Background: Gingival Overgrowth (GO) is a well documented and unwanted side effect that occurs mainly as a result of certain antiseizure, phenytoin. The aim of this study was to compare the effect of phenytoin on proliferation and production of IL1<i>β</i> and PGE2 in cultured human gingival fibroblasts (HGF) of children and adults. Materials and Methods: Normal HGFs were obtained from 4 healthy children and 4 adult and then were cultured with phenytoin (20 mg/ml). MTT test was used to evaluate the proliferation and ELISA to determine the level of IL1<i>β</i> and PGE2 production by HGFs. Analysis of proliferation were assessed by Independent T-Test and ANOVA analysis was used to assess the level of IL1<i>β</i> and PGE2 production with an a error level less than 0.05. Results: The proliferation of HGF was not affected significantly by phenytoin in both cultured fibroblast sources (P > 0.05). Phenytoin induced a significantly higher formation of IL1<i>β</i> and PGE2 in child’s HGFs as compared to adult’s HGFs (P < 0.05). Conclusion: The results suggest that different inflammatory responses and cytokine formation by child’s and adult’s HGFs are the probable key elements that cause different reactions of phenytoin therapy. More advanced and systematic studies are needed to verify these findings.
Medication-induced gingival overgrowth (GO) is a common subsequent of consuming anti-epileptic drug, im- munosuppressive drug, and calcium-channel-blockers [
Several mechanisms such as functional heterogeneity of the human gingival fibroblast, [
Brunius et al. have showed that phenytoin increases the production of interleukin-1β (IL-1β), and Prostaglan- din E2 (PGE2) which has been induced by TNF-α; and have suggested that PGE2 and IL-1β play a key role in pathogenesis of phenytoin-induced gingival overgrowth [
There is little known about the reasons for the differences in gingival overgrowth prevalence in pediatrics and adults, and also about the details of phenytoin and inflammatory mediators in these two groups. According to this lack of knowledge, prevalent usage of phenytoin in treatment of central nervous system diseases [
The pediatric samples were obtained from 4 healthy children, aging from 4 to 11 years, during a procedure of the mesiodense or impacted canine teeth whom have been referred due to orthodontic treatments. In addition, adult fibroblast samples were derived from 4 healthy adults who were submitted to crown lengthening surgery. The age range was 35 - 42 years. All of the persons were in good periodontal, oral and systemic health situation without any signs of inflammations in the site of biopsies. Pregnant women, addicted persons, systemic drug us- ers and any person with systemic diseases which might have any effects on periodontium were excluded from the study. Due to natural constraints in primary cell culture, some pediatric samples were lost during in the ex- periment, so that 22 adult samples and 19 pediatric samples were obtained in total. In both cases, a fragment of excess tissue was removed under local anesthesia at the moment of the surgery. Informed consent was obtained from each donor prior to the taking of samples and parent of each child was informed about the study and con- sent forms were signed by them. Experimental protocol was approved by the Ethics Committee in Shahid Be- heshti Medical University. The tissues were rinsed three times in sterile normal saline solution and transported in complete media including: Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco USA) supplemented with 10% fetal bovine serum (FBS; Gibco USA), 100 μg/ml streptomycin, 100 U/ml penicillin and 0.25 μg/ml Am- photericin B. Minced pieces of the tissue were explanted to 4 cm2 plates, and incubated at room temperature for 10 - 15 minutes and then the culture plate was flooded with complete media. Then samples were incubated at 37˚C in a humidified incubator containing 5% CO2. Samples were regularly controlled for contamination and cell growth, and were fed with fresh medium if necessary. When fibroblasts grew out from the explants, they were trypsinized and shifted to 25 cm 2 flasks (Nunc, Copenhagen, Denmark) for secondary culture. Fourth pas- sages of the cells were used for the experiments [
Gingival fibroblasts were seeded into 24-well plates (Nunc, Copenhagen, Denmark) at a density of 60 × 103 cells/well. For every individual fibroblast derived samples 6 wells were cultured but due to natural constraints in primary cell culture, several pediatric samples were lost during in the experiment, so that 22 adult samples and 19 pediatric samples were obtained in total. Wells were divided into control and experimental groups (3 wells for each group or triplicate). After 48 hours, the media of cell cultures was exchanged and Phenytoin (Sigma- Aldrich, St. Louis, MO, USA) (20 μg/ml) was added to the experimental wells and in control groups only com- plete media was added. Samples were then incubated at 37˚C in 95% humidified atmosphere containing CO2 for 48 hours.
