Concentrations Of Sodium Butyrate Research Articles

Flow cytometric and biochemical analysis of dose‐dependent effects of sodium butyrate on human endometrial adenocarcinoma cells

Sodium butyrate (SB) treatment was previously shown to produce seven-fold increases in estrogen hormone receptor binding sites of human endometrial adenocarcinoma (IK) cells. Flow cytometric analysis and histone gel electrophoresis were used to examine cell cycle, cell metabolism, and nuclear histone fractions in IK cells treated with different concentrations of SB. SB-treated cells stained with fluorochromes specific for DNA, RNA, or general protein were analyzed by flow cytometry (FCM). Changes in accessibility to three DNA stains and gel electrophoresis were used to analyze rearrangements in chromatin structure. SB caused an accumulation of cells in the G1 phase and inhibited DNA synthesis, but not cellular levels of RNA and protein. Hoechst accessibility to A-T rich regions on DNA was dramatically increased after removal of SB. H1 histones were dephosphorylated and core histones were acetylated during SB-treatment. Information obtained in these studies may be useful for correlating cellular and biochemical events with SB-induced increases in nuclear steroid hormone binding sites.

The differentiation-inducing agent sodium butyrate produces divergent effects on albumin and thyroxine-binding globulin synthesis by human hepatoblastoma-derived (Hep G2) cells

The addition of sodium butyrate, a differentiation-inducing agent, to the culture medium of human hepatoblastoma-derived (Hep G2) cells, produced a dose-dependent and time-dependent increase in albumin (ALB) and decrease in T4-binding globulin (TBG) synthesis and secretion. In the presence of 0.01 to 2.0 mM sodium butyrate, newly synthesized [35S]ALB progressively increased up to 139% of control cultures grown in the absence of sodium butyrate, whereas TBG synthesis was already slightly inhibited using the lowest concentrations of this agent and further diminished thereafter. The use of 5 mM and 10 mM sodium butyrate inhibited the synthesis of both proteins, probably as a consequence of toxic effects on cell cultures. The addition of 1 mM sodium butyrate for variable time intervals caused an increase in the amount of ALB recovered in the medium up to 146% after 72 h, and a decrease of TBG up to 44% of controls. These different effects on ALB and TBG occurred concomitantly with an inhibition of cell growth, as shown by the reduction in the cell number/flask compared to control cultures. At the highest sodium butyrate concentrations, a relevant impairment in the secretion of newly synthesized TBG, but not of ALB, also occurred. These divergent effects on ALB and TBG synthesis by Hep G2 cells might be related to biochemical differentiation induced by sodium butyrate in this tumoral cell system, suggesting that TBG synthesis is increased in Hep G2 cells because of their neoplastic nature.

Sodium butyrate selectively alters thyroid hormone receptor gene expression in GH3 cells.

High-affinity binding of thyroid hormone (T3) to nuclear receptors influences the transcriptional rate of specific genes. The carboxylic acid, sodium butyrate, is known to reduce nuclear binding of T3 in GH3 rat pituitary cels, and this effect correlates with inhibition of T3 responsiveness. Incubation of GH3 cells with 10 mM, but not 1 mM, sodium butyrate abolishes induction of growth hormone gene expression by T3. The relationship between thyroid hormone receptor (TR) depletion and inhibition of T3 action was further investigated by determining the effects of sodium butyrate on TR gene expression. GH3 cells contain mRNAs encoding at least three thyroid hormone receptors (TRs alpha 1, beta 1, and beta 2), as well as a related non-T3 binding repressor or T3 action (c-erbA alpha 2). Remarkably, 10 mM sodium butyrate reduces TR beta 2 mRNA levels by greater than 95% in the presence or absence of T3. The down-regulation of TR beta 2 mRNA occurs at a concentration of sodium butyrate between 5 and 10 mM and is maximal by 8 h of treatment. In contrast, TR beta 1 mRNA levels are unchanged. Furthermore, sodium butyrate has little effect on the mRNAs encoding TR alpha 1 and c-erbA alpha 2. Nuclear run-on assays indicated that sodium butyrate depletes TR beta 2 mRNA by rapidly repressing TR beta 2 gene transcription. In the presence of the transcriptional inhibitor, actinomycin D, the half-life of TR beta 2 mRNA was approximately 3 h in the presence or absence of sodium butyrate. Thus, the reduction in TR number induced by sodium butyrate is likely to be due to the transcriptionally mediated reduction in TR beta 2 mRNA. These data suggest that TR beta 2 has an important physiological role in the regulation of growth hormone gene expression by T3.

