Actions of Adjunctive Nutritional Antioxidants in Periodontitis and Prevalent Systemic Inflammatory Diseases.

Common risk markers for periodontitis and prevalent systemic comorbidities indicate similarities in their progression and molecular mechanisms involved. Resultant pro-oxidant disease profiles provide scope for attenuating their pathogeneses with appropriate adjunctive antioxidants. Levels of oxidative stress markers 8-hydroxy-deoxguanosine (8-HOdG) and malondialdehyde (MDA) are significantly higher in periodontitis and other chronic inflammatory conditions. There is a clear link between periodontitis and diseases associated with significant systemic inflammatory loading, such as metabolic syndrome. Micro- and macro-nutrients have proven to be effective in curbing molecular mechanisms that generate reactive oxygen and nitrogen species. A Mediterranean diet rich in fruits, vegetables, legumes, whole grain, nuts, fish, olive oil and red wine in moderation, could be attributed to the lower occurrence of cardiovascular disease, insulin resistance and other inflammatory diseases in this region. A significant number of naturally occurring flavonoids have been identified in these products. Flavonoids comprising flavonols, flavones and isoflavones are potent free radical scavengers, effective in inhibiting lipid peroxidation, with anti-atherosclerotic and antihypertensive effects.The phenolic compound oleocanthal isolated in virgin olive oil has similar anti-inflammatory actions to that of ibuprofen. The anti-atherogenic effects of MUFA and PUFA in nuts, enhance endothelial function by reducing total cholesterol, oxidized LDL, hs-CRP, sVCAM-1 levels, lipids, lipoproteins and inflammatory markers. Epigenetics influenced by environmental factors and interactions between genes and nutrients, are important considerations in influencing these effects. Using antioxidants as therapeutic adjuncts could enhance the antioxidant capacity of an inherent glutathione system and overcome oxidative effects, thereby mitigating therapeutic side-effects.

Anabolic potential of bone mineral in human periosteal fibroblasts using steroid markers of healing.

A deproteinized natural cancellous bone mineral (B) was studied in a cell culture model for its anabolic potential using two radiolabelled steroid substrates, 14C-testosterone (14C-T) and 14C-4-androstenedione (14C-4-A) independently; in the presence or absence of the anti-androgen finasteride (F) and minocycline (M). Culture medium was assayed for the biologically active metabolite 5 alpha-dihydrotestosterone (DHT) a marker of regenerative potential and wound healing. Confluent monolayer cultures of human periosteal fibroblasts were incubated in Eagle's minimum essential medium with each of the substrates 14C-T and 14C-4-A. Incubations were performed with previously established optimal concentrations of B5 (milligrams/ml), M25 (µg/ml) and F5 (µg/ml) alone and in combination (n=6) for 24h. The eluent was solvent extracted with ethyl acetate (2 ml x 2) and subjected to TLC in a benzene/acetone solvent system (4:1 v/v) for separation of metabolites; they were quantified using a radioisotope scanner. The yield of DHT was increased over controls in response to B and M with both substrates 14C-T and 14C-4-A by 1.7, 1.8-fold and 1.7, 1.6-fold respectively (n=6; p<0.001; one way ANOVA). Combined incubations of B and M resulted in similar yields. F inhibited DHT yields with both radiolabelled substrates by 2-3-fold (n=6; p<0.001) which was overcome by a combined incubation of F+B to values similar to those of controls (p<0.01). Documented pro-anabolic effects of minocycline were applicable as a standard for confirmation of responses to B. Significant increases in yields of DHT in response to B and M with both substrates indicate their anabolic potential in periosteal fibroblasts with implications for wound healing.

A role for non-antimicrobial actions of tetracyclines in combating oxidative stress in periodontal and metabolic diseases: a literature review.

