induced expression of CYP1A1 at both mRNA and protein levels, as well as its activity in human colon carcinoma HCT116 cells. The up-regulation of CYP1A1 expression/activity corresponded with an enhanced metabolism of BaP and formation of covalent DNA adducts induced by its ultimate genotoxic metabolite, BPDE. These effects were not limited to this particular cell line, because we made similar observations also when using other colon cell models, including AA/C1 and HT-29 cells, which represent colon cells derived at different stages of colon adenoma/carcinoma sequence. This suggests that, unlike in cells derived from the liver (Gradin et al. 1999; Wei et al. 2004), NaBt may co-regulate CYP1A1 induction in colon epithelial cells. A similar effect of NaBt was also confirmed in NCM460D cell line, originally derived from normal colon mucosa, where we observed significant up-regulation of CYP1A1 levels and EROD activity in cells treated with both NaBt and BaP. Finally, in non-proliferating differentiated human colon carcinoma Caco-2 cells, we observed that higher butyrate concentrations (3 mM) potentiated CYP1 induction. The differentiated non-proliferating Caco-2 cells appeared to be more sensitive to BaP or TCDD, and butyrate seemed to have less of an impact under these cultivation conditions. The higher sensitivity of differentiated Caco-2 cells to AhR ligands may correspond with the observation that kinetics of CYP1A1 mRNA induction depends on differentiation status of Caco-2 cells (Daujat et al. 1996). In general, colonocytes are exposed to higher levels of butyrate than, e.g. hepatocytes, since butyrate is rapidly metabolized by colonocytes and its levels in portal vein are already three orders of magnitude lower than within the colon lumen (Cummings et al. 1987). The role of butyrate in regulation of CYP1A1 levels could be thus different within the context of colon epithelium than in other organs, such as the liver.

The transcriptional regulation of CYP1A1 gene has been studied in a great detail over the past decades and the events at the CYP1A1 promoter, which are regulated upon binding of the active AhR seem to involve a number of transcriptional co-regulators, including HDACs and HATs (Beedanagari et al. 2010a; Wei et al. 2004; Whitlock 1999). Histone acetylation is a highly dynamic process, which is also controlled by diet and metabolism (Fan et al. 2015; Huang et al. 2015), and which typically leads to decondensation of chromatin associated with active transcription. It has been reported that p300 (a HAT enzyme) is recruited to the CYP1A1 enhancer upon the AhR binding (when activated by TCDD) to this region (Beedanagari et al. 2010a). Transcriptional induction of the murine Cyp1a1 gene (by BaP) leads to dissociation of HDAC1, a ubiquitous nuclear class 1 HDAC (Yang and Seto 2008), from Cypa1a1 enhancer and promoter (Schnekenburger et al. 2007; Wei et al. 2004). Importantly, both of these events seems to be linked with enhanced histone H3 and/or H4 acetylation within the promoter of human CYP1A1 gene, or within enhancer/promoter of the murine Cyp1a1 gene (Beedanagari et al. 2010a; Schnekenburger et al. 2007). In the present study, we found that butyrate significantly induced acetylation of both histone H3 (at Lys14) and histone H4 (at Lys16). This confirmed that even at the relatively low concentration of NaBt used in the present study (0.5 mM), a significant acetylation of these residues ensues. These particular histone post-translational modifications have been also identified within the mouse Cyp1a1 regulatory regions in mouse hepatoma cells exposed to millimolar NaBt concentrations (Schnekenburger et al. 2007). Moreover, the results of ChIP analysis indicated that NaBt also caused a decrease of the binding of HDAC1 to the human CYP1A1 enhancer. This supports the previously published hypothesis that HDAC1 functions as an active repressor of basal Cyp1a1 expression (Schnekenburger et al. 2007). Together with our observation that additional HDAC inhibitors, TSA and SAHA, exerted similar effects on induction of CYP1A1 expression/activity as NaBt, this indicates that the mechanism underlying the effects of butyrate on CYP1A1 transcriptional regulation likely depends on HDAC inhibition. Our results also suggest that the effects of butyrate are not linked to its ability to stimulate Wnt/β-catenin signaling (Bordonaro et al. 2008), since this effect was observed only at higher NaBt concentrations. Therefore, it seems unlikely that butyrate would modulate CYP1A1 induction in colon cells via this recently described mechanism of CYP1 regulation (Braeuning et al. 2011; Gerbal-Chaloin et al. 2014; Procházková et al. 2011; Vaas et al. 2014).

