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Biological

Butyric Acid Induces Apoptosis in Inflamed Fibroblasts

T. Kurita-Ochiai1,*, S. Seto4, N. Suzuki3, M. Yamamoto1, K. Otsuka3, K. Abe2 and K. Ochiai2

1 Department of Microbiology and Immunology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271-8587, Japan;
2 Department of Microbiology and
3 Department of Biochemistry, Nihon University School of Dentistry, Tokyo 101-8310, Japan; and
4 Department of Infectious Diseases, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431-3192, Japan

Correspondence: * corresponding author, ochiai.tomoko{at}nihon-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Butyric acid, an extracellular metabolite from periodontopathic bacteria, induces apoptosis in murine and human T- and B-cells, whereas intact gingival fibroblasts isolated from healthy humans are resistant to butyric-acid-induced apoptosis. We examined the susceptibility of inflamed gingival fibroblasts isolated from adult persons with periodontitis to butyric-acid-induced apoptosis. Butyric acid significantly suppressed the viability of inflamed gingival fibroblasts and induced apoptosis in a dose-dependent manner. The incubation of inflamed gingival fibroblasts with butyric acid induced DNA fragmentation and apoptotic changes such as chromatin condensation, hypodiploid nuclei, and mitochondrial injury. Furthermore, butyric-acid-induced apoptosis in inflamed gingival fibroblasts was reduced by caspase-3/7, -6, -8, and -9 inhibitors. Thus, inflamed gingival fibroblasts from adult persons with periodontitis appear to be highly susceptible to mitochondria- and caspase-dependent apoptosis induced by butyric acid, compared with healthy gingival fibroblasts.

Key Words: apoptosis • butyric acid • fibroblasts • periodontitis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Butyric acid, an extracellular metabolite from periodontopathic bacteria, induces apoptosis in murine and human T- and B-cells (Kurita-Ochiai et al., 1997, 1998, 2001). Emerging evidence indicates that the bacterial modulation of apoptosis is an important part of pathogenesis (Chen and Zychlinsky, 1994). Specific pathogens or their extracellular products may directly induce host cell apoptosis (Zychlinsky et al., 1992). For example, apoptosis has been observed in cultured CD4+ and CD8+ T-cells from persons with AIDS, as well as in activated peripheral blood lymphocyte cultures infected with HIV-1 (Terai et al., 1991; Meyaard et al., 1992). In chronic inflammatory disorders, persons with chronic hepatitis C have high hepatocyte loss due to apoptosis (Kronenberger et al., 2005). Helicobacter pylori infection, which causes chronic inflammation of the gastric mucosa, induces macrophage apoptosis (Cheng et al., 2005). Apoptosis is a key event in the regulation of the lifespan of terminally differentiated leukocytes and in chronic inflamed human gingival tissues. The in situ detection of apoptosis in human gingiva indicates that this process is relevant in controlling inflammation in periodontal disease (Jewett et al., 2000; Gamonal et al., 2001). Furthermore, the clinical significance of fibroblast apoptosis has recently been suggested by its linkage to the loss of attachment, an early feature of periodontitis that precedes the loss of bone (Ekuni et al., 2005). We previously demonstrated that the proliferation and apoptosis of human gingival fibroblasts isolated from periodontally healthy tissues are minimally affected by butyric acid treatment (Kurita-Ochiai et al., 2002). Therefore, we examined whether butyric acid induces cytotoxicity and apoptosis in gingival fibroblasts isolated from inflamed periodontal lesions of adult persons with periodontitis.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Highly purified acetic, butyric, isobutyric, propionic, valeric, isovaleric, succinic, pyruvic, oxalacetic, and malonic acids were purchased from Wako Pure Chemical Industries (Osaka, Japan). Solutions of fatty acids were diluted in alpha minimum essential medium ({alpha}-MEM; Invitrogen, Groningen, Netherlands) and adjusted to pH 7.2 with sodium hydroxide. The 3-(4,5-dimethyl-2-thiazolyl) -2,5-diphenyl tetrazolium bromide (MTT), 2-(6-amino-3-imino-3H-xanthen-9-yl) benzoic acid methyl ester (rhodamine 123), and 4' 6-diamino-2-phenylindole (DAPI) were purchased from Sigma (St. Louis, MO, USA). Synthetic peptides that act as inhibitors for caspase-3/7 (Ac-DEVD-CHO), caspase-8 (Ac-IETD-CHO), caspase-9 (Ac-LEHD-CHO), and caspase-6 (Ac-VEID-CHO) were obtained from the Peptide Institute (Osaka, Japan). These peptides were dissolved in DMSO (Sigma) and then diluted in phosphate-buffered saline (PBS).

