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Modulatory effects of indomethacin on androgen metabolism in human gingival and oral periosteal fibroblasts
Steroids 66 (2001) 857– 863
Modulatory effects of indomethacin on androgen metabolism in human gingival and oral periosteal fibroblasts A. Tilakaratnea, M. Sooryb,* a
b
Department of Periodontology, Faculty of Dental Sciences, University of Peradeniya, Sri-Lanka Division of Periodontology, Guy’s King’s & St. Thomas’ Dental Institute, King’s campus, Caldecot Road, London SE5 9RW, UK Received 20 September 2000; received in revised form 7 March 2001; accepted 8 March 2001
Abstract The non-steroidal anti-inflammatory agent indomethacin (I) suppresses gingival inflammation and alveolar bone resorption. Androgens particularly 5␣-dihydrotestosterone (DHT) have anabolic effects on connective tissue and bone matrices. Human oral periosteal fibroblasts (HPF) and gingival fibroblasts (HGF) instigate healing in inflammatory periodontal lesions. The aim of this investigation was to compare the modulatory effects of I on the metabolism of two androgen substrates in human oral periosteal and gingival fibroblasts in culture. Monolayer cultures of both cell types (5th–9th passage) were established in Eagle’s MEM and incubated with 14C-testosterone/14C-4androstenedione and serial concentrations of I (0.5–50 g/ml) for 24 h. The steroid metabolites were solvent extracted from the medium, separated by TLC and quantified using a radioisotope scanner. Both androgen substrates were metabolized mainly to DHT and 4-androstenedione/testosterone respectively, expressing 5␣-reductase and 17-hydroxysteroid dehydrogenase (17-HSD) activity in both HPF and HGF. There were 51% and 73% increases in the levels of DHT over controls, with HGF and HPF respectively (n ⫽ 6; n ⫽ 4, P ⬍ 0.01) in response to I at 1–5 g/ml, often reaching control values at 50 g/ml. The expression of 17-HSD activity showed less stimulation than the levels of DHT. Both androgen substrates were effective in this metabolic conversion, which is applicable to healing responses in both males and females in vivo. There were 57% increases (n ⫽ 4; P ⬍ 0.01) over controls, in the formation of androstanediol from 14C-4-androstenedione at 10 g of I, in HPF. This transformation may regulate androgen action in androgen-dependent tissue. In addition to its anti-inflammatory properties, indomethacin can contribute to anabolic reparatory responses, by increasing the expression of steroid metabolizing enzymes in gingival and periosteal fibroblasts, in the inflammatory periodontal lesion. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Steroid; Indomethacin; Fibroblasts; Inflammatory periodontal repair
1. Introduction Indomethacin belongs to the group of NSAIDs (nonsteroidal anti-inflammatory drugs) and is used in the treatment of musculoskeletal disorders such as rheumatoid arthritis, osteoarthritis, ankylosing spondylitis and also as an adjunct to periodontal therapy. The NSAIDs are effective inhibitors of the cyclo oxygenase pathway of the arachidonic cascade, resulting in decreased synthesis of prostaglandins. Higher levels of prostaglandins are associated with inflammed periodontal tissues [1– 4] and this may have an important role in the pathogenesis of periodontal diseases, by mediating inflammatory changes in the tissues. * Corresponding author. Tel.: ⫹44 20 7346 3492/30; fax: ⫹44 20 7346 3185. E-mail address: [email protected] (M. Soory).
Indomethacin is among the commonly used NSAIDs in clinical trials. By suppressing inflammation, and gingival crevicular fluid flow, these drugs can restrict the nutrient pool for pathogenic bacteria. The efficacy of indomethacin in the suppression of gingival inflammation and alveolar bone resorption has been studied [5] on a ligature induced periodontitis model in Beagle dogs. The results indicated that twice daily oral doses of indomethacin delayed the onset and suppressed the magnitude of the acute inflammatory reaction, and also reduced the amount of alveolar bone resorption during the study period. The effects of indomethacin on alveolar bone loss in experimental periodontitis have also been studied in the squirrel monkey [6]. The loss of alveolar bone height and mass which were seen in control animals were abolished in monkeys dosed with indomethacin. The anabolic effects of the androgen testosterone and its
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matrix stimulatory metabolite 5␣-dihydrotestosterone (DHT) on bone and connective tissue have been well documented [7–9]. The presence of inflammation can alter the expression of steroid hormone receptors, partly mediated by inflammatory cytokines, which can modulate the metabolism of circulating steroids and their effects on target tissues. When testosterone was incubated with homogenate, mitochondrial, microsomal and soluble fractions of healthy and inflammed gingiva from humans of both sexes, the metabolic activity was higher in the preparation from inflammed tissue, than in the samples from healthy gingiva [10,11]. In both types of tissue, testosterone was converted to DHT, suggesting that gingivae may be a target tissue for androgens. DHT is the most biologically active metabolite found in tissues when testosterone is metabolized, and it can contribute to growth and development of connective tissue and bone [7–9,12]. Such anabolic effects are more obvious when the normal synthesizing capacity of tissues is reduced. Thus, when a reparatory response is required, DHT can contribute to synthetic activity in fibroblasts and osteoblasts [12–14]. The expression of androgen receptors has been detected in a high proportion of periodontal and gingival tissues; also in fibroblasts derived from the same source [15]. Androgens inhibit prostaglandin production stimulated by interleukin-1 (IL-1), thus inhibiting bone resorption [16]. IL-1 found in inflammatory exudate has been shown to stimulate the synthesis of the potent androgen metabolite, DHT in both gingivae and periodontal ligament [17], thus contributing to repair processes in an environment of inflammation. IL-1 induced prostaglandin synthesis [16] and 5␣-reduction of androgen substrates in the periodontium [17], indicates a link between cytokines, inflammatory mediators and androgen metabolism. In view of these findings, the effect of the NSAID, indomethacin on androgen metabolism in fibroblasts derived from chronically inflammed gingivae and oral periosteum was thought to be a relevant area for investigation. Fibroblasts from these tissues were used, since they represent cells engaged in active matrix turnover [18]. Periosteal fibroblasts are engaged in wound healing and represent the source of cells for bone regeneration. Cells derived from an osteoblastic phenotype have been used to establish amplified tissue constructs, to aid tissue regeneration [19]. Hence it was relevant to use cultured periosteal fibroblasts which have considerable regenerative capacity, in this context, to demonstrate potential for tissue repair via the 5␣-reductase pathway. The aims of the investigation are to study the expression of steroid metabolizing enzymes in established monolayer cultures of human gingival and oral periosteal fibroblasts, in order to simulate the periodontium in vitro. The response at baseline and the effects of serial concentrations of indomethacin may provide information on healing, applicable to the inflammed periodontium.
