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Departments of
* Epidemiology and
Urology, The University of Texas M. D. Anderson Cancer Center, Houston, TX;
** The University of Texas School of Public Health, The University of Texas Health Science Center, Houston, TX; and
Scott Department of Urology, Baylor College of Medicine and Methodist Hospital, Houston, TX
2To whom correspondence should be addressed. E-mail: xwu{at}mdanderson.org.
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-radiation–induced DNA damage in the peripheral blood lymphocytes using the comet assay. Overall, intake of total carotenoids was lower (P < 0.01) for bladder cancer cases (mean ± SD: 1273.4 ± 688.9) compared with healthy controls (1501.3 ± 791.5). When categorized into quartiles, there was an inverse association between increasing levels of carotenoid intake and bladder cancer risk with greatest protective effect [odds ratio (OR) = 0.56, 95% CI, 0.37–0.85] in the quartile with the highest level of intake. Baseline and mutagen-induced DNA damage was significantly higher in cases than in controls; when analyzed jointly with carotenoid intake, high DNA damage and low carotenoid intake were associated with the highest risk. For example, with high baseline DNA damage and low total carotenoid intake, the OR was 3.08 (95% CI, 1.64–5.77); with high baseline DNA damage and high total carotenoid intake, the risk was somewhat attenuated (OR = 2.49, 95% CI, 1.28–4.84). The risk was decreased further for low baseline DNA damage and low total carotenoid intake (OR = 2.18; 95% CI, 1.13–4.22). This study provides evidence of a preventive role for carotenoids in bladder cancer, and these data may have important implications for cancer prevention, especially for individuals susceptible to DNA damage.
KEY WORDS: • comet assay • DNA damage • molecular epidemiology • smoking
Over 600 carotenoids have been found in nature; 
40 of these are present in the typical human diet. Of these 40 carotenoids, only 14 and some of their metabolites have been identified in blood and tissues (1). The most common carotenoids in the human diet are 
-carotene, ß-carotene, ß-cryptoxanthin, lutein, lycopene, and zeaxanthin (2). The first 3, -carotene, ß-carotene, and ß-cryptoxanthin, can be converted into retinol and are thus called provitamin A carotenoids. In contrast, lutein, lycopene, and zeaxanthin have no retinol activity and are called nonprovitamin A carotenoids (2).
Carotenoids have a range of diverse biologic functions and actions and have an essential role in human health. Carotenoids also appear to have a variety of chemopreventive actions. In particular, they have antioxidant potential and are capable of immunoenhancement; they can reduce chromosome aberrations, inhibit the formation of premalignant lesions, regulate gap-junction communication between cells, and can reduce cell proliferation and transformation (1–11). However, at present, limited data exist on the mechanism of their effect on DNA damage and repair.
Case-control and prospective studies have also shown an association between the consumption of foods rich in carotenoids and a relatively low incidence of various cancers, including cancers of the lung, bladder, stomach, prostate, head and neck, and liver (12–21). In a review by Ziegler et al. (12), intake of fruits and vegetables, a major source of carotenoids in the diet, was associated with a lower lung cancer risk in all 8 prospective studies and in 18 of the 20 retrospective studies examined. Similarly, a meta-analysis of diet and bladder cancer by Steinmaus et al. (13) revealed a relative risk of 1.40 (95% CI, 1.08–1.83) and 1.16 (95% CI, 1.01–1.34) for those consuming a diet low in fruits and vegetables, respectively. Several cohort studies also showed inverse associations between total fruit consumption and bladder cancer risk (14–17). In contrast, the Netherlands Cohort Study (22) and a large 14-y follow-up study of 16,477 Swedish twins (23) found no association between fruit and vegetable intake and bladder cancer risk. Indeed, in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study Group (24), there was an increased incidence of lung cancer in those groups taking ß-carotene either alone, or with vitamin E. A similar adverse effect was seen in the Beta-Carotene and Retinol Efficacy Trial (25), in which smokers and/or asbestos workers were given ß-carotene and vitamin A. In yet, a 3rd trial conducted in health care professionals, no effect (either beneficial or adverse) of ß-carotene supplementation was seen (26). The reason for these conflicting results is unclear; despite these ambiguous findings, however, there is evidence supporting the notion that the consumption of particular fruits and vegetables is useful for the prevention, and perhaps the treatment of cancer (27–32). Therefore, further investigation into the putative effects of carotenoids in reducing cancer risk is warranted.
