Title:Effects of butyrate on hepatic epigenetics and microsomal drug-metabolizing enzymes in chicken
The short chain fatty acid butyrate is one of the major end products of the anaerobic microbial fermentation of carbohydrates in the forestomachs of ruminants and in the large intestine of monogastric mammals, birds and humans. Butyrate is also a widely used feed additive as an alternative growth promoter due to its beneficial effects on growth performance, first of all in poultry and pig nutrition. This is of special importance due to the banning of the traditional antibiotic growth promoters in the European Union. Butyrate may provoke its effects on metabolism via many different, yet not completely defined pathways. One of those pathways is that butyrate is known as a histone deacetylase inhibitor, inducing histone hyperacetylation in vitro and playing a predominant role in the epigenetic regulation of gene expression and cell function. It was hypothesized in this study that butyrate, applied as feed additive, might cause similar in vivo modifications in the chromatin structure of the hepatocytes of chickens in the early post-hatch period. Further, it could influence the expression of certain genes and therefore modify the activity of hepatic microsomal drugmetabolizing cytochrome P450 (CYP) enzymes, resulting in pharmacoepigenetic interactions with simultaneously applied xenobiotics. Most experiments of this PhD study were performed in broiler chickens, because chickens are not only target species of butyrate administration as feed additive, but they can also serve as model for the investigation of butyrate’s actions. Regarding the intestinal effects of butyrate, this study aimed to compare the influence of butyrate on small intestinal histomorphology in chicken and rat, the latter as a model animal for monogastric mammals. In vivo studies were carried out in chicken to investigate the molecular mechanisms of butyrate’s epigenetic actions on the liver. Broiler chicks in the early post-hatch period received butyrate-supplemented diet (1.5 g/kg diet) or were treated once daily with orally administered bolus of butyrate following overnight fasting with two different doses (0.25 or 1.25 g/kg body weight per day) for five days. After slaughtering, cell organelles were separated by differential centrifugation from the livers and acetylation of hepatic core histones was screened from cell nuclei by western blotting. Effects of butyrate on CYP gene expression were tested at first in vitro on primary culture of chicken hepatocytes, followed by an in vivo trial with butyrate-fed chickens. The activity of the most important CYP enzymes was also monitored by aminopyrine N-demethylation, aniline hydroxylation and testosterone 6β-hydroxylation assays from the microsomal fractions of chickens. Furthermore, the interaction of butyrate and the macrolide antibiotic erythromycin was tested in vitro and finally also in vivo by studying the major pharmacokinetic parameters of erythromycin.