Carcinogenesis vol.26 no.8 pp.1414--1421, 2005
doi:10.1093/carcin/bgi082
Advance Access publication March 31, 2005
Cloudy apple juice decreases DNA damage, hyperproliferation and aberrant
crypt foci development in the distal colon of DMH-initiated rats
S.W.Barth1,, C.Fähndrich1, A.Bub1, H.Dietrich3,
B.Watzl1, F.Will3, K.Briviba1 and G.Rechkemmer1,2
1
Institute of Nutritional Physiology, Federal Research Centre for Nutrition
and Food, Haid-und-Neu-Strasse 9, 76131 Karlsruhe, Germany, 2Technical
University of Munich, Department of Food and Nutrition, 85354 FreisingWeihenstephan, Germany and 3Research Institute Geisenheim, Department of
Wine Analysis and Beverage Research, 65366 Geisenheim, Germany
To whom correspondence should be addressed. Tel: þ49 721 6625 408;
Fax: þ49 721 6625 404;
Email: [email protected]
Clear (CleA) and cloudy (CloA) apple juices containing
different amounts of analyzed procyanidins and pectin
were investigated for preventive effects of colon cancer
and underlying molecular mechanisms in F344 rats given
intraperitoneal injections of 1,2-dimethylhydrazine (DMH;
20 mg/kg body wt) once a week for 4 weeks. Rats received
either water (Cont), CleA or CloA (ad libitum) for 7 weeks
starting 1 week before the first DMH injection. CloA
inhibited DMH induced genotoxic damage in mucosa cells
of the distal colon compared with Cont as investigated by
single-cell microgel electrophoresis assay. The mean tail
intensity in mucosa cells of DMH-treated controls (Cont/
DMH: 6.1 0.9%) was significantly reduced by CloA
(2.4 0.8%; P 5 0.01) but not by CleA intervention
(4.1 1.2%; P 4 0.05). The crypt cell proliferation index
induced by DMH (Cont/NaCl: 10.0 0.7%; Cont/DMH:
19.9 1.0%; P 5 0.001) was significantly decreased by
CleA (15.7 0.7%; P 5 0.001) and CloA intervention
(11.9 0.4%; P 5 0.001). CloA but not CleA significantly
reduced the number of large aberrant crypt foci (ACF)
consisting of more than four aberrant crypts (AC) (Cont/
DMH: 37.4 5.4; CleA/DMH: 32.8 4.4, P 4 0.05; CloA/
DMH: 18.8 2.5 ACF; P 5 0.05) and the overall
mean ACF size in the distal colon (Cont/DMH: 2.31 0.09; CleA/DMH: 2.27 0.05; CloA/DMH: 2.04 0.03
AC/ACF; P 5 0.05). After treatment with DMH and/or
apple juices there were no changes in transcript levels
of colonic cyclooxygenase isoforms (COX-1, COX-2)
or glutathione-associated enzymes (GST-M2, g-GCS,
GST-P), the splenocyte natural killer cell activity and
plasma antioxidant status. However, CloA but not CleA
prevented the DMH-induced reduction of splenocyte
CD4/CD8 (T-helper cells to cytotoxic lymphocytes) ratio.
Since both formulations contained comparable concentrations and types of monomeric polyphenols, complex polyphenols or non-polyphenolic compounds, such as pectin
might be responsible for the stronger cancer-preventive
effect by CloA.
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.
Carcinogenesis vol.26 no.8 # Oxford University Press 2005; all rights reserved.
Introduction
Epidemiologic studies suggest that the consumption of vegetables and fruits is inversely associated with colorectal cancer
risk (1). Much of this protective effect of vegetables and fruits
has been attributed to biologically active secondary plant
metabolites, which are the non-nutrient plant constituents
such as the carotenoids, phenolic acids and flavonoids.
Until now, 46500 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 (10--12). 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.
1414
Cloudy apple juice decreases DNA damage
Beside the heterogeneous cloud fraction exclusive to CloA, the
juices further differ in their content of procyanidins and nonpolyphenolic 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.
Materials and methods
Apple juice preparations
The apple juices were produced in the Research Institute Geisenheim from the
2002 harvest. The following apple varieties were used: cv. Topaz (25%), cv.
