Toxicology Letters, 62 (1992) 255-262 0

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1992 Elsevier Science Publishers B.V. All rights reserved 0378-4274/92/$05.00

TOXLET 02776

Benzo[a]pyrene and nicotine impair epidermal growth factor mediated cellular functions of buccal mucosa

Shoou-Lih Wang, Chi-Ying Wu-Wang, Amalia Slomiany and Bronislaw L. Slomiany Research Center, New Jersey Dental School, University of Medicine and Dentistry of New Jersey, Newark, NJ (USA)

(Received 24 February 1992) (Accepted 4 June 1992) Key words: Epidermal growth factor; Receptor; Buccal mucosa; Benzo[a]pyrene; Nicotine

SUMMARY This study investigated the effect of two major ingredients in cigarette smoke, benzo[a]pyrene (BP) and nicotine, on epidermal growth factor (EGF) receptor binding and EGF-mediated cellular functions in rat buccal mucosa. Rat buccal tissue was incubated in DMEM in the absence (control) and presence of 10pM BP or nicotine for 2.5 h at 25°C. There were no significant differences in [“‘I]EGF binding to the buccal mucosal membranes between the control and treatment groups. Protein tyrosine kinase assay showed that EGF stimulated phosphorylation of a I70-kDa protein band in the controls, but not in the BP- and nicotine-treated samples. The basal [3H]thymidine incorporations were not significantly different between the groups. Nevertheless, addition of 5 nM EGF increased [3H]thymidine incorporation by 22% in the control, but not in the BP- or nicotine-treated group. The results demonstrate that BP and nicotine change the buccal mucosal functions associated with alteration of EGF receptor.

INTRODUCTION

It is well documented that cigarette smoking has adverse effects on human health [l-6]. The mouth acts as a primary target for tobacco smoke. Various studies indicated that smoking is associated with periodontal disease [7-lo], specific lip lesion, hyperkeratinization, leukoderma, leukoplakia and cancer of the oral mucosa [l l-141.

Correspondence to: S.L. Wang, Research Center, UMDNJ-NJ Bergen Street, Newark, NJ 07103-2400, USA.

Dental School, University Heights, 110

256

The mechanism by which cigarette smoking causes these untoward effects in the oral cavity remains poorly understood. Epidermal growth factor (EGF) is a mitogenic polypeptide secreted primarily by the submandibular glands [I 5-171. EGF provides cytoprotective function throughout the alimentary tract [18], including the oral cavity [19,20]. The mitogen interacts with the target cells via binding to a specific high affinity receptor located on the cellular membrane [l&17]. EGF receptors are present in various tissues of the oral cavity [21-231. In our previous investigations, we have characterized the biochemical properties of specific EGF receptors in both rat buccal mucosal cells 1191 and human buccal membrane [20]. The present study investigated the effect of two major components in cigarette smoke, benzo[a]pyrene (BP) and nicotine, on EGF binding and EGF receptor-mediated cellular functions in rat buccal mucosa. MATERIALS

AND PilETHODS

Mause submaxillary EGF, phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, bovine serum albumin (BSA) fraction V, dimethylsulfoxide (DMSO) and urethane were from Sigma Chemical Co., St. Louis, MO. [12’I]EGF (specific activity of 100 ,f.fCi/,ug)and [Y-~~P]ATP(spec. act. 3000 Ciimmoi) were obtained from Amersham Co., Arlington Heights, IL. ~3H]Thymidine (80 Ciimmol} was purchased from New England Nuclear (Boston, MA). Dulbecco’s Modified Eagle’s Medium (DMEM) was from Gibco, Grand Island, NY. BCA protein assay kit was from Pierce, Rockford, IL. Chemicals for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) were purchased from Bio-Rad, Rockville Centre, NY, Agarose-bound wheat germ agglutinin (WGA) was obtained from Vector Laboratories, Inc., Burlingame, CA. Pretreatment with BP or nicotine andpreparation

of buccal mucosal membrane

Adult male Sprague-Dawley rats (200-300 g) were housed in wire cages on a 12-h light cycle. Animals were anesthetized with 25% urethane (1 .Oml/l00 g body weight). Buccal mucosal tissue was dissected with a pair of surgical scissors and soaked in the ice-cold Ddbecco’s Modified Eagle’s Medium (DMEM). Tissue was minced with scissors and repetitively rinsed with DMEM. The minced tissue was then incubated in DMEM with or without 10 PM of BP or nicotine dissolved in DMSO at 25°C under the atmosphere of 95% 0,/S% CO, for 2.5 h. The control sample was incubated with DMSO (1% of DMEM, v/v) only, After incubation, the tissue was washed twice with homogenization buffer (0.25 M sucrose with 25 mM Tris-HCl buffer (pH 7.4) containing 1 mM PMSF, 100 KU/ml aprotinin and 1 pug/ml leupeptin). Cellular membrane fraction was prepared according to the method described by Hock and Hollenberg [24]. Homogenization was carried out with a Polytron homogenizer in an icechilled beaker for 30 s in the buffer. The homogenate was centrifuged at 600 x g for 15 min, and the supernatant was filtered through a double-layered gauze to remove

