147
Binding, Uptake, and Release of Nicotine by Human Gingival Fibroblasts Philip J. Hanes, George S. Schuster,f and Scott Lubas1 *
fibroblasts have reported an altered and attachment of fibroblasts to substrates and disturbances in protein synmorphology and thesis secretion. This altered functional and attachment response may be associated with changes in the cell membrane resulting from binding of the nicotine, or to disturbances in cell metabolism as a result of high intracellular levels of nicotine. The purpose of the present study, therefore, was to 1) determine whether gingival fibroblasts bound nicotine and if any binding observed was specific or non-specific in nature; 2) determine whether gingival fibroblasts internalized nicotine, and if so, at what rate; 3) determine whether gingival fibroblasts also released nicotine back into the extracellular environment; and 4) if gingival fibroblasts release nicotine intact or as a metabolite. Cultures of gingival fibroblasts were prepared from gingival connective tissue biopsies. Binding was evaluated at 4° C using a mixture of 3H-nicotine and unlabeled nicotine. Specific binding was calculated as the difference between 3H-nicotine bound in the presence and absence of unlabeled nicotine. The cells bound 1.44 (± 0.42) pmols/106 cells in the presence of unlabeled nicotine and 1.66 (± 0.55) pmols/106 cells in the absence of unlabeled nicotine. The difference was not significant. Uptake of nicotine was measured at 37° C after treating cells with 3H-nicotine for time periods up to 4 hours. Uptake in pmols/106 cells was 4.90 (± 0.34) at 15 minutes, 8.30 (± 0.75) at 30 minutes, 12.28 (± 2.62) at 1 hour and 26.31 (± 1.15) at 4 hours. The efflux or release of nicotine was evaluated after treating cells with 3H-nicotine for 4 hours and then measuring the amount of 3H-nicotine released into fresh non-radioactive medium over a 2-hour period by direct scintillation counting of aliquots of medium and by Chromatographie analysis of medium samples. Cells released nicotine back into the medium at a rate much slower than the rate of uptake, and the majority of nicotine released by the cells over the 2-hour period was in the form of nicotine rather than a metabolite. At earlier time points, a greater percentage of the 3H isotope separated chromatographically before 3H-nicotine, suggesting an association of the nicotine with intracellular vesicles or other intracellular components. The results of this study suggest that, although nicotine does bind to gingival fibroblasts, this binding is non-specific, and that the uptake of nicotine by these cells is continuous over 4 hours, providing high intracellular levels. In addition, gingival fibroblasts also release nicotine, apparently unmetabolized, back into the medium. However, an apparent association of the nicotine with intracellular components seems to result in the development of high intracellular levels of nicotine which may interfere with normal cellular functions. J Periodontol 1991; 62:147-152. Previous
studies of the effects of nicotine on
Key Words: Nicotine/adverse effects; fibroblasts; cell culture.
relationship between tobacco use and the prevalence severity of periodontal disease has long been debated. Although epidemiological studies have reported essentially The and
Department of Periodontics, School of Dentistry, Georgia, Augusta, GA. department of Oral Biology/Microbiology.
Medical
College
of
equal levels of periodontal disease in tobacco users and non-users,1-2 some investigations have described a greater prevalence of periodontal inflammation in smokers.3-5 Clinical observations and some epidemiological studies suggest that this positive correlation between smoking and peri-
odontal disease may be related to a greater accumulation of plaque and calculus in patients who smoke.6"9 However,
148
J Periodontol 1991
BINDING AND UPTAKE OF NICOTINE BY FIBROBLASTS
February
studies by Bastiaan and Waite10 and Feldmen et al." suggest that patients who smoke have less plaque accumulation than do non-smokers. Tobacco use, particularly smoking, has been strongly associated with an increased risk of cancer12-'6 and cardiovascular disease.12-17-18 These health risks may be the result of a tobacco associated impairment of normal immunological surveillance or defense mechanisms such as neutrophil and macrophage function,18-22 T-cell proliferation,23-24 and/ or the cellular immune response.23-25 The aforementioned disturbances in immune function reportedly associated with tobacco use could also contribute to the development and progression of plaque-induced chronic inflammatory periodontal disease. Many of the undesirable effects of tobacco have been attributed to nicotine, a major component of the particulate phase of tobacco smoke. Nicotine has been shown to induce vascular changes in gingival tissue26-27 similar to the exudative vasculitis described as characteristic of the initial lesion of periodontal inflammation.28 In in vitro systems, nicotine also suppresses DNA synthesis in lymphocytes and HeLa cells.24-29 As with other tissue responses, normal fibroblast function is critical for the maintenance of periodontal tissues and for optimal wound healing responses, and nicotine does have some effects on various fibroblast functions. For example, collagen production is apparently stimulated by nicotine; its secretion, however, is impaired.30 In addition, the growth and attachment of fibroblasts to substrates is impaired by nicotine.31 These effects could be related to the binding of nicotine to the cell membrane which could then interfere with membrane functions such as cell attachment and transport of materials between the intracellular and extracellular environments. Alternatively, the uptake of nicotine by the fibroblast may result in an interference with cell metabolic processes. In order to better define the effects of nicotine on the periodontal tissues, the present study evaluated the binding, uptake, and release of nicotine by gingival fibroblasts and whether the binding observed was specific or non-specific in nature.
successive washes with 0.1% EDTA and 0.25% trypsin. The cells were then transferred to 75 cm2 culture flasks and refed fresh medium. Cells were then incubated at 37° C under an atmosphere of 95% air/5% C02 until cultures
MATERIALS AND METHODS
nicotine.32-34
Cultures of Gingival Fibroblasts Primary cultures of gingival fibroblasts were obtained from gingival biopsies obtained during routine periodontal surgical procedures in the Department of Periodontics at the Medical College of Georgia School of Dentistry. Biopsies were obtained from adult patients, both male and female, with an original diagnosis of adult Periodontitis. Explants of gingival connective tissue were placed in culture dishes containing Eagles minimal essential medium (EMEM) supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, and 20% (v/v) fetal bovine serum. When cultures became confluent (7 to 10 days), the tissue expiant was discarded and the cells detached from the culture dish by
Uptake of Nicotine
reached
confluency.
