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Transdisciplinary Tobacco Use Research Center and Cancer Center, University of Minnesota, Minneapolis, Minnesota
Request for reprints: Stephen S. Hecht, University of Minnesota Cancer Center, Mayo Mail Code 806, 420 Delaware Street SE, Minneapolis, MN 55455. Phone: (612) 624-7604; Fax: (612) 626-5135. E-mail: hecht002{at}umn.edu
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Abstract |
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Introduction |
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23% of the U.S. adult population continues to smoke, and this figure has changed little since 1990 (3). There are over 1 billion smokers worldwide and total consumption of cigarettes is still increasing, having reached 5.5 trillion in 2000 (2, 4). For those people who are unwilling or unable to quit smoking, would a reduction in the number of cigarettes/day (CPD) decrease their risk for lung cancer? One way to address this question is to measure lung carcinogen dose in people who reduced their smoking. Polycyclic aromatic hydrocarbons (PAH) are firmly established as a pervasive group of environmental carcinogens, which are likely causes of human cancers. Occupational exposures to mixtures such as soots and tars, which are known to contain significant amounts of PAH, cause cancers of the skin and lung in humans (5, 6). There is little reason to doubt that PAH play a significant role in the etiology of cancers caused by cigarette smoke. PAH are present in cigarette smoke and many of these compounds are potent locally acting carcinogens (7, 8). Fractions of cigarette smoke condensate enriched in PAH are carcinogenic in laboratory animals (9). These fractions, together with tumor promoters and cocarcinogens, account for much of the carcinogenic activity of cigarette smoke condensate (9). Levels of PAH-DNA adducts are higher in tissues of smokers than nonsmokers in some studies (10). The types and locations of mutations in the p53 tumor suppressor gene isolated from human lung cancer are similar to those induced in vitro by diol epoxide ultimate carcinogens of PAH (11). Thus, PAH, together with tobacco-specific nitrosamines, are considered to be causes of lung cancer in smokers (2, 12). Elimination of these compounds from cigarette smoke would undoubtedly reduce lung cancer incidence.
Biomarkers of PAH uptake and metabolic activation provide information on human dose of this class of pulmonary carcinogens. These biomarkers include DNA adducts, protein adducts, and urinary metabolites (13, 14). Among these, urinary 1-hydroxypyrene (1-HOP), a metabolite of the noncarcinogen pyrene that is always a component of PAH mixtures, is an accepted biomarker of carcinogenic PAH dose (14–16). Therefore, in this study, we measured urinary 1-HOP in people who reduced their smoking.
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Subjects and Methods |
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Eligible subjects were asked to visit the research clinic for orientation and to obtain informed consent. They were also scheduled to return for a more thorough screening. During the orientation visit, subjects completed a tobacco use questionnaire and medical history form, which was reviewed by a physician.
Subjects who met inclusion criteria monitored their use of cigarettes and other tobacco products daily for 2 weeks to assess baseline tobacco use. Subjects were instructed that it was important to smoke cigarettes at their normal levels during this time. They returned the next week for the baseline visit (week –1), when they were randomly assigned to the reduction group or the waitlist group. Baseline measures were again taken at the subsequent visit (week 0) and those who were assigned to the waitlist group continued to smoke as usual for another 6 weeks while those smokers assigned to the reduction group began treatment.
Subjects assigned to the waitlist group were the basis for an analysis of biomarker longitudinal stability. This group maintained and monitored smoking for 8 weeks. The first 2 weeks coincided with the baseline for the reduction group and the following 6 weeks coincided with the reduction phase (see Fig. 1). Subjects in the waitlist group were assessed on all dependent measures during the first two baseline visits and at weeks 4 and 6 after the onset of "treatment" in the reduction group. They then entered the reduction phase after the 6-week visit.
Subjects assigned to the reduction group were expected to reduce their CPD by 25% in the first 2 weeks, 50% in the subsequent 2 weeks, and 75% in the next 2 weeks. They were given 4 mg nicotine gum and instructed on methods for reducing cigarette smoking. They kept track of times of gum use in their diaries. At each visit, if they were within two cigarettes of goals, they were advised to continue on gum unless they had trouble with adverse events associated with gum use. Subjects who had difficulty reaching the 50% reduction goals were offered additional nicotine replacement. If a subject was unable to approach the 50% goal (e.g., by less than two cigarettes), he or she was offered the option of using a 14 mg Nicoderm CQ patch (SmithKlineBeecham, Philadelphia, PA) in conjunction with the nicotine gum for a 2-week period until the 75% reduction period. At the end of the 50% reduction point, if a subject was unable to approach reduction goal or expressed concern about further reduction to 75%, an option of using a 21 mg Nicoderm CQ patch in conjunction with the nicotine gum was offered. In addition to the pharmacological therapies, subjects met with a trained counselor during the clinic visits for brief individual sessions lasting no more than 10 min. During these sessions, a specific, structured format was followed in which questions were asked about any difficulties meeting harm reduction goals, and suggestions for accomplishing the goals were given.
