Abstract
Leanness has been shown to be related to an increased risk of some cancer forms, including lung cancer. However, biological evidence supporting a causal link between leanness and carcinogenesis is limited. The authors investigated longitudinally the association between body mass index (BMI) and levels of urinary 8‐hydroxydeoxyguanosine (8‐OHdG), a marker of oxidative DNA damage, using data from 174 healthy employees who participated in a lifestyle intervention study. 8‐OHdG levels were measured using automated high‐performance liquid chromatography and adjusted for urinary creatinine levels. Analysis of repeated measurements using a random effects model detected a statistically significant inverse association between BMI and 8‐OHdG levels (P = 0.003); one unit decrease in BMI was associated with a 2.7% (95% confidence interval 0.9–4.4) increase in 8‐OHdG levels. The association was pronounced among men consuming less than 20 cigarettes per day (8.8% increase per unit decrease in BMI) and among non‐smoking men (3.7% increase). The results based on a longitudinal observation suggest that weight loss is associated with increased oxidative DNA damage, a state presumably related to an increased risk of cancer. (Cancer Sci 2007; 98: 1254–1258)
An inverse relationship between body mass index (BMI) and cancer risk has been found consistently for several organs, including lungs( 1 , 2 , 3 ) and esophagus,( 4 ) and the strength of association appears to differ according to smoking status. These data suggest that leanness may be related to an increased risk of cancer, and that the association may be modified by smoking status. However, controversy continues over the causal role of leanness in carcinogenesis.( 5 ) For instance, weight loss may be the result of preclinical cancer progression. Moreover, because smoking is related to lower BMI levels,( 6 ) the increased risk of cancer associated with leanness may reflect smoke‐induced weight loss. Biological evidence linking leanness to carcinogenesis may provide a clue to this issue.
Oxidative DNA stress is thought to play a major role in carcinogenesis,( 7 ) and increased levels of 8‐hydroxydeoxyguanosine (8‐OHdG), a marker of oxidative DNA damage, have been detected in the urine of smokers( 8 , 9 ) and in lung cancer tissue.( 10 ) Our previous analysis using baseline data of an intervention study( 11 ) as well as other cross‐sectional studies( 8 , 9 ) have reported an inverse association between BMI and urinary 8‐OHdG levels. Moreover, a modifying effect of smoking on their relationship was also suggested.( 8 , 11 ) These findings are in accordance with the leanness–cancer hypothesis; however, causal inference is limited by the cross‐sectional nature of the study design. We took 8‐OHdG measurements on two additional occasions during the follow‐up period for the participants in the above‐mentioned study.( 11 ) Here, we report a longitudinal association of BMI and body fat with 8‐OHdG levels.
Materials and Methods
Data were obtained from a worksite lifestyle intervention study in a Japanese city office.( 11 ) A total of 179 volunteers aged 28–57 years who did not have any history of cancer, stroke, ischaemic heart disease or diabetes were invited to participate in baseline, 4‐month and 12‐month health surveys. Five participants who did not participate in the follow‐up surveys and data for the remaining 174 subjects were used for the analysis. Written informed consent was obtained from each participant. The study protocol was approved by the ethics committee of Kyushu University.
Health‐related lifestyles were ascertained using a detailed questionnaire. Ever smokers were defined as those who smoked 100 cigarettes or more in their lifetime. Current smokers consuming cigarettes on a daily basis were asked about cigarette consumption a day. Subjects were divided into non‐smokers (including past smokers) or current smokers (including occasional smokers who consumed cigarettes less than on a daily basis). Weight was measured in a light cloth, and information about height was obtained from the record of the latest checkup. BMI was calculated as bodyweight in kilograms divided by the square of height in meters. Percentage body fat was measured using the bio‐impedance method (Body Scan, Tanita).
