Influence of phenytoin on cytoplasmic free Ca2+ level in human gingival fibroblasts

Abstract— Influence of 5,5-diphenylhydantoin (phenytoin;PHT) on the cytoplasmic free Ca²⁺ concentration, [Ca²⁺]ⁱ, was studied in fura 2 loaded adherent monolayers of human gingival fibroblasts derived from three patients before and after 9 months of PHT therapy. In the patient where gingival overgrowth developed during PHT medication (responder), addition of PHT to gingival fibroblasts derived before PHT medication induced a transient extracellular Ca²⁺ dependent increase in [Ca²⁺]ⁱ. In a non-responder patient, where gingival overgrowth did not develop during the same period of PHT therapy, addition of PHT to gingival fibroblasts derived before the start of medication did not significantly affect [Ca²⁺]_i. Under extracellular Ca²⁺ deficient conditions, addition of PHT to serum-starved fibroblasts derived from the two categories of patients before the medication resulted in an increase in [Ca²⁺]_i. In fibroblasts derived from the responder patient during PHT medication, in contrast to those from the non-responders (n = 2), the basal level of [Ca²⁺]_i was significantly decreased. The results indicate that, in the cases studied, there is a relationship between PHT induced alterations in [Ca²⁺]_i in gingival fibroblasts and the clinical development of gingival overgrowth.     

Effect of phenytoin on collagen accumulation by human gingival fibroblasts exposed to TNF-alpha in vitro

OBJECTIVE: Tumor necrosis factor (TNF)-alpha is associated with chronic gingival inflammation and reported to induce gingival overgrowth (GO), while phenytoin (PHT) is also known to be a causative agent of GO. We examined the synergistic effect of PHT and TNF-alpha on collagen metabolism in human gingival fibroblasts (HGFs). MATERIALS AND METHODS: HGFs were cultured with TNF-alpha and PHT. Quantitative real-time RT-PCR was employed to determine the mRNA levels for collagen, matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and integrin subunits. Cellular collagen endocytosis was determined using a flow-cytometry. RESULTS: The proliferation of HGFs was not affected by TNF-alpha or PHT individually, whereas both synergistically increased collagen accumulation in HGFs. Further, collagen mRNA expression was not increased by TNF-alpha or PHT, although together they markedly prevented cellular collagen endocytosis, associated with the suppression of alpha2beta1-integrin mRNA expression. The mRNA expression of MMP-1 and-2 was suppressed by PHT, while TIMP-1 mRNA expression was enhanced by both TNF-alpha and PHT. CONCLUSION: Our results suggest that TNF-alpha and PHT together cause impaired collagen metabolism by suppression of enzymatic degradation with MMPs/TIMP-1 and integrin-mediated endocytosis. These synergistic effects may also be involved in TNF-alpha- and PHT-induced collagen accumulation, leading to GO.     

Effect of phenytoin and nifedipine on collagen gene expression in human gingival fibroblasts

Phenytoin (PHT), a widely used anticonvulsant, and nifedipine (NF), an anti-anginal drug, cause clinically similar gingival overgrowths in some patients. The aim of this work was to investigate their effects on collagen and protein synthesis and cellular proliferation in normal human gingival fibroblasts in vitro. Gingival fibroblasts were cultured from biopsies taken from three healthy individuals during operations on maxillary canines and incubated with various concentrations of NF (100 and 200 ng/ml) and PHT (5 and 10 micrograms/ml) for up to 7 days. The results showed that NF and PHT have a specific effect in reducing total protein and collagen synthesis but do not influence cell proliferation in healthy gingival fibroblasts in vitro. In addition the level of mRNA for type I collagen was decreased after incubation of the cells with the drugs for 1 or 2 days. The decrease in the level of type I collagen mRNA seemed to be specific since the level of type IV collagenase mRNA used as a reference RNA did not decrease.     