Binding of Nicotine Cultures of gingival fibroblasts (passage 4-7) pooled from four patients were washed three times with phosphate buffered saline (PBS) (pH 7.2), harvested by scraping, and suspended in PBS. They were then centrifuged (1000 x g for 20 minutes), washed a second time in PBS, then resuspended in 4.0 ml of PBS. Cells were dispersed thoroughly using a vortex mixer, and cell viability and number were determined in an aliquot by trypan blue exclusion. Cell viability was greater than 95%. Cells were diluted in PBS to a concentration of 2.0 x 106 cells/ml and 0.25 ml aliquots containing 5 x 10s cells were placed into Eppendorf tubes. L-( )-[N-methyl-3H]-nicotine,+ specific activity 67.2 Ci/mmol, was obtained for experimental procedures and diluted to working concentration in DMSO such that the final concentration of DMSO was 0.1%, a concentration which has been shown to have no effect on cell viability and function.32 Ten (10) µ of either 3H-nicotine or a mixture of 3H-nicotine + unlabeled nicotine were added to the cell suspension in the Eppendorf tubes such that the final concentration of labeled nicotine in all samples was 0.1 µ , and the concentration of unlabeled nicotine in appropriate samples was 10 µ . The tubes were mixed on a vortex mixer then shaken in an ice bath at 4° C for 45 minutes. The reaction was terminated by adding 0.5 ml of ice cold PBS (ph 7.2) to each tube. Cells were harvested by centrifugation at 4° C for 5 minutes. The resulting cell pellet was then washed three times with PBS, centrifuging for 5 minutes each time. The final wash was decanted and the cells solubilized by incubation in 1.0 ml of 1% sodium dodecyl sulphate (SDS) in 10 mM dithiothreitiol for 2 hours at room temperature. Bound nicotine was assayed by scintillation counting in a Beckman scintillation counter. Specific binding was calculated as the difference between 3Hnicotine bound in the presence and absence of unlabeled —
Medium was removed from subconfluent cultures of gingival fibroblasts and replaced with 0.5 ml of 0.2 µ 3Hnicotine in EMEM. Cultures were placed back into the incubator at 37° C. At time intervals of 15, 30, 60, and 240 minutes, the 3H-nicotine containing medium was removed, the cells were washed three times with saline, and harvested by scraping. Harvested cells were suspended in 1 ml saline, then centrifuged, and washed three times and an aliquot counted. The remaining pellet was then solubilized in 1% SDS in 10 mM dithiothreitol for 2 hours at room temper*NEN Research Products, Boston, MA.
Volume 62 Number 2
ature.
3H-nicotine uptake
counting.
HANES, SCHUSTER, LUBAS was
assayed by
scintillation
Table 1:
Binding of
-Nicotine to
3H-nicotine in the medium assayed by scintilcounting. At the end of the 2-hour assay period, cells were harvested by scraping, washed in saline, and centrifuged three times and an aliquot counted. The resulting cell pellet was solubilized in 1% SDS in 10 mM dithiothreitol for 2 hours at room temperature and the remaining intracellular 3H-nicotine assayed by scintillation counting. amount of
lation
To determine whether the radioactive material released free nicotine, cultures of fibroblasts were grown and fed with radioactive medium as described above. The radioactive medium was then removed, the cells were washed three times with saline and fresh, non-radioactive EMEM medium (1.5 ml) was added to the culture dishes which were then returned to the incubator at 37° C. At time intervals of 10, 20, 40, 60, and 120 minutes, 350 µ aliquots of medium were removed from the culture dishes and 50 µ aliquots of media were assayed by scintillation counting. The remaining 300 µ of medium and an aliquot of the original radioactive medium were spotted on silica gel SG81 chromatography paper and separated using a chloroform:methanol:ammonium hydroxide (9:1.0:0.1) solvent system. At the end of the 2-hour assay period, the cells were harvested by scraping, washed in saline three times, spotted on chromatography paper, and the radioactive components separated as above. The resulting spots were located by autoradiography, cut out, and the radioactivity measured by scintillation counting.
(pmol/10''
Fibroblasts
cells) -
-
Sample Efflux of Nicotine and Metabolites Cultures containing 1 x 106 gingival fibroblasts were fed with 2 ml of EMEM supplemented with 0.1 µ 3H-nicotine and incubated at 37° C for 4 hours. The radioactive medium was then removed and the cells were washed three times with saline. One ml of fresh, non-radioactive medium was added to each dish and the cultures reincubated. At 15, 30, 60, and 120 minutes after refeeding, 100 µ aliquote of medium were removed from the culture dishes and the
Gingival
149
+
2.38 2.36 1.65 1.75 1.26 1.16 1.05
1.97 1.69 0.90 1.05 1.09
1.66 ± 0.55
1.44 ± 0.42
1 2 3 4 5 6 7
1.53 1.84
Cells were treated at 4°C with either labeled nicotine ( - ) or a mixture of labeled and unlabeled nicotine ( - + N). The amount of - bound per 10 cells in the presence and absence of unlabeled nicotine was compared using a r-test. The differences were not significant (r 0.8410, > 0.4), indicating that nicotine binding was non-specific. =
Uptake of Nicotine
by Human 30
Gingival Fibroblasts -
was
RESULTS of 3H-Nicotine to Gingival Fibroblasts Following 45 minutes of cold incubation gingival fibroblasts bound 1.44 ± 0.42 pmol/106 cells of -Nicotine in the presence of unlabeled nicotine, and 1.66 ± 0.55 pmol/106 cells of 3H-Nicotine in the absence of unlabeled nicotine (Table 1). There were no differences in the amount of 3H-Nicotine bound by the cells in the presence or absence of unlabeled
Binding
nicotine.
of 3H-Nicotine of Uptake nicotine by gingival fibroblasts appeared to be rapid and continuous over the 4 hours of incubation in medium containing 0.2 µ 3H-Nicotine (Fig. 1). Following 15 minutes of incubation, 4.9 ± 0.3 pmol/106 cells of
Uptake
O
30
60
90
120
150
180
210
240
minutes
Figure
1:
Uptake of nicotine was measured mean of 3 samples.
