Acta Veterinaria Hungarica DOI: 10.1556/AVet.2013.033

© 2013 Akadémiai Kiadó, Budapest

EPIGENETIC EFFECTS OF DIETARY BUTYRATE ON HEPATIC HISTONE ACETYLATION AND ENZYMES

OF BIOTRANSFORMATION IN CHICKEN

Gábor MÁTIS1*, Zsuzsanna NEOGRÁDY1, György CSIKÓ2, Péter GÁLFI2, Hedvig FÉBEL3, Katalin JEMNITZ4, Zsuzsanna VERES4, Anna KULCSÁR1, Ákos KENÉZ5

and Korinna HUBER5

1Department of Physiology and Biochemistry, Faculty of Veterinary Science, Szent István University, István u. 2, H-1078 Budapest, Hungary; 2Department of Pharmacology and

Toxicology, Faculty of Veterinary Science, Szent István University, Budapest, Hungary; 3Research Institute for Animal Breeding and Nutrition, Herceghalom, Hungary;

4Institute of Molecular Pharmacology, Research Centre of Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary; 5Department of Physiology,

University of Veterinary Medicine, Hanover, Germany

(Received 11 October 2012; accepted 11 December 2012)

The aim of the study was to investigate the in vivo epigenetic influences of dietary butyrate supplementation on the acetylation state of core histones and the activity of drug-metabolising microsomal cytochrome P450 (CYP) enzymes in the liver of broiler chickens in the starter period. One-day-old Ross 308 broilers were fed a starter diet without or with sodium butyrate (1.5 g/kg feed) for 21 days. After slaughtering, nucleus and microsome fractions were isolated from the ex-sanguinated liver by multi-step differential centrifugation. Histone acetylation level was detected from hepatocyte nuclei by Western blotting, while microsomal CYP activity was examined by specific enzyme assays. Hyperacetylation of hepatic histone H2A at lysine 5 was observed after butyrate supplementation, providing modifications in the epigenetic regulation of cell function. No significant changes could be found in the acetylation state of the other core histones at the acetylation sites examined. Furthermore, butyrate did not cause any changes in the drug-metabolising activity of hepatic microsomal CYP2H and CYP3A37 enzymes, which are mainly involved in the biotransformation of most xenobiotics in chicken. These data indicate that supplementation of the diet with butyrate proba-bly does not have any pharmacokinetic interactions with simultaneously applied xenobiotics.

Key words: Butyrate, histone hyperacetylation, cytochrome P450 enzymes, epigenetics, chicken

*Corresponding author; E-mail: matis.gabor@aotk.szie.hu; Phone: 0036 (1) 478-4158; Fax: 0036 (1) 478-4165

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Short-chain fatty acids (SCFA) are the major end-products of anaerobic microbial fermentation in the forestomachs of ruminants as well as in the large intestine of monogastric animals (Bergman, 1990). Among the different SCFA, n-butyrate is the most important energy source of the gastrointestinal mucosa, playing also a predominant role in the proliferation and differentiation of the gas-trointestinal epithelium (Gálfi and Neogrády, 2001), and providing a protective effect against colorectal cancer (Le Leu et al., 2007). It has a significant influ-ence on the balance of the intestinal microflora because of its selective antim-icrobial effect on most enteral pathogens (van Immerseel et al., 2005; Fernández-Rubio et al., 2008). These beneficial actions of butyrate made this SCFA a mole-cule of special interest as a feed additive (mainly as its sodium salt) to poultry diets, especially after the banning of antibiotics as growth promoters in the EU (Phillips, 2007).

It was described already by Gálfi and Bokori (1990) that dietary butyrate supplementation increased growth and had a positive influence on feed utilisa-tion in pigs. Hu and Guo (2007) reported that dietary butyrate supplementation caused increased body weight and weekly body weight gain in broiler chickens in the starter period.

Butyrate is efficiently metabolised by the intestinal epithelial cells (Velázquez et al., 1997), but a certain amount can be absorbed and transported into the liver by the portal vein (Bloemen et al., 2009). Butyrate is an important energy source of hepatocytes as a substrate of β-oxidation, but it is also a potent effector of hepatic metabolism (Beauvieux et al., 2001).

