1 Division of Human Nutrition, Wageningen University, Wageningen, The Netherlands and 2 Department of Gastroenterology and Hepatology, University Medical Centre Nijmegen, Nijmegen, The Netherlands
3 To whom correspondence should be addressed Email: pieter.vantveer{at}wur.nl
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Abstract |
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Abbreviations: CDNB, 1-chloro-,2,4 dinitrobenzene; CI, confidence interval; HNPCC, hereditary non-polyposis colorectal cancer; GSH, reduced glutathione; GST, glutathione S-transferase; p25, 25th percentile; p75, 75th percentile
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Introduction |
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Essential is the glutathione (GSH)/glutathione S-transferase (GST) detoxification system, which comprises antioxidant reduced GSH and GSTs (EC 2.5.1.18); a family of phase II enzymes, which, in humans, consists of four main subgroups: alpha(), mu(µ), pi(
) and theta(
).
Because this system plays an important role in detoxification of a broad range of carcinogens (3), high GST enzyme activity has been suggested as being beneficial to cancer prevention (4). Considering the low GST enzyme activity in the colon and rectum compared with tissues in which cancer occurs less frequently, we hypothesized previously that GST enzyme activity might be critically low and related to high rates of carcinogenesis in this organ (5,6).
Colonic GST enzyme activity and GST protein levels vary considerably between individuals (7,8), which may be related to differential susceptibility to colorectal cancer. Individuals with homozygous deletions of GSTM1 or GSTT1 (null genotype) do not have detectable GSTM1 or GSTT1 enzyme activity, respectively, and were postulated to be at higher risk of colorectal cancer. However, no consistent associations of GSTM1 and GSTT1 null polymorphisms with risk of colorectal cancer have been observed (9). Apart from inherited polymorphisms in GSTs, individuals may differ in GST enzyme activity due to differential exposure to bioactive compounds.
In vivo and in vitro studies have shown that a variety of dietary compounds or their metabolites can induce the GSH/GST detoxification system. These include glucosinolate metabolites and dithiolthiones present in brassica vegetables, diallyl sulfides present in allium vegetables, limonoids and flavonoids present in citrus fruits (3,1017), and butyrate produced by colonic fermentation of fibre (8).
Evidence for induction of the human rectal GSH/GST detoxification system was found in a crossover study among 10 volunteers consuming 300 g/day of cooked Brussels sprouts during 7 days. This yielded 30 and 15% increases of GST-alpha and GSTP1-1 protein levels, respectively. However, no effect upon GST enzyme activity was found (11), and taking 3 g/day of broccoli supplements for 14 days also did not influence GST enzyme activity in lymphocytes or colon mucosa (10).
Nonetheless, indications of the up-regulation of GST enzyme activity by brassica and allium vegetables were found in blood plasma, urine and saliva (1115). Moreover, duodenal GST- and GST-
protein levels were higher among subjects consuming vegetables at least four times a week, and antral GSTT1 protein levels were higher among subjects consuming fruits at least four times a week compared with those who consumed these products less frequently. However, no associations between frequency of consumption of fruits and vegetables and GST enzyme activity were found in these tissues (16).
Induction of the GSH/GST detoxification system by fruits and vegetables may partially account for the observed inverse associations between their consumption and risk of colorectal cancer (12,18). We investigated whether habitual consumption of fruits and vegetables, in particular of brassica and allium vegetables and citrus fruits, is positively associated with the following components of the rectal GSH/GST detoxification system: GST enzyme activity, GST-, GSTM1-1 and GSTP1-1 isoenzyme and GSH levels.
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Methods |
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Study population
Between December 1995 and February 1998, subjects undergoing colonoscopy or sigmoidoscopy in the outpatient clinic of the Department of Gastroenterology and Hepatology of University Medical Centre Nijmegen, The Netherlands, were recruited by their gastroenterologist. Eligible subjects were Dutch speaking Caucasians, between 18 and 75 years old at day of endoscopy and having no history of colorectal resection, polyposis coli, colorectal cancer or chronic inflammatory bowel disease. Subjects with and without adenomas, either with or without a family background of hereditary non-polyposis colorectal cancer (HNPCC) according to the Amsterdam I criteria (20) were included. All HNPCC family members were first-degree relatives of patients with colorectal or endometrial cancer, but were free of cancer themselves. Subjects who did not belong to a HNPCC family were excluded if diagnosed with colorectal adenoma >3 years prior to recruitment. The medical ethical committees of Wageningen University and University Medical Centre Nijmegen approved the study protocol.
