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Nutrition and Disease

Cruciferous Vegetables Reduce Morphological Markers of Colon Cancer Risk in Dimethylhydrazine-Treated Rats^1,2

Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN 55108

^* To whom correspondence should be addressed. E-mail: dgallahe{at}umn.edu.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Consumption of cruciferous vegetables has been associated with reduced colon cancer risk in human populations. However, little experimental evidence exists to support this association. Here, we report the effects of diets containing cruciferous vegetables on colon cancer risk. In Expt. 1, rats were fed a vegetable-free (basal) diet or diets containing different lyophilized cruciferous vegetables in concentrations between 4 and 10%. In Expt. 2, rats were fed the basal diet or diets containing 10–22.6% fresh cruciferous vegetables. Diets were fed for 2 wk (Expt. 1) or 3 wk (Expt. 2) before and 7 wk (Expt. 1) or 12 wk (Expt. 2) after administration of the colon carcinogen 1,2-dimethylhydrazine. Rats fed fresh vegetables were also injected with a low dose of carcinogen 18–24 h prior to termination. Groups fed lyophilized vegetables did not differ in aberrant crypt foci (ACF), sialomucin-producing foci, or mucin-depleted foci (MDF) numbers. However, all fresh vegetable diets significantly decreased ACF (40%) and MDF numbers. Activities of the hepatic phase I enzyme CYP2E1 did not differ among groups in either experiment. Hepatic glutathione S-transferase (GST) and quinone reductase activities did not differ among groups fed fresh vegetables, whereas the lyophilized cabbage diets decreased GST activity compared with the basal diet. Groups did not differ in apoptosis and cell proliferation labeling indices in colonic mucosa. This study indicates that fresh but not lyophilized cruciferous vegetables reduce colon cancer risk in rats. These results do not support changes in hepatic carcinogen metabolism or colonic crypt cytokinetics as a mechanism.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

Even though epidemiological studies show an inverse association between consumption of cruciferous vegetables and colon cancer risk, there is limited and conflicting evidence from animal studies to support this association. When fresh homogenized cabbage was fed to mice at 12.5% (wt:wt) at different stages of colon carcinogenesis, cabbage increased adenocarcinoma incidence when fed during initiation but decreased adenoma formation when fed during promotion (7). Red cabbage juice at 5% in the drinking water given to rats treated with aromatic amines as a carcinogen did not affect colonic precancerous lesion [aberrant crypt foci, (ACF)]³ numbers compared with the control group (8). Selenium-enriched broccoli has been shown to reduce ACF in F344 rats (9) and intestinal tumorigenesis in Min mice (10). However, the effects of nonenriched broccoli on an experimental model of colon cancer are unknown. To our knowledge, watercress feeding has not been evaluated in an animal model of colon cancer.

In this study, we have investigated whether diets containing watercress, green cabbage, red cabbage, and broccoli protect rats against formation of colonic ACF, a marker of cancer risk, induced by the colon carcinogen 1,2-dimethylhydrazine (DMH) when fed prior to, during, and after carcinogen administration. The study was divided in 2 experiments: in Expt. 1, lyophilized green cabbage, red cabbage, and watercress were fed. In Expt. 2, fresh green cabbage, watercress, and broccoli were fed, because each vegetable contains a different predominant glucosinolate: glucobrassicin for cabbage, gluconasturtiin for watercress, and glucoraphanin for broccoli. The concentration of vegetable in each diet was chosen to provide approximately the same total concentration of dietary glucosinolates, based on published values for glucosinolate content of these vegetables, thus providing a way to distinguish the effects of different types of glucosinolates on colon cancer risk. In addition to enumerating ACF, the primary markers of colon cancer risk, sialomucin-producing foci and mucin-depleted foci (MDF) were also measured as additional markers. Sialomucin-producing ACF have been proposed to be a better predictor of colon cancer risk than total ACF due to their higher rate of cell proliferation, higher degree of dysplasia, and increased distortion of the luminal opening compared with ACF-producing sulphomucins (11,12). MDF have recently been shown to be histologically more dysplastic than mucin-producing ACF and occur at the same order of magnitude as tumors, leading to the suggestion that these foci are the direct precursors of tumors (13).

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Rats and diets

Male Wistar rats, weighing 50–75 g, were purchased from Harlan Sprague Dawley. Animals were housed individually in wire-bottomed cages in rooms maintained at 20 ± 2°C with a relative humidity of 50 ± 10% and a 12-h-light/-dark cycle. This study was approved by the University of Minnesota Committee on Animal Care. Rats consumed diets and water ad libitum throughout the study.

