1 Department of Biosciences at Novum, Karolinska Institute, 141 57 Huddinge, Sweden,
2 Department of Pathology and Cytology, Karolinska Hospital, 171 76, Stockholm, Sweden and
3 Department of Medical Epidemiology, Karolinska Institute, 171 77 Stockholm, Sweden and Department of Urology, Sundsvall Hospital, 851 86 Sundsvall, Sweden
Abstract
Carcinogenic chemicals act through DNA damage and mitogenic effects. No established mechanism explains the cancer preventive effects, if any, of food items, such as vegetables and fruit. If such data were available, preferably on tumor-initiating genes, the evidence for the protective effects would become stronger. The von Hipple-Lindau (VHL) gene is the tumor suppressor gene predisposing to both sporadic renal cell carcinoma (RCC) and von Hippel-Lindau disease. We have earlier analyzed VHL mutations in RCCs from 102 Swedish patients identified in a case-control study and here examine associations between patient characteristics, including dietary habits and mutations, considering the type of mutation. The results are given as odds ratios (OR), separately for smokers and all patients. In univariate analysis, consumption of vegetables and citrus fruit decreased the frequency of VHL mutations among smokers and citrus fruit among all patients. In multivariate analysis of smokers' characteristics, welding fumes showed a risk of 5.63 for multiple VHL mutations. In smokers, citrus fruit decreased the OR of GC to AT mutations to 0.13 and that of multiple mutations to 0.17; vegetables decreased the OR for single mutations to 0.22. Among all subjects, welding fumes were a risk factor and citrus fruit a protective factor. Additionally, an intake of selenium protected against multiple mutations. The present results provide evidence that the intake of vegetables, selenium and particularly of citrus fruit protects the renal VHL gene from mutational insults that may be endogenous or common in a population. Even though most of the associations are biologically plausible, and vegetables and fruit were an a priori hypothesis, fortuitous results cannot be ruled out in this relatively small study.
Abbreviations: BMI, body-mass index; 95% CI, 95% confidence intervals; OR, odds ratio; RRC, renal cell carcinoma; SSCP, single-strand conformation polymorphism; VHL, von Hipple-Lindau.
Introduction
The mutational patterns in cancer-related and reporter genes have been measured with the aim of obtaining data on causes and mechanisms of mutations and cancer. This work has been promising in liver, lung and skin cancer, in which specific types of mutations in the p53 gene have been linked to etiological agents, aflatoxin B1, smoking and ultraviolet radiation, respectively (13). These are genotoxic factors that cause DNA damage, mutations and cell proliferation. Mutation studies could be potentially helpful even in identifying factors that decrease mutations. For example, the evidence for the protective effects of dietary factors in human cancer would be strengthened if data were available on the reduction of mutations in cancer-related genes from tumors (2,46). This is particularly timely because many findings on the protective effects of dietary items from case-control studies cannot be confirmed in, what are assumed to be, more reliable cohort studies (7). Furthermore, insight may be gained into the mechanism of action of such protective factors. The genes of study should preferentially be those involved in initiation of cancer formation, rather than those acting at later stages where metabolic and cellular imbalances per se may be the cause of mutations.
Many environmental factors are known to influence the risk for renal cell carcinoma (RCC), which accounts for ~2% of adult malignancies (8). Tobacco smoking is an established but modest risk factor with a relative risk of 1.4 (9). Other suggested risk factors include obesity (1012), some reproductive factors (13), some drugs, such as phenacetin, hypertension and/or its medication (14), and occupational exposure to trichloroethylene, gasoline, petroleum products, asbestos and iron processing fumes (8,1517). Many dietary factors have been considered in relation to RCC (18). Vegetables, fruit, vitamin C and carotenoids have shown protective effects whereas meat and milk products have been risk factors; however, the findings have not been uniform (1923).
