Life Sciences, Vol. Printed in the U S A
50, pp. 435-442
Pergamon P r e s s
'NORMAL' CIGARETTE SMOKING INCREASES FREE CORTISOL IN HABITUAL SMOKERS C. Kirschbaum*, S. Wrist*, C.J. Strasburger* * Department of Clinical and Physiological Psychology, University of Trier, Trier, Germany. ÷ Department of Internal Medicine, Klinikum Innenstadt, University of Munich, Munich, Germany. (Received in final form December 9, 1991)
Summarv In habitual smokers salivary cortisol responses to cigarette smoking were investigated. In the first study, 31 adults assigned to two experimental groups smoked either one or two cigarettes of their preferred brand. Mean salivary cortisol levels were significantly elevated after smoking of two cigarettes. In the second study, 10 smokers and 10 nonsmokers provided saliva samples at 20 min intervals over a 12-hr period. While environmental stimuli were paralleled in both groups overall cortisol output was significantly elevated in the smokers. These data suggest that 'normal' cigarette smoking can increase free cortisol levels. Tobacco is among the most widespread consummatory substances in modern societies. Although other pharmacological agents may also be involved, nicotine appears to play a crucial role in mediating pathophysiological and reinforcing consequences of smoking (1,2). Among other effects it is well documented that the concentration of numerous hormones change in response to acute nicotine exposure including serum levels of /3-endorphin, prolactin, luteinizing hormone, and vasopressin (3). As early as 1961, Htkfeld revealed first evidence for a stimulatory role of nicotine on hypothalamus-pituitary adrenal (HI'A) axis activity with increasing plasma cortisol and urinary 17-hydroxycorticosteroid levels following cigarette smoking (4). This finding was subsequently confirmed by several experiments in different laboratories (5-9) and the underlying mechanisms of nicotine-induced corticosteroid release have been partly elucidated. Nicotine probably stimulates hypothalamic cholinergic receptors leading to a release of corticotropin releasing hormone (CRH) which in turn stimulates ACTH secretion from pituitary corticotrophs with a subsequent cortisol release from the adrenal cortex (3). Alternatively, nicotine-induced vasopressin secretion may lead to ACTH release (10). Repeated nicotine consumption does not appear to prevent acute cortisol responses to smoking, since habitual smokers show dose-dependent cortisol increases following smoking (6,11-14). However, these effects have been observed in experimental settings using cigarettes with high nicotine content. In these studies cortisol levels increased only after smoking a minimum of
Dr. C. Kirschbaum, Dept. Clinical and Physiological Psychology, University of Trier, Building D, D-5500 Trier-Tarforst.
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press plc All rights r e s e r v e d .
436
Smoking and Free Cortisol
Vol. 50, No. 6, 1992
two 2.0 mg nicotine cigarettes within 10 minutes (12). Winternitz & Quillen (6) even asked their subjects to smoke eight 2.5 mg nicotine cigarettes within 2 hours to monitor HPA axis responses. Only in one study (15) a trend towards increasing cortisol levels was observed after smoking one usual-brand cigarette. Thus, the results available so far do not allow a definitive statement about the possible impact of "normal" smoking, i.e. subjects smoking their usually preferred brand of cigarettes with moderate nicotine content, on cortisol levels. Furthermore, to the best of the authors' knowledge only one study investigated the impact of cigarette smoking on overall cortisol excretion. Employing 24-hr urinary-free cortisol measures, Yeh & Barbieri (16) failed to observe statistically significant differences in cortisol excretion between smokers and nonsmokers. From these data one could draw the conclusion that cortisol is released only following high nicotine intake which appears to be rarely found under naturalistic conditions. Thus, cortisol levels of 'normal' smokers should parallel those of nonsmokers. Alternatively, it can be argued that the adrenal cortex may be stimulated by lower nicotine consumption, too, possibly leading to overall elevated mean cortisol levels. However, methodological problems like venipunctureinduced cortisol increases could have masked this low dose nicotine effect. Some of the methodological problems may be overcome by employing non-invasive measures of cortisol. With the advent of sensitive immunoassays cortisol levels can now be monitored stress-free from saliva samples closely reflecting the unbound cortisol fraction in blood (17-19). This technique allows for studies of adrenal activity under naturalistic conditions. Using the method of salivary cortisol determination we investigated the following questions: (a) Does 'normal' cigarette smoking, i.e. smoking the preferred brand of cigarettes, lead to elevated free cortisol levels? (b) If so, do smokers show higher mean cortisol concentrations than nonsmokers throughout the day given the same environmental stimulation? Methods Subjects and Experimental Protocols
Study 1: Investigating acute cortisol responses to smoking, 31 habitual smokers (18 females, 13 males; mean age 21.4 + 1.9 yrs) were studied. They had a mean dally cigarette consumption of 15 cigarettes and were smokers for at least six months. Detailed information about the study protocol was given and volunteers were paid for participation. They were not allowed to smoke for 1 hour prior to the experiment. All subjects were randomly assigned to one of two experimental conditions. After entering the laboratory at 4 p.m. the saliva sampling device "Salivette" (Sarstedt Inc, Rommelsdorf, F.R.G.) was introduced. This device mainly consists of a small cotton swab, on which the subjects gently chew for saliva flow stimulation. After 1-2 minutes the swab is removed and stored in a plastic tube for subsequent sample processing. This device does not alter salivary cortisol levels. For more details see (20). Two baseline saliva samples were obtained by the subjects themselves at -10 and -5 min. Thereafter, subjects in group #1 (n = 17; 7 males, 10 females; 5.0 + 1.9 yrs) smoked one cigarette and subjects in group #2 (n = 14; 6 males, 8 females; smoking for 5.9 + 2.2 yrs) smoked two cigarettes of their preferred brand. Nicotine content of the subjects' preferred cigarettes was 0.95 mg _ 0.20 (SD) per cigarette. Following smoking the volunteers rated their subjective well-being with respect to possible side effects of cigarette consumption, e.g.