Rectal biopsies were taken from 106 (82%) of the 130 eligible subjects. Twelve subjects did not return dietary and/or lifestyle questionnaires. Thus, the final study population consists of 94 subjects, including 44 members of 26 HNPCC families.
Data collection
After providing written informed consent, participants underwent endoscopy during which six biopsies from healthy mucosa were taken from rectal mucosa within 10 cm from the anal verge. Biopsy specimens were immediately frozen in liquid nitrogen and stored at 80°C. Additionally, 30 ml of EDTA blood was drawn and stored at 20°C.
Clinical information regarding the presence and characteristics of adenomas, HNPCC and indication for endoscopy was abstracted from medical records.
We requested that participants complete a questionnaire on lifestyle and socio-economic factors and a validated semi-quantitative food-frequency questionnaire (21). The questionnaire assesses consumption of fruits and vegetables with high reproducibility after a year (fruits, Spearman's r = 0.61 and 0.77; vegetables, r = 0.76 and 0.65 for males and females, respectively), while relative validity versus the means of 12 24-h recalls was moderate (fruits, Spearman's r = 0.56 and 0.38; vegetables, r = 0.56 and 0.31 for males and females, respectively) (21). Nutrient intake was calculated using the 1996 computerized version of the Dutch food composition table (22).
Laboratory assays
Rectal biopsies were processed into cytosolic fractions (11). Total GST enzyme activity was assayed by spectrophotometric determination of 1-chloro-2,4-dinitrobenzene (CDNB) conjugation with GSH (23). Total intracellular reduced glutathione (GSH) level was quantified by high performance liquid chromatography after reaction with monobromobimane using a modification of the method of Fahey and Newton (24,25). GST- (GSTA1-1, A1-2 and A2-2), GSTM1-1 and GSTP1-1 isoenzyme levels were determined by western blotting (11). GST enzyme activity and levels of isoenzymes and GSH were expressed per milligram of intracellular protein, as determined colorimetrically according to the method of Lowry et al. (26). Protein and enzyme activity measurements were done in duplicate. The within- and between-assay coefficients of variations were 1015% for protein and 510% for GST enzyme activity measurements, respectively.
As a result of limited tissue availability, not all biopsy samples could be analysed completely; this resulted in 78 samples analysed for GST enzyme activity, 94 for GSH, 91 for GST- and GSTP1-1 and 76 for GSTM1-1 isoenzyme levels.
DNA was extracted from 200 µl frozen whole blood (QIAamp blood kit, Qiagen Inc.), diluted to a concentration of 20 ng/µl and stored at 4°C until use. GSTM1 and GSTT1 genotypes were determined using a general multiplex polymerase chain reaction method followed by electrophoresis (27). A ß-globin gene fragment of 350 bp was present as a positive control.
Data analysis
Because all biopsies in which GSTM1-1 protein was not detected were obtained from GSTM1-null genotype carriers, we excluded these observations in analyses regarding this isoenzyme. We log-transformed GST-, GSTM1-1 and GSTP1-1 protein levels to yield approximately normally distributed variables. Nutrient, alcohol and fat intakes were adjusted, separately for men and women, for total energy intake using the residual method (28).
Spearman correlation coefficients were computed to quantify the strength of the association between GST isoenzymes, GSH and GST enzyme activity. Linear mixed models were fitted to associate dietary, lifestyle and medical determinants with GST enzyme activity, GSH and log-transformed GSTM1-1 and GSTP1-1 levels. In these models, family membership was incorporated as random intercept to account for the presence of relatives in the study population, and HNPCC and presence of adenomas at index endoscopy were included as indicator variables to account for the study design. Because no null polymorphism has been identified in any of the genes encoding GST-, we used Tobit regression (29), which explicitly allows for censored distributions of the dependent variable, to model this isoenzyme.
