Is Low Selenium Status a Risk Factor for Lung Cancer?

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1 American Journal of Epidemiology Copyright 1998 by The Johns Hopkins University School of Hygiene and Public Health All rights reserved Vol. 148, No. Printed in U.S.A Is Low Selenium Status a Risk Factor for Lung Cancer? Paul Knekt, 1 Jukka Marniemi, 2 Lyly Teppo, 3 Markku Heliovaara, 1 and Arpo Aromaa 1 The hypothesis that low selenium may in some circumstances be a factor for lung cancer was investigated in a case-control study nested within a longitudinal study. Serum samples from 9,1 cancer-free individuals were collected and stored at -20 C by the Finnish Mobile Clinic in and During follow-up until the end of 1991,95 of lung cancer were diagnosed. Selenium concentrations were determined from the serum samples of the and 190, individually matched for sex, age, and place of residence. Mean levels of serum selenium in and were 53.2 /ug/liter and 57.8 /xg/liter, respectively. The relative of lung cancer between the highest and lowest tertiles of serum selenium, adjusted for smoking, serum alpha-tocopherol, serum cholesterol, serum copper, serum orosomucoid, and body mass index (kg/m 2 ), was 0.41 (95% confidence interval (Cl) ). The association was stronger at lower levels (<5.9 mg/liter) of alpha-tocopherol (relative = 0.24, ). The association was also pronounced among current smokers and at higher levels of serum orosomucoid and serum copper. The relative for smokers who were twice ranked in higher selenium tertiles, at an interval of 4-7 years, in comparison with smokers who remained in the lowest fertile was 0.16 ( ). In accordance with the hypothesis, the findings suggest that very low selenium status may contribute to the of lung cancer. Am J Epidemiol 1998; 148: follow-up studies; lung; neoplasms; selenium; serum Results from observational epidemiologic studies have suggested that high intake of fruits and vegetables rich in carotenoids is associated with a decreased of cancer (1). Accordingly, it seemed obvious that the main carotenoid, beta-carotene, provides protection against cancer and that only confirmation from intervention trials was lacking. However, results from three recent large-scale, double-blind, randomized intervention trials (2-5) have shown no benefit from beta-carotene supplementation. Thus, the associations found in observational studies may not be due to beta-carotene, but possibly to other beneficial substances in fruits and vegetables or to health behavior associated with the use of these foods. It is also possible that the beneficial effects of some antioxidant micronutrients are not revealed in observational studies due to confounding or other method- Received for publication September 3, 1997, and accepted for publication March, Abbreviations: Cl, confidence interval; RR, relative ; SD, standard deviation. 1 National Public Health Institute, Helsinki, Finland. 2 Social Insurance Institution, Turku, Finland. 3 Finnish Cancer Registry, Helsinki, Finland. Reprint requests to Dr. Paul Knekt, National Public Health Institute, Mannerheimintie 166, 000 Helsinki, Finland. ological issues. One such micronutrient is selenium. By inhibiting cell proliferation, having effects on immune response, or through other mechanisms, selenium may have an anticarcinogenic potential as part of the cellular defense system against oxidative damage (6). Accordingly, we found a significant inverse association between serum selenium and subsequent cancer occurrence within the cohort studied in the Finnish Mobile Clinic Health Examination Survey (7). A similar result was reported in one other longitudinal observational study from the Netherlands (8), whereas other similar published studies (9-12) have found no significant associations. As the findings from two recent large-scale intervention studies are in agreement with the suggestion that selenium has a protective effect against lung cancer, either alone (6) or in combination with beta-carotene and alpha-tocopherol (13), it could be questioned whether the lack of association in the observational studies is due to methodological issues. Because more information is now available about the population of the Finnish Mobile Clinic Health Examination Survey (7), we decided to carry out a new study on the association between serum selenium and lung cancer to investigate in more detail under what conditions the association can be demonstrated. 975

