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Effect of smoking one cigarette on antioxidant metabolites in the saliva of healthy smokers
Archives of Oral Biology 44 (1999) 485±488
Eect of smoking one cigarette on antioxidant metabolites in the saliva of healthy smokers Bruno Zappacosta a,*, Silvia Persichilli a, Pasquale De Sole a, Alvaro Mordente b, Bruno Giardina a a
Istituto di Chimica e Chimica Clinica, CNR Centro Chimica dei Recettori, UniversitaÁ Cattolica del Sacro Cuore, Rome, Italy b Istituto di Chimica Biologica, UniversitaÁ Cattolica del Sacro Cuore, Rome, Italy Accepted 16 February 1999
Abstract Concentrations of glutathione, uric acid and total antioxidant activity, expressed as Trolox (a water-soluble vitamin E analogue) equivalent, were measured in the saliva of healthy non-smokers and smokers before and just after smoking a single cigarette. There was no statistically signi®cant dierence between smokers and non-smokers in uric acid concentrations and total radical-trapping antioxidant capacity, but glutathione concentrations were signi®cantly ( p < 0.05) higher in smokers. Smoking of a single cigarette induced a signi®cant reduction in glutathione concentration ( p < 0.05). Salivary antioxidant power may aect individual sensitivity toward tobacco stress. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Saliva; Antioxidants; Glutathione; Cigarette smoking
1. Introduction Many studies demonstrate the role of the balance between oxidants and antioxidant systems in physiological conditions and in disease pathogenesis (Machlin and Bendich, 1987; Halliwell, 1994). Saliva is a complex secretion whose components exert a well-documented role in health and disease (Federation Dentaire Internationale, 1992; Mandel, 1987). In addition to its
Abbreviations: ABD-F, ¯uoro-2; 1,3-benzoxadiazole-4-sulphonamide; ABTS, (2; 2'-azinobis-(3-ethylbenzothiazoline-6sulphonic acid); DTPA, diethylenetriaminepentaacetic acid; HPLC, high-performance liquid chromatography. * Corresponding author. Present address: Laboratorio di Chimica Clinica, Pol. A. Gemelli, L.go A. Gemelli, 8, 00168 Rome, Italy. Fax: +39-6-35501918.
lubricant properties (Hatton et al., 1985), saliva contains many biochemical systems known to be involved in soft-tissue repair, and many antibacterial components (McNabb and Tomasi, 1981; Tenovuo et al., 1986; 1987) including lysozyme, lactoferrin (Arnold et al., 1980) and salivary peroxidase (Tenovuo et al., 1981; Tenovuo and Pruitt, 1984; Carlsson, 1987). Furthermore, saliva contains various antioxidants, including uric acid, which contributes almost 70% of the total radical-trapping antioxidant capacity (Moore et al., 1994). The ®rst contact with cigarette smoke products occurs in the mouth. Cigarette smoking is reportedly associated with deleterious eects on oral tissues ranging from increases in periodontal disease and dental caries to oral carcinoma (Rivera-Hildago, 1986; Macgregor, 1989; Ferguson, 1998). Many studies clearly indicate the important role of tobacco smoking
0003-9969/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 0 3 - 9 9 6 9 ( 9 9 ) 0 0 0 2 5 - 4
486
B. Zappacosta et al. / Archives of Oral Biology 44 (1999) 485±488
in reducing the body's antioxidant content and emphasize the protective eect of antioxidant nutrients (Pryor, 1992; Anderson, 1991; Machlin and Bendich, 1987) in preventing tissue damage. We now present data on the eect of smoking a single cigarette on the antioxidant activity of saliva. In particular, we have measured the content of uric acid, the main salivary antioxidant, and that of total glutathione together with the total radical-trapping antioxidant capacity expressed as a Trolox (a water-soluble vitamin E analogue) equivalent. 2. Materials and methods 2.1. Participants Forty individuals [30±50 years of age; 20 non-smokers (9 males, 11 females) and 20 smokers (8 males, 12 females) with a daily consumption of 15±20 cigarettes] were enrolled in the study after obtaining their informed consent. All were healthy and without any sign of oral and respiratory diseases as determined from physical examination and history. 