Dietary fruit and vegetables protect against somatic mutation in vivo, but low or high intake of carotenoids does not

Fredrik Nyberg1,3, Sai-Mei Hou2, Göran Pershagen1 and Bo Lambert2

1 Division of Environmental Epidemiology, Institute of Environmental Medicine, Karolinska Institute, Box 210, SE-171 77 Stockholm
2 Center for Nutrition and Toxicology, Department for Biosciences, Novum, Karolinska Institute, Huddinge, Sweden

3 To whom correspondence should be addressed Email: fredrik.nyberg{at}imm.ki.se


    Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Epidemiological studies have demonstrated protective effects of vegetables and fruit on risk of cancer, but underlying mechanisms remain unclear. Intervention studies have in some cases contradicted previous epidemiological evidence, e.g. for beta-carotene supplementation and lung cancer, emphasizing the need for mechanistic data. We assessed in vivo mutagenic effects of several dietary items using the HPRT (hypoxanthine-guanine phosphoribosyl transferase) gene assay with T-lymphocytes from 312 individuals (158 lung cancer cases, 154 population controls), who provided information on diet and smoking habits. HPRT mutant frequency (MF) was significantly decreased in relation to intake of vegetables, citrus fruits and berries, respectively, as well as calculated vitamin C intake from diet. There was a significant U-shaped association with dietary carotenoid intake, with lowest MF near population average carotenoid intakes and higher mutation frequencies both at low and high intakes, and a similar borderline significant association was observed for beta-carotene. Our study is consistent with known diet–cancer associations and provides novel human in vivo mechanistic support for a cancer-protective effect of vegetables and fruit by modulation of somatic mutagenesis. Our results also provide support for the increase in lung cancer risk observed particularly in smokers in studies of beta-carotene supplementation.

Abbreviations: CI, confidence interval; HPRT, hypoxanthine-guanine phosphoribosyl transferase; MF, mutant frequency.


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Numerous epidemiological studies have demonstrated a protective effect of vegetable or fruit consumption on cancer risk (1,2). Blood levels of micronutrients and vitamins including beta-carotene have also been inversely correlated with cancer risk (2,3). More recently, however, large intervention studies with beta-carotene supplementation (47) found no clear protection against cancer or cardiovascular disease, and two studies were terminated because mortality or lung cancer incidence increased in the supplemented group (6,7). Subsequent analyses suggested that the deleterious effect might occur primarily among heavy current smokers and/or alcohol drinkers and asbestos-exposed subjects (8,9). According to one hypothesis this represents a pro-oxidant interaction effect of beta-carotene with such exposures (10,11). It could also be related to a high supplementation dose and unnaturally high serum levels (12).

The mechanisms underlying a cancer protection by fruit and vegetables are still uncertain. The antioxidative properties of a wide variety of fruit and vegetable micronutrients are considered to play a large role (1315). In experimental systems, several micronutrients including carotenoids have been shown to reduce DNA damage and to have antimutagenic and anticlastogenic effects (1619). These results suggest that the protective effect of fruit and vegetables against cancer may be due to their ability to reduce the somatic mutation rate. However, only limited evidence is available regarding dietary influences on human in vivo mutagenesis. Intracellular glutathione and ascorbate was shown in one study to correlate negatively with oxidative DNA damage (20) and although tomato consumption did not affect total plasma antioxidant capacity (21), oxidative damage as measured by the ‘comet’ assay appeared to be reduced by consumption of tomato, spinach or carrot; and the latter also reduced oxidative base damage in other studies (2224). Recently, vegetable and citrus fruit intake were shown to be inversely associated with mutations in the von Hipple-Lindau (VHL) tumour suppressor gene in tumour cells from patients with renal cell carcinoma, suggesting that such intake could protect the renal VHL gene against mutational insults that may be endogenous or common in the population (25).

The X-linked HPRT (hypoxanthine-guanine phosphoribosyltransferase) gene in T-lymphocytes has been used extensively as a reporter gene for human in vivo mutagenesis and biomarker for genetic effects of genotoxic exposures in human populations (26). The assay combines T-cell cloning with selection in medium containing 6-thioguanine, which allows the determination of HPRT mutant frequency (MF) and subsequent analysis of the mutations at the molecular level (27,28). Two major factors influencing the HPRT MF are donor age and smoking. Radiotherapy, chemotherapy, radiation accidents and occupational exposures to genotoxic chemicals have also been reported to increase the MF (review in ref. 26), and passive maternal exposure to tobacco smoke has been associated with characteristic HPRT deletions in cord blood T-cells (29). The limited data available on the influence of diet on human MF are inconclusive and based on small study samples (3032).