Vol. 50, No. 6, 1992
Smoking and Free Cortisol
437
nausea and tachycardia. Moreover, they obtained a total of 10 saliva samples post smoking at 5 minute intervals. Study 2: Ten smokers and 10 nonsmokers (mean age 22.2 + 2.9 yrs; 4 males, 16 females; smoking for 4.6 + 1.6 yrs) were chosen for participation in a study of circadian cortisol rhythms. All subjects were first year psychology students and they were all investigated on the same day. Ten pairs (8 pairs gender-matched, 2 pairs of different sex) each consisting of one smoking and one nonsmoking subject were formed. Since it was attempted to study the subjects under naturalistic conditions, smokers were not overnight-deprived. The two volunteers of a pair spent the whole day together in their natural environment from 9 a.m. to 9 p.m. Thus, they attended the same lectures, took the same meals at identical times etc. At 20-minute intervals they obtained saliva samples essentially in parallel over 12 hours. In order to ensure that a regular sampling regimen was followed they were provided with alarm clocks which gave a signal every 20 minutes reminding the subjects to obtain the next saliva sample. Except for intense physical exercise the subjects were not restricted in their activities which had to be recorded in a protocol. Likewise, smokers recorded the total number of cigarettes smoked as well as the precise time of smoking. Sample Handling and Cortisol Assay Saliva samples were kept at -20" C until being assayed. After thawing samples were centrifuged at 4000 rpm for 1 minute resulting in a clear saliva sample of low viscosity and a volume of approximately 1-1.5 ml. For biochemical analysis all samples of one experimental group (Study 1) were measured in a single assay in duplicates. Cortisol was determined from these samples with a serum cortisol RIA adapted for use with saliva. Assay protocol and evaluation have been described in detail elsewhere (21). Saliva samples from Study 2 were assayed using a delayed time-resolved fluorescence immunoassay (DELFIA) which has been described recently (22). In a previous experiment, results obtained with this DELFIA were highly correlated with results obtained with the RIA employed in Study 1 (r = 0.96, n = 148; (23). Data Analysis For statistical comparison of cortisol levels between and within subjects analyses of variance with repeated measures (ANOVAs), Wilcoxon's signed-rank tests and Mann-Whitney U-test were computed. In Study 1, a corfisol response to smoking was defined as an increase of salivary cortisol levels of at least 2.5 nmol/1 above individual baseline. This criterion was chosen according to Weitzman et al. (24). In total plasma cortisol measures they viewed an increase of at least 55.2 nmol/1 (equals 2 #g/dl) as a secretory episode. However, since in saliva only about 2-5 % of total plasma cortisol levels are found (25) our criterion appears to be even more strict in order to minimize false positive results. Results
None of the volunteers reported side-effectsof smoking like tachycardia or nausea. T w o subjects (one in each group) had baseline cortisol levels of ~ 20 nmol/l, indicating a considerable activation of the adrenal cortex immediately before the startof the experiment. With more than 3 standard deviationsabove the mean values of theirrespective group, the two subjects were excluded from subsequent statisticalanalyses. Figure I shows mean baseline and stimulated salivary cortisollevels after smoking either one or two cigarettes of the subjects' preferred brand (groups #I and #2). A N O V A results indicated a significantgroup × time effect (F = 4.61; P < 0.001). Wilcoxon tests revealed a dose-dependent effect of
438
Smoking and Free Cortisol
Vol. 50, No. 6, 1992
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40
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Time After Smoking (min) Fig. 1 Saliva cortisol levels ( ± SE) in response to smoking of one (Group #1) or two (Group #2) cigarettes of the subjects' preferred brand.