2.2. Chemicals Tri-n-buthylphosphine, Na2EDTA, myoglobin (horse heart), dimethylformamide, ABD-F, reduced glutathione, Trolox and DTPA were from Sigma (St Louis, MO, USA). Acetonitrile and ABTS were from Fluka (Buchs, Switzerland). All general chemicals and reagents were of the highest purity available. 2.3. Preparation of saliva samples Saliva samples were collected in Salivette disposable tubes (Sarstedt, Verona, Italy) according to the manufacturer's instructions. In brief, a cotton swab was placed under the tongue for 5 min and then centrifuged in the Salivette tube (1000 g for 10 min). All saliva samples were kept at 48C (Crioplast container; LP Italiana, Milan, Italy). Saliva samples were collected in the early morning, after fasting and before tooth brushing; smokers collected their second saliva sample immediately after smoking one cigarette, the ®rst in the day. The same brand of cigarette (mean nicotine content, 0.8 mg) was used for all participants. About 1.5 ml of saliva were collected and analysed for uric acid, glutathione and total radical-trapping antioxidant capacity activity. 2.4. HPLC apparatus The HPLC apparatus consisted of a Jasco PU-980 pump connected to a Jasco FP-920 ¯uorescence detec-
tor (Jasco, Tokyo, Japan) and equipped with an Alltech Allsphere ODS-2, 150 4.6 mm, 5.0-mm particle size column (Alltech Italia S.r.l., Milan, Italy) provided with a guard column packed with the same matrix as the separative column and equilibrated with 20% acetonitrile in 0.050 M phosphate buer, pH 3.0. 2.5. Determination of uric acid Uric acid was measured by an enzymatic colorimetric method (Uric acid plus; Boehringer Mannheim, Mannheim, Germany) in an automatic analyser (Hitachi 917). 2.6. Determination of glutathione Glutathione was measured by HPLC according to the method of Araki and Sako (1987). In brief, 300 ml of saliva was treated with 30 ml of 10% (v/v) tri-nbuthylphosphine in dimethylformamide for 30 min at 48C in order to reduce thiols and to decouple them from proteins. The solution was then mixed with 300 ml of a 10% trichloroacetic acid solution containing 1 mM Na2EDTA under vigorous vortexing, followed by centrifugation at 2000 g for 5 min; 250 ml of 1.125 M borate buer, 20 ml of 1.55 M NaOH and 20 ml of ABD-F (1 mg/ml) were then added to 100 ml of the clear supernatant. The mixture was incubated in a shaking water-bath for 10 min at 508C to accomplish complete derivatization of glutathione and other thiols. At the end of the reaction, the solution was cooled and 20 ml injected into the HPLC apparatus. Glutathione concentration was then measured ¯uorimetrically at excitation wavelength of 385 nm and emission wavelength of 515 nm. 2.7. Measurement of total antioxidant activity Total radical-trapping antioxidant capacity was measured according to Santini et al. (1997). This method is based on the quenching of ABTS radical cation (ABTS +) by antioxidants. ABTS (150 mmol/l), metmyoglobin (2.5 mmol/l) and saliva (25 ml) were mixed, and the reaction started by the addition of H2O2 (75 mmol/l). ABTS + formation was continuously monitored at 208C by the absorbance increase at 734 nm. The delay between the addition of H2O2 (time zero) and the onset of the absorbance increase (ABTS + formation) was measured. All reagents were dissolved in phosphate buer treated with Chelex-100 and containing DTPA 0.1 mmol/l to prevent any metal-catalysed oxidation. The assay was standardized using Trolox.
B. Zappacosta et al. / Archives of Oral Biology 44 (1999) 485±488 Table 1 Saliva concentrations (mM; median and 25±75% percentile range) of glutathione, uric acid and total radical-trapping antioxidant capacity (TRAP) of smokers and non-smokers
Glutathione Uric acid TRAP a
Non-smokers
Smokers
pa
1.2 (0.4±1.7) 186 (78±228) 350 (250±450)
3.3 (1.2±5.1) 213 (150±285) 293 (196±587)
0.04 NS NS
Signi®cance test, Mann±Whitney U-test.