Casual urine samples, collected mostly between 17:00 and 18:00 hours before dinner, were kept in tubes stored in a cooler box over night, and then frozen at –80ºC until analysis. Urinary samples were analyzed for 8‐OHdG using an automated high‐performance liquid chromatography (HPLC) system composed of anion‐exchange (HPLC‐1) and reverse‐phase (HPLC‐2) columns and an electrochemical detector.( 12 ) In short, the urinary 8‐OHdG level was determined using an apparatus in which pump 1 (Shiseido Nanospace SI‐2), the sampling injector (Gilson 231XL), the guard column for the HPLC‐1 (valve 1, pump 3), the HPLC‐1 column, the ultraviolet (UV) detector (Gilson UV/VIS‐155, 0.2 mm light‐path cell), the HPLC‐2 column (valve 2, loop, pump 2), and the electrochemical (EC) detector (ESA Coulochem 2) were connected. Urine samples were defrosted and 50 µL of each was mixed with the same volume of a dilution solution containing the ribonucleoside marker 8‐hydroxyguanosine (120 µg/mL) and 4% acetonitrile in a solution of 130 mM NaOAc (pH 4.5) and 0.6 mM H2SO4. The urine solutions were centrifuged at 13 000 r.p.m. for 5 min. A 20 µL aliquot of each supernatant injected into the first HPLC (MCI GEL CA08F, 7 µm, 1.5 × 150 mm, 2% acetonitrile in 0.3 mM sulfuric acid, 50 µL/min) from the sampling injector, via the guard column, and the chromatogram was recorded by the UV detector (245 nm). A aliquot of the fraction containing 8‐OHdG was automatically injected into the second HPLC column (Shiseido, Capcell Park C18, 5 µm, 4.6 × 250 mm, 10 mM sodium phosphate buffer [pH 6.7], 5% methanol, plus an antiseptic Reagent MB [100 µL/L], 1 mL/min). Finally, the 8‐OHdG was detected by an EC detector with a guard cell (5020) and an analytical cell (5011). Urinary creatinine levels of the same urine sample were measured simultaneously using anion exchange chromatography. The accuracy of the measurement, estimated from the recovery of an added 8‐OHdG standard, was 90–98%. When the same urine sample was analyzed three times, the variation of the data was within 7%.
8‐OHdG levels were adjusted for urinary creatinine levels and then log‐transformed before analysis. The Pearson correlation coefficient (r) was calculated for assessing the association between two continuous variables. To assess the longitudinal association between 8‐OHdG levels and BMI or percentage body fat over time, a random effects model was applied using the xtreg procedure (Stata), with age as a covariate. The random effects model was adopted because the repeated measurements of the subjects are mutually correlated, which violates the assumption of mutual independence of residuals in standard regression modeling. In the random effects model, the assumption of a fixed intercept of the linear model was relaxed and assumed to be randomly and normally distributed, whereas the regression coefficients of explanatory variables, or the effects of BMI or percentage boy fat and covariates on 8‐OHdG, were assumed to be fixed among subjects.( 13 ) Preliminary analysis revealed that the intervention was materially unrelated to changes in lifestyles, and thus no consideration was given to the intervention effect. The analysis was repeated according to sex and, in men, to smoking status. In the analysis according to smoking status, only data for men whose smoking status remained in the same category throughout the study period were used. Effect modification by sex or smoking was tested by adding a cross‐product term of BMI and each factor in the model. All statistical tests were two‐sided and were considered to be statistically significant at a P‐value less than 0.05. A point estimate and its 95% confidence interval were estimated using the maximum likelihood method. After taking an antilog of the regression coefficient, the percentage change of 8‐OHdG levels in the original scale was presented for 1 unit change in BMI or percentage body fat. All analyses were done with Stata, version 9.1.
Results
Of the 174 subjects, 37 (21%) were female. The mean (SD) age in years was 42 (7) for men and 39 (8) for women. Of the 137 men, 47 (34%) smoked at baseline and 23 (17%) consumed 20 cigarettes or more per day. Two women smoked occasionally at baseline. Smoking status (non‐smoker, smoker consuming less than 20 cigarettes per day, or smoker consuming 20 or more cigarettes per day) had changed in 10 men and 1 woman during the study period.