2. Experimental Radiolabelled androgens, 14C-testosterone and 14C-4androstenedione (specific radioactivity 58 Ci/mol) were obtained from Amersham International, Amersham, Bucks., UK. Organic solvents (benzene, acetone) for thin layer chromatography (TLC), ethyl acetate for extraction of metabolites and chloroform to re-dissolve the dried extract were all provided by Merck Ltd., Dagenham, Essex, UK. Indomethacin was obtained from Sigma Chemical Company Ltd., Poole, Dorset, UK. The incubation medium used was Eagle’s Minimum Essential Medium (MEM) with Lglutamine, antibiotic solution (penicillin and streptomycin) and sodium bicarbonate which were all provided by Gibco Ltd., Paisley, Scotland. Indomethacin used in the incubations were obtained from Sigma Chemical Co., Poole, Dorset, UK. 2.1. Cell culture techniques and analysis of androgen metabolites Chronically inflammed gingival and oral periosteal tissues were obtained from six and four periodontal patients respectively (equal numbers of males and females, aged 20 –50 years) undergoing regenerative or pocket elimination surgical procedures attending the Division of Periodontology, GKT Dental Institute (King’s Campus), London, UK (ethical approval was obtained from the institutional ethics committee, with written consent from the patients). All patients had completed initial phase periodontal treatment comprising scaling and root planing before these procedures and subsequent isolation of the gingival tissues from the sites with pocket depths of 6 – 8 mm (n ⫽ 6). Periosteal tissue was isolated at the base of a partial thickness mucosal flap, from the surface of the bone (2 mm3) [2], prior to regenerative techniques [20] requiring a vertically advanced flap procedure (n ⫽ 4). Previous workers have shown that although gingival tissues from healthy males metabolized testosterone better than that of healthy females, chronically inflammed gingiva from both sexes did not show any difference in testosterone metabolism [10,11]. Based on this evidence, the present study sample was not categorized for the sexes. But the samples were not pooled, maintaining individual cultures of cell-lines for control and test incubations. The gingival and periosteal tissues were minced into small fragments of approximately 1 mm3 and gingival (HGF) and periosteal fibroblasts (HPF) were established in primary culture in 25 cm2 tissue culture flasks. Serial passaging of primary cultures was carried out by partial digestion with 0.25% trypsin solution. Fibroblasts of the 4th–9th passage in confluent monolayer culture were used for the experiments. The contents of a fully confluent 25 cm2 flask (2.2 ⫻ 106 cells) were distributed into 24 wells of a multiwell dish, for each cell-line. The cells were allowed to
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become fully confluent in the multiwell dishes prior to experimentation to overcome mitogenic effects [21]. Duplicate incubations were performed for each individual cell-line of HGF (n ⫽ 6) or HPF (n ⫽ 4) in Eagle’s MEM, using 14C-testosterone/14C-4-androstenedione as substrate (0.025 and 0.01 Ci/ml, respectively), and serial concentrations of indomethacin (I; 0.5–50 g/ml). At the end of a 24 h incubation period in a humidified cell culture incubator at 37°C, the medium was solvent extracted with ethyl acetate (2 ml x 2) with added cold steroid standards. The extract was evaporated to dryness, re-dissolved in chloroform, spotted on TLC plates and the metabolites were separated in a benzene acetone solvent system (4:1 v/v). The separated metabolites were then quantified using a radioisotope scanner. The mean values and standard deviations for the yields of metabolites, from duplicates of each cell-line are shown in the Results section. The effects of serial concentrations of I are compared with controls, in the absence of testing agent. Significance testing was done using one way ANOVA. In order to make corrections for analytical losses, a separate set of experiments was done. Thirty two samples were set up in two groups of cell-free standards and cell-free incubations, with culture medium. The standards were subjected to extraction and evaporation procedures, prior to the addition of radiolabel, which was then quantified and served as the controls. The rest of the samples were incubated for 24 h after addition of the radiolabel, which were then analyzed and quantified. From the sample means, the percentage recovery was found to be 95.5%, accounting for losses during solvent extraction [22]. This correction was made for each of the samples. In addition, a quench correction was made for each sample, by comparing the total amount of radioactivity introduced with the total recovered per incubation. 2.2. Confirmation of the identity of metabolites The identity of the formed metabolites was confirmed using the mobility of cold standards added to the samples and disclosing them in iodine. The TLC plate was placed in a covered tank containing iodine crystals, and the iodinestained steroids were marked for comparison with the position and pattern of separated metabolites identified by the radioisotope scanner. Further confirmation of the authenticity of steroid metabolites was established by carrying out gas chromatography-mass spectrometry (g.c-m.s). 2.3. Characterization of DHT using g.c-m.s As DHT is the most significant biologically active metabolite in stimulating fibroblast matrix synthetic activity [9] it was characterized as follows. Several incubations were performed with human gingivae and unlabelled testosterone (10⫺6 mol/l). After extraction, the identity of 5␣-DHT as a metabolite in the dried extracts was confirmed by combined
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Fig. 1. The metabolic conversion of 14C-T in the presence or absence of indomethacin in gingival fibroblasts (HGF). Duplicate incubations of 6 cell-lines of HGF were performed with radiolabelled testosterone in multiwell plates for 24 h, with serial concentrations of indomethacin when the reaction was terminated by the addition of ethyl acetate. The metabolites were extracted, analysed and quantified. The mean values and standard deviations for the percentage yield of metabolites derived from control (C) and test incubations of 6 gingival cell lines are shown in Figs. 1 and 2.
gas chromatography-mass spectrometry (courtesy of Professor A.I. Mallet, St. Thomas’ Hospital, London, UK). The derivatised biologic material as the pentafluorobenzyloxime trimethylsilylether (PFBO/TMS) had a molecular ion [557] and mass spectral fragmentation pattern identical to those of authentic PFBO/TMS ether of DHT, but at lower levels, due to smaller concentrations of steroid. Characteristic ions were noted, for example at m/z values of 542 [M-15]⫹ due to loss of a methyl group; 467 [M-90]⫹ due to loss of TMS ether; 452 [M-90 –15]⫹ due to loss of TMS ether plus a methyl group and at an m/z value of 360, due to loss of the pentafluorobenzyloxime group. All these procedures have been described in detail [23].
3. Results All the samples used in the experiments metabolized the androgen substrates (14C-testosterone and 14C-4-androstenedione) to form DHT, 4-androstenedione (4-A)/testosterone (T) as major metabolites. Some 5␣-, 5-androstanediols and androstanedione were also produced. 3.1. Effects of serial concentrations of indomethacin on the metabolism of 14-C-testosterone by gingival fibroblasts (Fig. 1) The metabolic conversion of 14C-testosterone (430 pmol/incubation) as substrate, to DHT and 4-androstenedione by gingival fibroblasts, in response to serial concentrations of indomethacin is shown in Fig. 1. The mean value for the percentage conversion of substrate to each metabolite (derived from the means of duplicate incubations for each of 6 cell-lines) at different concentrations was compared with that of the control value. All the concentrations of indomethacin studied (0.5–50 g/ml) caused an increase in the levels of DHT compared to the controls, but this increase was greater in relation to the
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Fig. 2. The metabolic conversion of 14C-4-A in the presence or absence of indomethacin in gingival fibroblasts. Duplicate incubations were performed with HGF (n ⫽ 6) and radiolabelled 4-androstenedione in multiwell plates for 24 h, when the reaction was terminated by the addition of ethyl acetate. The metabolites were extracted, analysed and quantified.
smaller concentrations in the range. There was a gradual increase in the percentage levels of DHT from a baseline value of 2.8% (for controls) to 3.2% at the smallest concentration studied (0.5 g/ml), rising to a maximum level of 4% at concentrations of 5.0 and 8.0 g/ml of indomethacin (n ⫽ 6; P ⬍ 0.001; one way ANOVA). This was a 40% increase in the levels of DHT, over controls. There were decreased levels at subsequent concentrations, with a value of 3%, resulting in a 17% increase over controls at 15 and 20 g/ml, declining to control values, at higher concentrations. There was a gradual decrease in the synthesis of 4-A in response to the increasing concentrations of indomethacin (Fig. 1). The decreases were minimal in response to the smallest concentrations of indomethacin studied. However, at indomethacin concentrations of 30 –50 g/ml, there was 4.2–3.5% conversion to 4-A, compared with a baseline conversion of 9.7%. This was a 50 – 64% decrease in the yield of 4-A in response to indomethacin concentrations of 30 –50 g/ml, compared with control yields (n ⫽ 6; P ⬍ 0.05, as tested by ANOVA). The yields of diols increased from 2.6% (controls) to 3.3% at 3 g/ml of I, resulting in a 27% increase over control yields (n ⫽ 6; P ⬍ 0.05). 3.2. Effects of serial concentrations of indomethacin on the metabolism of 14C-4-androstenedione by gingival fibroblasts (Fig. 2) The metabolic conversion of 14C-4-androstenedione (172 pmol/incubation) as substrate, to DHT and T in response to indomethacin is shown in Fig. 2. The mean value for percentage yield of each metabolite from the substrate, at different concentrations of indomethacin (derived from duplicate values for each of 6 cell-lines) was compared with that of the mean control value. There was 2.7% conversion of the substrate to DHT at baseline, increasing to 4.4% in response to 1 g/ml of I, decreasing to 4% at 5 g/ml and reaching values similar to those of controls at 10 –50 g/ml. This was a 63% increase over control values, at 1 g/ml of indomethacin (n ⫽ 6; P ⬍
Fig. 3. The metabolic conversion of 14C-T in the presence or absence of indomethacin in periosteal fibroblasts (HPF). Duplicate incubations of 4 cell-lines of HPF were performed with radiolabelled testosterone in multiwell plates for 24 h, with serial concentrations of indomethacin when the reaction was terminated by the addition of ethyl acetate. The metabolites were extracted, analysed and quantified. The mean values and standard deviations for the percentage yield of metabolites derived from control (C) and test incubations of 4 periosteal cell lines are shown in Figs. 3 and 4.