487
In Table 2, the eect of smoking a single cigarette on the saliva concentration of glutathione, uric acid and total radical-trapping antioxidant capacity is shown. Statistical analysis showed a signi®cant reduction of glutathione ( p < 0.05). Because saliva is rich in peroxidase, which in presence of H2O2 and hypothiocyanite anion could interact with the ABTS radical formation, we also examined total radical-trapping antioxidant capacity in the absence of metmyoglobin and found none, clearly indicating that in our system the presence of hypothyocyanite and salivary peroxidase is without any eect.
2.8. Statistics Data are expressed as medians and range (25th±75th percentiles). As concentrations were not normally distributed we used the Mann±Whitney U-test to evaluate dierences between groups (see Table 1), and the Wilcoxon matched-pairs test for paired samples (see Table 2). 3. Results Table 1 shows the salivary concentrations of glutathione, uric acid and total radical-trapping antioxidant capacity in the smokers and non-smokers. No statistically signi®cant dierence was found for uric acid concentration and total radical-trapping antioxidant capacity, but the glutathione concentration was signi®cantly higher in smokers. Glutathione and uric acid concentrations in saliva are of the same order of magnitude as in plasma. As uric acid is the main component of total radical-trapping antioxidant capacity in saliva it should be positively correlated with total radical-trapping antioxidant capacity: the correlation between uric acid concentration and total radical-trapping antioxidant capacity of saliva was highly signi®cant (r 2=0.93), while no correlation was found for the glutathione concentration (r 2=0.4).
Table 2 Saliva concentrations (mM; median and 25±75% percentile range) of glutathione, uric acid and total radical-trapping antioxidant capacity (TRAP) of smokers before and after smoking a single cigarette
Glutathione Uric acid TRAP a
Before
After
pa
3.3 (1.2±5.1) 213 (150±285) 293 (196±587)
1.1 (0.4±2.7) 177 (120±219) 358 (249±412)
0.04 NS NS
Signi®cance test, Wilcoxon matched-pairs.
4. Discussion Free radicals and highly reactive oxygen species produced by dierent sources such as in¯ammatory cells or environment pollutants are important mediators for many human disorders (Halliwell, 1994; Machlin and Bendich, 1987), and it is well known that inhalation exposure to environmental carcinogens such as polycyclic aromatic hydrocarbons or cigarette smoke is associated with an increased risk of disease. Cigarette smoke contains free radicals and free radical generators in both gaseous and particulate phases that can cause tissue damage by reacting with polyunsaturated fatty acids at cellular membranes and nucleotides at the DNA level (Lee et al., 1989; Bankson et al., 1993). Antioxidants, by counteracting the harmful activities of free radicals, protect structural and tissue integrity. The antioxidant status of an individual is therefore of great importance. Oral structures and tissues are susceptible to the noxious eects of many irritating compounds from polluted air. In particular the stress on buccal tissues is especially increased during active smoking. Oral tissues are the ®rst target for gaseous and particulate products of cigarette smoking, and several oral in¯ammatory and degenerative diseases that can lead to neoplastic transformation are associated with smoking. Even just a few cigarettes induce increased concentrations of tobacco metabolites (nicotine and cotinine) in saliva, plasma and urine, and modify various biochemical and biological functions (Greenberg et al., 1984; Wang et al., 1992; Scherer and Richter, 1997). The oxidant± antioxidant systems are rapidly modi®ed, leading to important biological changes ranging from alterations of membrane lipids to damage to DNA bases, that constitute the molecular basis for many diseases (Halliwell, 1994). Our results demonstrate that even one cigarette reduces the concentration of glutathione in saliva, which, however, returns to the pre-smoking value after 1±2 hr (data not shown). This observation adds to the existing data showing that tobacco smoking or ex-