Table 1 shows the BMI, percentage body fat and 8‐OHdG levels during the study period. Mean BMI was higher among men than among women, whereas mean percentage body fat was higher among women than among men. There was no significant difference in the means of BMI and percentage body fat among the three surveys. At the 12‐month survey, 90% of subjects had a BMI within 5% of that measured at the baseline, whereas one‐half of the subjects showed a 5% or more change in percentage body fat.
Table 1.
Body mass index (BMI), percentage body fat and urinary 8‐hydroxydeoxyguanosine (8‐OHdG) levels and their changes during a 1‐year period
| Subjects | Variable | Baseline † | 4 months † | 12 months † | 1 year change † | Levels of change during 1 year (%) ‡ , § | ||
|---|---|---|---|---|---|---|---|---|
| Decreased | Stable | Increased | ||||||
| All subjects (n = 174) | BMI (kg/m2) | 23.9 (3.1) | 23.9 (3.0) | 23.8 (2.9) | –0.09 (0.94) | 6 | 90 | 5 |
| Body fat (%) | 22.8 (4.7) | 22.6 (4.6) | 22.4 (4.7) | –0.45 (1.85) | 32 | 50 | 18 | |
| 8‐OHdG (µg/g creatinine) | 3.9 (3.1, 5.1) | 3.6 (2.8, 4.6) | 3.8 (3.0, 5.2) | 0.04 (1.39) | 18 | 56 | 26 | |
| Men (n = 137) | BMI (kg/m2) | 24.6 (3.0) | 24.5 (2.4) | 24.4 (2.7) | –0.14 (0.98) | 6 | 91 | 4 |
| Body fat (%) | 21.8 (4.2) | 21.4 (4.0) | 21.3 (4.1) | –0.53 (1.92) | 36 | 46 | 18 | |
| 8‐OHdG (µg/g creatinine) | 3.9 (3.1, 5.0) | 3.6 (2.8, 4.6) | 3.9 (3.0, 5.2) | 0.04 (1.43) | 16 | 59 | 25 | |
| Women (n = 37) | BMI (kg/m2) | 21.5 (2.5) | 21.5 (2.5) | 21.6 (2.5) | 0.10 (0.75) | 5 | 86 | 8 |
| Body fat (%) | 26.6 (4.4) | 26.7 (4.3) | 26.5 (4.5) | –0.12 (1.58) | 16 | 68 | 16 | |
| 8‐OHdG (µg/g creatinine) | 3.9 (3.2, 5.2) | 3.4 (2.5, 4.6) | 3.8 (3.0, 5.5) | 0.01 (1.24) | 27 | 43 | 30 | |
| Non‐smoking men ¶ (n = 87) | BMI (kg/m2) | 24.2 (2.6) | 24.1 (2.6) | 24.0 (2.4) | –0.18 (0.97) | 6 | 90 | 5 |
| Body fat (%) | 21.3 (4.2) | 20.9 (4.0) | 20.6 (4.1) | –0.65 (2.11) | 38 | 43 | 19 | |
| 8‐OHdG (µg/g creatinine) | 3.6 (2.9, 4.7) | 3.6 (2.7, 4.5) | 3.6 (2.9, 4.8) | –0.04 (1.17) | 16 | 61 | 23 | |
| Smoking men ¶ (n = 40) | BMI (kg/m2) | 25.1 (3.6) | 25.0 (3.4) | 25.0 (3.1) | –0.09 (1.01) | 5 | 95 | – |
| Body fat (%) | 22.9 (4.1) | 22.5 (3.9) | 22.6 (4.0) | –0.34 (1.62) | 35 | 45 | 20 | |
| 8‐OHdG (µg/g creatinine) | 4.5 (3.6, 5.6) | 3.9 (3.2, 5.4) | 5.1 (3.8, 5.9) | 0.26 (1.94) | 15 | 55 | 30 | |
Median (interquartile range) was shown for 8‐OHdG levels at baseline, 4 months and 12 months; otherwise, the mean (SD) is shown.