0.01), decreasing to 52% at 5 g/ml and decreasing further to control values. The formation of testosterone from this substrate did not show much change in response to indomethacin. The baseline conversion of 2.6% increased to 2.9% at 0.5 and 1.0 g/ml of indomethacin. This was an 11% increase over controls. These increases were not significant statistically (as tested by ANOVA). All the other concentrations of indomethacin (ranging from 3.0 to 50 g/ml) caused an inhibitory effect on the synthesis of testosterone, with 2.2– 1.5% conversion from the substrate. The decreases in the yield of T varied between 13% and 45%, compared to the control value and were statistically significant (n ⫽ 6; P ⬍ 0.05, as tested by ANOVA). 3.3. The metabolic conversion of 14C-testosterone by oral periosteal fibroblasts in response to serial concentrations of indomethacin (Fig. 3) When 14C-testosterone was used as substrate (430 pmol/ incubation) with oral periosteal fibroblasts, the main metabolites formed were DHT, 4-androstenedione and the diols. The percentage of substrate converted to DHT ranged from 1.7% at baseline to 3.1% at 3 g/ml, decreasing to values slightly above controls at the highest concentrations of I. This was an 86% increase in the levels of DHT over controls, at 3 g/ml of I, decreasing to 80%, at 5 and 8 g/ml (n ⫽ 4; P ⬍ 0.01). The percentage conversion of the substrate to 4-A decreased from 4.9% at baseline to 1% at the highest concentration of I. This was a 20% decrease in the synthesis of 4-A, while there was stimulation of 5␣-reductase activity, decreasing further to 50% of control values at 10 g/ml and 25% of controls at 15–50 g/ml of I (n ⫽ 4; P ⬍ 0.01). The percentage yield of diols from the substrate increased from a baseline value (control) of 0.3% to 0.7% in response to 3 and 5 g/ml of I, decreasing to values similar to controls. This was a significant 2-fold increase (n ⫽ 4; P ⬍ 0.001) in
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Fig. 4. The metabolic conversion of 14C-4-A in the presence or absence of indomethacin in periosteal fibroblasts. Duplicate incubations were performed with HPF (n ⫽ 4) and radiolabelled 4-androstenedione in multiwell plates for 24 h, when the reaction was terminated by the addition of ethyl acetate. The metabolites were extracted, analysed and quantified.
the formation of the diols at 3–5 g/ml of I, compared with controls. 3.4. The metabolic conversion of 14C-4-androstenedione by oral periosteal fibroblasts in response to serial concentrations of indomethacin (Fig. 4) When 14C-4-androstenedione (172 pmol/ml) was incubated as the substrate with oral periosteal fibroblasts, the percentage yield of DHT increased from 1.5% at baseline (control) to 2.5% at a concentration of 5 g/ml of I, decreasing to control values at 10 –50 g/ml. This was a 60% increase in the levels of DHT over controls, at an indomethacin concentration of 5 g/ml (n ⫽ 4; P ⬍ 0.01). The percentage yield of testosterone from the substrate increased from 1.8% at baseline to 6.5% at 0.5– 8 g/ml of I. This was a 3.5-fold increase in the formation of testosterone at 0.5–10 g/ml of I, over control values (n ⫽ 4; P ⬍ 0.001). At indomethacin concentrations of 15–50 g/ml, 3-fold increases were maintained, with a 5.5% yield of testosterone from the substrate. There were also significant 2–3-fold increases in the formation of the diols at 1–10 g/ml of I (n ⫽ 4; P ⬍ 0.001), with a yield of 1.1% at baseline, increasing to 3.4% at 5 g/ml of I and 2% at 8 –10 g/ml of I.
4. Discussion In the present study, both androgens (testosterone and 4-androstenedione) were readily metabolized by gingival and oral periosteal fibroblasts in culture. The major pathways for the metabolism of 14C-testosterone and 14C-4androstenedione, appeared to be via 5␣-reductase and 17hydroxysteroid dehydrogenase (HSD) activity, resulting in the formation of DHT, 4-androstenedione and testosterone respectively. There were significant increases in the formation of the androstanediols by oral periosteal fibroblasts. A similar pattern of androgen metabolism has been shown in previous in vitro studies using inflammed human
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gingival tissues [13,17,24], periodontal ligament tissues [17], fibroblasts derived from healthy gingival tissue [25], fibroblasts derived from inflammed gingivae [26] and oral periosteum [20]. In inflammed gingivae, the pathway favouring 5␣-reduction of testosterone seems to be facilitated, rather than the synthesis of 4-A; this has been discussed in relation to drug-induced gingival overgrowth [27]. In this investigation, there was significant stimulation of the formation of physiologically active androgen metabolites from their substrates, in response to indomethacin by human gingival and oral periosteal fibroblasts. The response was significantly greater in oral periosteal fibroblasts, compared with gingival fibroblasts. There were also substantial yields of diols in these cultures, which could result in regulation of androgen action in androgen-dependent tissues. These metabolic conversions in gingival and periosteal fibroblasts could influence regenerative changes in the inflammed periodontium. Paracrine regulation of osteogenic differentiation may operate in the periosteum, an important tissue engaged in bone turnover. The formation of bone requires a pool of osteogenic progenitor cells with an extensive capacity for cell division and renewal, which can generate cells capable of traversing the osteoblast differentiation pathway [28]. Similarly, this investigation demonstrates significant modulation of DHT synthesis by indomethacin in periosteal fibroblasts, which reinforces the characteristics of this celltype. Other workers have demonstrated the capacity for synthesis of matrix [29] or mineralized tissue, in the osteoblastic phenotype, after serial passaging and in response to stimulatory agents [30,31]. The results obtained from cultures of chronically inflammed gingival and periosteal fibroblasts with regard to metabolism of androgen substrates and their response to indomethacin is suggestive of cells engaged in active matrix turnover, which is in keeping with documented evidence of their functions. For instance the rate of periosteal bone formation in rat skeletal bone was found to be enhanced in response to high doses of DHT [32]. These findings are relevant to this investigation, where enhanced expression of 5␣-reductase in periosteal fibroblasts in response to indomethacin is suggestive of androgen responsive protein synthesis in the periosteum. Adjunctive usage of indomethacin in the treatment of periodontal disease may be useful in this context. In general, the results of this investigation show that most concentrations of indomethacin studied, resulted in increased levels of DHT from both androgen substrates, although this effect was more pronounced and significant when testosterone was used as the substrate. This could give rise to significant anabolic effects. The results seen in Fig. 1 indicate a tendency toward increased levels of 5␣-DHT rather than 4-A synthesis in chronically inflammed gingival fibroblasts. Indomethacin is a potent inhibitor of 3␣-hydroxysteroid dehydrogenase, responsible for the conversion of DHT to androstanediol [33]. This could result in the build