Two series of models were fitted: a first to investigate the association between lifestyle and medical factors, and a second to address the association between consumption of fruits and vegetables with the aforementioned GSH/GST parameters. In the latter, total energy intake was included to control for potential confounding and to reduce the impact of under- and over-reporting of intake (28). To compensate for potential confounding, the following factors that potentially relate to both consumption of fruits and vegetables and to GST enzyme activity were added separately and mutually to the model: current and past smoking, sex and use of NSAIDs or paracetamol as indicator variables, and as continuous variables: age (years), body mass index (kg/m2) consumption of coffee, tea, (red) meat, energy-adjusted (saturated) fat, alcohol, red wine in grams per day and vitamin E (equivalents/day). Current smoking was the only factor that changed the regression coefficient of interest with >5 U in most models. To enhance comparability, we adjusted all models for this factor. Additionally, regression equations regarding GST isoenzymes were adjusted for energy-adjusted fat.
Fruits and vegetables were treated as continuous variables, tertiles and quadratic terms. Because Akaike's Information Criteria and likelihood ratio testsfor tertiles and quadratic terms, respectivelyshowed a similar fit to continuous terms, only models containing the latter are presented.
To evaluate hypothesized effect modification by GSTM1 and GSTT1 genotype and explore effect modification by smoking, age, sex, HNPCC and adenoma status, interaction terms were included and tested by Wald tests. Subsequently, to evaluate whether the observed associations may be attributed to fractions related to dietary fibre, vitamin A, vitamin C, ß-carotene and folate, we added these food components to the models. Finally, we checked whether influential outliers were present. This appeared not to be the case.
All tests of statistical significance were two-sided and considered to be significant at the level of 5%. The analyses were conducted using Statistical Analysis Software (SAS version 8.0, Sas Institute, Cary, NC).
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Results |
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No correlation was detected between GST isoenzymes, GSH and GST enzyme activity. However, after adjusting for HNPCC, adenoma status, smoking behaviour, sex, age, GSTM1 and GSTT1 genotype, a weak, negative correlation between GSH and GST enzyme activity appeared (r = 0.24, P = 0.049).
Table II describes GST enzyme activity according to GSTM1 and GSTT1 genotype, lifestyle and medical factors. Smokers had a higher GST enzyme activity compared with never smokers; former smoking subjects tended to have a higher GST enzyme activity compared with those who had never smoked, although the difference was not significant. Strikingly, the presence of adenomas at index endoscopy was associated with a 44.9 nmol/min/mg protein lower GST enzyme activity. Restricting the analysis to subjects having rectal adenomas increased this estimate to 57.7 nmol/min/mg protein [95% confidence interval (CI) = 6.1121.6], although statistical significance was lost. GST enzyme activity was not associated with age, sex, HNPCC, GSTM1 or GSTT1 genotype and history of adenomas. None of the associations was modified by GSTM1 or GSTT1 genotype (results not shown).
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Associations of fruits and vegetables with GST enzyme activity, according to GSTM1 genotype, are presented in Table III. Consumption of fruits, in particular citrus fruits, was positively associated with GST enzyme activity. Total consumption of vegetables was positively associated with GST enzyme activity among GSTM1-plus carriers, but not GSTM1-null carriers. This tended to be true for all subgroups except allium, although the modification was only statistically significant for total vegetables and brassica vegetables. No association between juices and GST enzyme activity could be detected.
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Consumption of citrus fruits, which was associated with total GST enzyme activity, was not associated with GST- [difference between high and low consumption: 9.7% (95% CI = 10.734.7)], GSTM1-1 [8.1%, 95% CI = (26.358.3)], GSTP1-1 [0.9%, 95% CI = (18.424.7)] and GSH [26.1%, 95% CI = (97.11796.3)].
Consumption of brassica vegetables, which was associated with GST enzyme activity, was also associated with GSTM1-1 protein levels [difference between high and low consumption: 67.5%, 95% CI = (6.8162.7)], but not with GST- [1.5%, 95% CI = (24.428.4)], GSTP1-1 [8.3%, 95% CI = (17.341.8)] and GSH [177.7%, 95% CI = (95.015405.8)]. Consumption of allium vegetables was not associated with GST enzyme activity, but was associated negatively with GSTP1-1 levels [difference between high and low consumption: 23.3%, 95% CI = (35.5; 8.6)]. Consumption of green leafy vegetables, which was also not associated with GST enzyme activity, was positively associated with GSTM levels [133.3%, 95% CI = (11.7387.4)], and negatively with GST-
levels [26.5%, 95% CI = (44.2; 3.3)]. These associations were not modified by GSTM1 and GSTT1 genotype, and remained after adding consumption of brassica vegetables to the model. Other associations of fruits and vegetables with GST isoenzyme and GSH levels were absent.