smoking: while mean cortisol levels were not elevated in group #1, smoking of two cigarettes lead to significantly increased cortisol concentrations in group #2. In Study II, the smokers had a mean cigarette consumption of 12.5 ___ 2.7 (SD; range 9-18) throughout the 12 hour-period under investigation. They smoked cigarettes with a mean nicotine content of 0.86 mg + 0.21 (SD; range 0.6-1). Salivary cortisol concentrations measured at 20-minutes intervals under naturalistic conditions are shown in Fig. 2. Describing the profile of mean cortisol levels, smokers had higher cortisol concentrations at 35 out of 37 measures compared to nonsmokers. While cortisol levels tended to parallel those of nonsmokers during the four lectures, smokers appeared to show increasing cortisol concentrations during the breaks (30 min) between two lectures. As indicated by their 16
• o
Smokers ( n = 1 0 ) Non Smokers ( n = l O )
14
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Time (hrs) Fig. 2 Mean circadian rhythm of saliva cortisol levels ( ± SE) in pairs of smokers and nonsmokers who spent one day together sampling saliva at 20 min intervals from 9:00 a.m to 9:00 p.m.
Vol. 50, No. 6, 1992
Smoking and Free Cortisol
439
protocols, this had been the primary time for cigarette smoking. In both groups lunch lead to increasing cortisol levels (P < 0.05) with a tendency towards larger increases in smokers. For the area under the cortisol curve the Mann-Whitney U-test revealed overall greater cortisol levels for smokers over the 12-hour period (P < 0.05). Discussion In past studies significant effects of cigarette smoking on the HPA axis have only been observed when subjects were studied under experimental conditions smoking 2 or more high nicotine cigarettes of at least 2.0 mg nicotine each (6,12). Such a situation appears to be rather artificial since these cigarettes often are not commercially available and few smokers consume several cigarettes within a time span of 10 minutes or less. Only few studies attempted to investigate the impact of 'normal' smoking behavior on cortisol levels. Cherek and coworkers (26) studied four male cigarette smokers under four experimental conditions (smoking yes/no, working yes/no) and obtained two cortisol values from each subject in each 2-hr session. In order to mimic normal smoking behavior the volunteers were provided with their preferred brand of cigarettes. With this experimental protocol it may not be surprising that Cherek et al. failed to find significant alterations in cortisol levels following smoking. In contrast to these data our results suggest that under 'normal' conditions too, cigarette smoking can lead to increased cortisol levels in habitual smokers. We observed significant elevations in subjects smoking their preferred brand of cigarettes. It is noteworthy that baseline cortisol levels measured in the two experimental groups in Study 1 appeared to be rather high for unstimulated afternoon measures compared to data obtained from a larger adult population (19,27). At least two reasons could have accounted for this situation. The first explanation might be that the high baseline levels in Study 1 may reflect a certain degree of stress response to the experimental setting. It is well documented that anticipation of a potentially distressing situation can stimulate the adrenal cortex to release cortisol (28-31). Therefore, in our study as well as in other experiments anticipatory responses have to be taken into account as a contaminating variable masking smaller changes in cortisol concentrations, e.g. after 'normal' cigarette smoking. Similarly, if blood samples are needed for cortisol analysis, the stress of venipuncture can induce additional nonspecific cortisol responses (29,32,33). These and other consequences of certain experimental settings may explain some of the negative findings reported in the literature on the correlation of acute effects of smoking low nicotine cigarettes on cortisol levels. An alternative explanation for the high baseline cortisol levels could be that smokers per se show elevated cortisol levels during this time of day. The results from Study 2 support this possibility. If 'normal' cigarette smoking can lead to acute changes in cortisol concentration, do habitual smokers show an overall enhanced output of cortisol throughout the day? Investigating this question Yeh and Barbieri (16) obtained one 24-hr urine specimen from smokers and nonsmokers and found comparable cortisol excretion in both groups. However, since a variety of external conditions (e.g. physical exercise, job stress, nutrition) known as potent modulators of HPA axis activity have not been controlled in their study, the negative results are difficult to interpret. In our present study of circadian cortisol rhythms we therefore attempted to exclude these external, intervening variables by establishing pairs each consisting of one smoker and one nonsmoker who spent one day together, performing essentially the same activities. Although all subjects spent 6 out of 12 hours investigated under nonsmoking conditions (i.e. lectures), and the smokers smoked rather low nicotine cigarettes, we found a