This review addresses the role of adjunctive tetracycline therapy in the management of periodontal diseases and its efficacy in reducing inflammatory burden, oxidative stress and its sequelae in patients with coexisting features of metabolic syndrome. Removal of the dimethylamine group at C4 of the tetracycline molecule reduces its antibiotic properties, enhancing its non-antimicrobial actions; this strategy has aided the development of several chemically modified tetracyclines such as minocycline and doxycycline, by altering different regions of the molecule for focused action on biological targets. Tetracyclines are effective in reducing inflammation by inhibiting matrix metalloproteinases, preventing excessive angiogenesis, inhibiting apoptosis and stimulating bone formation. There are important applications for tetracyclines in the management of diabetic, dyslipidaemic periodontal patients who smoke. The diverse mechanisms of action of tetracyclines in overcoming oxidative stress and enhancing matrix synthesis are discussed in this review.

Periodontal disease severity and systemic diseases prevalent in a Caribbean catchment area of patients.

OBJECTIVE: To describe associations between the prevalence of periodontal disease severity and co-existence of systemic disease(s) and a smoking habit amongst periodontal referrals in a Caribbean catchment area of patients. METHODS: A total of 100 patients completed a medical history questionnaire and were categorized for periodontal disease severity, using clinical and radiographic parameters for association with the prevalence of systemic diseases. RESULTS: Twenty-two per cent presented with moderate periodontal disease (M/F ratio: 1:2.7). 68% of patients examined presented with severe periodontal disease (M/F ratio: 1:1.35). Amongst patients of the same mean age of 48 years presenting with moderate or severe periodontal disease, there was a two-fold increase in the number of missing teeth, amongst patients with severe periodontal disease. In this category there was twice the proportion of smokers and twice the number of mobile teeth, compared with those with moderate periodontal disease. In addition, there was twice the prevalence of diabetics and three times the proportion of patients with combined systemic diseases amongst those with severe periodontal disease, compared with those presenting with moderate periodontal disease, who were predominantly hypertensive or had rheumatoid arthritis. A history of smoking, diabetes mellitus and a combined manifestation of systemic diseases appeared to be more prevalent amongst those with severe periodontal disease. All these findings were significant (p < 0.001). CONCLUSION: An association between severity of periodontal disease and co-existence of systemic diseases may have implications for a unified therapeutic strategy for health.

Steroid 5alpha-reductase activity of Treponema denticola.

INTRODUCTION: Previously we have shown that reference and freshly isolated Treponema denticola cultures possess 5alpha-reductase (5alpha-R) and 3beta- and 17beta-hydroxysteroid dehydrogenase activity. A gene matching the 3-oxo-5alpha-steroid 4-dehydrogenase family protein (gene ID: 2739284; locus tag: TDE2697) has been identified in T. denticola ATCC 35405. The aim of the work presented here was to optimize assay conditions and determine steroid substrate specificities for the 5alpha-R activity of T. denticola ATCC 33520. METHODS: 5alpha-R activity of cell-free preparations was assayed with radioactive steroid substrates. 5alpha-R-reduced products were identified using thin-layer chromatography and a radioisotope scanner. Assay conditions were optimized for co-factor, buffer and pH requirements. Apparent substrate specificities were determined for progesterone, 4-androstenedione, testosterone and corticosterone. The time-course for metabolism of radiolabelled progesterone and cholesterol substrates was investigated with anaerobic cultures. RESULTS: The optimum pH for 5alpha-R was 5.5 and the preferred co-factor was NADPH. The order of the steroids with respect to their 5alpha-R substrate specificities was (in descending order): progesterone, 4-androstenedione, testosterone and corticosterone. There are at least two intermediates in the synthesis of 5alpha-dihydrocholesterol from cholesterol. CONCLUSION: These results suggest that the 3-oxo-5alpha-steroid 4-dehydrogenase family protein gene of T. denticola codes for a functional protein that resembles mammalian 5alpha-R isoenzyme 2 with regard to co-factor requirement and pH optimum.

