1 Institute of Nutritional Physiology, Federal Research Centre for Nutrition and Food, Haid-und-Neu-Strasse 9, 76131 Karlsruhe, Germany, 2 Technical University of Munich, Department of Food and Nutrition, 85354 Freising-Weihenstephan, Germany and 3 Research Institute Geisenheim, Department of Wine Analysis and Beverage Research, 65366 Geisenheim, Germany
* To whom correspondence should be addressed. Tel: +49 721 6625 408; Fax: +49 721 6625 404; Email: stephan.barth{at}bfe.uni-karlsruhe.de
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Abstract |
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Abbreviations: ACF, aberrant crypt foci; AOM, azoxymethane; CleA, clear apple juice; CloA, cloudy apple juice; COX-1, cyclooxygenase-1; COX-2, cyclooxygenase-2; DMH, dimethylhydrazine; PGE2, prostaglandin E2; SCFA, short chain fatty acids
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Introduction |
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Until now, >6500 structurally different, naturally occurring flavonoids have been described. Various flavonoid compounds have been found to possess a range of cancer preventive activities including prevention of oxidative DNA damage (2), inhibition of carcinogen activation (3,4), induction of carcinogen detoxifying systems (5), interaction with cellular signalling pathways and modulation of gene expression controlling proliferation, differentiation and apoptosis of cancer cells (6).
Apples are a significant source of flavonoids in human diets in Europe (7,8) and epidemiological data correlated apple consumption with a reduced lung cancer risk (9). To initially characterize mechanisms by which apples may prevent cancer, studies using apple formulations derived from different apple tissues have shown antioxidative and antiproliferative activity in vitro (1012). Although no attempt has been made to identify the relevant bioactive component(s) in apples, the observed in vitro effects were discussed to be attributed either to the high concentrations of flavonoids in apple peel and flesh as well as apple procyanidins or non-flavonoid apple fibre such as apple pectin (13).
More detailed studies on the cancer preventive effects of different fruit or vegetable varieties have usually been conducted in complex animal models using either the lyophilized fruit or vegetable itself (14), juices (15), polyphenol enriched extracts (16,17) or specific constituents (18). However, comparable in vivo studies using apple preparations in rodent models for colon carcinogenesis are still lacking.
Besides genetic models of intestinal carcinogenesis (19), the rodent model of dimethylhydrazine (DMH)- or azoxymethane (AOM)- induced colon cancer has been widely used in studies to evaluate anticarcinogenic properties of dietary factors by analyzing colonocyte DNA damage (20), epithelial hyperproliferation (21), tumor/cell-cycle related gene expression (17), signal transduction (22), and initiation and growth of putative premalignant lesions called aberrant crypt foci (ACF) (14).
Owing to the strong in vitro support of cancer preventive activity by apple constituents, the aim of the present study was to investigate the potential effects of apple juice on markers of colon carcinogenesis in the rat model of DMH-induced carcinogenesis. As the variety of apple cultivars and procedures of apple processing significantly determine the content and spectrum of polyphenols and non-polyphenolic compounds in apple juices, we used polyphenol-rich apple varieties for the production of two different juice preparations. Both the cloudy (CloA) and the clear apple juice (CleA) contained high and comparable amounts of monomeric polyphenols. Beside the heterogeneous cloud fraction exclusive to CloA, the juices further differ in their content of procyanidins and non-polyphenolic dietary fibre ingredients, such as pectin. Using these apple juice preparations with different content of native apple compounds gives the opportunity to relate potential cancer preventive effects to the distinct composition of either CloA or CleA or both juices.
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Materials and methods |
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HPLC analysis of apple juice polyphenols
Apple polyphenols were separated on a 1090 HPLC/PDA system (Hewlett-Packard; Böblingen, Germany) equipped with a 250 x 4.6 mm Aqua 5 µm C18 column and protected with a 4 mm x 3 mm C18 ODS security guard (Phenomenex; Aschaffenburg, Germany). Gradient elution was applied with an acetonitrile/acetic acid gradient according to a previously published protocol (23). Detection wavelengths were 280, 320 and 360 nm. The juices were injected directly after centrifugation and 0.45 µm filtration with cellulose acetate membranes. Quantification was carried out using peak areas from external calibration curves. Analysis and polyphenol quantification were done immediately before experiments started. Further, the pectin content was analyzed according to a previously published protocol (24).