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up of DHT, due to its diminished metabolism, contributing to some of the anabolic effects of indomethacin. Consistent with this finding, there was a concomitant decrease in the levels of diol, accompanying the rise in DHT levels in human gingival fibroblasts, in our investigation. Thus, the effective levels of physiologically active androgens in gingivae in response to indomethacin, imply a suitable application for its usage as an adjunct to periodontal therapy. However the increased levels of DHT resulting from 5␣-reduction of androgens in response to indomethacin appeared to be dose dependent (Figs. 1 and 2) and thus, the presence of smaller concentrations of indomethacin would be more important for the above activity compared to the higher concentrations. This dose-dependent effect of indomethacin (at 0.5–5.0/8.0 g/ml) on 5␣-reduction of testosterone was very consistently seen with both androgen substrates, and both cell types. When the substrate (testosterone) was available at low levels, due to inadequate conversion of 14C-4-A to T, the metabolic conversion of T to DHT could only occur in small amounts. Indomethacin has been shown to enhance alkaline phosphatase activity and collagen synthesis in cultures of osteoblastic cells [34]. It may promote such growth promoting pathways by ligand-dependent or -independent stimulation of androgen receptors. Our preliminary investigations have shown that the alkaline phosphatase inhibitor levamisole reduced the stimulatory effects of indomethacin on 5␣reductase activity in cultured fibroblasts [35]. Other possible mechanisms include indomethacin-mediated growth factor actions via androgen receptors. Members of the steroid hormone receptor superfamily are transcriptional enhancers, when activated by their cognate steroid ligand [36]; demonstration of cross talk between polypeptide growth factors and steroid hormone receptors is relevant to transmission of signals to the nucleus, resulting in growth and differentiation. Our previous work reinforces these findings, regarding growth factor stimulation of 5␣-reductase expression in human gingival fibroblasts [24]. These mechanisms of action could apply to other NSAIDs, and may be linked to their anti-inflammatory potencies. Epidemiologic and clinical studies have suggested potentially beneficial effects of the adjunctive usage of NSAIDs on periodontal conditions. Patients receiving NSAIDs over a period of one year showed less gingival inflammation, shallower probing pocket depths and decreased loss of periodontal attachment, compared to a matched control group [37]. Similar results have been reported on a group of patients under long-term indomethacin therapy, compared to a healthy control group [38]. These studies may indicate that, indomethacin could have important effects on repair and healing in the periodontium. Therefore it is reasonable to suggest that, at least some of the contributory effects of indomethacin on tissue repair and healing in androgen target tissues could be attributed to its effects on the androgen metabolic pathway. The periodontal lesion is a unique wound healing model,
where repair is instigated against a hard avascular, highly mineralized denuded root surface and several adjunctive measures have been used to enhance this process. Due to anatomic limitations of healing in the diseased periodontium, adjunctive usage of materials such as indomethacin may simulate growth promoting substances in the inflammatory exudate and instigate repair. Some of the effects of indomethacin demonstrated in this cell culture model may be anticipated with the other NSAIDs. Acknowledgments The authors wish to acknowledge Dr. I. Tavares for usage of the radioisotope scanner in the Academic Department of Surgery (Rayne Institute, King’s Campus), Guy’s King’s & St. Thomas’ School of Medicine, London, UK. References [1] Goodson JM, Dewhirst FE, Brunetti A. Prostaglandin E2 levels, and human periodontal disease. Prostaglandins 1974;6:81–5. [2] El-Attar TM. PGE2 in human gingiva in health, and disease, and its stimulation by female sex steroids. Prostaglandins 1976;6:81–5. [3] El-Attar TM, Lin HS. Cyclic AMP, and prostaglandins in periodontal disease. In: Advances in prostaglandin, and thromboxane research. Samuelsson B, Ramwell PW, Paoletti R (editors). Newyard: Raven Press, 1980;8:1739 – 40. [4] Ohm K, Albers Von HK, Lisboa BP. Measurement of 8 prostaglandins in human gingival and periodontal disease using high pressure liquid chromatography and radioimmunoassay. J Periodont Res 1984; 19:501–11. [5] Nyman S, Schroeder HE, Lindhe J. Suppression of inflammation during experimental periodontitis in dogs. J Periodontol 1979;50: 450 – 61. [6] Weaks-Dybvig M, Sanavi F, Zander H, Rifkin BR. The effect of indomethacin on alveolar bone loss in experimental periodontitis. J Periodont Res 1982;17:90 –100. [7] Colvard DS, Eriksen EF, Keeting PE, Wilson EM, Lubahn DB, French FS, et al. Identification of androgen receptors in normal human osteoblast-like cells. Proc Natl Acad Sci USA 1989;86:854 –7. [8] Dassouli A, Manin M, Veyssiere G, Jean C. Androgens regulate expression of the gene coding for a mouse vas deferens protein related to the aldo-keto reductase superfamily in epithelial cell subcultures. J Steroid Biochem Mol Biol 1994;48:121– 8. [9] Normington K, Russell DW. Tissue distribution and kinetic characteristics of rat steroid 5␣-reductase isoenzymes. Evidence for distinct physiological functions. J Biol Chem 1992;267:19548 –54. [10] Ojanotko A, Neinstedt N, Harri P. Metabolism of testosterone by human healthy and inflammed gingivae in-vitro. Arch Oral Biol 1980;25:481– 4. [11] Sooriyamoorthy M, Gower DB. Phenytoin stimulation of 5␣-reductase activity in inflammed gingival fibroblasts. Med Sci Res 1989;17: 989 –99. [12] Kasperk CH, Wergedal JE, Farley JR, Linkhart T, Turner RT, Baylink DJ. Androgens directly stimulate proliferating bone cells in-vitro. Endocrinology 1989;124:1576 – 8. [13] Vittek J, Rappaport SC, Gordon GG, Munnangi PR, Southren AL. Concentration of circulating hormones and metabolism of androgens by human gingiva. J Periodontol 1979;50:254 – 64. [14] Sooriyamoorthy M, Gower DB. Hormonal influences on gingival tissue: relationship to periodontal disease. J Clin Periodontol 1989; 16:201– 8.
A. Tilakaratne, M. Soory / Steroids 66 (2001) 857– 863 [15] Parkar MH, Newman NH, Olsen I. Polymerase chain reaction (PCR) analysis of oestrogen and androgen receptor expression in human gingival and periodontal tissue. Arch Oral Biol 1996;41:979 – 83. [16] Pilbeam CC, Raisz LG. Effects of androgens on parathyroid hormone and IL-1 stimulated prostaglandin production in cultured neonatal mouse calvariae. Bone Miner 1990;5:1183– 8. [17] Kasasa SC, Soory M. The effect of interleukin-1 (IL-1) on androgen metabolism in human gingival tissue (HGT) and periodontal ligament (PDL). J Clin Periodontol 1996;23:419 –24. [18] Zhang M, Powers RM, Wolfinbarger L. A quantitative assessment of osteoinductivity of human demineralised bone matrix. J Periodontol 1997;68:1076 – 84. [19] Malikzadeh R, Hollinger JD, Buck D, Adams DF, Mc Allister BS. Isolation of human osteoblast-like cells and in vitro amplification for tissue engineering. J Periodontol 1998;69:1252– 62. [20] Soory M, Tilakaratne A. The effect of minocycline on the metabolism of androgens by human oral periosteal fibroblasts and its inhibition by finasteride. Archs Oral Biol 2000;45:257– 65. [21] Kahari VM, Heino J, Vuorio E. IL-1 increases collagen production, and mRNA levels in cultured skin fibroblasts. Biochem Biophys Acta 1987;929:142–7. [22] Soory M, Gower DB. The influence of prostaglandins on steroid conversions by human gingival fibroblasts. J Periodont Res 1998;33: 439 – 47. [23] Soory M. Bacterial steroidogenesis by periodontal pathogens and the effect of bacterial enzymes on steroid conversions by human gingival fibroblasts in culture. J Periodont Res 1995;30:124 –31. [24] Kasasa SC, Soory M. The synthesis of 5␣-dihydrotestosterone from androgens by human gingival tissues and fibroblasts in culture in response to TGF-␣ and PDGF. J Periodont Res 1996;31:312–21. [25] Sooriyamoorthy M, Harvey W, Gower DB. The use of human gingival fibroblasts in culture for studying the effects of phenytoin on testosterone metabolism. Arch Oral Biol 1988;33:353–9. [26] Soory M, Virdi H. The uptake of two androgen substrates by cultured gingival fibroblasts in response to minocycline and metabolic studies in a cell-free system. Arch Oral Biol 1999;44:215–22. [27] Sooriyamoorthy M, Gower DB. Drug-induced gingival overgrowth: clinical features and possible mechanisms. Med Sci Res 1989;17: 881– 4.