RCC is a manifestation in a rare cancer syndrome, von Hippel-Lindau disease, which is a monogenic disease caused by gene alterations in the von Hippel-Lindau (VHL) gene (2427). Evidence has been accumulated on the association of somatic VHL mutations with some 50% of sporadic RCC (25,2830). VHL mutations are thought to constitute the initiating events in renal tumorigenesis (25,30). Multiple and specific types of VHL mutations in RCC have been associated with exposure to the industrial solvent, trichloroethylene (31). This finding and animal data suggest that mutational patterns in the VHL gene may serve as an etiologic clue to factors causing renal cancer (32). A public database on VHL mutations has been established with 750 mutations currently (Universal VHL-Mutation Database, http://www.umd.necker.fr/).
We have carried out an analysis of VHL mutations from 102 cases of RCC that originated from a Swedish case-control study that was part of the collaborative international renal cancer studies cited above (29,33). Almost one-half (47%) of the patients had a VHL alteration and in many patients multiple mutations were identified. Because a wealth of potential etiological data were collected on these patients in the epidemiological studies (9,10,20), we analyze here their relationships to the detected VHL mutations, with a particular emphasis on those factors that were identified as risk or protective factors in the epidemiological studies on the same population. Our main hypothesis was to test whether the protective dietary factors act by reducing mutations in the VHL gene, a gatekeeper gene for renal cancer (34).
Materials and methods
Samples
The patients were identified from a previous case-control study on RCC (20). A total of 102 patients with RCC from eight hospitals in central Sweden were included in this study. All patients were male and 4177 years of age (mean age 62 years). The diagnosis was made during the time period from June 1, 1989 to December 31, 1991. With the help of a structured questionnaire by personal interview, detailed exposure information was obtained for the time prior to 1987, i.e. 2.55 years before diagnosis. The questions included demographic characteristics, body height and weight, physical activity, history of previous medication, reproductive history, medical history, tobacco use, alcohol consumption, dietary habits, occupation and occupational exposures (911,13,14,17,19,20).
The collection of dietary data has been described previously (19,20). Professional interviewers used structured questionnaires to collect information about the usual diet in the time before the year 1987. The frequency of consumption of 63 selected food items and beverages was recorded per day, week, month and year. In addition, a self-administered questionnaire was used to reconstruct dietary habits 20 years earlier. It was believed that the collected dietary data indicated mainly recent consumption by the subjects (20). For analysis, the data were classified in quartiles, in the form available for the present study. We dichotomized the data based on an a priori decision, as described under the Statistical analysis.
Archival paraffin-embedded samples of tumor and surrounding microscopically normal tissue were requested from pathology departments in the study areas. The slides of tumors were reviewed by a pathologist. Of 102 cancers, 86 were clear cell, 11 papillary, two chromophobe and three unclassified histology; 19 were grade 1, 51 grade 2 and 29 grades 3 and 4 combined, with three unclassified (33). A representative slide with a cancer area was selected from each case. Cancer was outlined with Indian ink on the slide. A 50 µm section was cut from the paraffin block and any non-neoplastic tissue was trimmed away from the section obtained.
DNA extraction and mutation detection
Genomic DNA samples were isolated from the paraffin-embedded sections and the VHL gene was amplified by five pairs of PCR primers as described previously (29, 33). Briefly, the PCR products were labeled by [32P]dCTP and analyzed in three conditions of electrophoresis for single-strand conformation polymorphism (SSCP). The shifted bands on the SSCP gel were sequenced by PCR-based sequencing on ABI 377. PCR and SSCP were repeated for the samples with positive findings, starting from the original DNA. The mutational analysis was completed before the present statistical analysis guaranteeing a complete independence.
Statistical analysis
Most of the variables were categorized in quartiles for analyses of the epidemiological studies (20). However, because of paucity of data we dichotomized the variables either by combining two top and two bottom quartiles [for potential risk factors, such as total energy intake, body-mass index (BMI), cholesterol, meat preparation] or by using the lowest quartile against three other quartiles (for potential protective factors, such as fruit, vegetables, selenium). The grouping of the quartiles was an a priori decision based on the unproven assumption that risk factors may act according to a linear doseresponse relationship, whereas protective factors may be non-linear, best observable at a true shortage. The cut-off values of the variables are shown in Tables IIIV.