Butyrate is also well known as an epigenetic factor influencing histone acetylation. Nucleosomes consist of DNA wrapped around a histone octamer comprised of dimers of core histones H2A, H2B, H3 and H4, also termed nu-cleosome core particles (NCP), which are connected by linker DNA sections (Arents et al., 1991). During posttranslational modifications of histones, the bal-ance of histone acetyltransferase (HAT) and histone deacetylase (HDAC) activ-ity determines the acetylation state of core histones (Rada-Iglesias et al., 2007). HAT binds acetyl groups to the originally positively charged lysine residues, while HDAC removes them. If HDAC is blocked by an inhibitor, it causes his-tone hyperacetylation in the N-terminal histone tails, which results in a modified, transcriptionally active structure of the NCP, therefore it influences the transcrip-tional pattern of certain genes (Davie, 2003; Gao et al., 2009).

HDACs (Class I and most of the Class II isoenzymes) are inhibited by bu-tyrate, causing hyperacetylated core histones in cell cultures (Davie, 2003). In contrast to the wide range of in vitro examinations (Candido et al., 1978), only very few data are available in the literature regarding the in vivo effects of bu-tyrate on histone acetylation, especially in chicken. A significant increase in total histone acetylation was reported in porcine caecal tissue after dietary supplemen-tation with the butyrate precursor lactulose (Kien et al., 2008).

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On the basis of its epigenetic action on chromatin structure, butyrate has also several metabolic effects, such as influencing the plasma insulin level (Neogrády et al., 1989) and insulin sensitivity of extrahepatic tissues (Gao et al., 2009).

Hepatic microsomal cytochrome P450 (CYP) monooxygenases are involved in the biotransformation of most xenobiotics (Anzenbacher and Anzerbacherová, 2001). The expression of some CYP genes is known to be affected by histone modifications (Baer-Dubowska et al., 2011). For example, the HDAC inhibitor trichostatin A caused modified expression of the CYP3A subfamily in cell cul-ture studies (Dannenberg and Edenberg, 2006). On the basis of these data we hy-pothesise that butyrate, like other HDAC inhibitors, can alter the activity of CYP and may have an impact on drug metabolism.

In chicken, the CYP1A and CYP2H subfamily, especially the isoenzymes CYP2H1 and CYP2H2 (which are orthologues to the mammalian CYP2B and CYP2C proteins) and CYP3A37 (a member of the CYP3A subfamily) are the most important CYPs in drug metabolism (Ourlin et al., 2000). They are inducible by the well-known enzyme inductor phenobarbital (PB) (Hansen and May, 1989).

The aim of this study was to screen the extent of acetylation at key sites of histones for the first time in broilers fed with butyrate in the early post-hatch pe-riod. Changes in acetylation were expected due to the functioning of butyrate as a HDAC inhibitor. Furthermore, the activity of the drug-metabolising micro-somal CYP2H and CYP3A37 enzymes was also examined by specific enzyme assays, applying widely-used test substrates (CYP2H/CYP3A37: aminopyrine N-demethylation, CYP2H: aniline hydroxylation, CYP3A37: testosterone 6β-hydroxylation) (Shimojo et al., 1993; Ourlin et al., 2000). Changes in CYP ac-tivities were expected to be associated with butyrate-provoked hyperacetylation of core histones. The finding whether butyrate modulates the activity of CYPs has special importance for future studies concerning the epigenetic mechanisms induced by butyrate and the possible relevant pharmacokinetic interactions be-tween butyrate and certain xenobiotics.

Materials and methods

Chemicals

Chemicals were purchased from Sigma-Aldrich (Munich, Germany) except when otherwise specified.

Animals and feeding

Thirty one-day-old broiler chicks of the Ross 308 strain (mixed gender), obtained from a commercial hatchery (Bábolna Tetra Company, Uraiújfalu, Hungary), were included in the experiment. Broilers were housed together in metal pens (five birds per pen) with a floor area of 1.5 m2 under controlled light

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(23 h/day from days 0 to 7; 18 h/day from days 8 to 14; 16 h/day from days 15 to 21). The temperature and other climatic circumstances were adjusted according to the requirements of the Ross technology (Ross, 2009).

The experimental chicks were randomised into three groups: ten chicks were fed with a control diet, free from any medication or chemical additives, formulated to the requirements of the starter period. The composition of the basal diet is shown in Table 1. Ten broilers were fed with the same feedstuff but sup-plemented with 1.5 g sodium butyrate/kg diet. Ten chickens received the control diet, but were treated with PB injection intracoelomally (phenobarbital sodium, Ph. Eur. 7.1, dissolved in sterile, pyrogen-free and endotoxin-free physiological saline solution) to induce CYP activity as a positive control. The applied dose was 80 mg/kg body weight once daily over the last three days of the experiment.