Dietary fibre, vitamin A, ß-carotene and folate did not affect the parameter estimates, whereas the effect of vitamin C could not be disentangled from the contribution of citrus fruits due to their high correlation (r = 0.76).
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Discussion |
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We requested subjects to report consumption of fruits and vegetables according to the year preceding endoscopy by filling out a food-frequency questionnaire. While the validity of the assessment was moderate, the reproducibility of the assessment was high. Although the assessment, like all assessments, is not perfect and may be subject to bias, this method allows assessing habitual consumption with minimal intrusion upon the participants. However, it is hard to determine whether we have assessed consumption in the time window relevant to GST induction. In rat hepatoma cells, GST induction was first observed 8 h after induction, with highest values after 2448 h (30). In humans, rectal GST induction is likely to be a delayed process, as bioactive compounds have to reach the intestinal tissue, either via blood or the gastrointestinal tract. Thereupon, the higher activity might persist for some time for biological reasons or prolonged exposure. Indeed, Szarka and co-workers reported that human rectal GST enzyme activity was stable over a 24 week interval in another cross-sectional study (31).
We included subjects undergoing endoscopy for medical reasons instead of healthy subjects. This is unlikely to cause problems as subjects with colorectal cancer and inflammatory bowel diseases were excluded since these conditions may influence metabolism and gene expression; meanwhile controlling for self-reported bowel complaints did not importantly alter the estimates. Nevertheless, given subject selection on HNPCC or adenoma status, we adjusted all parameter estimates for these conditions, although this did not influence them. Likewise, these selection criteria modify none of the examined associations.
Our study design allowed us to evaluate the association between GST enzyme activity and adenoma presence, sex, age, HNPCC and genetic polymorphisms in GSTs. In line with the postulated role of GST induction in cancer prevention and previously published results (31), the presence of adenomas at index endoscopy, was associated with a lower GST enzyme activity in normal rectal tissue compared with rectal tissue of patients without adenomas. Cancer-free members of HNPCC families had lower rectal GST- and GSH protein levels than those who did not belong to a HNPCC family. These differences, which could not be attributed to differences in GSTM1 and GSTT1 genotype, were unexpected and have not been reported before; therefore, these findings should be treated with caution. Interestingly, smoking was associated with a higher GST enzyme activity (Table II) and higher GST-
and GSTP1-1 protein levels. Indeed, rodent studies showed that several polycyclic aromatic hydrocarbons present in cigarette smoke induce GST enzyme activity as part of an adaptive response mechanism to chemical stress (3).
The main hypothesis concerned the association between fruits, vegetables and GST enzyme activity, isoenzyme levels and GSH. First, fruits, in particular citrus fruits, were associated positively with GST enzyme activity. This might be attributed to the coumarin auraptene and to limonoids (highly oxidized triterpenoids), which are present in high concentrations in citrus fruit tissues (17,32). Indeed, auraptene increased GST enzyme activity in rat colon tissue (33) and limonoids induced GST enzyme activity in small intestinal mucosa and liver of mice (34). However, auraptene and limonoids are also present in citrus fruit juices (17), which were not associated with GST enzyme activity in this study. As processed juices comprise the larger part of the intake of juices, compounds that are lost during the processing of citrus fruits to juices may be responsible for these findings. Additionally, bioavailability of phytochemicals might differ between whole fruits and fruit juices. Other compounds present in citrus fruits may also account for the higher GST enzyme activity, such as citrus flavonoids and D-limonene (35). The latter enhanced colonic GST enzyme activity in rats (36), albeit at doses that far exceed human exposure.
Secondly, consumption of vegetables was positively associated with GST enzyme activity among those with the GSTM1-plus genotype, but not those with the GSTM1-null genotype. This association was most evident for brassica vegetables, which are rich in glucosinolates. Isothiocyanates may be primarily responsible for this GST inducing capacity (37), although glucosinolate content may depend on species, maturity and processing of the plants (38).