Bohnapfel (17.5%), cv. Winterrambour (22.5%), cv. Bittenfelder (15%) and
mixed table-apple varieties (20%). The fruits were crushed in a hammer mill
(Bellmer; Niefern, Germany) and extracted in an HP-L 200 horizontal press
(Bucher; Niederweningen, Switzerland). The resulting juice was centrifuged
with a separator (SA R 3036, Westfalia, Oelde, Germany) and finally pasteurized (85 C, 30 s). After a further separation, the CloA was hot-filled (85 C)
into 0.75 l glass bottles. CleA was produced from CloA by pectinase (50 ml/kg,
Fructozym P, Erbsl€
oh, Geisenheim, Germany) treatment. After depectinization, the juice was cross-flow filtered (Bucher-Abcor, Niederweningen,
Switzerland, Supercor membrane, nominal cutoff 18 kDa) and hot-filled.
Bottled apple juices were stored at 4 C until further use.
HPLC analysis of apple juice polyphenols
Apple polyphenols were separated on a 1090 HPLC/PDA system (HewlettPackard; B€
oblingen, Germany) equipped with a 250 4.6 mm Aqua 5 mm C18
column and protected with a 4 mm 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 mm 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 ¼ 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 12--12 h light--dark 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 ¼ 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 ¼ 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 ¼ 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 mM Na2HPO4 2H2O, 278 mM K2HPO4 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 ¼ 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 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 RT--PCR 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 25--30 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 959--982
and 1683--1662 of the rat COX-2 cDNA (GenBank accession
no. NM_017232.2), nt 1091--1110 and 1775--1752 of the rat COX-1
cDNA (accession no. NM_017043.1), nt 904--925 and 1758--1736 of rat
g-GCS (accession no. NM_012815.2), nt 1--20 and 768--749 of rat GST-M2
(accession no. M13590.1), nt 2--22 and 480--460 of GST-P cDNA (accession
no. NM_012577.1) and nt 71--94 and 570--547 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 1
TBE buffer and ethidium bromide-stained bands were visualized and quantified using an automated computer-based image analysis system (QuantityOne/
FluorS Imager; Biorad, M€unchen, 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 ¼ 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 100 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 mm 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), DNAsynthesizing 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, Gr€unberg,
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 400 magnification under a Zeiss Axiovert
100 microscope. Well-oriented crypts (n ¼ 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 RPMI1640 culture medium containing 50 ml/l of heat-inactivated fetal bovine
serum, L-glutamine (2 mmol/l), penicillin (1 105 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).
1415
S.W.Barth et al.
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 ¼ 5--7/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 55%.
Statistical analysis
All data are presented as the mean SEM. Statistical analysis was accomplished using ANOVA and Newman--Keuls multiple group comparison test to
identify significant differences between groups. P-values 50.05 were considered significant.
Results
General observations
During the experiment, body weight curves were similar in all
groups independent of DMH treatment or intervention with
either of the different apple juices. Without considering the
available energy from the apple pectin the mean daily caloric
intake by the apple juice intervention was 23.6 kJ (CleA;
21.5 ml/animal/day) and 27.8 kJ (CloA; 22.9 ml/animal/day)
per animal. Rats fed on apple juices compensated the increased
juice-derived energy by a decreased food intake compared
with Cont receiving tap water (P 5 0.001, Cont: 17.7 1.9 g, CleA: 15.5 1.0 g and CloA: 15.0 0.9 g), which
resulted in a comparable daily total energy intake (Cont:
215.3 kJ, CleA: 212.7 kJ and CloA: 210.8 kJ) and growth
rate independent of the treatment group.
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.
Genotoxicity
DNA damage in colon mucosa cells isolated from rats receiving injections of 0.9% NaCl instead of DMH was low as
evaluated by the comet assay (Figure 1). Juice intervention
had no effect in groups treated with NaCl. The treatment with
DMH induced a significant increase in DNA damage in the
water group (Cont) when compared with Cont/NaCl. This
strong genotoxic effect of DMH was significantly reduced by
the intervention with CloA (P 5 0.01) but not with CleA (P 4
0.05). The level of DNA damage in the group of CloA/DMH
did not significantly differ from that of CloA/NaCl (Figure 1).