Table 1

Composition and calculated nutrient content of the basal diet of broilers

Item

Ingredient Corn, g/kg 593.7 Soybean meal, g/kg 310.0 Corn gluten meal, g/kg 50.0 Limestone, g/kg 15.0 Monocalcium phosphate, g/kg 18.5 NaCl, g/kg 4.0 Vitamin-mineral mixture1, g/kg 6.0 L-Lysine HCl, g/kg 1.8 DL-Methionine, g/kg 1.0

Calculated nutrient content CP, g/kg 212.2 Ether extract, g/kg 29.4 Crude fibre, g/kg 25.3 Ash, g/kg 65.9 AMEn, MJ/kg 11.9 Lysine, g/kg 11.9 Methionine, g/kg 4.9 Methionine + Cysteine, g/kg 8.6 Calcium, g/kg 11.6 Available phosphorus, g/kg 4.5

1Provided (per kilogram of diet as fed): Se, 0.24 mg; Fe, 135 mg; Mn, 136 mg; Cu, 22 mg; Zn, 110 mg; I, 1.2 mg; retinyl acetate, 4.14 mg; cholecalciferol, 0.075 mg; α-tocopherol, 26.85 mg, choline chloride, 360 mg; menadione, 3.0 mg; riboflavin, 7.0 mg; cobalamin, 0.03 mg; niacin, 40 mg; pantothenic acid, 12 mg; folic acid, 1.0 mg; pyridoxine, 5.0 mg

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Feed and water were provided ad libitum for all groups. The correct so-dium butyrate content of the feedstuff and the elevation of blood butyrate con-centration in the butyrate-fed animals were confirmed by gas chromatography (Shimadzu GC 2010, Japan) using a 30 m (0.25 mm i.d.) fused silica column (Nukol column, Supelco Inc., Bellefonte, PA, USA). The animal feeding experi-ments fully complied with legislation on research involving animal subjects ac-cording to the European Communities Council Directive of 24 November 1986 (86/609/EEC). All procedures were approved by the Local Animal Test Committee of the Faculty of Veterinary Science, Szent István University, Budapest, Hungary.

On day 21, the body weight of the chicks was recorded, then 12 h post feeding all birds were slaughtered in anaesthesia with carbon dioxide by decapi-tation. The duration of the experiment has been chosen so as to ensure optimal observation of both the possible epigenetic influence of butyrate and the induci-bility of CYP enzyme activity.

Liver sampling and separation of subcellular organelles

Immediately after slaughtering the liver was flushed with chilled physio-logical saline solution through the portal vein and was excised after exsanguina-tion. Subcellular organelles were isolated according to the protocol of van der Hoeven and Coon (1974).

The total protein concentration of isolated microsome suspension samples was determined by bicinchoninic acid (BCA) protein assay (Walker, 1996) for further standardisation of microsomal CYP enzyme activities. Measurements were conducted on 96-well-plates using a BCA Protein Assay Kit (Fisher Scien-tific, Rockford, IL, USA), according to the manufacturer’s protocol. Bovine se-rum albumin (included in the kit) was used as standard. Each measurement was carried out in triplicates.

All cell nucleus and microsome preparations were shock-frozen in liquid nitrogen and were stored at –80 °C until further examinations. Cell nuclei were isolated only from control and butyrate-fed chickens, while a microsome fraction was prepared also from PB-treated animals.

Histone isolation and Western blot analysis

Purified histone extract was isolated using a Histone Purification Mini Kit (Active Motif, Carlsbad, CA, USA) from the cell nucleus fraction according to the manufacturer’s protocol. During the whole purification procedure kit re-agents prevented further deacetylase activity to ensure the same acetylation status as in vivo. The yield of total core histone proteins was quantified by meas-uring the absorbance at 230 nm.

Electrophoresis and Western blotting were performed as described in the instructions of the applied Acetyl Histone Antibody Sampler Kit (Cell Signaling,

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MA, USA). According to the total core histone concentration, samples were di-luted with an SDS- and mercaptoethanol-containing loading buffer (supplemented with 50 mM dithiothreitol), sonicated for 15 sec to reduce the viscosity of sam-ples, and proteins were denatured by heating at 95 °C for 5 min. Histones were separated by SDS-PAGE on polyacrylamide (4–20%) precast gradient gels (Bio-rad Laboratories, CA, USA). Separated proteins were transferred onto nitrocellu-lose membranes (0.2 μm pore size, Biorad Laboratories, CA, USA) by tank blot-ting (blotting time 1 h). To confirm the blotting performance, gels were stained with Coomassie Brilliant Blue.

