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(CANCERRESEARCH52, 5906-5912, November1, 19921
ABSTRA@
The effectof increasingamountsof wheatbran (0, 5, 10,
20%)inAIN-76 semisynthetic diet on colonic luminal short chain
fatty acids,epithelial cell histone acetylation, and cytokinetics,
was studied for 2weeks In groups of 10 male Sprague-Dawley rats.
Luminal contentswere removedfrom the colon at sacrifice, quick
frozen, and analyzed forshort chain fatty acids by gas-liquid
chromatography. Histone acetyladon wasassessedin cells
isolatedfromthe same @nim*Is.Cellproliferation was measured after a
short pulse in vivo with I3Hlthymldine.Colonicluminal butyrate
levelswere lowerin the 0 and 20% fibergroups, and higher in the 5
and 10% fiber groups. In contrast, cellpruliferadon, as determined
by labeling index, was higher in the 0 and20%fiber groups, and
lowerin the 5 and 10%fiber groups. This resultedIn a significant
inverse correlation between luminal butyrate levels andcolonic cell
proliferation. In addition, there was a positive linear conelatlon
between luminal butyric acid levels and colon epithelial cell
histone acetylation. From these data it was concluded that colonic
butyratelevels can be modulated by the addition of wheat bran to
the diet andthat butyrate can modulate DNA synthesis (calculated as
labeling index) in the proliferative compartments of colonic
crypts. The localization of dividing cells was unchanged and no
induction of terminal differentiatlon was detectable (contrary to
what has been observed fortransformed cells in culture).
INTRODUCTION
Colonic epithelial cell proliferation and differentiation
havebeen well characterized both in normal (1—3)and in
pathological conditions (4, 5). Recently it was suggested that the
measurements of these parameters in gastrointestinal cells
mightassist studies of nutritional intervention attempting to
inhibitneoplasia (6).
Many recent epidemiological (7—9)and experimental
(10—14)studies have focused on the role of dietary fibers and
theirgastrointestinal metabolites on colonic epithelial cell
physiology and carcinogenesis. The widely divergent results of
thesestudies could be explained, in part, by taking into account
differences in fiber composition, particularly the fermentable
cornponents (15). Among the main fecal byproducts of
fermentablefibers are (SCFA)3 (16, 17). One ofthem, butyric acid,
has beendescribed to produce quite striking effects on all the
tested celllines of neoplastic or transformed origin (18, 19).
These effects
Received8/10/92; accepted8/24/92.The costs of publicationof this
article weredefrayedin part by the paymentof
pagecharges.This articlemust
thereforebeherebymarkedadvertisementin accordance with 18
U.S.C.Section 1734solelyto indicatethis fact.
I Supported in part by Associazione Italiana per Ia Ricerca sul
Cancro (AIRC
1990-92) (L. C. B.), American Cancer Society Grant SIG-7A (M.
L), and AmericanInstituteforCancerResearch(J. R. L.).
2 To whom requests for reprints should be addressed, at
Department of ChemicalCarcinogenesis,Istituto Nazionaleper Ia
Ricercasul Cancro,1ST,VialeBenedetto XV no. 10, 16132Genoa,
Italy.
3 The abbreviations used are: AIN-76, American Institute
ofNutrition semisyntheticdiet
SCFA,shortchainfattyacidsAACC,AmericanAssociationofCerealChemists
dThd, thymidine.
are seen even at concentrations obtainable in vivo, with
fecaland cellular concentrations being correlated (20, 21). In
fact, inseveral instances the expression of constitutive genes was
increased (22, 23) together with a few usually silent genes
(23—25). The net result was a marked inhibition of both DNA
andprotein synthesis (18, 19, 26), resulting in a preferential
blockof the cell cycle in early G1 (26), eventually leading to
terminalcellular differentiation (18, 19). Butyric acid also has an
effecton several enzymatic activities (18, 19, 27—29),including
histone deacetylases (30). This class of enzymes is inhibited
evenby trace amounts of butyric acid with resultant histone
hyperacetylation (30). Acetylation of the lysine residues in the
basicamino terminal region of the histones neutralizes their
positivecharges, weakening the interaction with the phosphate group
ofthe DNA strand, and therefore opening the structure of thebasic
unit of the chromatin, the nucleosome (31, 32). Association with
hyperacetylated histones has been shown to be a necessary but
insufficient condition for the expression of a genesequence (33,
34). It also makes the DNA more prone to carcinogen damage and
damage repair (35, 36).