Pancreatic Islet A2B5- and 3G5-Reactive Gangliosides are Markers of Differentiation in Rat Insulinoma Cells

Rat insulinoma (RIN) cells, in comparison with adult islet cells, are relatively undifferentiated. They secrete low amounts of islet hormones, are unresponsive to glucose, and display pluripotency. A minority of RIN cells react with monoclonal antibodies A2B5 and 3G5 which recognize complex gangliosides on normal islet cells. In order to determine whether the expression of A2B5- or 3G5-reactive gangliosides is modulated during differentiation RIN cells were cultured with various concentrations of sodium butyrate (NaB), a known inducer of cellular differentiation. Expression of A2B5- and 3G5-reactive gangliosides was determined by indirect immunofluorescence and flow cytofluorimetry. NaB exposure resulted in a dose-dependent decrease in cell proliferation over 5 days of 1.5-, 2.9-, and 17.5-fold at 0.5, 1.0 and 3.0 mM, respectively, and a distinct change in cellular morphology. Cells exposed to NaB displayed prominent neurite-like projections. At 3 mM NaB, insulin secretion increased 7.9-fold and the percentage of cells expressing A2B5- and 3G5-reactive gangliosides increased by up to 4.4- and 5.5-fold, respectively. The expression of A2B5- or 3G5-reactive gangliosides per cell also increased, by 2.4- and 1.3-fold, respectively, at 3 mM NaB. These findings demonstrate that the expression of cell surface A2B5- and 3G5-reactive gangliosides is not static but increases with cell differentiation. NaB-treated RIN cells may serve as a model to study the role of gangliosides in the function and lineage relationships of islet cells.

Determination of mating-type conversion rates of heterothallic Saccharomyces cerevisiae with the fluctuation assay

The fluctuation assay of Luria and Delbruck was adapted for the exact determination of mating-type interconversion rates of semi-sterile heterothallic strains of Saccharomyces cereviae. These rates varied between 10−7 and 10−6, depending on the individual strain, and were enhanced in a dose-dependent manner for cells growing in the presence of increasing concentrations of sodium butyrate. Under our experimental conditions, the distribution of alpha cells over populations of a(ste2) cells corresponded to a Poissonian distribution (within sample errors); that over populations of a(ste14)-cells did not differ significantly from the distribution calculated by Lea and Coulsen.

Changes in X-Ray Sensitivity and Glutathione Content of Human Colon Tumor Cells after Exposure to the Differentiation-Inducing Agent Sodium Butyrate

Clone A human colon cancer cells were exposed to concentrations of sodium butyrate (NAB, 0-2 mM) for three passages in vitro, and responses to either graded single doses or split doses of 250 kVp X rays were determined. The survival data were fit to the single-hit, multitarget model of inactivation. For the graded single dose experiments, we found that NAB produced a decrease in the magnitude of the quasi-threshold (Dq) parameter after a concentration of about 0.9 mM was exceeded. Similarly, in split dose experiments, the amount of sublethal damage recovery (SLDR) was reduced in a concentration-dependent manner as shown by a decrease in the Dq parameter. However, the inhibition of SLDR occurred with no apparent threshold NAB concentration. NAB did not affect potentially lethal damage recovery. Paradoxically, increasing concentrations of NAB produced an exponential increase in the intracellular glutathione content, which could be blocked by exposure of the cells to buthionine sulfoximine (BSO). BSO treatment of NAB-adapted cells led to additional cell killing, again most noted by changes in the Dq parameter. We postulate that these responses are associated with NAB-induced changes in chromatin structure, particularly the association between DNA and nucleosomal histones H3 and H4.