Histones were identified by immunodetection using antibodies of the Ace-tyl Histone Antibody Sampler Kit: after blocking with 5% fat-free milk solution for 2 h, membranes were incubated overnight with primary antibodies recognis-ing histones H2A (1:1000), H2B (1:500), H3 (1:1000), H4 (1:500) and their ac-etylated forms. Each acetyl histone antibody was specific for the target histone modified at one specific acetylation site (H2A and H2B: Lys 5, H3: Lys 9, H4: Lys 8). Detection of the primary antibody was performed using an anti-rabbit secondary antibody (1:2000) coupled with horseradish peroxidase. Bands were detected by the Chemidoc XRS enhanced chemiluminescence system (Biorad Laboratories, CA, USA). Membranes were finally stained with Indian ink to de-tect the separated proteins. Band intensities were quantified by the Quantity One 1-D Analysis software (Biorad Laboratories, CA, USA), and results were stan-dardised to the bands stained with Indian ink to ensure equal loading. Acetyla-tion ratios were determined considering relative protein expression levels of each histone and its acetylated form.

Enzyme assays on hepatic microsomal CYP activity

Aminopyrine N-demethylation assay. Microsomal CYP2H/CYP3A37 ac-tivity was determined by the aminopyrine N-demethylation assay, in which for-maldehyde production could be measured by the spectrophotometric method of Nash (1953). The enzyme assay was carried out according to the modified protocol of García-Agúndez et al. (1990). The reaction mixture contained an NADPH+H+-regenerating cofactor mixture, prepared from 0.5 mM NADPH+H+ (Reanal Pri-vate Co. Ltd., Budapest, Hungary), 50 mM glucose 6-phosphate, 4 IU/l glucose 6-phosphate dehydrogenase, 5 mM MgCl2 (Reanal Private Co. Ltd., Budapest, Hungary) and 50 mM semicarbazide.

One hundred µl microsome suspension was incubated with 200 µl cofactor mixture and 900 µl 0.05 M phosphate buffer (pH 7.4) in the presence of different concentrations (0, 1.25, 2.5, 5, 10 mM) of dimethylamino-antipyrine (amino-pyrine) for 10 min at 37 °C. The reaction was stopped by adding 200 µl 20% tri-chloroacetic acid. The mixture was centrifuged with 4,500 g for 10 min and 800 µl of the supernatant was added to 400 µl Nash reagent (0.16 ml acetyl ace-tone and 0.24 ml concentrated acetic acid in 20 ml of 4 M ammonium acetate so-

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lution). The mixture was incubated at 60 °C for 30 min, cooled down on ice and the absorbance was measured spectrophotometrically at 415 nm against reagent blank. The results were corrected for an inhibited blank per each substrate con-centration (inhibited previously by adding 20% trichloroacetic acid). Formalde-hyde standard curves were determined under the same conditions as used for mi-crosomal activity measurements; each sample was examined in triplicates. Fi-nally, mean specific enzyme activity, Vmax and KM values were calculated and compared between groups. All results were standardised according to the total protein concentration of microsome samples.

Aniline hydroxylation assay. CYP2H activity was measured by the aniline hydroxylation assay. The enzyme assay was carried out according to the modified protocol of Murray and Ryan (1982). The reaction mixture was prepared with the same composition and the assay was conducted under the same conditions as for the aminopyrine N-demethylation assay, using different concentrations (0, 1.25, 2.5, 5, 10 mM) of aniline hydrochloride. After centrifugation of the mixture, 400 µl 10% Na2CO3 solution and 400 µl alkaline phenol solution (0.8% phenol solution in 0.2 M NaOH) was incubated with 400 µl supernatant at 37 °C for 30 min, cooled down on ice and the absorbance was measured spectrophotomet-rically at 605 nm against reagent blank. 4-aminophenol standard curves were also prepared; each sample was examined in triplicates. Finally, mean specific en-zyme activity, Vmax and KM values were calculated similarly to the aminopyrine N-demethylation assay. All results were standardised according to the total pro-tein concentration of microsome samples and were corrected for an inhibited blank per each substrate concentration.