In this paper studies are presented involving the modulationof
colonic epithelial cell proliferation by variable contents offiber
in the diet. We thought it would be helpful in understanding
molecular mechanisms involved, to use a very well-definedtype of
semisynthetic diet (AIN-76) (37) with the addition of 0,5, 10, and
20% of a very well-characterized fiber: AACC hardred wheat bran.
The amount and composition of fecal SCFAwere carefully monitored
and the level of histone acetylationwas analyzed for each
individual rat. Furthermore, the proliferative index was determined
for 50 crypt columns/rat.
MATERIAlS AND METHODS
Diet. The A!N-76 semisyntheticstandard diet (59.6% glucose;23.6%
casein vitamin-free; 0.4% D,L-methlonrne;8% corn oil USP;0.4%
choline bitartrate; 2% AIN-76 vitamin mix; 6% AIN-76 mineralmix,
Dyets, Inc., Bethlehem, PA) was the basal diet for all studies
(37).Note that glucose is substituting for starch, which may escape
digestionin the small intestine and act like dietary fiber, and had
to be eliminatedas a variable.The diet was used in its standard
formula or was uniformlydiluted by the addition ofO, 5, 10, or 20%
wheat bran (AACC certifiedhard red wheat bran, St. Paul, MN). The
wheat bran supplement wasanalyzed by the AACC and contained 42.7%
total dietary fiber (Association of Official Analytical Chemists
Method 43.Al4). Diets werefreshly prepared every other day and
stored at 4C.
Animals. In each diet study a group oftwenty 100-g male rats
(5pmgue Dawley Charles River, Italy) was singly housed. The
distance fromthe wire grid floor to the cage base was 10 cm to
prevent rats fromreaching their feces. They received the diet for 2
weeks with food andwater ad libitum. During the study rats were
weighed weeklyand monitored daily for food intake. Weight gain was
calculated as the difference between initial and final body weight.
Energy intake was evaluatedfrom the food intake by using
physiological fuel values of4.0 KcaI/g for
5906
Modulation of Colonic Epithelial Cell Proliferation, Histone
Acetylation,and Luminal Short Chain Fatty Acids by Variation of
Dietary Fiber(Wheat Bran) in Rats'
Lidia C. Boffa,2 Joanne R. Lupton, Maria R. Mariani, Marcello
Ceppi, Harold L Newmark,Alessandra Scalmati, and Martin Lipkin
Department of Chemical Carcinogenesis, Istitulo Nazionale per Ia
Ricerca su! Cancro, 1ST, 16132 Genoa, Italy fL. C. B., M. R. M., M.
C.]; Irving WeinsteinLaborato?y for Gastrointestinal Cancer
Prevention, Gastroenterology and Nutrition Service, Memorial
Sloan-Kettering Cancer Center, New York, New York 10021(A. S., H. L
N., M. LI; and FacultyofNutrition, TexasA&M
University,CollegeStation, Texas77843-2471(1. R. Li
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MODULATION OF COLONIC EPITHELIAL CELLS BY DIETARY FIBER
protein and carbohydrate and 9.0 Kcal/g for lipids, while
dietary fiberwas considered to provide 0 calories. Daily energy
intake was calculatedby multiplying the energy density of each diet
by the respective dailyfood intake. Food intake, energy intake, and
body weight were determined on 20 rats/diet group. Data were
analyzed by one wayanalysis ofvariance. Where variance was
significant (P < 0.05), means were saparated by using Duncan's
test (38). At the end of the experiments, ratswere sacrificed by
cervical dislocation between 11 n.m. and 12 noon tominimize the
effects of diurnal variation.
At the time of sacrifice the colon was quickly resected,
openedlengthwise, and the feces were removed, frozen, and stored at
—20°Cuntil analysis for short chain fatty acids. The colon was
placed immediately into ice-cold isotonic saline. Histones from 12
rats/diet groupwere analyzed and cell proliferation indexes were
assessed in the memaining 8 animals.
Cell Proliferation !ndexes. One h prior to sacrifice 8
animals/groupwere given injections of [3HJthymidine (Amersham
International,United Kingdom; 2 Ci/mmol) 1 @@Ci/gof body weight.