440
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Vol. 50, No. 6, 1992
significantly larger mean corfisol level in smokers compared to their nonsmoking partners. With respect to absolute cortisol concentrations it has to be stressed that in our study smokers showed only moderately increased cortisol levels throughout the day when compared to nonsmokers. It could be assumed, however that larger differences in overall cortisol output are prominent in heavy smokers who are not restricted in their daily cigarette consumption by the environment. Moreover, cortisol levels should probably be monitored in smokers also before the first cigarette of the day in future studies. This may lead to a more definitive answer to the question whether cortisol levels are chronically elevated in smokers. Given a possible chronical hypersecretion of cortisol in habitual smokers, which physiological significance may this have? Besides other substances being released following smoking, nicotine-induced cortisol secretion may act directly upon central nervous structures in order to change processes like attention, memory, or mood. Such effects have been observed in several studies (34-37) probably reflecting some of the biochemical changes which may reflect the "gratifications of smoking" (1). In contrast, adverse effects of chronically elevated cortisol levels may include suppression of parameters of the immune system like natural killer cell activity (38) or a reduced responsiveness of the HPA axis to a variety of stimuli. In fact, Sellini and coworkers (8) revealed first evidence for attenuated cortisol responses in smokers. After lysin-8-vasopressin injection they observed smaller cortisol increases in smokers, however similar reactions were obtained following insulin induced hypoglycemia. In view of the hypothesis that glucocorticoids prevent the organism from overshooting defense reactions in stressful situations (39) chronic smoking thus could indirectly threaten homeostasis by blocking the g l u ~ r t i c o i d feedback loop. This intriguing hypothesis should be carefully investigated in future studies. Acknowledzement We are indebted to Prof. Dr. D. Hellhammer for rigorously supporting this study and to Mrs. I. Rummel-Friihauf and A. Fritzen for technical expertise and their invaluable help. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
S. SCHACHTER, Ann. Int. Med. 88 104-114 (1978). O.F. POMERLEAU and J. ROSECRANS, Psychoneuroendocrinology. 14 407-423 (1989). K. FUXE, K. ANDERSSON, P. ENEROTH, A. HARFSTRAND and L.F. AGNATI, Psychoneuroendocrinology. 14 19-41 (1989). B. HOKFELT, Acta Medica Scandinavica 369(Suppl) 123-124 (1961). P.E. CRYER, M.W. HAYMOND, J.V. SANTIAGO and S.D. SHAH, N. Engl. J. Med. 295 573-577 (1976). W.W. WlNTERNITZ and D. QUILLEN, J. Clin. Pharmacol. 17 389-397 (1977). V.V. GOSSAIN, N.K. SHERMA, L. SRIVASTAVA, A.M. MICHELAKIS and D.R. ROVNER, Am. J. Med. Sci. 291 325-327 (1986). M. SELLINI, S. BACCARINI, E. DIMITRIADIS, M.P. SARTORI and C. LETIZIA, Medicina. Firenze. 9 194-196 (1989). C.S. POMERLEAU, O.F. POMERLEAU, K. MCPHEE and E.M. MORRELL, Br. J. Addict. 85 1309-1316 (1990). O.F. POMERLEAU, J.B. FERTIG, L.E. SEYLER and J. JAFFE, Psychopharmacology Berlin. 81 61-67 (1983).
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11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
32. 33. 34. 35. 36.
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P. HILL and E.L. WYNDER, Am. Heart J. 87 491-496 (1974). J.N. WILKINS, H.E. CARLSON, H. VAN VUNAKIS, M.A. HILL, E. GRITZ and M.E. JARVIK, Psychopharmacology Berlin. 78 305-308 0982). L.E.J. SEYLER, J. FERTIG, O. POMERLEAU, D. HUNT and K. PARKER, Life Sci. 34 57-65 (1984). L.E.J. SEYLER, O.F. POMERLEAU, J.B. FERTIG, D. HUNT and K. PARKER, Pharmacol. Biochem. Behav. 24 159-162 (1986). O.F. POMERLEAU and C.S. POMERLEAU, Ciba. Found. Symp. 152 225-235 (1990). J. YEH and R.L. BARBIERI, Fertil. Steril. 52 1067-1069 (1989). R.F. VINING and R.A. MCGINLEY, Crit. Rev. Clin. Lab. Sci. 23 95-146 (1986). D. RIAD-FAHMY, G.F. READ, R.F. WALKER and K. GRIFFITI-IS, Endocr. Rev. 3 367-395 (1982). C. KIRSCHBAUM and D.H. HELLHAMMER, Neuropsychobiology 22 150-169 (1989). D.H. HELLHAMMER, C. KIRSCHBAUM and L. BELKIEN, Advanced Methods in Psychobiology, J.N. Hingtgen, D.H. Hellhammer and G. Huppmann (eds), 281-289, Hogrefe, Toronto (1987). C. KIRSCHBAUM, C.J. STRASBURGER, W. JAMMERS and D.H. HELLHAMMER, Pharmacol. Biochem. Behav. 