Bowel Mucosa-associated Lymphoid Tissue in Experimental Red

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RESEARCH PAPERS
Experimental Pathology and Health Sciences
2008; 2 (2): zz-zz
Bowel Mucosa-associated Lymphoid Tissue in
Experimental Red Wine, De-alcoholized Red Wine,
Ethanol and DMH administration
Tiago F. Rama1, Isabel S. Carneiro1, Ana Rita P. Fonseca1, José C. B. Silva1, Karen Cavalcanti1
and António M. S. Cabrita1,2
1 Experimental Pathology Institute - Faculty of Medicine - University of Coimbra
2 CIMAGO
ABSTRACT: The majority of colorectal cancers may owe their appearance to environmental factors, so a large
portion of the disease is theoretically avoidable. The implementation of preventive strategies depends on the identification of such factors. Consumption of large amounts of alcoholic beverages may increase the risk of some
cancers (i.e., those of the upper gastrointestinal tract, the liver, the colorectum and the female breast).
Nevertheless, there is growing evidence of health benefits of red wine consumption, probably due to its nonalcoholic components.
Many authors have reported that lymphoid noduli of GALT (gut-associated lymphoid tissue) play a promotional
role in the tumour formation in the large bowel in rats and possibly in humans. Lymphatic nodules can occasionally become more prominent in the bowel mucosa of rats, as a result, for instance, of the administration of the
carcinogen Dimethylhydrazine (DMH).
The main goal of the present work was to study bowel mucosa-associated lymphoid tissue (through lymphoid tissue morphometry, measuring the total area and percentage of that area occupying the mucosa) after an 8-week
experimental administration of DMH, red wine (RW, alone and with DMH), ethanol (E5%) and de-alcoholized
red wine (DRW) to Wistar male rats with 8 weeks of age, divided in 5 groups of 20 elements.
No significant correlation was found between the total area and its percentage occupying the mucosa (%A-M),
considering all cases. Consequently, a greater occupation of the mucosa doesn't depend on larger total areas of
lymphoid tissue. Lymphoid tissue areas that are limited to submucosa appear to be of a different nature from those
which have a mucosal component.
Considering all cases, a significant difference between groups was found both concerning total area (F=12,053,
p<0,01) and %A-M (F=5,587, p<0,01). Excluding the cases without occupation of the mucosa, the results were
similar. In both comparisons, the total area in the DRW group was significantly superior to those of DMH,
DMH+RW and RW. However, the %A-M was significantly inferior in the DRW comparing to the E5% group (all
cases considered) and to the DMH group (%A-M=0 excluded).
These results suggest that different stimulus can be responsible for the presence of lymphoid nodules in the
mucosa and there may be different biopathologic meanings correlated to the aetiological agent. A characterization
of the cell populations present at the lymphoid sites in each group would probably shed some light on the differences found..
KEYWORDS: DMH, Lymphoid tissue, Bowel Mucosa.
Tiago F. Rama
INTRODUCTION
Colorectal cancer (CRC) ranks is the fourth
most frequently diagnosed cancer worldwide, representing 9.4% of all incident cancer in men and 10.1%
in women. However, it is not equally common throughout the world. If the westernised countries (North
America; those in northern, southern, and western
Europe; Australasia; and New Zealand) are combined,
colorectal cancer represents 12.6% of all incident cancer in westernised countries in men and 14.1% in
women. The number of new cases of colorectal cancer
worldwide has been increasing rapidly since 1975 (when
it was 500 000). Colorectal cancer is the second commonest cause of death from any cancer in men in the
European Union.[1]
Given that many cases present late, the prognosis is generally poor. Hopefully, clarification of the
underlying disease processes will lead to more effective
intervention and a lower mortality in the future.
The macroscopic appearance of CRC lesions
may be that of a polypoid vegetating mass or of a flat
infiltrating lesion. Most of these tumours are adenocar33
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cinomas (96%) that, in some cases, show a mucinous
component. Nevertheless, colorectal tumours cover a
wide range of premalignant and malignant lesions.
