Phenytoin-induced Gingival Overgrowth Research Articles

18‑α‑glycyrrhetinic acid induces apoptosis in gingival fibroblasts exposed to phenytoin.

Phenytoin (PHT)-induced gingival overgrowth is caused by the increased proliferation and reduced apoptosis of gingival fibroblasts in inflammatory gingiva. Licorice has long been used as a component of therapeutic preparations. It inhibits cell proliferation, induces cell apoptosis and has anti-inflammatory effects. 18-α-glycyrrhetinic acid (18α-GA), the active compound in licorice, promotes apoptosis in various types of cells. The present study determined whether 18α-GA affects apoptosis in gingival fibroblasts exposed to PHT. The present study aimed to establish a basis for the therapeutic application of 18α-GA to treat the gingival overgrowth induced by PHT. Human gingival fibroblasts from healthy donors were cultured to semi-confluence and then stimulated in serum-free DMEM containing PHT with or without 18α-GA for subsequent experiments. Apoptotic cells were detected by ELISA. Analysis of the distribution of cell cycle phases and the apoptotic cell population was performed by flow cytometry. The expression levels of mRNAs and proteins of apoptotic regulators were measured using reverse transcription-quantitative PCR and western blotting, respectively. Caspase (CASP) activities were assessed by an ELISA. Treatment with 18α-GA markedly increased the number of apoptotic cells, reduced BCL2 mRNA expression, increased CASP2 and receptor (TNFRSF)-interacting serine-threonine kinase 1 (RIPK1) domain containing adaptor with death domain, Fas (TNFRSF6)-associated via death domain, RIPK1, tumor necrosis factor receptor superfamily; member 1A, TNF receptor-associated factor 2, CASP2, CASP3 and CASP9 mRNA expression, and also upregulated the protein expression levels and activities of caspase-2, caspase-3 and caspase-9. These results demonstrated that 18α-GA induced apoptosis through the activation of the Fas and TNF pathways in the death receptor signaling pathway in gingival fibroblasts treated with PHT. 18α-GA exhibited therapeutic potential for the treatment of PHT-induced gingival overgrowth.

Dental management of a child patient with phenytoin-induced gingival overgrowth: report of a case with a brief review of literature

Drug-induced gingival overgrowth is medication-related gingival overgrowth commonly associated with certain systemic medications. Gingival overgrowth is a common adverse effect of phenytoin therapy. The condition is more frequently seen in children as compared to adults, affects both genders equally, and may vary from mild to severe. It may be associated with difficulty in mastication, delayed eruption of teeth, tissue trauma, and secondary inflammation. It can be managed by both surgical and nonsurgical therapies. A case of dental management of phenytoin-induced gingival enlargement in a 7-year-old girl is presented with a brief literature review.

Phenytoin-induced gingival overgrowth.

Phenytoin-induced gingival overgrowth.

Suplementasi asam folat untuk mengurangi pembesaran gingiva akibat terapi fenitoin: a scoping review

Background: Epilepsy is a neurological condition that around 70 million people worldwide have. Phenytoin is the most common anti-epileptic therapy for controlling epilepsy and preventing its recurrence. The side effect of phenytoin therapy includes gingival overgrowth which often causes psychological and aesthetical problems, interferes with normal mastication, and worsen the gingival inflammation. There are surgical and non-surgical options to treat phenytoin-induced gingival overgrowth, one of the non-surgical options to treat phenytoin-induced gingival overgrowth. One of the non-surgical treatment for phenytoin-induced gingival overgrowth is folic acid supplementation, which has been studied to prevent the adverse side effects of phenytoin therapy despite the variative results. Objective: To discover the current theory depiction about the effect of folic acid supplementation on gingival overgrowth due to phenytoin therapy. Method: The type of the research is descriptive observational research and method of scoping review. Journals obtained using PCC (population, concept, and context) with population of patients on phenytoin therapy, using concept of preventing phenytoin-induced gingival overgrowth using agent supplementation, and context of folic acid. The inclusion and exclusion criteria filtered by boolean search on two databases, PubMed and Google Scholar from 1987 to 2020. Results: A total of 5 journals found and all of them are included in a table with the results of the studies discussing about folic acid supplementation in phenytoin-induced gingival overgrowth. Conclusion: Folic acid supplementation was found to delay the onset, reduce the incidence and severity as well as the risk of recurrence after gingivectomy on phenytoin-induced gingival overgrowth.