In Expt. 1, rats were divided into 4 dietary groups (n = 15–16 per group) upon arrival as follows: basal diet (modified AIN-93G, vegetable free), green cabbage diet (10% wt:wt of lyophilized green cabbage, variety Quisto), red cabbage diet (10% wt:wt of lyophilized red cabbage, variety Ruby Perfection), and watercress diet (4% wt:wt of lyophilized watercress) (composition of the diets is shown in Table 1). The 2 varieties of cabbage fed in this experiment were provided by Dr. Vince Fritz of the Department of Horticultural Science, University of Minnesota. Upon harvest, the cabbage was stored at –18°C and, prior to lyophilization, the frozen tissue was broken into smaller pieces inside a cold chamber to prevent thawing. Growth chamber-grown watercress was provided by Lynette Wong and Dr. Albert Markhart, also of the Department of Horticultural Science, University of Minnesota. Following harvest, the plant tissue was immediately frozen in liquid nitrogen and stored at –70°C. The lyophilized cabbage and watercress were powdered and stored at –18°C for up to 6 mo prior to the start of the experiment.

Diets were freshly prepared every week and stored at –80°C. Fresh diet was given to the animals daily.

Experimental design

    Expt. 1. The experimental diets were fed for 2 wk prior to the first carcinogen treatment. DMH (Sigma-Aldrich) was administered subcutaneously, once per week for 2 consecutive weeks at a dose of 50 mg/kg body weight. After the second DMH injection, animals continued to be fed the experimental diets for an additional 7 wk prior to termination. Body weights and food intake were recorded periodically throughout the study.

    Expt. 2. The experimental diets were fed for 3 wk prior to the first carcinogen treatment. DMH was administered subcutaneously, once per week for 2 consecutive weeks at 50 mg/kg body weight, followed by an additional 12 wk of feeding the experimental diets. Approximately 18–24 h prior to termination, rats received another DMH injection of 15 mg/kg body weight. This is a time postinjection of maximal colonic apoptosis (15), but too short a time to influence aberrant crypt (AC) development.

Determination of ACF and mucin-producing ACF

Animals were anesthetized with isoflurane and colons removed and flushed with PBS. The colons were cut open along the longitudinal median, fixed flat in 10% buffered formalin, and stored at 4°C. AC and ACF were counted on 2- x 5-cm sections of the distal colon and stained with methylene blue using a modification of the method described by Bird (16). After ACF determination, colons were kept in 10% formalin solution and later processed in high-iron diamine Alcian blue staining for visualization of ACF mucin type (11). We scored colons for MDF at 40x according to criteria described by Caderni et al. (13). Briefly, to be considered mucin-depleted, a focus had to show absence or minimal presence of mucin in addition to fulfilling 2 of the following: 1) distortion of the opening of the lumen compared with normal surrounding crypts; 2) elevation of the lesion above the surface of the colon; and 3) multiplicity >3. Representative images of the mucin-staining patterns of ACF are shown in Figure 1.

View larger version (74K): [in this window] [in a new window]   FIGURE 1  Representative images of mucin staining patterns in colonic ACF. (A) ACF expressing both sulfomucin (maroon-colored spots) and sialomucin (blue spots). (B) ACF expressing only sialomucin. (C) Mucin-depleted ACF.

Liver cytosolic and microsomal fractions were obtained as previously described (6). Microsomal isolation was performed as previously described (17). Protein concentration of microsomal and cytosolic fractions was determined based on the Bradford method (18).

Determination of liver enzymes

    Cytochrome P450 2E1. Activity of cytochrome P450 2E1 (CYP 2E1) was assayed by a kinetic method for p-nitrophenol hydroxylase activity (19), as modified by Reinke and Moyer (20). Enzyme activity was calculated using an extinction coefficient of 3.57 (mmol/L)^–1 cm^–1 for p-nitrocatechol.

    Glutathione S-transferase activity. Total glutathione S-transferase (GST) activity was measured spectrophotometrically using 1-chloro-2,4-dinitrobenzene as the substrate (21). GST activity was calculated using an extinction coefficient of 9.6 (mmol/L)^–1 cm^–1 for the 1-chloro-2,4-dinitrobenzene-reduced glutathione conjugate.