Testosterone 6β-hydroxylation assay. CYP3A37 activity was determined by testosterone 6β-hydroxylation assay. All reactions were performed in total volumes of 200 µl. The incubation mixtures consisted of testosterone (0.5 mM), hepatic microsomal protein (1 mg/ml), NADPH+H+ (1 mM) and a NADPH+H+-generating system in 0.1 M Tris-HCl buffer (pH 7.4). The reactions were stopped after incubation for 15 min at 37 °C by the addition of 200 µl ice-cold methanol. Samples were centrifuged at 13,000 rpm for 10 min at 4 °C and the supernatants were analysed by HPLC. The chromatographic method described by Lutz et al. (2002) was employed with some modifications. Potassium phosphate buffer (10 mM) at pH 3.2, containing methanol (57:43), was used as a mobile phase. Following isocratic separation of metabolites, the column was rinsed of remain-ing substrate as well as other hydrophobic components by employing gradients reaching 90% of organic solvent and then the column was re-equilibrated. The flow rate was 3 ml/min, and the detector was set to 254 nm. Chromatography was performed on a Merck Hitachi HPLC system using an L-6200 A pump, an L-4250 UV detector and a D-6000 A interface (Darmstadt, Germany) with a 100 × 4.6 mm Chromolith Performance C18 (Merck) chromatographic column for ana-lytical separation.

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Statistical analysis

For the statistical analysis of data the R 2.14.0 software was used. Non-parametric Mann-Whitney’s test was used for comparison of results and Pear-son’s test of correlation was performed in order to analyse correlations between the measured parameters. All tests were two-tailed, the level of significance was considered at P < 0.05, and 0.05 ≤ P < 0.10 was considered to be a near-significant trend. Data are presented as means ± SEM.

Results

Dietary butyrate supplementation significantly (P < 0.001) improved the body weight in the first two weeks after hatching (day 7: control group: 124.6 ± 3.4 g, butyrate-treated group: 157.5 ± 4.2 g; day 14: control group: 303.5 ± 8.8 g, butyrate-treated group: 334.3 ± 7.9 g), in harmony with the results of Hu and Guo (2007), but this effect was attenuated at the end of the experiment (day 21: control group: 636.7 ± 16.8 g, butyrate-treated group: 666.4 ± 15.1 g; P = 0.029).

Analysis of hepatic histone acetylation Screening of important acetylation sites at core histones revealed that bu-

tyrate caused a significantly increased ratio in histone H2A acetylation at Lys 5 (P = 0.038) in broiler chickens fed with butyrate during the early post-hatch pe-riod. In contrast, dietary butyrate had no significant influence on the acetylation state of core histones H2B, H3 and H4 at the examined acetylation sites (H2B: P = 0.397, H3: P = 0.181, H4: P = 0.382). The quantitative results and the repre-sentative bands obtained by Western blotting are shown in Fig. 1.

Enzyme assays on hepatic microsomal CYP activity Aminopyrine N-demethylation assay. No significant difference was ob-

served between the average aminopyrine N-demethylation activity (P = 0.541), nor between Vmax (P = 0.436) and KM (P = 0.114) values of hepatic microsomal CYP2H/CYP3A37 enzymes of butyrate-fed and control broiler chickens. PB treatment caused notable enzyme induction with significantly increased mean specific activity (P = 0.006), Vmax (P = 0.003) and KM values (P = 0.011) (Table 2 and Fig. 2.A).

Aniline hydroxylation assay. Butyrate supplementation did not alter the aniline hydroxylation activity of hepatic microsomal enzymes, which indicated the activity of the CYP2H subfamily. There was no significant difference in av-erage enzyme activity (P = 0.321), Vmax (P = 0.370) and KM (P = 0.673) values between control and butyrate-fed chickens. PB treatment significantly induced the mean enzyme activity (P < 0.001) and increased Vmax (P = 0.028) values, while KM tended to decrease (P = 0.050) (Table 2 and Fig. 2.B).

