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
Fekadu Kassie1,5,*,
Maria Uhl1,*,
Sylvie Rabot2,
Bettina Grasl-Kraupp1,
Ruud Verkerk3,
Michael Kundi4,
Monika Chabicovsky1,
Rolf Schulte-Hermann1 and
Siegfried Knasmüller1
1 Institute of Cancer Research, Borschkegasse 8A, 1090 Vienna, Austria,
2 National Institute for Agronomic Research, Unit on Ecology and Physiology of the Digestive Tract, Jouy-en Josas, France,
3 Product Design and Quality Management Group, University of Wageningen, The Netherlands and
4 Institute of Environmental Hygiene, University of Vienna, Austria
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Abstract
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The aim of this study was to investigate the chemopreventive effects of widely consumed cruciferous vegetables, namely Brussels sprouts and red cabbage towards 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced preneoplastic lesions [liver glutathione-S-transferase placental positive (GST-P+) foci and colonic aberrant crypt foci (ACF)]. Male F344 rats were treated with IQ (100 mg/kg bw/g) on 10 alternating days and received drinking water supplemented with Brussels sprouts and red cabbage juices (5% v/v) before and during the carcinogen treatment. From each vegetable two different cultivars were tested. Brussels sprouts reduced the frequency of IQ-induced aberrant foci in both organs (4152% in the colon and 2767% in the liver). Also, Brussels sprouts drastically diminished (8591%) the size of liver GST-P+ foci, but no such effect was seen in the colon. With red cabbage, the size of liver GST-P+ foci was markedly reduced (4183%) whereas the foci frequency was only moderately decreased (1950%). No protection was seen in the colon after treatment with red cabbage. Cooking (10 min, 100°C) of the vegetables had no influence on their protective effects. The stronger chemoprotective effects of Brussels sprouts may be due to the fact that the overall glucosinolate contents were substantially (23-fold) higher than those of the cabbage cultivars, but it was not possible to attribute the reduction of preneoplastic lesions to specific glucosinolates. The activities of hepatic UDP-glucuronosyltransferase form 2 (UDPGT-2) and cytochrome P4501A2 were increased by both vegetables. The induction effect of Brussels sprouts on the activity of UDPGT-2 was more marked than that of the red cabbage cultivars, suggesting that increased glucuronidation of IQ may account for the reduction of the preneoplastic lesions. Our findings support the assumption that Brassica vegetables protect against the carcinogenic effects of heterocyclic amines.
Abbreviations: ACF, aberrant crypt foci; GST-P, glutathione-S-transferase placental form; HA, heterocyclic amine; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; ITC, isothiocyanate; UDPGT, UDP glucuronosyltransferase.
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Introduction
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Numerous epidemiological studies suggest that consumption of cruciferous vegetables is inversely related with the incidence of various forms of cancer, in particular with colorectal cancer (14). One of the risk factors for the development of colon cancer may be exposure to heterocyclic aromatic amines (HAs), which are formed during the cooking of meat and fish (5). Therefore, we hypothesized that the cancer protective properties of cruciferous vegetables might be due to their chemopreventive effects against HAs. Indeed, we found recently that garden cress (Lepidium sativum), a cruciferous plant and its constituents reduce the induction of DNA damage and aberrant crypt foci (ACF) formation by 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline [IQ, (6)], a member of HA which causes colon and liver tumors in rats (5).
ACF and hepatic glutathione-S-transferase (GST-P+) foci are early preneoplastic lesions in colon and liver, respectively, which may give rise to the formation of tumors (710). The initiation and further development of carcinogen-induced ACF and GST-P+ could be prevented by dietary constituents (1113).
The major shortcoming of ACF assay models for the identification of chemoprotective agents against HAs is the low foci yield, which hinders the detection of anticarcinogenic effects (14). In an earlier study we observed an increase in the number of IQ-induced colonic ACF by a modified (high fatlow fibre) diet (15).
The chemopreventive effects of cruciferous vegetables towards dietary carcinogens such as nitrosamines and polycyclic aromatic hydrocarbons have been attributed to isothiocyanates and indoles (1621), which are formed upon hydrolysis of the parent glucosinolates by the enzyme myrosinase. Any process that damages plant tissues leads to the release of myrosinase. Cooking procedures, however, lead to inactivation of the enzyme (22) thereby reducing the release of breakdown products and the protective properties of the vegetables.