The large majority of colorectal malignancies
develop from adenomatouse polyps. These can be
defined as well demarcated masses of epithelial dysplasia
with uncontrolled crypt cell division. The earliest phases
of colorectal carcinogenesis initiate in the normal
mucosa, with a generalised disorder of cell replication,
and with the appearance of clusters of enlarged crypts
(aberrant crypts) showing proliferative, biochemical and
biomolecular abnormalities.
The human colonic epithelium is an actively
proliferating and self-renewing system; in normal conditions the replicative zone is confined to the lower 3/4 of
the colonic crypts, while it is absent in the upper portions and in the surface epithelium. Thus, cells migrate
towards the most superficial regions of the glands and
are then extruded from the mucosal surface; the whole
process takes 4-6 days. In the early stages of colorectal
carcinogenesis, epithelial cells become unable to repress
DNA synthesis during migration from the lower to the
upper portions of the crypt, and develop an enhanced
capacity to proliferate. As a result, the proliferative zone
expands, and S-phase cells can be observed throughout
the entire length of the gland. [2]
So far, various genes and proteins that play a
role in colorectal carcinogenesis have been identified among these, the APC gene is thought to be crucial.
APC is a tumour suppressor gene which is inactivated in
Familiar Adenomatous Polyposis (FAP). There have
been identified various APC protein functions affecting:
cellular adhesion and motility; cell cycle progression;
crypt fission; cell migration; apoptosis - all those being
altered when an APC mutation is present. Moreover,
APC gene has two domains that interact and are
involved in down-regulation of beta-catenin gene; the
majority of tumourigenic APC mutation results in loss
of these areas and consequently loss of degradation sites
of the oncogene. [3]
Beta-catenin is a multifunctional protein which
plays a dual role in the cell. It was first identified as a protein associated with E-cadherin in maintaining cell-tocell interaction. It also acts as transcription factor in the
Wnt signal transduction pathway. Under normal conditions, beta-catenin is under rigorous control of the
upstream regulators of the Wnt-signalling cascade; the
APC gene is active, beta-catenin is degraded and the Wnt
signalling pathway is inhibited. [4]
It is now well recognized that mutant APC proteins cannot down-regulate beta-catenin, leading to aberrant cytoplasm accumulation of this protein and deregulation of beta-catenin signalling - the free protein interacts with the hTCF-4 protein, forming a complex which
enters the nucleus and promotes abnormal transcription
of a variety of oncogenes (i.e. myc), leading to carcinogenesis. [3,4,5]
Inactivation of APC has been reported in most
sporadic colorectal tumours (80%) and it is considered
an early event in colorectal tumourigenesis. There is an
34
evidence that a minority of sporadic CCR tumours lack
APC gene mutation and shown to possess somatic
mutation in beta-catenin gene.[3]
Recently, the MYH gene has been associated
with multiple colorectal tumours. It participates in the
DNA base-excision-repair, avoiding mutations in other
genes, namely APC. In a study that analysed the incidence of germ-line MYH mutations in selected
Portuguese families recorded in a hereditary tumour
registry, and evaluated the risk of colo-rectal cancer in
this syndrome, found a large frequency of biallelic
MYH mutations (69%) in APC mutation negative
patients belonging to families with attenuated polyposis. [6]
Nevertheless, it is known for sure that much
still remains to unravel in the genomic field of colorectal carcinogenesis. Moreover, colorectal cancer is widely believed to be an environmental disease, with "environmental" defined broadly to include a wide range of
ill defined cultural, social, and lifestyle practices. As
much as 70.80% of colorectal cancers may owe their
appearance to such factors; this clearly identifies colorectal cancer as one of the major neoplasms in which
causes may be rapidly identified, and a large portion of
the disease is theoretically avoidable. The move from
theoretically avoidable causes to implementation of
preventive strategies depends on the identification of
risk factors [1].
Table 1. Chemical compounds that can be found on red wine.
Wine is produced through the alcoholic fermentation of fresh grapes (crushed - or not), or of
grape juice. There is a huge variety of wines, each with
different chemical composition and properties. The
complex chemical composition of red wine is summarized in table 1 [46].