Expression of carcinoembryonic antigen by adenoma and carcinoma derived epithelial cell lines: possible marker of tumour progression and modulation of expression by sodium butyrate

We have used an indirect immunofluorescence assay to demonstrate the cell membrane expression of carcinoembryonic antigen (CEA) by a pre-malignant colorectal adenoma derived epithelial cell line (PC/AA) and three colorectal carcinoma cell lines (HT29, PC/JW and PC/JW/FI). The results obtained indicated that CEA may be used as a marker for tumour progression up to the point of malignant transformation, after which the selection for anaplastic variants during continuous in vitro culture may result in the subsequent reduction of cell membrane CEA expression. The percentage of PC/AA cells expressing cell membrane CEA increased from 23.1% of diploid early passage (passage 18) cells to 56.0% of aneuploid late passage (passage 58) cells. Although non-tumorigenic, the proportion of PC/AA cells expressing cell membrane CEA at late passage corresponded to that for the PC/JW carcinoma line (56.2%) and is further evidence for the progression of PC/AA in culture. A 3T3 feeder-independent variant of PC/JW (PC/JW/FI) demonstrated a similar percentage of CEA-positive cells as the parental line for the first 21 passages without feeder support, but by passage 27 without 3T3 feeders only 35.3% of cells stained positive. This could be restored to 62.0% by continuous treatment with sodium butyrate (2 mM). A differential growth response to sodium butyrate was noted for the pre-malignant adenoma cell line PC/AA and the carcinoma lines HT29 and PC/JW/FI. Concentrations of sodium butyrate (2 mM) that killed early passage PC/AA cells allowed the late passage PC/AA cells and the carcinoma lines to proliferate, raising the possibility of sodium butyrate acting as a tumour promotor in the human colorectum.

Effects of differing concentrations of sodium butyrate on 1,2-dimethylhydrazine-induced rat intestinal neoplasia

The incidence, distribution, size, and histopathology of small and large bowel tumors induced by parenteral administration of 1,2-dimethylhydrazine were examined in rats given 1% or 2% sodium butyrate dissolved in drinking water. Although previous in vitro reports on colon cancer cell lines have suggested that sodium butyrate might have a role to play as a chemotherapeutic “differentiating agent,” the results of this in vivo study indicate that sodium butyrate treatment enhanced the development of colonic neoplasia and was associated with increased fecal butyric acid concentrations. In contrast, no changes were seen in the incidence of small bowel tumors, luminal butyric acid concentrations, mucosal morphology, or brush-border enzyme activities (i.e., sucrase, alkaline phosphatase). This study suggests that dietary butyrate has an important, possibly indirect, regulatory role in carcinogenesis associated with an experimental animal model of colonic neoplasia.

DNA hypermethylation in sodium butyrate-treated WI-38 fibroblasts.

Sodium butyrate is very often used to alter gene expression in cultured cells. In this study, we examined the effects of this compound on various cellular events in WI-38 human embryonic lung fibroblasts in culture. During a 16-20-h treatment at sodium butyrate concentrations of between 5 and 20 mM, no adverse effects on cell morphology were observed. However, cell division and DNA synthesis were reversibly inhibited, the latter by 85, 80, and 70% at sodium butyrate concentrations of 5, 10, and 20 mM, respectively. Although overall protein synthetic activity was not significantly affected, RNA synthesis decreased to 76% of the control values at a sodium butyrate concentration of 5 mM. Butyrate treatment also caused hypermethylation of DNA cytosines as determined by differential digestion by MspI/HpaII restriction endonucleases and by high performance liquid chromatography analysis of the DNA. The 5-methylcytosine content of the DNA in untreated WI-38 fibroblasts was 2.94 +/- 0.46% of total cytosine residues, while in cultures treated with 5, 10, and 20 mM sodium butyrate, these values were 5.76 +/- 0.28, 5.91 +/- 0.37, and 6.8 +/- 0.44%, respectively. An interesting feature is that this hypermethylation occurred in DNA which was synthesized in the presence of sodium butyrate (newly synthesized) as well as in DNA which had been synthesized before butyrate administration (pre-existing DNA). The hypermethylated state was conserved only in the former situation, since the methylcytosines were rapidly lost in the subsequent generation in the latter case. It would therefore appear that methylcytosines are maintained after cell replication only if they are generated on newly synthesized DNA.