Fibroblast Cultures
Human gingival fibroblasts were isolated from attached gingiva. Attached gingiva was obtained from the hard palate area, 5 mm from the gingival margin, from three adult volunteers (two males and one female, from 23 to 32 yrs of age) with clinically healthy periodontium, and from four adult persons (one male and three females, from 22 to 42 yrs of age) with periodontitis. Informed consent was obtained from all donors for tissue resection. The protocol for the human study was approved by the ethics committee of the Nihon University School of Dentistry, Tokyo, Japan.

The collected human gingiva was cut into small pieces, placed in 35-mm2 tissue culture dishes, and cultured in complete medium consisting of {alpha}-MEM supplemented with 10% heat-inactivated fetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. The healthy gingival fibroblasts, termed N23, N32, and GF7, were isolated from human gingival tissues obtained from periodontally healthy adults. The inflamed gingival fibroblasts, termed F22-G, F33-G, M38-G, and F42-G, were isolated from persons with adult periodontitis. The established line of healthy gingival fibroblasts, Gin-1, was obtained from the American Type Culture Collection (Rockville, MD, USA). Fibroblasts were cultured at 37°C in a 5% (v/v) CO2 atmosphere in complete medium consisting of {alpha}-MEM supplemented with 10% heat-inactivated fetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin, and were used for assays at the fifth and eighth passages.

Cell Viability Assay
Fibroblasts (1.0 x 104 cells/well) were treated in 0.1 mL of complete medium with specific doses of butyric acid (1.25–5 mM) or fatty acids (5 mM) in flat-bottomed 96-well plates. After 48 hrs, cell viability was measured by the MTT assay, as described previously (Kurita-Ochiai et al., 2001, 2002).

Detection of Apoptosis
Fibroblasts (5 x 104 cells/well) were cultured in 1 mL of complete medium in 24-well tissue-culture plates (Falcon; Becton Dickinson Labware, Lincoln Park, NJ, USA) with 5 mM butyric acid. For the apoptosis inhibition assay, the cells were pre-treated for 2 hrs with 10 µM of each caspase inhibitor before the addition of butyric acid. For nuclear staining, the cells were stained with 2 µg/mL DAPI in PBS and counterstained with 7 µg/mL hydroethidine in PBS (Poly Sciences, Warrington, PA, USA). The cell morphology was visualized by fluorescence microscopy at 200x magnification. The numbers of apoptotic and non-apoptotic cells were counted under a fluorescent microscope (BX50; Olympus, Tokyo, Japan) with the aid of an eyepiece graticule (24 mm2; Olympus).

Gel Electrophoresis
Fibroblasts (5 x 104 cells/well) were cultured in 1 mL of complete medium in 24-well tissue-culture plates with or without 5 mM butyric acid. After 20 hrs, gel electrophoresis was performed, as described previously (Kurita-Ochiai et al., 1997, 1998).

Measurement of Mitochondrial Membrane Potential
Changes in mitochondrial membrane potential in butyric-acid-exposed fibroblasts were measured by the uptake of lipophilic cation rhodamine 123 into mitochondria. After fibroblasts were incubated with butyric acid, the cells were re-suspended in 1 mL of 10 µg/mL rhodamine 123 for 30 min at room temperature, washed twice with PBS, and re-suspended in PBS. The samples were analyzed for fluorescence (FLI detector, filter 530/30 nm band pass) with a FACSCalibur flow cytometer (Becton Dickinson, Sunnyvale, CA, USA). Histograms were analyzed with CellQuest software (Becton Dickinson).