In this report, we have studied the effect of diet on the mutation frequency in the HPRT gene in human T-lymphocytes, as a marker for human in vivo somatic mutagenesis. The study represents an effort to provide a mechanistic link for the association between diet and cancer risk in humans. The study population includes non-smoking and smoking lung cancer patients and matched population controls, including a comparatively large number of never-smokers and females. We have reported previously results on lung cancer risk in this population in relation to genetic polymorphisms (GSTM1 and NAT2), environmental tobacco smoke (ETS) exposure, and diet (3335), as well as on HPRT MF in relation to age, smoking, case-control status and genetic polymorphisms (36,37).


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Study subjects
There were 312 subjects with MF data (158 lung cancer cases and 154 matched population controls; 83 men and 229 women) of whom 154 were never-smokers, 47 former, 38 recent and 73 current smokers. The source population included all persons >=30 years old, resident in Stockholm County. Details of the study population and selection criteria have been described (33,36,38).

Briefly, we recruited cases during 1992–1995 at the three major county hospitals, asking subjects to participate when diagnosed with lung cancer but not yet subject to radiotherapy or chemotherapy. A selection of ever-smoker cases was frequency matched to never-smoker cases and subsequently population controls were frequency matched to cases by hospital catchment area, gender, age group and smoking category (33,36,38).

Of 370 recruited subjects, 349 provided adequate blood samples and HPRT MF could be analysed for 329. Ten invalid MF analyses (no positive clones) and seven outlier values with confidence interval (CI) exceeding 5-fold the MF (related to low cloning efficiency or too few positive or seeded wells) were discarded, leaving 312 valid MF values (36).

Dietary and other exposure data
We obtained detailed exposure data by personal interview, usually in conjunction with blood collection (33). The questionnaire covered active and passive smoking, residential and working histories, and a food frequency assessment focusing on the year prior to the interview of 19 foods or food groups, mainly items rich in vitamin A, beta-carotene and vitamin C, as detailed elsewhere (35). Response alternatives were: daily or almost daily, several times per week, weekly, monthly, less than monthly, never. The six categories were also collapsed into three as close as possible approximate tertiles of the exposure distribution among all subjects. For cheese and beverages, daily slices/servings were also reported and the three-category variable was defined as less than daily, once daily and several servings daily; in addition a variable for approximate quintiles of calculated daily servings was created. Combined intake of cabbages, tomato and green vegetables, either excluding (Vegetable Index A) or including carrots (Vegetable Index B), was classified as low, medium or high. A similar Fruit Index included citrus fruits and other fruits (35).

We calculated intakes of beta-carotene, total carotenoids, retinol, vitamin A and vitamin C, by summing over all questionnaire items (35,38). Weights for intake frequency were multiplied with number of portions, portion sizes and nutrient contents/100 g to obtain the daily amount of each nutrient from each item (38). For comparability, nutrient cut-points were based on previously determined quintiles among the extended set of never-smoking population controls, as used in related analyses of diet and lung cancer risk (35).

Determination of HPRT MF
Peripheral blood was used for direct isolation of lymphocytes (Polymorphprep, Pharmacia, Sweden). All analyses were made with coded samples and in batches containing one time-matched sample from each subgroup of cases/controls and smoker/non-smoker to minimize variation over time. We determined HPRT MF using T-cell cloning as described (27,39). The results have been reported previously (36). Briefly, two 96 well non-selection plates received one or two target lymphocytes and 2 x 104 lethally X-irradiated lymphoblastoid (RJK853) feeder cells per well in growth-medium enriched with T-cell growth factor (TCGF; 20% conditioned medium). Selection plates received 2 x 104 target lymphocytes and 1 x 104 feeder cells per well, in TCGF-enriched medium containing 2 µg/ml 6-thioguanine (Sigma, Germany). After 2 weeks, clonal cell growth was scored visually. Cloning efficiency and MF were calculated from the proportion of negative wells assuming a Poisson distribution (27), as described (36).

Statistical methods
We used linear regression to investigate the influence of various factors on the MF (ln-transformed). For low beta (<0.1) such a logarithmic model has an approximately linear slope which is interpretable as a proportional increase in outcome per unit exposure; above that the increase is more clearly non-linear (36). P-values (two-sided test) or 95% CI are given as appropriate.