488
B. Zappacosta et al. / Archives of Oral Biology 44 (1999) 485±488
posure to air pollutants decreases the antioxidant defence mechanisms of the upper airways or mouth (Housley et al., 1995; Kelly et al., 1996; Piythilake et al., 1994; Pryor, 1992). Housley et al. (1995) showed a reduction of urate concentration in nasal lavage following in vitro ozone exposure. About 4% of their participants were almost completely devoid of nasal uric acid. As our method for glutathione analysis measures both the reduced and the oxidized forms, the decrease of saliva glutathione concentration immediately after smoking one cigarette is probably due to the formation of conjugates between glutathione and compounds reacting with the sulphur atom of glutathione. If this analysis is correct, it could follow that individuals with already low pre-smoking glutathione concentrations are the most prone to the noxious eects of cigarette smoking. References Anderson, R., 1991. Assessment of the roles of vitamin C, Vitamin E and beta-carotene in the modulation of antioxidant stress mediated by cigarette smoke-activated phagocites. Am. Clin. Nutr 53 (1 Suppl), 358S±361S. Araki, A., Sako, Y., 1987. Determination of free and total homocysteine in human plasma by high-performance liquid chromatography with ¯uorescence detector. J. Chromatogr 422, 43±52. Arnold, R.R., Brewer, M., Gauthier, J.J., 1980. Bactericidal activity of human lactoferrin: sensitivity of a variety of microorganisms. Infect. Immun 28, 893±898. Bankson, D.D., Kenstin, M., Rifai, N., 1993. Role of free radicals in cancer and atheriosclerosis. Clin. Lab. Med 13, 463±480. Carlsson, J., 1987. Salivary peroxidase: an important part of our defense against oxygen toxicity. J. Oral. Pathol 16, 412±416. Federation Dentaire Internationale, 1992. Saliva: its role in health and disease. Int. Dent. J 42, 291±304. Ferguson, D.B., 1998. The eects of tobacco usage on saliva ¯ow rate and composition and on susceptibility to dental caries. Oral Disease 4, 60±63. Greenberg, R.A., Haley, N.J., Etzel, R.A., Loda, F.A., 1984. Measuring the exposure of infants to tobacco smoke. Nicotine and cotinine in urine and saliva. N. Engl. J. Med 310 (17), 1075±1078. Halliwell, B., 1994. Free radicals, antioxidants, and human disease: curiosity, cause, or consequence. The Lancet 344, 721±724. Hatton, M.N., Loomis, R.E., Levine, M.J., Tabak, L.A., 1985. Masticatory lubrication. Biochem. J 230, 817±820. Housley, D.G., Mudway, I., Kelly, F.J., Eccles, R., Richards, R.J., 1995. Depletion of urate in human nasal lavage fol-
lowing in vitro ozone exposure. Int. J. Biochem. Cell Biol 27, 1153±1159. Kelly, F.J., Blomberg, A., Frew, A., Holgate, S.T., Sandstrom, T., 1996. Antioxidant kinetics in lung lavage ¯uid following exposure of humans to nitrogen dioxide. Am. J. Respir 154, 1700±1705. Lee, C.K., Brown, B.G., Rice Jr, W.Y., Doolittle, D.J., 1989. Role of oxygen free radicals in the induction of sister chomatid exchanges by cigarette smoke. Environ. Mol. Mutagen 13, 54±59. Machlin, L.J., Bendich, A., 1987. Free radical tissue damage. FASEB J 1, 441±445. Macgregor, I.D.M., 1989. Eects of smoking on oral ecology. A review of the literature. Clin. Prev. Dent 11, 3±7. Mandel, I.D., 1987. The function of saliva. J. Dent. Res 66, 623±627. McNabb, P.C., Tomasi, T.B., 1981. Host defense mechanisms at mucosal surfaces. Ann. Rev. Microbiol 35, 477±496. Moore, S., Calder, K.A., Miller, N.J., Rice Evans, C.A., 