Percentage body fat at the 12‐month survey was not available for one non‐smoking man who was thus excluded in the corresponding analyses.
BMI and percentage body fat: decreased ≤–5%, stable >–5% and <5%, increased ≥5%; 8‐OHdG: decreased ≤–20%, stable >–20% and <20%, increased ≥20%.
¶ Only 127 men who remained in the same smoking category throughout the study period are presented.
The sum of percentages may not equal 100 due to rounding error.
The levels of 8‐OHdG ranged from 1.2 to 11.4 µg/g creatinine (median, 3.9 µg/g creatinine), 1.1–9.4 µg/g creatinine (median, 3.6 µg/g creatinine), and 1.0–14.1 µg/g creatinine (median, 3.8 µg/g creatinine) at the baseline, 4‐month and 12‐month surveys, respectively. Lower levels of 8‐OHdG at 4 months were observed among women and smoking men but not among non‐smoking men. At the 12‐month survey, 26% subjects showed a 20% or more increase in 8‐OHdG compared to the baseline value, whereas 18% showed a 20% or more decrease.
In men, smokers had higher levels of 8‐OHdG than non‐smokers. The association between smoking and 8‐OHdG levels was examined longitudinally using a random effects model with smoking status at each survey included as an independent variable. Compared to non‐smokers, current smokers who consumed less than 20 cigarettes per day had a 16% higher mean of 8‐OHdG levels, and those who consumed 20 cigarettes or more per day had a 40% higher mean of 8‐OHdG levels.
Three measurements of 8‐OHdG during the 1‐year period were highly correlated with each other; the correlation coefficients between the baseline and 4‐month surveys was 0.80, and that between the baseline and 12‐month surveys was 0.79. Among the 77 subjects who had experienced little change in BMI (within 2%) and remained in the same smoking category at 12 months, the correlation coefficient between the baseline and 12‐month surveys was 0.85.
Figure 1 shows the relationship between percentage change in 8‐OHdG levels and BMI between the baseline and 12‐month surveys. A significant inverse association was observed between the two parameters (r = –0.20, P = 0.007). The correlation was stronger in non‐smoking men (r = –0.27) and male smokers consuming less than 20 cigarettes per day (r = –0.33). In contrast, a change in 8‐OHdG levels was not associated with a change in percentage body fat (r = –0.09, P = 0.26).
Figure 1.
Scatterplots of percentage change in 8‐hydroxydeoxyguanosine (8‐OHdG) against percentage change in body mass index (BMI) between the baseline and 12‐month surveys. Percentage change was calculated as the difference between the values at baseline and the 12‐month survey divided by the baseline value; minus indicates decrease, whereas plus indicates increase. Data for all of the study subjects (n = 174) are plotted. The curve shows the prediction for the change in 8‐OHdG (%) based on estimation of a fractional polynomial of the change in BMI (%) by a model with two power terms.
Longitudinal association of BMI and percentage body fat with 8‐OHdG levels is shown in Table 2. BMI was significantly inversely related to the levels of 8‐OHdG among all subjects; 8‐OHdG levels increased by 2.7% with a 1 unit decrease in BMI. The inverse association was statistically significant in men, whereas it was not significant in women; the interaction between sex and BMI did not reach statistical significance. Among men who remained in the same smoking category during the study period, the association between BMI and 8‐OHdG levels was statistically significant among current smokers consuming less than 20 cigarettes per day (8.8% increase in 8‐OHdG levels per unit decrease in BMI) and among non‐smokers (3.7% increase), whereas it was not significant among current smokers consuming 20 cigarettes or more a day (2.5% increase). The interaction between smoking status and BMI was not statistically significant (P for interaction = 0.11). The association between percentage body fat and 8‐OHdG levels was less clear, although there was non‐significant inverse relationship between them.
Table 2.