In the present study we investigated if commonly consumed cruciferous vegetables are protective towards IQ. Juices from two cultivars each of Brussels sprouts and red cabbage were added to the drinking water (5%, v/v) of rats before and during treatment with IQ. Four months after the last treatment with the carcinogen, the frequencies of IQ-induced hepatic glutathione-S-transferase (GST-P+) foci and colonic aberrant crypt foci (ACF) were determined. In order to assess the effect of cooking on the protection of the vegetables, juices were prepared from raw and cooked vegetables and used in the assay. Moreover, to find out which glucosinolates are responsible for the protective effects, we determined the glucosinolate contents of the different vegetables and tried to correlate the results to the effects seen in the aberrant foci experiments. On the basis of our recent findings with garden cress (6), we hypothesized that the mechanism behind the protective effects of cruciferous plants towards IQ is induction of UDP-glucuronosyltransferase (UDPGT), an important enzyme in the detoxification of HAs (2325) that can be induced by dietary constituents (26). To ascertain this assumption, we measured the effects of the different vegetables on the activities of UDPGT isozymes and compared the effects with the results obtained in the chemoprevention studies. Additionally, the impact of the juices on cytochrome P4501A2, an enzyme that catalyses the activation HAs (23), was investigated.
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Materials and methods
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Chemicals
Methylene blue, bovine serum albumin, chloramphenicol, 4-methyl umbelliferone (4-MU) and methoxyresorufin were supplied by Sigma (St Louis, MO, USA). Bradfords reagent was purchased from Biorad (Munich, Germany). 2-Amino-3-methyl-imidazo[4,5-f]quinoline was purchased from Toronto Research Chemicals (Toronto, Canada). GST pi polyclonal antibody was bought from Novocastra (Newcastle, UK).
Juice preparation
Brussels sprouts (Brassica oleracea L. var. gemmifera Cyrus and B.oleracea L. var. gemmifera Maximus) and red cabbage (B.oleracea L. var. capitata subvar. Rubra Roxy and B.oleracea L. var. capitata subvar. Rubra Reliant) were obtained from Novartis Seeds BV (Enkhuizen, The Netherlands). The vegetables were grown on the field and stored at 4°C in the dark after being harvested. Juices were prepared freshly every day from raw and cooked (100°C, 10 min in 100 ml water) materials. After discarding the outer leaves, the vegetables were cut into small pieces and juiced with a commercial machine (Elin T3232). The juices were diluted 1:20 either with tap water (raw vegetables) or cooking water (cooked vegetables). On the basis of preliminary experiments in which the palatability of the juices had been investigated, a 5% (v/v) concentration in the drinking water was chosen for the main experiments.
Analyses of glucosinolates
The glucosinolate contents of the vegetables were determined according to Wathelet et al. (27) and Spinks et al. (28) with slight modifications. Fresh plant tissue was extracted in boiling methanol (70%) in a water bath at 70°C for 20 min. Subsequently, the extract was centrifuged (1000 g, 10 min) and the supernatant collected. The pellet was re-extracted twice following the same procedure. Aliquots of the supernatants were loaded onto ion-exchange mini-columns (DEAE Sephadex A-25) and the glucosinolates were desulphated on-column as described by Helboe et al. (29). The desulphoglucosinolates were eluted with water and separated by gradient system high performance liquid chromatography (Thermo Separation Products) using a Nova Pak C18 (5 mm) reverse phase column (3.9 x 159 mm; Waters Corporation, USA). The solvent programme consisted of water for 1 min and a linear gradient over 20 min to water/acetonitrile 80/20 (flow 1.0 ml/min). The desulphoglucosinolates were monitored by UV-absorption at 229 nm and quantified against the internal standard glucotropaeolin. Identification of the individual glucosinolates was done by comparing retention times with pure standards and with a standard rapeseed reference material (BCR 367, Commission of the European Community Bureau of References, Brussels, Belgium).
Animals and treatment
All experiments were carried out with 3-week-old male Fischer 344 rats (body weight, 133157 g). The animals were purchased from Charles River Inc. (Borchen, Germany) and housed in groups of three in plastic cages under standard conditions (24 ± 1°C, humidity 50 ± 5%, 12 h light/dark cycle).
After 1 week of acclimatization, rats were switched from Purina rat chow (Soest, Germany) to a modified (high fatfibre free) AIN 76 diet (SDS, Witham, UK) on which they remained for the duration of the experiment. The rats were randomized into 10 groups (eight animals/group), namely (i) negative control; (ii) IQ control; and (iii) combined treatment groups (IQ and vegetables). The juices were given to the rats 5 days before and during the treatment with IQ, which was administered by gavage on 10 alternate days in corn oil (100 mg/kg bw/day). The control group received corn oil and pure tab water. Sixteen weeks after the last IQ treatment, the animals were killed by CO2 asphyxiation.