How genetic variation was analyzed in phenytoin-induced gingival enlargement using single-nucleotide polymorphism of candidate gene CYP2C9?

Background:Phenytoin-induced gingival overgrowth is an adverse drug reaction affecting few individuals, on phenytoin therapy for its antiepileptic effect. Analysis of genetic variation of CYP2C9*2 gene was done to identify the action of metabolic enzyme cytochrome P 450 on this drug. The main background of this publication is a quick review about one of the molecular techniques used to identify the single-nucleotide polymorphism (SNP) using polymerase chain reaction coupled with restriction fragment length polymorphism (PCR-RFLP).Materials and Methods:Deoxyribonucleic acid (DNA) was extracted from 5 ml of venous blood withdrawn from the individual, who had gingival overgrowth following phenytoin therapy. DNA was isolated, using the phenol-chloroform method. Isolated DNA was used for SNP analysis of CYP2C9*2 presentation. The basic procedure used for SNP analysis in our case was PCR-RFLP.Results:Genetic variation of CYP2C9*2 in our case was homomutant.Conclusion:The etiology of phenytoin-induced gingival overgrowth is always an enigma, but it is now becoming clearer that a multifactorial role may be involved in the cause. One of the factors analyzed was polymorphism of CYP2C9*2 gene and it was found to be homomutant in our case. Adverse drug reaction can be minimized, by either reducing the drug dosage or drug substitution. However, larger scale genome-wide study has to be carried out to confirm one of the etiopathogenesis as mutation of the CYP2C9 gene, in phenytoin-induced gingival overgrowth.

Multiple Functions of Lysyl Oxidase Like-2 in Oral Fibroproliferative Processes

Gingival overgrowth is a side effect of certain medications, including calcium channel blockers, cyclosporin A, and phenytoin. Phenytoin-induced gingival overgrowth is fibrotic. Lysyl oxidases are extracellular enzymes that are required for biosynthetic cross-linking of collagens, and members of this enzyme family are upregulated in fibrosis. Previous studies in humans and in a mouse model of phenytoin-induced gingival overgrowth have shown that LOXL2 is elevated in the epithelium and connective tissue in gingival overgrowth tissues and not in normal tissues. Here, using a novel LOXL2 isoform-selective inhibitor and knockdown studies in loss- and gain-of-function studies, we investigated roles for LOXL2 in promoting cultures of human gingival fibroblasts to proliferate and to accumulate collagen. Data indicate that LOXL2 stimulates gingival fibroblast proliferation, likely by a platelet-derived growth factor B receptor-mediated mechanism. Moreover, collagen accumulation was stimulated by LOXL2 enzyme and inhibited by LOXL2 inhibitor or gene knockdown. These studies suggest that LOXL2 could serve as a potential therapeutic target to address oral fibrotic conditions.

Curcumin inhibits TGF-β1-induced connective tissue growth factor expression through the interruption of Smad2 signaling in human gingival fibroblasts.

Many fibrotic processes are associated with an increased level of transforming growth factor-β1 (TGF-β1). TGF-β1 can increase synthesis of matrix proteins and enhance secretion of protease inhibitors, resulting in matrix accumulation. Connective tissue growth factor (CTGF) is a downstream profibrotic effector of TGF-β1 and is associated with the fibrosis in several human organs. Curcumin has been applied to reduce matrix accumulation in fibrotic diseases. This study was aimed to evaluate whether curcumin could suppress TGF-β1-induced CTGF expression and its related signaling pathway involving in this inhibitory action in primary human gingival fibroblasts. The differences in CTGF expression among three types of gingival overgrowth and normal gingival tissues were assessed by immunohistochemistry. Gingival fibroblast viability in cultured media with different concentrations of curcumin was studied by MTT assay. The effect of curcumin on TGF-β1-induced CTGF expression in primary human gingival fibroblasts was examined by immunoblotting. Moreover, the proteins involved in TGF-β1 signaling pathways including TGF-β1 receptors and Smad2 were also analyzed by immunoblotting. CTGF was highly expressed in fibroblasts, epithelial cells and some of endothelial cells, smooth muscle cells, and inflammatory cells in phenytoin-induced gingival overgrowth tissues rather than in those of hereditary and inflammatory gingival overgrowth tissues. Moreover, CTGF expression in the epithelial and connective tissue layers was higher in phenytoin-induced gingival overgrowth tissues than in normal gingival tissues. Curcumin was nontoxic and could reduce TGF-β1-induced CTGF expression by attenuating the phosphorylation and nuclear translocation of Smad2. Curcumin can suppress TGF-β1-induced CTGF expression through the interruption of Smad2 signaling.