Periodontal disease severity and systemic diseases prevalent in a Caribbean catchment area of patients / Severidad de la enfermedad periodontal y enfermedades sistémicas en una zona de captación de pacientes en el Caribe

OBJECTIVE: To describe associations between the prevalence of periodontal disease severity and co-existence of systemic disease(s) and a smoking habit amongst periodontal referrals in a Caribbean catchment area of patients. METHODS: A total of 100 patients completed a medical history questionnaire and were categorized for periodontal disease severity, using clinical and radiographic parameters for association with the prevalence of systemic diseases. RESULTS: Twenty-two per cent presented with moderate periodontal disease (M/F ratio: 1:2.7). 68% of patients examined presented with severe periodontal disease (M/F ratio: 1:1.35). Amongst patients of the same mean age of 48 years presenting with moderate or severe periodontal disease, there was a two-fold increase in the number of missing teeth, amongst patients with severe periodontal disease. In this category there was twice the proportion of smokers and twice the number of mobile teeth, compared with those with moderate periodontal disease. In addition, there was twice the prevalence of diabetics and three times the proportion of patients with combined systemic diseases amongst those with severe periodontal disease, compared with those presenting with moderate periodontal disease, who were predominantly hypertensive or had rheumatoid arthritis. A history of smoking, diabetes mellitus and a combined manifestation of systemic diseases appeared to be more prevalent amongst those with severe periodontal disease. All these findings were significant (p < 0.001). CONCLUSION: An association between severity of periodontal disease and co-existence of systemic diseases may have implications for a unified therapeutic strategy for health.

OBJETIVO: Describir las asociaciones entre la prevalencia de la enfermedad periodontal y la co-existencia de enfermedades sistémicas y el hábito de fumar entre los casos periodontales referidos en una zona de captación caribeña de pacientes. MÉTODOS: Un total de 100 pacientes contestó un cuestionario de historia médica y fueron clasificados en correspondencia con la severidad de la enfermedad periodontal, usando parámetros clínicos y radiográficos para la asociación con la prevalencia de las enfermedades sistémicas. RESULTADOS: El veintidós por ciento presentó la enfermedad periodontal moderada (proporción M/F: 1:2.7). El 68% de los pacientes examinados presentó la enfermedad periodontal severa (proporción M/F: 1:1.35). Entre los pacientes de la misma edad promedio de 48 años que se presentaron con la enfermedad periodontal moderada o severa, hubo un aumento doble en el número de dientes perdidos entre los pacientes con la enfermedad periodontal severa. En esta categoría hubo una doble proporción de fumadores y dos veces el número de dientes móviles, en comparación con aquellos que presentaban enfermedad periodontal moderada. Además, hubo dos veces más prevalencia de diabetes y una proporción tres veces mayor de pacientes con enfermedades sistémicas combinadas entre aquellos que presentaban enfermedad periodontal severa, en comparación con aquellos que tenían la enfermedad periodontal moderada – quienes eran predominantemente hipertensos o padecían de artritis reumatoide. Antecedentes de hábito de fumar, diabetes y una manifestación combinada de enfermedades sistémicas resultaron ser más prevalentes entre los que padecían la enfermedad periodontal severa. Todos estos hallazgos fueron significativos (p < 0.001). CONCLUSIÓN: Una asociación entre la severidad de enfermedad periodontal y la co-existencia de enfermedades sistémicas puede tener implicaciones en una estrategia terapéutica unificada para la salud.

Antioxidant effects of glutathione and IGF in a hyperglycaemic cell culture model of fibroblasts: some actions of advanced glycaemic end products (AGE) and nicotine.

The aim of this investigation was to establish potential oxidative effects of glucose, advanced glycation end products (AGE) and nicotine (N) in a fibroblast cell culture model using the anti-oxidants glutathione (G) and insulin like growth factor (IGF). Assays of androgen metabolites were used as biomarkers of healing in this context. Confluent monolayer cultures of human gingival fibroblasts were established in 24 well multiwell plates and incubated in Eagle's MEM for 24h using two radiolabelled androgen substrates 14C-testosterone/14C-4-androstenedione. The established effective concentrations of G1000, glutathione and AGE were used alone and in combination with nicotine and insulin-like growth factor. The medium was then solvent extracted for steroid metabolites, evaporated to dryness and subjected to thin layer chromatography in a benzene acetone solvent system 4:1 v/v for separation of formed metabolites. The metabolites were quantified, using a radioisotope scanner. Significant reduction in the yields of DHT in response to G1000, AGE and nicotine (n=6; p <0.003) were overcome by glutathione (n=6; p <0.002). The stimulatory effect of IGF when combined with AGE was further enhanced by the antioxidant effect of glutathione (n=6; p <0.003). Glucose, AGE and nicotine had a significant inhibitory effect on the yields of the androgen biomarker DHT, overcome by the antioxidant glutathione and IGF, suggestive of an oxidant role for the former agents and an anti-oxidant one for the latter. These agents affected yields of androgen metabolites, biomarkers of oxidative stress and repair, with potential implications on healing in uncontrolled diabetic smokers.