Initially we examined MF averages across dietary categories to detect patterns and interactions and used dummy (indicator) regression variables for categories to determine approximate exposure–response shape without assumptions (40). For further analyses with continuous variables, questionnaire items were scored 0–5 for the six categories, or 0, 0.115, 0.345, 1, 3 and 6 (times/week). Where daily servings were available, the calculated daily servings variables were used directly, or quintile variables, scored 0–4. In exploratory analyses with these variables, categorical variables scored 0–5 were generally more significant and stronger predictors than variables scored with consumption frequencies. Similarly for nutrients, individual continuous scoring or category median value scoring were not superior to scoring the categories 0–4, although results were similar. We therefore used simple categorical scoring (0–5, 0–4) as our primary approach. The vegetable and fruit indexes were scored 0–2. For beverages and cheese, average daily servings calculated from questionnaire data was used and its logarithm showed better fit (ln[value + 1]). We investigated suggested non-linearity by adding a quadratic term. For total carotenoids and beta-carotene the quadratic term provided a significant improvement.


    Results
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Major effects of diet on MF
The study population includes non-smoking and smoking lung cancer patients and matched population controls, including a comparatively large number of never-smokers and females (Table I). The associations between one dietary variable at a time and HPRT MF are shown in Tables II and III. Table II can be examined to determine an approximate shape of exposure–response without modelling assumptions. There was a clear negative association between geometric mean MF and Vegetable Index A (intake of green leafy vegetables, cabbages and cauliflower, and tomatoes, but excluding carrots). Several other trends were also apparent, including a non-linear trend for beta-carotene and total carotenoids and a negative association between vitamin C intake and HPRT MF. All patterns or trends were very similar when using age-adjusted MF instead (not shown).


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Table I. Characteristics of study population

 

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Table II. Geometric meana of HPRT MF (x10-6) and number of subjects (n) within categories of dietary variables (total n = 312)

 

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Table III. Linear regression analyses of the influence of dietary variables taken one at a time on the HPRT MF (unit increase in lnMF per category or unit of exposure)a

 
Table III presents the estimated coefficients and CIs for all dietary variables from separate linear regression models, with ln MF as the outcome variable and adjusted for age and smoking category. As shown in Table II, a high intake of vegetables seemed to decrease MF also in these multivariate analyses. Vegetable index A (excluding carrots) was a strong and highly significant predictor (P = 0.02). The association was similar in cases and controls, smokers and never-smokers (Figure 1A). Vegetable Index B (including carrots) showed a weaker but essentially similar relationship. Other dietary intakes that significantly decreased MF were citrus fruits and berries. Among nutrients, high vitamin C intake decreased MF significantly in congruence with the results for citrus fruits, with similar pattern in smokers and never-smokers (Figure 1B). Total carotenoids were associated with lower MF at average intake than at low or high intakes, with a significant quadratic term. Although this significant U-shaped relationship was similar among smokers and never-smokers, it appeared somewhat stronger among smokers, and the result for beta-carotene was very similar (Figure 1C and D). More unexpectedly, significantly higher MF with increasing levels of cheese intake was observed (Table III).



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Fig. 1. (A) Influence of intake of vegetables (green vegetables, cabbages and cauliflower, tomatoes) on HPRT MF (geometric mean). (B) Influence of intake of vitamin C on HPRT MF (geometric mean). (C) Influence of intake of total carotenoids on HPRT MF (geometric mean). (D) Influence of intake of beta-carotene on HPRT MF (geometric mean).

 
We evaluated the confounding of and interaction with other variables known to influence or suspected of influencing the MF (36), including age, smoking habits, case status (conceptually a marker for mutagen susceptibility), gender and GSTM1 and NAT2 genotype (possible genetic susceptibility markers). The latter four were not included in subsequent models, as they did not act as confounders, i.e. did not substantially affect estimates for the dietary variables. The evaluation of interactions between all our dietary variables against the six variables listed above resulted in less rather than more statistically significant interactions than expected by chance. As supporting data for such interactions is also lacking, we chose to include only main effects in the final models.