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*

0

50

100

150

200

H2A H2B H3 H4

y(%

) ControlButyrate

Fig. 1. Relative acetylation of hepatic core histones in broiler chicken with dietary butyrate

supplementation (1.5 g/kg diet), compared to those of controls (considered as 100%). Data are presented as mean ± SEM (n = 10/group). Representative bands are also shown as obtained by

Western blotting; in each case, open bars refer to the control (C), and the closed ones to the butyrate-fed group (B). The upper row indicates the relative protein expression levels of H2A, H2B, H3 and

H4, respectively, while the bands specific for acetylated histones can be seen below. Western blots were done in duplicate for H2A in all animals to confirm the result. *Significant difference, P < 0.05

Table 2

Effects of dietary butyrate supplementation on the kinetic properties of cytochrome P450 (CYP) enzymes in broiler chicken1

Enzyme action Responsible CYP

Kinetic parameter Control Butyrate3 PB4

Aminopyrine N-demethylation CYP2H/3A37 Vmax (μmol/mg/min)

KM (mM) 0.116 ± 0.0180.574 ± 0.184

0.144 ± 0.0490.869 ± 0.210

0.380 ± 0.057**

1.734 ± 0.117*

Aniline hydroxylation CYP2H Vmax (μmol/mg/min)

KM (mM) 0.090 ± 0.0138.282 ± 2.246

0.072 ± 0.0166.531 ± 1.288

0.144 ± 0.016*

4.165 ± 0.391†

1Values are shown as means ± SEM; n = 10/group; 3Butyrate = diet supplemented with 1.5 g/kg diet sodium butyrate; 4PB = chickens received 80 mg/kg body weight phenobarbital intracoelomally on days 18–21; ∗Significant difference, P < 0.05, ∗∗Significant difference, P < 0.01, †Near-significant trend, P < 0.1

Testosterone 6β-hydroxylation assay. Similarly, butyrate did not influence the hepatic microsomal CYP3A37 activity in broiler chicken, tested by the testosterone 6β-hydroxylation assay (P = 0.436), while PB treatment resulted in a highly signifi-cant, approximately seven times higher mean enzyme activity (P < 0.001) (Fig. 2.C).

A significant positive correlation was found between aniline hydroxylation (CYP2H) and testosterone 6β-hydroxylation (CYP3A37) activity of the chicken liver microsomes (P < 0.001). Similarly, aminopyrine N-demethylation and testos-terone 6β-hydroxylation were also positively correlated (P = 0.025). Interestingly, a significant positive correlation could also be found between the acetylation state

H2A H2B H3 H4

200

150

100

50

0

Rel

ativ

e hi

ston

e ac

etyl

atio

n (%

)

Control

Butyrate

H2A

Ac H2A

H2B

Ac H2B

H3

Ac H3

H4

Ac H4

C B C B C B C B

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of H3 histone and microsomal CYP3A37 activity (P = 0.045). However, all of these correlations could be observed both in butyrate-fed and control chickens.

Aminopyrine N-demethylation

**

0

50

100

150

200

250

Control Butyrate PB

nmol

/min

per

mg

prot

ein

A

Aniline-hydroxylation

***

0

10

20

30

40

50

60

70

Control Butyrate Phenobarbital

nmol

/min

per

mg

prot

ein

B

Testosterone 6β-hydroxylation

***

010

20

30

40

5060

70

80

Control Butyrate PB

pmol

/min

per

mg

prot

ein

C

Fig. 2. A. Average amount of formaldehyde produced in the aminopyrine N-demethylation assay

(nmol per min per mg microsomal protein), indicating the mean specific activity of hepatic micro-somal CYP2H/CYP3A37 isoenzymes. B. Average amount of 4-aminophenol produced in the ani-line hydroxylation assay (nmol per min per mg microsomal protein), indicating the mean specific

activity of hepatic microsomal CYP2H isoenzyme. C. Average amount of 6β-hydroxy-testosterone produced in the testosterone 6β-hydroxylation assay (pmol per min per mg microsomal protein), indicating the mean specific activity of hepatic microsomal CYP3A37 isoenzyme. All tests were

carried out with hepatic microsome fractions of broiler chickens fed with control or butyrate-supplemented (1.5 g/kg diet) diet or treated with phenobarbital (PB). Data are presented as mean ±

SEM (n = 10/group). **Significant difference, P < 0.01, ***Significant difference, P < 0.001

Aniline hydroxylation

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Discussion

Analysis of hepatic histone acetylation

Butyrate-induced in vitro hyperacetylation of histone H2A was described also by Tobisawa et al. (2010) on a mouse colonic epithelial cell line. H2A has the largest number of subtypes among all core histones, 13 variants have been identified by Bonenfant et al. (2006). It was reported by Ishibashi et al. (2009) that the acetylation of H2A variants played a critical role in conformational changes and decreased stability of the NCP, working synergistically with acety-lation of the rest of core histone N-terminal tails. According to another study (Brower-Toland et al., 2005), H2A and H2B acetylation also plays an important role in decreasing the histone-DNA interactions in the acetylated NCP, making the chromatin transcriptionally active.