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Results
The types of identified VHL mutations among 102 RCC patients are shown in Table I; 47 patients had any mutation, and 17 of these presented with a multiple mutation, i.e. two or more distinct alterations. The most common type of mutation was a GC to AT transition (given as G to A in Tables IIV
), which was found in 25 tumors. Most of these mutations (14/22 = 56% of patients and 49% of mutations) were at CpG sites, and all but three of the mutations were in clear cell carcinomas (33).
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In the early analysis we noted that, although smoking was not a risk factor for mutations (OR 0.95, 95% CI 0.412.21), it appeared to change the outcome of many variables. However, because non-smokers only numbered 32, and 15 with mutations, there was no possibility of testing whether it was a true effect modifier. We therefore decided to give the multivariate results of Tables III and IV separately only for smokers and all individuals but emphasizing that the dataset are not independent.
The variables of interest from Table II were individually tested for specific types of mutations among smokers in multivariate models in Table III
, adjusting for age, education and BMI. Welding fumes were a risk factor particularly for multiple mutations (OR 5.65, 95% CI 1.3922.93). The protective effects of vegetables were noted for patients with any, and single mutations. The effects of citrus fruit influenced all types of mutations; the protective effect was strongest for GC to AT transitions (OR 0.13, 95% CI 0.030.57).
A similar analysis on all subjects gave essentially identical results for welding fumes and for citrus fruit (Table IV). Vegetables showed no significant effect. Selenium (at >26 µg/day) emerged as a protective factor for multiple mutations, with an OR of 0.27 (95% CI 0.070.97) and for GC to AT transition, with an OR of 0.31 (95% CI 0.101.02, borderline significance, P = 0.05). We tested the effect of welding fumes in models where vegetables and fruit were also entered, with essentially unchanged results. For example, the OR was 4.58 (P = 0.04) for multiple mutations when citrus fruit was included in the model.
Discussion
Molecular epidemiology studies use human populations for molecular analysis to establish risks factors and mechanisms for diseases (1,35). One area has been to catalog mutations in tumors in order to deduce underlying mechanisms from the observed mutational patterns. The largest number of tumor-specific mutations (over 14 000) has been collected on the p53 gene, and some etiological conclusions have been arrived at (13). Yet the large number of potential exposures to agents that damage DNA in a non-specific way have limited the approach. Furthermore, p53 mutations are thought to be relatively late events in many cancers, in which the mutations may signal cellular imbalances in growing tumors rather than tumor-initiating events (2). The mutational data are deposited to the databases without etiological data, thus not allowing tests for specific hypothesis. Among the studies where dietary data have been collected, p53 mutations have been decreased in gastric cancer and unchanged in colon cancer in regard to the intake of vegetables (4,5). The VHL mutation database with 750 mutations is an important source of data for RCC (Universal VHL-Mutation Database, http://www.umd.necker.fr/).
The present study has several special features that should increase its validity. First, it was nested into the Swedish part of a well-conducted international case-control study. All diet and exposure data originated from this study. As many results from Swedish studies have also been reported separately, the concordance of risk/protective factors for RCC and mutations can be readily compared. Secondly, the VHL is a gatekeeper gene for RCC and the mutations may signal early events in tumorigenesis. Together with the German study on exposure to trichloroethylene (31), the present study is the only one relating etiological data with the prevalence of VHL mutations. Thirdly, because the epidemiological data were available on the same population, we primarily tested a priori hypothesis rather than generated new ones by random testing, which would cause a severe multiple comparisons problem. We will discuss the biological plausibility of the findings later. The results from mutation analysis were reported before the present statistical analysis, excluding any unintentional bias in the interpretation of the mutation data (29,33). All the mutation results have been rechecked from the staring material in order to exclude artefactual assignments. However, if such were present, they would bias the results towards null, no effect.