2 976 Knekt et al. MATERIALS AND METHODS During the period , the Finnish Mobile Clinic Health Examination Survey carried out multiphasic health examinations in 12 municipalities from different parts of Finland (14). Altogether, 17,551 men and women aged 19 years or more were invited to participate in the study. The participation rate was 90 percent. All participants completed a pr ed questionnaire which was checked at the baseline examination. The questionnaire yielded information about smoking status and alcohol consumption. Subjects were classified according to smoking status as never smokers, ex-smokers, smokers of cigars and pipe only, and smokers of <15 cigarettes a day, cigarettes a day, and ^25 cigarettes a day. Body height and weight were measured, and body mass index (weight (kg)/height (m) 2 ) was calculated. Serum samples were drawn, the cholesterol concentration was determined, and the serum samples were stored at 20 C. Information concerning subsequent incidence of lung cancer, available through the nationwide Finnish Cancer Registry (15), was linked to the data. Reporting to this registry has been obligatory since The registry receives information from hospitals, physicians, pathology laboratories, and death certificates, and practically every cancer case is reported to the registry. After exclusion of persons from which no frozen serum samples existed and persons who did not suffer from cancer during the baseline examination, the population consisted of 9,1 individuals. During a follow-up period of a maximum of 19 years up to the end of 1991, 95 of lung cancer (International Classification of Diseases, Seventh Revision, codes ) were diagnosed. Of the lung cancer, 90 were men and five were women. Among the, mean age (standard deviation (SD)) was 58 (9) years at baseline, 66 (9) years at diagnosis, and 74 (9) years at the end of follow-up. A case-control study design, nested within the cohort, was chosen. Two were selected for each cancer case by individual matching for sex, age, and place of residence. The were drawn from the same municipality as the case, and age was matched as closely as possible. Of the, 67 were fitted exactly and 19 differed by a maximum of 1-2 years. Of the remaining nine sets, one of the two differed by 3-9 years. Matching for place of residence also controlled for season of drawing the serum samples and the duration of storage of serum samples. A total of 190 were selected. For 240 individuals, the serum samples were kept frozen at 20 C until 1995, when they were thawed for analysis of selenium, alpha-tocopherol, retinol, copper, ferritin, and orosomucoid. The serum samples for the remaining 45 individuals were thawed and refrozen once during the storage. Serum samples for each case and its matched were analyzed independently of case-control status, of which the laboratory personnel were unaware. The concentration of serum selenium was determined by the graphite furnace technique utilizing the Zeeman background correction (16, 17). Alpha-tocopherol and retinol concentrations were determined simultaneously by high performance liquid chromatography (18). Serum copper and serum zinc were analyzed by a direct flame atomic absorption spectrophotometric method (Varian SpectrAA-640, Melbourne, Australia) (19). Serum ferritin was measured with a method based on the micro particle enzyme immunoassay technology using the IMX Ferritin assay kit (Abbott Diagnostics, Weisbaden- Delkenheim, Germany), and serum orosomucoid was measured with immunoturbidimetry (Orion Diagnostica, Espoo, Finland). The coefficients of variation for the determinations were: 9.7 percent for selenium, 3.9 percent for alpha-tocopherol, 2.3 percent for retinol, 4.3 percent for orosomucoid, 3.8 percent for copper, and 4.6 percent for zinc. Because the serum samples were stored for over 20 years, the effects of storage on the serum concentrations of selenium and alpha-tocopherol were evaluated. Serum samples were analyzed in 1985, refrozen, and reanalyzed years later. The reliability coefficient for serum selenium was high, 0.87, which suggests that the serum selenium concentrations were stable during storage (figure la). In accordance with results from other studies (20, 21), there was loss of alpha-tocopherol during storage. The mean level decreased significantly from 9.92 mg/liter to 7.72 mg/ liter (p < 0.001). Accordingly, the reliability coefficient for alpha-tocopherol was lower, 0.66 (figure lb). However, the correlation coefficient between serum cholesterol determined from fresh serum samples at the baseline and serum alpha-tocopherol determined from the same serum samples after 20 years' storage was 0.52, which suggests that the relative levels of alpha-tocopherol had persisted. Thus, the alphatocopherol determinations can be regarded as reliable enough for a study such as this. In , at an average of 5.7 years (range 4-7 years) before the baseline examination, the individuals of the present study had participated in a health examination survey (22). Selenium and alpha-tocopherol concentrations were determined from serum samples taken during that survey, and the biologic variations of these variables were evaluated by comparing the concentrations observed among in both surveys (figure 2). As in a previous study (23), the long-term