    NAD(P)H:quinone reductase activity. Quinone reductase (QR) activity was measured using 2,6-dichloroindophenol as the substrate according to the method by Ernster (22) as modified by Benson et al. (23). Enzyme activity was calculated using an extinction coefficient of 21 (mmol/L)^–1cm^–1.

Immunohistochemistry

A 0.8-cm section of the distal end of the colon was cut, paraffin embedded, and sectioned into 6-µm sections for immunohistochemical determination of apoptotic and cell proliferation labeling indices. Apoptotic cells were identified using a commercial kit (ApoTag Peroxidase In Situ Oligo Ligation kit, Chemicon International). Cell proliferation was determined by detection of proliferating cell nuclear antigen (PCNA), using a PCNA-specific mouse antibody, and an ABC detection kit (Chemicon International). Both the apoptotic labeling index and the cell proliferation labeling index were calculated as the ratio of the number of labeled cells to the total number of cells present in one side of a colonic crypt, multiplied by 100. A total of 25 crypts per animal were counted.

Statistical methods

Values in the text are means ± SEM. Means were compared among groups by 1-way ANOVA followed by Student's t test for the inspection of group differences. P < 0.05 was used as the critical level of significance. Linear regressions were calculated using the least squares method. All statistical analyses were carried out using the SAS System for Windows, release 9.1 (SAS Institute).

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Diet groups did not differ in final body weight or food intake during Expt. 1. In Expt. 2, rats fed the fresh broccoli diet had a significantly lower final body weight than rats fed the basal diet. Rats fed fresh green cabbage consumed significantly more food than the other groups (Table 3), likely due to the lower energy density of this diet.

View larger version (25K): [in this window] [in a new window]   FIGURE 2  Number of ACF expressing both sialomucin and sulfomucin, expressing only sialomucin, or expressing no mucin (mucin depleted) per cm2 of rat distal colon fed the basal diet or diets containing fresh broccoli, green cabbage, or watercress at 14.4, 22.6, or 10% of the diet, respectively. Values are means ± SE, n = 15–16. Means without a common letter differ, P < 0.05. (A) Number after feeding lyophilized cruciferous vegetables (Expt. 1). (B) Number after feeding fresh cruciferous vegetables (Expt. 2).

    Phase I and phase II liver enzymes. The activities of CYP 2E1 did not differ among the groups in either Expt. 1 or 2 (range of means, 24.2–29.8 and 119.3–145.5 nmol·min^–1·mg protein^–1, respectively). In Expt. 1, GST activity was lower in rats fed the green (39.3 ± 4.0) and red cabbage (37.8 ± 3.6) diets than in those fed the basal diet (49.8 ± 3.9) (P < 0.05). GST activity did not differ among the groups in Expt. 2 (range of means, 9.6–12.0). QR activity did not differ in Expt. 1 (range of means, 12.1–14.6) or Expt. 2 (range of means, 535.3–651.0). In Expt. 2, in which a low dose of carcinogen was administered 18–24 h before termination, CYP2E1 and QR activities were much greater and GST activities much less than in Expt. 1.

    Apoptosis and cell proliferation. The groups did not differ in the effects of fresh vegetables on the apoptotic labeling index (range of means, 1.99–3.05%), cell proliferation labeling index (range of means, 14.0–18.3%), or the apoptotic labeling index:cell proliferation labeling index ratio (range of means, 0.17–0.29) in rat distal colon.

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
In a previous study, we examined the effects of feeding indole-3-carbinol, a secondary metabolite of the glucosinolate glucobrassicin, and phenethyl isothiocyanate, a metabolite of gluconasturtiin, to carcinogen-treated rats (17). Indole-3-carbinol decreased ACF number and tended to reduce MDF, whereas phenethyl isothiocyanate was without effect. The rationale in the present study for feeding watercress, cabbage, and broccoli rather than secondary metabolites of glucosinolates, such as isothiocyanates and indoles, was 2-fold. First, humans are exposed to these products via consumption of whole cruciferous vegetables. Second, cruciferous vegetables contain a mixture of many glucosinolates. The secondary metabolites of most of these are unavailable in quantities sufficient for feeding studies, yet they may have important chemopreventive properties. For example, it has been shown that watercress contains glucosinolates other than gluconasturtiin, whose secondary metabolites, such as 7-methylsulfinylheptyl and 8-methylsulfinyloctyl isothiocyanates, have been found to induce phase II enzymes in cell culture (24). Only by feeding cruciferous vegetables can the complex mixture of glucosinolates be provided.