Sigmoid colonswere fixed in 10% buffered formalin acetate, pH
6.9—7.1,and stabilizedwith 1.5% methanol (39). Paraffin sections
were processed in a blindedfashion for autoradiography and were
developed and stained with hematoxylin and eosin (39). In glands
longitudinally sectioned from baseto lumen, the total number of
cells per “cryptcolumn―(each side of thelength of the crypt)
and the number and position of labeled cells werescored (40). For
each individual rat the labeling index is defined as thetotal
number of [3H]dThd-labeledepithelial cells (in all the 50
cryptsassayed) divided by the total number ofcells counted in the
same cryptcolumn. This index was calculated not only for the total
crypt but alsofor each of its five proliferative compartments of
equal size (compartment 1 at the base ofthe crypt and compartment 5
at the crypt surface)(40). The significance of any apparent change
in cell number, labeledcells, and labeling index of a group of
rats, with the variation of thepercentage of wheat bran supplement
to the diet, was estimated byconventional analysis (using all the
individual parameters in eachgroup) with an updated version of a
program based on the MannWhitney test (41).
Analysis of Level of Histone Acetylation. Histones were
preparedfrom colon epithelial cells of 12 rats ofeach group. Colons
were rinsedin KC1 (25 mM), MgCl2 (5 mM), phenylmethylsulfonyl
fluoride/1,2-epoxy-3-(p-nitrophenoxy)propane (0.1 mM), sodium
butyrate (5 mM),Tris-HCI buffer (50 mM), pH 7.2. Epithelial cells
were scraped from themucosal layer and placed in chilled
buffer-sucrose(0.32 M).Nuclei wereprepared as previously described
(42). Histones were extracted fromwashed nuclei with 0.25
MHC1,precipitated with 9 volumes of acetoneat 4°C,and freed from
nonhistone proteins and nucleic acid fragmentsby ion-exchange
chromatography on Bio-Rex 70 (Bio-Rad) as alreadydescribed (43).
Electrophoretic separation of histone acetylated forms,mainly H4
and H3, was obtained on gels containing 15%polyacrylamide, 5.5%
acetic acid, 8 Murea, and 0.2% Triton X-100 (43). Gels werestained
with 0.25% Coomassie Brilliant Blue-R and analyzed directlywith a
scanning laser spectrophotometer (LKB 2202 Ultrascan) connected to
a recorder-integrator (LKB).The statistical significanceof
thedifference between the amount of each acetylated forms of
histone H4and its total level of acetylation in the different
dietary groups wasevaluated in Table 5, using Duncan's means
separation (38).
Analysis of Short Chain Fatty Acids. Rat focal samples were
storedat —20Cuntil use. They were then placed in liquid nitrogen
and groundto a fine powder in a chilled mortar and pestle. To
approximately 0.3 gof powdered sample weighed accurately in a
1.5-mI microcentrifugetube, 500 @tlof 3 mM ethylbutyric acid
(internal standard) in 70%ethanol were added. Each tube was then
vortexed, stored briefly at 4°C,and subsequently shaken for 20 mm
(44). Samples were then centrifuged for 20 mm at 11,500 x g at
4°Cand 100 @lof the resultingsupernatant were combined with 100
gdof 3 mr@iheptanoic acid in 70%ethanol (45). Immediately before
injection in the gas chromatograph,20 @dof 10% H3P04 were added to
the mixture and 1 Mlwas analyzed(46, 47). All solutions and samples
were kept refrigerated during use.Chromatographic determinations
were performed in a gas chromatograph Hewlett-Packard 5890 Series
II, with flame ionization detector
equipped with a 30-m, 0.53-mm inside diameter,
HP-FFA-PTA-TPAcapillary column (Hewlett-Packard) and a I-rn,
0.53-mm inside diameter, deactivated glass capillary precolumn
(Supelco) (48). A HewlettPackard Series II Integrator was used to
plot and integrate data. Thechromatographic conditions used were:
injector at 165°C,detector at220°C,column flow, 2.21 ml/min
helium, make-up flow, 28 mL/min N2,flame, 430 mi/mm air, 30 ml/min
H2. Samples were concentrated onthe column head at 80°Cfor 30 s.