Animals
Male Fischer 344 (F344) rats (n = 90; 118 g on arrival) were obtained from a licensed animal supplier (Harlan Winkelmann, Borchen, Germany) and housed in a temperature- and humidity-controlled animal unit under ambient temperature of 21 ± 2°C and a 1212 h lightdark cycle. The rats were allowed to acclimatize for 9 days during which they consumed ad libitum tap water. Owing to the described effects of isoflavones on AOM-induced development of ACF (25) a standard rat diet free of soyprotein was used for the present study (Ssniff; Soest, Germany) and was further HPLC-analyzed to confirm the absence of potential isoflavone contamination (data not shown). The basal diet consisted of 3.3% fat, 19.0% protein, 4.9% fibre, 3.1% minerals and 56.7% carbohydrates (12.2 kJ/g).
Experimental protocol
After a period of 10 days from arrival, the rats were randomly assigned to three treatment groups (n = 30/group) receiving either tap water (control; Cont), CleA or CloA ad libitum until the end of the experiment. Juices were provided daily and juice or water consumption was recorded. Juices had the same ascorbic acid content (59 mg/l) immediately before filling into the drinking bottles. Body weight was recorded twice every week. One week after starting the juice intervention, half (n = 15) of the intervention group received intraperitoneal injections of either DMH (20 mg/kg body wt) or 0.9% NaCl four times at 1-week intervals. Three weeks after the last DMH injection, animals were killed by decapitation and biological samples were prepared and processed as detailed below for the respective analytical methods.
The experimental protocol was performed in accordance with the guidelines of the ethics committee responsible for the administrative district of Karlsruhe.
Single cell microgel electrophoresis assay (comet assay)
Immediately after decapitation of the animals (n = 5/group), the entire colon was rapidly removed and divided into proximal and distal portions; the limit of the proximal portion was defined by the fish bone pattern. The distal colon was placed in 37°C HBSS (137 mM NaCl, 5.4 mM KCl, 337 µM Na2HPO4 x 2H2O, 278 µM K2HPO4 x 3H2O, 5.6 mM glucose, 4.2 mM NaHCO3 and 20 mM HEPES). Subsequently, mucosa cells from the distal colon sample were isolated and analyzed using the procedure described by Wollowski et al. (26).
Fifty cells of each slide (150 cells per animal) were analyzed using the imaging software of Perceptive Instruments (Halstead, UK). The amount of damaged DNA was expressed as the percentage of DNA in the tail (tail intensity).
mRNA analysis
The distal colons of n = 5 animals/group were cleared of the contents by flushing with 37°C prewarmed 0.9% NaCl in diethylpyrocarbonate-treated water and mucosa cells were collected in 1.8 ml of phosphate buffered-saline (1 x PBS) by carefully scraping the luminal colon mucosa with a glass slide. Total RNA was extracted from the cells using the guanidine isocyanate/acid phenol method (27). The reverse transcription (RT) and the polymerase chain reaction (PCR) were performed in a PCR thermocycler (PTC 200, MJ Research Inc., Waltham, MA) by using the Ready-to-Go RTPCR kit in accordance with the kit manual (Amersham Pharmacia Biotech, Freiburg, Germany). PCR cycling conditions were: initial denaturation at 95°C for 5 min, subsequent amplification over 2530 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 60 s, final extension at 72°C for 5 min. The respective sequences used for generation of sense and antisense primers were nt 959982 and 16831662 of the rat COX-2 cDNA (GenBank accession no. NM_017232.2), nt 10911110 and 17751752 of the rat COX-1 cDNA (accession no. NM_017043.1), nt 904925 and 17581736 of rat -GCS (accession no. NM_012815.2), nt 120 and 768749 of rat GST-M2 (accession no. M13590.1), nt 222 and 480460 of GST-P cDNA (accession no. NM_012577.1) and nt 7194 and 570547 of rat GAPDH cDNA (accession no. XM_235039.2) used as internal standard for normalization. Primer sequences for amplification of the GSH-related enzyme cDNAs were taken from a previously published protocol (17).