257

debris before recentrifugation at 10 000 x g for 30 min. The resulting supernatant was centrifuged at 40 000 x g for 40 min, and the pellet was washed with 50 mM Tris-HCl buffer (pH 7.6) containing 1 mM PMSF, 100 KU/ml aprotinin and 1 ,@ml leupeptin. The final pellet was resuspended in the same buffer and stored at -70°C in 0.2-ml aliquots until analysis. Protein was determined by the BCA protein assay kit. [“‘IJEGF-binding

assay

Standard [‘251]EGF-binding assay was conducted by a modified method of Rao et al. [25] and as described before [20]. Briefly, 250 ,ug membrane protein was incubated in duplicate with [12’I]EGF (40 000 cpm, 0.18 nM) in the absence or presence of unlabeled EGF for 1 h at 25°C. Membrane-bound [“‘I]EGF were separated by centrifugation, the pellet was washed with ice-cold buffer and recentrifuged. The resultant pellets were counted in a Packard Gamma counter. EGF receptor purljkation

Glycoprotein receptors were purified by a WGA affinity chromatography [26]. Membrane protein was solubilized in 50 mM HEPES, 10 mM MgSO,, 1 mM PMSF and 1% Triton X-l 00 (pH 7.6). The solubilized protein was recycled three times over a column containing 3.5 ml agarose-bound WGA at 4°C. The column was washed with 40 ml of washing buffer (50 mM HEPES, 150 mM NaCl, and 0.1% Triton X-100 (pH 7.6)) and the receptors were eluted form the column with 10 ml of the same buffer containing 0.3 M N-acetylglucosamine and collected into l.O-ml fractions. The eluates were monitored for protein by ultraviolet spectrometry at 280 nm. Three eluates with highest absorbance at 280 nm were combined and the protein concentration determined by using BCA protein assay kit. EGF receptor phosphorylation

EGF receptor phosphorylation was performed by a modification [26-281 of the method of Lowe et al. [29]. Lectin-purified membrane protein (12 lug) was preincubated in the presence or absence of 1.0 ,uM EGF in a final volume of 80 ~1 50 mM HEPES buffer (pH 7.6), with 10 mM MgSO, and 1 mM PMSF. After 30 min at 25°C phosphorylation was initiated by the addition of 20 ~1 reaction mixture to give final concentrations of 5 ,uM [32P]ATP (spec. act., 20@i/nmol), 1 mM CTP, 3 mM MnCl,, 20 mM MgCl,, and 2 mM sodium vanadate. After 3 min at 4°C the reaction was terminated by the addition of an equal volume of stopping solution (0.17 M Tris-HCl, 10% SDS, and 100 mM dithiothreitol (pH 6.8)) and heating at 100°C for 10 min. Aliquots (120 ~1) were analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. The density of phosphorylated protein band was determined by a laser densitometer (Biomed Instruments, Inc., Fullerton, CA). Macromolecule

synthesis

in buccal mucosal tissue

Experiments were conducted as described [28,30]. Minced buccal mucosal tissue (0.1 g) in 2 ml of DMEM or DMEM containing 10 ,uM of BP or nicotine was

258 TABLE I SPECIFIC BINDING OF [‘251]EGF TO THE MUCOSAL MEMBRANE BUCCAL TISSUE PRETREATED WITH BP OR NICOTINE DMSO (control)

1.81 f 0.04

[?]EGF

PREPARATION

FROM

bound (fmol/mg protein)

BP (10pM)

nicotine (10 ,uM)

1.88 + 0.39 (NS)

1.9 f 0.18 (NS)

a Data represent mean f SE of three experiments each performed in triplicate (NS, no significant difference between control and treated groups, Student’s t-test).

incubated with [3H]thymidine (2 @X/plate) in the presence or absence of 5 nM of EGF at 25°C under the atmosphere of 95% 0,/5% CO, for 2.5 h. After incubation, tissue was washed twice with 10 ml of the same medium and homogenized with a polytron homogenizer in 2 ml of the lysis buffer (10 mM NaCl, 1.5 mM MgCl,, 1% NP-40, 10 mM Tris-HCl (pH 7.4) and 10% glycerol). The homogenate was incubated at 25°C overnight, centrifuged and the supernatant was dialyzed extensively against distilled water, and counted. RESULTS

Table I shows the specific binding of [lz51]EGF to the buccal mucosal membrane with and without BP or nicotine pretreatment. There were no significant differences in [‘251]EGF receptor binding between the control and BP- or nicotine-treated groups. To examine whether BP or nicotine treatment altered the EGF receptor kinase activity, the membrane samples were solubilized and subjected to purification by WGA affinity chromatography. Such lectin-purified samples were then used to study the EGF-stimulated autophosphorylation of the EGF receptor. EGF stimulated the phosphorylation of a 170-kDa protein in the control group, but no EGF-stimulated autophosphorylation was observed in the BP- or nicotine-treated groups (Fig. 1). The effects of EGF on DNA synthesis was studied by examining the [3H]thymidine incorporation to buccal tissue. BP or nicotine treatment did not change the basal (-EGF) incorporation of [3H]thymidine (Table II). In the presence of 5 nM EGF there was a 22% increase of thymidine incorporation in the control samples. However, there was no significant increase of the EGF-stimulated thymidine incorporation in the BP- or nicotine-treated group. DISCUSSION