Statistical Analysis
Multiple-group comparisons were made by one-way analysis of variance (ANOVA), followed by post hoc intergroup comparisons with the Bonferroni-Dunn test. Where appropriate, we used Student’s t test to assess the statistical significance of differences between two groups.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Butyric Acid on the Viability of Healthy and Inflamed Gingival Fibroblasts
The viability of healthy gingival fibroblasts Gin-1, N23, N32, and GF7 in the presence of butyric acid was slightly lower than, but not significantly different from, that observed for untreated cells (Fig. 1AGo). In contrast, butyric acid markedly decreased the viability of inflamed gingival fibroblasts F22-G, F33-G, M38-G, and F42-G in a dose-dependent manner. As little as 1.25 mM butyric acid resulted in 40–48% cell death, compared with control cultures (P < 0.01). Maximal inhibition by butyric acid was observed at a dose of 5 mM (88–94% vs. control, P < 0.01). Interestingly, other volatile organic acids, such as propionic, valeric, and isovaleric acids, also suppressed inflamed gingival fibroblast viability (30–40%) compared with healthy gingival fibroblasts (4–14%; P < 0.01; Fig. 1BGo). In contrast, inflamed and healthy gingival fibroblasts showed weak sensitivity to the volatile fatty acids acetic acid and isobutyric acid, as well as to the non-volatile fatty acids succinic, pyruvic, oxalacetic, and malonic acids (Figs. 1B, 1CGo).


Figure 1
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  		Figure 1. Effects of short-chain fatty acids on the viability of gingival fibroblasts. Healthy (Gin-1, N23, N32, and GF7) and inflamed (F22-G, F33-G, M38-G, and F42-G) gingival fibroblasts were cultured with (A) the indicated concentrations of butyric acid, (B) 5 mM volatile fatty acids, or (C) 5 mM non-volatile fatty acids. Cell viability was determined by the MTT assay and expressed as a percentage of the absorbance obtained in the absence of fatty acids. The results are expressed as the mean ± standard error of 3 independent experiments. *P < 0.01 vs. untreated control cells (white bar).

 
Butyric Acid Induces Apoptosis in Inflamed Gingival Fibroblasts
The induction of apoptosis by butyric acid was indicated by DAPI staining of the nuclei, electrophoresis of the fragmented DNA, and changes in the mitochondrial membrane potential. When inflamed gingival fibroblasts F22-G, F33-G, M38-G, and F42-G were incubated with 1.25, 2.5, or 5 mM butyric acid for 24 hrs and stained with fluorescent probes, the number of apoptotic cells increased linearly in a dose-dependent manner (Fig. 2AGo). Furthermore, when the cells were cultured with 5 mM butyric acid for various time periods, the number of apoptotic cells increased in all 4 types of inflamed gingival fibroblasts in a time-dependent manner (Fig. 2BGo). By 24 hrs, apoptotic cells accounted for ~ 80% of the total cell population in inflamed gingival fibroblast cultures (Fig. 2BGo). The healthy gingival fibroblasts Gin-1, N23, and GF7 did not exhibit laddering patterns indicative of fragmented DNA in the presence of 5 mM butyric acid (Fig. 2CGo, lanes 2, 4, and 6). In contrast, typical oligonucleosomal ladders were observed when the inflamed gingival fibroblasts F22-G, M33-G, M38-G, and M42-G were incubated with 5 mM butyric acid (Fig. 2CGo, lanes 8, 10, 12, and 14). When we examined the morphological changes in the nuclei of 5 mM butyric-acid-treated inflamed M38-G fibroblasts by DAPI staining, several nuclei appeared condensed and partitioned into several nodules, whereas no such nuclei were found in healthy N23 fibroblasts (Fig. 2DGo). Similar results were observed with the other healthy and inflamed gingival fibroblasts.


Figure 2
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  		Figure 2. Butyric-acid-induced apoptosis in inflamed gingival fibroblasts. Healthy (Gin-1, N23, N32, and GF7) and inflamed (F22-G, F33-G, M38-G, and F42-G) gingival fibroblasts were cultured (A) with the indicated concentrations of butyric acid for 24 hrs and (B) in the presence of 5 mM butyric acid for the indicated time periods. Harvested cells were stained with DAPI. The results are expressed as the mean ± standard error of 3 independent experiments. (C) Agarose gel electrophoresis of DNA. Healthy (Gin-1, N23, and GF7) and inflamed (F22-G, F33-G, M38-G, and F42-G) gingival fibroblasts were cultured in the absence (lanes 1, 3, 5, 7, 9, 11, and 13) or presence (lanes 2, 4, 6, 8, 10, 12, and 14) of 5 mM butyric acid (BA). (D) Nuclear morphology of gingival fibroblasts treated with butyric acid. HGF (N23, upper panels) and IGF (M38-G, lower panels) were cultured in the absence (left panels) or presence (right panels) of 5 mM butyric acid (BA) and stained with DAPI. The results are representative of 3 independent experiments. Scale bars, 10 µm.