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[28] Owen TA, Aronow M, Shalhoub V, Barone LM, Wilming L, Tassinari MS, Kennedy MB, Pockwinse S, Lian JB, Stein GS. Progressive development of the rat osteoblast phenotype in vitro. Reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. J Cell Physiol 1999;143:420 –30. [29] Aarden EM, Wassenaar AM, Alblas MJ, Nijweide PJ. Immunocytochemical demonstration of extracellular matrix protein in isolated osteocytes. Histochem Cell Biol 1996;106:495–501. [30] Sukhu B, Rotenberg B, Binkert C, Kohno H, Zohar R, Mc Culloch CA, Tenenbaum HC. Tamoxifen attenuates glucocorticoid action on bone formation in vitro. Endocrinology 1997;138:3269 –75. [31] Larzowski DA, Fraher LJ, Hodsman A, Steer B, Modowski D, Hein VK. Regional variation of insulin-like growth factor-1 gene expression in mature rat bone and cartilage. Bone 1994;15:563–76. [32] Coxam V, Boman BM, Meecham M, Roth CM, Miller MA, Miller SC. Effects of dihydrotestosterone alone and in combination with oestrogen on bone mineral density, bone growth and formation rates in ovariectomised rats. Bone 1996;19:107–14. [33] Penning TM, Mukharji I, Barrows S, Talalay P. Purification and properties of a 3␣-hydroxysteroid dehydrogenase of rat liver cytosol and its inhibition by anti-inflammatory drugs. Biochem J 1984;222: 601–11. [34] Kajii T, Suzuki K, Yoshikawa M, Imai T, Matsumoto A, Nakanura S. Long-term effects of prostaglandin E2 on the mineralisation of a clonal osteoblastic cell-line (MC3T3–E1). Arch Oral Biol 1999;44: 233– 41. [35] Soory M, Tilakaratne A. Alkaline phosphatase activity and indomethacin mediated androgen action in fibroblasts. J Dent Res (Special issue) 2000;79:312. [36] Ignar-Trowbridge DM, Teng CT, Ross KA, Parker MG, Korach KS, Mc Lachlan JA. Peptide growth factors elicit oestrogen receptordependent transcriptional activation of an oestrogen responsive element. Molec Endocrinol 1993;7:992– 8. [37] Waite IM, Saxton CA, Young A, Wagg BJ, Corbett M. The periodontal status of subjects receiving non-steroidal anti-inflammatory drugs. J Periodont Res 1981;16:100 – 8. [38] Feldman RS, Szeto B, Chaucey HH, Goldhaber P. Non-steroidal anti-inflammatory drugs in the reduction of human alveolar bone loss. J Clin Periodontol 1983;10:131– 6.
Modulatory effects of indomethacin on androgen metabolism in human gingival and oral periosteal fibroblasts A. Tilakaratnea, M. Sooryb,* a
b
Department of Periodontology, Faculty of Dental Sciences, University of Peradeniya, Sri-Lanka Division of Periodontology, Guy’s King’s & St. Thomas’ Dental Institute, King’s campus, Caldecot Road, London SE5 9RW, UK Received 20 September 2000; received in revised form 7 March 2001; accepted 8 March 2001
Abstract The non-steroidal anti-inflammatory agent indomethacin (I) suppresses gingival inflammation and alveolar bone resorption. Androgens particularly 5␣-dihydrotestosterone (DHT) have anabolic effects on connective tissue and bone matrices. Human oral periosteal fibroblasts (HPF) and gingival fibroblasts (HGF) instigate healing in inflammatory periodontal lesions. The aim of this investigation was to compare the modulatory effects of I on the metabolism of two androgen substrates in human oral periosteal and gingival fibroblasts in culture. Monolayer cultures of both cell types (5th–9th passage) were established in Eagle’s MEM and incubated with 14C-testosterone/14C-4androstenedione and serial concentrations of I (0.5–50 g/ml) for 24 h. The steroid metabolites were solvent extracted from the medium, separated by TLC and quantified using a radioisotope scanner. Both androgen substrates were metabolized mainly to DHT and 4-androstenedione/testosterone respectively, expressing 5␣-reductase and 17-hydroxysteroid dehydrogenase (17-HSD) activity in both HPF and HGF. There were 51% and 73% increases in the levels of DHT over controls, with HGF and HPF respectively (n ⫽ 6; n ⫽ 4, P ⬍ 0.01) in response to I at 1–5 g/ml, often reaching control values at 50 g/ml. The expression of 17-HSD activity showed less stimulation than the levels of DHT. Both androgen substrates were effective in this metabolic conversion, which is applicable to healing responses in both males and females in vivo. There were 57% increases (n ⫽ 4; P ⬍ 0.01) over controls, in the formation of androstanediol from 14C-4-androstenedione at 10 g of I, in HPF. This transformation may regulate androgen action in androgen-dependent tissue. In addition to its anti-inflammatory properties, indomethacin can contribute to anabolic reparatory responses, by increasing the expression of steroid metabolizing enzymes in gingival and periosteal fibroblasts, in the inflammatory periodontal lesion. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Steroid; Indomethacin; Fibroblasts; Inflammatory periodontal repair
1. Introduction Indomethacin belongs to the group of NSAIDs (nonsteroidal anti-inflammatory drugs) and is used in the treatment of musculoskeletal disorders such as rheumatoid arthritis, osteoarthritis, ankylosing spondylitis and also as an adjunct to periodontal therapy. The NSAIDs are effective inhibitors of the cyclo oxygenase pathway of the arachidonic cascade, resulting in decreased synthesis of prostaglandins. Higher levels of prostaglandins are associated with inflammed periodontal tissues [1– 4] and this may have an important role in the pathogenesis of periodontal diseases, by mediating inflammatory changes in the tissues. * Corresponding author. Tel.: ⫹44 20 7346 3492/30; fax: ⫹44 20 7346 3185. E-mail address: [email protected] (M. Soory).
Indomethacin is among the commonly used NSAIDs in clinical trials. By suppressing inflammation, and gingival crevicular fluid flow, these drugs can restrict the nutrient pool for pathogenic bacteria. The efficacy of indomethacin in the suppression of gingival inflammation and alveolar bone resorption has been studied [5] on a ligature induced periodontitis model in Beagle dogs. The results indicated that twice daily oral doses of indomethacin delayed the onset and suppressed the magnitude of the acute inflammatory reaction, and also reduced the amount of alveolar bone resorption during the study period. The effects of indomethacin on alveolar bone loss in experimental periodontitis have also been studied in the squirrel monkey [6]. The loss of alveolar bone height and mass which were seen in control animals were abolished in monkeys dosed with indomethacin. The anabolic effects of the androgen testosterone and its
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matrix stimulatory metabolite 5␣-dihydrotestosterone (DHT) on bone and connective tissue have been well documented [7–9]. The presence of inflammation can alter the expression of steroid hormone receptors, partly mediated by inflammatory cytokines, which can modulate the metabolism of circulating steroids and their effects on target tissues. When testosterone was incubated with homogenate, mitochondrial, microsomal and soluble fractions of healthy and inflammed gingiva from humans of both sexes, the metabolic activity was higher in the preparation from inflammed tissue, than in the samples from healthy gingiva [10,11]. In both types of tissue, testosterone was converted to DHT, suggesting that gingivae may be a target tissue for androgens. DHT is the most biologically active metabolite found in tissues when testosterone is metabolized, and it can contribute to growth and development of connective tissue and bone [7–9,12]. Such anabolic effects are more obvious when the normal synthesizing capacity of tissues is reduced. Thus, when a reparatory response is required, DHT can contribute to synthetic activity in fibroblasts and osteoblasts [12–14]. The expression of androgen receptors has been detected in a high proportion of periodontal and gingival tissues; also in fibroblasts derived from the same source [15]. Androgens inhibit prostaglandin production stimulated by interleukin-1 (IL-1), thus inhibiting bone resorption [16]. IL-1 found in inflammatory exudate has been shown to stimulate the synthesis of the potent androgen metabolite, DHT in both gingivae and periodontal ligament [17], thus contributing to repair processes in an environment of inflammation. IL-1 induced prostaglandin synthesis [16] and 5␣-reduction of androgen substrates in the periodontium [17], indicates a link between cytokines, inflammatory mediators and androgen metabolism. In view of these findings, the effect of the NSAID, indomethacin on androgen metabolism in fibroblasts derived from chronically inflammed gingivae and oral periosteum was thought to be a relevant area for investigation. Fibroblasts from these tissues were used, since they represent cells engaged in active matrix turnover [18]. Periosteal fibroblasts are engaged in wound healing and represent the source of cells for bone regeneration. Cells derived from an osteoblastic phenotype have been used to establish amplified tissue constructs, to aid tissue regeneration [19]. Hence it was relevant to use cultured periosteal fibroblasts which have considerable regenerative capacity, in this context, to demonstrate potential for tissue repair via the 5␣-reductase pathway. The aims of the investigation are to study the expression of steroid metabolizing enzymes in established monolayer cultures of human gingival and oral periosteal fibroblasts, in order to simulate the periodontium in vitro. The response at baseline and the effects of serial concentrations of indomethacin may provide information on healing, applicable to the inflammed periodontium.