The mechanism of phenytoin-induced gingival overgrowth on mitogen-activated protein kinase pathway

The mechanism of phenytoin-induced gingival overgrowth on mitogen-activated protein kinase pathway

Lack of pathogenic mutations in SOS1 gene in phenytoin-induced gingival overgrowth patients

ObjectiveGingival overgrowth is a side effect associated with some distinct classes of drugs, such as anticonvulsants, immunosuppressants, and calcium channel blockers. One of the main drugs associated with gingival overgrowth is the antiepileptic phenytoin, which affects gingival tissues by altering extracellular matrix metabolism. It has been shown that mutation of human SOS1 gene is responsible for a rare hereditary gingival fibromatosis type 1, a benign gingival overgrowth. The aim of the present study is to evaluate the possible contribution of SOS1 mutation to gingival overgrowth-related phenotype. DesignWe selected and screened for mutations a group of 24 epileptic patients who experienced significant gingival overgrowth following phenytoin therapy. Mutation scanning was carried out by denaturing high-performance liquid chromatography analysis of the entire coding region of the SOS1 gene. Novel identified variants were analyzed in-silico by using Alamut Visual mutation interpretation software, and comparison with normal control group was done. ResultsMutation scanning of the entire coding sequence of SOS1 gene identified seven intronic variants and one new exonic substitution (c.138G>A). The seven common intronic variants were not considered to be of pathogenic importance. The exonic substitution c.138G>A was found to be absent in 100 ethnically matched normal control chromosomes, but was not expected to have functional significance based on prediction bioinformatics tools. ConclusionsThis study represents the first mutation analysis of the SOS1 gene in phenytoin-induced gingival overgrowth epileptic patients. Present results suggest that obvious pathogenic mutations in the SOS1 gene do not represent a common mechanism underlying phenytoin-induced gingival overgrowth in epileptic patients; other mechanisms are likely to be involved in the pathogenesis of this drug-induced phenotype.

Phenytoin-induced gingival overgrowth caused by death receptor pathway malfunction.

In this study, we investigated the role of phenytoin (PHT) in death receptor-induced apoptosis of gingival fibroblasts to clarify the mechanism of PHT-induced gingival overgrowth. Human gingival fibroblasts were cultured to semiconfluence and treated with PHT (0.025, 0.1, 0.25, and 1.0μM) for 48h, and then, the apoptotic cell numbers were relatively determined by absorptiometry. After 24h of 0.25μM PHT treatment, caspase activity was measured by absorptiometry, apoptotic and cell cycle phase distribution was analyzed by flow cytometry, expression levels of apoptotic genes were quantified by real-time qPCR, and expression of apoptotic proteins was detected by Western blot analysis. After 48h of 0.25μM PHT treatment, appearance of apoptotic cells was detected by TUNEL assay. PHT treatment decreased the proportion of apoptotic cells in gingival fibroblasts compared to a serum-free control culture in response to the protein changes as follows: PHT upregulated c-FLIP and, in turn, downregulated FADD, caspase-8, and caspase-3; PHT upregulated c-IAP2 and downregulated TRAF2; PHT downregulated caspase-9 and caspase-3 via decreased RIPK1 activity and increased Bcl-2 activity. PHT-induced gingival overgrowth may result from the above-mentioned mechanisms involving apoptosis inhibition in gingival fibroblasts.

