folate malabsorption across intestinal brush bordermembrane during experimental alcoholism Abid Hamid1, Nissar Ahmad Wani1, Satyavati Rana2, Kim Vaiphei3, Akhtar Mahmood4and Jyotdeep Kau
Trang 1folate malabsorption across intestinal brush border
membrane during experimental alcoholism
Abid Hamid1, Nissar Ahmad Wani1, Satyavati Rana2, Kim Vaiphei3, Akhtar Mahmood4and
Jyotdeep Kaur1
1 Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Histopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
4 Department of Biochemistry, Panjab University, Chandigarh, India
Keywords
alcoholism; brush border membrane; crypt–
villus axis; methylation; reduced folate
carrier
Correspondence
J Kaur, Department of Biochemistry,
Postgraduate Institute of Medical Education
and Research, Chandigarh 160 012, India
Fax: +91 172 2744401 ⁄ 2745078
Tel: +91 172 2747585 5181
E-mail: jyotdeep2001@yahoo.co.in
(Received 1 August 2007, revised 6 October
2007, accepted 17 October 2007)
doi:10.1111/j.1742-4658.2007.06150.x
Folate plays a critical role in maintaining normal metabolic, energy, differ-entiation and growth status of all mammalian cells The intestinal folate uptake is tightly and diversely regulated, and disturbances in folate homeo-stasis are observed in alcoholism, attributable, in part, to intestinal mal-absorption of folate The aim of this study was to delineate the regulatory mechanisms of folate transport in intestinal absorptive epithelia in order to obtain insights into folate malabsorption in a rat model of alcoholism The rats were fed 1 gÆkg)1 body weight of ethanol daily for 3 months A reduced uptake of [3H]folic acid in intestinal brush border membrane was observed over the course of ethanol administration for 3 months Folate transport exhibited saturable kinetics and the decreased intestinal brush border membrane folate transport in chronic alcoholism was associated with an increased Km value and a low Vmax value Importantly, the lower intestinal [3H]folic acid uptake in ethanol-fed rats was observed in all cell fractions corresponding to villus tip, mid-villus and crypt base RT-PCR analysis for reduced folate carrier, the major folate transporter, revealed that reduced folate carrier mRNA levels were decreased in jejunal tissue derived from ethanol-fed rats Parallel changes were observed in reduced folate carrier protein levels in brush border membrane along the entire crypt–villus axis In addition, immunohistochemical staining for reduced folate carrier protein showed that, in alcoholic conditions, deranged reduced folate carrier localization was observed along the entire crypt–vil-lus axis, with a more prominent effect in differentiating crypt base stem cells These changes in functional activity of the membrane transport sys-tem were not caused by a general loss of intestinal architecture, and hence can be attributed to the specific effect of ethanol ingestion on the folate transport system The low folate uptake activity observed in ethanol-fed rats was found to be associated with decreased serum and red blood cell folate levels, which might explain the observed jejunal genomic hypomethy-lation These findings offer possible mechanistic insights into folate mal-absorption during alcoholism
Abbreviations
BBM, brush border membrane; BBMV, brush border membrane vesicle; LAP, leucine aminopeptidase; RBC, red blood cell;
RFC, reduced folate carrier; SAM, S-adenosyl methionine.