Epithelial proliferation
In all the animal groups analyzed, BrdU was exclusively incorporated in crypt cell nuclei located at the bottom of the colon
crypts. Neither of the juices affected the low proliferation
1416
Table I. HPLC analysis of polyphenolic compounds and pectin in
CleA and CloA
Polyphenols
CleA
mg/la (mg/kg
body wtb)
CloA
mg/la (mg/kg
body wtb)
Procyanidin B1
Procyanidin B2
Epicatechin
Phloretin-20 -galactoside
Phloretin-20 -xyloglucoside
Phlorizine
Chlorogenic acid
3-Coumaroyl quinic acid
Caffeic acid
4-Coumaroyl quinic acid
Quercetin-3-rutinoside
Quercetin-3-galactoside
Quercetin-3-glucoside
Quercetin-3-rhamnoside
Total
4.2
15.5
17.5
6.0
60.9
23.9
154.0
1.9
2.3
75.1
0.8
1.9
1.1
3.2
368.3
5.8
20.9
17.9
6.3
59.8
19.7
155.9
1.9
0.6
76.7
0.5
1.7
1.0
2.5
371.2
Pectin
220 (22.63)
(0.43)
(1.59)
(1.80)
(0.62)
(6.28)
(2.46)
(15.83)
(0.20)
(0.24)
(7.72)
(0.08)
(0.20)
(0.11)
(0.33)
(37.89)
(0.64)
(2.29)
(1.96)
(0.69)
(6.55)
(2.16)
(17.08)
(0.21)
(0.07)
(8.40)
(0.05)
(0.19)
(0.11)
(0.27)
(40.67)
834 (91.38)
a
Data represent the mean of three independent HPLC measurements.
The mean daily polyphenol uptake during the experiment as detailed in
parentheses is calculated with 209 g as the mean body weight and a mean
daily juice intake of 21.5 ml CleA and 22.9 ml CloA during the intervention.
b
Fig. 1. Comet assay results of genotoxic DNA damage expressed as the
mean tail intensity (mean SEM; n ¼ 5/group) analyzed in mucosal cells of
the distal colon. The treatment with DMH resulted in a significant 43-fold
increase in genotoxic DNA damage in mucosal cells of the control (Cont)
group (P 5 0.001). Intervention with CleA did not inhibit DMH increased
damage (P 4 0.05), whereas CloA led to a significant (P 5 0.01)
suppression of DMH-mediated genotoxicity reaching the level of
CloA/NaCl.
index in colon crypts of NaCl treated control animals
(Figure 2). Compared with the Cont/NaCl group, DMH significantly increased the epithelial proliferation in the Cont group
(P 5 0.001). This hyperproliferative effect of DMH observed,
was significantly reduced by intervention with CleA (P 5
0.001) and almost abolished by CloA (P 5 0.001). Furthermore, the proliferation lowering effect of CloA was significantly stronger when compared with CleA (P 5 0.001; CloA/
DMH versus CleA/DMH). However, when compared with the
respective controls receiving NaCl instead of DMH, the proliferative activity in the colon epithelium of DMH-treated
groups with either juice intervention was still significantly
elevated (P 5 0.001; CleA/DMH versus CleA/NaCl; P 5
0.05, CloA/DMH versus CloA/NaCl).
Cloudy apple juice decreases DNA damage
Aberrant crypt foci (ACF)
There were no ACF in the distal colon of any of the groups
treated with NaCl. CleA given to the DMH-treated animals
had no effect either on the number of ACF (Figure 3A) or the
estimated number of AC per ACF (Figure 3C). In contrast, the
total number of ACFs per colon was consistently reduced in
animals with CloA intervention when compared with the
Cont/DMH group (Figure 3A). This effect, however, was
only statistically significant for the largest ACFs (4 crypts/
foci) with an ~50% reduction of ACF number when compared
with either the Cont (Figure 3D; 37.4 5.4 versus 18.8 2.5;
P 5 0.05) or the CleA group (32.8 4.4 versus 18.8 2.5;
P 5 0.05). As a result of reduced number of large ACFs, the
intervention with CloA significantly reduced the mean ACF
multiplicity when compared with Cont (Figure 3C; 2.3 0.1
versus 2.0 0.03; P 5 0.05).