Although basal levels of CYP1 enzymes in human colon have been suggested to be low (Bieche et al. 2007; Choudhary et al. 2005), it has been demonstrated that CYP1A1 expression (or expression of additional AhR-regulated enzymes) and EROD activity can be induced in human colon tissue slices (van de Kerkhof et al. 2008). Cyp1a1 is induced in mouse colon in vivo by both BaP and TCDD (Uno et al. 2008), and it has been shown to be inducible in human colon by pharmacological AhR inducer omeprazole (McDonnell et al. 1992). This indicates that CYP1A1 may contribute to the AhR-inducible metabolism of BaP in colon. Here, we found that the induction of CYP1A1 expression and/or activity in several distinct models of human colon cells, including cells derived from normal human colon mucosa, was associated with an increased formation of the covalent DNA adducts. We have recently demonstrated that CYP1A1 plays a major role in formation of genotoxic BaP metabolites in HCT116 cells (Kabátková et al. 2015), which has been indicated also by other studies (Hockley et al. 2008; Wohak et al. 2016). An increased rate of BaP metabolism that was observed in cells co-treated by NaBt was confirmed also by analysis of BaP metabolites, which showed that levels of both direct BPDE precursor (BaP-7,8-DHD) and its products (BaP-tetrols) are significantly higher in BaP/NaBt-treated cells than in cells exposed only to BaP. Together, these data suggest that CYP1A1 could play a significant role in metabolism of BaP in human colon cells. However, it is important to note that in vivo, both inducibility and functional role of CYP1A1 in BaP metabolism may differ from in vitro cultures, where factors including cell proliferation rate and differentiation might further modulate CYP1A1 induction/activity. In their studies using Cyp1a1 knockout mice, Uno et al. have shown that higher Cyp1a1 levels may increase metabolic clearance of BaP in wild-type animals and thus reduce formation of BPDE-DNA adducts in various organs, suggesting that Cyp1a1 could be more important in BaP detoxification in gastrointestinal tract (Uno et al. 2001, 2006). Therefore, coordinate induction of CYP1A1 by BaP and butyrate might serve to reduce formation of BPDE-DNA adducts in vivo, thus contributing to protective role of butyrate against PAHs in colon tissue. Therefore, the results of the present study should be interpreted with caution, as it will be necessary to establish its in vivo relevance for metabolism of PAHs in human colon epithelium.

In summary, the data presented here suggest that the effects of BaP can be significantly modulated by butyrate in various types of colon epithelial cell models in vitro. Increased acetylation of histones that is elicited by this compound, may, via chromatin remodeling, contribute to induction of CYP1A1 expression and its activity in colon cells. This may in turn alter metabolism of BaP. Therefore, more attention should be paid to the mechanisms, thorough which butyrate may contribute to the transcriptional control of the CYP1A1 expression/activity in human colon epithelium, since these may alter detoxification and/or bioactivation of some dietary carcinogens. Importantly, more studies are necessary to establish the relevance of the observed phenomenon in vivo, and to determine the precise role of butyrate in control of metabolism of BaP or related dietary carcinogens within colon epithelium.

Notes

Acknowledgements

This study was supported by the Czech Science Foundation (Project No. 13-09766S to A.K.), the Czech Ministry of Agriculture (RO 0515 to M.M.) and Internal Grant Agency of the Ministry of Health of the Czech Republic (NT14599-3/2013 to J.K.). The authors acknowledge the assistance provided by the Research Infrastructure NanoEnviCz, supported by the Ministry of Education, Youth and Sports of the Czech Republic under Project No. LM2015073. The authors wish to thank prof. Chris Paraskeva (School of Cellular and Molecular Medicine, University of Bristol, Bristol, UK) for kindly providing AA/C1 cell line. The expert technical assistance of Radek Fedr, Iva Lišková and Martina Urbánková is gratefully acknowledged.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

204_2016_1887_MOESM1_ESM.pdf (24 kb)
Supplementary material 1 (PDF 23 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Ondřej Zapletal
    • 1
    • 2
  • Zuzana Tylichová
    • 1
    • 2
  • Jiří Neča
    • 3
  • Jiří Kohoutek
    • 3
  • Miroslav Machala
    • 3
  • Alena Milcová
    • 4
  • Michaela Pokorná
    • 4
  • Jan Topinka
    • 4
  • Mary Pat Moyer
    • 5
  • Jiřina Hofmanová
    • 1
  • Alois Kozubík
    • 1
  • Jan Vondráček
    • 1
  1. 1.Department of Cytokinetics, Institute of BiophysicsThe Czech Academy of SciencesBrnoCzech Republic
  2. 2.Department of Experimental Biology, Faculty of ScienceMasaryk UniversityBrnoCzech Republic
  3. 3.Department of Chemistry and ToxicologyVeterinary Research InstituteBrnoCzech Republic
  4. 4.Department of Genetic Ecotoxicology, Institute of Experimental MedicineThe Czech Academy of SciencesPragueCzech Republic
  5. 5.INCELL Corporation LLCSan AntonioUSA

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