Studies of the association between red wine
consumption and cancer in humans are in their initial
stages. There is a clear association between chronic
alcohol consumption and the development of cancers
of the upper gastrointestinal tract, the liver, the colorectum [23,26,27,28,29] and the female breast [24, 25].
Even though other constituents of the wine may also
be responsible for its noxious effects, the deleterious
effects of alcoholic beverages on the human body have
been attributed to ethanol and his metabolites, particularly to acetaldehyde. During cancer initiation, ethanol
increases the activation of various pro-carcinogens
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present in alcoholic beverages, tobacco smoke, diet and
industrial chemicals to carcinogens through the induction of cytochrome P450 2E1 (CYP2E1). Ethanol is
metabolized by alcohol dehydrogenase (ADH) to
acetaldehyde, which is a carcinogen and binds to DNA.
This metabolism is modified by polymorphisms in the
genes that encode ADH and acetaldehyde dehydrogenase (ALDH), yielding various amounts of acetaldehyde.
In addiction, ethanol is also oxidized by CYP2E1, again
producing acetaldehyde but also reactive oxygen species
(ROS). ROS lead to lipid peroxidation, which produces
compounds that bind to DNA to form mutagenic
adducts. During cancer promotion, ethanol and acetaldehyde alter methyl transfer, leading to DNA hypomethylation that could change the expression of oncogenes
and tumour-suppressor genes. Ethanol also decreases
levels of retinoic acid owing to increased CYP2E1-mediated metabolism, leading to generation of toxic metabolites that are associated with changes in cell cycle behaviour and cellular hyper-regeneration. Finally, ethanolassociated immune suppression may facilitate tumour
cell spread [23].
Although consumption of large amounts of
alcoholic beverages may increase the risk of some cancers, there is growing evidence that the health benefits of
red wine are related to its nonalcoholic components namely the polyphenols, which are found in the skin and
seeds of grapes. During the fermentation process the
alcohol produced dissolves the polyphenols. These have
been found to have antioxidant properties, protecting
cells from oxidative damage caused by free radicals.
Cellular damage caused by free radicals has been implicated in the development of cancer. Research on the
antioxidants found in red wine has shown that they may
help inhibit the development of certain cancers. Red
wine particularly contains high levels of resveratrol, a
type of polyphenol called a phytoalexin, that, besides
being an antioxidant, has been shown to: (1) reduce
tumor incidence in animals by affecting one or more
stages of cancer development; (2) inhibit growth of
many types of cancer cells in culture; (3) reduce activation of NF kappa B, a protein that affects cancer cell
growth and metastasis; and (4) reduce inflammation.
Recent evidence from animal studies suggests this antiinflammatory compound may be an effective chemopreventive agent in three stages of the cancer process: initiation, promotion and progression [30 - 37]. Some curious studies verified that wine and other alcoholic drinks
may reduce the mutagenic action of some food components [38]. Certain flavonoids - such as myricetin and
quercetin - when combined with cooked food mutagenics reduce the mutagenicity of these substances. Other
flavonoids inhibit substances that bind to colonic cells
DNA. Matsukawa Y. et al, in 1997, demonstrated a colon
cancer chemopreventive potential of quercetin [39].
Before that, in 1996, Pereira M. et al demonstrated the
opposite result. [40]. Many questions about these issues
remain without a definitive answer, and there is no doubt
that more studies are needed.
Many authors [17, 41-45] have reported that
lymphoid noduli of GALT (gut-associated lymphoid tissue) play a promotional role in the tumour formation in
the large bowel in rats and possibly in humans. Hardman
and Cameron [7] concluded from their research on rats
that pre-existing lymphoid noduli in the wall of the large
bowel of DMH-treated rats are strongly promotional to
carcinogenesis in nearby colonic crypt epithelium.