Gene expression
The treatment with DMH only showed the tendency to
increase COX-2 mRNA levels but did not significantly change
mucosal cyclooxygenase isoforms COX-1 and COX-2 mRNA
levels in the distal colon of control animals. Furthermore, none
of the juices used for intervention leads to significant gene
expression changes in groups treated with NaCl or DMH
(Table II). Moreover, besides mRNAs of cyclooxygenases,
we also analyzed transcripts coding for the glutathionerelated enzymes gGCS, GST-P and GST-M2, which were
previously shown to be modulated by black tea and red wine
polyphenols applied in an AOM post-initiation protocol (17).
Of the GSH-related enzyme mRNAs analyzed, the gene
expression of GST-M2, gGCS and GST-P was not affected
by DMH. Furthermore, none of the apple juices showed any
effect on gene expression levels of GSH-related enzymes
(Table II).
Fig. 2. Proliferation index in the distal colon as the percentage of
BrdU-positive cells (mean SEM) determined within 25 randomly chosen
crypts per animal. The treatment with DMH significantly increased the
percentage of BrdU positive cells (mean SEM) within the crypts of the
water receiving control (Cont) group (P 5 0.001). When compared with
Cont, the intervention with CleA or CloA significantly (P 5 0.001) reduced
hyperproliferation by DMH with a stronger antiproliferative effect of CloA
(P 5 0.001; CloA/DMH versus CleA/DMH).
B
Cont/DMH
CleA/DMH
CloA/DMH
200
150
C
600
2.4
550
2.3
500
AC/ACF
ACF / colon
250
AC/colon
A
450
400
350
2.2
2.1
300
100
*
2.0
250
D
100
≥4 AC
ACF≥
50
ACF
40
75
30
*
ACF
20
10
50
Cont/DMH
CleA/DMH
CloA/DMH
0
*
*
25
0
1
2
3
4
5
6
7
>8
AC/ACF
Fig. 3. Analysis of ACF in the distal colon mucosa of DMH-treated animals (mean SEM; n ¼ 5/group). The total number of ACFs (A) as well as the total
number of aberrant crypts per colon (B) remained unchanged by intervention with either the clear (CleA) or cloudy apple juice (CloA). (D) As further detailed with
respect to ACF size (AC/ACF) the constant number of ACFs irrespective of juice intervention was restricted to small sized ACFs (AC/ACF 5 3) while
the mean number of larger ACFs (AC/ACF 4) was significantly reduced by CloA but not CleA (D, insert; P 5 0.05). This resulted in an overall decrease
of ACF size by CloA (C, P 5 0.05).
1417
S.W.Barth et al.
Table II. Normalized mucosal transcript levels of cyclooxygenase (COX) isoforms and glutathione associated enzymes in the distal colon
Cont/NaCl
COX-1
COX-2
GST-M2
gGCS
GST-P
0.43
0.39
0.42
0.72
0.59
0.07
0.08
0.06
0.07
0.06
Cont/DMH
0.55
0.48
0.53
0.69
0.64
0.04
0.09
0.02
0.06
0.04
CleA/NaCl
0.54
0.46
0.55
0.80
0.70
0.05
0.06
0.05
0.07
0.08
CleA/DMH
0.50
0.38
0.48
0.75
0.59
0.02
0.05
0.02
0.07
0.05
CloA/NaCl
0.48
0.36
0.48
0.70
0.63
0.04
0.02
0.02
0.06
0.01
CloA/DMH
0.45
0.31
0.57
0.67
0.64
0.09
0.06
0.10
0.06
0.03
Results are presented as the mean SEM on n ¼ 5 animals per group.
Immunological parameters
To evaluate the potential effects of the apple juice preparations
on parameters of systemic immunity which might be implicated in eliminating tumor cells, we analyzed the lytic activity of NK cells isolated from spleen and quantitated cytotoxic
CD4-positive and CD8-positive subsets of spleen lymphocytes.
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 5 0.05) or CleA (P 5 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 4
0.05) and a significantly elevated CD4:CD8 ratio when compared with the Cont and also the CleA groups receiving DMH
(P 5 0.05).
Antioxidant status
As determined with the TEAC assay, the plasma total antioxidant capacity was not modified by DMH or any of the
juices used for interventions (data not shown). However, results of the FRAP assay showed slightly but not significantly
elevated ferric reducing capacity in groups receiving CleA
when compared with the respective water receiving controls
(Figure 5). Furthermore, this increase was significant for the
CloA/DMH group when compared with the Cont/DMH group
(P 5 0.05).