Modulation of androgen metabolism by phenytoin, oestradiol and tamoxifen in human gingival fibroblasts.

OBJECTIVES: The aim of this investigation is to study androgen metabolism in gingival fibroblasts in response to phenytoin, oestradiol and the antioestrogen tamoxifen, in order to establish the possible role of hormones in the aetiopathogenesis of phenytoin-induced gingival overgrowth. MATERIALS AND METHODS: Six cell lines of human gingival fibroblasts were established in monolayer culture in Eagle's minimum essential medium. Duplicate incubations were performed independently with radiolabelled testosterone and 4-androstenedione, respectively (14C-T/14C-4-A), with optimal concentrations of phenytoin, oestradiol and tamoxifen alone and in combination. At the end of a 24-h incubation period, the medium was solvent extracted for steroid metabolites, which were separated by thin layer chromatography and quantified using a radioisotope scanner. RESULTS: The substrates were metabolised mainly to the diols, 5alpha-dihydrotestosterone (DHT) and 4-androstenedione or testosterone, with the two substrates used. The trends were that phenytoin and oestradiol significantly elevated the yields of the androgens DHT, diols and 4-A/testosterone from both substrates while tamoxifen inhibited the stimulatory effects of oestradiol and phenytoin alone and in combination (n=6; p<0.01, one-way anova). CONCLUSION: Specific hormone-mediated activity in response to phenytoin could contribute to the pathogenesis of gingival overgrowth, which can be decreased by the anti oestrogen tamoxifen.

Effects of alkaline phosphatase and its inhibitor levamisole on the modulation of androgen metabolism by nicotine and minocycline in human gingival and oral periosteal fibroblasts.

UNLABELLED: The aim of this investigation was to establish the implications of nicotine, minocycline, alkaline phosphatase (AP) and its inhibitor levamisole (L) on tissue turnover in human gingival and periosteal fibroblasts (HGF, HPF) using [14C]-testosterone as substrate. Monolayer cultures of HGF and HPF established from four patients were incubated in duplicate with serial and optimal concentrations of nicotine and minocycline, alone and in combination, for 24h in Eagle's MEM, with the substrate [14C]-testosterone. Further experiments were carried out on HPF only, to investigate the effects of alkaline phosphatase (AP) and its inhibitor levamisole (L) on the metabolism of [14C]-testosterone, followed by the effects of L on the modulatory actions of nicotine. The cell-conditioned medium was then solvent-extracted, analysed and quantified for steroid metabolites using a radioisotope scanner. At low concentrations, nicotine stimulated the synthesis of the physiologically active androgen 5alpha-dihydrotestosterone (DHT) from [14C]-testosterone, with inhibition at higher concentrations (n=4; P<0.01). Minocycline stimulated the synthesis of DHT, with decreased yields in the presence of nicotine (n=4; P<0.01), but greater than with nicotine alone. Alkaline phosphatase significantly enhanced the synthesis of androgen metabolites by HPF (n=4; P<0.01), with inhibition in response to L alone and in combination with AP, to less than control values (n=4; P<0.01). L also caused further inhibition in the yields of androgen metabolites when incubated with nicotine, implying that some of the inhibitory effects of nicotine could be due to inhibition of AP activity. CONCLUSION: This investigation has shown that nicotine can inhibit the formation of matrix-stimulatory steroid metabolites in fibroblasts, partly due to inhibition of AP activity. Minocycline is a useful adjunct, in reducing the inhibition of androgen metabolism caused by nicotine.

Anabolic potential of fibroblasts from chronically inflamed gingivae grown in a hyperglycemic culture medium in the presence or absence of insulin and nicotine.