Sodium butyrate selectively inhibits host cell glycoprotein synthesis in human fibroblasts infected with cytomegalovirus

Host cell as well as viral DNA synthesis in human fibroblasts infected with human cytomegalovirus was found to be largely resistant even to high concentrations of sodium butyrate. Likewise, production of viral progeny was reduced by 1-2 orders of magnitude but not abolished. On the other hand, the drug allowed (modified) glycosylation only of viral polypeptides whereas that of host proteins was suppressed. Immunofluorescence studies on living cells suggested that butyrate may interfere with processing and intracellular transport of virus-specific surface membrane antigens.

Modulation of in vitro myogenesis by submillimolar concentrations of sodium butyrate

The effects of submillimolar concentrations of sodium butyrate on the differentiation of cultured chick myoblasts have been studied. The continuous presence of 0.5 mM butyrate inhibited myoblast fusion, creatine kinase (CK) isozyme transition, and synthesis of total RNA and protein until the 4th day of myogenesis, after which the fusion index reached control values and total CK activity was elevated. The latter continued to exhibit daily increases over control levels, largely reflecting activity of the MM-CK muscle-specific isozyme which increased to twice the control level by the 8th day. Similar but less striking patterns of early inhibition followed by stimulation were observed for total protein content and synthesis of total protein and RNA. On the other hand, DNA content was slightly but significantly depressed in treated cultures at all times. Butyrate treatment did not reverse 5′-bromodeoxyuridine (BUdR) inhibition of MM-CK differentiation. It was also noted that continuous treatment with 0.5 mM butyrate prevented the unexplained sporadic deterioration of myotubes sometimes observed at the 4th day.

Sodium butyrate stimulates DNA repair in UV-irradiated normal and xeroderma pigmentosum human fibroblasts.

Histone acetylation, DNA replicative synthesis, UV-induced DNA repair synthesis, and UV-induced endonuclease-sensitive sites were measured in normal human fibroblasts and xeroderma pigmentosum fibroblasts (complementation groups A, C, and D) following exposure to sodium butyrate. In all four cell types, treatment with millimolar concentrations of sodium butyrate resulted in a hyperacetylation of the core histones. Furthermore, following an exposure of 20 mM sodium butyrate for 48 h, the extent of hyperacetylation was the same in each cell type. In agreement with previous reports, we observed a marked decrease in DNA replicative synthesis in each cell type following increasing times of exposure to sodium butyrate. On the other hand, we observed a marked increase in DNA repair synthesis occurring during early times after UV irradiation in normal cells and in two of the xeroderma pigmentosum cell strains (groups C and D). This increase appeared to correlate with the increase in the highest acetylated form of histone H4. Furthermore, the total number of endonuclease-sensitive sites (i.e. prior to the onset of repair) induced by UV radiation was the same in both butyrated-treated and untreated normal cells over the dose range of 0-20 J/m2. However, the initial rate of removal of these sites increased in butyrate-treated normal cells. These results indicate that sodium butyrate stimulates the initial rate of nucleotide excision repair in both normal and (partially) repair-deficient human cells at concentrations where the histones are maximally hyperacetylated.

Butyrate-induced histone hyperacetylation in human and mouse cells: estimation of putative sites of histone acetylation in vivo.