 
Mitochondria and Caspase Dependency of Butyric-acid-induced Apoptosis in Inflamed Gingival Fibroblasts
Butyric acid markedly decreased the mitochondrial membrane potential in inflamed F22-G and M38-G fibroblasts by 80 and 75%, respectively, compared with untreated cells, whereas no marked decrease in the mitochondrial membrane potential was observed in butyric-acid-treated healthy gingival fibroblasts (Fig. 3AGo). To examine the caspase dependency of butyric-acid-induced apoptosis in inflamed gingival fibroblasts, we cultured F22-G and M38-G with 5 mM butyric acid in the presence of synthetic peptides that inhibit the activation of caspase-3/7 (Ac-DEVD-CHO), caspase-6 (Ac-VEID-CHO), caspase-8/6 (Ac-IETD-CHO), and caspase-9 (Ac-LEHD-CHO). In the presence of Ac-DEVD-CHO and Ac-VEID-CHO, the number of apoptotic cells in F22-G and M38-G was significantly decreased compared with that in peptide-free butyric-acid-treated controls (46–48% for DEVD-CHO and 47–48% for VEID-CHO vs. controls; P < 0.01; Fig. 3BGo). When the cells were cultured with Ac-IEHD-CHO and Ac-LEHD-CHO, butyric-acid-induced apoptosis in inflamed gingival fibroblasts was further reduced by ~ 60% compared with that in butyric-acid-treated control cells (P < 0.01). The addition of individual synthetic peptides alone, which inhibited the activation of caspase, did not induce fibroblast apoptosis. Furthermore, the final concentration of the solvent DMSO in the culture medium was 0.1%, and this low concentration did not exert any affect on the fibroblasts.


Figure 3
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  		Figure 3. Mitochondrial membrane potential (MMP) and effects of caspase inhibitors on the butyric-acid-induced apoptosis of inflamed gingival fibroblasts. (A) Healthy (Gin-1 and N23) and inflamed (F22-G and M38-G) gingival fibroblasts were treated with 5 mM butyric acid, and the MMP was measured as the fluorescence emitted by rhodamine 123 taken up by the mitochondria. Data are expressed as a percentage of the control (mean ± standard error [SE]). *P < 0.01 vs. corresponding control cells (Gin 1 and N23). (B) Inflamed gingival fibroblasts (F22-G and M38-G) were pre-treated with caspase inhibitors and then treated with 5 mM butyric acid. Harvested cells were stained with DAPI. *P < 0.01 vs. inhibitor-free butyric-acid-treated control cells. The results are expressed as the mean ± SE of 3 independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Butyric acid, a virulence factor common to Porphyromonas gingivalis, Prevotella loescheii, and Fusobacterium nucleatum (Kurita-Ochiai et al., 1995), suppressed inflamed gingival fibroblast viability in a dose-dependent manner. Furthermore, other volatile fatty acids, such as propionic, valeric, and isovaleric acids, also suppressed inflamed gingival fibroblast viability. Because the volatile fatty acids found in the culture filtrates consisted primarily of butyric and isovaleric acids for P. gingivalis, propionic, butyric, and isovaleric acid for Prev. loescheii, and butyric acid for F. nucleatum (Kurita-Ochiai et al., 1995), and because these commercial volatile fatty acids significantly depressed inflamed gingival fibroblast viability, these bacterial metabolites may be relevant in periodontal disease.