Determination of preneoplastic lesions in colon and liver
The livers of the killed animals were weighed and sections fixed in freshly prepared Carnoys solution and processed as described (30). GST-P+ foci (
3 cells) were identified by anti-placental GST stain under a light microscope. Their numbers were calculated per cm2 evaluated tissue section and at least 1 cm2 per animal was evaluated with an automatic image analyser (Lucia, Nikon; Meerbusch, Germany).
For the determination of ACF, the colon was removed and cleaned with Ringers solution, then cut open along the longitudinal median and fixed flat in 10% buffered formalin (pH 7.5) for 24 h. The samples were stained in 0.2% methylene blue and the number of ACF/colon and the number of crypts/focus were evaluated for the entire length of the colon from each rat at a 60-fold magnification as described by Bird (31).
Enzyme measurements
The effects of the vegetables on enzyme activities were monitored in a separate experiment. The rats (4/group) received for 1 week 5% (v/v) fresh juices of raw and cooked plant material. On the eighth day, the animals were killed and liver and colon microsomes prepared according to Ryand et al. (32). Protein levels of the microsomes were determined by the method of Bradford (33). UDPGT-1 and UDPGT-2 activities were measured by using 4-methyl umbelliferone and chloramphenicol as substrates according to Lilienblum et al. (34) and Young and Lietmann (35) with slight modifications, respectively. Cytochrome P4501A2 activity was determined using methoxyresorufin as a substrate according to Lubet et al. (36) at an excitation of 550 nm and an emission wavelength of 585 nm.
Statistical analysis
The numbers of ACF as well as the number and area of GST-P+ foci were compared using ANOVA and linear contrast after homogenizing the variance using logarithmic transformation. Induction of P4501A2 and UDPGT-2 activity was analysed with ANOVA following Dunnets multiple comparison test.
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Results
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Glucosinolate content of the vegetables
The overall glucosinolate content of Brussels sprouts cultivars was 23-fold higher than those of the red cabbage varieties (Table I
). The level of the different glucosinolates varied widely not only between the different vegetables but also between the different cultivars of the same vegetable. Sinigrin dominated in Brussels sprouts Cyrus (68% of the total glucosinolate content) whereas Maximus was additionally rich in iberin. In the red cabbage cultivars, sinigrin and glucoraphanin were the most abundant glucosinolates in Roxy and Reliant, respectively.
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Table I. Glucosinolate content of Brussels sprouts (Cyrus and Maximus) and red cabbage (Roxy and Reliant)a
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Effect of treatment on body weight and relative liver weight
Table II
shows that none of the treatments had an influence on either body weight or liver weight of the animals. Also, supplementation of the drinking water with juices had no significant effect on food consumption.
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Table II. Consumption of Brussels sprouts and red cabbage juices, body weight gain of rats and relative liver weight
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Effect of the vegetables on hepatic GST-P+ foci
All vegetables caused a reduction in the number and size of hepatic GST-P+ foci but the effects of the different cultivars varied over a broad range (Table III
). Brussels sprouts were consistently more effective than red cabbage. The strongest effect was measured with raw Cyrus, which reduced the number and size of foci by 67% and 91%, respectively. Interestingly, all juices caused a more pronounced reduction in the size than in the frequency of the foci. No significant differences were found between the protective properties of juices from cooked and raw vegetables.
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Table III. Influence of red cabbage (Roxy and Reliant varieties) and Brussels sprouts (Cyrus and Maximus varieties) juices on IQ-induced liver GST-P+ foci in the F344 rata
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Effect of the vegetables on colonic aberrant crypt foci
IQ caused a marked induction of ACF (Table IV
). The majority of these lesions was located in the distal part of the colon (Figure 1
). Both cultivars of Brussels sprouts decreased the number of IQ-induced ACF markedly, and the extent of reduction was similar (~50%) in all parts of the colon. With the red cabbage cultivars, only a slight, statistically non-significant reduction of the foci number was found. In contrast to the findings in the liver, the crypt multiplicity, which reflects the size of the foci was not decreased by any of the vegetables (Table IV
).