In contrast to the large amount of in vitro results, very few reports can be found about in vivo trials on HDAC inhibitors, especially in chicken. The results of this study are partly in agreement with those of another in vivo experiment, where a significant increase in total histone acetylation was reported in porcine caecal tissue after dietary supplementation with lactulose, a precursor of butyrate production by anaerobic gut bacteria (Kien et al., 2008). In the same study, a de-creased total histone acetylation ratio was found after caecal infusion of butyrate, so the effect on histone acetylation may depend also on the endogenous or ex-ogenous origin of HDAC inhibitors.

No significant changes could be observed in the recent study on the acety-lation state of core histones H2B, H3 and H4 at the acetylation sites examined. Interestingly, the observed in vivo epigenetic effects of butyrate on hepatic his-tone acetylation are partly in contrast with its in vitro action, because butyrate caused hyperacetylation of H3 and H4 in all examined vertebrate cell lines, while H2A and H2B were affected in certain rat-derived cell cultures (Candido et al., 1978). The applied primary antibodies recognised the most frequent individual acetylation possibility of each histone (H2A and H2B: Lys 5, H3: Lys 9, H4: Lys 8) close to the N-terminal end of the molecule. However, there are still some other lysine residues in all core histones which may be potential targets of HDAC inhibitors (Zhang et al., 2002). The study of Davie (2003) also underlines the importance of the acetylation site, as well as the actual phase of the cell cycle in the histone-hyperacetylating effect of butyrate. The critical role of HATs was reported by Rada-Iglesias et al. (2007), who found decreased histone acetylation caused by butyrate treatment in cell lines (HepG2 and HT-29), according to its pleiotropic effect on HAT and HDAC. In association with these reports, lack of histone hyperacetylation at the above-mentioned acetylation sites in H2B, H3 and H4 may also be in connection with the modified activity of HATs.

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The observed hyperacetylation of hepatic histone H2A, caused by dietary butyrate supplementation, may modify the expression of certain genes, such as those of microsomal CYP enzymes involved in biotransformation.

Enzyme assays on hepatic microsomal CYP activity

Very few data have been presented in the literature on the effect of drugs or other xenobiotics on CYP enzymes in chicken. Zhang et al. (2011) reported that enrofloxacin decreased the expression of CYP1A and CYP3A subfamilies, while marbofloxacin decreased the expression of only CYP1A in broilers. Induc-tion of CYP3A and CYP2H1 gene expression by PB was described by Paolini et al. (1997) and Davidson et al. (2001). In agreement with these results, we also found CYP2H and CYP3A37 isoenzymes to be well inducible by PB.

The observed correlations are also of special interest because of the recog-nised direct connection between changes at the level of histone proteins and those of the CYP enzyme activity. Baer-Dubowska et al. (2011) underlined the influence of histone acetylation on gene expression of several members of the CYP2 family. In harmony with our results, Li et al. (2009) reported that altera-tions in histone H3 acetylation are involved in the expression of the CYP3A sub-family in the adult mouse.

On the basis of our results, dietary butyrate supplementation with the ap-plied dose did not affect the microsomal CYP2H and CYP3A37 activity of the liver, so there might be no remarkable pharmacokinetic consequences of the bu-tyrate addition. However, possible effects of dietary butyrate on other CYP sub-families cannot be excluded.

Acknowledgements

The authors would like to express their special thanks to Kathrin Hansen (De-partment of Physiology, University of Veterinary Medicine, Hanover, Germany) for her kind help in the Western blot examinations and to Éva Fejes and Zsolt Wagner (Institute of Pharmacodynamics, Faculty of Pharmacology, Semmelweis University, Budapest, Hungary) for their help in the microsome isolation. The excellent assistance of Kata An-nus, Tünde Benedek, Daniella Nyáry and Dániel Pleva (Department of Physiology and Biochemistry, Faculty of Veterinary Science, Szent István University, Budapest, Hun-gary) in the care for the animals and in the sampling is gratefully acknowledged.

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