3 Low Selenium Status and Lung Cancer 977 R = 0.87 a) Selenium (/jg/l) 40 b) a-tocopherol (mg/l) R = ID FIGURE 1. Reproducibility of a) serum selenium and b) serum alpha-tocopherol determined from frozen (-20 C) serum samples thawed in 1985 (x-axis) and 1995 (y-axis). (Number of samples: selenium, 2, and alpha-tocopherol, 144; reliability coefficient = 0.87 for selenium and 0.66 for alpha-tocopherol; selenium mean levels (SD) = 64.8 ptg/liter (18.2) and 57.3 ptg/liter (20.5), respectively; alpha-tocopherol mean levels = 9.92 mg/liter (3.86) and 7.72 mg/liter (3.22), respectively). biologic persistence of selenium was low, with a reliability coefficient of 0.39 (figure 2a). Although the mean concentration of serum alpha-tocopherol was 13 percent less at follow-up, the corresponding coefficient for alpha-tocopherol was higher at 0.59 (figure 2b). Individuals in the lowest fertile of serum selenium in both surveys were defined as having a persistently low status. The relative of lung cancer between quartiles of serum selenium was estimated by the conditional logistic model (24). Adjustments for potential confounding factors and estimation of effect modification were performed by including the variables in the model. Tests for significance were carried out by the likelihood ratio test based on the model. The reliability and long-term persistence of serum selenium and serum alpha-tocopherol were estimated with the intraclass correlation coefficient (25). RESULTS The strongest association between potential factors and occurrence of lung cancer was observed for smoking (table 1). Significantly higher mean levels of serum orosomucoid and serum copper, and nonsignificantly lower levels of body mass index and serum selenium, were observed in than in. There was a significant inverse gradient between serum selenium level and lung cancer incidence (table 2). The smoking-adjusted relative between the highest and lowest tertiles of the micronutrient was 0.44 (95 percent confidence interval (CI) ). Further adjustment for serum alpha-tocopherol, serum cholesterol, serum copper, serum orosomucoid, and body mass index did not notably alter the results. The association persisted after exclusion of that occurred during the first 2 years of follow-up (adjusted relative (RR) = 0.41, 95 percent CI ). The association between serum alpha-tocopherol and of lung cancer was not significant. Study of the simultaneous effect of serum selenium and serum alpha-tocopherol on lung cancer incidence showed that there was an inverse association between serum selenium and lung cancer only at low levels of serum alpha-tocopherol (table 3). The adjusted relative was 0.29 (95 percent CI ) between higher and low serum selenium levels at serum alphatocopherol concentrations <5.9 mg/liter. The corresponding value at higher levels of serum alphatocopherol was 0.61 (95 percent CI ). The interaction was nonsignificant, however. The strength of association between serum selenium and lung cancer incidence varied between categories

4 978 Knekt et al. R = 0.39 a) Selenium 18 R = 0.59 b) a-tocopherol (mg/l) Q> 5 V) re a> 5 I CO First measurement I 1</> re I c o o Q> CO First measurement FIGURE 2. Long-term persistence of a) serum selenium and b) alpha-tocopherol determinations based on serum samples taken 4-7 years apart. First measurement (x-axis), second measurement (y-axis). ( samples: selenium, 153, and alpha-tocopherol, 161; reliability coefficient = 0.39 for selenium and 0.59 for alpha-tocopherol; selenium mean levels (SD) = 58.9 /xg/liter (22.9) and 58.0 /xg/liter (22.7), respectively; alpha-tocopherol mean levels = 7.60 mg/liter (2.82) and 6.63 mg/liter (2.40), respectively). of different variables: there was a significant inverse association among current smokers but not among nonsmokers (table 4). Similarly, an inverse association was observed at high levels of serum orosomucoid and serum copper, but not at lower levels of these two variables (table 4). The interaction was significant for serum orosomucoid (p = 0.02) and almost significant for serum copper (p 0.06). The strength of association was also dependent on the length of follow-up (table 5). The relative of lung cancer in the lowest tertile of serum selenium for the 20-year follow-up was significantly less than only for the first years of follow-up. The hypothesis that the real association between serum selenium and lung cancer among smokers may be hidden in observational epidemiologic studies because of low long-term persistence of serum selenium was tested by comparing the predictive values of low TABLE 1. Mean levels of different variables in lung cancer and their, Finnish Mobile Clinic Health Survey, Variable Males* (%) Current smokers (%) Age (years),* mean (SDf) Body mass index (kg/m 2 ), mean (SD) Alcohol consumption (g/day), mean (SD) Serum cholesterol (mg/dl), mean (SD) Serum ferritin (fig/liter), mean (SD) Serum orosomucoid (mg/liter), mean (SD) Serum copper (nmol/liter), mean (SD) Serum zinc ( imol/liter), mean (SD) Serum retinol (ng/iiter), mean (SD) Serum alpha-tocopherol (mg/liter), mean (SD) 92 Serum selenium (jjg/liter), mean (SD) 91 * Matching factor t SD, standard deviation Cases (8.8) 25.1 (3.8) 212(251) 280 (50) 91.2(65.2) 865 (239) 18.7(3.1) 12.5 (2.8) 609 (120) 6.77 (2.89) 53.2 (24.3) Controls (8.6) 25.8 (2.6) 209 (240) 272 (31) 81.9(44.2) 788 (190) 17.4 (2.9) 12.4 (1.6) 609 (2) 6.78(1.82) 57.8(16.9) p value for difference 0 <