2. Experimental Radiolabelled androgens, 14C-testosterone and 14C-4androstenedione (specific radioactivity 58 Ci/mol) were obtained from Amersham International, Amersham, Bucks., UK. Organic solvents (benzene, acetone) for thin layer chromatography (TLC), ethyl acetate for extraction of metabolites and chloroform to re-dissolve the dried extract were all provided by Merck Ltd., Dagenham, Essex, UK. Indomethacin was obtained from Sigma Chemical Company Ltd., Poole, Dorset, UK. The incubation medium used was Eagle’s Minimum Essential Medium (MEM) with Lglutamine, antibiotic solution (penicillin and streptomycin) and sodium bicarbonate which were all provided by Gibco Ltd., Paisley, Scotland. Indomethacin used in the incubations were obtained from Sigma Chemical Co., Poole, Dorset, UK. 2.1. Cell culture techniques and analysis of androgen metabolites Chronically inflammed gingival and oral periosteal tissues were obtained from six and four periodontal patients respectively (equal numbers of males and females, aged 20 –50 years) undergoing regenerative or pocket elimination surgical procedures attending the Division of Periodontology, GKT Dental Institute (King’s Campus), London, UK (ethical approval was obtained from the institutional ethics committee, with written consent from the patients). All patients had completed initial phase periodontal treatment comprising scaling and root planing before these procedures and subsequent isolation of the gingival tissues from the sites with pocket depths of 6 – 8 mm (n ⫽ 6). Periosteal tissue was isolated at the base of a partial thickness mucosal flap, from the surface of the bone (2 mm3) [2], prior to regenerative techniques [20] requiring a vertically advanced flap procedure (n ⫽ 4). Previous workers have shown that although gingival tissues from healthy males metabolized testosterone better than that of healthy females, chronically inflammed gingiva from both sexes did not show any difference in testosterone metabolism [10,11]. Based on this evidence, the present study sample was not categorized for the sexes. But the samples were not pooled, maintaining individual cultures of cell-lines for control and test incubations. The gingival and periosteal tissues were minced into small fragments of approximately 1 mm3 and gingival (HGF) and periosteal fibroblasts (HPF) were established in primary culture in 25 cm2 tissue culture flasks. Serial passaging of primary cultures was carried out by partial digestion with 0.25% trypsin solution. Fibroblasts of the 4th–9th passage in confluent monolayer culture were used for the experiments. The contents of a fully confluent 25 cm2 flask (2.2 ⫻ 106 cells) were distributed into 24 wells of a multiwell dish, for each cell-line. The cells were allowed to
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become fully confluent in the multiwell dishes prior to experimentation to overcome mitogenic effects [21]. Duplicate incubations were performed for each individual cell-line of HGF (n ⫽ 6) or HPF (n ⫽ 4) in Eagle’s MEM, using 14C-testosterone/14C-4-androstenedione as substrate (0.025 and 0.01 Ci/ml, respectively), and serial concentrations of indomethacin (I; 0.5–50 g/ml). At the end of a 24 h incubation period in a humidified cell culture incubator at 37°C, the medium was solvent extracted with ethyl acetate (2 ml x 2) with added cold steroid standards. The extract was evaporated to dryness, re-dissolved in chloroform, spotted on TLC plates and the metabolites were separated in a benzene acetone solvent system (4:1 v/v). The separated metabolites were then quantified using a radioisotope scanner. The mean values and standard deviations for the yields of metabolites, from duplicates of each cell-line are shown in the Results section. The effects of serial concentrations of I are compared with controls, in the absence of testing agent. Significance testing was done using one way ANOVA. In order to make corrections for analytical losses, a separate set of experiments was done. Thirty two samples were set up in two groups of cell-free standards and cell-free incubations, with culture medium. The standards were subjected to extraction and evaporation procedures, prior to the addition of radiolabel, which was then quantified and served as the controls. The rest of the samples were incubated for 24 h after addition of the radiolabel, which were then analyzed and quantified. From the sample means, the percentage recovery was found to be 95.5%, accounting for losses during solvent extraction [22]. This correction was made for each of the samples. In addition, a quench correction was made for each sample, by comparing the total amount of radioactivity introduced with the total recovered per incubation. 2.2. Confirmation of the identity of metabolites The identity of the formed metabolites was confirmed using the mobility of cold standards added to the samples and disclosing them in iodine. The TLC plate was placed in a covered tank containing iodine crystals, and the iodinestained steroids were marked for comparison with the position and pattern of separated metabolites identified by the radioisotope scanner. Further confirmation of the authenticity of steroid metabolites was established by carrying out gas chromatography-mass spectrometry (g.c-m.s). 2.3. Characterization of DHT using g.c-m.s As DHT is the most significant biologically active metabolite in stimulating fibroblast matrix synthetic activity [9] it was characterized as follows. Several incubations were performed with human gingivae and unlabelled testosterone (10⫺6 mol/l). After extraction, the identity of 5␣-DHT as a metabolite in the dried extracts was confirmed by combined
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Fig. 1. The metabolic conversion of 14C-T in the presence or absence of indomethacin in gingival fibroblasts (HGF). Duplicate incubations of 6 cell-lines of HGF were performed with radiolabelled testosterone in multiwell plates for 24 h, with serial concentrations of indomethacin when the reaction was terminated by the addition of ethyl acetate. The metabolites were extracted, analysed and quantified. The mean values and standard deviations for the percentage yield of metabolites derived from control (C) and test incubations of 6 gingival cell lines are shown in Figs. 1 and 2.
gas chromatography-mass spectrometry (courtesy of Professor A.I. Mallet, St. Thomas’ Hospital, London, UK). The derivatised biologic material as the pentafluorobenzyloxime trimethylsilylether (PFBO/TMS) had a molecular ion [557] and mass spectral fragmentation pattern identical to those of authentic PFBO/TMS ether of DHT, but at lower levels, due to smaller concentrations of steroid. Characteristic ions were noted, for example at m/z values of 542 [M-15]⫹ due to loss of a methyl group; 467 [M-90]⫹ due to loss of TMS ether; 452 [M-90 –15]⫹ due to loss of TMS ether plus a methyl group and at an m/z value of 360, due to loss of the pentafluorobenzyloxime group. All these procedures have been described in detail [23].