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Table IV. Influence of red cabbage (Roxy and Reliant) and Brussels sprouts (Cyrus and Maximus) juices on IQ-induced colonic ACF frequency and crypt multiplicity in F344 ratsa
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Fig. 1. Distribution of ACF in the colon of rats treated with either IQ alone or IQ and cooked Brussels sprouts Cyrus. The colon was removed intact by severing at the caeco-colonic and colonic-rectal junctions and processed as described in the Materials and methods section. The sections designated as proximal, middle and distal part of the colon represent the first, second and third fraction of the total length.
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Induction of cytochrome P450 1A2 and UDPGT
The effects of the juices on the activity of hepatic cytochrome P4501A2 and UDPGT-2 are summarized in Table V
. Raw and cooked Maximus, raw Cyrus and raw Reliant increased the activity of hepatic UDPGT-2 significantly, whereas only raw and cooked Maximus increased the activity of hepatic P4501A2. None of the vegetables modified the activity of hepatic UDPGT-1 (data not shown). The activities of UDPGT 1 and UDPGT 2 in the colon were
90% lower than those measured in the liver and there were no significant differences between the control and treatment groups (data not shown).
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Table V. Effect of red cabbage (Roxy and Reliant varieties) and Brussels sprouts (Cyrus and Maximus varieties) juices on the activities of hepatic cytochrome P4501A2 and UDPGT-2 in F344 ratsa
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Discussion
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According to our knowledge this is the first report on the prevention of HA-induced preneoplastic lesions by commonly consumed cruciferous vegetables. These observations support our earlier assumption, which was based on the results obtained with garden cress (6), that cruciferous plants are protective towards the carcinogenic effects of cooked food mutagens.
By using a modified diet composed of high fat but without fibre, it was possible to study the prevention of IQ-induced preneoplastic lesions simultaneously in liver and colon, the main target organs for tumor induction by HAs of the aminoimidazoazarene group.
All four cultivars reduced the size and number of GST+ foci in the liver, albeit to a different extent. The maximum reduction in the foci frequency was around 50%, whereas the foci size was decreased by up to 90% (Table III
). These findings indicate that the vegetables prevent not only the formation of initiated cells but also their clonal expansion. Earlier studies indicated that Brassica vegetables and their constituents inhibit the induction of hepatic preneoplastic foci by aflatoxin and diethylnitrosamine (3739) and Hirose and his coworkers (40) were the first who reported that phenetyl-ITC, a breakdown product of gluconastruitiin found in some cruciferous plants, reduces the number of hepatic GST+ foci induced by a HA (2-amino-6-methyldipyrido[1,2-a:3,2-d]imidazole) in rats.
In the colon, a different pattern of protection was seen as compared to that observed in the liver (Table IV
) in that Brussels sprouts but not red cabbage were protective and none of the juices reduced the multiplicity of the crypts, which reflects the size of the foci. This observation suggests that the vegetables prevent only the formation but not the development of the preneoplastic lesions in this organ. At present, although a few studies on the protective effects of specific Brassica constituents towards HA-induced ACF are available (41,42), no data concerning chemoprevention by Brassica vegetables are available.
Our findings indicate that the chemoprotective properties of Brussels sprouts and red cabbage involve inhibition of the formation (in both liver and colon) as well as development (only in liver) of preneoplastic lesions. One of the most important mechanisms of chemoprotection is induction of phase II enzymes, which detoxify DNA-reactive metabolites and thereby inhibit the formation of initiated cells (43,44). It has been postulated by Sparnins and Wattenberg (45) two decades ago that induction of glutathione S-transferase (GST) by constituents of Brassica vegetables and other dietary constituents accounts for their anticarcinogenic effects towards polycyclic aromatic hydrocarbons. However, the role of this enzyme in the detoxification of HAs is unclear (23). In previous experiments with garden cress we failed to detect an induction of GST, but the reduction of IQ-induced ACF formation was paralleled by a pronounced induction of UDPGT form 2 (6). Therefore, we hypothesized that the enhancement of the activity of this enzyme might be causally related to the protective effects. Indeed, the results of the present experiments support this assumption. Both red cabbage and Brussels sprouts enhanced the activity of UDPGT-2 (Table V
) but the effect of the latter was stronger than the former. Human and rat UDGPT isozymes were reported to play an important role in the detoxification of HAs (25,46) and the activity of these enzymes is known to be induced by cooked Brussels sprouts (47) and glucosinolates (26). Brassica vegetables can also induce glucuronidation reactions in humans. Pantuck et al. (48) reported an increase in the urinary excretion of paracetamol glucuronides in man after consumption of Brussels sprouts and cabbage. Also, recently, Knize and his cowokers (49) reported that consumption of broccoli increases the excretion of glucuronidation products of PhIP. Another possible mechanism of protection is the inhibition of enzymes which are involved in the activation of HAs. On the basis of results of in vitro experiments with bacteria, it was postulated that the antimutagenic effects of juices of Brassicas and other vegetables against IQ might involve inhibition of cytochrome P450 isozymes (50,51). An identical mode of action was postulated by Barcelo et al. (52) who found pronounced inhibition of IQ-induced mutagenicity by sulforaphane in a cell line expressing human CYP1A. The results of the present study, however, show that under in vivo conditions no such inhibitory effects take place. In contrast, even a moderate induction of MROD, a marker of P4501A2 which plays a key role in the activation of IQ and other HAs (23) was observed with some of the juices (Table V
). This latter finding is in agreement with the result of an earlier investigation (53).