To date, few studies have evaluated the joint effects of carotenoid intake and latent genetic instability. Therefore, using subjects enrolled in an ongoing bladder cancer case-control study, we evaluated self-reported intake of carotenoids and latent genetic instability as assessed by the comet assay, which incorporates a diverse variety of DNA repair mechanisms from which DNA repair capacity can be inferred indirectly. We hypothesized in this study that individuals with low carotenoid intake and increased genetic instability would be at increased risk for bladder cancer.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Epidemiologic data. All study participants provided informed consent and completed a 45-min personal interview administered by M. D. Anderson staff interviewers. The interview elicited various information including demographics and smoking history. The questionnaire consisted of a fixed script and included introductory and transitional statements; all interviewers were trained in the use of probes. Approval for the use of human subjects was obtained from the M. D. Anderson, Methodist Hospital, and Kelsey-Seybold institutional review boards. For this specific analysis, approval was also obtained from The University of Texas Committee for the Protection of Human Subjects.
An individual who had smoked at least 100 cigarettes in his or her lifetime was defined as an ever smoker. Ever smokers included former smokers, current smokers, and those who had quit smoking within the previous year. For case subjects, a former smoker was a person who had quit smoking at least 1 y before diagnosis; for controls, a former smoker was a person who had quit smoking at least 1 y before the interview.
Dietary carotenoid assessment. M. D. Anderson staff interviewers also administered a separate 60-min FFQ to assess diet during the year before diagnosis in the cases or the year before enrollment in the study in the controls. The FFQ was derived from a modified version of the Health Habits and History Questionnaire (HHHQ)3 developed by the National Cancer Institute (33). The questionnaire includes a detailed food-frequency list including 135 food and beverage items, an open-ended section, and other dietary behavior questions such as vitamin and supplement use, dining at restaurants, food preparation methods, and ethnic foods commonly consumed in the Houston area. The validity and reliability of this questionnaire was documented previously (34,35). The DIETSYS + Plus (version 5.9) dietary analysis program, designed to accompany the HHHQ (36), was used for double key entry of all of the completed FFQs, with updated carotenoid values in accordance with the published values by Holden et al. (37).
For this analysis, carotenoid intake was adjusted by the daily energy intake (1000 kJ/d), and 3 separate carotenoid summary measures of daily intake [µg/(kJ · d)] were generated, 1 for total carotenoids, 1 for the provitamin A carotenoids, and 1 for the nonprovitamin A carotenoids. The total carotenoid intake was an estimate of the 6 major carotenoids that are at the highest concentration in blood in the U.S. population: -carotene, ß-carotene, ß-cryptoxanthin, lutein, lycopene, and zeaxanthin (2,37,38). The provitamin A carotenoid intake was comprised of the summation of -carotene, ß-carotene, and ß-cryptoxanthin and the nonprovitamin A carotenoid intake was comprised of the summation of lutein, lycopene, and zeaxanthin.
Tissue cultures and comet assay.
At the conclusion of the questionnaire interview, a 40-mL blood sample was collected into coded heparinized tubes. As previously described (39), blood tissue cultures were established immediately upon receipt of the blood sample and incubated for 72 h before the comet assay. Three cell cultures were set up for each study subject for separate measurements of baseline (without a mutagen challenge) and benzo[a]pyrene diol epoxide (BPDE)- and -radiation-induced comets.
We slightly modified the comet assay (39) under alkaline conditions from the original method described by Singh et al. (40). Briefly, a cell culture was mixed with agarose gel and adhered to a microscope slide. The cells were lysed by submersing the slides in freshly prepared lysis buffer for 1 h at 4°C to remove all of the cellular proteins. The slides were next placed in alkali buffer (pH > 12.0) to denature and unwind the DNA and express the alkali-labile sites. To separate the damaged DNA from the nuclei, a constant electric current was applied; the slides were then neutralized, fixed in 100% methanol, and stored in the dark at room temperature until ready for analysis. Ethidium bromide was used as the fluorescent dye that binds to the DNA, which allowed for the quantification of DNA damage.
Consecutive comet cells (n = 50) were manually selected and automatically quantified using the Komet 4.0.2 (Kinetic Imaging) imaging software attached to a fluorescent microscope. The Olive tail moment (41) was used as the parameter for DNA damage and is calculated by the imaging system software. The averages of the Olive tail moment for each subject were calculated for the baseline and mutagen-induced comets.
Statistical analysis.