5 Low Selenium Status and Lung Cancer 979 TABLE 2. s for lung cancer between tertiles of serum selenium and serum alpha tocopherol, Finnish Mobile Clinic Health Survey, Variable Serum selenium fertile (ng/liter) < >60.6 p for trend Smoking-adjusted analysis t Total model* Serum alpha-tocopherol fertile (mg/liter) < >7.6 p for trend The total model includes serum selenium, serum alpha-tocopherol, serum cholesterol, serum copper, serum orosomucoid, smoking, and body mass index, t Cl, confidence interval. TABLE 3. s (RR) for lung cancer between higher and tow levels of serum selenium and serum alpha-tocopherol, Finnish Mobile Clinic Health Survey, Serum selenium (ng/liter) <45.5 >45.5 No. of No. of Smoking-adjusted analysis Serum alpha-tocopherol (ng/liter) <5.9 RR t RR > No. of No. of RR 0.24 Total model* Serum alpha-tocopherol (ng/liter) <5.9 RR > * The total model includes serum selenium, semm alpha-tocopherol, serum cholesterol, serum copper, serum orosomucoid, smoking, and body mass index. f Cl, confidence interval. TABLE 4. s of lung cancer between higher and low levels of serum selenium* by categories of different variables, Finnish Mobile Clinic Health Survey, Variable Smoking Nonsmoker Current Orosomucoid, (mg/liter) <880 >880 Copper (nmol/liter) <15.8 > Smoking-adjusted analysis j Total modelt * Low level serum selenium is <45.5 jig/liter. t The total model includes serum selenium, serum alpha-tocopherol, serum cholesterol, serum copper, serum orosomucoid, smoking, and body mass index. i Cl, confidence interval. serum selenium levels measured 4-7 years before the beginning of the follow-up, at the baseline examination, with low serum selenium levels that persisted at both stages (table 6). The weakest prediction was observed for smokers with low serum selenium concentrations measured before the baseline stage (RR = 0.81, 95 percent Cl ). The corresponding value for persons with low levels only at the baseline

6 980 Knekt et al. TABLE 5. s of lung cancer between tertiles of serum selenium for different follow-up periods, Finnish Mobile Clinic Health Survey, Follow-up (years) Serum selenium < > Smoklng-adjusted analysis t Total model* < > * The total model includes serum selenium, serum alpha-tocopherol, serum cholesterol, serum copper, serum orosomucoid, smoking, and body mass index. f Cl, confidence interval. TABLE 6. s of lung cancer between higher and low levels of serum selenium measured at different stages in smokers, Finnish Mobile Clinic Health Survey, Time of measurement Stage 1: 4-7 years before baseline Stage 2: at baseline Stages 1 and 2 Category Lowf Higher Low$ Higher Both low Higher * Cl, confidence interval. t Serum selenium <47.5 ng/liter at stage 1. $ Serum selenium <45.5 ng/liter at stage 2. Serum selenium <47.5 ng/liter at stage 1 and <45.5 u.g/liter at stage 2. examination proper was 0.43 (95 percent Cl ). The strongest prediction (RR = 0.16, 95 percent Cl ) was observed for persons with persistently low serum selenium concentration. DISCUSSION We observed an inverse association between serum selenium concentration and lung cancer incidence. The association persisted after exclusion of that occurred during the first 2 years of follow-up, which suggests that the low serum selenium levels may not be due to occult lung cancer. The association also persisted after adjustment for several potential lung cancer factors, and is thus apparently not due to confounding by these factors. Our finding is in agreement with the finding of one intervention trial (6), which suggests that selenium provides protection against lung cancer. It is also in agreement with the findings of two nested case-control studies (7, 8). The lack of association in several observational studies (9-12) may be due to methodological issues. First, selenium may exert its protective effect only * when the basic level is low in relation to the oxidative stress (7). Second, the association may be present only in subgroups of effect-modifying factors and thus is not revealed in total populations. Third, selenium may provide protection only in certain circumstances or in combination with other antioxidants. Finally, associations estimated from one single measurement of serum selenium may underestimate the real effect, because of low long-term persistency. The serum selenium level of the present population was about 60 percent of the levels generally observed in other Western countries (26). Moreover, the Linxian intervention trial (27), which reported a suggestive protective effect of a supplement consisting of selenium, alpha-tocopherol, and beta-carotene, was carried out in an area with very low selenium levels. This suggests that the effect of selenium possibly exists only at low levels. On the other hand, the intervention trial by Clark et al. (6) used considerably higher doses of selenium. Accordingly, it seems that there must be some other factors which modify the possible effect of selenium. No significant reduction in lung cancer was associated with higher serum alpha-tocopherol