over
4 hours. Each
point
represents the
nicotine had been taken up by the cells. By 30 minutes, this amount had increased to 8.3 ± 0.75 pmol/10ft cells. Uptake continued over the following 90 minutes and more than doubled between 1 hour (12.3 ± 2.6 pmol/106 cells) and 2 hours of incubation (26.3 ± 1.15 pmol/106 cells). Efflux of 3H-Nicotine and Metabolites The efflux or release of 3H-Nicotine that had been taken up by the gingival fibroblasts back into the culture medium over the 2-hour period of assay (Fig. 2) never approached the rate or amount of uptake observed. After 15 minutes of incubation, the rate of 3H-Nicotine release was 3.92 x 10"4 pmol/106 cells/minute. The rate of release slowed over 10"4 pmol/106 cells/ the following 45 minutes to 2.70 10"4 pmol/106 cells/ minute at 30 minutes and 0.82 minute at 1 hour. Between 1 and 2 hours, the rate of ni10~4 cotine release increased again to the rate of 2.35 At each hours. time cells/minute 2 at pmol/106 point, the amount of 3H-Nicotine released back into the medium was less than 0.0001 of the corresponding uptake value. Chromatographie analysis of medium samples taken over
J Periodontol
150
BINDING AND UPTAKE OF NICOTINE BY FIBROBLASTS
Spots analogous to the suspected isotope impurities products observed in the control sample were also identified in each of the medium samples taken at the various time points. When the gingival fibroblasts were harvested and chromatographed, only 30% of the isotope separated out as free 3H-Nicotine. Approximately 32% remained at the chromatograph's origin, and the remaining 38% was evenly earlier time points.
Efflux of Nicotine
or
by Human Gingival Fibroblasts 10.0
«
7.0
February 1991
-
h
distributed between the other two spots.
0.0 *-1-1-1-1-1-1-10 30 60 90 120
minutes
Figure 2: Efflux or release of nicotine was measured over a 2-hour period following a 4-hour pretreatment of cells with -Nicotine. Each point represents a mean of 3 samples. the 2 hours of assay suggested that the majority of the 3H released into the culture medium was in the form of 3HNicotine (Table 2). Approximately 88% of the control sample of 3H-Nicotine + medium separated out as 3H-Nicotine. Other spots comprising 11.9% of the control sample were also located, possibly corresponding to either radioactive impurities, oxidation products, or to a binding of the nicotine to some component(s) of the medium, possibly a serum component. At each of the time points evaluated, the largest percentage of the isotope separated out as 3HNicotine, and this percentage increased dramatically with the passage of time from 49.4% at 10 minutes to 74.0% at 120 minutes. There were some differences observed in the patterns of separation of the media samples taken at the various time points compared to the control sample. However, these differences were of lesser magnitude at each subsequent time point with the final 120 minute medium sample separating in a pattern similar to the control. A larger percentage of the isotope appeared to remain at the chromatograph's origin in each of the medium samples compared to the control (9.9 to 16.5% compared to 1.5%). In addition, relatively large percentages of the isotope separated before the 3H-Nicotine in media samples taken at the
Table 2: Efflux of -Nicotine and Metabolites CPM
Sample Origin Spot 1 Spot 2 Spot 3 -
10 Minutes 223 101 99 292 698
(15.8) (7.1) (7.0) (20.7) (49.4)
20 Minutes 221 (16.1) 129 (9.4) 130 (9.5) 126 (9.2) 770 (56.0)
(% total counts)
40 Minutes
60 Minutes
222 123 126 120 755
125 86 68 86 772
(16.5) (9.1) (9.4) (8.9) (56.1)
DISCUSSION Numerous studies have described the effects of nicotine and smoking on epithelial tissue,13-16 but the effects of these agents on elements of the connective tissue have not been as thoroughly investigated, particularly in regard to the connective tissue components of the periodontium. Although tobacco products contain other biologically active components such as carbon monoxide, nitrogen, carbon dioxide, nitrates, nitrosamines, and polycyclic hydrocarbons, nicotine has several biological effects which could contribute to the development of an inflammatory lesion in the periodontal tissues. In view of the fact that nicotine is known to readily penetrate the epithelial barriers of skin and oral mucosa,35 to suppress immunological defense mechanisms,22-23-25 and to alter fibroblast function in vitro,30-31 a potential role for tobacco in the etiology of periodontal disease is apparent, as is a potential deleterious effect of tobacco components on normal wound healing responses such as those involving the fibroblast. The gingival fibroblast is an important component of the periodontal connective tissues involved both in wound healing responses and in normal metabolic processes. Cultured gingival fibroblasts in the present study were shown to both bind and take up nicotine at relatively rapid rates. Although nicotine has been shown to bind specifically to receptors and cell membranes of neural and brain tissue,36-37 the binding of nicotine to gingival fibroblasts in the present study was non-specific in nature. In regard to nicotine uptake by these cells, the amount of nicotine in the intracellular compartment reached a level of 26.3 pmol/ 106 cells after 4 hours of exposure at 37° C, compared to 1.66 pmol/106 cells after 45 minutes at 4°C. This temperature restricts the nicotine to binding to the cell membrane,
(11.0) (7.6) (6.0) (7.6) (67.9)
120 Minutes 142 86 61 85
(9.9) (6.0) (4.2) (5.9) 1,062 (74.0)
Cells 177 70 71 72 168
(31.7)
(12.5)
(12.7) (12.9) (30.1)
-
+
Med
5,876 (1 25,424 (6 3,021 (0 12,756 (3 348,860 (88
Efflux or release of -Nicotine and/or its metabolites was determined at various time intervals after labeling by Chromatographie separation in aliquots of medium and from harvested cells. Spots were located by autoradiography, cut out, and assayed by scintillation counting. The counts per minute (CPM) in each spot and the percent of the total radioactivity in each spot are indicated. Cells radioactivity remaining cell associated. - + Med original radioactive medium used for pretreatment of cells. =
=
Volume 62 Number 2 across the cell membrane. At 37° C, however, normal cellular physiological functions permit transport or diffusion across the cell membrane. Hence, the increase in 3H-Nicotine uptake at 37° C represents a prob-
limiting translocation
