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3 TNO Quality of Life, Business Unit Biosciences, 4 TNO Quality of Life, Business Unit Quality and Safety, Zeist, The Netherlands; 5 RIKILT- Institute of Food Safety, Wageningen, The Netherlands; and 6 Wageningen University and Research Centre, Division of Toxicology, Wageningen, The Netherlands
* To whom correspondence should be addressed. E-mail: ashwin.dihal{at}tno.nl.
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ABSTRACT |
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 TOP ABSTRACT IntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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0.05). Rats supplemented with 40 g rutin/kg diet had only 30% of the (3'-O-methyl-) quercetin concentration of 10 g quercetin/kg diet-fed rats (P < 0.001). In conclusion, quercetin, but not rutin, at a high dose reduced colorectal carcinogenesis in AOM-treated rats, which was not reflected by changes in ACF-parameters. The lack of protection by rutin is probably due to its low bioavailability.
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Introduction |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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5% (1). Epidemiological studies suggest that intake of fruits and vegetables (2), dietary fiber (3), and flavonoids (4) are inversely correlated with the CRC incidence. One of the major flavonoid representatives in the human diet is quercetin, which in its natural form is glycosylated and can be found in fruits and vegetables, including blueberries, onions, curly kale, broccoli, and leek (5). Once ingested, conjugated quercetin can be released by intracellular and membrane-bound ß-glycosidases of small intestinal cells (6). In the colon, on the other hand, quercetin glycosidic bonds can be hydrolyzed by local ß-glycosidases produced by bacteria, leading to colonic release and absorption of quercetin (7). This is also the case with rutin (quercetin-3-O-ß-rutinoside), the major quercetin glycoside found in tea. Worldwide, CRC shows a high annual incidence (1,000,000 patients) and mortality (530,000) (8). Therefore, biomarkers that reveal colorectal carcinogenesis at an early stage are urgently needed for prevention of colorectal tumors. Such biomarkers were first described in 1981 by Shamsuddin et al. (9,10) and designated aberrant crypts in 1987 by Bird (11). However, the validity of these histo-pathological aberrations as predictors of colon carcinogenesis is still a matter of debate (12–14). It has been suggested that histological lesions showing accumulation of ß-catenin in colonic crypts may be more relevant indicators of colorectal carcinogenesis (15). In mice, these ß-catenin accumulated crypts (BCA-C) demonstrate increased cell proliferation and are positively correlated with the development of colorectal tumors. Gene mutations in ß-catenin, among others, may prevent phosphorylation of the ß-catenin protein, which is required for its degradation and can lead to its cytoplasmic accumulation (16). Consequently, cytoplasmic ß-catenin can migrate into the nucleus and may target cell proliferation, differentiation, and apoptosis (1,17). Therefore, accumulation of free ß-catenin is thought to be an early event in colorectal carcinogenesis (16).
Because quercetin supplements are commercially available and claimed to be health promoting, the aim of the present study was to investigate the effects of quercetin aglycone, i.e. the sugar-free flavonoid, and its major dietary source, rutin, on (biomarkers of) colorectal carcinogenesis. Based on previous studies (18,19), it was hypothesized that quercetin might inhibit development of CRC in azoxymethane (AOM)-treated rats.
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Materials and Methods |
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Interim autopsy. At 8 wk post-AOM, 8 AOM-treated and 6 untreated rats/diet group were killed in the morning without overnight food deprivation. Rats were anesthetized by inhalation of 5% isoflurane combination with O2 and N2O (1: 1). After collection of abdominal aortic blood, quercetin and its metabolites in plasma of untreated rats were stabilized by 5.6 mmol/L ascorbate (Merck) (20).
Following excision, colorectums were cut longitudinally and rinsed with 70% ethanol at 4°C. Colorectums of AOM-treated rats were fixed between filter paper and stored in 70% ethanol at 4°C prior to the aberrant crypt foci (ACF) counts and finally embedded in paraffin.
Final autopsy.
At wk 38 post-AOM, rats were killed as described above, with modifications. Among tumor-bearing rats, the tumor number and maximum diameter were recorded. Large tumors (diameter 
5 mm) were removed and split diagonally (13). The luminal part, as well as small tumors (diameter <5 mm), were fixed in RNAlater, for mRNA analysis to be reported elsewhere. Therefore, tumor classification as being adenomas or carcinomas was assessed only in large tumors.
ACF in unsectioned colons. To visualize ACF, colorectums of AOM-treated rats killed at the interim autopsy were stained for 10 min with 0.1% (w:v) methylene blue dissolved in 70% ethanol. ACF were recognized as single or multiple enlarged crypts, with altered luminal openings and thickened epithelium, in comparison with surrounding normal crypts (11). ACF numbers and multiplicity, i.e. the number of aberrant crypts per ACF, were scored.