Longitudinal analysis † of the relationship between urinary 8‐hydroxydeoxyguanosine (8‐OHdG) levels to body mass index (BMI) and percentage body fat
| Subjects | n | % change in 8‐OHdG per change in body mass index (95% CI) | % change in 8‐OHdG per change in percentage body fat (95% CI) |
|---|---|---|---|
| All subjects | 174 | –2.7 (–4.4, –0.9) | –0.9 (–2.0, 0.1) |
| Men | 137 | –3.4 (–5.2, –1.4) | –1.1 (–2.4, 0.1) |
| Women | 37 | –2.0 (–7.3, 3.5) | –0.7 (–3.8, 2.4) |
| Non‐smoking men ‡ | 87 | –3.7 (–6.1, –1.2) | –1.3 (–2.7, 0.4) |
| Smoking men ‡ | 40 | –3.6 (–6.7, – 0.3) | –1.6 (–4.0, 0.9) |
| <20 cigarettes per day ‡ | 19 | –8.8 (–13.5, –3.8) | –3.3 (–6.8, 0.4) |
| ≥20 cigarettes per day ‡ | 21 | –2.5 (–6.5, 1.7) | –1.1 (–4.4, 2.4) |
Random effects model.
Only men who remained in the same smoking category throughout the study period were analyzed.
Discussion
Epidemiological studies have suggested that leanness is associated with an increased risk of some cancer forms. However, the leanness–cancer association is attributable to preclinical diseases, and this sets a limit to causal inference from epidemiological findings. Biomarker studies may provide a clue to the mechanism linking leanness to cancer risk.
The source of urinary 8‐OHdG may be the hydrolysis of 8‐OH‐dGTP by the nucleotide sanitization enzyme MTH1, the nucleotide excision repair of DNA, and the apoptosis of oxidatively damaged cells.( 14 , 15 ) It remains to be established whether an increase in urinary 8‐OHdG levels is associated with either higher levels of oxidized DNA or nucleotide precursors, or an enhanced capacity of 8‐OHdG repair enzymes. However, urinary excretion of 8‐OHdG is believed to reflect a general average risk of promutagenic oxidative adducts in the DNA of all tissues and organs,( 14 ) which is supported by epidemiological studies( 8 , 9 , 11 ) showing a considerable increase in the levels of urinary 8‐OHdG associated with smoking, a convincing carcinogenic factor.
In the present study, changes in BMI were significantly inversely associated with changes in 8‐OHdG levels. Although previous cross‐sectional studies (including ours( 11 )) have reported an inverse association between BMI and 8‐OHdG levels, such an association is attributable to confounding factors and may not be reproduced at an individual level. In this regard, our present findings from a longitudinal study are important because they provide direct evidence that a change in 8‐OHdG level is associated with a change in BMI. A pilot investigation also showed that leanness was associated with higher levels of aromatic DNA adducts among smokers.( 16 ) The leanness–cancer association in epidemiological studies is thus biologically plausible.
Two cross‐sectional studies( 8 , 11 ) reported a stronger association between BMI and 8‐OHdG levels among smokers than among non‐smokers, suggesting a modifying effect of smoking. In the present analysis, however, the results were somewhat different; there was a suggestion of stronger association between BMI and 8‐OHdG among light smokers and non‐smokers than among heavy smokers. We have no plausible explanation for this. Interestingly, the relationship between cigarette consumption and BMI was U‐shaped, showing lowest BMI levels among light smokers.( 6 ) We speculate that the differential effects of smoking on weight according to cigarette consumption may lead to a difference in the magnitude of the association between BMI and oxidative stress even among smokers. Epidemiological data are also conflicting on this point; some studies found a more pronounced leanness–cancer association among smokers compared to non‐smokers,( 2 , 3 ) whereas others reported a stronger association among non‐smokers.( 1 ) Likewise, we observed a somewhat stronger association between BMI and 8‐OHdG in men than in women. There was virtually no association between percentage fat mass and 8‐OHdG levels (Table 2), and women had a considerably greater proportion of fat mass than men (Table 1). Thus, the sex difference in the association between BMI and 8‐OHdG may be ascribed to that in body composition. However, as none of the subgroup analyses stated above reached statistical significance, a larger study is required to confirm whether the extent of association between BMI and oxidative DNA damage differs according to smoking status or sex.