The findings from Expt. 1 indicate that diets containing lyophilized watercress, green, or red cabbage were not protective against the formation of ACF. In studies using the aromatic amine 2-amino-3-methylimidazo{4,5-f}quinoline as the colon carcinogen, feeding rats cooked or raw red cabbage juice in drinking water before and during carcinogen treatment provided no protection against formation of ACF (8,25). Thus, cruciferous vegetables, when processed by either lyophilizing or juicing, seem to be ineffective in reducing colon cancer risk in animal models.

In contrast, our study found that watercress, green cabbage, and broccoli, incorporated into diets fresh and immediately frozen, were highly effective in reducing the number of total ACF and ACF producing sialomucin and sulfomucin in rats; fewer ACF tended to produce only sialomucin in rats fed the broccoli diet relative to the those fed the basal diet (Expt. 2). MDF number was also significantly lower in rats fed fresh cabbage and broccoli, with a trend for reduction in those fed watercress compared with the basal diet (P = 0.057). The difference between the 2 experiments is unlikely to be due to the additional week of feeding prior to carcinogen administration and longer feeding afterwards, because isothiocyanates and indole-3-carbinol have been shown to affect phase I enzymes within hours to days after exposure (26,27) and ACF number in the 2 basal groups did not differ (P = 0.19). As the vegetable-containing diets contained approximately the same total content of glucosinolates, and these cruciferous vegetables contain different predominant glucosinolates, our results suggest that the glucosinolates may not differ dramatically in their chemoprotective properties. Although rats fed the broccoli diet had significantly lower final body weights than rats fed the basal diet, final body weight did not explain the variations in ACF number among diet groups, suggesting that differences in ACF number were due to dietary differences, not differences in final body weight.

In Expt. 1, the quantity of vegetables consumed by the rats, on a fresh weight basis, ranged from 18.4 g/d for watercress to 30.7 g/d for red and green cabbage. In Expt. 2, the quantity of fresh vegetables consumed ranged from 3 g/d for watercress to 6.8 g/d for green cabbage. Thus, fresh vegetables were effective at reducing colon cancer risk at a dietary concentration of approximately one-fifth of that of the lyophilized vegetables, in fresh weight equivalents. The reason for a lack of effect of lyophilized vegetables on ACF number in Expt. 1 may have been instability of the glucosinolates during the lyophilization process or the loss of myrosinase activity during lyophilization, which would have prevented the conversion of glucosinolates to their respective secondary metabolites. Regardless, the method of processing of cruciferous vegetables should be considered in future studies of their chemopreventive effects.

CYP2E1 is a phase I enzyme involved in the activation of several carcinogens and is necessary for activation of DMH to its carcinogenic metabolite (28). No change in CYP2E1 enzyme activity was observed after feeding cruciferous vegetables, either lyophilized or fresh, compared with the basal diet. We have previously observed hepatic CYP 2E1 activity did not change after long-term feeding of indole-3-carbinol or phenethyl isothiocyanate (17). In a study feeding 10% lyophilized broccoli for 7 d (29), a significant increase in hepatic CYP 2E1 protein was reported. The reasons for the inconsistent findings between this study and those of others are not apparent, although the length of feeding in the studies described above was much shorter than in our study. Further studies are needed to resolve the issue of the effect of glucosinolates on CYP activities.

Phase II enzymes are generally involved in detoxification of reactive compounds, including carcinogens, by increasing their polarity and facilitating excretion. However, studies of the effects of lyophilized cabbage-containing diets on hepatic GST activity in rodents have been inconsistent (30,31). When 20% lyophilized cabbage was fed to mice for 2 wk, a nonsignificant reduction in hepatic GST activity was observed (31). In contrast, rats fed 25% lyophilized cabbage for 21 d showed a significant increase in hepatic GST activity (30). In the present study, in Expt. 1, rats fed the lyophilized green cabbage and red cabbage diets had significantly decreased hepatic GST activity compared with the control group. However, GST activity did not differ in Expt. 2 using fresh vegetables. Garden cress juice, gavaged to rats for 3 d, did not affect either hepatic or colonic GST activity (32). Thus, cruciferous vegetables in a fresh form seem not to affect GST activity, whereas in their lyophilized form, their affect on GST activity remains uncertain.