The temperature was increased at70°C/mmto a temperature of 145C
for 5 mm. A temperature gradientof 5°C/mmwas continued until the
final temperature of 185°Cwasreached. After the analysis the
column was allowed to regenerate at200°Cfor 5 mm and the
temperature was reduced for the next run.Short chain fatty acid
standards containing 2-ethylbutyric acid, heptanoic acid, and H3P04
were made fresh, stored at —70°C,and chromatographed at the
beginning and end of daily sample runs. Blankscontaining only the
internal standard solutions in 70% ethanol werealso chromatographed
daily. Response factors and retention times forthe individual fatty
acids weredetermined from the standard runs. Fattyacid composition
of samples was calculated from the appropriate standard response
factor. Data were obtained from a sample of 10 rats/dietary group.
Statistical evaluation of the difference in amount ofSCFAs for each
group was performed by using Duncan's mean separation.
Mathematical and Statisdcal Analysis of Data. Cellular labeling
in
dexes were fitted in a logistic regression model according to
the equation (49):
J@=exp(fl,x1)/1 +exp(Z$,x1)
where I@is the labeling index, x1are the covariates (specific
group ofeach individual; the fecal butyric acid concentration) and
@,the parameters, derived from the data, that indicate how I, is
modified withdifferent values ofx1. Data analysis was performed
with GLIM package(50). This model is the most suitable for the
statistical analysis oflabeling index (as percentage) since it is
based on a binomial distribution. It has been used in order to
evaluate the diet, butyric acid, and theirconcurrent effect on
[email protected] importance of each variable is evaluatedaccording to its
deviance, defined as the difference between the vanability not
accounted for by the model in the presence and absence ofthe
variable studied (Table 4). The deviance has approximatively
thetrend of a x2 with degrees of freedom equal to the number of
independent parameter taken into consideration during model
fitting. The percentage of variability accounted for by the model
and covariates considered was estimated with the pseudo R2 test
(51).
In the study of a possible correlation between level of histone
acetylation and fecal butyric acid concentration, the mean value of
eachgroup was fitted in a linear regression model by using the
acetylationlevel as the dependent variable. The significance (P) of
the model wasalso evaluated (52).
RESULTS
Effect of Diet on Food and Energy Intake and Weight Gain.Table 1
summarizes results of food and energy intake andweight gain in
groups ofrats ingesting 0, 5, 10, and 20% dietarywheat bran. At all
fiber supplementation levels, rats consumeda greater weight of food
than when wheat bran was not added tothe diet. This was presumably
due to the diet energy dilutionwith fiber supplementation. Energy
densities of the diet were:4.05 kcal/g, 0%; 3.93 kcal/g, 5%; 3.80
Kcal/g, 10%; 3.57Kcal/g, 20%. When food energy values are
calculated, allgroups of rats excluding the one with highest level
of fibersupplementation, consumed equivalent amounts of energy.This
was true in both the first and second week. Initial bodyweights
were similar across groups. Body weights at the end ofthe first and
second week and weight gains are in the same rankorder as energy
intakes with 5 > 0 > 10 > 20% wheat bran after1week. After
2 weeks there was no difference between the 0 and
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Table I Effectofdiet onfood and energyintake and rat
bodyweightValuesare expressed as mean ±SEM on a sample size of
20rats/group.Means
within a rownot sharing the samesuperscriptare
significantlydifferentatP
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Table 3 Mean amount, in p.w/gwetweight,ofluminal SCFAin
groupsoflO rats maintained with AIN-76 diet supplemented with
differentamountsofwheatbranResults
are means ±SEM on a sample size of 10 rats/group. Values
ineachrownot sharing a common superscript are
significantlydifferent by onewayanalysis
of variance by using Duncan's means separation (P <
0.05)(52).Wheat
bran0%
5% 10%20%AcidAcetic
22.20 ±2.6― 30.30 ±2.9― 28.90 ±1.4― 82.70
±7.6°Propionic5.30 ±2.6― 10.20 ±0.8° 9.60 ±1.1° 7.70
±@Isobutyric0.07 ±0.05c 0.99 ±0.09 0.67 ±0.08k' 0.26
±0.09cButyric1.15 ±0.lSc 7.28 ±0.86°-―9.53 ±l.18a 6.23
±0.81―Isovalenc0.22±0.l0'@ l.34±0.ID
0.86±0.12―1.89±1.65°―Valeric0.13 ±0.07c 1.17 ±0.12° 0.76
±0.09― 0.16 ±O.06cTotal
29.10 ±3.2c 5131 ±39b 50.36 ±2.5― 98.93 ±7.1°
Table 4 Evaluationofeffects ofeach covariateon labeling
indexesas calculatedby the model that takes into account the 2
covariates and theirinteractionCovariate
flu SEPGroup
1 (O%)@ —2.04700 0.06033 20%) were hyperacetylated.