Regulation of epidermal growth factor receptor metabolism in gingival fibroblasts by phenytoin in vitro

Normal human gingival fibroblasts derived from five children between 8 and 12 yr of age were cultured under serum-free conditions in the presence of epidermal growth factor (EGF) either alone or in combination with 5,5-diphenylhydantoin (phenytoin; PHT). DNA-synthesis, binding of EGF to its cell-surface receptor and internalisation of EGF-receptor-ligand complexes were studied. In normal gingival fibroblasts treated solely with EGF for 48 h, DNA synthesis increased significantly, as in cells treated solely with PHT. When EGF binding data was calculated according to Scatchard, it was found that the number of EGF receptors in fibroblasts increased significantly after PHT treatment. The number of EGF-receptors in untreated gingival fibroblasts varied from 147,000 to 170,000 receptors per cell whereas in PHT-treated fibroblasts the range was from 181,000 to 280,000. The study indicates that PHT regulates EGF-receptor metabolism in human gingival fibroblasts by increasing the number of cell-surface EGF-receptors which may contribute to the alteration of gingival connective tissue observed in patients undergoing PHT medication.     

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.     

The pharmacokinetics of phenytoin in gingival crevicular fluid and plasma in relation to gingival overgrowth

Abstract The aim of this study was to determine whether phenytoin (PHT) could be detected in gingival crevicular fluid (GCF), and to relate its concentration to both plasma level and degree of gingival overgrowth. 23 patients medicated with phenytoin for at least 6 months were clinically examined for signs of periodontal disease and gingival overgrowth. 12 patients out of these demonstrated clinically significant overgrowth and their plaque scores and gingival inflammation were greater than for the non-overgrowth group (p<0.001). Phenytoin concentrations were determined by high performance liquid chromatography, and was detected in GCF. There was a significant correlation between the GCF and plasma phenytoin concentrations (p<0.05), but it was not related to the extent of gingival overgrowth. Inflammation increased the GCF volume, but was not a determinant of GCF phenytoin concentration. It is concluded that effusion of phenytoin into GCF is regulated by the plasma levels of the drug, but its concentration in GCF is not related to the incidence of gingival overgrowth.     

Effect of phenytoin on interleukin-1β production in human gingival fibroblasts challenged to tumor necrosis factor αin vitro

Effects and interaction of tumor necrosis factor α (TNFα) and the antiepileptic drug phenytoin (PHT) on interleukin-1β (IL-1β) production as well as on prostaglandin E₂ (PGE₂) formation were studied in gingival fibroblasts in vitro. TNFα, in contrast to PHT, dose-dependently stimulated the production of cell-associated IL-1β. The stimulatory effect of TNFα on IL-1β production was accompanied by enhanced PGE₂ formation. When PHT and TNFα were added simultaneously, the drug potentiated the stimulatory effect of TNFα on both IL-Iβ production and PGE₂ formation. The major PHT metabolite, p-HPPH, did not affect IL-1β production, either alone or in combination with TNFα. The production of IL-1β induced by TNFα and the combination of TNFα and PHT was further enhanced in the presence of the prostaglandin endoperoxide (PGH) synthase inhibitors, indomethacin and flurbiprofen. The PHT-mediated enhancement of TNFα-induced IL-1β production and PGE₂ formation in gingival fibroblasts may be an important link in the pathogenesis of gingival overgrowth induced by PHT.     

Synthesis of sulfated glycosaminoglycans by human gingival fibroblasts from phenytoin-induced gingival overgrowth in vitro

Abstract – The in vitro synthesis of sulfated glycosaminoglycans (GAGs) was studied in gingival fibroblasts from two patients exhibiting phenytoin(PHT)-induced gingival overgrowth, i.e. pseudopockets, which required surgical excision, from one patient on PHT medication not exhibiting pseudopockets and from two normal controls. The results showed that the newly synthesized GAGs were distributed to the culture medium, to a pericellular pool and to the cell fraction. Gingival fibroblasts from the PHT-induced gingival overgrowth showed a significantly increased incorporation of ³⁵SO₄^2- into GAGs compared to the other strains, and this, increase was mainly confined to the dermatan sulfate fraction. These results are in accordance with our previous biochemical studies where increased amounts of GAGs were found in gingival biopsies from the PHT-induced lesion.     

