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Home , Calcium channel blocker, Nisoldipine

261

Journal of Oral Science, Vol. 43, No. 4, 261-268, 2001

Comparative study of -channel blockers on cell

proliferation, DNA and collagen syntheses, and EGF receptors of cultured gingival fibroblasts derived from human

, and responders

Hiroko Matsumoto•˜, Ichinari Noji•˜, Yoshiaki Akimoto•õ and Akira Fujii•˜

§Department of and †Second Department of Oral Surgery, Nihon University School of Dentistry at Matsudo, Chiba 271-8587

(Received 6 September and accepted 4 December 2001)

Abstract: Our previous study indicated that Key words: calcium-channel blockers; gingival fibroblasts derived from patients reactive to nifedipine overgrowth; human gingival fibroblasts might be susceptible to the other calcium-channel (HGF). blockers (nicardipine, and ) in terms of cell proliferation, DNA synthesis, and collagen synthesis. Thus, the present investigation was designed Introduction to clarify the cross-reactivity among dihydropyridine Many case reports have implicated nifedipine, one of calcium-channel blockers (nifedipine, nicardipine, and the dihydropyridine calcium-channel blockers, as a cause nisoldipine). Human gingival fibroblasts derived from of gingival overgrowth (first reported by Ramon et al. (1) seven, two, and one patients who developed gingival and Lederman et al. (2)). The incidence of gingival overgrowth as a result of nifedipine, nicardipine, and overgrowth due to nifedipine has been reported to be 6.5% nisoldipine , respectively, were examined (3), more than 10% (4), 13.7% (5), 15% (6), and 20% (7). in terms of the effect of calcium-channel blockers This unwanted has also been reported in patients (nifedipine, diltiazem, verapamil, and nicardipine) on taking three other calcium-channel blockers; diltiazem cell proliferation, DNA synthesis, collagen synthesis, (8-10), verapamil (11-13), and nicardipine (5,14). We and the number of epidermal growth factor (EGF) have recently found a patient who developed gingival receptors. was used as a positive control. With overgrowth as a result of nisoldipine . Excellent most of the calcium-channel blockers and phenytoin, reviews on -induced gingival overgrowth are available fibroblasts from patients reactive to nifedipine and (15-17). Nifedipine is increasingly used in the treatment nicardipine medications gave a better cell proliferation of pectoris and and is the most rate, DNA synthesis, and an increased number of EGF frequently cited causal agent in calcium-- receptors, compared to non-drug-treated control. induced gingival overgrowth (4). However, this was not the case for calcium-channel We have previously demonstrated that the fibroblasts blockers tested in fibroblasts from patients reactive to from patients reactive to nifedipine gave trends toward better nisoldipine medication. (J. Oral Sci. 43, 261-268, 2001) cell proliferation rates, DNA synthesis, and collagen synthesis than those from non-reactive patients in the

Correspondence to Dr. Akira Fujii, Department of presence of 1 ƒÊM of calcium-channel blockers (nifedipine, Pharmacology, Nihon University School of Dentistry at diltiazem, nicardipine, and verapamil) or phenytoin (17). Matsudo, 2-870-1 Sakaecho-Nishi, Matsudo, Chiba 271-8587, Therefore, it is possible that fibroblasts from reactive Japan patients may be also susceptible to the other calcium- Tel: +81-47-360-9344 channel blockers, which indicates that those patients who Fax: +81-47-360-9348 E-mail address: [email protected] developed gingival overgrowth because of nifedipine 262

medication may also develop it in response to other Ltd., Japan). The fibroblasts used for experiments calcium-channel blockers. In order to clarify this cross- proliferated in the logarithmic phase between the 5th and susceptibility, we have isolated gingival fibroblasts from the 8th passage. patients reactive to nifedipine (seven strains), nicardipine (two strains), and nisoldipine (one strain), and tested the Cell-proliferation assay effect of calcium-channel blockers (nifedipine, diltiazem, The cell-proliferation assay was carried out by a nicardipine, and verapamil) and phenytoin on cell previously reported method (17). All ten strains of proliferation rate, DNA synthesis, collagen synthesis, and fibroblasts were subjected to the experiment in the following the number and Kd value of epidermal growth factor manner: Fibroblasts (approximately 3•~104) in 500 ƒÊl of