BACKGROUND: Impaired fibroblast function due to hyperglycemia shows reversal in response to insulin. The aim of this investigation was to use a hyperglycemic cell-culture model to study the anabolic products of androgen metabolism in fibroblasts in response to insulin and nicotine. METHODS: Human gingival fibroblasts were derived from chronically inflamed gingivae of six nondiabetic periodontal patients with no history of smoking. Six cell lines were established in monolayer culture in 24 well multiwell plates, and duplicate incubations were performed with each cell line for all three experiments. Eagle's minimum essential medium was used in a range of individual experiments, with radiolabeled testosterone as substrate, in the presence or absence of (1) glucose (1 to 4,000 microg/ml); (2) insulin (1 to 100 microg/ml) independently; (3) an effective concentration of glucose (500 microg/ml) with serial concentrations of insulin (1 to 100 microg/ml); and (4) effective concentrations of nicotine (250 microg/ml), glucose, and their combinations in response to insulin (5 microg/ml). The controls contained no agents other than the radiolabeled substrate. At the end of a 24-hour incubation period, the medium was solvent extracted with ethyl acetate, and androgen metabolites were separated by thin-layer chromatography and were quantified using a radioisotope scanner. RESULTS: The androgen substrate 14C-testosterone was metabolized mainly to 5alpha-dihydrotestosterone (DHT) and 4-androstenedione. (1) Glucose at a concentration of 500 microg/ml reduced yields of DHT by 36% (n = 6; P < 0.01). (2) Insulin caused a small but significant inhibition of DHT in normoglycemic cells. (3) Serial concentrations of insulin significantly counteracted the inhibitory effects of glucose on the yields of DHT (n = 6; P < 0.01). (4) The independent inhibitory effects of nicotine and glucose on metabolic yields of DHT were marginally more pronounced in combination but significantly overcome in the presence of insulin. CONCLUSION: Human gingival fibroblasts obtained from chronically inflamed tissue of nondiabetic patients demonstrated that the inhibitory effects of glucose and nicotine on androgen metabolism can be overcome by insulin, in varying degrees.

Androgen metabolic response to indomethacin and the alkaline phosphatase inhibitor levamisole in fibroblasts.

OBJECTIVES: The aim of this investigation is to study the effects of indomethacin (I) and the alkaline phosphatase (ALP) inhibitor levamisole (L) on androgen 5alpha-reductase expression in gingival and periosteal fibroblasts, in the context of repair in the periodontium. Chronically inflamed human gingival fibroblasts (HGF) were used to demonstrate the comparative effects of L on HGF and human oral periosteal fibroblasts (HPF). MATERIAL AND METHODS: Monolayer cultures of six cell lines of HPF of the fifth to ninth passage were incubated in duplicate with 14C-testosterone/14C-4-androstenedione as substrates in Eagle's MEM; I was added at concentrations of 1 and 3 microg/ml in the presence or absence of the established inhibitory concentration of 30 microg/ml L and incubated for 24 h. The medium was solvent extracted for radioactive metabolites, separated by thin layer chromatography and quantified. RESULTS: L caused 50% inhibition of 5alpha-reductase and 17beta-hydroxysteroid dehydrogenase activity in HGF. In HPF, 5alpha-reductase expression was enhanced by I with both substrates, by 65-76% (n = 6; p<0.01), inhibited by 30-50% (n = 6; p<0.01) with L and restored to control values in combination. CONCLUSION: Yields of androgen metabolites may be linked to ALP activity, with implications on healing, during adjunctive treatment of inflammatory periodontal disease with I.

Hormone mediation of immune responses in the progression of diabetes, rheumatoid arthritis and periodontal diseases.