In the study ofa possible correlation between level of
histoneacetylation in colon epithelial cells and fecal butyric acid
concentration, it was found that the mean value of each group(Table
5) can be fitted in a regression model where acetylationlevel is
the dependent variable. The slope of the linear relationwas 10.04
with SE of 4.52 and P < 0.05; the correlation therefore is
significant and positive.
DISCUSSION
In the literature there are several experimental
(10—17)andepidemiological (7—9)reports on the influence of
fiber (amountand composition) in the diet on colon epithelial cell
metabolismand carcinogenesis. In many cases the results are
conflicting orcontradictory.
In this paper a clarification of some of the issues was
attempted by using a well described model system: inbred
rats,standardized housing conditions, well defined semisynthetic
diets supplemented with different percentages of
thoroughlycharacterized fiber, and the experiments were ended at
the sametime of the day.
In previous experiments we have seen that when diets
areuniformly diluted by the addition of the fiber supplement
therats merely consumed a greater weight of diet in order to
compensate. Since diets are designed on an energy basis, if rats
haveequivalent energy intakes they will have equivalent intakes of
allnutrients except fiber.
In this study the highest fiber group (20%) did not eat
asufficiently greater weight of food to entirely compensate forthe
energy dilution (Table 1). Thus this group of rats had alower
energy intake than the other three groups; this was reflected also
in a lower weight gain (Table 1). Although this is acomplicating
issue in our study, we do not feel that it shouldaffect the
interpretation of our results. In fact, in almost everydiet-large
bowel cancer study (53, 54), the group ofanimals thatgains the
least weight would develop the lowest number oftumors. In our case,
however, the “marker―for cancer risk, anincrease in cell
proliferation, was highest not lowest in thisgroup. It should also
be noted that a 20% wheat bran-supplemented diet is not really a
20% fiber-supplemented diet butrather an 8.5% fiber diet (since
wheat bran is only 42.7% dietary
acetate followed the same pattern as total SCFA concentrations;
however, the 5 and 10% levels ofsupplementation did notproduce
acetate values that were significantly higher than controls. In
contrast, levels of propionate, isobutyrate, and valerateincreased
over control values with 5 and 10% but returned tocontrol level
with 20% wheat bran supplementation in the diet.There was no
significant diet effect on levels of isovalerate.
Butyrate was the SCFA ofinterest because ofits known effecton
histone deacetylase (30). Its concentration increased in thecolonic
lumen with increasing amounts ofwheat bran up to the10%
supplementation level, but the butyrate concentration ofthe 20%
fiber dietary group was back to the 5% level (Table 3).
Mathematical Analysis of Data. On the basis of the
logisticregression model we have established that all the
covariatesavailable have a significant effect on I(labeling index),
althoughthe percentage of variability they account for when
evaluatedwith the pseudo R2 test, was found to be only 47%.
The evaluation of the effect of each covariate on the
labelingindex I, is analyzed in Table 4 through the (fl@)in the
equationgiven in “Materialsand Methods.― All groups of rats
with adifferent diet determine a highly significant (P < 0.001)
andinversely proportional effect to the labeling index (e.g.,
—2.04700). Butyric acid too has an inversely proportional
effectover I, but with a lower significance. The concurrent effect
ofthe two variables though, is in part modulated by a factor
ofinteraction between the different groups of rats and butyricacid.
This interaction has a directly proportional effect on
thedefinition off,, with a significance that varies within the
groups(from significant P < 0.05 to nonsignificant P = 0.8)
(Table 4).