The suppression of preneoplastic foci development by the vegetables may be described to induction of apoptosis. A number of recent articles report induction of apoptosis by constituents of cruciferous vegetables in vitro (5456) and in vivo (57). In a study by Smith et al. (12), the reduction of dimethylhydrazine-induced ACF in rats was reduced by sinigrin, one of the most abundant glucosinolates in Brassica vegetables, and was paralleled by a strong increase in the apoptosis rate. Therefore, the authors postulated that inhibition of ACF formation is due to a selective deletion of damaged stem cells in the crypts through apoptosis.
The chemical analysis of the vegetables shows that the level of individual glucosinolates differ strongly, even between cultivars of the same vegetable (Table I
). Nevertheless, no pronounced differences were seen in the biological effects of different cultivars. Thus, neither the inhibition of preneoplastic lesions nor alterations of the enzymatic activities appear to depend on the level of individual glucosinolates. Rather the overall glucosinolate content, which was substantially (23-fold) higher in the Brussels sprouts than in red cabbage, seems to play a more important role in regard to chemoprotection towards IQ and enzyme induction. This assumption is supported by the report of Nho and Jeffery (58) in which synergistic upregulation of different phase II enzymes was found when a combination of glucosinolate derivatives was used.
The stability of glucosinolate degradation products, constituents believed to be responsible for the chemoprotective effect of cruciferous vegetables (1621), has been addressed in earlier reports. de Vos and Blijleven (59) have found that alkyl or alkenyl isothiocynanates are volatile except those bearing a sulfoxyl terminal side chain and indole moiety such as 3-methylsulfinylpropyl isothiocyanate (iberin isothiocyanate) and 3-indolylyacetonitrile. Sulforaphane (4-methylsulfinylbutyl isothiocyanate) was reported to be a more stable and less volatile compound during processing and cooking (60) whereas allyl isothiocyanate was very volatile although it can also be further decomposed to non-volatile compounds under cooking conditions (61). In a preliminary experiment in which we analysed juices prepared from raw Brussels sprouts for breakdown products, we found iberin, sulforaphane, goitrin and indole-3-carbinol (Verkerk et al., in preparation).
In spite of the fact that the plant enzyme myrosinase, which catalyses the formation of isothiocyanates and indoles from glucosinolates is inactivated by heating, we found no marked difference between the protective effects of cooked and raw vegetables. This might be due to the formation of anticarcinogenic breakdown products of glucosinolates in the gastrointestinal tract by representatives of the intestinal flora (21) and/or by hydrolysis under acidic conditions in the stomach (57).
In many in vivo chemoprevention studies with HAs, including those with constituents of Brassica vegetables, the doses of putative protective compounds used were by far higher than the levels present in the human diet (58,59). In the present study, the amounts of juice which caused protective effects were quite small (0.60.7 ml/day/rat, body weight 250 g) and correspond to consumption of 180210 ml juice/day/person (body weight 75 kg). The same amount of bioactive juice is contained in regular size vegetable meals (300400 g). This comparison supports the assumption that Brassica vegetables may protect humans against the carcinogenic risks of HAs. The inverse relationship between the incidence of colon cancer and consumption of Brassica vegetables, which has been observed in a number of epidemiological studies (14), may be due to these effects.
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Notes
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5 To whom requests for reprints should be addressed at: Institute of Cancer Research, Borschkegasse 8A, 1090 Vienna, Austria Email: profeka{at}yahoo.com 
* Contributed equally. 
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Acknowledgments
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This study was supported by EU-funds (EFGLU and HC-AMINES) to S.K, S.R and R.V.
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Received June 7, 2002;
revised October 14, 2002;
accepted October 21, 2002.