34 747-751 (1989). R. DRF_~SF~IDORFER, C. KIRSCHBAUM, W.G. WOOD and C.J. STRASBURGER, J. Clin. Chem. Clin. Biochem. 28 653 (1991). C.J. STRASBURGER, R. DRF~SENDORFER and W.G. WOOD, J. Clin. Chem. Clin. Biochem. 28 663 (1991). E.D. WEITZMAN, D. FUKUSHIMA, C. NOGEIRE, H. ROFFWARG, T.F. GALLAGHER and L. I-IELLMAN, J. Clin. Endocdnol. Metab. 33 14-22 (1971). R.E. POLAND and R.T. RUBIN, Life Sci. 30 177-181 (1982). D.R. CHEREK, J.E. SMITH, J.D. LANE and J.T. BRAUCHI, Clin. Pharmacol. Ther. 32 765-768 (1982). J. BRANDTST,~DTER, B. BALTF~-GOTZ, C. KIRSCHBAUM and D.H. HELLHAMMER, J. Psychosom. Res. 35 173-185 (1991). J.W. MASON, Psychosom. Meal. 30 Suppl:576-Suppl:607 (1968). J.W. MASON, L.H. HARTLEY, T.A. KOTCHEN, E.H. MOUGEY, P.T. RICKETrS and L.G. JONES, Psychosom. Med. 35 406-414 (1973). H. BEN-ARYEH, R. ROLL, L. KAHANA, E. MALBERGER, R. SZARGEL and D. GUTMAN, Int. J. Psychosom. 32 3-8 (1985). H. LEHNERT, J. BEYER, P. WALGER, R. MURISON, C. KIRSCHBAUM and D.H. HELLHAMMER, Frontiers in Stress Research, H. Weiner, I. Florin, R. Murison and D.H. HeUhammer (eds), 257-260, Huber, Toronto (1989). R.M. ROSE and M.W. HURST, J. Human. Stress. 1 22-36 (1975). W. HUBERT, M. MOLLER and E. NIESCHLAG, Horm. Res. 32 198-202 (1989). P.J. O'CONNOR, W.P. MORGAN, J.S. RAGLIN, C.M. BARKSDALE and N.H. KALIN, Psychoneuroendocrinology. 14 303-310 (1989). J. BORN, V. HITZLER, R. PIETROWSKY, P. PAUSCHINGER and H.L. FEHM, Neuropsychobiology 20 145-151 (1989). D. VON ZERSSEN, P. DOERR, H.M. EMRICH, R. LUND and K.M. PIRKE, Eur. Arch. Psychiatry Neurol. Sci. 237 36-45 (1987).
442
37.
38. 39.
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I.M. BLACKBURN, L.J. WHALLEY, J.E. CHRISTIE, A. SHERING, M. FOGGO, J. BENNIE, D. FARRER, G. WATTS, H. WILSON and G. FINK, J. Affective Disord. 13 31-43 (1987). M.P. NAIR, Z.A. KRONFOL and S.A. SCHWARTZ, Clin. Immunol. Immunopathol. 54 395-409 (1990). A. MUNCK, P.M. GUYRE and N.J. HOLBROOK, Endocr. Rev. 5 25-44 (1984).
50, pp. 435-442
Pergamon P r e s s
'NORMAL' CIGARETTE SMOKING INCREASES FREE CORTISOL IN HABITUAL SMOKERS C. Kirschbaum*, S. Wrist*, C.J. Strasburger* * Department of Clinical and Physiological Psychology, University of Trier, Trier, Germany. ÷ Department of Internal Medicine, Klinikum Innenstadt, University of Munich, Munich, Germany. (Received in final form December 9, 1991)
Summarv In habitual smokers salivary cortisol responses to cigarette smoking were investigated. In the first study, 31 adults assigned to two experimental groups smoked either one or two cigarettes of their preferred brand. Mean salivary cortisol levels were significantly elevated after smoking of two cigarettes. In the second study, 10 smokers and 10 nonsmokers provided saliva samples at 20 min intervals over a 12-hr period. While environmental stimuli were paralleled in both groups overall cortisol output was significantly elevated in the smokers. These data suggest that 'normal' cigarette smoking can increase free cortisol levels. Tobacco is among the most widespread consummatory substances in modern societies. Although other pharmacological agents may also be involved, nicotine appears to play a crucial role in mediating pathophysiological and reinforcing consequences of smoking (1,2). Among other effects it is well documented that the concentration of numerous hormones change in response to acute nicotine exposure including serum levels of /3-endorphin, prolactin, luteinizing hormone, and vasopressin (3). As early as 1961, Htkfeld revealed first evidence for a stimulatory role of nicotine on hypothalamus-pituitary adrenal (HI'A) axis activity with increasing plasma cortisol and urinary 17-hydroxycorticosteroid levels following cigarette smoking (4). This finding was subsequently confirmed by several experiments in different laboratories (5-9) and the underlying mechanisms of nicotine-induced corticosteroid release have been partly elucidated. Nicotine probably stimulates hypothalamic cholinergic receptors leading to a release of corticotropin releasing hormone (CRH) which in turn stimulates ACTH secretion from pituitary corticotrophs with a subsequent cortisol release from the adrenal cortex (3). Alternatively, nicotine-induced vasopressin secretion may lead to ACTH release (10). Repeated nicotine consumption does not appear to prevent acute cortisol responses to smoking, since habitual smokers show dose-dependent cortisol increases following smoking (6,11-14). However, these effects have been observed in experimental settings using cigarettes with high nicotine content. In these studies cortisol levels increased only after smoking a minimum of
Dr. C. Kirschbaum, Dept. Clinical and Physiological Psychology, University of Trier, Building D, D-5500 Trier-Tarforst.