Trang 2The mechanism of folate transport is under extensive
investigation because mammals require the ingestion
and absorption of preformed folates in order to
meet their needs for one-carbon moieties to sustain
key biosynthetic reactions [1] In addition, the
cellu-lar concentration of folate cofactors, in different
oxidative states, governs the intricate network of
methylation reactions of DNA, RNA, proteins and
phospholipids [2] The most well-characterized folate
transporter, the reduced folate carrier (RFC), is an
integral membrane protein of 65 kDa that
medi-ates the cellular uptake of reduced folmedi-ates and
anti-folates, and is ubiquitously expressed in tissues [3],
consistent with its integral role in tissue folate
homeostasis [4]
Cellular folate concentrations are influenced by
folate availability, cellular folate transport efficiency,
folate polyglutamylation and turnover, specifically
through degradation [1] These processes have been
found to provide a potential means of ensuring
ade-quate levels of RFC transcripts and protein in
response to tissue requirements for folate cofactors or
exogenous tissue or cell-specific signals [5,6]
Deficiency of folate is highly prevalent throughout
the world [7] Moreover, alcohol-associated folate
deficiency has become a major health problem
world-wide [8,9], and can develop because of dietary
inade-quacy, intestinal malabsorption, altered hepatobiliary
metabolism and increased renal excretion [10,11]
However, it is a well-established fact that the
pri-mary effect of ethanol on folate metabolism is
reflected in intestinal malabsorption [12,13] Previous
studies have demonstrated that both initial
deconju-gation and subsequent transport of monoglutamic
folate are impaired in alcoholics [14] However, the
exact molecular mechanism regulating intestinal
folate transport in alcoholism is not yet clear
There-fore, the aim of this study was to elucidate the
mechanisms of regulation of folate malabsorption
during chronic alcoholism Under chronic alcoholic
conditions, the kinetic constants of the folate
trans-port process in intestinal brush border membrane
(BBM) were calculated, and the mRNA and protein
expression of a major folate transporter, RFC, was
studied The investigation of the regulation of folate
transport via RFC expression in absorptive epithelia
may aid in the development of future therapeutic
strategies targeting the regulatory protein In
addi-tion to alcoholism, folate malabsorpaddi-tion has also
been reported to occur in several intestinal diseases,
congenital disorders of the folate transport system,
drug interactions and intestinal resection, and may
involve similar mechanisms
Results
There was no significant decrease in body weight of ethanol-fed rats relative to the control group during the course of the experiment At the time of killing, the mean body weights of rats in control and ethanol-fed groups were 201 ± 8 and 196 ± 9 g, respectively
Estimation of blood alcohol levels
In order to establish the suitability of the rat model for studies on experimental alcoholism using our experimental set-up, the blood alcohol level was a pre-requisite parameter It was found that the alcohol level was 88% higher (P < 0.001) in the chronic ethanol-fed group than in the control group The mean blood alcohol levels were 15.04 ± 1.96 and 1.77 ± 0.34 mgÆdL)1 in the ethanol-fed and control groups, respectively
Purity of membrane vesicles The membrane vesicle preparations were evaluated for purity by biochemical, morphological and functional criteria The specific activities of alkaline phosphatase and sodium–potassium adenosine triphosphatase (Na+,K+-ATPase) were studied to check the purity of BBM vesicles (BBMVs) A 12–15-fold increase in alka-line phosphatase activity was observed in isolated BBMVs, with a minimum activity of Na+,K+-ATPase, relative to the respective homogenates Transmission electron micrographs revealed sealed and intact vesicles without contamination of subcellular organelles, and were similar in the two groups of rats with ‘right side out’ orientation (Fig 1A,B) The functional integrity of intestinal BBMVs was checked using [14C]d-glucose uptake, which revealed a transient overshoot of the intravesicular glucose concentration over its equilib-rium uptake in the presence of a sodium gradient (data not shown) [3H]Folic acid transport, measured by incubating BBMVs for various time intervals, was found to be at a maximum at 30 s in both control and ethanol-fed groups, as described previously [15] For further experiments, a 30 s time interval was chosen for the determination of the initial uptake Moreover, [3H]folic acid uptake revealed no significant difference between fresh and frozen vesicles In the control group, the uptake was observed to be 36.20 ± 3.20 and 35.29 ± 2.20 pmolÆ(30 s))1Æmg)1 protein in fresh and frozen BBMVs, respectively, in comparison with 19.69 ± 1.90 and 19.06 ± 2.81 pmolÆ(30 s))1Æmg)1 protein in the ethanol-fed group Therefore, for further studies, frozen reconstituted vesicles were used