(EGF) receptors in those gingival fibroblasts. Dulbecco's modified Eagle media (DMEM) supplemented

with 10% fetal calf serum (FCS) and agents

Materials and Methods (streptomycin 100 ƒÊg/ml, penicillin G 100 U/ml, and Cells amphotericin B 0.2 ƒÊgml, DMEM-10) were allowed to

Cultures of fibroblast-like cells were established from settle in a 24-well plate (Falcon tissue culture plate 3047) gingival specimens of seven, two, and one patients who for 24 h. Cells were washed and the medium was replaced develop gingival overgrowth as a result of nifedipine , with DMEM containing 1% FCS and the same anti- nicardipine, and nisoldipine medications , respectively microbial agents as above (DMEM-1) for 24 h. Cells were (Table 1). All the specimens were obtained during again washed, a fresh 500 ƒÊ1 of DMEM-1 poured and 20 gingivectomy, clearance of remaining teeth, or orthopedic ul of 25 ƒÊM calcium-channel blockers (nifedipine, surgery of the alveolar ridge from the patient who gave diltiazem, verapamil, nicardipine; Sigma Chemical Co ., informed consent, under a protocol approved by the St. Louis, MO, U.S.A.) and phenytoin (Sigma Chemical

Committee on Studies involving Human Beings of Nihon Co., St. Louis, MO, U.S.A.) were added to make the final University School of Dentistry at Matsudo. Disappearance concentration of 1ƒÊM in 4 wells each and kept for 48 h . or decreased severity of the overgrowth after discontinuance Our previous dose-response experiment revealed that 1 ƒÊM of medication was regarded as indicating a responsive of nifedipine gave the best growth in fibroblasts from state. Isolation and culture of gingival fibroblasts were nifedipine-reactive patients (17). Cells were then washed performed by the methods described previously (17,18). again and treated in the same manner as above except that Homogeneity of fibroblasts was determined by flow DMEM-10 was used.Cells were harvested using 0 .25% cytometry (FACS Vantage, Nippon Becton Dickinson Co. trypsin and 0.02% EDTA in Dulbecco's phosphate buffered

Table 1 Source of cell strains 263

saline (DPBS) and the population count was performed ml centrifuge tube and then 1.5 ml of 10% TCA containing using a Coulter Counter ZM (Coulter Electronics, Ltd., 0.4% tannic acid and 0.0576% L-proline was added. The

Luton, England). The number of cells in the absence of tube was centrifuged at 11,000 •~g for 3 min and the served as the control. precipitated residue was washed three times with 750 ƒÊl of the same 10% TCA as above. The precipitates were then

DNA synthesis washed with 750ƒÊl of ethylether:ethanol (1:3, v/v) and the

DNA synthesis was determined using the method residue was air dried and dissolved in 300ƒÊl of 0.3 M NaOH previously reported (17) with a slight modification. at 37°C for 30 min. After neutralization with 300 ƒÊl of 0.3 Specifically, fibroblasts (approximately 6•~103 cells) in M HCl, the solution was divided into three portions (200