The crucial role of the immune response is common to diabetes mellitus (DM), rheumatoid arthritis (RA) and periodontal disease. This review identifies advances in this field and exciting paradigms in their management. Uncontrolled hyperglycaemia in diabetic patients results in the formation of advanced glycation end products (AGEs), which are detrimental to cell structure and function. Altered host resistance such as defective migration of PMN, impaired phagocytosis and an exaggerated inflammatory response to microbial products also compromises healing in uncontrolled diabetic patients, further compromised in smokers. Nicotine has well documented effects on the immune response, cell adhesion proteins and apoptosis which affect the severity of disease presentation and response to treatment. Rheumatoid arthritis is a multifactorial disease that results in severe destruction of synovial cartilage and bone. Local secretion of large amounts of TNF-alpha and IL-1 due to activation of immunocompetent cells characterises the pathophysiology of RA. This has lead to the emergence of TNF-alpha inhibitors such as etanercept and infliximab in its management. Periodontal disease has a microbial aetiology. But it is similar to RA, in its cyclical pattern of destruction associated with high levels of pro-inflammatory cytokines, which can persist after removal of the antigenic stimulus. Non steroidal anti-inflammatory agents (NSAIDs) have been used as an adjunct to mechanical removal of bacterial antigen, in the management of periodontal disease. The non-reproductive functions of steroid hormones include effects on immunocompetent cells, fibroblasts and osteoblasts, which affect the initiation and progression of inflammatory diseases. Hormone replacement therapy could be another facet in a multifaceted treatment approach in these patients, where indicated.

Phenytoin-mediated androgen metabolism in gingival fibroblasts. Effects of the antiandrogen finasteride and the alkaline phosphatase inhibitor levamisole.

OBJECTIVES: This investigation attempts to identify the role of the alkaline phosphatase inhibitor levamisole (L) and the antiandrogen finasteride (F) on 5alpha-reductase activity in gingival fibroblasts, to elucidate mechanisms for phenytoin-induced gingival overgrowth. MATERIAL AND METHODS: Human gingival fibroblasts were incubated with Eagle's MEM and 14C-testosterone/14C-4-androstenedione as substrates; effective concentrations of phenytoin (Ph), levamisole (L) and finasteride (F), alone and in combinations of (Ph + F) (Ph + L) were added to the incubate. After 24 h, the medium was analysed for steroid metabolites and quantified using a radioisotope scanner. RESULTS: The metabolites isolated were 5alpha-dihydrotestosterone (DHT), 4-androstenedione (4-A) or testosterone (T) from each substrate. With 14C-T as substrate, Ph stimulated DHT synthesis by 1.7-fold, while F and L inhibited this activity by 1.8-fold and 34%, respectively (n = 6; P < 0.001). The combination of Ph + F reduced yields by 2.7-fold compared with Ph alone and Ph + L reduced DHT synthesis by 2.4-fold compared with Ph alone (n = 6; P < 0.001). When 14C-4-androstenedione was used as substrate, similar trends were identified. CONCLUSION: These results suggest that the alkaline phosphatase inhibitor levamisole and the 5alpha-reductase inhibitor finasteride can substantially decrease the yields of DHT in fibroblasts, stimulated by phenytoin. This could be a potential target for reducing the gingival overgrowth caused by phenytoin.

The modulation of androgen metabolism by estradiol, minocycline, and indomethacin in a cell culture model.

BACKGROUND: This investigation attempts to clarify the proanabolic effects of minocycline and indomethacin by studying their effects on androgen metabolism and mediation by estradiol. A cell culture model was used with androgen substrates because of the proanabolic effects of androgen metabolites. METHODS: Monolayer cultures of human gingival fibroblasts (HGF) derived from 6 patients were incubated in duplicate with 14C- testosterone or 14C-4-androstenedione as substrates and optimal concentrations of estradiol (E1,3 microgram/ml) and minocycline (M25 microgram/ml) or indomethacin (I, 1 microgram/ml) alone and in combination (E1,3+11 or E1,3+M25 microgram/ml); similar experiments were carried out with human oral periosteal fibroblasts (HPF), M, I, E, and the combinations. At the end of a 24-hour incubation period in Eagle's MEM, the medium was solvent extracted with ethyl acetate and the metabolites were separated by TLC in a benzene:acetone solvent system (4:1 v/v). The separated metabolites were quantified using a radioisotope scanner. RESULTS: Both androgens were metabolized to 5alpha-dihydrotestosterone (DHT) and 4-androstenedione (4-A) or testosterone (T) at baseline and in response to the agents tested, by HGF and HPF. With HGF, there were significant increases in the yields of DHT and 4-A or T in response to M, E, and M+E, resulting in 50% to 2.4-fold increases in these metabolites over control incubations (n = 6; P<0.01). The responses to I and combinations of I+E were similar. HPF also demonstrated significant increases of 29% to 4-fold in the yields of androgen metabolites in response to M, E, and M+E (n = 6; P<0.01). I and E similarly increased the yields of androgen metabolites, alone and in combination. CONCLUSIONS: Adjunctive periodontal treatment with minocycline or indomethacin can contribute to hormone-modulated anabolic responses in males and females in gingival and periosteal fibroblasts derived from a chronically inflamed source.