So far, only one study examined consumption of brassica vegetables in relation to rectal GST enzyme activity. In this study, 10 human volunteers consumed 300 g/day of Brussels sprouts for 7 days. No effect upon rectal GST enzyme activity and GSH levels was found, while GSTA and GSTP1-1 levels increased by 30 and 15%, respectively (11). In our study, consumption of brassica vegetables was not associated with GST- and GSTP1-1 levels. However, it was associated with GSTM level, which is in line with the observed association between brassica vegetables and GST enzyme activity among GSTM1-plus genotype carriers.
Furthermore, the synthetic dithiolethione oltiprazdithiolethiones are a class of chemical compounds, which also occur naturally in brassica vegetablesincreased colonic mucosal GST enzyme activity at single oral doses of 125 and 250, but not at 500 or 1000 mg/m2, in 24 subjects having a family history of colorectal cancer or a personal history of colorectal polyps or carcinomas (39). However, this could not be confirmed in two other trials (40,41). Other studies have evaluated the effect of brassica vegetables upon GST enzyme activity in other tissues (1115), but because of organ-specific patterns of expression (7) and differences in bioavailability of bioactive compounds able to induce GST, it is hard to compare their results to those of the present study.
Although compounds of allium vegetables did positively affect the GSH/GST detoxification system in many in vitro or in vivo studies (3), as well as in a human study on lymphocytes (12), we observed a negative association between allium vegetables and GSTP1-1 levels. However, a previous study in which six healthy volunteers were given 250 g/day of mixed vegetables (among which allium vegetables) for a period of 3 weeks reported decreased GSTP1 protein levels in five of the six subjects, which is in line with our observation (42).
Assessing overall GST enzyme activity, no correlation between GST isoenzyme levels and GST enzyme activity appeared. This could be partially attributed to our substrate CDNB, which reacts with moderate activity to GSTA1, GSTA2 and GSTP1, and with high activity to GSTM1 and GSTM2 (3,43). The GST-µ and GST- classes belong to the most important GST isoenzymes in the rectum (11). While this makes CDNB a good general substrate, it is not highly specific to a particular isoenzyme, which might be related to the lack of correlation between GST isoenzymes and GST enzyme activity. Besides, GSTM2 protein levels were not assessed, while GSTM2 contributes to GST enzyme activity. As GSTM2 is expressed in the human colon and is inducible (8), induction of GSTM2 might further explain the lack of correlation. Alternatively, the assessment of GST enzyme activity might be slightly biased, as GSTs also serve as binding and transport proteins, resulting in a temporary loss of enzyme activity (3,43).
Associations with fruits and vegetables appeared to be different for GST isoenzymes and GST enzyme activity. For citrus fruits, we found an association with GST enzyme activity, but this could not be attributed to any of the assessed isoenzymes. Theoretically, we cannot exclude the possibility that citrus fruits induce GSTM2 exclusively. However, it seems more likely that effects on specific isoenzymes are too small to be detected, whereas effects on GST enzyme activitywhich reflects isoenzyme levels of GSTA, GSTM and GSTP1-1 in combination with their specific activitycan be detected. This might also explain why consumption of green leafy vegetables was associated with GSTM1-1 and GST- levels, while no association with GST enzyme activity was found. As these associations have not been examined before, the actual nature of the relationships remains to be established.
The underlying idea of this study is that high GST enzyme activity is believed to be beneficial to cancer prevention. GST enzyme activity in normal mucosa along the gastrointestinal tract was inversely correlated with tumour incidence (6); inhibition of rat bowel carcinogenesis was shown to correlate with induction of GST (33); and compounds that induce GST generally counteract cytogenetic damage (44). However, it should be noted that certain food components [e.g. sulforaphane and indole 3-carbinol present in broccoli (45,46)] may affect phase I as well as phase II enzymes; the balance between induction and inhibition of different detoxification systems is of utmost relevance (47).
In conclusion, our study shows positive associations of habitual levels of consumption of fruits and vegetables with rectal GST enzyme activity. This is especially true for citrus fruits and brassica vegetables, but positive associations with the latter only existed for GSTM1-plus individuals.
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Acknowledgments |
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References |
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