Primers were tested for linearity over cycle number and analyses were all carried out in the linear portion of the curve, thus allowing semi-quantitative analysis of mRNA amount. PCR products were run on 1.5% agarose gel in 1x TBE buffer and ethidium bromide-stained bands were visualized and quantified using an automated computer-based image analysis system (QuantityOne/FluorS Imager; Biorad, München, Germany). Quantities of each PCR product were normalized by dividing the average gray level of the signal by that of the corresponding GAPDH amplicon.
Assays of ACF and colonic cell proliferation
To investigate the proliferative activity of epithelial cells, n = 5 rats per group received a 30 mg/kg intraperitoneal injection of 5-bromodeoxyuridine (BrdU) 1 h before killing. After excision of the distal colon and carefully flushing with prewarmed 0.9% NaCl, the mucosa was pinned flat on a paraffin wax block in a Petri dish, mucosal side up and fixed in 4% PBS-buffered formalin solution for minimum 1 h (BrdU assay) or 16 h (ACF assay).
The distal 5 mm strip of the colonic mucosa specimen was resected for BrdU assay and after a further overnight fixation the remaining colonic mucosa not utilized for proliferation assay was rinsed in PBS, stained with 0.2% methylene blue for 5 min, and examined under an inverse microscope (Zeiss Axiovert 100) at 100x magnification. Crypts were considered aberrant, if they were visibly enlarged and protruding when compared with surrounding crypts, having elongated openings and increased pericryptal zones. The number of ACFs observed per distal colon and the number of aberrant crypts per focus were recorded.
For processing the BrdU assay the resected strip was fixed and dehydrated, embedded in Paraplast plus (VWR International, Bruchsal, Germany) and cut into 5 µm thick sections (Microm HM 500 O, Heidelberg, Germany) mounted on silanized glass slides and dried overnight at room temperature. After heating the sections at 97°C for 30 min in 10 mM sodium citrate (pH 6.0), DNA-synthesizing epithelial cells were identified by immunohistochemistry with an anti-BrdU monoclonal antibody (DAKO Cytomation, Glostrup, Denmark) diluted 1:200 and with the Vectastain Elite ABC kit (Alexis, Grünberg, Germany). Immunocomplexes were visualized with diaminobenzidine. Labeled cells were recognizable under the microscope by the dark-brown nuclear pigment. Tissue sections were then counterstained for nuclear staining with hematoxylin and examined at 400x magnification under a Zeiss Axiovert 100 microscope. Well-oriented crypts (n = 25 per animal) with the lumen visible from the bottom to the mucosal surface and with a single layer of cells along each crypt column were selected for counting BrdU labelled and BrdU unlabelled epithelial cells randomly selected from entire tissue sections.
Immunological analysis
Immune cell suspensions from resected spleens were prepared with RPMI-1640 culture medium containing 50 ml/l of heat-inactivated fetal bovine serum, L-glutamine (2 mmol/l), penicillin (1 x 105 U/l), streptomycin (100 mg/l) and HEPES (25 mmol/l); all components were purchased from Life Sciences (Eggenstein-Leopoldshafen, Germany). Splenocytes were isolated as previously described (28).
The expression of cell-surface markers on the immune cells of spleen was investigated by immunofluorescence using phycoerythrin-conjugated mouse anti-rat monoclonal antibodies to CD4 (Caltag, Hamburg, Germany) and fluorescein-conjugated mouse anti-rat monoclonal antibodies to CD8 (Caltag) with appropriate isotype controls. Samples were analyzed with a FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany).
Natural killer (NK) cell activity of immune cells derived from spleen was assessed by flow cytometry. The mouse Moloney leukemia cell line, YAC-1, was used as target cell line (28).
Antioxidant status
Total plasma antioxidant activity in rats (n = 57/group) was determined by using the FRAP assay with minor modifications as decribed before (29) and the improved decolorization TEAC assay (30). Interassay and intraassay variability were <5%.