3. Results All the samples used in the experiments metabolized the androgen substrates (14C-testosterone and 14C-4-androstenedione) to form DHT, 4-androstenedione (4-A)/testosterone (T) as major metabolites. Some 5␣-, 5-androstanediols and androstanedione were also produced. 3.1. Effects of serial concentrations of indomethacin on the metabolism of 14-C-testosterone by gingival fibroblasts (Fig. 1) The metabolic conversion of 14C-testosterone (430 pmol/incubation) as substrate, to DHT and 4-androstenedione by gingival fibroblasts, in response to serial concentrations of indomethacin is shown in Fig. 1. The mean value for the percentage conversion of substrate to each metabolite (derived from the means of duplicate incubations for each of 6 cell-lines) at different concentrations was compared with that of the control value. All the concentrations of indomethacin studied (0.5–50 g/ml) caused an increase in the levels of DHT compared to the controls, but this increase was greater in relation to the
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Fig. 2. The metabolic conversion of 14C-4-A in the presence or absence of indomethacin in gingival fibroblasts. Duplicate incubations were performed with HGF (n ⫽ 6) and radiolabelled 4-androstenedione in multiwell plates for 24 h, when the reaction was terminated by the addition of ethyl acetate. The metabolites were extracted, analysed and quantified.
smaller concentrations in the range. There was a gradual increase in the percentage levels of DHT from a baseline value of 2.8% (for controls) to 3.2% at the smallest concentration studied (0.5 g/ml), rising to a maximum level of 4% at concentrations of 5.0 and 8.0 g/ml of indomethacin (n ⫽ 6; P ⬍ 0.001; one way ANOVA). This was a 40% increase in the levels of DHT, over controls. There were decreased levels at subsequent concentrations, with a value of 3%, resulting in a 17% increase over controls at 15 and 20 g/ml, declining to control values, at higher concentrations. There was a gradual decrease in the synthesis of 4-A in response to the increasing concentrations of indomethacin (Fig. 1). The decreases were minimal in response to the smallest concentrations of indomethacin studied. However, at indomethacin concentrations of 30 –50 g/ml, there was 4.2–3.5% conversion to 4-A, compared with a baseline conversion of 9.7%. This was a 50 – 64% decrease in the yield of 4-A in response to indomethacin concentrations of 30 –50 g/ml, compared with control yields (n ⫽ 6; P ⬍ 0.05, as tested by ANOVA). The yields of diols increased from 2.6% (controls) to 3.3% at 3 g/ml of I, resulting in a 27% increase over control yields (n ⫽ 6; P ⬍ 0.05). 3.2. Effects of serial concentrations of indomethacin on the metabolism of 14C-4-androstenedione by gingival fibroblasts (Fig. 2) The metabolic conversion of 14C-4-androstenedione (172 pmol/incubation) as substrate, to DHT and T in response to indomethacin is shown in Fig. 2. The mean value for percentage yield of each metabolite from the substrate, at different concentrations of indomethacin (derived from duplicate values for each of 6 cell-lines) was compared with that of the mean control value. There was 2.7% conversion of the substrate to DHT at baseline, increasing to 4.4% in response to 1 g/ml of I, decreasing to 4% at 5 g/ml and reaching values similar to those of controls at 10 –50 g/ml. This was a 63% increase over control values, at 1 g/ml of indomethacin (n ⫽ 6; P ⬍
Fig. 3. The metabolic conversion of 14C-T in the presence or absence of indomethacin in periosteal fibroblasts (HPF). Duplicate incubations of 4 cell-lines of HPF were performed with radiolabelled testosterone in multiwell plates for 24 h, with serial concentrations of indomethacin when the reaction was terminated by the addition of ethyl acetate. The metabolites were extracted, analysed and quantified. The mean values and standard deviations for the percentage yield of metabolites derived from control (C) and test incubations of 4 periosteal cell lines are shown in Figs. 3 and 4.
0.01), decreasing to 52% at 5 g/ml and decreasing further to control values. The formation of testosterone from this substrate did not show much change in response to indomethacin. The baseline conversion of 2.6% increased to 2.9% at 0.5 and 1.0 g/ml of indomethacin. This was an 11% increase over controls. These increases were not significant statistically (as tested by ANOVA). All the other concentrations of indomethacin (ranging from 3.0 to 50 g/ml) caused an inhibitory effect on the synthesis of testosterone, with 2.2– 1.5% conversion from the substrate. The decreases in the yield of T varied between 13% and 45%, compared to the control value and were statistically significant (n ⫽ 6; P ⬍ 0.05, as tested by ANOVA). 3.3. The metabolic conversion of 14C-testosterone by oral periosteal fibroblasts in response to serial concentrations of indomethacin (Fig. 3) When 14C-testosterone was used as substrate (430 pmol/ incubation) with oral periosteal fibroblasts, the main metabolites formed were DHT, 4-androstenedione and the diols. The percentage of substrate converted to DHT ranged from 1.7% at baseline to 3.1% at 3 g/ml, decreasing to values slightly above controls at the highest concentrations of I. This was an 86% increase in the levels of DHT over controls, at 3 g/ml of I, decreasing to 80%, at 5 and 8 g/ml (n ⫽ 4; P ⬍ 0.01). The percentage conversion of the substrate to 4-A decreased from 4.9% at baseline to 1% at the highest concentration of I. This was a 20% decrease in the synthesis of 4-A, while there was stimulation of 5␣-reductase activity, decreasing further to 50% of control values at 10 g/ml and 25% of controls at 15–50 g/ml of I (n ⫽ 4; P ⬍ 0.01). The percentage yield of diols from the substrate increased from a baseline value (control) of 0.3% to 0.7% in response to 3 and 5 g/ml of I, decreasing to values similar to controls. This was a significant 2-fold increase (n ⫽ 4; P ⬍ 0.001) in
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Fig. 4. The metabolic conversion of 14C-4-A in the presence or absence of indomethacin in periosteal fibroblasts. Duplicate incubations were performed with HPF (n ⫽ 4) and radiolabelled 4-androstenedione in multiwell plates for 24 h, when the reaction was terminated by the addition of ethyl acetate. The metabolites were extracted, analysed and quantified.
the formation of the diols at 3–5 g/ml of I, compared with controls. 3.4. The metabolic conversion of 14C-4-androstenedione by oral periosteal fibroblasts in response to serial concentrations of indomethacin (Fig. 4) When 14C-4-androstenedione (172 pmol/ml) was incubated as the substrate with oral periosteal fibroblasts, the percentage yield of DHT increased from 1.5% at baseline (control) to 2.5% at a concentration of 5 g/ml of I, decreasing to control values at 10 –50 g/ml. This was a 60% increase in the levels of DHT over controls, at an indomethacin concentration of 5 g/ml (n ⫽ 4; P ⬍ 0.01). The percentage yield of testosterone from the substrate increased from 1.8% at baseline to 6.5% at 0.5– 8 g/ml of I. This was a 3.5-fold increase in the formation of testosterone at 0.5–10 g/ml of I, over control values (n ⫽ 4; P ⬍ 0.001). At indomethacin concentrations of 15–50 g/ml, 3-fold increases were maintained, with a 5.5% yield of testosterone from the substrate. There were also significant 2–3-fold increases in the formation of the diols at 1–10 g/ml of I (n ⫽ 4; P ⬍ 0.001), with a yield of 1.1% at baseline, increasing to 3.4% at 5 g/ml of I and 2% at 8 –10 g/ml of I.