7 Low Selenium Status and Lung Cancer 981 concentrations in the present study. Although several case-control studies have found significantly lower vitamin E levels in lung cancer than in (28), the more reliable prospective studies found mainly nonsignificant associations (29-35). Studies that have suggested a protective effect were those by Menkes et al. (9) and the placebo group follow-up study of the ATBC intervention trial (2). It has been suggested (36) that vitamin E reduces oxidative damage seen in selenium deficiency. In accordance with that hypothesis, we found a stronger inverse association between serum selenium and lung cancer in the subpopulation of individuals with low serum levels of alpha-tocopherol. The results from the Linxian intervention trial are also in accordance with the hypothesis, because a combination of selenium, alpha-tocopherol, and beta-carotene apparently provided protection against lung cancer (13). However, other intervention trials have found that alpha-tocopherol (2) or beta-carotene (2, 4, 5) without selenium do not provide protection. The suggestion that antioxidants combined provides protection is supported by the results from a previous study of the Finnish Mobile Clinic Health Examination Survey cohort (37). In that study, there was an inverse association between the combined index of alphatocopherol, beta-carotene, retinol, and selenium and occurrence of lung cancer. This effect was pronounced in nonsmokers. As expected, we observed a very high of lung cancer among smokers. Furthermore, among lung cancer patients, we found elevated levels of the acute phase protein orosomucoid, and of serum copper, which was mainly transported by another acute phase protein, ceruloplasmin (38). These associations are also in agreement with previous findings (39-41). Our analysis of the association between serum selenium concentration and lung cancer in subgroups of these variables suggests that the possible effect of selenium on lung cancer could be modified by the effects of certain factors, or described by potential indicators of elevated lung cancer or progressing disease. Accordingly, among current smokers, the of lung cancer in the highest tertile of serum selenium was only 25 percent of the in the lowest tertile, whereas no inverse association was observed in nonsmokers. Similarly, the potential benefit of serum selenium was observed only at high levels of serum orosomucoid and serum copper. Thus, the lack of association between serum selenium and lung cancer in some studies may possibly be due to a different distribution of such factors. The finding by Clark et al. (6) of a protective effect of selenium supplementation among individuals with skin cancer is in agreement with this suggestion. The association between serum selenium and lung cancer was strongly dependent on the length of followup. During the first years of follow-up, there was an 84 percent reduction in the in the highest quartile compared with the lowest quartile, whereas no significant reduction was observed during later years. This result can be explained by the low long-term persistence of selenium status observed both by us and others (23). Our finding of a strong increase among persons with a persistently low serum selenium level is also in agreement with that hypothesis. Several limitations in studies such as the present investigation should be taken into account when interpreting the results. First, we can not exclude the possibility that the association that we observed between serum selenium and lung cancer may be due to other dietary factors or to health behavior associated with dietary patterns. Second, although the results were adjusted for the main factor for lung cancer, smoking, there may be other confounding factors that were not considered in this study. Third, because the data on smoking were based on a questionnaire with broad categories and because smoking behavior may have changed during the long follow-up, residual confounding may have remained even after adjustment for smoking. Finally, the sera had been stored for over 20 years, and some of the sera samples had once been thawed and refrozen before the determinations were made. This has decreased the serum alpha-tocopherol levels, and the reduction may have been influenced by the presence of other serum components. The information available, however, suggests that the reduction has been similarly distributed over the whole distribution of alpha-tocopherol values. In summary, individuals with a low serum selenium concentration had an elevated of lung cancer. The inverse association was pronounced among smokers and among persons with high levels of serum orosomucoid or serum copper. Higher serum selenium was associated with a reduced only at low alphatocopherol levels. The association between serum selenium and lung cancer was weak during later years of the follow-up, apparently because of strong biologic variations in the selenium values. Accordingly, persons with a persistently low serum selenium level had a pronounced of developing lung cancer. In conclusion, the results suggest that different methodological issues may conceal the association between selenium status and lung cancer occurrence in observational studies, and that low selenium status in some circumstances may be a factor in causing lung cancer.

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