100 ƒÊl of DMEM-10 were allowed to settle in a 96-well ul each), the first portion being placed in a scintillation vial, the second portion digested by collagenase, and the third plate (Falcon tissue culture plate 3072) for 24 h. The adherent cells were washed and the medium was replaced portion acting as control for the second portion. Collagenase with DMEM-1 and then 4 ƒÊl of 25 ƒÊM calcium-channel digestion was performed by addition of 50 ƒÊlitube (400 blockers and phenytoin were added in the same manner U/tube) of collagenase (type VII, Sigma Chemical Co., St. as above except in 6 wells each and kept for 48 h. The Louis, MO, U.S.A.) solution dissolved in 1.4 M HEPES medium was changed again with DMEM-1 and calcium- buffer (pH 7.4) for 2 h at 37°C. The second and third channel blockers and phenytoin were added. 3H-thymidine portions were then treated with 650 ƒÊl of 10% TCA, centrifuged at 11,000 •~g for 3 min, and the supernatant (22.2 kBq, Amersham Life Science, Tokyo, Japan) was added to each well 24 h after the last medium change and was placed in the scintillation vial. All three fractions then incorporated into the cells for 24 h. During this pulse- were measured by liquid scintillation counting using label period, cells were in the S-phase stage and Ultima Gold (Packard Japan, Tokyo, Japan) as a scintillator. The radioactivities of the ones with non-drug treatment proliferation was very low because the medium (DMEM- 1) contained only 1% of FCS. After the treatment using served as the control.

0.25% trypsin and 0.02% EDTA in DPBS, cells were harvested with a cell collector (Micromate 196, Packard EGF assay

Japan, Tokyo, Japan), and the resulting radioactivities on EGF receptor assay was performed by the method the disc were measured using a direct beta counter (Matrix reported by King and Cuatrecasas (19) and Modeer et al.

96, Packard Japan, Tokyo, Japan). Radioactivity of the cell (20,21) with a slight modification. Specifically, fibroblasts in the absence of drug served as a control. (approximately 3•~104 cells) in 1.5 ml of DMEM-10 were allowed to settle in a 6-well plate (Falcon tissue culture

Collagen synthesis plate 3046) for 24 h. The adherent cells were washed and

Collagen synthesis was determined by the method the medium was replaced with DMEM-1 and then 60 ƒÊl described previously (17) with a slight modification. of 25ƒÊM calcium-channel blockers and phenytoin were

Specifically, fibroblasts (approximately 3•~104 cells) in added in the same manner as above in 3 wells each and

500 ƒÊl of DMEM-10 were allowed to settle in a 24-well kept for 24 h. The medium was discarded and the monolayer fibroblasts in a 6-well culture plate were washed 3 times plate (Falcon tissue culture plate 3047) for 24 h. The adherent cells were washed and the medium was replaced with 500 ƒÊl each of cold DPBS, and then replaced with cold Hank's solution (400ƒÊl) substituted with 0.1% bovine with DMEM-1 and then 20 ƒÊl of 25ƒÊM calcium-channel blockers and phenytoin were added in the same manner serum albumin. Calcium-channel blockers and phenytoin as above in 4 wells each and kept for 48 h. The medium were added to the solution to make the final concentration was changed again with DMEM-1 and calcium-channel of 1 ƒÊM, and then different concentration of unlabeled EGF blockers and phenytoin were added in the same manner (0-1250 ng/ml in cold DPBS) were added into the well and as above. 3H-proline (92.5 kBq, Amersham Life Science, incubated for 10 min at 4°C. Next, radioiodinated EGF (125I-

Tokyo, Japan) was added to each well 24 h after the last EGF; 1.8 kBq) was added into the well, and incubated for medium change and then incorporated into the cell for 24 a further 3h at 4•Ž. Following the incubation, the well was h. During this pulse-label period, cells were in the S- washed 3 times with cold DPBS and then dissolved into 500 ƒÊl of 0.5 M NaOH. The radioactivity was determined phase stage and proliferation was very low because the medium (DMEM-1) contained only 1% FCS. Cells were by gamma counting (COBRA, Packard Japan, Tokyo, washed three times with 500ƒÊl/well of cold DPBS and then Japan). The experiment was performed three times and the dissolved into 150 ƒÊl/well of 0.3 M NaOH at 37•Ž for 1 data are the mean of three trials. Bmax (Bmax1 and Bmax2) h. The solution from two wells was transferred into a 2 and Kd (Kd1 and Kd2) values were obtained in the Scatchard 264

analysis. Data are expressed as relative Bmax1 and Bmax2 and Kd1 and Kd2 for high affinity and low affinity receptors, respectively, compared to non-drug-treated control.