NH4C1 and protein metabolism in human gingival fibroblasts

Abstract – The biologic effect of ammonia was studied in cultures of fibroblasts isolated from human gingiva. NH_CI in the range 2–20 mu was found to exhibit a concentration dependent growth inhibitory effect, with a delayed action wllich was most pronounced at low concentrations. Concomitant with the growth inhibitor)'effect a significant cellular accumulation of protein was evident. No effect on protein synthesis in general, as measured by G-protine incorporation, was found, whereas some inhibitory effect on collagen biosynthesis was indicated. Secretion of C-collagen and other labeled proteins was not affected. The only pronounced effect of ammonia on metabolism of the l4C-labeled proteins was an inhibition of the intracellular degradation of newly synthesized collagen, the lysosomes being suggested as the site for this degradation.     

Enhanced prostaglandin biosynthesis in human gingival fibroblasts isolated from patients treated with phenytoin

Prostaglandin E2 (PGE2) formation was studied in human gingival fibroblasts derived from three epileptic patients before and after 9 months of phenytoin (PHT) therapy. Interleukin 1 (IL-1 alpha; 0.3-6.0 ng/ml), (IL-1 beta; 10-1000 pg/ml) and tumour necrosis factor (TNF alpha; 0.01-0.1 microgram/ml) dose-dependently stimulated the formation of PGE2 in 24 h cultures. In fibroblasts, derived after 9 months of PHT therapy, IL-1 alpha, IL-1 beta and TNF alpha induced a significantly higher formation of PGE2 compared to that in fibroblasts derived before PHT therapy. IL-1 beta induced a significantly higher release also of 3H-arachidonic acid (3H-AA) from prelabelled PHT fibroblasts compared to that in prelabelled gingival fibroblasts isolated before the drug therapy. Addition of exogenous AA caused a spontaneous increase of PGE2 formation in PHT fibroblasts compared to that in fibroblasts isolated before the PHT treatment. The results indicate that PHT medication results in an upregulation of prostanoid formation in gingival fibroblasts partly due to an increased phospholipase A2 activity and partly due to an increased cyclooxygenase activity.     

Inhibitory effect of NH4Cl on secretion of collagen in human gingival fibroblasts

Abstract – The general protein synthesis in human gingival fibroblasts as measured by ¹⁴C-proline incorporation was only moderately inhibited by 10 mM NH₄Cl during incubation for 36 h. The proportion secreted as noncollagen protein and recovered from the cellular fraction as collagen was not significantly affected, whereas a pronounced inhibitory effect on the secretion of collagen was evident after 24 h. This effect was dose dependent, with a significant inhibition of collagen secretion even at 2 mM ammonia. The applied concentrations of NH₄Cl had no significant effect on the hydroxylation of prolyl residues in collagen. Ammonia had no inhibitory effect on the secretion of fibronectin, another major secretory protein from fibroblasts. When comparing different lysosomotropic agents; NH₄Cl, chloroquine and methylamine, the most prominent effect was consistently found to be an inhibition of the secretion of collagen.     

The pathogenesis of drug-induced gingival overgrowth

Abstract Gingival overgrowth is a well-documented unwanted effect, associated with phenytoin, cyclosporin. and the calcium channel blockers. The pathogenesis of drug-induced gingival overgrowth is uncertain, and there appears to be no unifying hypothesis that links together the 3 commonly implicated drugs. In this review, we consider a multifactorial model which expands on the interaction between drug and/or metabolite, with the gingival fibroblasts. Factors which impact upon this model include age. genetic predisposition, pharmacokinetic variables, plaque-induced inflammatory and immunological changes and activation of growth factors. Of these, genetic factors which give rise to fibroblast heterogeneity, gingival inflammation, and pharmacokinetic variables appear to be significant in the expression of gingival overgrowth, A more thorough understanding of the pathogenesis of this unwanted effect will hopefully elucidate appropriate mechanisms for its control.     