1994. Antioxidant activity of saliva and periodontal disease. Free Radic. Res 21, 417±425. Piythilake, C.J., Macaluso, M., Hine, R.J., Richards, E.W., Krumdieck, C.L., 1994. Local and systemic eects of cigarette smoking on folate and vitamin B-12. Am. J. Clin. Nutr 60, 559±566. Pryor, W.A., 1992. Biological eects of cigarette smoke, wood smoke, and the smoke from plastics: the use of electron spin resonance. Free Radic. Biol. Med 13, 659±676. Rivera-Hildago, F., 1986. Smoking and periodontal disease: a review of the literature. J. Periodontal 57, 617±624. Santini, S.A., Marra, G., Giardina, B., Cotroneo, P., Mordente, A., Martorana, G.E., Manto, A., Ghirlanda, G., 1997. Defective plasma antioxidant defenses and enhanced susceptibility to lipid peroxidation in uncomplicated IDDM. Diabetes 46, 1853±1858. Scherer, G., Richter, E., 1997. Biomonitoring exposure to environmental tobacco smoke (ETS): a critical reappraisal. Hum. Exp. Toxicol 16 (8), 449±459. Tenovuo, J., Pruitt, K.M., 1984. Relationship of the human salivary peroxidase system to oral health. J. Oral. Pathol 13, 573±584. Tenovuo, J., Mansson-Rahemtulla, B., Pruitt, K.M., Arnold, R.R., 1981. Inhibition of dental plaque acid production by the salivary lactoperoxidase system. Infect. Immun 34, 208±214. Tenovuo, J., Lehtonen, O.P., Aaltonen, A.S., Vilja, P., Tuohimaa, P., 1986. Antimicrobical factors in whole saliva of human infants. Infect. Immun 51, 49±53. Tenovuo, J., Grahn, E., Lehtonen, O.P., Hyyppa, T., Karhuvaara, L., Vilja, P., 1987. Antimicrobical factors in saliva: ontogeny and relation to oral health. J. Dent. Res 66, 475±479. Wang, S.L., Milles, M., Wu-Wang, C.Y., Mardirossian, G., Leung, C., Slomiany, A., Slomiany, B.L., 1992. Eect of cigarette smoking on salivary epidermal growth factor (EGF) and EGF receptor in human buccal mucosa. Toxicology 75 (2), 145±157.
Eect of smoking one cigarette on antioxidant metabolites in the saliva of healthy smokers Bruno Zappacosta a,*, Silvia Persichilli a, Pasquale De Sole a, Alvaro Mordente b, Bruno Giardina a a
Istituto di Chimica e Chimica Clinica, CNR Centro Chimica dei Recettori, UniversitaÁ Cattolica del Sacro Cuore, Rome, Italy b Istituto di Chimica Biologica, UniversitaÁ Cattolica del Sacro Cuore, Rome, Italy Accepted 16 February 1999
Abstract Concentrations of glutathione, uric acid and total antioxidant activity, expressed as Trolox (a water-soluble vitamin E analogue) equivalent, were measured in the saliva of healthy non-smokers and smokers before and just after smoking a single cigarette. There was no statistically signi®cant dierence between smokers and non-smokers in uric acid concentrations and total radical-trapping antioxidant capacity, but glutathione concentrations were signi®cantly ( p < 0.05) higher in smokers. Smoking of a single cigarette induced a signi®cant reduction in glutathione concentration ( p < 0.05). Salivary antioxidant power may aect individual sensitivity toward tobacco stress. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Saliva; Antioxidants; Glutathione; Cigarette smoking
1. Introduction Many studies demonstrate the role of the balance between oxidants and antioxidant systems in physiological conditions and in disease pathogenesis (Machlin and Bendich, 1987; Halliwell, 1994). Saliva is a complex secretion whose components exert a well-documented role in health and disease (Federation Dentaire Internationale, 1992; Mandel, 1987). In addition to its
Abbreviations: ABD-F, ¯uoro-2; 1,3-benzoxadiazole-4-sulphonamide; ABTS, (2; 2'-azinobis-(3-ethylbenzothiazoline-6sulphonic acid); DTPA, diethylenetriaminepentaacetic acid; HPLC, high-performance liquid chromatography. * Corresponding author. Present address: Laboratorio di Chimica Clinica, Pol. A. Gemelli, L.go A. Gemelli, 8, 00168 Rome, Italy. Fax: +39-6-35501918.