Quantification of quercetin and its metabolites in plasma.
Quercetin metabolites in pooled plasma samples of AOM-untreated rats killed at the interim autopsy were deconjugated by the Helix pomatia enzyme (Sigma) with ß-glucuronidase 7500 kU/L) and sulfatase (500 kU/L) activity (20). Quercetin and its metabolites were analyzed by HPLC with Coularray detection and quantified with calibration curves for quercetin, 3'-O-methyl-quercetin (isorhamnetin, Roth), and 4'-O-methyl-quercetin (tamarixetin, Extrasynthese). Plasma concentrations of rats fed 1 and 10 g quercetin/kg diet were published previously (20).
Immuno-histochemistry. After alcohol fixation and paraffin embedding, we performed immuno-histochemistry on the middle of the distal colon because of the highest ACF frequency. Per rat, 35 colon sections were cut en face at 4 µm (15) for hematoxylin and eosin staining (every 1st slide) as a reference for adjacent slides stained for total ß-catenin (every 2nd slide) and Ki67 (every 3rd slide). After blocking endogenous peroxidase (30 min in 0.3% [v:v] H2O2 in methanol) and heat-induced antigen retrieval (15 min in 1 mmol/L EDTA-NaOH, pH 8.0 at 95–98°C), sections were incubated with 25% (v:v) goat serum in PBS (15 min). Subsequently, sections were incubated with primary antibodies against total ß-catenin (Neomarkers) for 30 min (4000x PBS dilution, rabbit polyclonal antibody) or the proliferation marker Ki67 (Neomarkers) for 10 min (200x PBS-dilution, rabbit monoclonal antibody). Sections were then incubated with the secondary Powervision Poly horseradish peroxidase labeled polyclonal antibody (Immuno Vision Technologies) for 30 min. Horseradish peroxidase activity was visualized by incubation with Vector NovaRED (Vector Laboratories) for 10 min. Finally, sections were weakly counterstained with hematoxylin for 45 s.
ß-Catenin accumulated crypts (BCA-C) and ß-catenin accumulated ACF (BCA-ACF) were recognized as crypts or ACF, respectively, showing nucleic and/or cytoplasmic ß-catenin accumulation (15). The number of lesions, multiplicity, and grade of ß-catenin accumulation was scored, the latter expressed as excessive ß-catenin staining intensity (low, intermediate, or high) relative to normal crypts. To distinguish ACF in unsectioned and en face sectioned colons, the term en face ACF is used for the latter.
In Ki67-stained slides, the number of positive and negative nuclei were scored per ACF and expressed as the proliferation index (22). Large colorectal tumors (diameter 5 mm) were classified in hematoxylin and eosin stained slides (13).
Statistics.
We analyzed the dose-dependent effect of quercetin on ACF multiplicity and tumor incidence with the Cochran-Mantel-Haenszel test. Tumor incidences among different groups were compared with one another using the chi-square test. We calculated correlations between dietary quercetin and plasma concentrations of quercetin and its metabolites with the Pearson's correlation coefficient. Quercetin-mediated effects on CRC precursors, assessed by immuno-histochemistry in rats fed a 0 or 10 g quercetin/kg diet, were analyzed using the nonparametric Mann-Whitney U test to account for unequal variances. Plasma concentrations of quercetin and 3'-O-methyl-quercetin were compared using the 1-way ANOVA, followed by the t test with the overall error term to compare all groups. Mean values are presented as means ± SD and differences were significant if P 0.05 (2-tailed).
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Results |
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900 mg/(kg body wt·d) at wk 2, declining to 450 mg/(kg body wt·d) at wk 38 post-AOM. The corrected quercetin intake by untreated rats fed diets containing 1 g/kg and 0.1 g quercetin/kg were 10% and 1%, respectively, of the group fed 10 g quercetin/kg. AOM-treated rats fed the 40 g rutin/kg diet consumed 3500 mg rutin/(kg body wt·d) at wk 2, declining to 1700 mg/(kg body wt·d) at wk 38. In the course of the experiment, the corrected dietary intakes of quercetin and rutin did not differ between AOM-treated and untreated rats. In total, 4 AOM-treated rats were down in health or moribund as a result of colon tumors (2 rats), a zymbal gland tumor (1 rat), and a small intestinal tumor (1 rat), most likely caused by AOM toxicity. Because these rats were killed 3–4 mo before completion of the study, they were excluded from further analyses to ensure proper time-matched comparisons between groups.