Cyclosporin A upregulates prostaglandin E2 production in human gingival fibroblasts challenged with tumor necrosis factor alpha in vitro

The effect of Cyclosporin A (CsA) on prostaglandin E₂ (PGE₂) production in human gingival fibroblasts challenged with tumor necrosis factor alpha (TNF-α) was studied. TNF-α (1-100 ng/ml) dose-dependently stimulated PGE₂; formation in 24 h cultures. CsA (1-100 ng/ml) did not induce PGE₂; formation itself but potentiated TNF-α induced PGE; formation in gingival fibroblasts in a manner dependent on the concentrations of both CsA and TNF-α. TNF-α (10 ng/ml) stimulated the release of [³H]-arachidonic acid (A.A) from prelabelled fibroblasts that was potentiated by CsA (100 ng/ml). Addition of exogenous unlabelled AA (5-20 μM/ml) to the cells resulted in enhanced PGE₂: formation that was not potentiated by CsA (100 ng/mi). Furthermore. CsA (100 ng/ml) did not further increase the level of cyclooxygenase-2 mRNA induced by TNF-α (10 ng/ml). although PGE₂ formation was enhanced. The results indicate that CsA and TNF-α act in concert on PGE₂ formation in gingival fibroblasts. which may be of importance in the pathogenesis of gingival overgrowth induced by the drug.     

lnterleukin-1β and phenytoin reduce α1 (1) procollagen mRNA expression in human gingival fibroblasts

Effects of and interactions between interleukin-1β (IL-1 β) and phenytoin (PHT) on α1 (I) procollagen gene and protein expression in human gingival fibroblasts and its relation to prostaglandin E₂ (PGE₂) formation were studied. IL-1β (300 pg/ ml) reduced the steady-state level of αl(I) procollagen mRNA by 50% and decreased the amount of procollagen I by 35%. PHT (10 μg/ml) reduced the level of α1(I) procollagen mRNA by 40% but the amount of procollagen I in the medium was unchanged. In combination with IL-1β, PHT potentiated the inhibitory effect of IL-1β on αl(I) procollagen mRNA level that was accompanied by an increased PGE₂ formation. Preincubation with indomethacin (10^-6m) partially reduced the inhibitory effect of IL-1β as well as of IL-1β in combination with PHT on the mRNA level of αl(I) procollagen. The inhibitory effect of PHT was unaffected by indomethacin treatment. Addition of exogenous PGE₂ (≥10 nm) dose-dependently reduced steady-state level of α1(I) procollagen mRNA as well as the amount of procollagen 1. The study indicates that IL-1 reduces the expression of αl(I) procollagen mRNA in human gingival fibroblasts partly by a prostaglandin endoperoxide (PGH) synthase-mediated pathway and partly by a PGH-synthase independent pathway, whereas PHT reduces α1(I) procollagen gene expression by a PGH-synthase independent pathway. The potentiation of the inhibitory effect of IL-1 induced by PHT was mediated mainly by a PGH-synthase dependent pathway.     