Statistical methodology Statistical analyses on the data for fibroblasts obtained from nifedipine (seven strains) reactive patients on the means of 3-5 multiple experiments were carried out by the multiple analysis of variance (multiple ANOVA) and the significance was established on the basis of the Newman- Keuls test at P < 0.05.

Results Fig. 1 The effect of calcium-channel blockers and phenytoin

Homogeneity of fibroblasts on cell proliferation of 7 strains of fibroblasts from Each group of cell-strains used in the present experiment nifedipine-reactive patients, 2 strains from nicardipine- were subjected to flow cytometry and were each found to reactive patients, and 1 strain from a nisoldipine- give homogeneous samples in terms of the appearance of reactive patient. Cells were grown 48 h in DMEM-10 SC-F (difference of size and characteristics of cell surface) in the presence of 1 ƒÊM of calcium-channel blockers and SC-S (difference of inner substance) . or phenytoin. Data are presented as the mean ratio to non-drug-treated controls. NIF, nifedipine; DIL,

Cell proliferation diltiazem; VER, verapamil; NIC, nicardipine; PHT , The effect of calcium-channel blockers and phenytoin phenytoin.•¡, Fibroblasts derived from nifedipine- reactive patients; •¬, Fibroblasts derived from on relative growth rates (number of cells in experiment nicardipine-reactive patients; • , Fibroblasts derived /number of cells in non-drug-treated control) in nifedipine, from a nisoldipine-reactive patient. nicardipine, and nisoldipine responders are summarized in Fig. 1. With all calcium-channel blockers and phenytoin, fibroblasts from subjects reactive to nifedipine showed better growth than ones reactive to nicardipine and nisoldipine. Fibroblasts from subjects reactive to nicardipine showed better growth by nifedipine and those reactive to nisoldipine by nicardipine and phenytoin . All fibroblasts from subjects reactive to calcium-channel blockers showed better growth with all calcium-channel blockers and phenytoin than non-drug-treated controls (the relative growth rate was greater than 1). No statistical difference was found among the calcium-channel blockers in the subjects reactive to nifedipine. Fig. 2 The effect of calcium-channel blockers and phenytoin DNA synthesis on DNA synthesis in monolayer cultures of 7 strains The effect of calcium-channel blockers and phenytoin of fibroblasts from nifedipine-reactive patients , 2 strains from nicardipine-reactive patients on relative [3H]-thymidine incorporation rates (radioactivity , and 1 strain from a nisoldipine-reactive patient. Cells were kept in in experiment/radioactivity in non-drug-treated control) are DMEM-1 and pulse-labeled with [31-1]-thymidine for summarized in Fig. 2. With all calcium-channel blockers 24 h in the presence of 1ƒÊ M of calcium-channel and phenytoin, except for nicardipine in fibroblasts from blockers or phenytoin. Data are presented as the mean nifedipine-reactive patients, all fibroblasts from subjects ratio to non-drug-treated control. NIF, Nifedipine; reactive to calcium-channel blockers showed greater [3H]- DIL, diltiazem; VER, verapamil; NIC , nicardipine; thymidine incorporation than non-drug-treated controls (the PHT, phenytoin.•¡, Fibroblasts derived from relative [3H]-thymidine incorporation rate was greater nifedipine-reactive patients; •¬, Fibroblasts derived than 1). No statistical difference was found among the from nicardipine-reactive patients; • ,Fibroblasts calcium-channel blockers in the subjects reactive to derived from a nisoldipine-reactive patient . nifedipine. 265