The association of 8‐OHdG with percentage fat mass was weaker than that with BMI. This suggests that overall body mass may be a better predictor of oxidative DNA damage than fat mass. However, this inference may be limited because percentage body fat measured using the bio‐impedance method is subject to the conditions under which the measurement is made, and because it does not specify the location of fat deposition, either subcutaneous or visceral, which may determine the health impacts.
The mechanism by which weight loss induces oxidative DNA damage is not clear. The present subjects were healthy workers and, as our previous data showed,( 11 ) the inverse relationship between BMI and 8‐OHdG was observed up to BMI of 27 kg/m2. Thus, nutritional deficiency or reduced antioxidant capacity related to weight loss is not plausible. Oxidative DNA damage may be linked to weight loss through the mechanism of reactive oxygen species production. If weight loss is induced by an increase in energy expenditure or metabolic rate, there may be an elevation in mitochondrial production of reactive oxygen species in the cells,( 14 ) thereby leading to higher levels of oxidative stress. Exposure to substances like nicotine, which increases cellular metabolism,( 17 ) may lead to an increase in internal reactive oxygen species production as well as weight loss. Individual variation in susceptibility to such substances would produce an inverse association between bodyweight and oxidative DNA damage, even among individuals having similar exposure levels.
In theory, oxidative DNA damage can be involved in the pathogenic process of any cancer form. However, because the leanness–cancer association has been reported for lung cancer( 1 , 2 , 3 ) and squamous cell esophageal cancer,( 4 ) the oxidative DNA damage appears to be specifically related to tobacco smoke‐related carcinogenesis or squamous cell cancers. Non‐smokers, having potential exposure to various carcinogens including environmental tobacco smoke, are also at risk of developing these types of cancer through the same mechanism.
The advantages of our study are longitudinal design with repeated samplings from a healthy working population and the use of an automated HPLC method in measuring 8‐OHdG levels. The enzyme‐linked immunosorbent assay method is simple and cost efficient, but it produces two to four times higher values compared with those measured using HPLC, probably due to cross‐reactions with substances having similar structure to 8‐OHdG.( 18 ) Most HPLC methods developed so far are not be suitable for epidemiological research because of the complicated manual procedures involved (reviewed in( 12 )). The method we used is able to analyze large samples with reasonable reproducibility.( 12 ) In fact, the 8‐OHdG levels measured at three points in time during the 12‐month period were highly correlated each other, and the correlation was even higher among individuals who remained in the same levels of smoking and BMI over the study period. This suggests that urinary 8‐OHdG is quite stable over time, at least a 1‐year period, if no major changes occur that influence 8‐OHdG levels. However, the use of creatinine‐adjusted 8‐OHdG levels in assessing their relationship to BMI may be a concern because creatinine levels reflect muscle mass, which is closely related to age and sex. However, the age range of the study subjects was not wide and the analysis limited only to men showed an even stronger association. Moreover, the results were materially unchanged when no adjustment was made for creatinine levels (data not shown).
In conclusion, the present study based on a longitudinal observation found that levels of urinary 8‐OHdG, a biomarker of oxidative DNA damage, are inversely associated with BMI. Considering the potentially important contribution of oxidative DNA damage to carcinogenesis, this data lends support to the epidemiological finding of an increased risk of cancer associated with low BMI. Future studies should explore the mechanisms linking increased oxidative DNA damage to leanness and clarify cancer forms in which the risk of cancer increases as weight decreases.
Acknowledgments
This work was supported by the Third Term Comprehensive 10‐year Strategy for Cancer Control from the Ministry of Health, Labour and Welfare, Japan. Ms Tamami Hatano and the Safety and Health Division of Kitakyushu City Office are thanked for their help in conducting the survey, and Ms Sayumi Yamasaki is thanked for her skillful assistance.
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