Tobacco smoke has been demonstrated to correlate highly with several diseases of buccal mucosa [7-141. However, the cellular mechanisms by which smoking induces oral disease remains poorly explored. Evidence from our previous studies

259

Control

BP

- EGF + EGF

- EGF + EGF

NIC - EGF

+ EGF

f

I

Fig. 1.Autoradiogram of SDS-polyacrylamide gels shows EGF stimulated phosphorylation of proteins in solubilized, lectin-purified membrane of control buccal mucosa. No EGF-stimulated protein phosphorylation was observed in either BP- or nicotine-treated sample.

[19,20,31,32] and that by others [21-231 support the important role of EGF and its receptor in maintaining the health of the oral cavity. To investigate whether ingredients of cigarette smoke alter EGF receptor in buccal mucosa, we adopted a rapid in vitro method, in which BP or nicotine in DMSO was co-incubated with the buccal mucosal tissue. The results obtained indicate that ingredients of cigarette smoke impair the EGFmediated cellular function in buccal mucosa. BP is a tumor initiator [5,33] at the dose levels found in tobacco tar. The basic fraction of cigarette tar contains 6@-80% nicotine which has effects attributable to the BP-induced carcinogenesis [5,33-351. The dose (10 ,uM) of nicotine used in this study was selected based on the data of Van TABLE II EFFECT OF BP OR NICOTINE ON EGF-MEDIATED

[3H]THYMIDINE INCORPORATION

[3H]Thymidine incorporation (cpm/mg protein)

Without EGF With EGF Specific effect of EGF

DMSO (control)

BP (IOyM)

nicotine (IOpM)

31 367 + 996 38 536 f 793

32 262 f 990 33 445 + 980

32 774 + 150 33 904 + 1024

7168 + 333

1183 + 162

* Data represent means i SE of three experiments each performed in dupficate.

I161 1: 337

260

Vunakis et al. [36] and Haley et al. [37], who demonstrated that the salivary cotinine level (major metabolite of nicotine) of the smoker reaches the 150-550 ng/ml level within 15-90 min after cigarette smoking. The same concentration of BP was also chosen for this study. Our previous study [38] has also shown a BP dose-dependent effect on EGF receptor in human placental cell cultures with the maximum effects at a BP concentration of 10 PM. The biochemical features of the EGF receptor are known to be altered by various environmental chemicals [26,38-401. Our recent studies [3 1,321 have demonstrated that ethanol treatment decreases EGF receptor binding, impairs EGF receptor autophosphorylation and reduces EGF-stimulated cellular functions in rat buccal mucosa. In the present study, BP or nicotine treatment did not alter the [‘251]EGF binding to the buccal mucosal membrane. These data are consistent with our previous finding showing that EGF receptor binding in human placenta was also not altered by cigarette smoking [26]. The specific [lz51]EGF binding to the buccal mucosal membrane was lower than that reported previously [19,31,32]. The lower specific binding most likely resulted from the higher non-specific binding, possibly caused by the presence of DMSO in the culture medium. Since EGF receptor protein tyrosine kinase is very important for the EGF-mediated cellular functions [16,17,26,38,40], we further purified the buccal mucosal membrane for the study of EGF receptor autophosphorylation. There was no visible phosphorylated protein band with a molecular mass of 170 kDa in the BP- or nicotine-treated group seen in the control samples. It has been reported that DMSO affects the EGF stimulation of autophosphorylation [41,42]. In our study, although DMSO might have been removed during the extensive wash and WGA purification, the co-incubation of mucosal cell with DMSO might have desensitized the EGF receptor and, thus, decreased the potency of EGF-stimulated autophosphorylation. Therefore, in order to enhance the EGF-stimulated phosphorylated protein band, the X-ray film was exposed for 6 weeks at -70°C which resulted in a darker background. In agreement with the protein tyrosine kinase study, EGF-stimulated DNA synthesis in the BP- or nicotine-treated sample was negligible as compared to the control sample. The extent of basal incorporation (-EGF) of [3H]thymidine was not significantly different among the groups. These data indicate that mucosal tissue in culture medium was biologically active regardless of the treatment. Research into the biochemical mechanisms that underlie the effect of smoking on buccal EGF receptor may provide a better understanding of the factors which determine susceptibility of buccal mucosa to insults induced by this noxious agent. Whether the cigarette smoke-associated alterations of EGF receptor in buccal tissue are related to oral mucosal diseases and cancer needs further investigations. ACKNOWLEDGEMENT

This work was supported by NIH grant 2507RR06004-02 (BRSG).

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Benzo[a]pyrene and nicotine impair epidermal growth factor mediated cellular functions of buccal mucosa.

This study investigated the effect of two major ingredients in cigarette smoke, benzo[a]pyrene (BP) and nicotine, on epidermal growth factor (EGF) rec...
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