Periodontal Management of Phenytoin-Induced Gingival Overgrowth

Phenytoin can induce excessive gingival overgrowth that hampers oral function and compromises effective oral hygiene and aesthetics. A 39-year-old male was referred for dental treatment with several complaints, especially upper and lower gingival overgrowth that hindered speech and swallowing. Generalized deep probing pocket depths and bone loss were detected. A diagnosis of gingival overgrowth, associated with phenytoin use and chronic periodontitis, was established. The treatment plan consisted of conservative management involving dental education, motivation, and meticulous instruction in oral hygiene, together with scaling and root planning. During the re-evaluation, a marked reduction in the clinical parameters was noted. Surgical therapy was also performed in order to reduce the pocket depths. The histological diagnosis was consistent with drug-induced gingival hyperplasia, and supportive periodontal therapy was proposed. At this writing, the patient was undergoing follow-up projected to continue for a total of 4 years. Phenytoin-induced gingival overgrowth can be managed through the use of periodontal procedures and oral hygiene.

Severe Anti-epileptic Drug-induced Gingival Overgrowth in a Physically Disabled Patient.

Anti-epileptic drugs are considered to be the main drugs associated with gingival overgrowth. The co-administration of phenytoin and other anti-epileptic drugs, which increases the risk of phenytoin-induced gingival overgrowth, has been previously reported. However, no report has been done considering the new generation of anti-epileptic drug topiramate and its association with gingival overgrowth. High levels of dental plaque and calculus have also been reported as being a critical risk factor in the development and severity of drug-induced gingival overgrowth. Thus, this case report highlights the occurrence of severe gingival overgrowth and generalized periodontitis in a physically disabled patient with epilepsy who had been taking phenytoin and topiramate drugs for 10 years. It also emphasizes the importance for both medical and dental professionals to reduce the severity and impact of drug-induced gingival overgrowth.

Prevention of Phenytoin-Induced Gingival Overgrowth by Lovastatin in Mice

Drug-induced gingival overgrowth is caused by the antiseizure medication phenytoin, calcium channel blockers, and ciclosporin. Characteristics of these drug-induced gingival overgrowth lesions differ. We evaluate the ability of a mouse model to mimic human phenytoin-induced gingival overgrowth and assess the ability of a drug to prevent its development. Lovastatin was chosen based on previous analyses of tissue-specific regulation of CCN2 production in human gingival fibroblasts and the known roles of CCN2 in promoting fibrosis and epithelial to mesenchymal transition. Data indicate that anterior gingival tissue overgrowth occurred in phenytoin-treated mice based on gross tissue observations and histomorphometry of tissue sections. Molecular markers of epithelial plasticity and fibrosis were regulated by phenytoin in gingival epithelial tissues and in connective tissues similar to that seen in humans. Lovastatin attenuated epithelial gingival tissue growth in phenytoin-treated mice and altered the expressions of markers for epithelial to mesenchymal transition. Data indicate that phenytoin-induced gingival overgrowth in mice mimics molecular aspects of human gingival overgrowth and that lovastatin normalizes the tissue morphology and the expression of the molecular markers studied. Data are consistent with characterization of phenytoin-induced human gingival overgrowth in vivo and in vitro characteristics of cultured human gingival epithelial and connective tissue cells. Findings suggest that statins may serve to prevent or attenuate phenytoin-induced human gingival overgrowth, although specific human studies are required.

What is the Effect of Folic Acid Supplementation on the Incidence or Reduction of PhenytoinInduced Gingival Overgrowth? A Systematic Review

Background: Several published studies have reported both positive and negative effects regarding the administration of folic acid (FA) on either prevention, or reduction of phenytoin (PHT)-induced gingival overgrowth (PIGO). Several other published studies have evaluated the effect of FA supplementation upon gingival health. Objective: The primary objective is a systematic assessment review of studies evaluating the efficacy of FA therapy for the prevention or reduction of PIGO. Secondary objectives entail evaluation of the FA and seizure control, on the mechanism of Drug-IGO, and on FA supplementation and gingival health. Materials and methods: Electronic databases (a Medline via PubMed database, and Cochrane Central Register of Controlled Trials) search was conducted; other sources were searched such as Google Scholar and clinicaltrials.gov. Reference lists of retrieved articles were searched. Randomized placebo-controlled trials (RCTs) assessing the effect of topical or systemic administration of folic acid on gingival enlargement in phenytoin patients were included. Two reviewers (HH, RB) performed the study search, data extraction, and risk of bias assessment. The literature was reviewed regarding PHT-FA interaction, and FA supplementation to promote gingival health. Results: The main research was through Pubmed search, and done on February, 15, 2015. It resulted in eight items. Only five items were considered for screening. The other three items were not interventional. One extra title was found on Google Scholar website. The total RCTs included for the systematic review were six trials. Studies regarding FA and gingival health were also evaluated which produced another four items. Conclusion: FA supplementation may improve the gingival health status, and may delay the onset of PICO, as there are only at present a limited number of studies with a limited number of subjects, further studies are necessary to confirm or deny the positive effects of FA therapy. In PHT-treated epileptic patients properly managed, FA supplementation has a very limited risk in interfering with seizure control. Due to very limited toxicity and the potential for promoting gingival health, future FA supplementation therapy studies should be considered,

