Butyrate alters expression of cytochrome P450 1A1 and metabolism of benzo[a]pyrene via its histone deacetylase activity in colon epithelial cell models
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.
KeywordsCYP1A1 Butyrate Polycyclic aromatic hydrocarbons DNA adducts Histone deacetylases Colon epithelial cells
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
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.
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.
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 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.
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.
NaBt increases levels of major BaP metabolites and formation of DNA adducts in HCT116 cells
NaBt co-stimulates EROD activity and formation of DNA adducts in additional models of colon cells
The enhanced CYP1A1 expression is not linked to stimulation of Wnt/β-catenin signaling
HDAC inhibitors increase expression and activity of CYP1A1, as well as formation of covalent DNA adducts in HCT116 cells
NaBt increases histone H3 and H4 acetylation and decreases HDAC1 binding to CYP1A1 enhancer
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.
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.
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Conflict of interest
The authors declare that they have no conflict of interest.
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