Fig. 4. The ratio of CD4 to CD8 positive subsets of spleen lymphocytes
was determined in groups treated either with DMH or NaCl combined with
water (Cont), clear (CleA) or cloudy (CloA) apple juice (mean SEM;
n ¼ 4/group). The CD4:CD8 ratio was significantly reduced by DMH in Cont
and CleA groups (P 5 0.05). The intervention with CloA prevented
the DMH-induced drop in CD4:CD8 leading to a significantly elevated
CD4:CD8 when compared with the DMH receiving Cont and CleA groups,
respectively (P 5 0.05).
Discussion
Previous in vitro studies have suggested that polyphenolic
compounds in apple have cancer-preventive properties since
bioactivity toward cellular parameters including proliferation, apoptosis and differentiation was shown in different cell
lines (13). However, until now in vivo data on protective
effects using crude apple preparations were not available. In
the present in vivo study, we have identified the cancer preventive potential of a cloudy apple juice which reduced genotoxicity, hyperproliferation and the development of aberrant
crypt foci by DMH, with a further modulation of immune
parameters. This effect was stronger for CloA than for CleA
(antiproliferation) or exclusive to CloA (antigenotoxicity,
ACF development, systemic immune parameter).
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
1418
Fig. 5. Results of the ferric reducing antioxidant capacity (FRAP) showed
a weak but not statistically significant elevation by the CleA group. As
compared with the water (Cont) receiving DMH group, the CloA group
showed a significant increase in ferric reducing capacity in the group
treated with DMH (P 5 0.05).
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
Cloudy apple juice decreases DNA damage
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 720--1200 mg pectin in 12--20 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 procyanidinenriched 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 postinitiation 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 preinitiation 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
1419
S.W.Barth et al.
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 DMHmediated 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
1420
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.
Acknowledgements
The technical staff of the Research Institute Geisenheim and the Institute of
Nutritional Physiology are acknowledged for the valuable technical help. This
study was supported by the project grant—01 EA0105—from the Federal
Ministry of Education and Research of Germany.
Conflict of Interest Statement: None declared.
References
1. Terry,P., Giovannucci,E., Michels,K.B., Bergkvist,L., Hansen,H.,
Holmberg,L. and Wolk,A. (2001) Fruit, vegetables, dietary fiber, and risk
of colorectal cancer. J. Natl Cancer Inst., 93, 525--533.
2. Bub,A., Watzl,B., Blockhaus,M., Briviba,K., Liegibel,U., M€uller,H., PoolZobel,B.L. and Rechkemmer,G. (2003) Fruit juice consumption modulates
antioxidative status, immune status and DNA damage. J. Nutr. Biochem.,
14, 90--98.
3. Likhachev,A., Anisimov,V., Parvanova,L. and Pozharisski,K. (1985)
Effect of exogenous beta-glucuronidase on the carcinogenicity of 1,2dimethylhydrazine in rats: evidence that carcinogenic intermediates form
conjugates and act through their subsequent enzymatic release.
Carcinogenesis, 6, 679--681.
4. Vasquez,H. and Strobel,H. (2001) Role of cytochrome P450 in DNA
damage produced by treatment of colon cells with 1,2-dimethylhydrazine.
Int. J. Oncol., 18, 553--557.
5. Canivenc-Lavier,M.C.,
Vernevaut,M.F.,
Totis,M.,
Siess,M.H.,
Magdalou,J. and Suschetet,M. (1996) Comparative effects of flavonoids
and model inducers on drug-metabolizing enzymes in rat liver.
Toxicology, 114, 19--27.
6. Wenzel,U., Herzog,A., Kuntz,S. and Daniel,H. (2004) Protein expression
profiling identifies molecular targets of quercetin as a major dietary
flavonoid in human colon cancer cells. Proteomics, 4, 2160--2174.
7. Arts,I.C., Hollman,P.C., Feskens,E.J., Bueno de Mesquita,H.B. and
Kromhout,D. (2001) Catechin intake and associated dietary and lifestyle
factors in a representative sample of Dutch men and women. Eur. J. Clin.
Nutr., 55, 76--81.
8. Knekt,P., Jarvinen,R., Seppanen,R., Hellovaara,M., Teppo,L., Pukkala,E.
and Aromaa,A. (1997) Dietary flavonoids and the risk of lung cancer and
other malignant neoplasms. Am. J. Epidemiol., 146, 223--230.