Statistical analysis
All data are presented as the mean ± SEM. Statistical analysis was accomplished using ANOVA and NewmanKeuls multiple group comparison test to identify significant differences between groups. P-values <0.05 were considered significant.
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Results |
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Polyphenol and pectin content of apple juice preparations
Table I shows the concentrations of polyphenolic compounds present in CleA and CloA. Both apple juice preparations contained a similar mixture and quantity of dihydrochalcones, hydroxycinnamic acids and quercetin glycosides. When focused on the contents of procyanidins B1 and B2, CloA contained 30% more procyanidins B1/B2 than CleA (Table I). The composition and the amounts of the different polyphenols reflect the typical pattern of mixed cider apple juices. Further analysis showed that the pectin concentration in CloA is
4-fold that of CleA (Table I), which is due to the enzymatic pectin degradation and the ultrafiltration during the processing of clear juices.
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There was no statistically significant effect on NK cell activity of either the DMH treatment or the apple juice intervention in any of the groups (data not shown).
The CD4:CD8 ratio was significantly reduced by DMH in the groups with water (Cont; P < 0.05) or CleA (P < 0.05) when compared with the respective controls treated with NaCl (Figure 4). This reduction of CD4:CD8, which was due to increased CD4 and decreased CD8, was not observed in the DMH group receiving CloA, leading to a normalization of CD4:CD8 when compared with the Cont/NaCl group (P > 0.05) and a significantly elevated CD4:CD8 ratio when compared with the Cont and also the CleA groups receiving DMH (P < 0.05).
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Discussion |
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Since DNA damage is recognized as the initial step in chemical carcinogenesis, the blocking of DNA damage would be the first line of defense against cancer caused by carcinogens. Furthermore, it is assumed that DNA-alkylation may also be important in human cancer formation (31) and thus antigenotoxic effects observed in chemical carcinogenesis by DMH or AOM in rodent models might be representative for the human system. Accordingly, by using comet assay based detection of DNA single strand breaks, our results showed that CloA but not CleA attenuated the genotoxic effect of DMH in enterocytes of the distal colon.
The apple juice preparations used in the current study contained similar amounts of well characterized monomeric polyphenols. However, CloA provided a higher content of procyanidin B1/B2 and of pectin and an additional cloud fraction lacking in CleA. These differences might explain the greater antigenotoxic effect exerted by CloA.
Pectin as the main dietary fibre source, present in cloudy apple juices has been shown to be a good fermentation substrate for intestinal bacteria releasing distinct spectra of short chain fatty acids (SCFA) (acetate, proprionate, butyrate) (32). SCFA exert a trophic function on colonic mucosa (33) and further influence mucosal proliferation and apoptosis in the colon (34,35). However, when using a mixture of SCFA potentially arising in the colon from bacterial fermentation of pectin, DNA breaks by H2O2 could not be inhibited in rat colonic epithelial cells in vitro (36).
DMH is an indirect carcinogen, which is metabolized by P450 2E1 and 1A2 isoforms to methyldiazonium to exert its carcinogenic effect by giving rise the main DMH adduct O6-methylguanine (37). Other results suggested that beside the alkylating effect on DNA, radical oxygen species may also play a role in DMH carcinogenesis (38). However, the involvement of superoxide anion radical as reported by this study using short-term protocols which terminate 24 h after a single carcinogen application (38) appear to be a part of the very initial mechanism of DMH initiation since comparable effects were not observable by comet assays in context with our study terminating 3 weeks after the final DMH dose (K.Briviba, unpublished data). Interestingly, in our experimental design significantly elevated DNA single strand breaks in the DMH treated water group could be detected as late as 3 weeks after the final DMH application. These observations might be due to the relatively high total DMH dose (80 mg/kg) which result in a persistence of adduct formation as already shown for a susceptible mouse strain treated with variable doses of DMH (39).