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press plc All rights r e s e r v e d .
436
Smoking and Free Cortisol
Vol. 50, No. 6, 1992
two 2.0 mg nicotine cigarettes within 10 minutes (12). Winternitz & Quillen (6) even asked their subjects to smoke eight 2.5 mg nicotine cigarettes within 2 hours to monitor HPA axis responses. Only in one study (15) a trend towards increasing cortisol levels was observed after smoking one usual-brand cigarette. Thus, the results available so far do not allow a definitive statement about the possible impact of "normal" smoking, i.e. subjects smoking their usually preferred brand of cigarettes with moderate nicotine content, on cortisol levels. Furthermore, to the best of the authors' knowledge only one study investigated the impact of cigarette smoking on overall cortisol excretion. Employing 24-hr urinary-free cortisol measures, Yeh & Barbieri (16) failed to observe statistically significant differences in cortisol excretion between smokers and nonsmokers. From these data one could draw the conclusion that cortisol is released only following high nicotine intake which appears to be rarely found under naturalistic conditions. Thus, cortisol levels of 'normal' smokers should parallel those of nonsmokers. Alternatively, it can be argued that the adrenal cortex may be stimulated by lower nicotine consumption, too, possibly leading to overall elevated mean cortisol levels. However, methodological problems like venipunctureinduced cortisol increases could have masked this low dose nicotine effect. Some of the methodological problems may be overcome by employing non-invasive measures of cortisol. With the advent of sensitive immunoassays cortisol levels can now be monitored stress-free from saliva samples closely reflecting the unbound cortisol fraction in blood (17-19). This technique allows for studies of adrenal activity under naturalistic conditions. Using the method of salivary cortisol determination we investigated the following questions: (a) Does 'normal' cigarette smoking, i.e. smoking the preferred brand of cigarettes, lead to elevated free cortisol levels? (b) If so, do smokers show higher mean cortisol concentrations than nonsmokers throughout the day given the same environmental stimulation? Methods Subjects and Experimental Protocols
Study 1: Investigating acute cortisol responses to smoking, 31 habitual smokers (18 females, 13 males; mean age 21.4 + 1.9 yrs) were studied. They had a mean dally cigarette consumption of 15 cigarettes and were smokers for at least six months. Detailed information about the study protocol was given and volunteers were paid for participation. They were not allowed to smoke for 1 hour prior to the experiment. All subjects were randomly assigned to one of two experimental conditions. After entering the laboratory at 4 p.m. the saliva sampling device "Salivette" (Sarstedt Inc, Rommelsdorf, F.R.G.) was introduced. This device mainly consists of a small cotton swab, on which the subjects gently chew for saliva flow stimulation. After 1-2 minutes the swab is removed and stored in a plastic tube for subsequent sample processing. This device does not alter salivary cortisol levels. For more details see (20). Two baseline saliva samples were obtained by the subjects themselves at -10 and -5 min. Thereafter, subjects in group #1 (n = 17; 7 males, 10 females; 5.0 + 1.9 yrs) smoked one cigarette and subjects in group #2 (n = 14; 6 males, 8 females; smoking for 5.9 + 2.2 yrs) smoked two cigarettes of their preferred brand. Nicotine content of the subjects' preferred cigarettes was 0.95 mg _ 0.20 (SD) per cigarette. Following smoking the volunteers rated their subjective well-being with respect to possible side effects of cigarette consumption, e.g.