lubricant properties (Hatton et al., 1985), saliva contains many biochemical systems known to be involved in soft-tissue repair, and many antibacterial components (McNabb and Tomasi, 1981; Tenovuo et al., 1986; 1987) including lysozyme, lactoferrin (Arnold et al., 1980) and salivary peroxidase (Tenovuo et al., 1981; Tenovuo and Pruitt, 1984; Carlsson, 1987). Furthermore, saliva contains various antioxidants, including uric acid, which contributes almost 70% of the total radical-trapping antioxidant capacity (Moore et al., 1994). The ®rst contact with cigarette smoke products occurs in the mouth. Cigarette smoking is reportedly associated with deleterious eects on oral tissues ranging from increases in periodontal disease and dental caries to oral carcinoma (Rivera-Hildago, 1986; Macgregor, 1989; Ferguson, 1998). Many studies clearly indicate the important role of tobacco smoking
0003-9969/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 0 3 - 9 9 6 9 ( 9 9 ) 0 0 0 2 5 - 4
486
B. Zappacosta et al. / Archives of Oral Biology 44 (1999) 485±488
in reducing the body's antioxidant content and emphasize the protective eect of antioxidant nutrients (Pryor, 1992; Anderson, 1991; Machlin and Bendich, 1987) in preventing tissue damage. We now present data on the eect of smoking a single cigarette on the antioxidant activity of saliva. In particular, we have measured the content of uric acid, the main salivary antioxidant, and that of total glutathione together with the total radical-trapping antioxidant capacity expressed as a Trolox (a water-soluble vitamin E analogue) equivalent. 2. Materials and methods 2.1. Participants Forty individuals [30±50 years of age; 20 non-smokers (9 males, 11 females) and 20 smokers (8 males, 12 females) with a daily consumption of 15±20 cigarettes] were enrolled in the study after obtaining their informed consent. All were healthy and without any sign of oral and respiratory diseases as determined from physical examination and history. 2.2. Chemicals Tri-n-buthylphosphine, Na2EDTA, myoglobin (horse heart), dimethylformamide, ABD-F, reduced glutathione, Trolox and DTPA were from Sigma (St Louis, MO, USA). Acetonitrile and ABTS were from Fluka (Buchs, Switzerland). All general chemicals and reagents were of the highest purity available. 2.3. Preparation of saliva samples Saliva samples were collected in Salivette disposable tubes (Sarstedt, Verona, Italy) according to the manufacturer's instructions. In brief, a cotton swab was placed under the tongue for 5 min and then centrifuged in the Salivette tube (1000 g for 10 min). All saliva samples were kept at 48C (Crioplast container; LP Italiana, Milan, Italy). Saliva samples were collected in the early morning, after fasting and before tooth brushing; smokers collected their second saliva sample immediately after smoking one cigarette, the ®rst in the day. The same brand of cigarette (mean nicotine content, 0.8 mg) was used for all participants. About 1.5 ml of saliva were collected and analysed for uric acid, glutathione and total radical-trapping antioxidant capacity activity. 2.4. HPLC apparatus The HPLC apparatus consisted of a Jasco PU-980 pump connected to a Jasco FP-920 ¯uorescence detec-
tor (Jasco, Tokyo, Japan) and equipped with an Alltech Allsphere ODS-2, 150 4.6 mm, 5.0-mm particle size column (Alltech Italia S.r.l., Milan, Italy) provided with a guard column packed with the same matrix as the separative column and equilibrated with 20% acetonitrile in 0.050 M phosphate buer, pH 3.0. 2.5. Determination of uric acid Uric acid was measured by an enzymatic colorimetric method (Uric acid plus; Boehringer Mannheim, Mannheim, Germany) in an automatic analyser (Hitachi 917). 2.6. Determination of glutathione Glutathione was measured by HPLC according to the method of Araki and Sako (1987). In brief, 300 ml of saliva was treated with 30 ml of 10% (v/v) tri-nbuthylphosphine in dimethylformamide for 30 min at 48C in order to reduce thiols and to decouple them from proteins. The solution was then mixed with 300 ml of a 10% trichloroacetic acid solution containing 1 mM Na2EDTA under vigorous vortexing, followed by centrifugation at 2000 g for 5 min; 250 ml of 1.125 M borate buer, 20 ml of 1.55 M NaOH and 20 ml of ABD-F (1 mg/ml) were then added to 100 ml of the clear supernatant. The mixture was incubated in a shaking water-bath for 10 min at 508C to accomplish complete derivatization of glutathione and other thiols. At the end of the reaction, the solution was cooled and 20 ml injected into the HPLC apparatus. Glutathione concentration was then measured ¯uorimetrically at excitation wavelength of 385 nm and emission wavelength of 515 nm. 2.7. Measurement of total antioxidant activity Total radical-trapping antioxidant capacity was measured according to Santini et al. (1997). This method is based on the quenching of ABTS radical cation (ABTS +) by antioxidants. ABTS (150 mmol/l), metmyoglobin (2.5 mmol/l) and saliva (25 ml) were mixed, and the reaction started by the addition of H2O2 (75 mmol/l). ABTS + formation was continuously monitored at 208C by the absorbance increase at 734 nm. The delay between the addition of H2O2 (time zero) and the onset of the absorbance increase (ABTS + formation) was measured. All reagents were dissolved in phosphate buer treated with Chelex-100 and containing DTPA 0.1 mmol/l to prevent any metal-catalysed oxidation. The assay was standardized using Trolox.