All statistical analyses were performed using the Intercooled Stata 8.0 statistical software package (Stata Corporation). The 
2 test was used to test for differences between the cases and the controls in the distribution of gender, ethnicity, smoking status, income, and categorical variables for BMI (kg/m2), age, and pack-years smoked. The means ± SD were calculated for continuous variables, and Student’s t test was used to test for differences between the cases and controls in terms of age, pack-years smoked, BMI, dietary intake, and Olive tail moments. Odds ratios (ORs) and 95% CI were calculated as estimates of relative risk. Unconditional multivariate logistic regression analysis was performed to control for possible confounding by age, gender, ethnicity, smoking status, pack-years smoked, and total energy (kJ/d), where appropriate. Formal tests of multiplicative interaction were made using a multiplicative interaction term included in the multivariate model. To estimate the joint effects of carotenoid intake and DNA damage, as assessed by the comet assay, the continuous variables (i.e., carotenoids and comet tail moments) were treated as categorical variables by dichotomizing the data based on their control distributions so as to maintain reasonable numbers of cases and controls in the high and low risk strata. Carotenoid intake and the baseline- and BPDE-induced tail moments were dichotomized at the 50th percentile of the value in controls, whereas the -radiation–induced tail moment was dichotomized at the 75th percentile. Individuals whose values fell above the 50th percentile for carotenoids and baseline- and BPDE-induced tail moments and 75th percentile for -radiation–induced tail moment percentile were coded as being at risk. All statistical tests were two-sided.
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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75% of the bladder cancer patients and 74% of the control subjects (Table 1); Caucasians constituted 91% of the cases and 89% of the controls. The cases and the controls did not differ in age (P = 0.28), gender (P = 0.55), ethnicity (P = 0.82), or BMI (P = 0.12). Among the cases, 24% were current smokers compared with 8.6% for the controls, and cases smoked more than the controls. Comet data reflecting DNA damage were available on a subset of subjects in this study population. The baseline level of DNA damage was significantly higher in the cases than in the controls. Similarly, the cases had higher levels of BPDE- and -radiation–induced DNA damage compared with the controls. The CV could not be calculated because the blood cultures were assayed only once; however, in a previous study (42) using Epstein-Barr virus–transformed cell lines assayed in triplicate, the CV was 12.1 at baseline for the cell line from a healthy control and from a head and neck cancer patient. For BPDE-induced damage using concentrations of 1.5–3.0 µmol/L, the CVs ranged from 7.3 to 16.2 and 4.9 to 23.6 for the healthy controls and head and neck cancer patients, respectively.
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-carotene intake (P = 0.12) (Table 3). When carotenoid intake was stratified by gender, male cases reported lower intakes of ß-carotene, ß-cryptoxanthin, lutein, lycopene, zeaxanthin, nonprovitamin A carotenoids, and total carotenoids than did the male controls. In contrast, women with bladder cancer reported a lower intake of ß-carotenoid, provitamin A carotenoids, nonprovitamin A carotenoids, and total carotenoids than did the female controls.
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20 pack-years), individuals with low carotenoid intake had higher ORs than those with high carotenoid intake. These patterns were consistent for all 3 carotenoid summary measures.
Because both the baseline and BPDE-induced DNA damage were normally distributed, these 2 variables were dichotomized at the 50th percentile for the categorical analyses. However, the -induced DNA damage by case-control status revealed a slightly nonnormal distribution; therefore, we dichotomized the data at the 75th percentile to maintain adequate numbers of cases and controls in the high and low risk strata. When the tail moment was dichotomized, elevated tail moments were associated with significantly elevated ORs for both baseline DNA damage (OR = 1.67, 95% CI, 1.10–2.53) and -radiation–induced damage (OR = 1.57, 95% CI, 1.00–2.43), after controlling for age, gender, ethnicity, smoking status, and pack-years smoked (Table 6). The OR associated with BPDE-induced damage was elevated to a similar degree but was of borderline significance (OR = 1.46, 95% CI, 0.96–2.22).
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Overall, bladder cancer patients reported a significantly lower intake for each of the individual carotenoids compared with the healthy controls, although the difference in -carotene intake was not significant. Furthermore, except for lycopene, women had a higher intake of each carotenoid compared with men. It is not clear whether these observations indicate that women in this study ate a healthier diet because only subtle differences existed between men and women in their intake of total protein, total fat, saturated fat, and total carbohydrates, as well as in their average BMI (data not shown). It is difficult, however, to draw firm conclusions from these findings because women were underrepresented, comprising only 25% of the cases and 26% of the controls.