able internalization of the isotope by these cells. The presence of nicotine in the fibroblast's intracellular compartment is likely to disrupt normal cellular metabolic processes such as collagen synthesis and protein secretion30 and may result in a disruption of normal wound healing responses.38 Raulin et al.31 reported a disturbance of normal fibroblast morphology and attachment to substrates after exposure to nicotine. These findings were associated with an increased number of intracellular vacuoles. The release of nicotine and the nature of components seen in the present study suggest that the vacuolization observed by Raulin et al.31 may be related to the packaging of nicotine or its metabolic products into vacuoles or vesicles prior to excretion or metabolism. In this regard, gingival fibroblasts in the present study released nicotine back into the medium initially at a rate of 3.92 x 10"4 pmol/106 cells/minute following 15 minutes of incubation. This relatively rapid initial rate of release may have been due to nicotine bound to the surface of the cell but not yet internalized, as suggested by the uptake data (Fig. 1). The reduced rate of release at 30 and 60 minutes may have possibly been due to the time required for the fibroblast to metabolize the nicotine or package the nicotine (or metabolic products) into vesicles or vacuoles prior to release. The increasing rate of release by 120 minutes may have been a result of the presence of high levels of intracellular nicotine and the cells' attempt to eliminate it. The high levels of intracellular nicotine in these fibroblasts would be the anticipated outcome of the marked disparity in the rates of 3H-Nicotine uptake and efflux, with the recycling of released 3H-Nicotine contributing very little to the process in view of the extremely small amounts of 3H-Nicotine actually released by these cells. It is apparent that the levels of cell associated nicotine can bê quite high, reaching levels of 12 to 26 pmol of nicotine per million cells after 1 hour of exposure (Fig. 1). In the Chromatographie analysis of effluent nicotine in the present study, the percentage of isotope detected as "free" nicotine ranged from 49% at 10 minutes to 74% at 2 hours. The tendency for a greater proportion of the labeled component to either remain at or closer to the chromatograph's origin than the nicotine at earlier time points suggests that a certain proportion is associated with a cell or medium component which would impede its movement on Chromatographie analysis. This observation likewise suggests that these fibroblasts are packaging nicotine into vesicles or vacuoles prior to release, or that the nicotine may be associating with the small vesicles normally produced by cells in culture and released into the medium. Once this is "saturated," more is released as free nicotine either from the cells or from the vesicles. The original radioactive medium shows similar spots, but the proportions are not as great, suggesting that at least a portion of the isotope as-
HANES, SCHUSTER, LUBAS
151
sociates with medium, but non-cellular components. We are unable to ascertain at this time how much of the slower moving components may be bound nicotine metabolites versus unmetabolized nicotine. The Chromatographie analysis of the nicotine content of the fibroblasts at the end of the 2 hour efflux of nicotine showed only 30% of the intracellular isotope to separate out as free 3H-nicotine. Of the remaining 70%, 32% remained at the origin with the remainder separating before the 3H-nicotine. An association of the isotope with cell components would likely result in a reduction in movement on the 3H-nicotine on the chromatography paper as was observed in the present study. It further suggests that the majority of the intracellular nicotine remains intracellular, where it can affect cell metabolism or function. The results of this study indicate that: 1) nicotine does bind to gingival fibroblast cell membranes and that the binding that occurs is non-specific in nature; 2) nicotine is taken up by gingival fibroblasts at a rapid and continuous rate over a 4-hour period of assay; 3) nicotine taken up is also released by gingival fibroblasts, but at a rate much slower than the rate of uptake; and 4) of the nicotine that remains with the cells after 2 hours, about 1/3 is present as free and/or unmetabolized nicotine.
Acknowledgments This study was supported by Smokeless Tobacco Research REFERENCES 1. Ludwick W, Massler
gingivitis
to
Grant Number 0056 from the Council, New York, NY.
M. Relation of dental caries experience and in males 17-21 years old. J Dent Res
cigarette smoking
1952; 31:319-322. 2. Lilienthal , Amerena U, Gregory G. An epidemiologica! study of chronic periodontal disease. Arch Oral Biol 1065; 10:553-566. 3. Arno A, Waerhaug J, Lovdahl A, Schei O. Incidence of gingivitis as related to sex, occupation, tobacco consumption, toothbrushing, and age. Oral Surg Oral Med Oral Pathol 1958; 11:587-595. 4. Burt BA. Diet and dental health; a study of relationships. United States, 1971-1974. Washington DC: Government Printing Office, National Center for Health Statistics Series 11, no. 225; 1982. 5. Ismail AI, Burt BA, Eklund SA. Epidemiological patterns of smoking and periodontal disease in the United States. J Am Dent Assn 1983; 106:617-621. 6. Brandtzaeg P, Jamison HC. A study on periodontal health and oral hygiene in Norwegian army recruits. J Periodontol 1964; 35:302307. 7. Sheiham A. Periodontal disease and oral cleanliness in tobacco smokers. J Periodontol 1971; 42:259-263. 8. Ainamo J. The seeming effect of tobacco consumption on the occurrence of periodontal disease and dental caries. Suom Hammaslaak Toimi 1971; 67:87-94. 9. Rivera-Hidalgo F. Smoking and periodontal disease—A review of the literature. J Periodontol 1986; 57:617-624. 10. Bastiaan RJ, Waite IM. Effects of tobacco smoking on plaque development and gingivitis. J Periodontol 1978; 49:480-482. 11. Feldmen RS, Bravacos JS, Rose CL. Association between smoking different tobacco products and periodontal disease indexes. / Periodontol 1983; 54:481-487. 12. DHEW. Healthy People: The Surgeon General's report on Health
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24. 25.
BINDING AND UPTAKE OF NICOTINE BY FIBROBLASTS Promotion and Disease Prevention. Washington DC: Government Printing Office, DHEW Publication No. (PHS) 79-55071; 1979. Schuller HM. Cell type specific, receptor-mediated modulation of growth kinetics in human lung cancer cell lines by nicotine and tobacco-related nitrosamines. Biochem Pharmocol 1989; 38:3439-3442. Sankaranarayanan R, Duffy SW, Padmakumary G, Day NE, Padmanabhan TK. Tobacco chewing, alcohol and nasal snuff in cancer of the gingiva in Karala, India. Br J Cancer 1989; 60:638-643. Merletti F, Boffetta , Ciccone G, Mashberg A, Terracini . Role of tobacco and alcoholic beverages in the etiology of cancer of the oral cavity/oropharynx in Torino, Italy. Cancer Res 1989; 49:49194924. Trichopoulos D, Katsouyanni K. Oral contraceptives, tobacco smoking, and breast cancer risk. Lancet 1989; 2(8655):158-159. Perkins KA, Epstein LH, Jennings JR, Stiller RD. The cardiovascular effects of nicotine during stress. Psychopharmacology 1986; 90:373378. Benowitz NL, Jacob P, Yu L. Daily use of smokeless tobacco: Systemic effects. Ann Intern Med 1989; 111:112-116. Eichel , Shahriek HA. Tobacco smoke toxicity, loss of human oral leukocyte function and fluid cell metabolism. Science 1969; 166:14241428. Warr GA, Martin RR. In vitro migration of human alveolar macrophages: Effects of cigarette smoking. Infect Immun 1973; 8:222-227. Holt PG. Differential toxicity of tobacco smoke to various cell types including those of the immune system. AustJ Exp Bio! Med Sci 1974; 52:211-214. Kenney EB, Kraal JH, Saxe SR, Jones J. The effect of cigarette smoke on human oral polymorphonuclear leukocytes. J Periodont Res 1977; 12:227-234. Holt PG, Keast D. Environmentally induced changes in immunological function: Acute and chronic effects of inhalation of tobacco smoke and other atmospheric contaminants in man and experimental animals. Bacterio! Rev 1977; 41:205-216. Neher GH. Nicotine-induced depression of lymphocyte growth. Toxico! Appi Pharmacol 1974; 27:253-258. Warr GA, Martin RR. Response of human pulmonary macrophages to migration inhibition factor. Am Rev Respir Dis 1973; 108:371373.