B. Zappacosta et al. / Archives of Oral Biology 44 (1999) 485±488 Table 1 Saliva concentrations (mM; median and 25±75% percentile range) of glutathione, uric acid and total radical-trapping antioxidant capacity (TRAP) of smokers and non-smokers
Glutathione Uric acid TRAP a
Non-smokers
Smokers
pa
1.2 (0.4±1.7) 186 (78±228) 350 (250±450)
3.3 (1.2±5.1) 213 (150±285) 293 (196±587)
0.04 NS NS
Signi®cance test, Mann±Whitney U-test.
487
In Table 2, the eect of smoking a single cigarette on the saliva concentration of glutathione, uric acid and total radical-trapping antioxidant capacity is shown. Statistical analysis showed a signi®cant reduction of glutathione ( p < 0.05). Because saliva is rich in peroxidase, which in presence of H2O2 and hypothiocyanite anion could interact with the ABTS radical formation, we also examined total radical-trapping antioxidant capacity in the absence of metmyoglobin and found none, clearly indicating that in our system the presence of hypothyocyanite and salivary peroxidase is without any eect.
2.8. Statistics Data are expressed as medians and range (25th±75th percentiles). As concentrations were not normally distributed we used the Mann±Whitney U-test to evaluate dierences between groups (see Table 1), and the Wilcoxon matched-pairs test for paired samples (see Table 2). 3. Results Table 1 shows the salivary concentrations of glutathione, uric acid and total radical-trapping antioxidant capacity in the smokers and non-smokers. No statistically signi®cant dierence was found for uric acid concentration and total radical-trapping antioxidant capacity, but the glutathione concentration was signi®cantly higher in smokers. Glutathione and uric acid concentrations in saliva are of the same order of magnitude as in plasma. As uric acid is the main component of total radical-trapping antioxidant capacity in saliva it should be positively correlated with total radical-trapping antioxidant capacity: the correlation between uric acid concentration and total radical-trapping antioxidant capacity of saliva was highly signi®cant (r 2=0.93), while no correlation was found for the glutathione concentration (r 2=0.4).
Table 2 Saliva concentrations (mM; median and 25±75% percentile range) of glutathione, uric acid and total radical-trapping antioxidant capacity (TRAP) of smokers before and after smoking a single cigarette
Glutathione Uric acid TRAP a
Before
After
pa
3.3 (1.2±5.1) 213 (150±285) 293 (196±587)
1.1 (0.4±2.7) 177 (120±219) 358 (249±412)
0.04 NS NS
Signi®cance test, Wilcoxon matched-pairs.