After the 6-week reduction period, subjects who maintained baseline smoking rate or had increased smoking were discontinued from nicotine replacements and followed. Other subjects who demonstrated some reduction in smoking were advised to either sustain or reduce their level of smoking reduction to 
50% of their baseline smoking or to quit. Subjects were given nicotine replacement for another 6 weeks, during which time they were advised to use nicotine products as needed to sustain reduction or to quit, but with the goal of gradually reducing their use of nicotine gum over the 6-week period. After 12 weeks of being supplied with nicotine replacement products, they purchased their own products, if necessary. If at any time during or after the 6-week treatment sessions the subject reported wanting to quit, a target date was established, self-help treatment manuals were dispensed, and brief counseling was provided. Follow-up sessions were held at 8, 12, and 26 weeks after the initiation of reduction. Most of the dependent measures were assessed at 12 and 26 weeks.
Urine samples were collected to assess 1-HOP and anatabine. Carbon monoxide (CO) was determined in expired air. Subjective measures included a tobacco daily diary in which subjects were asked to record date and time of each cigarette and nicotine gum and the use of any other tobacco product. In addition, a tobacco use questionnaire concerned with current tobacco use status, number of 24 h quit attempts, and duration of abstinence during these quit attempts was also administered at the clinic visits. No attempt was made to monitor diet or environmental exposure to PAH. Cash was paid at each visit to maximize compliance. The amount paid per visit was dependent on the procedures for that visit.
Subjects were included in the biochemically verified groups if their anatabine levels were reduced by 30% or more at weeks 8 and 12 compared with baseline (17). Anatabine measurements were performed on all subjects who reported having reduced CPD by 40% or more during weeks 4–12 after baseline or by 40% or more by week 4 and 70% or more during weeks 6–12 after baseline. This validation test was applied only to subjects whose baseline level of anatabine was >3.5 ng/ml. In subjects whose urinary anatabine concentration was below this level, exhaled CO was used as a biomarker, and they were included in the biochemically verified groups if their CO levels were reduced by 30% or more at weeks 8 and 12 compared with baseline.
Biomarker Measurements
Alveolar CO was measured at each visit with the Bedfont Micro Smokerlyzer (Bedfont, Medford, NJ). Subjects were asked to hold their breath for 15 s and expire into the device.
First morning urine voids (80 ml) were collected in 4 oz. (118 ml) polyethylene specimen collection jars (Fisher Scientific Co., Pittsburgh, PA). Four 4.5 ml aliquots were transferred to 5 ml cryotubes (Corning, Inc., Acton, MA). One tube was reserved, while the other three were used for biomarker analyses. The urine remaining in the cup was also saved. Each sample was given a unique bar code label. All containers were maintained at –20°C until analysis. Anatabine was determined by a modification of a published method using 5-ethylnornicotine as internal standard (17). Further details of the method will be described separately. Cotinine and its glucuronide (total cotinine) were quantified as described (18). The relative SD for assay precision was 8.7%. 1-HOP in urine was determined by an improved method, which will be described separately.(19) The relative SD for assay precision was 4.13%, and added and measured levels of 1-HOP correlated (R = 0.999). 1-HOP occurs predominantly as conjugates in human urine, but these are hydrolyzed during the first step of the analysis. The data reported here represent the total of free and conjugated 1-HOP and are called 1-HOP in this article.
Statistical Analysis
Statistical analysis was primarily used to determine if a reduction in number of CPD would lead to a reduction in 1-HOP. Because the study was designed with each subject decreasing his or her CPD over time, time effects were examined. A linear mixed model for repeated measures was used to examine these time effects (20). Different covariance structures were considered and the structure with the lowest Akaike's information criterion value (21, 22) was selected in each model. 1-HOP was analyzed on the log scale to satisfy normality and constant variance assumptions. Confidence intervals for 1-HOP at each time point were constructed on the log scale and then back transformed to the original scale. Confidence intervals for percentage reduction in 1-HOP were constructed on the exponential scale and then back transformed to the original scale.
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Results |
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The number of subjects recruited into the study was 151. Of these, 102 were randomized to the reduction group and 49 to the waitlist group. Of the participants who were randomized to the reduction group, none dropped out prior to baseline measurement and 37 dropped out for various reasons during treatment (by week 12). Of the subjects in the waitlist group, 3 dropped out during the waitlist portion of the study and 13 dropped out during the reduction phase of the study. Ninety-eight of 151 subjects completed treatment through week 12 and 99 subjects achieved 40% reduction in CPD at both weeks 4 and 6. Sixty-five percent of all the participants who completed treatment were followed for 6 months.