With most calcium-channel blockers and phenytoin, Collagen synthesis fibroblasts from subjects reactive to nifedipine showed less The effects of calcium-channel blockers and phenytoin collagen synthesis than non-drug-treated controls (the on the relative [31-1]-prolineincorporation rate (radioactivity relative [31-1]-prolineincorporation rate was smaller than in experiment/radioactivity in non-drug-treated control) in 1). Only phenytoin treatment on fibroblasts reactive to the collagenase digestible collagen are shown in Fig. 3. nifedipine and nisoldipine gave greater collagen synthesis. No statistical difference was found among the calcium- channel blockers in the subjects reactive to nifedipine.

Assay for EGF The effects of calcium-channel blockers and phenytoin on relative number of the high affinity EGF receptors (Bmax 1) and the low affinity EGF receptors (Bmax2) (number in experiment/number in non-drug-treated control) are summarized in Fig. 4. With all calcium-channel blockers and phenytoin, fibroblasts from subjects reactive to nifedipine and nicardipine had greater relative numbers of Fig. 3 The effect of calcium-channel blockers and phenytoin Bmaxi (Fig. 4A). Only with verapamil and phenytoin, on collagenase digestible collagen synthesis in fibroblasts from subjects reactive to nisoldipine showed monolayer cultures of 7 strains of fibroblasts from greater relative numbers of Bmax1. In case of Bmax2, nifedipine-reactive patients, 2 strains from nicardipine- fibroblasts from subjects reactive to nifedipine and reactive patients, and 1 strain from a nisoldipine- nicardipine showed greater relative numbers of Bmax2 reactive patient. Cells were kept in DMEM-1 and (Fig. 4B). Only with nifedipine and verapamil, fibroblasts pulse-labeled with [3H]-proline for 24 h in the presence of 1 ƒÊM of calcium-channel blockers or phenytoin. Data from subject reactive to nisoldipine showed a greater

presented as the mean ratio to non-drug-treated control. relative number of Bmax2. Thus, both fibroblasts from NIF, Nifedipine; DIL, diltiazem; VER, verapamil; subjects reactive to nifedipine and nicardipine were affected NIC, nicardipine; PHT, phenytoin.•¡, Fibroblasts to increase the number of Bmax1 and Bmax2 by calcium- derived from nifedipine-reactive patients; •¬, channel blockers and phenytoin. Fibroblasts derived from nicardipine-reactive The effects of calcium-channel blockers and phenytoin patients; • , Fibroblasts derived from a nisoldipine- on relative affinities of high and low affinity EGF receptors reactive patient. (Kd1 and Kd2, respectively) (Kd of experimental/Kd of non-

B A

Fig. 4 The effect of calcium-channel blockers and phenytoin on the number of high affinity EGF receptors (A) and low affinity EGF receptors (B) in the monolayer cultures of 7 strains of fibroblasts from nifedipine-reactive patients, 2 strains from nicardipine-reactive patients, and 1 strain from a nisoldipine-reactive patient. Cells were kept in cold Hank's solution substituted

with 0.1% bovine serum albumin. Calcium-channel blockers and phenytoin were added to the solution to make the final concentration of 1 ƒÊM, and then different concentrations of unlabeled EGF (0-1250 ng/ml in cold 20ƒÊl of DPBS) were

added and incubated for 10 min at 4•Ž. Next, 125I-EGF was added and incubated for a further 3 h at 4 •Ž. Data presented as the mean ratio to non-drug-treated control. NIF, Nifedipine; DIL, diltiazem; VER, verapamil; NIC, nicardipine; PHT,

phenytoin. •¡ Fibroblasts derived from nifedipine-reactive patients; •¬, Fibroblasts derived from nicardipine-reactive patients; • ,Fibroblasts derived from a nisoldipine-reactive patient. 266