Phenytoin-induced gingival overgrowth: A case report

Drug-induced gingival enlargement manifests as an abnormal growth of the gingiva due to an adverse drug reaction in patients treated with anticonvulsants, immunosuppressants and calcium channel blockers. The enlargement affects the normal oral hygiene practice and may even interfere with masticatory functions. It may gradually becomes a source of pain and could lead to disfigurement and cosmetic problem, interfere with mastication and speech, impede effective tooth cleaning or force the teeth out of alignment.It is a serious concern for both the patient and the clinician due to unaesthetic appearance and formation of new niches for Periodonto-pathogenic bacteria. Among the anticonvulsants, Phenytoin has most frequently been described to be associated with Gingival Enlargement. This case report presents gingival hyperplasia as a side effect of Phenytoin use in a 17-year-old female patient on this drug for the last 1 year.

Comprehensive Mutation Analysis of PIK3CA, p14ARF, p16INK4a and p21Waf1/Cip1Genes is Suggestive of a Non- Neoplastic Nature of Phenytoin Induced Gingival Overgrowth

Dilantin sodium (phenytoin) is an antiepileptic drug, which is routinely used to control generalized tonic clonic seizure and partial seizure episodes. A few case reports of oral squamous cell carcinomas arising from regions of phenytoin induced gingival overgrowth (GO), and overexpression of mitogenic factors and p53 have presented this condition as a pathology with potential to transform into malignancy. We recently investigated the genetic status of p53 and H-ras, which are known to be frequently mutated in Indian oral carcinomas in GO tissues and found them to only contain wild type sequences, which suggested a non-neoplastic nature of phenytoin induced GO. However, besides p53 and H-ras, other oncogenes and tumor suppressors such as PIK3CA, p14ARF, p16INK4a and p21Waf1/Cip1, are frequently altered in oral squamous cell carcinoma, and hence are required to be analyzed in phenytoin induced GO tissues to be affirmative of its non-neoplastic nature. 100ng of chromosomal DNA isolated from twenty gingival overgrowth tissues were amplified with primers for exons 9 and 20 of PIK3CA, exons 1α, 1β and 2 of p16INK4a and p14ARF, and exon 2 of p21Waf1/Cip1, in independent reactions. PCR amplicons were subsequently gel purified and eluted products were sequenced. Sequencing analysis of the twenty samples of phenytoin induced gingival growth showed no mutations in the analyzed exons of PIK3CA, p14ARF, p16INK4a and p21Waf1/Cip1. The present data indicate that the mutational alterations of genes, PIK3CA, p14ARF, p16INK4a and p21Waf1/Cip1 that are frequently mutated in oral squamous cell carcinomas are rare in phenytoin induced gingival growth. Thus the findings provide further evidence that phenytoin induced gingival overgrowth as a non-neoplastic lesion, which may be considered as clinically significant given the fact that the epileptic patients are routinely administered with phenytoin for the rest of their lives to control seizure episodes.

Role of nuclear factor kappa-B in phenytoin-induced gingival overgrowth

This study investigates the expression of transcription factor nuclear factor-kappa B (NF-κB) and its relation to various cellular mediators that act in the pathogenesis of phenytoin-induced gingival overgrowth. Eighteen epileptic patients had phenytoin-induced gingival overgrowth (PHT-GO), 20 patients with plaque-induced gingivitis (Gingivitis), and 20 periodontally and systemically healthy individuals (Control) were included in this study. The expression of activated NF-κB subunits (p50 and p65), IL-1β, TNF-α and TGFβ-1 levels were examined in the gingival sections obtained from each participant. The results demonstrated a significantly higher expression of p65 in fibroblasts in PHT-GO group with respect to Gingivitis (P<0.05) and control groups (P<0.01). However, we found no statistically significant differences between PHT-GO and Gingivitis groups according to the immunohistochemical staining in macrophages (P>0.05). Immune-reactive TGFβ-1 levels in the gingival connective tissue cells were statistically higher in PHT-GO group with respect to Gingivitis group(P<0.05). Statistically significant correlations were found between the HI and activated TGFβ-1 and p65 levels in PHT-GO group. The results of this study showed that NF-κB is activated in PHT-related gingival overgrowth. This study may provide a basis for future research into specific NF-κB inhibition for preventing of the side effects of this drug.