Cartman et al. [8] found a significant overall correlation
between the numerical distribution of GALT and overt
tumours along the length of the large bowel in mice,
adding further support to the conclusion that lymphoid
nodules (GALT), specifically those in the distal colon,
are promotional to the carcinogenic process in nearby
colonic crypt epithelium. Do lymphoid nodules promote
carcinogenesis in nearby colonic crypt epithelium in
humans? Lymphoid nodules are known to be present in
the wall of the large bowel in humans [9, 10]. Langman
and Rowland [11] reported that the density of large
bowel lymphoid follicles in humans is 1.5 to 2 times
higher in the rectum than in the rest of the large bowel,
regardless of age or sex. This lymphoid nodule distribution in humans correlates with the higher incidence of
adenocarcinomas and adenomas found in the rectum, as
compared to the rest of the large bowel of humans [1215], and further supports the hypothesis that lymphoid
nodules are promotional to carcinogenesis in nearby
colonic crypt epithelium. Support for the idea that lymphoid cells promote colon carcinogenesis in humans
also stems from the following findings: (a) 56% of
microscopic adenomas found in colonic mucosa resected from colon cancer patients were located over lymphoid follicles [16]; (b) in humans, the colonic crypts
near lymphoid follicles have cells which were less differentiated than were the cells in those colonic crypts which
were further away from the lymphoid follicles [17]; and
(c) inflammation of the large bowel increases risk for
colorectal cancer. It has been reported that there is an
increased incidence of cancer in persons who: (a) have
chronic infection of Schistosoma japonicum in the large
bowel [18]; (b) have chronic inflammatory disease of the
large bowel, such as Crohn's disease [9]; and (c) have
ulcerative colitis [10]. Thus, these observations from
mice [8], from humans [11,19-21], and from rats [7] all
support the hypothesis that lymphatic nodules are promotional to large bowel carcinogenesis.
Moreover, lymphatic nodules can occasionally
become more prominent in the mucosa; Cabrita et al.
[22] showed that the experimental administration of the
carcinogen Dimethylhydrazine (DMH) to Wistar male
rats increased the bowel lymphoid tissue present in the
mucosa (while the control group, which was kept with
no manipulation, showed no lymphoid tissue occupying
the mucosa at all). That is, lymphoid tissue which normally did not occupied the mucosa, did so when this carcinogen was administrated.
The objective of the present work was to study
bowel mucosa-associated lymphoid tissue (for that purpose it was done lymphoid tissue morphometry, measuring the total area and percentage of that area occupying the mucosa) after the experimental administration of
35
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DMH, red wine, ethanol and de-alcoholized red wine to alcoholized red wine; E5% - 5% ethanol; %M - percentage of
cases with areas limited to the mucosa; %A-M - percentage of
Wistar male rats.
MATERIALS AND METHODS
In this study were used Wistar male rats with 8
weeks of age, in five groups of 20 elements Group I:
administration of DMH, 15mg/Kg, twice a week, during 8 weeks Group II: administration of red wine (RW),
3.57ml/Kg five times a week (DMH+RW), during 8
weeks Group III: administration of 5% ethanol (E5%)
in the drinking water, ad libitum Group IV: administration of de-alcoholized red wine (DRW), during 8 weeks
Group V: administration of DMH, 15mg/Kg and red
wine 3.57ml/Kg five times a week, during 8 weeks.
In each group, all animals were sacrificed at 8th
week and a complete necropsy was performed. In the
colon, aberrant crypts and macroscopic normal areas
were evaluated by macroscopy and histology. Tissue
samples were collected for light microscopy study based
on lymphoid tissue morphometry. Photos were taken
and then analysed through SigmaScan Pro(C), measuring
total areas of lymphoid tissue (in pixel unit) and the percentage of the total area present in the mucosa. The
results were statistically treated through SPSS(C). The
Pearson Correlation was used to compare the total area
with and its percentage occupying the mucosa (%A-M),
first considering all cases, then excluding the cases without mucosa occupation, and finally repeating the same
comparisons within each group. The T-test was performed (first for the total amount of cases and then for
each group) to compare the total areas of cases with
occupation of the mucosa to the ones without mucosa
occupation. One-way anovas were performed to compare groups both in terms of total area and %A-M - the
first considered all cases, the second excluded the cases
without occupation of the mucosa, and the third included only the cases in which the area is limited to the submucosa. In all three cases the Levene's statistic showed
an absence of homogeneity of variances; as a result, in
order to retrieve information about group-to-group
comparisons, the Post-Hoc Games-Howell was done for
the first two comparisons (as for the third, since there
were groups with less than two cases, a Post-Hoc couldn't be performed). Throughout the entire statistical
analysis, the degrees of significance considered were
p<0.01 and p<0.05.