Increased bFGF level in the serum of patients with phenytoin-induced gingival overgrowth

Abstract. To elucidate the involvement of bFGF (basic fibroblast growth factor) in the pathogenesis of phenytoin-induced gingival overgrowth, we measured the concentration of bFGF in the serum of 36 epileptic patients taking phenytoin and in 94 normal volunteers by enzyme-linked immunosorbent assay technique. The concentration of phenytoin in serum was determined by high-performance liquid chromatography. In 34 of 36 patients taking phenytoin in this investigation, apparent gingival overgrowth was noticed. The mean concentration of bFGF was 33.9±18.5 pg/ml in the overgrowth group and 10.6±5.2 pg/ml in the volunteer group ( p <0.01). The serum phenytoin level did not correlate ( r =0.22, p = 0.2) with the degree of gingival overgrowth but there was a significant correlation ( r =0 38, p =0.023) between the degree of gingival overgrowth and the serum bFGF level. However, no correlation was observed among age, daily phenytoin dose, total phenytoin dose, duration of phenytoin therapy, serum phenytoin level, or serum bFGF level. The results suggested that enhanced serum bFGF level was implicated in the pathogenesis of phenytoin-induced gingival overgrowth.     

Cytotoxic effects of gingival retraction cords on human gingival fibroblasts in vitro

The objective of this study was to determine the cytocompatibility of three different extracts of gingival retraction cords and to compare the cytotoxic effect of these materials on human gingival fibroblasts. Gingival retraction cords impregnated with aluminium sulphate (Gingi-Aid), dl-adrenaline HCl (Gingi-Pak) and non-drug-impregnated cord (Gingi-Plain) were eluted with culture medium for 10 min and 24 h. Cytotoxicity was judged using a tetrazolium bromide reduction assay. Our data demonstrated that gingival retraction cords applied alone almost completely inhibited cell viability (P < 0.05). In addition, the results also showed that the eluates from aluminium sulphate-impregnated cord, dl-adrenaline HCl-impregnated cord and non-drug-impregnated cord were cytotoxic to primary human gingival fibroblast cultures (P < 0.05). The cell viability of incubation of gingival fibroblasts containing 10-min eluates of aluminium sulphate, dl-adrenaline HCl and non-drug-impregnated cord was 61, 21 and 70%, respectively. The cell viability of incubation of gingival fibroblasts containing 24 h eluates of aluminium sulphate, dl-adrenaline HCl and non-drug-impregnated cord was 68, 58 and 72%, respectively. It was found that dl-adrenaline HCl-impregnated gingival retraction cord was the most toxic gingival retraction cord among the materials tested in all cultures (P < 0.05). The cytotoxicity decreased in an order of dl-adrenaline HCl-impregnated cord > aluminium sulphate-impregnated cord > non-drug-impregnated cord. The extent or degree of the cytotoxicity depended on the materials tested. Gingival retraction cords have significant potential for gingival toxicity. Careful management of gingiva retraction cords would lower the risk of potential gingival tissue damage during clinical application procedure and thus increase the success of prosthodontic procedures.     

Synergistic enhancement of collagenous protein synthesis by human gingival fibroblasts exposed to nifedipine and TNF-alpha in vitro

BACKGROUND: Gingival overgrowth occurs in patients receiving nifedipine. Gingival inflammation may be an etiologic factor. METHODS: Gingival fibroblasts were either exposed to (i) 0-500 ng/ml TNF-alpha or 10(-7) M nifedipine or (ii) 0-500 ng/ml TNF-alpha + 10(-7) M nifedipine for 7 days. 3H-proline was used to quantify collagenous protein synthesis. RESULTS: Both TNF-alpha and 10(-7) M nifedipine significantly decreased cell proliferation, and 10(-7) M nifedipine + 500 ng/ml TNF-alpha reversed these effects. Collagenous protein synthesis was significantly reduced by TNF-alpha and was significantly enhanced by either 10(-7) M nifedipine or 5-500 ng/ml TNF-alpha + 10(-7) M nifedipine. CONCLUSIONS: Our data report that nifedipine reverses the primary effects of TNF-alpha on collagenous protein synthesis. Patients with gingivitis could be susceptible to gingival overgrowth during nifedipine therapy as a result of synergistic effects of these agents on fibroblast metabolism, which occurs irrespective of reduced cell numbers.