Effect of Diet on Histone H4 Acetylatlon. Total purified histone
preparations from colon epithelial cells of rats maintamed with 0,
5, 10, and 20% wheat bran-supplemented diet,and a colon
carcinoma-derived cell line HT29, were separatedinto their
components by gel electrophoresis according to theirlevel of
acetylation. Histone H4 acetylated forms can be easily
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Table 5 Hisione H4 acetylation in rat colon epithelial cells:
correlation with percentage ofwheat bran in the dietResults are
means ±SEM on a sample size of 12 rats/group. Values in each
column not sharing a common superscript are significantly
different
of variance by using Duncan's means separation (P < 0.05)
(52).byone wayanalysisAcetyl
groups―%
ofwheat bran 0 1 2 34Totale0
8.6 ±0.8° ND173.8 ±0.8°10.0±0.7c 8.0 ±0.7―137.4
±0.8―8.5±0.7°
11.8±0.7°144.5±0.9°@6.5±0.7― 12.1±0.8°132.5
±0.9°'6.4±0.8'@ 4.8 ±0.9'@95.1 ±0.8°
Table 6
Correlationbetweencellproliferation,histoneacetylation,andbutyricacidconcentration
in colonic epithelial cells ofrats maintained on
dietswithdifferenifiber
supplements%a
jb Acetylation―BAd0
0.130±0.013 73.8±0.81.15±0.1550.066 ±0.005 137.4 ±0.8 7.28
±0.86100.057 ±0.004 144.5±0.9 9.53 ±1.18200.084 ±0.006 132.5
±0.9 6.23 ±0.81a
Percentageofwheat bransupplementedto the AIN-76 basicdiet ±SE.b
Labeling index of total colon epithelial cells in a homogeneous
group ofrats(number
of[3Hjthymidine-labeledcells/totalnumberofcells
scored)±SE.CGlobal level of histone H4 acetylation ± SE.
d Luminal BA (butyric acid) concentration, in @a.i/gwet weight,
in groupof10rats maintained on diet supplemented with different
amount of fiber(seefootnote
a).
MODULATIONOF COLONICEPITHELIALCELLSBY DIETARYFIBER
44.2 ±0.9― 46.4 ±0.9― 0.8 ±0.7cS 23.9±0.8° 40.4±1.0°
17.7±0.8°
10 24.0 ±0.8° 39.5 ±0.9° 16.2 ±0.8°20 28.2±0.9@
4l.8±0.9c 11.4±0.9―HT29 42.1 ±1.0°' 36.7 ±0.9@'
10.0±0.8b
d Amount of non-, mono-, di-, ui-, tetraacetylated forms as
percentage of total histone H4 (average of 4 determinations).e
Global acetylation level of histone H4 = 1 x % mono- + 2 x % di- +
3 x % tn- + 4 x % tetraacetylated form ±SE.1Not detectable or
within SE.
fiber). The 20% wheat bran-supplemented diet would correspond to
a human diet of approximately 43 g/day. This isdefinitely a high
fiber diet but not “nonphysiological―nor “inordinately
high.―
Before describing an overall evaluation of the data we wouldlike
to point out two peculiar effects of the fiber-free diet.
Theabsence of fiber seems to cause a marked hyperplastic effect
oncolonic crypt epithelial cells (Table 2). This type of effect
hasbeen previously achieved, in this model system, only with
anutritional stress diet (55). Furthermore, even in the absence
ofknown precursors of fermentable material in the diet,
luminalbutyric acid concentration was relatively high (1. 15
@LM/gwetweight; Table 3), leaving its origin so far
undetermined.
When we analyzed the cellular parameters of the groups ofrats in
our studies, we noted that variation in results did notcorrelate
linearly with the percentage of wheat bran in the diet.In
particular, an increase in the percentage of wheat bran from0 to
10% led to a progressive decrease of [3H]dThd-labeledcrypt column
cells and labeling index, but a 20% supplementcauses an inversion
of the trend (Table 2).
Butyric acid is the only one of the SCFAs produced by
fiberfermentation, whose concentration has an effect that could
account, at least in part, for the above noted labeling
indexes(Tables 3 and 4). Since a mathematical evaluation of an
appropriately chosen logistic regression model showed that the
interpolation of the covariate analyzed (Table 4) can justify
only47% of the experimental distribution of labeling indexes,
thismeans that other covariates are involved in the definition of
theevent. Although these covariates are necessary for the
accuratedefinition of the model, it is unlikely that a single one
of themhas a determinant role as a modulator of cellular
proliferation.