the total area that occupies the mucosa; %S - percentage of
cases with areas limited to the submucosa; %S+M - percentage
of cases with areas both in the mucosa and submucosa).
Table 3. Only cases with mucosa (M) occupation considered.
Only in the DMH+RW group a negative significant correlation
between total area and %A-M was found; nevertheless a, with
the exception of the E5% group, all correlations present negative values. (DMH - dimethylhydrazine; RW - red wine; DRW
- de-alcoholized red wine; E5% - 5% ethanol; %A-M - percentage of the total area that occupies the mucosa).
No significant difference was found between
the total areas of the cases in which mucosa is occupied
and that of those without mucosa occupation (t=1.41,
p=0.16). In the table 4 are summarized the results for
each group separately.
Table 4. T-test performed for each group, comparing total
areas of cases with occupation of the mucosa to the ones without mucosa occupation. In the E5% group, the areas of the
cases with mucosa occupation are significantly larger than
those of the cases limited to the submucosa. In the DRW
group, the difference is in the limit of significance. (DMH dimethylhydrazine; RW - red wine; DRW - de-alcoholized red
wine; E5% - 5% ethanol).
Finally, groups were compared in terms of
total area and %A-M (tables 5-6). First, all cases were
included, and a significant difference between groups
was found both in what concerned total area (F=12.053,
p<0.01 ) and %A-M (F=5.587, p<0.01).
Table 5. Post-Hoc Games-Howell group comparison after
one-way anova was applied (first factor: total area - F=12.053,
RESULTS
p<0.01; second factor: percentage of the area occupying the
No significant correlation was found between mucosa - F=5.587, p<0.01); all cases were included. (DMH the total area and its percentage occupying the mucosa dimethylhydrazine; RW - red wine; DRW - de-alcoholized red
(%A-M), considering all cases (r = -0.182, p=0.070). wine; E5% - 5% ethanol; “<” - smaller; “>” - larger).
However, the same correlation, when the cases without
mucosa occupation were excluded, was negative and
strongly significant (r = -0.314, p<0.01). The tables 2
and 3 summarize these correlations within each group.
Table 2. All cases considered. Only in the E5% group a positive significant correlation between total area and %A-M was
found. (DMH - dimethylhydrazine; RW - red wine; DRW - de36
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Second, the cases without occupation of the
mucosa were excluded, and again a significant difference between groups was found, both in what concerned total area (F=9.213, p<0.01) and %A-M
(F=6.810, p<0.01).
Table 6. Post-Hoc Games-Howell group comparison after
one-way anova was applied (first factor: total area - F=9.213,
p<0.01; second factor: percentage of the area occuping the
mucosa - F=6.810, p<0.01); cases without mucosa occupation were excluded. (DMH - dimethylhydrazine; RW - red
wine; DRW - de-alcoholized red wine; E5% - 5% ethanol;
Figure 3. Lymphoid tissue. Colonic mucosa, H&E, 200X in
the original.
“<” - smaller; “>” - larger).
Third, only the cases in which the area is limited to the submucosa were included, and no significant
differences between groups were found concerning the
total area (F=1.751; p=0.183). As there were groups
with less than two cases, a Post-Hoc couldn't be performed.
Figure 1. Colonic mucosa, H&E, 100X in the original.
Figure 2. Lymphoid tissue. Ceccum, H&E, 100X in the
original.
Figure 4. Lymphoid tissue. Colonic mucosa, H&E, 100X in
the original.