9. Feskanich,D.,
Ziegler,R.G.,
Michaud,D.S.,
Giovannucci,E.L.,
Speizer,F.E., Willett,W.C. and Colditz,G.A. (2000) Prospective study of
fruit and vegetable consumption and risk of lung cancer among men and
women. J. Natl Cancer Inst., 92, 1812--1823.
10. Ding,M., Lu,Y., Bowman,L., Huang,C., Leonard,S., Wang,L.,
Vallyathan,V., Castranova,V. and Shi,X. (2004) Inhibition of AP-1 and
neoplastic transformation by fresh apple peel extract. J. Biol. Chem., 279,
10670--10676
11. Eberhardt,M.V., Lee,C.Y. and Liu,R.H. (2000) Antioxidant activity of
fresh apples. Nature, 405, 903--904.
12. Liu,R.H. and Sun,J. (2003) Antiproliferative activity of apples is not due to
phenolic-induced hydrogen peroxide formation. J. Agric. Food Chem., 51,
1718--1723.
13. Boyer,J. and Liu,R.H. (2004) Apple phytochemicals and their health
benefits. Nutr. J., 3, 5--19.
14. Femia,A.P., Caderni,G., Ianni,M., Salvadori,M., Schijlen,E., Collins,G.,
Bovy,A. and Dolara,P. (2003) Effect of diets fortified with tomatoes or
Cloudy apple juice decreases DNA damage
onions with variable quercetin-glycoside content on azoxymethaneinduced aberrant crypt foci in the colon of rats. Eur. J. Nutr., 42, 346--352.
15. Tanaka,T., Kohno,H., Murakami,M., Shimada,R., Kagami,S., Sumida,T.,
Azuma,Y. and Ogawa,H. (2000) Suppression of azoxymethane-induced
colon carcinogenesis in male F344 rats by mandarin juices rich in betacryptoxanthin and hesperidin. Int. J. Cancer, 88, 146--150.
16. Caderni,G., Remy,S., Cheynier,V., Morozzi,G. and Dolara,P. (1999)
Effect of complex polyphenols on colon carcinogenesis. Eur. J. Nutr.,
38, 126--132.
17. Luceri,C., Caderni,G., Sanna,A. and Dolara,P. (2002) Red wine and black
tea polyphenols modulate the expression of cycloxygenase-2, inducible
nitric oxide synthase and glutathione-related enzymes in azoxymethaneinduced F344 rat colon tumors. J. Nutr., 132, 1376--1379.
18. Femia,A.P., Caderni,G., Buzzigoli,C., Cocca,E., Salvadori,M, and
Dolara,P. (2001) Effect of simple phenolic compounds on
azoxymethane-induced aberrant crypt foci in rat colon. Nutr. Cancer,
41, 107--110.
19. Corpet,D.E. and Pierre,F. (2003) Point: from animal models to prevention
of colon cancer. Systematic review of chemoprevention in Min mice and
choice of the model system. Cancer Epidemiol. Biomarkers Prev., 12,
391--400.
20. Nozawa,H., Yoshida,A., Tajima,O., Katayama,M., Sonobe,H.,
Wakabayashi,K. and Kondo,K. (2004) Intake of beer inhibits
azoxymethane-induced colonic carcinogenesis in male Fischer 344 rats.
Int. J. Cancer, 108, 404--411.
21. Sengupta,A., Ghosh,S. and Das,S. (2003) Tea can protect against aberrant
crypt foci formation during azoxymethane induced rat colon carcinogenesis. J. Exp. Clin. Cancer Res., 22, 185--191.
22. Davidson,L.A., Brown,R.E., Chang,W.C., Morris,J.S., Wang,N.,
Carroll,R.J., Turner,N.D., Lupton,J.R. and Chapkin,R.S. (2000)
Morphodensitometric analysis of protein kinase C beta(II) expression in
rat colon: modulation by diet and relation to in situ cell proliferation and
apoptosis. Carcinogenesis, 21, 1513--1519.
23. Schieber,A., Keller,P. and Carle,R. (2001) Determination of phenolic
acids and flavonoids of apple and pear by high-performance liquid
chromatography. J. Chromatogr. A, 910, 265--273.