In order to assess the amount of inflammatory mediators produced by gingival fibroblasts, supernatant fluid of each control and experimental well was collected. The concentration of PGE2, IL-1β was determined by En- zyme-Linked Immunosorbent assay (ELISA) using the ELISA kits [PGE2 (R & D systems, Minneapolis, MN, USA Cat No. KGE004B)]. IL-1β (R & D systems, Minneapolis, MN, USA Cat No. DLB50). ELISA assay was performed according to the manufacturer’s instructions. Sample absorbances were analyzed using an ELISA reader for IL-1β at 450 nm and PGE2 at 405, and the concentration of each sample determined by comparing with a standard related curve.
Gingival fibroblasts were seeded into 96-well plates (Nunc, Copenhagen, Denmark) at a density of 5 × 103 cells/ well, and were cultured in a 200 μl medium. For every individual fibroblast derived samples 6 wells were cul- tured. After 48-hour incubation, the samples were divided into control and experimental groups (3 wells for each group or triplicate). Phenytoin was added to the experimental wells. MTT stock solution [tetrazolium salt 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Merk, Darmstadt, Germany)] was prepared in phosphate-buffer saline (PBS; Sigma-Aldrich, St. Louis, MO, USA) in proportion to 5 mg/ml. After 48 hours of incubation, the medium was replaced with 200 μl of a fresh medium containing a final concentration of MTT salt as 0.5 mg/ml. Cells were then incubated at 37˚C in 95% humidified atmosphere containing CO2 for 4 hours. Acidic Isopropanol ( 0.04 M Hal in absolute Isopropanol) was used to dissolve the crystals of formazan produced by the living cells. The quantity of color related to dissolve formazan crystals was then measured at a wave- length of 570 nm with ELISA plate reader (Anthos 2020 Australia). Optical density is directly proportional to the number of living cells.
Data which has been released by ELISA were then tested by Kolmogrov Simonov test to evaluate normal dis- tribution of adult and pediatric gingival cell population. (Mean value of triplicate was calculated and every indi- vidual experiment and related control data were compared). ANOVA was used for statistical evaluation. Results between the two groups were compared by student’s T-test. p value < 0.05 was considered statistically signifi- cant.
Rate of fibroblast proliferation in both adults and pediatrics groups increased in the presence of phenytoin. How- ever, these changes did not reach a significant level. The difference between phenytoin-induced proliferation in adults group and in pediatrics group was not significant (Graph 1).
Phenytoin did not cause a significant raise in synthesis of IL-1β in adult fibroblasts. In contrast, the pheny- toin-induced synthesis of IL-1β significantly increased in pediatric fibroblasts (p < 0.05). The change in produc- tion of IL-1β caused by presence of phenytoin was significantly higher in pediatrics than that in adults group (p < 0.05) (Graph 2).
In the adults group, the level of synthesized PGE2 decreased in presence of phenytoin, but this reduction did not reach a significant level. The production of PGE 2 in pediatrics group significantly increased (p < 0.05). The change in levels of synthesized PGE 2 in pediatrics was significantly higher than that in adults (p < 0.05) (Graph 3).
In this study, no significant increase was perceived in the rate of proliferation of pediatrics and adults fibroblasts after exposure to phenytoin. Also, the rate of proliferation was not significantly different between adults and pe- diatrics. In agreement with our results, Hassel et al. [
Graph 1. Rate of proliferation (mean + standard deviation) in adult and pediatric fibroblasts in both control group and phenytoin-treated group.
Graph 2. The expression of released IL-1β (mean + standard deviation) in adult and pediatric fibroblasts in both control group and phenytoin-treated group.
Graph 3. The expression of released PGE2 (mean + standard deviation) in adult and pediatric fibroblasts in both control group and phenytoin-treated group.