Because the human diet is typically quite heterogeneous, we chose to explore the intake of 3 separate carotenoid summary measures to assess the association of carotenoid intake with bladder cancer risk. This approach was also taken because there is evidence that provitamin A carotenoids possess biologic properties not shared by nonprovitamin A carotenoids; therefore, the provitamin A carotenoids may have a distinct effect on bladder cancer risk. As expected, cases had a lower intake of all 3 carotenoid summary measures (Table 3). By categorizing individuals into discrete groups, such as quartiles, we were able to estimate risk for a given level of carotenoid intake and attempted to identify possible thresholds of risk (Table 4). There tended to be an inverse association between cancer risk and increasing carotenoid intake. However, the data were unable to clearly demonstrate a threshold of carotenoid intake at which the greatest effect on risk occurred, although the general trend suggested convincingly that higher levels of carotenoid intake were associated with lower bladder cancer risk. Specifically, high total carotenoid and nonprovitamin A carotenoid intake were associated with a 44 and 43% decrease in bladder cancer risk, respectively, and a borderline nonsignificant 29% decrease in risk was found for a high intake of provitamin A carotenoids. There were no substantial differences in risk between men and women when the data were dichotomized by gender (data not shown).
Although prospective and retrospective studies (12–21) reported a lower risk of bladder cancer in subjects with high consumption of fruit and vegetables, 2 large prospective studies found no such relation (22,23); in a recent review by Zeegers et al. (18), the authors concluded that fruit consumption is likely associated with a small decrease in risk, whereas total vegetable intake probably has no effect on bladder cancer. Recently, a nested case-control study (43) and a large case-control study (20) found carotenoids to be protective for bladder cancer. After adjusting for pack-years of smoking, however, the ORs weakened and were not significant in the nested case-control study (43) but remained significant, in agreement with our findings, in the case-control study (20). On a whole, these conflicting results are a perplexing issue; however, attaining definitive evidence on the actual effects of specific dietary factors on risks of cancer is challenging because all forms of studies have limitations, and in most situations, no single form of evidence will provide definitive conclusions regarding diet and cancer associations. Thus, as Key et al. (21) recommend, the best conclusions will be based on careful and critical evaluation of all forms of evidence.
The association between carotenoid intake and smoking status is of interest. Analysis of the independent effects of smoking revealed that current smokers were at a 4.8-fold increased risk, whereas former smokers were at a 2-fold increased risk (data not shown). Further, regardless of carotenoid intake, current and heavy smokers were at a greater risk than former and never smokers, and there was a reduced risk in high carotenoid intake vs. low carotenoid intake for each stratum of smoking. Hence, these data demonstrate main effects for both smoking and carotenoid intake as well as effect modification in the joint effect analyses as these 2 factors are modified across strata. Due to the putative benefits associated with dietary carotenoids, it would be expected that high carotenoid intake would be necessary to exert protective effects even in the presence of smoking. Dietary carotenoids may prevent free radical–induced damage to DNA and counter the effects of cigarette smoke, which contributes to total oxidative stress (44,45). On average, active smokers have 25% lower circulating concentrations of carotenoids than never smokers (46), which may explain the substantial increase in bladder cancer risk, regardless of carotenoid intake, among smokers in our study. Also, Nomura et al. (43) recently reported that currently smoking healthy controls from a nested case-control had lower serum levels of carotenoids than nonsmokers. Handelman et al. (47) also showed that ß-carotene, ß-cryptoxanthin, lutein, lycopene, and zeaxanthin degrade in vitro when exposed to the gas phase of cigarette smoke. Furthermore, 2 clinical trials found ß-carotene supplementation to be associated with an increased risk of lung cancer among current smokers (24,25). Of interest, Palozza et al. (19) recently reported that in the presence of cigarette smoke condensate, ß-carotene induces in vitro DNA oxidative damage. It is difficult to draw any conclusions about those previous clinical trials compared with the present study because they examined a different cancer end point, utilized carotenoid supplementation as opposed to dietary assessment, and the study population comprised only smokers. If dietary carotenoids indeed have chemopreventive activity, adequate quantities would have to be consumed in the diet to lessen the adverse effects of cigarette smoking.