Of the individuals who achieved 40% reduction at weeks 4 and 6, the mean (SD) age was 46.4 (9.44, range 20–66) years, number of cigarettes smoked at baseline was 23.7 (5.75, range 15.3–45.6), total cotinine level at baseline was 5380 (2900, range 804–15,300) ng/ml, duration of cigarette use was 28.3 (9.9, range 3.0–49) years, Fagerstrom Tolerance Questionnaire score was 4.44 (1.50, range 0–7.0), and mean number of quit attempts was 3.4 (5.6, range 0–50). When compared against those who dropped from the study, individuals who completed treatment were 5 years older (P = 0.0052) and started to smoke daily later (18.3 versus 16.6, P = 0.0089) and smoked 3.4 years longer (P = 0.06). There was no difference between these two groups in number of cigarettes smoked at baseline, number of years smoked, cotinine level, or Fagerstrom Tolerance Questionnaire score.
The study design called for staged reductions in CPD, 25% in the first 2 weeks after baseline, 50% in weeks 2–4, and 75% in weeks 4–6, followed by maintenance at this level, but not all participants could follow this protocol. Therefore, we defined subgroups of smokers who reduced their CPD to differing extents. The first subgroup reduced CPD by 40% or more during weeks 4–6 after baseline. The second reduced CPD by 40% or more during weeks 4–12 after baseline. The third reduced CPD by 40% or more at week 4 and by 70% or more during weeks 6–12 after baseline.
Levels of 1-HOP/ml urine and 1-HOP/mg creatinine in the urine of smokers who decreased CPD by 40% or more during weeks 4–6 after baseline, based on self-report, are summarized in Table 1A. Effects of reduction in CPD on 1-HOP/mg creatinine are shown in Fig. 2A. Significant reductions in 1-HOP concentrations were observed at all time points. The reductions in 1-HOP/mg creatinine at weeks 4 and 6 were 25% and 32% from baseline. Some smokers in this group relapsed after 6 weeks and were not able to maintain 40% reduction in CPD. Twelve percent (11 of 95) of subjects had relapsed at week 8, 21% (19 of 89) at week 12, and 56% (46 of 82) at week 26. Still, average percentage reductions in CPD at weeks 8, 12, and 26 after baseline were 66%, 63%, and 40%, respectively. The corresponding reductions in 1-HOP/mg creatinine at these time points were 29%, 25%, and 30%.
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Results for smokers who reduced their cigarette consumption by 40% at week 4 and by 70% at weeks 6–12 after baseline, based on self-report, are summarized in Table 1D and Fig. 2D. Although reductions in CPD were greater in this group than in the others discussed above, reductions in 1-HOP were generally less and were significant only at the 4-week time point. Anatabine measurements verified the reported reductions in 17 subjects in this group, but the reductions in 1-HOP in these subjects were not markedly different from those in the whole group.
Results of the analyses of the waitlist group were abstracted and are presented in Fig. 3. The difference between this analysis and those presented above is that reduction levels were compared with data for four baseline visits. There was no significant difference among levels of 1-HOP in urine during the four baseline visits. Mean reductions in CPD of 41%, 65%, 64%, 60%, and 34% were observed at weeks 4, 6, 8, 12, and 26 after baseline. There were significant reductions in mean 1-HOP/mg creatinine of 21%, 28%, 25%, and 31% at weeks 4, 8, 12, and 26 following baseline, respectively, and a nonsignificant reduction of 19% at week 6.
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Nineteen of our subjects achieved 50% or greater reduction of 1-HOP/mg creatinine and 1-HOP/ml urine at week 12 compared with their baseline level. The mean baseline level of 1-HOP in these subjects (group 1) was 2.50 pmol/mg creatinine, significantly greater (P < 0.0001) than the mean baseline level of 1.44 pmol/mg creatinine in 99 smokers (group 2) who did not achieve this reduction. Mean baseline CPD (23.9 in group 1 and 23.4 in group 2) were not significantly different, nor were mean percentage reductions in CPD (40.9 in group 1 and 31.1 in group 2).