The mechanism by which CloA and CleA impair the initial genotoxic event of DMH can only be hypothesized. It cannot be deduced from our results, whether the protection from DMH-mediated initial oxidation or prolonged DNA-adduct formation is due to interaction with mucosal/bacterial glucuronidase (3), DMH activating P450 isoenzymes (4), an increased rate of 8-OhdG repair or antioxidant enzyme activity (38,40). In this context, the unchanged levels of GST-related enzyme expression as analyzed in the present study might exclude an apple juice mediated induction of phase II detoxification by those isoforms, which were shown to be involved in the chemopreventive activity of red wine and black tea polyphenols (17). Moreover, free radical scavenging during the time of DMH treatment might also be excluded as the underlying mechanism which determined the antigenotoxic effect of the juices. Although the apple juices of the present study represented a significant antioxidant capacity of 5.5 mM trolox equivalent, the trolox equivalent plasma antioxidant capacity (TEAC) in the rats remained unchanged irrespective of apple juice intervention. This might be based on the low bioavailability of apple polyphenols as already suggested for humans (41).
In the rodent DMH model, DNA alkylation leading to DNA damage has been shown to be closely associated with increased epithelial proliferation, which taken together result in enhanced tumor induction in the distal colon (39). In the present study, in accordance with the observed antigenotoxic activity of both apple juice preparations, the DMH-induced hyperproliferation was significantly inhibited by the intervention with CloA and to a significantly lesser extent with CleA.
It is well documented, that pectin as the major dietary fibre component in apple affects epithelial hyperproliferation in different animal models of colon carcinogenesis (22,42). In these studies pectin was provided at 6% by weight in the experimental diets corresponding to an estimated total daily uptake of 7201200 mg pectin in 1220 g diet per animal. According to our juice analysis the mean daily pectin uptake per animal was 19.1 mg and 4.73 mg of apple pectin by CloA and CleA uptake, respectively. Owing to the relatively low pectin dosage provided by the juices when compared with pectin containing diets, we suppose that the observed antiproliferative effect of the juice preparations is not solely mediated by pectin but the relatively higher amount of pectin in CloA might further promote antiproliferative mechanisms when compared with CleA.
Consistent with our in vivo results of antiproliferative capacity by apple juice preparations, apple polyphenol extracts exhibited strong antiproliferative activity in vitro (12). As recently characterized by others, this in vitro antiproliferative activity of apple polyphenolic compounds might be related to procyanidins rather than flavonoid monomers. A procyanidin-enriched apple extract showed growth inhibiting properties in a human metastatic cell line without any antiproliferative effect of comparable concentrations of flavonoid monomers (43). These data suggest that in our in vivo experiment the apple procyanidins might be also responsible for the significant reduction of colonocyte proliferation. However, no in vivo data are available on the antiproliferative capacity of apple procyanidins. Until now, in vivo studies have favored enriched extracts derived from grapes (16) or grape seeds (44) and showed distinct results depending on different extract composition or intervention protocols used. Applied in a post-initiation protocol, chronic administration of complex wine extracts containing significant amounts of procyanidins did not affect epithelial proliferation in the colon (16). When applied in a short-term pre-initiation experiment, a daily dose of grape seed procyanidin extract or purified grape seed procyanidin B2 significantly reduced AOM-mediated colonic proliferation as analyzed by PCNA labelling (44). Procyanidin B2 is a major procyanidin compound of different apple cultivars (45) and is also found as a major constituent in the apple juices used for the present study. Interestingly, the estimated dosage of 2 mg/kg body weight of pure procyanidin B2 which significantly reduced the AOM-mediated epithelial hyperproliferation (44) is comparable to the procyanidin dosage by the average daily uptake of the apple juices used in the present experiment.