Many other biological mechanisms also exist to explain the chemopreventive actions of carotenoids. ß-Carotene, -carotene, lutein, and lycopene improve cellular gap-junction communication, which would improve homeostasis, cell-cell communication, and restrict the clonal expansion of initiated cells (48,49). Additionally, many carotenoids act as antioxidants by quenching free radicals and reactive oxygen species (31,49–51). ß-Carotene alone was shown to reverse carcinogenesis in the preinvasive stages of cancer and prevent cancer progression by affecting cellular differentiation and proliferation and improving immune response (51,52). -Carotene was shown to reduce the number of lung tumors in mice to 30% compared with a control group (53), prevent lipid peroxidation, inhibit the formation and uptake of carcinogens (51), and inhibit the growth of human neuroblastoma cell lines in vitro (50). ß-Cryptoxanthin also exhibited chemopreventive properties by quenching singlet oxygen (54,55), antipromoter actions (56), stimulating the expression of the RB and p73 genes (57), and protecting against mutagen-induced cancer (58). Short-term tomato intake, a rich source of lycopene, was shown in humans to decrease lipid peroxides (59), DNA strand breaks in circulating lymphocyte DNA (60), and oxidative DNA damage (61). Another possible source of chemopreventive activity from carotenoids may arise from vitamin A activity in the provitamin A carotenoids. Vitamin A has been associated with a variety of chemopreventive roles, such as the modulation of gene expression and enhancement of immune function, inhibition of kinase C activity in cancer cells, reduced in vitro expression of c-myc and H-ras oncogenes, induced cell differentiation, and growth inhibition in cancer cell lines (62–66).
Previous studies also showed that carotenoid supplementation decreased DNA damage in vitro and in vivo (4–10). For example, the lymphocytes of human subjects after carotenoid supplementation showed resistance to in vitro induced oxidative DNA damage (8). Similarly, a ß-carotene-retinol supplement given to Filipino betel chewers decreased the proportion of buccal mucosal cells with micronuclei to one third, whereas a control group that received no supplements did not change (9). Similarly, van Poppel et al. (10) showed that supplementation with ß-carotene in heavy smokers was associated with a 27% decrease in the frequency of micronuclei in exfoliated lung cells in sputum relative to micronuclei levels in a placebo group.
Various assays can be applied to measure DNA damage and repair. Previous case-control studies, including the current study, utilized the comet assay to show that latent genetic instability is associated with an increased risk for cancer (39,42,67–73). The comet assay, also called the single-cell gel electrophoresis assay, is a relatively simple high-throughput laboratory method that measures DNA damage in individual cells in vitro. Separate mutagen challenges were selected for this study because each mutagen induces a specific type of DNA damage and provides information on possible deficiencies in different DNA repair pathways. Specifically, -radiation induces single- and double-strand breaks that are repaired by base excision and/or double-strand break repair pathway(s), whereas BPDE induces DNA adducts that are repaired by the nucleotide excision repair pathway.
As expected, the peripheral blood lymphocytes from cases had higher levels of DNA damage than those from controls, both at baseline and after BPDE and -radiation exposure in vitro. Although carotenoid supplementation was shown to decrease DNA damage in vitro and in vivo (4–10), when we compared DNA damage for high carotenoid intake vs. low carotenoid intake, there was only a nonsignificant reduction in DNA damage for healthy controls (data not shown). Previous studies (4–10) that observed a reduction in DNA damage utilized carotenoid supplementation, which may have direct consequences for reducing DNA damage in laboratory assays.
When the data for carotenoid intake and DNA damage were analyzed jointly, individuals with a low carotenoid intake and high DNA damage were at the greatest risk for bladder cancer, but encouragingly, the results suggested that a high carotenoid intake reduces this risk among individuals susceptible to DNA damage. Although these observations were not consistent for all analyses, these inconsistencies likely pertain to sample size because DNA damage data were available for only a subset of the study population.
Limitations and sources of bias should be considered, including the use of FFQ data to estimate dietary carotenoid intake, and potential selection and recall biases. Dietary carotenoid intake is also significantly correlated with folate (data not shown); thus, we cannot exclude the possibility that this protective effect from carotenoids may have resulted in part from other micronutrient sources. Smoking may be another possible source of bias because smokers were shown to have less "healthy" dietary habits (74). Sample size was also an issue. Formal tests of multiplicative interaction were not significant (data not shown) because this sample size was underpowered to assess interaction, yet it was still reasonable to examine joint effects. Moreover, in the joint effects analyses, we were limited by sample size to dichotomizing the carotenoid and comet data.
In conclusion, although there was some difference in the degree of protection conferred by total carotenoids, provitamin A carotenoids, and nonprovitamin A carotenoids, this study provides further support for a chemopreventive role for carotenoids by demonstrating that a high intake of carotenoids was associated with an overall decrease in bladder cancer risk and also among individuals susceptible to induced DNA damage.
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FOOTNOTES |
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3 Abbreviations used: BPDE, benzo[a]pyrene diol epoxide; HHHQ, Health Habits and History Questionnaire; OR, odds ratio.
Manuscript received 3 June 2004. Initial review completed 8 July 2004. Revision accepted 21 September 2004.
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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