In this study, liver QR activity did not differ among any groups in either experiment. To our knowledge, QR activity has not previously been measured in an experimental model of colon cancer fed watercress and cabbage. We were unable to measure QR activity in colon tissue, which has a much higher QR activity than liver, because colon tissue was needed for quantification of ACF and determination of ACF mucin type. However, results from Hwang and Jeffery (6), who measured QR activity in both liver and colon of rats fed broccoli processed 4 different ways, indicate that QR activity at these 2 sites is highly correlated (R² = 0.90; our calculation from their mean values). Thus, the lack of differences among groups in QR activity in the liver in the present experiment likely indicates a similar lack of differences in the colon.

CYP 2E1 activity in Expt. 2, in which rats were given a low-dose carcinogen treatment 18–24 h before termination and collection of liver tissue, was 4-fold greater compared with Expt. 1. This suggests that DMH is a potent inducer of CYP 2E1. Rats subjected to acute exposure to DMH (Expt. 2) also had significantly lower GST activity and greater QR activity compared with Expt. 1. We are not aware of other studies examining the acute effects of DMH on these enzymes. Additional studies may be warranted to determine the changes in enzyme activity under different acute carcinogen treatments.

In Expt. 2, cell proliferation and apoptosis were measured 18–24 h after DMH administration. This approach has been utilized by others (34,35) to increase the sensitivity of these assays, given that DMH administration causes an immediate stimulation of apoptosis and suppression of proliferation (35). We found no changes in apoptotic or cell proliferation labeling indices among the groups. Similarly, neither broccoli nor Brussels sprouts altered the PCNA labeling index (36). This is in contrast with the results of Smith et al. (15), who found that feeding 400 mg/kg diet of the isothiocyanate sinigrin 6 h after carcinogen administration reduced apoptosis in rats 18–48 h later. Our results, however, suggest that changes in colonic crypt cytokinetics may not be involved in the protection against colon cancer risk by long-term feeding of fresh cruciferous vegetables.

In our recent study in which we fed purified indole-3-carbinol and the phenethyl isothiocyanate to carcinogen-treated rats, using the same experimental design employed in the present experiment, ACF was reduced by indole-3-carbinol at a dietary concentration of 1.36 mmol/kg, whereas phenethyl isothiocyanate was not effective at a dietary concentration as high as 3.37 mmol/kg (24). In this study, all 3 fresh vegetables significantly reduced ACF number at a dietary concentration estimated to provide 95 mg glucosinolates/kg (0.23 mmol/kg), suggesting the possibility that the mix of the parent glucosinolates may be more potent in chemoprevention than the molar equivalent of the isolated secondary metabolites or that there may be additional bioactive compounds that contribute to this effect. For example, it has been reported that red cabbage anthocyanins reduced DMH-induced colorectal carcinogenesis when fed at 5% of the diet (37). Regardless, the mechanism by which these cruciferous vegetables exert their protection does not seem to involve changes in carcinogen metabolism or colon crypt cytokinetics. Thus, additional studies are needed to determine the mechanisms by which fresh cruciferous vegetables reduce colon cancer risk.

	ACKNOWLEDGMENTS

 
The authors thank Dr. Gary Gardner for sharing important observations regarding instability of glucosinolates, Dr. Vince Fritz for providing the cabbage for Expt. 1, and Lynette Wong and Dr. Albert Markhart for providing the watercress for Expt. 1.

	FOOTNOTES

 
¹ Supported by the Minnesota Research Fund (formerly the SOTA TEC Fund) and the Minnesota Agricultural Experimental Station.

² Author disclosures: A. Y. Arikawa and D. D. Gallaher, no conflicts of interest.

³ Abbreviations used: AC, aberrant crypt; ACF, aberrant crypt foci; CYP 2E1, cytochrome P450 2E1; DMH, 1,2-dimethylhydrazine; GST, glutathione S-transferase; MDF, mucin-depleted foci; PCNA, proliferating cell nuclear antigen; QR, quinone reductase.

Manuscript received 19 July 2007. Initial review completed 19 August 2007. Revision accepted 17 December 2007.