Models of the influence of several dietary components on the HPRT MF
As citrus fruits and berries contribute largely to vitamin C intake and the same is true for vegetable variables and total carotenoids, these variables cannot be combined in the same statistical model. In Table IV, we therefore evaluate two different combined dietary models. In both model I and model II, the inclusion of both the citrus fruit/vitamin C and the vegetable/total carotenoid components result in slight attenuation of the other component, indicating a limited mutual confounding. However, both components are significant (or borderline significant) when the other is removed (models Ia/Ib and IIa/IIb), and both appear to be associated with important effects. Thus, despite some loss of significance, the main models I and/or II appear to most adequately capture the relevant dietary components.


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Table IV. Combined multivariate linear regression models of the influence of diet on the HPRT MF (unit increase in lnMF per category or unit of exposure)a

 

    Discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
The results of this study demonstrate decreased HPRT MF related to intake of vegetables, as well as citrus fruits and berries. Calculated intake of vitamin C accordingly also decreased the MF, but for carotenoids from diet a U-shaped association was found with lowest MF near average carotenoid intakes.

Previous studies on diet and HPRT MF are sparse and based on small samples. In one study of 52 healthy males aged 50–59 years the MF decreased with increasing plasma vitamin C, especially in smokers (30), consistent with our results on dietary vitamin C. Another study of 70 women aged 32–68 years with breast masses found significant positive correlations between MF and fibre, vitamin A, copper and iron intake and negative correlations for fat and alcohol (31). A third study of 83 healthy individuals aged 20–80 years found a positive correlation between MF and body mass index and per cent energy intake from total carbohydrate, starch and fat, but no correlation between MF and estimated antioxidant intake (32). In an animal model, however, supplementation of diet for 4–6 weeks with antioxidants (a mixture of vitamins C, E, beta-carotene, rutin, selenium, zinc) reduced the HPRT MF in splenocytes of gamma-irradiated mice (18).

Various factors may have contributed to inconsistencies between studies. The quality and type of exposure information is important. We collected data by personal interview, using a food frequency questionnaire developed in a previous study (41). The questionnaire included 19 items, several focusing directly on food groupings, which provided good coverage of the nutrients studied (35). For group items, the components were explicitly listed. We did not collect information on dietary supplements, partly because such data often have low validity, as we found in the previous study, where such data were collected but could not be used (41). In addition, supplements are probably not an important source for beta-carotene (42) so this issue may have relevance mainly for our vitamin C results. Furthermore, if measurement error due to unmeasured supplementation is non-differential, an attenuation of the estimate between total nutrient intake and HPRT MF would be expected; if it is not, some bias is possible, as in any study when all sources of exposure cannot be adequately characterized. Although such information was not systematically collected, no organ transplants were noted, nor did autoimmune disease or drugs that affect the immune system appear to be common in the study population. All in all, these factors, if present, appear unlikely to have impacted on the dietary results.

Our study population included both lung cancer cases and population controls. We did not, however, see any significant interactions with case-control status, nor confounding by case-control status. Furthermore, previous analyses in this sample (36) and other data (43) have not demonstrated differences in MF between cases and controls. Regarding the possibility of differential dietary reporting from cases and controls, the contrasting results for citrus fruits and juices, which were not protective against lung cancer, as compared with other fruits, which were protective in previously reported case-control analyses (35) speak against cases or controls systematically over- or under-reporting intake of conceived ‘healthy’ foods in general. As cases may alter dietary habits near diagnosis and present habits have been shown to influence reporting of earlier habits under some circumstances (44,45), misreporting can occur even when interview occurs close to diagnosis. Therefore, we specifically noted important dietary changes in the last 6 months and corrected for these. Few cases reported such changes and then generally very recently.

In mechanistic terms, the lower HPRT MF levels we consistently saw with intake of vegetables, fruit and berries provide general support for the hypothesis that a protective effect against cancer mediated by these foods may be due to their ability to reduce the somatic mutation rate. This is in line with recent results showing vegetable and citrus fruit intake to be inversely associated with mutations in the VHL tumour suppressor gene (25). The U-shaped association between total carotenoids (and similarly beta-carotene) and in vivo HPRT MF is intriguing. Carotenoids (usually beta-carotene) have consistently been associated with decreased lung cancer risk in epidemiological studies (3). Nevertheless, intervention studies with beta-carotene supplementation (47) found no clear protective effects (3), but instead an increased lung cancer risk which seems to affect primarily heavy current smokers and/or alcohol drinkers and asbestos-exposed subjects (8,9). Even in these studies, however, subjects with higher baseline beta-carotene serum levels or consumption of beta-carotene-rich foods had lower risk of lung cancer, confirming other epidemiological data (10). For comparison, median dietary intake of beta-carotene in our study was similar to dietary intake in the ATBC study (~2000 µg/day), which in turn had similar baseline beta-carotene serum levels to the CARET study (~0.2 µg/ml) (6,7,46), suggesting that serum levels in our study are also likely to have been around this level. Serum levels in these studies after intervention were 10–15-fold higher.