Verruciform Xanthoma in a Severe Context of Phenytoin-Induced Gingival Overgrowth: a Case Report

Introduction: Phenytoin was involved in drug-induced gingival overgrowth, with a prevalence rate reaching 50%. In the literature, tumorigenic implication has been discussed with this kind of hyperplasia with many controversies. Since 1971, this is the first report, to our knowledge, to combine phenytoin hyperplasia with the occurrence of verruciform xanthoma; the aim behind it being the need to differentiate this lesion from classical hyperplasia.Case presentation: A 23-year-old man presented with an advanced case of gingival overgrowth due to prolonged intake of phenytoin. Oral examination showed a total coverage of the tooth structure, with one unusual cauliflower-like lesion. A surgical treatment was performed, and a biopsy was taken from the suspected area. Histopathologic examination revealed the diagnosis of verruciform xanthoma. No recurrence of the disease was evidenced after 12 months of therapy.Conclusion: The definitive pathogenesis of these two entities is still discussed, and looking at the l...

Inhibition of G1 cell cycle arrest in human gingival fibroblasts exposed to phenytoin

Gingival overgrowth is caused in response to the antiepileptic drug phenytoin (PHT). PHT-induced gingival overgrowth is characterized by the proliferation of fibroblasts and increased collagen formation in gingiva. Fibroblast proliferation is regulated through the cell cycle. Thus, in the present study, we examined the effects of PHT on the cell cycle, the expression of cell cycle control proteins and the proliferation in human gingival fibroblasts (hGFs). Cells were stimulated in serum-free DMEM with or without 0.25 μm PHT. Subsequently, the cell cycle phase distribution and the protein expression after 24 h and the cell proliferation after 24, 48 and 72 h were evaluated. PHT significantly inhibited synchronization at the G₀/G₁ phase of the cell cycle in hGFs through serum starvation. Stimulation with PHT for 48 and 72 h significantly induced a proliferative response in hGFs. PHT decreased the expression of the Cdk-inhibitory proteins p21 and p27 and increased the levels of the S phase-promoting proteins phospho-Thr160-Cdk2 and phospho-Ser807/811-Rb in serum-free DMEM. The inhibition of G₁ cell cycle arrest in hGFs may result from an increase in phosphorylated Cdk2 and Rb proteins and decreased levels of p21 and p27 proteins by PHT. The gingival overgrowth may be caused by the failure of the G1 cell cycle arrest in GFs exposed to PHT.

Functional Aesthetic Treatment of Patient With Phenytoin-Induced Gingival Overgrowth

Gingival overgrowth (GO) may be related to the frequent use of certain medications, such as cyclosporin, phenytoin (PHT), and nifedipine, and is therefore denominated drug-induced GO. This article reports a case of a patient who with chronic periodontitis made use of PHT and presented generalized GO. A 30-year-old man with GO was referred to the clinic of the Universidade Estadual Paulista, Brazil. The complaint was poor aesthetics because of the GO. The patient had a medical history of a controlled epileptic state, and PHT was administered as an anticonvulsant medication. The clinical examination showed generalized edematous gingival tissues and presence of bacterial plaque and calculus on the surfaces of the teeth. The diagnosis was GO associated with PHT because no other risk factors were identified. Treatment consisted of meticulous oral hygiene instruction, scaling, root surface instrumentation, prophylaxis, and daily chlorhexidine mouth rinses. After this stage, periodontal surgery was performed, and histopathologic evaluation was made. The patient has been under control for 3 years after the periodontal surgery, and up to the present time, there has been no recurrence. It can be concluded that PHT associated with the presence of irritants favored gingival growth and that the association of nonsurgical and surgical periodontal therapies was effective in the treatment of GO. Besides, motivating the patient to maintain oral hygiene is a prerequisite for the maintenance of periodontal health.