	LITERATURE CITED

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 

1. American Cancer Society. Cancer facts and figures 2006. Atlanta: American Cancer Society; 2006.

2. Verhoeven DT, Goldbohm RA, van Poppel G, Verhagen H, van den Brandt PA. Epidemiological studies on brassica vegetables and cancer risk. Cancer Epidemiol Biomarkers Prev. 1996;5:733–48.[Abstract/Free Full Text]

3. Fahey JW, Zalcmann AT, Talalay P. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry. 2001;56:5–51.[Medline]

4. Verhoeven DT, Verhagen H, Goldbohm RA, van den Brandt PA, van Poppel G. A review of mechanisms underlying anticarcinogenicity by brassica vegetables. Chem Biol Interact. 1997;103:79–129.[Medline]

5. Verkerk R, Dekker M. Glucosinolates and myrosinase activity in red cabbage (Brassica oleracea L. var. Capitata f. rubra DC.) after various microwave treatments. J Agric Food Chem. 2004;52:7318–23.[Medline]

6. Hwang ES, Jeffery EH. Effects of different processing methods on induction of quinone reductase by dietary broccoli in rats. J Med Food. 2004;7:95–9.[Medline]

7. Temple NJ, Basu TK. Selenium and cabbage and colon carcinogenesis in mice. J Natl Cancer Inst. 1987;79:1131–4.[Medline]

8. Kassie F, Uhl M, Rabot S, Grasl-Kraupp B, Verkerk R, Kundi M, Chabicovsky M, Schulte-Hermann R, Knasmuller S. Chemoprevention of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced colonic and hepatic preneoplastic lesions in the F344 rat by cruciferous vegetables administered simultaneously with the carcinogen. Carcinogenesis. 2003;24:255–61.[Abstract/Free Full Text]

9. Finley JW, Davis CD, Feng Y. Selenium from high selenium broccoli protects rats from colon cancer. J Nutr. 2000;130:2384–9.[Abstract/Free Full Text]

10. Davis CD, Zeng H, Finley JW. Selenium-enriched broccoli decreases intestinal tumorigenesis in multiple intestinal neoplasia mice. J Nutr. 2002;132:307–9.[Abstract/Free Full Text]

11. Jenab M, Chen J, Thompson LU. Sialomucin production in aberrant crypt foci relates to degree of dysplasia and rate of cell proliferation. Cancer Lett. 2001;165:19–25.[Medline]

12. Caderni G, Giannini A, Lancioni L, Luceri C, Biggeri A, Dolara P. Characterisation of aberrant crypt foci in carcinogen-treated rats: association with intestinal carcinogenesis. Br J Nutr. 1995;71:763–9.

13. Caderni G, Femia AP, Giannini A, Favuzza A, Luceri C, Salvadori M, Dolara P. Identification of mucin-depleted foci in the unsectioned colon of azoxymethane-treated rats: correlation with carcinogenesis. Cancer Res. 2003;63:2388–92.[Abstract/Free Full Text]

14. McNaughton SA, Marks GC. Development of a food composition database for the estimation of dietary intakes of glucosinolates, the biologically active constituents of cruciferous vegetables. Br J Nutr. 2003;90:687–97.[Medline]

15. Smith TK, Lund EK, Johnson IT. Inhibition of dimethylhydrazine-induced aberrant crypt foci and induction of apoptosis in rat colon following oral administration of the glucosinolate sinigrin. Carcinogenesis. 1998;19:267–73.[Abstract/Free Full Text]

16. Bird RP. Observation and quantification of aberrant crypts in the murine colon treated with a colon carcinogen: preliminary findings. Cancer Lett. 1987;37:147–51.[Medline]

17. Plate AYA, Gallaher DD. Effects of indole-3-carbinol and phenethyl isothiocyanate on colon carcinogenesis induced by azoxymethane in rats. Carcinogenesis. 2006;27:287–92.[Abstract/Free Full Text]

18. Bradford M. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54.[Medline]

19. Allis JW, Robinson BL. A kinetic assay for p-nitrophenol hydroxylase in rat liver microsomes. Anal Biochem. 1994;219:49–52.[Medline]

20. Reinke LA, Moyer MJ. p-Nitrophenol hydroxylation. A microsomal oxidation which is highly inducible by ethanol. Drug Metab Dispos. 1985;13:548–52.[Abstract]

21. Habig W, Pabst M, Jakoby W. Glutathione S-transferases. J Biol Chem. 1974;249:7130–9.[Abstract/Free Full Text]

22. Ernster L. DT diaphorase. In: Estabrook RW, Pullman ME, editors. Methods in enzymology. New York: Academic Press; 1967. p. 309–17.