4. Discussion In the present study, both androgens (testosterone and 4-androstenedione) were readily metabolized by gingival and oral periosteal fibroblasts in culture. The major pathways for the metabolism of 14C-testosterone and 14C-4androstenedione, appeared to be via 5␣-reductase and 17hydroxysteroid dehydrogenase (HSD) activity, resulting in the formation of DHT, 4-androstenedione and testosterone respectively. There were significant increases in the formation of the androstanediols by oral periosteal fibroblasts. A similar pattern of androgen metabolism has been shown in previous in vitro studies using inflammed human
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gingival tissues [13,17,24], periodontal ligament tissues [17], fibroblasts derived from healthy gingival tissue [25], fibroblasts derived from inflammed gingivae [26] and oral periosteum [20]. In inflammed gingivae, the pathway favouring 5␣-reduction of testosterone seems to be facilitated, rather than the synthesis of 4-A; this has been discussed in relation to drug-induced gingival overgrowth [27]. In this investigation, there was significant stimulation of the formation of physiologically active androgen metabolites from their substrates, in response to indomethacin by human gingival and oral periosteal fibroblasts. The response was significantly greater in oral periosteal fibroblasts, compared with gingival fibroblasts. There were also substantial yields of diols in these cultures, which could result in regulation of androgen action in androgen-dependent tissues. These metabolic conversions in gingival and periosteal fibroblasts could influence regenerative changes in the inflammed periodontium. Paracrine regulation of osteogenic differentiation may operate in the periosteum, an important tissue engaged in bone turnover. The formation of bone requires a pool of osteogenic progenitor cells with an extensive capacity for cell division and renewal, which can generate cells capable of traversing the osteoblast differentiation pathway [28]. Similarly, this investigation demonstrates significant modulation of DHT synthesis by indomethacin in periosteal fibroblasts, which reinforces the characteristics of this celltype. Other workers have demonstrated the capacity for synthesis of matrix [29] or mineralized tissue, in the osteoblastic phenotype, after serial passaging and in response to stimulatory agents [30,31]. The results obtained from cultures of chronically inflammed gingival and periosteal fibroblasts with regard to metabolism of androgen substrates and their response to indomethacin is suggestive of cells engaged in active matrix turnover, which is in keeping with documented evidence of their functions. For instance the rate of periosteal bone formation in rat skeletal bone was found to be enhanced in response to high doses of DHT [32]. These findings are relevant to this investigation, where enhanced expression of 5␣-reductase in periosteal fibroblasts in response to indomethacin is suggestive of androgen responsive protein synthesis in the periosteum. Adjunctive usage of indomethacin in the treatment of periodontal disease may be useful in this context. In general, the results of this investigation show that most concentrations of indomethacin studied, resulted in increased levels of DHT from both androgen substrates, although this effect was more pronounced and significant when testosterone was used as the substrate. This could give rise to significant anabolic effects. The results seen in Fig. 1 indicate a tendency toward increased levels of 5␣-DHT rather than 4-A synthesis in chronically inflammed gingival fibroblasts. Indomethacin is a potent inhibitor of 3␣-hydroxysteroid dehydrogenase, responsible for the conversion of DHT to androstanediol [33]. This could result in the build
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up of DHT, due to its diminished metabolism, contributing to some of the anabolic effects of indomethacin. Consistent with this finding, there was a concomitant decrease in the levels of diol, accompanying the rise in DHT levels in human gingival fibroblasts, in our investigation. Thus, the effective levels of physiologically active androgens in gingivae in response to indomethacin, imply a suitable application for its usage as an adjunct to periodontal therapy. However the increased levels of DHT resulting from 5␣-reduction of androgens in response to indomethacin appeared to be dose dependent (Figs. 1 and 2) and thus, the presence of smaller concentrations of indomethacin would be more important for the above activity compared to the higher concentrations. This dose-dependent effect of indomethacin (at 0.5–5.0/8.0 g/ml) on 5␣-reduction of testosterone was very consistently seen with both androgen substrates, and both cell types. When the substrate (testosterone) was available at low levels, due to inadequate conversion of 14C-4-A to T, the metabolic conversion of T to DHT could only occur in small amounts. Indomethacin has been shown to enhance alkaline phosphatase activity and collagen synthesis in cultures of osteoblastic cells [34]. It may promote such growth promoting pathways by ligand-dependent or -independent stimulation of androgen receptors. Our preliminary investigations have shown that the alkaline phosphatase inhibitor levamisole reduced the stimulatory effects of indomethacin on 5␣reductase activity in cultured fibroblasts [35]. Other possible mechanisms include indomethacin-mediated growth factor actions via androgen receptors. Members of the steroid hormone receptor superfamily are transcriptional enhancers, when activated by their cognate steroid ligand [36]; demonstration of cross talk between polypeptide growth factors and steroid hormone receptors is relevant to transmission of signals to the nucleus, resulting in growth and differentiation. Our previous work reinforces these findings, regarding growth factor stimulation of 5␣-reductase expression in human gingival fibroblasts [24]. These mechanisms of action could apply to other NSAIDs, and may be linked to their anti-inflammatory potencies. Epidemiologic and clinical studies have suggested potentially beneficial effects of the adjunctive usage of NSAIDs on periodontal conditions. Patients receiving NSAIDs over a period of one year showed less gingival inflammation, shallower probing pocket depths and decreased loss of periodontal attachment, compared to a matched control group [37]. Similar results have been reported on a group of patients under long-term indomethacin therapy, compared to a healthy control group [38]. These studies may indicate that, indomethacin could have important effects on repair and healing in the periodontium. Therefore it is reasonable to suggest that, at least some of the contributory effects of indomethacin on tissue repair and healing in androgen target tissues could be attributed to its effects on the androgen metabolic pathway. The periodontal lesion is a unique wound healing model,
where repair is instigated against a hard avascular, highly mineralized denuded root surface and several adjunctive measures have been used to enhance this process. Due to anatomic limitations of healing in the diseased periodontium, adjunctive usage of materials such as indomethacin may simulate growth promoting substances in the inflammatory exudate and instigate repair. Some of the effects of indomethacin demonstrated in this cell culture model may be anticipated with the other NSAIDs. Acknowledgments The authors wish to acknowledge Dr. I. Tavares for usage of the radioisotope scanner in the Academic Department of Surgery (Rayne Institute, King’s Campus), Guy’s King’s & St. Thomas’ School of Medicine, London, UK. References [1] Goodson JM, Dewhirst FE, Brunetti A. Prostaglandin E2 levels, and human periodontal disease. Prostaglandins 1974;6:81–5. [2] El-Attar TM. PGE2 in human gingiva in health, and disease, and its stimulation by female sex steroids. Prostaglandins 1976;6:81–5. [3] El-Attar TM, Lin HS. Cyclic AMP, and prostaglandins in periodontal disease. In: Advances in prostaglandin, and thromboxane research. Samuelsson B, Ramwell PW, Paoletti R (editors). Newyard: Raven Press, 1980;8:1739 – 40. [4] Ohm K, Albers Von HK, Lisboa BP. Measurement of 8 prostaglandins in human gingival and periodontal disease using high pressure liquid chromatography and radioimmunoassay. J Periodont Res 1984; 19:501–11. [5] Nyman S, Schroeder HE, Lindhe J. Suppression of inflammation during experimental periodontitis in dogs. J Periodontol 1979;50: 450 – 61. [6] Weaks-Dybvig M, Sanavi F, Zander H, Rifkin BR. The effect of indomethacin on alveolar bone loss in experimental periodontitis. J Periodont Res 1982;17:90 –100. [7] Colvard DS, Eriksen EF, Keeting PE, Wilson EM, Lubahn DB, French FS, et al. Identification of androgen receptors in normal human osteoblast-like cells. Proc Natl Acad Sci USA 1989;86:854 –7. [8] Dassouli A, Manin M, Veyssiere G, Jean C. Androgens regulate expression of the gene coding for a mouse vas deferens protein related to the aldo-keto reductase superfamily in epithelial cell subcultures. J Steroid Biochem Mol Biol 1994;48:121– 8. [9] Normington K, Russell DW. Tissue distribution and kinetic characteristics of rat steroid 5␣-reductase isoenzymes. Evidence for distinct physiological functions. J Biol Chem 1992;267:19548 –54. [10] Ojanotko A, Neinstedt N, Harri P. Metabolism of testosterone by human healthy and inflammed gingivae in-vitro. Arch Oral Biol 1980;25:481– 4. [11] Sooriyamoorthy M, Gower DB. Phenytoin stimulation of 5␣-reductase activity in inflammed gingival fibroblasts. Med Sci Res 1989;17: 989 –99. [12] Kasperk CH, Wergedal JE, Farley JR, Linkhart T, Turner RT, Baylink DJ. Androgens directly stimulate proliferating bone cells in-vitro. Endocrinology 1989;124:1576 – 8. [13] Vittek J, Rappaport SC, Gordon GG, Munnangi PR, Southren AL. Concentration of circulating hormones and metabolism of androgens by human gingiva. J Periodontol 1979;50:254 – 64. [14] Sooriyamoorthy M, Gower DB. Hormonal influences on gingival tissue: relationship to periodontal disease. J Clin Periodontol 1989; 16:201– 8.
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