Phenytoin stimulated pediatrics fibroblasts to produce more levels of IL1β. However, in the adults group, no significant change was perceived. The study of Modeer et al. [
In this study, after exposure to phenytoin, pediatric fibroblasts produced higher levels of PGE2; while in adults group, the PGE2 production decreased. Levels of produced PGE2 by treated pediatric fibroblasts were sig- nificantly higher than those by treated adult’s fibroblasts. These results are consistent with Modeer et al., who have shown the increase in PGE2 production of treated fibroblasts in both in vitro [
It seems that several mechanisms such as increased inflammatory activities and altered immunological pro- cesses and disturbed homeostasis of cytokines, as well as secretion of inflammatory mediators as TNF-α, PGE2; and IL-1β, could affect the gingival fibroblasts and cause some disturbances in ECM homeostasis, in cell proli- feration, and in homeostasis of connective tissue proteins. Studies have reported that phenytoin causes distur- bance in cell population, which subsequently results in alternations in production of growth factors and receptors such as EGF and PDGF, and stimulates production of IL-1β and PGE2. These phenomena, in turn, causes gin- gival overgrowth [
IL-1β, a member of pleiotropic cytokines, is known to be influent on immunological processes, fibroblast’s growth, and inflammatory lesions, and to be capable of stimulating collagenase and prostaglandins such as hya- luronic acid [
These results support the hypothesis that IL-1β plays an important role in regulating ECM in gingival tissue. However, the reports on this issue are inconsistent, e.g. Gonzales et al. have shown that phenytoin raises the in- hibitive effect of IL-1β [
PGE2 is another inflammatory mediator that takes part in regulating the production of collagen and in ECM turn over. Yucel et al. [
Recent studies have proposed different processes for drug-induced inflammatory trends. For example, one possible explanation is the role of pathogen-sensitive sensors such as Toll-like receptors (TLR) [
According to our literature review, this study compared the effect of phenytoin on fibroblasts is investigated through two age groups for the first time. According to epidemiological studies, age is a determinant factor in the occurrence of gingival overgrowth. Doufexi et al. [
Regarding to our results, the levels of two important inflammatory mediators was increased in pediatric group, and no significant difference was perceived in the cell proliferation. These results suggest that more prevalence of gingival overgrowth in pediatrics in comparison with adults might be attributed to the different immunologi- cal trends in pediatrics, as well as levels and responses of inflammatory cytokines. Unlike this study, the study of Sooriyamorthy did not resulted in a relationship between age and clinical features, a phenomenon which was attributed to the unique phonotype of fibroblasts or to androgynous metabolism influences. Monolayer cultures from gingival fibroblasts can clearly metabolize labeled testosterone into its active metabolite, 5α-dihydrotes- tosteron. Levels of this metabolite increases in presence of phenytoin [
Dental plaque is another factor in phenytoin- and cyclosporine-induced gingival overgrowth especially in younger patients. Studies have suggested that the prevalence and intensity of gingival overgrowth is positively correlated with dental plaque [
Bonding of plasma proteins is another probable cause of difference in overgrowth of gingiva in pediatrics and adults. There is neither linear relationship between the duration nor intensity of treatment and the levels of plasma proteins. Reduction in the levels of these proteins and their bonding to phenytoin causes the drug to dis- perse in the tissues, especially in the tissues with high affinity to phenytoin, e.g. gingival fibroblast tissues [
The discussed reasons for different response of gingival fibroblasts to phenytoin in pediatrics and adults are briefly demonstrated in
The present study is the only research that has compared the effects of phenytoin on gingival fibroblasts in children and adults, however, further studies are necessary to confirm the discussed results. Unlike other studies,
this study was designed to investigate different aspects of drug influence. This study was conducted without cell lines genetic manipulations or retentive materials, features that are potential advantages of this study.
The different results of variant studies could be attributed to different sources of fibroblasts, the location of biopsies, age of patients or small population of samples. The natural heterogeneity of fibroblasts in response to phenytoin might be another reason for such differences. In order to prevent such issues in this study, those cul- tures, among cell lines extracted from different patients, were chosen that had shown rather similar responses to phenytoin, and the coefficient of correlation among different wells was calculated at the end [
No significant difference was perceived between proliferations of these two groups. The significant increase in production of IL-1β and PGE 2 in pediatrics as compared to adults might be attributed to genetic differences and to conditional factors such as gingival plaque and preexisting gingival inflammation.
There is no conflict of interests. Authors would like to thank research council of dental school, Shahid Beheshti University of Medical sciences for its financial support of this study.