Our results suggest that even within ordinary dietary habits of a normal population, the protective effect of carotenoids on in vivo mutagenicity may be most apparent at moderate carotenoid intakes and decrease both at high and at low carotenoid levels. This provides a biological basis in humans in vivo for the unexpected observations in the supplementation studies. The U-shaped relationship also has some experimental in vitro support (47). We found no significant interaction with smoking although the data did suggest the U-shape was more prominent in ever smokers. This provides some supports for the hypothesis that dose-dependent auto-oxidation and possibly additional oxidation of carotenoids by cigarette smoke may provide a basis for increased in vivo mutagenicity by overwhelming the basic anti-oxidant effect of carotenoids at normal levels (10). If the cancer promoting effect observed after a relatively short follow-up, as in the intervention studies, is due to direct mutagenic effect on lung cells, a cancer excess might be expected to show up in subjects with a large cell population close to final neoplastic transformation, such as current smokers with cigarette exposure of relatively high intensity (9).

Another mechanistic hypothesis is that the increased cancer risk could be related to high supplementation dose and resulting unnaturally high serum levels (12), possibly via co-carcinogenic properties of beta-carotene such as increase in carcinogen-activating CYP450 enzymes (11) or perturbation of cell membrane organization (47). In the present study, the food frequency questions concern intake over the last year, which is an appropriate time range for the effect on MF. In studies of cancer, use of this type of food frequency data involves the further assumption that it may represent intake over decades. Validation has shown reasonable correlation in terms of ranking the subjects, but less validity for assessing absolute intake levels. Persons with extremely high or low exposures during the last year are nonetheless unlikely to have had as extreme exposures constantly over decades. Consequently, we may well expect less misclassification of relevant exposure in this study when the time scale of the outcome, HPRT MF, is more congruent with the exposure assessment time scale.

Instead of food frequency assessment of dietary intake, direct measurement of serum levels of nutrients could be advocated and has some advantages. However, due to various factors such as liver buffering of serum levels for retinol, saturation of vitamin C plasma levels at intakes >1000 mg/day, high temporal and other inter-individual variation in measurements of nutrients (especially vitamin C) and often higher correlation between food frequency measurements of dietary intake and serum levels than between two serum levels measured some time apart (48), it is doubtful that such measurements are superior for assessing nutrient intake or status over a time period of months or a year for the nutrients studied in this investigation.

Our result for cheese intake is intriguing. It may be a chance finding. Another possibility is that it could represent an effect of fat, if reported cheese intake is a marker for high fat consumption in our population. Unfortunately, our questionnaire was not designed to measure fat consumption. Fat is an important source of calories in most human populations, and laboratory animals receiving reduced calorie but nutritionally adequate diet have extended life spans, lowered incidence of neoplasms and lower HPRT MF (49,50).

The dietary effects on HPRT MF shown in this study are of important magnitude in relation to age and smoking effects on MF (36). For example, lnMF increases by ~1.1% (i.e. beta {approx} 0.011) per year of age, which can be compared with the beta-coefficients in Tables III and IV (for example beta = -0.07 or ~7% decrease per category of citrus consumption; Table III).

In conclusion, our study is consistent with known diet-cancer associations and provides novel human in vivo mechanistic support for a cancer-protective effect of vegetables and fruit by modulation of somatic mutagenesis. Our results regarding carotenoids including beta-carotene imply that care should be taken in the use of single nutrients or dietary components to promote health. The old recommendation of a ‘mixed diet’ seems to have a scientific foundation in that single components may act best at a certain optimal level and in concert with other components. A mixed diet is also in itself generally a guarantee against inadvertently consuming too much of any one specific micronutrient. A broad intake of vegetables and fruit appears to protect against excessive somatic mutation rates.


    Acknowledgments
 
The study received financing from the Swedish Environmental Protection Agency, the Swedish Cancer Society and Swedish Match.


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 

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Received September 11, 2002; revised October 28, 2002; accepted February 4, 2003.