It is well described that the growth inhibiting activity of plant polyphenols is often accompanied by enhanced apoptosis (46) which represents a parameter depending on cellular concentrations of COX-2 derived prostaglandin E2 (PGE2). Alterations in COX-2 activity and PGE2 concentrations determine resistance to apoptosis or apoptosis promotion (47). Phytochemicals with the potential as chemopreventive agents show COX-2 inhibiting activities (17). In the present pre-initiation experiment COX-2 mRNA level in the colonic mucosa of Cont was slightly but not statistically significantly elevated by DMH when compared with Cont/NaCl. This might be on account of the short-term protocol giving rise to early stages of ACFs without COX-2 overexpression when compared with the normal colon mucosa (48). Moreover, none of the apple juices further reduced the level of mucosal COX-2 mRNA after DMH treatment. Thus, the present finding of constant COX-2 expression in the colon mucosa irrespective of treatment with DMH and/or intervention with apple juices suggests that the observed alterations of colonic proliferation might not be directly associated with or mediated by changes in basal COX-2 expression.
In addition, we also analyzed effects of apple juice intervention on the initiation and growth of ACF. We assessed ACFs by recording their total number and the number of ACFs consisting of four or more aberrant crypts, since it has been postulated that mainly the larger ACF are more predictive for tumor development than the total number of ACF (49). However, more recently it has been considered that neither number nor size of ACFs are suitable biomarkers for colorectal cancer and thus, other ACF-related characteristics, such as mucin content (50) or beta-catenin expression (51) may be more predictive to carcinogenesis. Furthermore, it could be demonstrated that more than the general histopathological features of ACFs, the gene expression profiles are distinct and predictive for high- or low-risk ACFs with different tumorigenic potential (52). Although we are aware about the inconsistency of ACFs in their predictive value for growing out into later adenoma and further carcinoma stages, we could clearly show that the uptake of CloA but not CleA reduced the number of ACFs in the distal colon. This CloA effect was significant for ACFs which contained four or more ACs with an 50% reduction of large sized ACFs and a significantly reduced ACF multiplicity suggesting that the large ACFs might be more responsive to intervention with CloA.
In addition to the role of pectin in modulating proliferation and apoptosis in rat colon epithelium (22,42), diets high in fibre (e.g. 10% pectin) significantly suppressed AOM induction of ACF development (53). However, owing to the relatively low dosage of pectin provided daily by the juices in the present study, it is unclear whether pectin or other juice components such as procyanidins are primarily responsible for the anticarcinogenic properties. Others have recently reported that a procyanidin enriched extract purified from apple reduced the total number of colonic ACFs by 50% without affecting ACF multiplicity when applied in drinking water at a final concentration of 0.01% over 5 weeks post-initiation (43). Monomeric apple polyphenols did not show any significant effects in the ACF assay at comparable doses. Although we did not analyze the amount of polymeric procyanidins (degree of polymerization 3) in juices, the mean daily dose of procyanidin B1/B2 by CloA uptake exceeded that of the CleA groups by
45%.
Furthermore, besides these parameters locally restricted to the distal colon, we could also show that CloA had an impact on the immune system. As already demonstrated for AOM (28), DMH also significantly reduced the spleen CD4:CD8 ratio in control animals owing to a drop in CD4 and a rise in CD8 positive spleenocytes. In humans, it has been demonstrated for different types of cancers, that an increase in peripheral CD8 and a decrease in CD4 positive T-cells is generally accompanied by malignant tumor progression and further, is a valuable prognostic marker with respect to tumor recurrence or survival rate (54). As a consequence, a normalization of DMH-mediated decreased CD4:CD8 ratio through CloA intake would imply a modulation of immunological processes associated with tumor promotion and progression.
In summary, the data presented here provide evidence that primarily CloA reduced colonic precarcinogenic events in rats initiated for colon carcinogenesis with DMH. At present, we only can speculate about the components in CloA which could be responsible for the anticancer effects including antigenotoxic, antiproliferative and growth inhibiting properties on ACFs and immunomodulatory effects. Many apple ingredients, shown to modify cancer related processes in vitro have already been identified. However, it is difficult to evaluate the relative importance of individual constituents for the anticancer effects of the complex apple juices seen in the present study in vivo. We suggest, that the strong effect obtained by CloA is mainly due to the distinct diversity of polyphenols and non-polyphenolic compounds. These might alter their cancer preventive properties and their capacity to modulate immune parameters by antagonistic, additive and/or synergistic mechanisms.
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Acknowledgments |
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Conflict of Interest Statement: None declared.
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References |
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