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Discussion |
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This is the first study to quantify 1-HOP in the urine of smokers who reduced their CPD. Several other studies have examined the effects of reduced smoking on biomarkers. Benowitz et al. (27) investigated 13 people who smoked an average of 37 CPD at baseline and reduced to 15, 10, or 5 CPD for 3–4 days. Nicotine in blood, carboxyhemoglobin, and urinary mutagenicity were reduced but to a lesser extent than expected based on reduction in CPD. Several studies have measured reductions in CO in people who reduced their CPD, with results similar to those observed here (28). Hurt et al. (29) quantified biomarkers in a study of 23 subjects who used a nicotine inhaler to reduce smoking. Reductions in smoking were not associated with consistent reductions in 4-aminobiphenyl hemoglobin adducts or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in urine. NNAL glucuronides in urine were reduced at one time point and plasma thiocyanate levels increased. We have also measured NNAL, NNAL glucuronides, and NNAL plus NNAL glucuronides (total NNAL) in the urine of the subjects who participated in the present study (30). The data for percentage reduction in total NNAL (based on NNAL/mg creatinine) are summarized in Table 2. The observed percentage reductions of total NNAL and 1-HOP were generally quite similar (columns 4 and 5), with the exception of the smokers summarized in Table 1D in whom reductions in total NNAL were substantially greater than those of 1-HOP. Unlike 1-HOP, NNAL and NNAL glucuronides are tobacco-specific biomarkers, being metabolites of the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. When people stop smoking, their urinary total NNAL gradually decays and is ultimately not detectable (18). Therefore, although the observed reductions of total NNAL and 1-HOP were similar, the theoretical percentage reduction was often greater for 1-HOP than total NNAL (Table 2, columns 7 and 8)).
The reason for the lower than expected reductions in 1-HOP levels in the smokers summarized in Table 1D and Fig. 1D is unknown. One possible explanation relates to compensation, which may be greater in the smokers who reduced their CPD by 70% or more than in those who reduced by only 40% or more. If this were the case, however, we would have expected similar effects on levels of both total NNAL and 1-HOP, whereas in fact the observed percentage reductions were quite different (Table 2, columns 4 and 5)). It is also possible that there may have been undetected relapse to higher numbers of CPD than reported by some individuals in this group because reduction was biochemically verified in only 17 of these 30 subjects. However, the substantial reductions in total NNAL observed in this group are not consistent with that explanation. The lower than expected reductions of urinary 1-HOP may simply relate to other sources of exposure, which were fortuitously greater in this relatively small group.
While large reductions in urinary 1-HOP were not consistently observed in this study, 19 of our subjects did achieve 50% reduction at the 12-week time point and 31 achieved 30% reduction at the 26-week time point. Considering that most urinary 1-HOP other than that derived from smoking may result from dietary exposure (22, 23), these individuals likely substantially reduced their respiratory uptake of PAH. Because PAH are likely to contribute significantly to lung cancer in smokers, reductions such as these could be important, especially when one considers the magnitude of the smoking and lung cancer problem. If methods could be devised to achieve these reductions in a larger percentage of smokers, this could be a path toward cancer reduction in people who cannot stop smoking. Our results demonstrate that the group of 19 subjects who reduced by 50% or more at week 12 had significantly higher baseline 1-HOP than other subjects in the study but similar baseline CPD. This suggests that certain individuals who smoke intensely, thereby increasing pyrene dose, may particularly benefit from a smoking reduction strategy, although cessation is clearly a better option.
A strength of this study was the use of anatabine for biochemical verification of self-reported smoking. Because our subjects used nicotine replacement therapy, we could not employ cotinine measurements to evaluate changes in smoking. Anatabine is a tobacco alkaloid, which is present in cigarette smoke but not in medicinal nicotine products and can therefore be used for verification (17). In general, the results we obtained for subjects in whom reduced smoking was biochemically verified were similar to those from self-report (Table 1, B and C; Fig. 1, B and C). However, anatabine is not a perfect validation tool because its levels can be affected by compensation (e.g., large reductions in CPD might still produce only small reductions in anatabine). Another strength of this study was the waitlist group in which we had four baseline measurements of 1-HOP. The mean baseline values for 1-HOP were not significantly different from each other. However, the results shown in Fig. 3 clearly demonstrated a significant reduction in 1-HOP levels at most time points during the reduction phase compared with baseline. A weakness of this study is the 1-HOP biomarker itself. While the analytical procedure is robust in terms of precision and accuracy (19), 1-HOP is unfortunately not tobacco specific. This complicates interpretation of the data particularly because smoking only increases urinary 1-HOP levels by 2–3-fold. Unfortunately, there are no known tobacco-specific PAH compounds, so 1-HOP may still be one of the most practical biomarkers for evaluating uptake of this class of carcinogens.
In summary, our results demonstrate that substantial reductions in 1-HOP can be achieved in some people by reduction in CPD, but there was not a consistent relationship between these parameters. For most of our subjects, the observed percentage reductions in 1-HOP were relatively modest. Interpretation of the effects of smoking on urinary 1-HOP levels is complicated by sources of exposure to pyrene other than tobacco products.
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Acknowledgments |
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Footnotes |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 9/30/03; revised 12/22/03; accepted 1/16/04.
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