Human and mouse cells in culture were treated with various concentrations of sodium butyrate. Acid-extracted histones of control and butyrate-treated cells were analyzed by two-dimensional gel electrophoresis. All core histones of the control cells contained modified forms. All core histones of the butyrate-treated cells were hyperacetylated. Depending on the number of acetylation sites per molecule, each histone or histone variant exhibited a characteristic number of acetylated forms. This number was the same for each histone common in human and mouse cells treated with butyrate. Histones 2A.1, 2A.2, and 2A.X have two sites of inner acetylation; 2A.Z has 3; 2B's have 5; and each one of the H3 variants as well as H4 have 4.

The Regulation of Lipogenesis by Cyclic Nucleotides in Intact Hepatocytes Prepared by a Simplified Technique

Abstract An improved, simplified procedure is described for the isolation of liver cells from rat or chicken. Livers were perfused in situ and then incubated in a solution of collagenase and hyaluronidase. Greater than 50% of the liver mass was converted into a suspension of intact parenchymal cells which excluded a vital stain and were undamaged when viewed with the electron microscope. The cells actively incorporated labeled precursors into lipids and proteins without specific cofactor requirements in both simple, ionic, and complex culture media. The chicken hepatocytes synthesized lipids and proteins for several days in suspension culture, but suspended rat hepatocytes had minimal biosynthetic activity after 24 hours. Both dibutyryl cyclic AMP (0.1 mm) and adenosine 3' : 5'-monophosphate (cyclic AMP) (0.1 mm) produced a similar striking inhibition of [1-14C]acetate incorporation into lipids in chicken hepatocytes. Dibutyryl cyclic AMP was less inhibitory to lipid synthesis by rat liver cells and cyclic AMP had no effect on this process. The inhibitory actions of dibutyryl cyclic AMP were probably not due to isotopic dilution of the label within the cells. Neither cyclic nucleotide had a lipolytic effect in vitro on hepatocyte lipids prelabeled in vivo. The inhibitory action of dibutyryl cyclic AMP on lipogenesis was significantly greater than the effect of twice the concentration of sodium butyrate, and 5'-AMP did not diminish hepatic lipid synthesis. It is postulated that hepatic lipid synthesis is inhibited specifically by cyclic nucleotides, and that hormonal stimulation of adenylate cyclase regulates hepatic lipid synthesis.

Fatty acid oxidation and esterification in isolated rat hepatocytes: regulation by dibutyryl adenosine 3′,5′-cyclic monophosphate

Isolated rat hepatocytes rapidly utilized [(14)C]palmitate and, in particular, synthesized large amounts of neutral lipids from palmitate. Incorporation into cellular lipids occurred at a linear rate proportional to the medium concentration of fatty acids. Oxidation of [(14)C]palmitate to CO(2) increased with time and was much slower than palmitate esterification. Since [(14)C]acetate and [(14)C]glucose were oxidized to CO(2) at a linear rate, the lag in fatty acid oxidation to CO(2) did not involve enzymatic steps subsequent to acetate formation. The relative contribution of palmitate to esterification and to CO(2) formation depended upon the molar ratio of palmitate to albumin (v) and the length of incubation. Dibutyryl cyclic AMP (1 mM) reduced the oxidation of palmitate and acetate to CO(2) by about 50 and 90%, respectively, but did not alter palmitate esterification. However, equivalent concentrations of sodium butyrate produced similar decreases in CO(2) formation. Dibutyryl cyclic AMP (1 mM) also stimulated palmitate oxidation to water-soluble products, principally ketone bodies, by 50-100%. Sodium butyrate exerted no effect, while monobutyryl cyclic AMP and cyclic AMP both stimulated this pathway significantly. These results indicate that both v and dibutyryl cyclic AMP regulate the metabolism of fatty acids by isolated hepatocytes and suggest that hormonal stimulation of adenyl cyclase controls hepatic lipid metabolism.