24. Will,F., Bauckhage,K. and Dietrich,H. (2000) Apple pomace liquefaction
with pectinases and cellulases—analytical data of the corresponding
juices. Eur. Food Res. Technol., 211, 291--297.
25. Gee,J.M., Noteborn,H.P., Polley,A.C. and Johnson,I.T. (2000) Increased
induction of aberrant crypt foci by 1,2-dimethylhydrazine in rats fed diets
containing purified genistein or genistein-rich soya protein.
Carcinogenesis, 21, 2255--2259.
26. Wollowski,I., Ji,S.T., Bakalinsky,A.T., Neudecker,C. and PoolZobel, B.L. (1999) Bacteria used for the production of yogurt inactivate
carcinogens and prevent DNA damage in the colon of rats. J. Nutr., 129,
77--82.
27. Chomszinski,P. and Sacchi,N. (1987) Single step method of RNA isolation
by acid guanidium thiocyanate-phenol-chloroform extraction. Anal.
Biochem., 162, 156--159.
28. Roller,M., Femia,A.P., Caderni,G., Rechkemmer,G. and Watzl,B. (2004)
Intestinal immunity of rats with colon cancer is modulated by
oligofructose-enriched inulin combined with Lactobacillus rhamnosus
and Bifidobacterium lactis. Br. J. Nutr., 92, 931--938.
29. Bub,A., Watzl,B., Abrahamse,S.L., Delincee,H., Adam,S., Wever,J.,
M€
uller,H. and Rechkemmer,G. (2000) Moderate intervention with
carotenoid-rich vegetable products reduces lipid peroxidation in men.
J. Nutr., 130, 2200--2206.
30. Re,R., Pellegrini,N., Proteggente,A., Pannala,A., Yang,M. and RiceEvans,C. (1999) Antioxidant activity applying an improved ABTS
radical cation decolorization assay. Free Radic. Biol. Med., 26,
1231--1237.
31. Povey,A.C., Hall,C.N., Cooper,D.P., O’Connor,P.J. and Margison,G.P.
(2000) Determinants of O(6)-alkylguanine-DNA alkyltransferase activity
in normal and tumour tissue from human colon and rectum. Int. J. Cancer,
85, 68--72.
32. Sembries,S., Dongowski,G., Jacobasch,G., Mehrlander,K., Will,F. and
Dietrich,H. (2003) Effects of dietary fibre-rich juice colloids from apple
pomace extraction juices on intestinal fermentation products and
microbiota in rats. Br. J. Nutr., 90, 607--615.
33. Roediger,W.E. (1989) Short chain fatty acids as metabolic regulators of
ion absorption in the colon. Acta Vet. Scand. Suppl., 86, 116--125.
34. Fukunaga,T., Sasaki,M., Araki,Y., Okamoto,T., Yasuoka,T., Tsujikawa,T.,
Fujiyama,Y. and Bamba,T. (2003) Effects of the soluble fibre pectin on
intestinal cell proliferation, fecal short chain fatty acid production and
microbial population. Digestion, 67, 42--49.
35. Hu,Y., Martin,J., Le Leu,R. and Young,G.P. (2002) The colonic response
to genotoxic carcinogens in the rat: regulation by dietary fibre.
Carcinogenesis, 23, 1131--1137.
36. Abrahamse,S.L., Pool-Zobel,B.L. and Rechkemmer,G. (1999) Potential of
short chain fatty acids to modulate the induction of DNA damage and
changes in the intracellular calcium concentration by oxidative stress in
isolated rat distal colon cells. Carcinogenesis, 20, 629--634.
37. Rogers,K.J. and Pegg,A.E. (1977) Formation of O6-methylguanine by
alkylation of rat liver, colon, and kidney DNA following administration of
1,2-dimethylhydrazine. Cancer Res., 37, 4082--4087.
38. Lodovici,M., Casalini,C., De Filippo,C., Copeland,E., Xu,X., Clifford,M.
and Dolara,P. (2000) Inhibition of 1,2-dimethylhydrazine-induced oxidative DNA damage in rat colon mucosa by black tea complex
polyphenols. Food Chem. Toxicol., 38, 1085--1088.
39. Jackson,P.E., O’Connor,P.J., Cooper,D.P., Margison,G.P. and Povey,A.C.
(2003) Associations between tissue-specific DNA alkylation, DNA repair
and cell proliferation in the colon and colon tumour yield in mice treated
with 1,2-dimethylhydrazine. Carcinogenesis, 24, 527--533.