However, butyric acid alone does not have a distinct function in inflamed gingival fibroblasts. For instance, general apoptosis-inducing factors, such as genistein, staurosporin, and H-7, also induced the apoptosis of inflamed gingival fibroblasts, but not of healthy fibroblasts (data not shown). Ochiai and Kurita-Ochiai (2003) demonstrated that butyric acid induces apoptosis in monocytes/macrophages at levels similar to those in T- and B-cells; however, epithelial cells and fibroblasts are not as sensitive. Therefore, healthy gingival fibroblasts are also resistant to apoptosis induced by volatile fatty acids. Because inflamed gingival fibroblasts showed apoptosis sensitivity to volatile fatty acids, the inflamed cells might have been sensitive to some type of stress signal. This may be a non-specific type of cell death. Therefore, a similar phenomenon may be caused by stress factors, other than volatile fatty acids, that originate in bacteria or in humans. However, the elevation of butyric acid levels as a metabolic product following the proliferation of bacteria may also be relevant to cell death, because non-volatile fatty acids, which are similar metabolic products, did not affect the viability of inflamed fibroblasts.

Based on the results of previous investigations that detected 13.3–26.8 mM butyric acid in culture filtrates from P. gingivalis, Prev. loescheii, and F. nucleatum (Kurita-Ochiai et al., 1995), and showed that the butyric acid concentration in subgingival plaque from a site of periodontitis can reach 14.4–20 mM (Margolis et al., 1988; Naleway et al., 1989), and that its concentration in periodontal pockets is correlated with the severity of periodontal disease (Botta et al., 1985), butyric acid was proposed to be an important virulence factor in these periodontopathogens. Therefore, we cultured gingival fibroblast cells with 1.25–5 mM butyric acid, which is relevant to the amounts released by bacteria in periodontal disease.

Butyric-acid-stimulated inflamed gingival fibroblasts underwent apoptosis via a specific form of programmed cell death characterized by internucleosomal DNA digestion, as indicated by gel electrophoresis. Cell death was also associated with chromatin condensation, which was detected by DAPI staining. A significant decrease in mitochondrial membrane potential was also observed in butyric-acid-treated inflamed gingival fibroblasts. It appears that mitochondrial damage induces caspase activity: Mitochondrial damage results in the activation of caspase-9, which leads to the activation of caspase-3, which in turn can cleave death substrates such as nuclear lamins (Lazebnik et al., 1995), PARP (Lazebnik et al., 1994), fodrin (Cryns et al., 1996), and ICAD (Sakahira et al., 1998). There are two major pathways through which apoptosis is induced. One pathway involves death receptors and is exemplified by Fas-mediated caspase-8 activation; the other involves stress- or mitochondria-mediated caspase-9 activation. The butyric-acid-induced apoptosis of inflamed gingival fibroblasts was reduced by inhibitors of caspase-3/7, -6, -8, and -9. This suggests that caspase-3, -6, -7, -8, and -9 each play an essential role in the butyric-acid-induced apoptosis of inflamed gingival fibroblasts. Therefore, in thebutyric-acid-induced apoptosis of inflamed gingival fibroblasts, death-receptor-mediated and stress-mediated signals appear to cause caspase activation. However, because caspase inhibitors did not completely inhibit the inflamed gingival fibroblast apoptosis induced by butyric acid, factors other than caspases may also play an important role, together with caspases, in butyric-acid-induced apoptosis in inflamed gingival fibroblasts.

Adult periodontitis is a chronic inflammatory disease that results from the interaction between specific Gram-negative bacteria and the host. Previous studies have reported the occurrence of apoptotic cells in gingival biopsies from persons with periodontitis (Koulouri et al., 1990; Sawa et al., 1999). Therefore, butyric-acid-induced apoptosis of gingival fibroblasts isolated from persons with adult periodontitis could contribute to the mechanisms involved in the destruction of the supporting tissues of the teeth.

In conclusion, inflamed gingival fibroblasts from persons with adult periodontitis were highly susceptible to mitochondria- and caspase-dependent apoptosis induced by butyric acid, compared with healthy gingival fibroblasts. This is the first demonstration that butyric acid can induce apoptosis in gingival fibroblasts, possibly contributing to the destruction of gingival tissues in inflamed periodontal lesions.


   ACKNOWLEDGMENTS
 
We thank Dr. Kuniyoshi Kikuchi for excellent technical assistance. This work was supported by a grant from the Tsuchiya Culture Promotion Foundation and by a scientific research grant-in-aid (19390537) from the Ministry of Education, Science, and Culture of Japan.

Received for publication July 26, 2005. Revision received October 19, 2007. Accepted for publication October 2, 2007.


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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Journal of Dental Research, Vol. 87, No. 1, 51-55 (2008)
DOI: 10.1177/154405910808700108
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