ACF in unsectioned colons.
All AOM-treated rats killed at wk 8 post-AOM (n = 8/group) developed ACF found mainly in the distal colon. Quercetin did not affect ACF, because ACF numbers in control rats (45.8 ± 20.0) were comparable to rats fed a 0.1- (59.4 ± 18.3), 1- (42.3 ± 13.8), or 10 g quercetin/kg diet (49.6 ± 17.8). In addition, ACF numbers among rutin-treated rats (42.9 ± 14.9) were comparable with the controls. Quercetin did not affect the ACF multiplicity (range: 1–8), even when ACF were classified as small (3 aberrant crypts/focus) and large (4 aberrant crypts/focus) (23,24) (data not shown).
ß-Catenin accumulation. Two subpopulations of en face ACF were found that showed either ß-catenin immuno-reactivity restricted to the cell membrane, as for the normal adjacent crypts (Fig. 1A), or immuno-reactivity in the nucleus and/or cytoplasm (Fig. 1D). The second class of putative CRC biomarkers, BCA-C (Fig. 1G), did not show an enlarged crypt lumen or thickened epithelium relative to the surrounding normal crypts, in contrast to ACF and BCA-ACF.
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Compared with controls, the number of en face ACF, regardless of the ß-catenin accumulation status, was not affected by the 10 g quercetin/kg diet (0 vs. 10 g quercetin/kg diet: 10.3 ± 4.4 vs. 10.5 ± 6.7, n = 8/group). The same was true for ACF with (1.4 ± 1.1 vs. 1.8 ± 1.3) and without (9.0 ± 4.1 vs. 9.0 ± 6.2) ß-catenin accumulation, consistent with observations in methylene blue-stained ACF in unsectioned colons (45.8 ± 20 vs. 49.6 ± 17.8).
Multiplicities of ACF, BCA-ACF, and BCA-C did not differ between rats fed the treatment with the 10 g quercetin/kg diet and controls, even when a distinction was made between small (3 aberrant crypts/focus) and large (4 aberrant crypts/focus) lesions (data not shown). This result agrees with methylene blue-stained ACF in unsectioned colons.
Proliferation. The proliferation index within the total number of en face ACF, regardless of the ß-catenin accumulation status, decreased (P < 0.05) from 99.0 ± 0.5% (n = 5/group) in control rats to 80 ± 16.5% in 10 g quercetin/kg diet-treated rats. In ACF without ß-catenin accumulation, the proliferation index tended (P = 0.060) to be lower in rats fed the 10 g quercetin/kg diet (79.8 ± 18.5%) than in controls (98.7 ± 1.1%). The relatively few ACF with ß-catenin accumulation also tended to be lower (P = 0.07) in rats fed the 10 g quercetin/kg diet (82.1 ± 18.9%) than in controls (99.8 ± 0.4%).
Colorectal tumors. At 38 wk post-AOM, untreated rats fed the control diet or diets supplemented with quercetin or rutin had not developed colorectal tumors. The tumor incidence, i.e. the number of tumor-bearing rats expressed as a percentage of the total number of rats, was 50% in AOM-treated rats fed the control diet and was dose dependently (P < 0.05) decreased by dietary quercetin (0 vs. 10 g quercetin/kg diet: P < 0.05, Table 1). The dose dependent decrease in tumor incidence in quercetin-fed rats at wk 38 did not correlate with the ACF numbers scored in unsectioned colons obtained at wk 8. Both the tumor multiplicity (the mean number of tumors per rat after exclusion of tumor negative rats) and tumor diameter were negatively correlated with the dietary quercetin concentration (both: r = –0.98, P < 0.05). The tumor incidence, multiplicity, and size were not affected by the 40 g rutin/kg diet.
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0.99, P < 0.01). Interestingly, rats fed the 40 g rutin/kg diet, which equals 20 g quercetin aglycone/kg diet, had only 30% of quercetin and 3'-O-methyl-quercetin relative to rats fed the 10 g quercetin/kg diet (P < 0.001).
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0.05). Plasma concentrations of tamarixetin (4'-O-methyl-quercetin) were below the detection limit for all flavonoid-supplemented groups.
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Discussion |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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4 crypts/focus) are reliable CRC biomarkers (23,24). The lack of a correlation between the number of methylene blue-stained ACF and the tumor incidence has been described (12–14). Wijnands et al. investigated the effect of a 40 g rutin/kg diet in a similar AOM model and found comparable tumor incidences in controls and rutin-treated rats (13), as was the case in our study. Because we found no relation between ACF and the tumor incidence, we hypothesized that a specific ACF subclass might be related to CRC. Therefore, ACF were immuno-histochemically classified into lesions with and without accumulation of the CRC-related protein ß-catenin. However, neither of these ACF subclasses was influenced by quercetin. Taken together, ACF cannot be considered general biomarkers for CRC.