drug-treated control) are summarized in Fig. 5. Most of non-reactive patients (17). The same trend was found in relativeKdis in fibroblasts from subjects reactive to fibroblasts from patients reactive to nicardipine medication, nifedipine, nicardipine, and nisoldipine showed smaller but not in fibroblasts from the patient reactive to nisoldipine. relativeKd1, except in the one from subjects reactive to Thus, nifedipine and nicardipine might act in quite a nisoldipine, for nifedipine and nicardipine showed greater similar manner to gingival fibroblasts obtained from relativeKd1 (Fig. 5A). All Kd2s in fibroblasts from subjects patients reactive to nifedipine or nicardipine medication. reactive to nifedipine were greater than those of non-drug- The statistical analysis of the data for fibroblasts obtained treated controls (Fig. 5B). No statistical differences were from the nisoldipine-reactive patient was not performed found among the calcium-channel blockers in the subjects in the present study because of the limitation of number reactive to nifedipine in Bmax and Kd values. of strains available. Nishikawa etal. (23) reported that both responder and Discussion non-responder cells to nifedipine were not significantly This is our first and a rare case of gingival overgrowth sensitive to EGF on DNA synthesis as compared to the found in a patient receiving nisoldipine (22). At present, control cells. We demonstrated in this investigation that only one case was found out of 76 nisoldipine-receiving responder cells to nifedipine and nicardipine gave an patients. Since nifedipine, nicardipine, and nisoldipine increasing number of cell-surface EGF receptors when they are all dihydropyridine calcium-channel blockers (Fig . were treated with calcium-channel blockers and phenytoin 6), it wasofinterest to investigate the influence of calcium- than those of non-drug-treated controls. Although we did channel blockers (nifedipine, diltiazem, verapamil, and not compare fibroblasts from reactive and non-reactive nicardipine) and phenytoin on fibroblasts from patients patients, the treatment with all calcium-channel blockers reactive to nifedipine, nicardipine, and nisoldipine in order or phenytoin enhanced reactivity to EGF in fibroblasts from to find cross reactivity. Fibroblasts from subjects reactive reactive patients. to nifedipine and nicardipine showed almost the same Modeer and Andersson (20) reported that phenytoin trend to calcium-channel blockers and phenytoin in terms regulated EGF receptor in human gingival ofincreased proliferation rate and DNA synthesis. However, fibroblasts by increasing the number of cell-surface EGF fibroblasts from the subject reactive to nisoldipine showed receptors which might contribute to the alteration of a different pattern. As suggested previously, with calcium- gingival connective tissue observed in patients undergoing channel blockers and phenytoin , fibroblasts from patients phenytoin medication.Modeer et al. (21) also reported that reactive to nifedipine medication gave better cell in fibroblasts derived from a patient who developed gingival proliferation rates and DNA synthesis than ones from overgrowth during phenytoin medication (responder) as

A B

Fig. 5 The effect of calcium-channel blockers and phenytoin on affinity of high affinity EGF receptors (Kd1; A) and low affinity EGF receptors (Kd2; B) in monolayer cultures of 7 strains of fibroblasts from nifedipine-reactive patients, 2 strains from nicardipine-reactive patients, and 1 strain from a nisoldipine-reactive patient . Cells were kept in cold Hank's solution substituted with 0.1% bovine serum albumin. Calcium-channel blockers and phenytoin were added to the solution to make the final concentration of 1 ƒÊM, and then different concentrations of unlabeled EGF (0 -1250 ng/ml in cold 20 ƒÊl of DPBS) were added and incubated for 10 min at 4•Ž. Next, 125I-EGF was added and incubated for a further 3 h at 4•Ž . Data presented as the mean ratio to non-drug-treated control . NIF, Nifedipine; DIL, diltiazem; VER, verapamil; NIC , nicardipine; PHT, phenytoin.•¡, Fibroblasts derived from nifedipine-reactive patients; •¬, Fibroblasts derived from nicardipine-reactive