Vol. 50, No. 6, 1992
Smoking and Free Cortisol
437
nausea and tachycardia. Moreover, they obtained a total of 10 saliva samples post smoking at 5 minute intervals. Study 2: Ten smokers and 10 nonsmokers (mean age 22.2 + 2.9 yrs; 4 males, 16 females; smoking for 4.6 + 1.6 yrs) were chosen for participation in a study of circadian cortisol rhythms. All subjects were first year psychology students and they were all investigated on the same day. Ten pairs (8 pairs gender-matched, 2 pairs of different sex) each consisting of one smoking and one nonsmoking subject were formed. Since it was attempted to study the subjects under naturalistic conditions, smokers were not overnight-deprived. The two volunteers of a pair spent the whole day together in their natural environment from 9 a.m. to 9 p.m. Thus, they attended the same lectures, took the same meals at identical times etc. At 20-minute intervals they obtained saliva samples essentially in parallel over 12 hours. In order to ensure that a regular sampling regimen was followed they were provided with alarm clocks which gave a signal every 20 minutes reminding the subjects to obtain the next saliva sample. Except for intense physical exercise the subjects were not restricted in their activities which had to be recorded in a protocol. Likewise, smokers recorded the total number of cigarettes smoked as well as the precise time of smoking. Sample Handling and Cortisol Assay Saliva samples were kept at -20" C until being assayed. After thawing samples were centrifuged at 4000 rpm for 1 minute resulting in a clear saliva sample of low viscosity and a volume of approximately 1-1.5 ml. For biochemical analysis all samples of one experimental group (Study 1) were measured in a single assay in duplicates. Cortisol was determined from these samples with a serum cortisol RIA adapted for use with saliva. Assay protocol and evaluation have been described in detail elsewhere (21). Saliva samples from Study 2 were assayed using a delayed time-resolved fluorescence immunoassay (DELFIA) which has been described recently (22). In a previous experiment, results obtained with this DELFIA were highly correlated with results obtained with the RIA employed in Study 1 (r = 0.96, n = 148; (23). Data Analysis For statistical comparison of cortisol levels between and within subjects analyses of variance with repeated measures (ANOVAs), Wilcoxon's signed-rank tests and Mann-Whitney U-test were computed. In Study 1, a corfisol response to smoking was defined as an increase of salivary cortisol levels of at least 2.5 nmol/1 above individual baseline. This criterion was chosen according to Weitzman et al. (24). In total plasma cortisol measures they viewed an increase of at least 55.2 nmol/1 (equals 2 #g/dl) as a secretory episode. However, since in saliva only about 2-5 % of total plasma cortisol levels are found (25) our criterion appears to be even more strict in order to minimize false positive results. Results
None of the volunteers reported side-effectsof smoking like tachycardia or nausea. T w o subjects (one in each group) had baseline cortisol levels of ~ 20 nmol/l, indicating a considerable activation of the adrenal cortex immediately before the startof the experiment. With more than 3 standard deviationsabove the mean values of theirrespective group, the two subjects were excluded from subsequent statisticalanalyses. Figure I shows mean baseline and stimulated salivary cortisollevels after smoking either one or two cigarettes of the subjects' preferred brand (groups #I and #2). A N O V A results indicated a significantgroup × time effect (F = 4.61; P < 0.001). Wilcoxon tests revealed a dose-dependent effect of
438
Smoking and Free Cortisol
Vol. 50, No. 6, 1992
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Time After Smoking (min) Fig. 1 Saliva cortisol levels ( ± SE) in response to smoking of one (Group #1) or two (Group #2) cigarettes of the subjects' preferred brand.
smoking: while mean cortisol levels were not elevated in group #1, smoking of two cigarettes lead to significantly increased cortisol concentrations in group #2. In Study II, the smokers had a mean cigarette consumption of 12.5 ___ 2.7 (SD; range 9-18) throughout the 12 hour-period under investigation. They smoked cigarettes with a mean nicotine content of 0.86 mg + 0.21 (SD; range 0.6-1). Salivary cortisol concentrations measured at 20-minutes intervals under naturalistic conditions are shown in Fig. 2. Describing the profile of mean cortisol levels, smokers had higher cortisol concentrations at 35 out of 37 measures compared to nonsmokers. While cortisol levels tended to parallel those of nonsmokers during the four lectures, smokers appeared to show increasing cortisol concentrations during the breaks (30 min) between two lectures. As indicated by their 16
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Time (hrs) Fig. 2 Mean circadian rhythm of saliva cortisol levels ( ± SE) in pairs of smokers and nonsmokers who spent one day together sampling saliva at 20 min intervals from 9:00 a.m to 9:00 p.m.