4. Discussion Free radicals and highly reactive oxygen species produced by dierent sources such as in¯ammatory cells or environment pollutants are important mediators for many human disorders (Halliwell, 1994; Machlin and Bendich, 1987), and it is well known that inhalation exposure to environmental carcinogens such as polycyclic aromatic hydrocarbons or cigarette smoke is associated with an increased risk of disease. Cigarette smoke contains free radicals and free radical generators in both gaseous and particulate phases that can cause tissue damage by reacting with polyunsaturated fatty acids at cellular membranes and nucleotides at the DNA level (Lee et al., 1989; Bankson et al., 1993). Antioxidants, by counteracting the harmful activities of free radicals, protect structural and tissue integrity. The antioxidant status of an individual is therefore of great importance. Oral structures and tissues are susceptible to the noxious eects of many irritating compounds from polluted air. In particular the stress on buccal tissues is especially increased during active smoking. Oral tissues are the ®rst target for gaseous and particulate products of cigarette smoking, and several oral in¯ammatory and degenerative diseases that can lead to neoplastic transformation are associated with smoking. Even just a few cigarettes induce increased concentrations of tobacco metabolites (nicotine and cotinine) in saliva, plasma and urine, and modify various biochemical and biological functions (Greenberg et al., 1984; Wang et al., 1992; Scherer and Richter, 1997). The oxidant± antioxidant systems are rapidly modi®ed, leading to important biological changes ranging from alterations of membrane lipids to damage to DNA bases, that constitute the molecular basis for many diseases (Halliwell, 1994). Our results demonstrate that even one cigarette reduces the concentration of glutathione in saliva, which, however, returns to the pre-smoking value after 1±2 hr (data not shown). This observation adds to the existing data showing that tobacco smoking or ex-
488
B. Zappacosta et al. / Archives of Oral Biology 44 (1999) 485±488
posure to air pollutants decreases the antioxidant defence mechanisms of the upper airways or mouth (Housley et al., 1995; Kelly et al., 1996; Piythilake et al., 1994; Pryor, 1992). Housley et al. (1995) showed a reduction of urate concentration in nasal lavage following in vitro ozone exposure. About 4% of their participants were almost completely devoid of nasal uric acid. As our method for glutathione analysis measures both the reduced and the oxidized forms, the decrease of saliva glutathione concentration immediately after smoking one cigarette is probably due to the formation of conjugates between glutathione and compounds reacting with the sulphur atom of glutathione. If this analysis is correct, it could follow that individuals with already low pre-smoking glutathione concentrations are the most prone to the noxious eects of cigarette smoking. References Anderson, R., 1991. Assessment of the roles of vitamin C, Vitamin E and beta-carotene in the modulation of antioxidant stress mediated by cigarette smoke-activated phagocites. Am. Clin. Nutr 53 (1 Suppl), 358S±361S. Araki, A., Sako, Y., 1987. Determination of free and total homocysteine in human plasma by high-performance liquid chromatography with ¯uorescence detector. J. Chromatogr 422, 43±52. Arnold, R.R., Brewer, M., Gauthier, J.J., 1980. Bactericidal activity of human lactoferrin: sensitivity of a variety of microorganisms. Infect. Immun 28, 893±898. Bankson, D.D., Kenstin, M., Rifai, N., 1993. Role of free radicals in cancer and atheriosclerosis. Clin. Lab. Med 13, 463±480. Carlsson, J., 1987. Salivary peroxidase: an important part of our defense against oxygen toxicity. J. Oral. Pathol 16, 412±416. Federation Dentaire Internationale, 1992. Saliva: its role in health and disease. Int. Dent. J 42, 291±304. Ferguson, D.B., 1998. The eects of tobacco usage on saliva ¯ow rate and composition and on susceptibility to dental caries. Oral Disease 4, 60±63. Greenberg, R.A., Haley, N.J., Etzel, R.A., Loda, F.A., 1984. Measuring the exposure of infants to tobacco smoke. Nicotine and cotinine in urine and saliva. N. Engl. J. Med 310 (17), 1075±1078. Halliwell, B., 1994. Free radicals, antioxidants, and human disease: curiosity, cause, or consequence. The Lancet 344, 721±724. Hatton, M.N., Loomis, R.E., Levine, M.J., Tabak, L.A., 1985. Masticatory lubrication. Biochem. J 230, 817±820. Housley, D.G., Mudway, I., Kelly, F.J., Eccles, R., Richards, R.J., 1995. Depletion of urate in human nasal lavage fol-
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