40. Inagake,M., Yamane,T., Kitao,Y., Oya,K., Matsumoto,H., Kikuoka,N.,
Nakatani,H., Takahashi,T., Nishimura,H. and Iwashima,A. (1995)
Inhibition of 1,2-dimethylhydrazine-induced oxidative DNA damage by
green tea extract in rat. Jpn. J. Cancer Res., 86, 1106--1111.
41. Lotito,S.B. and Frei,B. (2004) Relevance of apple polyphenols as
antioxidants in human plasma: contrasting in vitro and in vivo effects.
Free Radic. Biol. Med., 36, 201--211.
42. Umar,S., Morris,A.P., Kourouma,F. and Sellin,J.H. (2003) Dietary pectin
and calcium inhibit colonic proliferation in vivo by differing mechanisms.
Cell Prolif., 36, 361--375.
43. Gosse,F., Guyot,S., Roussi,S., Lobstein,A., Fischer,B., Seiler,N. and
Raul,F. (2005) Chemopreventive properties of apple procyanidins on
human colon cancer-derived metastatic SW620 cells and in a rat model of
experimental carcinogenesis. Carcinogenesis. 26, 1291--1295.
44. Nomoto,H., Iigo,M., Hamada,H., Kojima,S. and Tsuda,H. (2004)
Chemoprevention of colorectal cancer by grape seed proanthocyanidin
is accompanied by a decrease in proliferation and increase in apoptosis.
Nutr. Cancer, 49, 81--88.
45. Alonso-Salces,R.M., Barranco,A., Abad,B., Berrueta,L.A., Gallo,B. and
Vicente,F. (2004) Polyphenolic profiles of Basque cider apple cultivars
and their technological properties. J. Agric. Food Chem., 52, 2938--2952.
46. Galati,G., Teng,S., Moridani,M.Y., Chan,T.S. and O’Brien,P.J. (2000)
Cancer chemoprevention and apoptosis mechanisms induced by dietary
polyphenolics. Drug Metabol. Drug Interact., 17, 311--349.
47. Tsujii,M. and DuBois,R.N. (1995) Alterations in cellular adhesion and
apoptosis in epithelial cells overexpressing prostaglandin endoperoxide
synthase 2. Cell, 83, 493--501.
48. Takahashi,M. and Wakabayashi,K. (2004) Gene mutations and altered
gene expression in azoxymethane-induced colon carcinogenesis in
rodents. Cancer Sci., 95, 475--480.
49. Caderni,G., Giannini,A., Lancioni,L., Luceri,C., Biggeri,A. and Dolara,P.
(1995) Characterisation of aberrant crypt foci in carcinogen-treated rats:
association with intestinal carcinogenesis. Br. J. Cancer, 71, 763--769.
50. Femia,A.P., Dolara,P. and Caderni,G. (2004) Mucin-depleted foci (MDF)
in the colon of rats treated with azoxymethane (AOM) are useful
biomarkers for colon carcinogenesis. Carcinogenesis, 25, 277--281.
51. Mori,H., Yamada,Y., Kuno,T. and Hirose,Y. (2004) Aberrant crypt foci
and beta-catenin accumulated crypts; significance and roles for colorectal
carcinogenesis. Mutat. Res., 566, 191--208.
52. Nambiar,P.R., Nakanishi,M., Gupta,R. et al. (2004) Genetic signatures of
high- and low-risk aberrant crypt foci in a mouse model of sporadic colon
cancer. Cancer Res., 64, 6394--6401.
53. Rao,C.V., Chou,D., Simi,B., Ku,H. and Reddy,B.S. (1998) Prevention of
colonic aberrant crypt foci and modulation of large bowel microbial
activity by dietary coffee fiber, inulin and pectin. Carcinogenesis, 19,
1815--1819.
54. McMillan,D.C., Fyffe,G.D., Wotherspoon,H.A., Cooke,T.G. and
McArdle,C.S. (1997) Prospective study of circulating T-lymphocyte
subpopulations and disease progression in colorectal cancer. Dis. Colon
Rectum, 40, 1068--1071.
Received January 25, 2005; revised February 28, 2005;
accepted March 22, 2005
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