23. Benson A, Hunkeler M, Talalay P. Increase of NAD(P)H:quinone reductase by dietary antioxidants: possible role in protection against carcinogenesis and toxicity. Proc Natl Acad Sci USA. 1980;77:5216–20.[Abstract/Free Full Text]

24. Rose P, Faulkner K, Williamson G, Mithen R. 7-Methylsulfinylheptyl and 8-methylsulfinyloctyl isothiocyanates from watercress are potent inducers of phase II enzymes. Carcinogenesis. 2000;21:1983–8.[Abstract/Free Full Text]

25. Uhl M, Kassie F, Rabot S, Grasl-Kraupp B, Chakraborty A, Laky B, Kundi M, Knasmuller S. Effect of common Brassica vegetables (Brussels sprouts and red cabbage) on the development of preneoplastic lesions induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in liver and colon of Fischer 344 rats. J Chromatogr B Analyt Technol Biomed Life Sci. 2004;802:225–30.[Medline]

26. Guo Z, Smith TJ, Wang E, Eklind KI, Chung FL, Yang CS. Structure-activity relationships of arylalkyl isothiocyanates for the inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone metabolism and the modulation of xenobiotic-metabolizing enzymes in rats and mice. Carcinogenesis. 1993;14:1167–73.[Abstract/Free Full Text]

27. Xu M, Schut HA, Bjeldanes LF, Williams DE, Bailey GS, Dashwood RH. Inhibition of 2-amino-3-methylimidazo[4,5-f]quinoline-DNA adducts by indole-3-carbinol: dose-response studies in the rat colon. Carcinogenesis. 1997;18:2149–53.[Abstract/Free Full Text]

28. Sohn OS, Ishizaki H, Yang CS, Fiala ES. Metabolism of azoxymethane, methylazoxymethanol and N-nitrosodimethylamine by cytochrome P450IIE1. Carcinogenesis. 1991;12:127–31.[Abstract/Free Full Text]

29. Vang O, Jensen H, Autrup H. Induction of cytochrome P-450IA1, IA2, IIB1, IIB2 and IIE1 by broccoli in rat liver and colon. Chem Biol Interact. 1991;78:85–96.[Medline]

30. Whitty JP, Bjeldanes LF. The effects of dietary cabbage on xenobiotic-metabolizing enzymes and the binding of aflatoxin B1 to hepatic DNA in rats. Food Chem Toxicol. 1987;25:581–7.[Medline]

31. Sparnins VL, Venegas PL, Wattenberg LW. Glutathione S-transferase activity: enhancement by compounds inhibiting chemical carcinogenesis and by dietary constituents. J Natl Cancer Inst. 1982;68:493–6.[Medline]

32. Kassie F, Rabot S, Uhl M, Huber W, Qin HM, Helma C, Schulte-Hermann R, Knasmuller S. Chemoprotective effects of garden cress (Lepidium sativum) and its constituents towards 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ)-induced genotoxic effects and colonic preneoplastic lesions. Carcinogenesis. 2002;23:1155–61.[Abstract/Free Full Text]

33. Hughes R, Rowland IR. Stimulation of apoptosis by two prebiotic chicory fructans in the rat colon. Carcinogenesis. 2001;22:43–7.[Abstract/Free Full Text]

34. Hambly RJ, Saunders M, Rijken PJ, Rowland IR. Influence of dietary components associated with high or low risk of colon cancer on apoptosis in the rat colon. Food Chem Toxicol. 2002;40:801–8.[Medline]

35. Ijiri K. Apoptosis (cell death) induced in mouse bowel by 1,2-dimethylhydrazine, methylazoxymethanol acetate and gamma rays. Cancer Res. 1989;49:6342–6.[Abstract/Free Full Text]

36. O'Neill IK, Loktionov A, Manson MM, Ball H, Bandaletova T, Bingham SA. Comparison of metabolic effects of vegetables and teas with colorectal proliferation and with tumour development in DMH-treated F344 rats. Cancer Lett. 1997;114:287–91.[Medline]

37. Hagiwara A, Yoshino H, Ichihara T, Kawabe M, Tamano S, Aoki H, Koda T, Nakamura M, Imaida K, et al. Prevention by natural food anthocyanins, purple sweet potato color and red cabbage color, of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-associated colorectal carcinogenesis in rats initiated with 1,2-dimethylhydrazine. J Toxicol Sci. 2002;27:57–68.[Medline]

38. Reeves PG, Nielsen FH, Fahey GC Jr. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr. 1993;123:1939–51.[Abstract/Free Full Text]

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