Dexamethasone-mediated androgen metabolism in human gingival and oral periosteal fibroblasts.

Dexamethasone modulates the effects of other hormones and mediates cell function; the periodontium is a target tissue for androgens. It was therefore relevant to investigate the modulation of androgen metabolism by dexamethasone in cultured human gingival (HGF) and oral periosteal fibroblasts (HPF). Each cell line was incubated in Eagle minimum essential medium with [(14)C]testosterone/[(14)C]4-androstenedione as substrates and serial concentrations of dexamethasone (0.5-50 microg/ml), for 24h; the medium was solvent-extracted, analyzed and quantified for steroid metabolites. In response to dexamethasone, both HGF (n=6) and HPF (n=4) showed up to two-fold increases in the formation of 5alpha-dihydrotestosterone and 4-androstenedione (P<0.01, one-way ANOVA), and 3.6- to 5-fold increases in the formation of testosterone (P<0.001), from [(14)C]4-androstenedione, with some inhibition at higher concentrations. Dexamethasone stimulated the formation of physiologically active androgen metabolites in a dose-dependent manner. These metabolites might therefore contribute to dichotomous effects in connective tissues of the periodontium, dependent on effective concentrations of dexamethasone.

Modulatory effects of indomethacin on androgen metabolism in human gingival and oral periosteal fibroblasts.

The non-steroidal anti-inflammatory agent indomethacin (I) suppresses gingival inflammation and alveolar bone resorption. Androgens particularly 5 alpha-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 (5(th)-9(th) passage) were established in Eagle's MEM and incubated with 14C-testosterone/14C-4-androstenedione and serial concentrations of I (0.5-50 microg/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 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase (17 beta-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 microg/ml, often reaching control values at 50 microg/ml. The expression of 17 beta-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 microg 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.

The effects of human mast-cell products and of phenytoin on androgen 5alpha-reductase expression in human gingival fibroblasts.

The aim was to elucidate mechanisms for phenytoin-induced gingival overgrowth, using effects of type 1 human mast cell (HMC-1) supernatant and histamine on the expression of 5alpha-reductase in human gingival fibroblasts (HGF), and the effects of phenytoin on this activity. Duplicate incubations of HGF in Eagle's minimum essential medium (MEM) were performed with [14C] testosterone and serial concentrations of mast-cell histamine (1-100 microgram/ml)/HMC-1 culture supernatant at serial dilutions of 1-100 microl/ml for 24 h. Other experiments employed optimal concentrations of HMC-1 supernatant (10 microl/ml) or histamine (8 microgram/ml) and phenytoin (1 and 5 microgram/ml), alone and in combination, using two androgen substrates, [14C] testosterone and [14C] 4-androstenedione. At the end of a 24-h incubation the medium was solvent-extracted for steroid metabolites, analysed and quantified in a radioisotope scanner. HMC-1, histamine (n=3) and phenytoin (n=6) significantly increased the synthesis of dihydrotestosterone and 4-androstenedione by up to 80% (P<0.01); the combination of HMC-1 and phenytoin caused two-fold increases (n=6; P<0.01). The incubations with histamine alone and in combination with phenytoin showed significant stimulation of dihydroxytestosterone and the diols alone and in combination, which was less pronounced in combination. This investigation demonstrates significant stimulation of 5alpha-reductase activity in human gingival fibroblasts by mast-cell supernatant and a specific product histamine, alone and in combination with phenytoin. Androgen 5alpha-reductase-mediated anabolic actions in connective tissue are well documented. The findings suggest a novel hypothesis that mast-cell mediated androgen action in the gingiva in response to phenytoin could contribute to gingival overgrowth.

Effects of the anti-androgen finasteride on the modulatory actions of oestradiol on androgen metabolism by human gingival fibroblasts.