February 1991
Shephard BC, Hirsch RS. The effects of intraarterial and nicotine on gingival circulation. Oral Surg Oral Med OralPathol 1981; 52:577-582. Clark NG, Shephard BC. The effects of epinephrine and nicotine on gingival blood flow in the rabbit. Arch Oral Biol 1984; 29:789-793. Page RC, Schroeder HE. Pathogenesis of inflammatory periodontal disease. A summary of current work. Lab Invest 1976; 33:235-249. Altmann H, Weniger , Dolejs I. Influence of nicotine on DNA metabolism. Klin Wochenschr (Suppl II) 1984; 62:101-104. Chamson A, Frey J, Hívért M. Effects of tobacco smoke extracts on collagen biosynthesis by fibroblast cell cultures. J Toxicol Environ Health 1982; 9:921-932. Raulin LA, McPherson JC, McQuade MJ, Hanson BS. The effect of nicotine on the attachment of human fibroblasts to glass and human root surfaces in vitro. J Periodontol 1988; 59:318-325. Cabot MC. Effects of cellular phospholipid modification on phorbol diester binding. Cancer Res 1983; 43:4233-4238. Schuster GS, Erbland JF, Wyrick SD, Singh BB. Phorbol ester binding to oral epithelial cells in the presence of retinóte acid or N-nitrosonornicotine. Cytobios 1988; 54:53-60. Solanki V, Slaga TJ. Specific binding of phorbol ester tumor promoters to intact primary epidermal cells from Sencar mice. Proc Nati Acad Sci (USA) 1981; 78:2549-2553. Taylor P. Ganglionic stimulating and blocking agents. In: Goodman LS, Oilman A, eds. The Pharmacological Basis of Therapeutics, 7th ed. New York: Macmillan Company; 1985:218. Cairns NJ, Wonnacott S. ^H-Nicotine binding sites in fetal human brain. Brain Res 1988; 475:1-7. Reavill C, Jenner , Kumar R, Stolerman IP. High affinity binding of -nicotine to rat brain membranes and its inhibition by analogues of nicotine. Neuropharmacology 1988; 27:235-241. Preber H, Bergstrom J. The effect of non-surgical treatment on periodontal pockets in smokers and non-smokers. J Clin Periodontol 1986; 13:319-323.
26. Clarke NG,
epinephrine
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35.
36. 37.
38.
Send reprint requests to: Dr. Philip J. Hanes, Department of Periodontics, School of Dentistry, Medical College of Georgia, 1459 Laney Walker Boulevard, Augusta, GA 30912. Accepted for publication August 7, 1990.
Binding, Uptake, and Release of Nicotine by Human Gingival Fibroblasts Philip J. Hanes, George S. Schuster,f and Scott Lubas1 *
fibroblasts have reported an altered and attachment of fibroblasts to substrates and disturbances in protein synmorphology and thesis secretion. This altered functional and attachment response may be associated with changes in the cell membrane resulting from binding of the nicotine, or to disturbances in cell metabolism as a result of high intracellular levels of nicotine. The purpose of the present study, therefore, was to 1) determine whether gingival fibroblasts bound nicotine and if any binding observed was specific or non-specific in nature; 2) determine whether gingival fibroblasts internalized nicotine, and if so, at what rate; 3) determine whether gingival fibroblasts also released nicotine back into the extracellular environment; and 4) if gingival fibroblasts release nicotine intact or as a metabolite. Cultures of gingival fibroblasts were prepared from gingival connective tissue biopsies. Binding was evaluated at 4° C using a mixture of 3H-nicotine and unlabeled nicotine. Specific binding was calculated as the difference between 3H-nicotine bound in the presence and absence of unlabeled nicotine. The cells bound 1.44 (± 0.42) pmols/106 cells in the presence of unlabeled nicotine and 1.66 (± 0.55) pmols/106 cells in the absence of unlabeled nicotine. The difference was not significant. Uptake of nicotine was measured at 37° C after treating cells with 3H-nicotine for time periods up to 4 hours. Uptake in pmols/106 cells was 4.90 (± 0.34) at 15 minutes, 8.30 (± 0.75) at 30 minutes, 12.28 (± 2.62) at 1 hour and 26.31 (± 1.15) at 4 hours. The efflux or release of nicotine was evaluated after treating cells with 3H-nicotine for 4 hours and then measuring the amount of 3H-nicotine released into fresh non-radioactive medium over a 2-hour period by direct scintillation counting of aliquots of medium and by Chromatographie analysis of medium samples. Cells released nicotine back into the medium at a rate much slower than the rate of uptake, and the majority of nicotine released by the cells over the 2-hour period was in the form of nicotine rather than a metabolite. At earlier time points, a greater percentage of the 3H isotope separated chromatographically before 3H-nicotine, suggesting an association of the nicotine with intracellular vesicles or other intracellular components. The results of this study suggest that, although nicotine does bind to gingival fibroblasts, this binding is non-specific, and that the uptake of nicotine by these cells is continuous over 4 hours, providing high intracellular levels. In addition, gingival fibroblasts also release nicotine, apparently unmetabolized, back into the medium. However, an apparent association of the nicotine with intracellular components seems to result in the development of high intracellular levels of nicotine which may interfere with normal cellular functions. J Periodontol 1991; 62:147-152. Previous
studies of the effects of nicotine on
Key Words: Nicotine/adverse effects; fibroblasts; cell culture.
relationship between tobacco use and the prevalence severity of periodontal disease has long been debated. Although epidemiological studies have reported essentially The and
Department of Periodontics, School of Dentistry, Georgia, Augusta, GA. department of Oral Biology/Microbiology.