patients; • , Fibroblasts derived from a nisoldipine-reactive patient. 267 well as in the fibroblasts derived from a patient where receptors on negative cooperativity. Although the nature gingival overgrowth did not develop (non-responder), the of EGF receptor was not clarified in the present study, the affinity of the EGF receptor for EGF was not significantly further study might be necessary to clarify it. These findings changed. However, phenytoin medication resulted in a suggest that fibroblasts obtained from subjects reactive to down-regulation of EGF receptor metabolism in fibroblasts a specific calcium-channel blocker showed cross-reactivity derived from a responder patient, whereas in the non- to the other calcium-channel blockers. Thus, care should responder patient EGF receptor metabolism was up- be taken when one changes from one calcium-channel regulated. In case of nifedipine and nicardipine, the number blocker to another. of EGF receptors was increased in fibroblasts from patients reactive to nifedipine and nicardipine with calcium-channel Acknowledgments blockers and phenytoin. The present data support the This investigation was supported in part by a Grant-in- former explanation that suggests an increase in EGF Aid for Scientific Research C, 09671908, from the Ministry receptors. Since the relative Bmax was generally greater of Education, Science, and Culture, Japan, a Research than 1 (the ratio of drug treated/non-drug-treated was Grant, C-90-21, by a Suzuki Grant for Joint Research, 1995, greater than 1) in the present study, it was not likely that from Nihon University School of Dentistry at Matsudo, calcium-channel blockers cause down-regulation in and by a Grant, 1994, from The Tsuchiya Foundation, fibroblasts obtained from reactive patients. The relative Kdis Matsudo, Japan. in fibroblasts from subjects reactive to nifedipine, nicardipine and nisoldipine were generally smaller then References those of non-drug-treated controls. On the contrary, Kd2s 1. Ramon, Y., Behar, S., Kishon, Y. and Engelberg, I.S. in fibroblasts from subjects reactive to nifedipine were (1984) Gingival hyperplasia caused by nifedipine greater than those of non-drug-treated controls. This - A preliminary report . Int. J. Cardiol. 5, 195-204 indicates the presence of two different kinds of EGF 2. Lederman, D., Lumerman, H., Reuben, S. and Freedman, P.D. (1984) Gingival hyperplasia associated with nifedipine therapy. Report of a case. Oral Surg. Oral Med. Oral Pathol. 57, 620-622 3. Katsumi, Y., Takahara, M., Watanabe, Y., Muto, T., Atsuta, F., Tsuchiya, H., Hanazawa, Y., Takahashi, K., Uchiyama, S., Kanazawa, H. and Sato, K. (1991) Statistical study of incidence of gingival hyperplasia induced by hypotensive drugs (Ca channel blockers). J. Jpn. Stomatol. Soc. 40, 169-178 (in Japanese) 4. Seymour, R.A. (1991) blockers and gingival overgrowth. Br. Dent. J. 170, 376-379 5. Akimoto, Y., Uda, A., Omata, H., Shibutani, J., Nishimura, H., Kaneko, K., Kawana, M., Hashimoto, T., Matsumoto, H., Fujii, A., Kaneda, T. and Yamamoto, H. (1995) Gingival hyperplasia caused by nicardipine. Jpn. J. Clin. Pharmacol. Ther. 26, 211-212 (in Japanese) 6. Barak, S., Engelberg, I.S. and Hiss, J. (1987) Gingival hyperplasia caused by nifedipine. Histopathologic findings. J. Periodontol. 58, 639-642 7. Barclay, S., Thomason, J.M., Idle, J.R. and Seymour, R.A. (1992) The incidence and severity of nifedipine- induced gingival overgrowth. J. Clin. Periodontol. 19, 311-314 8. Colvard, M.D., Bishop, J., Weissman, D. and Gargiulo, A.V. (1986) Cardizem induced gingival hyperplasia: a report of two cases. Periodontal. Case Fig. 6 Structure of nifedipine and related compounds. Rep. 8, 67-68 268

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