5 alpha-Reduction of androgen substrates results in the formation of the biologically active androgen 5 alpha-dihydrotestosterone (DHT), while 17 beta-hydroxysteroid dehydrogenase metabolises androgen substrates to 4-androstenedione or testosterone. The aim here was to study the effect of the anti-androgen finasteride on 5 alpha-reduction of androgens by human gingival fibroblasts (HGF) and its modulation by oestradiol-17 beta. Duplicate cultures of HGF were incubated with [14C]testosterone/[14C]4-androstenedione in Eagle minimum essential medium (n=6) in the presence or absence of oestradiol-17 beta (O) or finasteride (F; 0.1-3 microg/ml) for 24 h. The steroid metabolites were analysed and quantified using a radioisotope scanner. With [14C]testosterone as substrate, oestradiol stimulated the formation of DHT by 63% (n=6; P<0.01). In contrast, finasteride inhibited this activity by 61% (n=6; P<0.01). The combination of O+F produced 43% less inhibition than finasteride alone (n=6; P<0.01). There were 200-300% increases in the formation of 4-androstenedione in response to O and F, being less pronounced in combination. Oestradiol stimulated the formation of DHT from [14C]4-androstenedione by 300-600% and finasteride reduced the yield of DHT by 40-64%; there was less inhibition in combination with O. There were 300-700% increases in the formation of testosterone in response to F and O alone and in combination (n=6; P<0.01). Oestradiol-induced stimulation of 5 alpha-reductase activity on androgen substrates by HGF is suggestive of hormone modulatory mechanisms in the healing periodontium of both sexes. Its inhibition by finasteride is suggestive of type 2 isoenzyme activity, confirming target-tissue functions in the gingiva.

Effects of hormonal contraceptives on the periodontium, in a population of rural Sri-Lankan women.

AIMS: Women using hormonal contraceptives can be considered to be a 'risk group' for periodontal disease, due to prolonged, sustained serum levels of oestrogens and progesterone. This investigation aims to study the effects of hormonal contraceptives on periodontal tissues. METHODS: 32 women using hormonal contraceptives for less than 2 years, 17 for 2-4 years and a matched control group of 39 non-users were selected for the study. They were clinically examined for plaque levels (plaque index: PLI), gingival condition (gingival index: GI) and loss of periodontal attachment (LA). RESULTS: Contraceptive users of less than 2 years and 2-4 years duration (n=32, n= 17 respectively) and non-users (n=39) had similar oral hygiene levels; yet the contraceptive users had a significantly higher level of gingival inflammation, compared to the non-users (p<0.001; 1-way ANOVA). Usage of hormonal contraceptives for 2-4 years (n= 17) caused a significantly higher LA (p<0.001) compared to that of controls (n=39). CONCLUSIONS: Usage of contraceptive preparations containing oestrogen and progesterone resulted in hormonal changes similar to those seen in pregnancy, associated with increased prevalence of gingivitis. There was significantly higher LA with prolonged usage of hormonal contraceptives, compared with controls.

Periodontal disease status during pregnancy and 3 months post-partum, in a rural population of Sri-Lankan women.

OBJECTIVES: The aim of this investigation was to study the effects of pregnancy on the periodontium, in a rural population of Sri-Lankan women. METHODS: The study group consisted of 47 pregnant women and 47 non-pregnant women who served as matched controls. All subjects were examined for plaque (plaque index: PLI), gingival condition (gingival index: GI) and loss of periodontal attachment (LA) levels, 4 x during the study, at 3-monthly intervals. RESULTS: Despite similar scores for plaque levels in both pregnant and non-pregnant women, the GI of pregnant women was significantly increased, during the 1st and 2nd trimesters compared to the controls (p<0.01, 2-way ANOVA). During the 3rd trimester, GI was further increased (p<0.001), but dropped at 3 months post-partum. Values for LA did not show significant differences from that of controls, during any of the stages of pregnancy. CONCLUSIONS: The results of this study show that pregnancy had an effect only on the gingivae and not on periodontal attachment levels. The effects of oestrogen and progesterone could give rise to a more florid response to the irritant effects of plaque, resulting in severe gingivitis.