Medical
College
of
equal levels of periodontal disease in tobacco users and non-users,1-2 some investigations have described a greater prevalence of periodontal inflammation in smokers.3-5 Clinical observations and some epidemiological studies suggest that this positive correlation between smoking and peri-
odontal disease may be related to a greater accumulation of plaque and calculus in patients who smoke.6"9 However,
148
J Periodontol 1991
BINDING AND UPTAKE OF NICOTINE BY FIBROBLASTS
February
studies by Bastiaan and Waite10 and Feldmen et al." suggest that patients who smoke have less plaque accumulation than do non-smokers. Tobacco use, particularly smoking, has been strongly associated with an increased risk of cancer12-'6 and cardiovascular disease.12-17-18 These health risks may be the result of a tobacco associated impairment of normal immunological surveillance or defense mechanisms such as neutrophil and macrophage function,18-22 T-cell proliferation,23-24 and/ or the cellular immune response.23-25 The aforementioned disturbances in immune function reportedly associated with tobacco use could also contribute to the development and progression of plaque-induced chronic inflammatory periodontal disease. Many of the undesirable effects of tobacco have been attributed to nicotine, a major component of the particulate phase of tobacco smoke. Nicotine has been shown to induce vascular changes in gingival tissue26-27 similar to the exudative vasculitis described as characteristic of the initial lesion of periodontal inflammation.28 In in vitro systems, nicotine also suppresses DNA synthesis in lymphocytes and HeLa cells.24-29 As with other tissue responses, normal fibroblast function is critical for the maintenance of periodontal tissues and for optimal wound healing responses, and nicotine does have some effects on various fibroblast functions. For example, collagen production is apparently stimulated by nicotine; its secretion, however, is impaired.30 In addition, the growth and attachment of fibroblasts to substrates is impaired by nicotine.31 These effects could be related to the binding of nicotine to the cell membrane which could then interfere with membrane functions such as cell attachment and transport of materials between the intracellular and extracellular environments. Alternatively, the uptake of nicotine by the fibroblast may result in an interference with cell metabolic processes. In order to better define the effects of nicotine on the periodontal tissues, the present study evaluated the binding, uptake, and release of nicotine by gingival fibroblasts and whether the binding observed was specific or non-specific in nature.
successive washes with 0.1% EDTA and 0.25% trypsin. The cells were then transferred to 75 cm2 culture flasks and refed fresh medium. Cells were then incubated at 37° C under an atmosphere of 95% air/5% C02 until cultures
MATERIALS AND METHODS
nicotine.32-34
Cultures of Gingival Fibroblasts Primary cultures of gingival fibroblasts were obtained from gingival biopsies obtained during routine periodontal surgical procedures in the Department of Periodontics at the Medical College of Georgia School of Dentistry. Biopsies were obtained from adult patients, both male and female, with an original diagnosis of adult Periodontitis. Explants of gingival connective tissue were placed in culture dishes containing Eagles minimal essential medium (EMEM) supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, and 20% (v/v) fetal bovine serum. When cultures became confluent (7 to 10 days), the tissue expiant was discarded and the cells detached from the culture dish by
Uptake of Nicotine
reached
confluency.
Binding of Nicotine Cultures of gingival fibroblasts (passage 4-7) pooled from four patients were washed three times with phosphate buffered saline (PBS) (pH 7.2), harvested by scraping, and suspended in PBS. They were then centrifuged (1000 x g for 20 minutes), washed a second time in PBS, then resuspended in 4.0 ml of PBS. Cells were dispersed thoroughly using a vortex mixer, and cell viability and number were determined in an aliquot by trypan blue exclusion. Cell viability was greater than 95%. Cells were diluted in PBS to a concentration of 2.0 x 106 cells/ml and 0.25 ml aliquots containing 5 x 10s cells were placed into Eppendorf tubes. L-( )-[N-methyl-3H]-nicotine,+ specific activity 67.2 Ci/mmol, was obtained for experimental procedures and diluted to working concentration in DMSO such that the final concentration of DMSO was 0.1%, a concentration which has been shown to have no effect on cell viability and function.32 Ten (10) µ of either 3H-nicotine or a mixture of 3H-nicotine + unlabeled nicotine were added to the cell suspension in the Eppendorf tubes such that the final concentration of labeled nicotine in all samples was 0.1 µ , and the concentration of unlabeled nicotine in appropriate samples was 10 µ . The tubes were mixed on a vortex mixer then shaken in an ice bath at 4° C for 45 minutes. The reaction was terminated by adding 0.5 ml of ice cold PBS (ph 7.2) to each tube. Cells were harvested by centrifugation at 4° C for 5 minutes. The resulting cell pellet was then washed three times with PBS, centrifuging for 5 minutes each time. The final wash was decanted and the cells solubilized by incubation in 1.0 ml of 1% sodium dodecyl sulphate (SDS) in 10 mM dithiothreitiol for 2 hours at room temperature. Bound nicotine was assayed by scintillation counting in a Beckman scintillation counter. Specific binding was calculated as the difference between 3Hnicotine bound in the presence and absence of unlabeled —
Medium was removed from subconfluent cultures of gingival fibroblasts and replaced with 0.5 ml of 0.2 µ 3Hnicotine in EMEM. Cultures were placed back into the incubator at 37° C. At time intervals of 15, 30, 60, and 240 minutes, the 3H-nicotine containing medium was removed, the cells were washed three times with saline, and harvested by scraping. Harvested cells were suspended in 1 ml saline, then centrifuged, and washed three times and an aliquot counted. The remaining pellet was then solubilized in 1% SDS in 10 mM dithiothreitol for 2 hours at room temper*NEN Research Products, Boston, MA.
Volume 62 Number 2
ature.
3H-nicotine uptake
counting.
HANES, SCHUSTER, LUBAS was
assayed by
scintillation
Table 1:
Binding of
-Nicotine to
3H-nicotine in the medium assayed by scintilcounting. At the end of the 2-hour assay period, cells were harvested by scraping, washed in saline, and centrifuged three times and an aliquot counted. The resulting cell pellet was solubilized in 1% SDS in 10 mM dithiothreitol for 2 hours at room temperature and the remaining intracellular 3H-nicotine assayed by scintillation counting. amount of
lation
To determine whether the radioactive material released free nicotine, cultures of fibroblasts were grown and fed with radioactive medium as described above. The radioactive medium was then removed, the cells were washed three times with saline and fresh, non-radioactive EMEM medium (1.5 ml) was added to the culture dishes which were then returned to the incubator at 37° C. At time intervals of 10, 20, 40, 60, and 120 minutes, 350 µ aliquots of medium were removed from the culture dishes and 50 µ aliquots of media were assayed by scintillation counting. The remaining 300 µ of medium and an aliquot of the original radioactive medium were spotted on silica gel SG81 chromatography paper and separated using a chloroform:methanol:ammonium hydroxide (9:1.0:0.1) solvent system. At the end of the 2-hour assay period, the cells were harvested by scraping, washed in saline three times, spotted on chromatography paper, and the radioactive components separated as above. The resulting spots were located by autoradiography, cut out, and the radioactivity measured by scintillation counting.