Figure 5. Lymphoid tissue. Rectum, H&E, 100X in the original.
Figure 6. Lymphoid tissue. Rectum, H&E, 100X in the original.
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DISCUSSION
There are certain aspects which we need to
have in mind:
1. the reduced number of the sample, more marked in
some groups than others, and more meaningful amongst
the cases without involvement of the mucosa;
2. possibly resulting from the latter, we frequently found
ourselves in a situation of lack of homogeneity of variances, which forced us to apply more conservative PostHoc algorithms, carrying an eventual loss of associations;
3. it was technically impossible to analyze and compare
the lymphoid tissue alterations incidence in each group only the observation of serial cuts would make it possible to affirm beyond doubt that a given specimen did not
have lymphoid areas in the total extend of his colon.
Consequently, we are not able to declare that any of the
compounds administered causes a greater/lesser number
of lesions than the others;
4. the alcohol percentage in the red wine is superior to
5%, what could create biases when comparing these two
groups.
The most significant findings of the present
work were the following:
1. considering all cases in which lymphoid tissue occupies the mucosa, there was a strongly significant negative
correlation (r =-0.31, p<0.01) between the total area and
the percentage on the mucosa. The same type of correlation (r =-0.69, p=0.013) was found in the group of
DMH+RW.
2. comparing the total area in the cases without mucosa
occupation to the total area of those that do, there is no
significant difference between the total areas of the cases
in which mucosa is occupied and that of those without
mucosa occupation (t=1.41, p=0.16). However, there is
a very significant difference in the alcohol group; and, in
the DRW group, the difference is in the limit of significance.
3. there is a significant difference between groups in
what concerns either total area and percentage of
mucosa occupation, both considering all cases and only
the cases with occupation of the mucosa.
4. no significant difference was found between groups
concerning total areas when only cases limited to the
submucosa were considered.
5. comparing the groups and considering all the cases,
we found that the total area in the DRW group was significantly superior to those of DMH, DMH+RW and
RW. However, the percentage occupying the mucosa was
significantly inferior in the DRW comparing to the E5%
group.
6. comparing the groups and considering only the cases
with mucosa occupation, we found again that the total
area in the DRW group was significantly superior to
those of DMH, DMH+RW and RW. However, the percentage occupying the mucosa was significantly inferior
in the DRW comparing to the DMH group.
It seems that a greater occupation of the
mucosa doesn't depend on larger total areas of lymphoid
tissue; in fact, in some cases the opposite situation
38
occurs. Lymphoid tissue areas that are limited to submucosa appear to be of a different nature from those which
have a mucosal component.
The total areas found in the DRW group are
generally superior to those of the DMH, DMH+RW
and RW groups, either including or excluding the areas
limited to the submucosa. Nevertheless, in the DRW
group the percentage of the total area that is on the
mucosa is smaller than that of the E5% group (considering all cases) and that of the DMH group (excluding
the cases limited to the submucosa). Are the lymphoid
areas observed after administration of DRW of a different kind? Does this different behaviour reflects a beneficial effect of the non-alcoholic components of the red
wine - assuming that a greater invasion of the mucosa is
the result of the action of a carcinogen? Or, if the latter
is false, does this behaviour represents, by the contrary, a
more noxious one? Does ethanol - and ethanol only accounts for the lack of repercussion of red wine over
the characteristics of lymphoid areas when co administrated with DMH? Is it also the responsible for an
absence of differences between the results of RW and
DMH results, and a significant difference between RDW
and RW cases?
CONCLUSION
The results of the study suggest that different
stimulus can be responsible for the presence of lymphoid nodules in the mucosa and there may be different
biopathologic meanings correlated to the aetiological
agent.
Only a characterization of the cell populations
present at the lymphoid sites after the administration of
each compounds tested would probably reveal the
answer to the questions posted above.
ACKNOWLEDGEMENTS
We thanks to the kind collaboration in the lab
work of Ana Rafael Msci, Elisa Patricio, Margarida
Menezes and David Ferreira. This study was supported
by a CIMAGO grant.
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