Butyric acid is known as an inducer of terminal differentiation
of cultured cells of neoplastic origin (18, 19). Among theeffects
produced it causes a partial block of cells in early G1,with
consequent inhibition of DNA synthesis. An inhibition ofthis type
could be involved in modulating the labeling index ofcolonic crypt
cells of different groups of rats (Table 6). The
correlation coefficient between labeling index (1) and
luminalbutyrate concentration is strongly negative (—0.98; P <
0.01). Itshould be noted though that an opposite effect has been
previously observed at very low butyric acid concentrations (in
therange of 0.1 mM) in some normal primary cells in culture
(56).
Cell accumulation (hyperplasia) in the colon of the
fiber-freediet group of rats could not be accounted for simply by
theabsence ofthe brush-offeffect ofundigested fibrous material.
Infact, hyperplasia appears, even if to a lesser extent, also in
thegroup with the highest wheat bran supplementation (20%).
Thiseffect could be accounted for to some extent by the difference
ofcrypt cell proliferation in the different groups (Table 2).
The state of morphological differentiation of colonic epithehal
cells was not studied extensively, but no change was observed: this
type of evaluation might show variations in differentiated cell
distribution throughout the 5 crypt compartmentswith different
proliferative capacity (Table 2).
Butyric acid is also a modulator of chromatin primary
(nucleosome) structure because of its ability to inhibit
histonedeacetylation, therefore loosening the interaction between
theDNA and these structural proteins. In colonic epithelial cells
ofdifferent groups ofrats the level ofhistone acetylation
appearedto be modulated by the cellular concentration of butyric
acidthat is in equilibrium with its fecal concentration (Table 6)
(44).The correlation coefficient between the level of histone
acetylation and butyric acid concentration in each group is
positive(0.96; P < 0.05), while the labeling index (1) is
negative (—0.97;P < 0.05) (Table 6). This means that butyric
acid concentrationcontrols to some extent not only colonic
epithelial cell primarystructure (and therefore its accessibility
to carcinogen damageand damage repair), but also cell
proliferation.
In the study of a possible correlation between luminal
butyricacid concentration and histone acetylation, the mean value
ofthis last parameter was conventionally evaluated for eachgroup.
In fact a fairly large number of rats is needed to obtainand
analyze an electrophoretic separation ofthe different acetylated
forms. Consequently there is an averaging effect of thedata and the
level ofacetylation cannot be utilized as a covariatein Table
4.
A complicating factor in the interpretation of the results
arethe data for the 20% wheat bran diet group. There is no
satisfactory explanation for the increase in cell number per
crypt,labeled cells, and labeling index (corresponding to a lower
concentration ofluminal butyric acid and consequent lower degreeof
histone acetylation), with an increase of the wheat bran supplement
from 10 to 20%. However, several parameters shouldbe taken into
account: the variation in concentration of luminalbutyric acid
itself and the decreased transit time through theintestine, with
consequent shorter exposure of fecal material tointestinal flora
and epithelial cells to the fermentation products.
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MODULATION OF COLONIC EPITHELIAL CELLS BY DIETARY FIBER
These findings have further implications for both rodent
andhuman studies. They can also explain some data in the
literature, correlating amount of sodium butyrate to
1,2-dimethylhydrazine-induced rat intestinal neoplasia (57).
Thus the 5% wheat bran diet decreased both colonic epithehal
cell hyperproliferation and hyperplasia, compared with
thefiber-free diet. However, when wheat bran content was
furtherenriched to 10 and 20%, increased hyperplasia and
hyperproliferation gradually recurred. These findings may help to
explain previous similar data in rodent models (10,
58—60).Theyalso substantiate the hypothesis that complexities may
arise inclinical human fiber trials (61) in which subjects are
expected tobegin clinical trials consuming different amounts of
fiber,thereby contributing to lower, higher, or unchanged
proliferative indices which are expected to be more uniform.
Unfortunately, differences of this type have sometimes tended to
belumped together in the past.
From the results obtained in this study we postulate that,while
diets containing moderate amounts of fiber may haveprotective
effects on cell proliferation, differentiation, and carcinogenesis,
fiber-free diets and diets supplemented with toomuch fiber would
have the potential to promote colon carcinogenesis.
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