(pmol/10''
Fibroblasts
cells) -
-
Sample Efflux of Nicotine and Metabolites Cultures containing 1 x 106 gingival fibroblasts were fed with 2 ml of EMEM supplemented with 0.1 µ 3H-nicotine and incubated at 37° C for 4 hours. The radioactive medium was then removed and the cells were washed three times with saline. One ml of fresh, non-radioactive medium was added to each dish and the cultures reincubated. At 15, 30, 60, and 120 minutes after refeeding, 100 µ aliquote of medium were removed from the culture dishes and the
Gingival
149
+
2.38 2.36 1.65 1.75 1.26 1.16 1.05
1.97 1.69 0.90 1.05 1.09
1.66 ± 0.55
1.44 ± 0.42
1 2 3 4 5 6 7
1.53 1.84
Cells were treated at 4°C with either labeled nicotine ( - ) or a mixture of labeled and unlabeled nicotine ( - + N). The amount of - bound per 10 cells in the presence and absence of unlabeled nicotine was compared using a r-test. The differences were not significant (r 0.8410, > 0.4), indicating that nicotine binding was non-specific. =
Uptake of Nicotine
by Human 30
Gingival Fibroblasts -
was
RESULTS of 3H-Nicotine to Gingival Fibroblasts Following 45 minutes of cold incubation gingival fibroblasts bound 1.44 ± 0.42 pmol/106 cells of -Nicotine in the presence of unlabeled nicotine, and 1.66 ± 0.55 pmol/106 cells of 3H-Nicotine in the absence of unlabeled nicotine (Table 1). There were no differences in the amount of 3H-Nicotine bound by the cells in the presence or absence of unlabeled
Binding
nicotine.
of 3H-Nicotine of Uptake nicotine by gingival fibroblasts appeared to be rapid and continuous over the 4 hours of incubation in medium containing 0.2 µ 3H-Nicotine (Fig. 1). Following 15 minutes of incubation, 4.9 ± 0.3 pmol/106 cells of
Uptake
O
30
60
90
120
150
180
210
240
minutes
Figure
1:
Uptake of nicotine was measured mean of 3 samples.
over
4 hours. Each
point
represents the
nicotine had been taken up by the cells. By 30 minutes, this amount had increased to 8.3 ± 0.75 pmol/10ft cells. Uptake continued over the following 90 minutes and more than doubled between 1 hour (12.3 ± 2.6 pmol/106 cells) and 2 hours of incubation (26.3 ± 1.15 pmol/106 cells). Efflux of 3H-Nicotine and Metabolites The efflux or release of 3H-Nicotine that had been taken up by the gingival fibroblasts back into the culture medium over the 2-hour period of assay (Fig. 2) never approached the rate or amount of uptake observed. After 15 minutes of incubation, the rate of 3H-Nicotine release was 3.92 x 10"4 pmol/106 cells/minute. The rate of release slowed over 10"4 pmol/106 cells/ the following 45 minutes to 2.70 10"4 pmol/106 cells/ minute at 30 minutes and 0.82 minute at 1 hour. Between 1 and 2 hours, the rate of ni10~4 cotine release increased again to the rate of 2.35 At each hours. time cells/minute 2 at pmol/106 point, the amount of 3H-Nicotine released back into the medium was less than 0.0001 of the corresponding uptake value. Chromatographie analysis of medium samples taken over
J Periodontol
150
BINDING AND UPTAKE OF NICOTINE BY FIBROBLASTS
Spots analogous to the suspected isotope impurities products observed in the control sample were also identified in each of the medium samples taken at the various time points. When the gingival fibroblasts were harvested and chromatographed, only 30% of the isotope separated out as free 3H-Nicotine. Approximately 32% remained at the chromatograph's origin, and the remaining 38% was evenly earlier time points.
Efflux of Nicotine
or
by Human Gingival Fibroblasts 10.0
«
7.0
February 1991
-
h
distributed between the other two spots.