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protocols, this had been the primary time for cigarette smoking. In both groups lunch lead to increasing cortisol levels (P < 0.05) with a tendency towards larger increases in smokers. For the area under the cortisol curve the Mann-Whitney U-test revealed overall greater cortisol levels for smokers over the 12-hour period (P < 0.05). Discussion In past studies significant effects of cigarette smoking on the HPA axis have only been observed when subjects were studied under experimental conditions smoking 2 or more high nicotine cigarettes of at least 2.0 mg nicotine each (6,12). Such a situation appears to be rather artificial since these cigarettes often are not commercially available and few smokers consume several cigarettes within a time span of 10 minutes or less. Only few studies attempted to investigate the impact of 'normal' smoking behavior on cortisol levels. Cherek and coworkers (26) studied four male cigarette smokers under four experimental conditions (smoking yes/no, working yes/no) and obtained two cortisol values from each subject in each 2-hr session. In order to mimic normal smoking behavior the volunteers were provided with their preferred brand of cigarettes. With this experimental protocol it may not be surprising that Cherek et al. failed to find significant alterations in cortisol levels following smoking. In contrast to these data our results suggest that under 'normal' conditions too, cigarette smoking can lead to increased cortisol levels in habitual smokers. We observed significant elevations in subjects smoking their preferred brand of cigarettes. It is noteworthy that baseline cortisol levels measured in the two experimental groups in Study 1 appeared to be rather high for unstimulated afternoon measures compared to data obtained from a larger adult population (19,27). At least two reasons could have accounted for this situation. The first explanation might be that the high baseline levels in Study 1 may reflect a certain degree of stress response to the experimental setting. It is well documented that anticipation of a potentially distressing situation can stimulate the adrenal cortex to release cortisol (28-31). Therefore, in our study as well as in other experiments anticipatory responses have to be taken into account as a contaminating variable masking smaller changes in cortisol concentrations, e.g. after 'normal' cigarette smoking. Similarly, if blood samples are needed for cortisol analysis, the stress of venipuncture can induce additional nonspecific cortisol responses (29,32,33). These and other consequences of certain experimental settings may explain some of the negative findings reported in the literature on the correlation of acute effects of smoking low nicotine cigarettes on cortisol levels. An alternative explanation for the high baseline cortisol levels could be that smokers per se show elevated cortisol levels during this time of day. The results from Study 2 support this possibility. If 'normal' cigarette smoking can lead to acute changes in cortisol concentration, do habitual smokers show an overall enhanced output of cortisol throughout the day? Investigating this question Yeh and Barbieri (16) obtained one 24-hr urine specimen from smokers and nonsmokers and found comparable cortisol excretion in both groups. However, since a variety of external conditions (e.g. physical exercise, job stress, nutrition) known as potent modulators of HPA axis activity have not been controlled in their study, the negative results are difficult to interpret. In our present study of circadian cortisol rhythms we therefore attempted to exclude these external, intervening variables by establishing pairs each consisting of one smoker and one nonsmoker who spent one day together, performing essentially the same activities. Although all subjects spent 6 out of 12 hours investigated under nonsmoking conditions (i.e. lectures), and the smokers smoked rather low nicotine cigarettes, we found a
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significantly larger mean corfisol level in smokers compared to their nonsmoking partners. With respect to absolute cortisol concentrations it has to be stressed that in our study smokers showed only moderately increased cortisol levels throughout the day when compared to nonsmokers. It could be assumed, however that larger differences in overall cortisol output are prominent in heavy smokers who are not restricted in their daily cigarette consumption by the environment. Moreover, cortisol levels should probably be monitored in smokers also before the first cigarette of the day in future studies. This may lead to a more definitive answer to the question whether cortisol levels are chronically elevated in smokers. Given a possible chronical hypersecretion of cortisol in habitual smokers, which physiological significance may this have? Besides other substances being released following smoking, nicotine-induced cortisol secretion may act directly upon central nervous structures in order to change processes like attention, memory, or mood. Such effects have been observed in several studies (34-37) probably reflecting some of the biochemical changes which may reflect the "gratifications of smoking" (1). In contrast, adverse effects of chronically elevated cortisol levels may include suppression of parameters of the immune system like natural killer cell activity (38) or a reduced responsiveness of the HPA axis to a variety of stimuli. In fact, Sellini and coworkers (8) revealed first evidence for attenuated cortisol responses in smokers. After lysin-8-vasopressin injection they observed smaller cortisol increases in smokers, however similar reactions were obtained following insulin induced hypoglycemia. In view of the hypothesis that glucocorticoids prevent the organism from overshooting defense reactions in stressful situations (39) chronic smoking thus could indirectly threaten homeostasis by blocking the g l u ~ r t i c o i d feedback loop. This intriguing hypothesis should be carefully investigated in future studies. Acknowledzement We are indebted to Prof. Dr. D. Hellhammer for rigorously supporting this study and to Mrs. I. Rummel-Friihauf and A. Fritzen for technical expertise and their invaluable help. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
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