Another putative CRC biomarker analyzed is the BCA-C. Among BCA-C, quercetin significantly decreased ß-catenin accumulation at an intermediate grade, whereas none of the ACF-related variables were affected. Although quercetin only partially affected BCA-C-related variables, these results suggest that the BCA-C might be better biomarkers for CRC than ACF. Hata et al. (15) provided evidence that BCA-C, but not ACF, are reliable biomarkers in AOM-induced colorectal carcinogenesis. These authors described increasing ACF numbers but decreasing tumor numbers toward the proximal colon. In contrast, occurrence of BCA-C and colorectal tumors increased toward the rectum and were correlated with one another.
Quercetin not only dose dependently decreased the incidence of colorectal tumors but also reduced the tumor multiplicity and size of AOM-induced colorectal tumors. The quercetin-mediated decrease in tumor incidence is consistent with Deschner et al. (18), who fed AOM-treated CF1 mice with 1–20 g quercetin/kg diet for 50 wk. In our study, the protective effect evoked by quercetin might be caused by decreased cell proliferation, as was indicated by the dose-dependent decrease in tumor size and the significant decrease in proliferation index in ACF. Another mechanism involved might be induction of apoptosis by quercetin (19).
Rats were supplemented with a 40 g rutin/kg diet (20 g quercetin aglycone/kg diet) to compensate for rutin's low bioavailability (25,26). Therefore, calculated concentrations of quercetin and its metabolites in the 40 g rutin/kg diet-treated group should be twice that of rats fed the 10 g quercetin/kg diet, which is in contrast to our findings. Compared with rats fed the 10 g quercetin/kg diet, rutin-supplemented rats showed only 30% of the quercetin and 3'-O-methyl-quercetin concentrations. Limiting factors involved in bioavailability of the quercetin moiety in rutin are probably intracolonic quercetin degradation and limited colonic absorption (7), the latter as a result of a relatively low absorptive area in the colon (5).
In this study, corrected quercetin intake by rats supplemented with the lowest quercetin concentration of the 0.1 g quercetin/kg diet was 4.5–9.0 mg quercetin/(kg body wt/d) throughout the experiment. Linear extrapolation of this dietary intake by rats to a human weighing 60 kg would equal daily supplementation with 0.3–0.5 g quercetin, which approximates the daily intake of 1 quercetin supplement of 500 mg. The CRC incidence was significantly decreased in AOM-treated rats fed the 10 g quercetin/kg diet, which after extrapolation to a human weighing 60 kg would equal supplementation with 30–50 g quercetin aglycone/d. This amount is an increase of 2,000–3,000 times the regular dietary quercetin intake of an estimated 16.3 mg/d when expressed as aglycone (27). One of the major quercetin sources in the human diet is rutin (7), which in this experiment seems to be the inactive quercetin isoform with respect to reduction of colorectal carcinogenesis. Thus, when assuming similarity in AOM-induced development of CRC in rats and colorectal carcinogenesis in humans, inhibition of human colorectal carcinogenesis cannot be achieved by regular dietary intake of conjugated quercetin or by intake of supplements containing quercetin aglycone. Quercetin's beneficial effect might occur only at unrealistically high concentrations equaling daily intakes of 60–100 quercetin pills of 500 mg each.
In conclusion, inhibition of the tumor incidence by quercetin at wk 38 was not associated with the ACF number or multiplicity at wk 8 or with the ACF classification based on the absence or presence of ß-catenin accumulation. Therefore, the ACF are not reliable biomarkers for CRC. On the other hand, ß-catenin accumulation in BCA-C was partially decreased by quercetin. Therefore, BCA-C might be more relevant CRC biomarkers than BCA-ACF, but this requires further research. In contrast to quercetin aglycone, rutin lacks anticarcinogenic activity, probably as a result of its low bioavailability.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supplemental Table 1 is available with the online posting of this paper at jn.nutrition.org.
7 Abbreviations used: AOM, azoxymethane; ACF, aberrant crypt foci; BCA-ACF, ß-catenin accumulated aberrant crypt foci; BCA-C, ß-catenin accumulated crypts; CRC, colorectal cancer.
Manuscript received 28 February 2006. Initial review completed 20 April 2006. Revision accepted 15 August 2006.
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LITERATURE CITED |
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