0.0 *-1-1-1-1-1-1-10 30 60 90 120
minutes
Figure 2: Efflux or release of nicotine was measured over a 2-hour period following a 4-hour pretreatment of cells with -Nicotine. Each point represents a mean of 3 samples. the 2 hours of assay suggested that the majority of the 3H released into the culture medium was in the form of 3HNicotine (Table 2). Approximately 88% of the control sample of 3H-Nicotine + medium separated out as 3H-Nicotine. Other spots comprising 11.9% of the control sample were also located, possibly corresponding to either radioactive impurities, oxidation products, or to a binding of the nicotine to some component(s) of the medium, possibly a serum component. At each of the time points evaluated, the largest percentage of the isotope separated out as 3HNicotine, and this percentage increased dramatically with the passage of time from 49.4% at 10 minutes to 74.0% at 120 minutes. There were some differences observed in the patterns of separation of the media samples taken at the various time points compared to the control sample. However, these differences were of lesser magnitude at each subsequent time point with the final 120 minute medium sample separating in a pattern similar to the control. A larger percentage of the isotope appeared to remain at the chromatograph's origin in each of the medium samples compared to the control (9.9 to 16.5% compared to 1.5%). In addition, relatively large percentages of the isotope separated before the 3H-Nicotine in media samples taken at the
Table 2: Efflux of -Nicotine and Metabolites CPM
Sample Origin Spot 1 Spot 2 Spot 3 -
10 Minutes 223 101 99 292 698
(15.8) (7.1) (7.0) (20.7) (49.4)
20 Minutes 221 (16.1) 129 (9.4) 130 (9.5) 126 (9.2) 770 (56.0)
(% total counts)
40 Minutes
60 Minutes
222 123 126 120 755
125 86 68 86 772
(16.5) (9.1) (9.4) (8.9) (56.1)
DISCUSSION Numerous studies have described the effects of nicotine and smoking on epithelial tissue,13-16 but the effects of these agents on elements of the connective tissue have not been as thoroughly investigated, particularly in regard to the connective tissue components of the periodontium. Although tobacco products contain other biologically active components such as carbon monoxide, nitrogen, carbon dioxide, nitrates, nitrosamines, and polycyclic hydrocarbons, nicotine has several biological effects which could contribute to the development of an inflammatory lesion in the periodontal tissues. In view of the fact that nicotine is known to readily penetrate the epithelial barriers of skin and oral mucosa,35 to suppress immunological defense mechanisms,22-23-25 and to alter fibroblast function in vitro,30-31 a potential role for tobacco in the etiology of periodontal disease is apparent, as is a potential deleterious effect of tobacco components on normal wound healing responses such as those involving the fibroblast. The gingival fibroblast is an important component of the periodontal connective tissues involved both in wound healing responses and in normal metabolic processes. Cultured gingival fibroblasts in the present study were shown to both bind and take up nicotine at relatively rapid rates. Although nicotine has been shown to bind specifically to receptors and cell membranes of neural and brain tissue,36-37 the binding of nicotine to gingival fibroblasts in the present study was non-specific in nature. In regard to nicotine uptake by these cells, the amount of nicotine in the intracellular compartment reached a level of 26.3 pmol/ 106 cells after 4 hours of exposure at 37° C, compared to 1.66 pmol/106 cells after 45 minutes at 4°C. This temperature restricts the nicotine to binding to the cell membrane,
(11.0) (7.6) (6.0) (7.6) (67.9)
120 Minutes 142 86 61 85
(9.9) (6.0) (4.2) (5.9) 1,062 (74.0)
Cells 177 70 71 72 168
(31.7)
(12.5)
(12.7) (12.9) (30.1)
-
+
Med
5,876 (1 25,424 (6 3,021 (0 12,756 (3 348,860 (88
Efflux or release of -Nicotine and/or its metabolites was determined at various time intervals after labeling by Chromatographie separation in aliquots of medium and from harvested cells. Spots were located by autoradiography, cut out, and assayed by scintillation counting. The counts per minute (CPM) in each spot and the percent of the total radioactivity in each spot are indicated. Cells radioactivity remaining cell associated. - + Med original radioactive medium used for pretreatment of cells. =
=
Volume 62 Number 2 across the cell membrane. At 37° C, however, normal cellular physiological functions permit transport or diffusion across the cell membrane. Hence, the increase in 3H-Nicotine uptake at 37° C represents a prob-
limiting translocation
able internalization of the isotope by these cells. The presence of nicotine in the fibroblast's intracellular compartment is likely to disrupt normal cellular metabolic processes such as collagen synthesis and protein secretion30 and may result in a disruption of normal wound healing responses.38 Raulin et al.31 reported a disturbance of normal fibroblast morphology and attachment to substrates after exposure to nicotine. These findings were associated with an increased number of intracellular vacuoles. The release of nicotine and the nature of components seen in the present study suggest that the vacuolization observed by Raulin et al.31 may be related to the packaging of nicotine or its metabolic products into vacuoles or vesicles prior to excretion or metabolism. In this regard, gingival fibroblasts in the present study released nicotine back into the medium initially at a rate of 3.92 x 10"4 pmol/106 cells/minute following 15 minutes of incubation. This relatively rapid initial rate of release may have been due to nicotine bound to the surface of the cell but not yet internalized, as suggested by the uptake data (Fig. 1). The reduced rate of release at 30 and 60 minutes may have possibly been due to the time required for the fibroblast to metabolize the nicotine or package the nicotine (or metabolic products) into vesicles or vacuoles prior to release. The increasing rate of release by 120 minutes may have been a result of the presence of high levels of intracellular nicotine and the cells' attempt to eliminate it. The high levels of intracellular nicotine in these fibroblasts would be the anticipated outcome of the marked disparity in the rates of 3H-Nicotine uptake and efflux, with the recycling of released 3H-Nicotine contributing very little to the process in view of the extremely small amounts of 3H-Nicotine actually released by these cells. It is apparent that the levels of cell associated nicotine can bê quite high, reaching levels of 12 to 26 pmol of nicotine per million cells after 1 hour of exposure (Fig. 1). In the Chromatographie analysis of effluent nicotine in the present study, the percentage of isotope detected as "free" nicotine ranged from 49% at 10 minutes to 74% at 2 hours. The tendency for a greater proportion of the labeled component to either remain at or closer to the chromatograph's origin than the nicotine at earlier time points suggests that a certain proportion is associated with a cell or medium component which would impede its movement on Chromatographie analysis. This observation likewise suggests that these fibroblasts are packaging nicotine into vesicles or vacuoles prior to release, or that the nicotine may be associating with the small vesicles normally produced by cells in culture and released into the medium. Once this is "saturated," more is released as free nicotine either from the cells or from the vesicles. The original radioactive medium shows similar spots, but the proportions are not as great, suggesting that at least a portion of the isotope as-
HANES, SCHUSTER, LUBAS
151
sociates with medium, but non-cellular components. We are unable to ascertain at this time how much of the slower moving components may be bound nicotine metabolites versus unmetabolized nicotine. The Chromatographie analysis of the nicotine content of the fibroblasts at the end of the 2 hour efflux of nicotine showed only 30% of the intracellular isotope to separate out as free 3H-nicotine. Of the remaining 70%, 32% remained at the origin with the remainder separating before the 3H-nicotine. An association of the isotope with cell components would likely result in a reduction in movement on the 3H-nicotine on the chromatography paper as was observed in the present study. It further suggests that the majority of the intracellular nicotine remains intracellular, where it can affect cell metabolism or function. The results of this study indicate that: 1) nicotine does bind to gingival fibroblast cell membranes and that the binding that occurs is non-specific in nature; 2) nicotine is taken up by gingival fibroblasts at a rapid and continuous rate over a 4-hour period of assay; 3) nicotine taken up is also released by gingival fibroblasts, but at a rate much slower than the rate of uptake; and 4) of the nicotine that remains with the cells after 2 hours, about 1/3 is present as free and/or unmetabolized nicotine.
Acknowledgments This study was supported by Smokeless Tobacco Research REFERENCES 1. Ludwick W, Massler
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Send reprint requests to: Dr. Philip J. Hanes, Department of Periodontics, School of Dentistry, Medical College of Georgia, 1459 Laney Walker Boulevard, Augusta, GA 30912. Accepted for publication August 7, 1990.