The type of mutations may give some etiological clues. The most common mutation, GC to AT transition, was found in 25 patients (53% of patients with mutations and also 53% of all mutations, counting all 84 mutations). This is the most common mutation type also in the VHL mutation database but it represents only 24% of all the listed events (http://www.umd.necker.fr/). Most of these mutations in our material (56% of patients and 49% of mutations) took place at CpG dinucleotides, in line with the VHL mutation database, where 51% of GC to AT mutations target CpG dinucleotides. In the p53 gene CpG sites are involved only between 5 and 40% of all GC to AT mutations, depending on the organ (2). In the p53 database (R4, April 2000, http://www.iarc.fr/) only 79 kidney cancers were recorded. However, among those 15 of 29 (52%) were GC to AT mutations at CpG sites, suggesting that kidney cancer deviates from other cancers in p53 mutations regarding the unusually high share of CpG sites. The CpG dinucletides are vulnerable to deamination of methylated C to T, giving rise to a G to C mutation. Oxidative stress, particularly nitric oxide enhances the rate of deamination and these types of mutations are associated with endogenous mutagenic processes (1,2,3638). Frequent multiple mutations in the VHL gene were detected here and in previous studies, including patients exposed to the industrial solvent, trichloroethylene (30,31).
The main hypothesis of the study was to explore the mechanisms for the assumed protective effects of fruit and vegetable consumption for renal cancer. Both citrus fruit (consumed at >420 g/month, equivalent of five small oranges) and vegetables provided protection against the prevalence of VHL mutations in renal tumors. The effect of citrus fruit was stronger among smokers than all subjects; the effect of vegetables among all subjects was not significant. The apparently differential results between smokers and all subjects were not due to direct mutation-inducing effects of smoking because the OR for mutation among smokers was close to unity, 0.95. However, smokers are known to have a lower intake and (independent of intake) serum level of vitamin C, which suggests that smoking may be an effect modifier (39). In the Swedish epidemiological study on RCC, the effect of citrus fruit intake was noted only among non-smokers, similar to the effect of total fruit consumption; no effect of vegetables was noted (20). However, in a study from Los Angles, citrus fruit had no strong effect when adjusted for other nutrients, and smoking had no effect modification (22). Such apparently conflicting data may be due to study populations, methods or genuine discrepancies, because of small effects caused by nutritional factors. Citrus fruit intake caused the largest reduction in the prevalence of GC to AT transitions, the type of mutation connected to endogenous mutagenic events, as discussed above. Although the protective effect of vegetables was on any and single mutations, they also reduced GC to AT mutations because these were the most common type of all mutations.
Intake of selenium afforded a protection against multiple mutations and against GC to AT transitions, the latter with a borderline significance. Selenium is a cofactor in the family of glutathione peroxidases and thioredoxin reductase that metabolize reactive oxygen species, and it can induce apoptosis, providing mechanistic pathways for its cancer preventive effects (40). However, the data on selenium and cancer in humans have been limited to certain cancers and the data on RCC are lacking (6,18,41,42). One reason for the inconsistent results may be that it is difficult to assess the intake of selenium through food frequency data, probably also weakening our estimates for selenium (18). The intake of selenium is considered to be low in Sweden, ranging from 10 to 70 µg/day (43). In our analysis the cut-off point for selenium intake was 26 µg/day, which is well below authoritative intake recommendations (18).
Several occupational exposures have been associated with an excess of RCC, including asbestos exposure in many studies (17). In the present study only exposure to welding fumes showed an increase in the prevalence of mutations, specifically multiple mutations. The results were not modified when vegetable and fruit variables were entered in the models for welding fumes. However, as this was one of many occupational exposures tested, chance cannot be ruled out. The international study on RCC, in which the Swedish study was a part of, found no risk among welders (17). However, welders have been shown to be at a risk of RCC according to a large Nordic record linkage study (44). Interestingly, the exposure to trichloroethylene associated particularly with the number of multiple VHL mutations in the previous study (31).
The present data provide molecular evidence that some fruits and vegetables reduce mutations in the VHL gene in RCC. The data are coherent in pointing to the types of mutations that are thought to be caused by active oxygen species and that can be inactivated by antioxidants such as vitamin C present in citrus fruit and other fruits and vegetables. The effect of selenium may have the same mechanism. The larger effects noted among smokers are also consistent because they have lower serum vitamin C levels than non-smokers. Even though the main results correspond to the a priori hypothesis and are biologically plausible, chance findings can only be ruled out in an independent study.
Notes
4 To whom correspondence should be addressed Email: kari.hemminki{at}cnt.ki.se
Acknowledgments
The study was supported by the Swedish Cancer Society.
References