Trang 3[3H]Folic acid uptake
For all the assays, except folic acid transport during
the course of the study, BBMVs were isolated at the
end of 3 months of treatment
Folic acid transport during the course of the study
Folic acid uptake into BBMVs from control and
etha-nol-fed rats was studied at 1.5, 2 and 3 months during
the course of chronic ethanol dosing Ethanol-fed rats
showed a decrease in [3H]folic acid transport, of the
order of 24, 55 and 62%, respectively, relative to the
control group (Fig 2) Thus, malabsorption of folate
was observed over the entire course of ethanol
treat-ment of 3 months
Determination of the kinetic constants of [3H]folic acid
uptake in BBMVs
The effect of substrate concentration on [3H]folic acid
transport in BBMVs from control and ethanol-fed rats
after 3 months of treatment was determined by varying
the [3H]folic acid concentration from 0.125 to 1.50 lm
(i.e within the physiological range) When transport was plotted versus substrate concentration (Fig 3), the curve showed a plateau at about 1.00 lm in both groups From 0.125 to 1.0 lm of folic acid, the uptake was 21–39% less in the ethanol-fed group (P < 0.01,
P < 0.001) From the data, the kinetic constants Km and Vmax for folic acid transport were determined from the Lineweaver–Burk plot (Fig 3, inset) The Km values for control and ethanol-fed groups were found
to be 0.90 ± 0.08 and 1.53 ± 0.09 lm (P < 0.01), respectively The Vmax values for control and ethanol-fed groups were found to be 100 ± 5.60 and
83 ± 3.65 pmolÆ(30 s))1Æmg)1 protein (P < 0.05), respectively
Folate transport across the crypt–villus axis
of the intestine The cell fractions (F1–F9) were isolated from the small intestine of both groups of rats at the end of
Fig 1 Electron micrographs (· 60 000) of
representative BBMVs with uniform shape
showing sealed outer surfaces and ‘right
side out’ orientation: (A) control group;
(B) ethanol-fed group.
0
10
20
30
40
Control
Ethanol
***
Fig 2 [ 3 H]Folic acid transport in intestinal BBMVs at different
intervals during the course of ethanol administration An incubation
buffer of pH 5.5 and a [ 3 H]folic acid concentration of 0.5 l M
were used for uptake measurements Each data point is the
mean ± standard deviation of eight separate uptake determinations
carried out in triplicate ***P < 0.001 versus control.
10 20 30 40 50
60
Control Ethanol
***
[S] (µ M )
***
**
***
**
Fig 3 [ 3 H]Folic acid uptake in intestinal BBMVs as a function of substrate concentration (inset Lineweaver–Burk plot) Uptake was measured by varying the [ 3 H]folic acid concentration from 0.125 to 1.50 l M in an incubation medium of pH 5.5 after incubating BBMVs for 30 s Each data point is the mean ± standard deviation of eight separate uptake determinations carried out in triplicate **P < 0.01,
***P < 0.001 versus control.
Trang 43 months of treatment, and were characterized by an
approximate eight-fold decrease in specific activity of
the villus cell marker enzyme alkaline phosphatase
from F1 (villus tip) to F9 (crypt base) (data not
shown) In addition, isolated epithelial cells were
characterized by measuring the DNA content and
[3H]thymidine incorporation into DNA of various
cell fractions, as described previously [16] On the
basis of the distribution patterns of the cell markers,
the nine cell fractions were grouped as villus tip
(F1–F3), mid-villus (F4–F6) and crypt (F7–F9) cells,
representing differentiated, differentiating and
prolif-erating enterocytes, respectively Folate transport
from the respective BBMVs was studied It was
observed to be 24% higher at the villus tip than
at the crypt base (P < 0.01) in the control
group; this increase was found to be 33% in the
ethanol-fed group (P < 0.001) Ethanol feeding
resulted in a significant decrease in folate transport
along the entire crypt–villus axis, the decrease being
at a maximum (50%) at the crypt base (data not
shown)
Expression of mRNA corresponding to RFC
in the intestine
The finding that the folic acid uptake process has an
apparent Km value in the micromolar range [17]
strongly suggests that the process is carrier mediated
In order to elucidate the mechanism of reduced folate
transport in chronic alcoholism, transcriptional and
translational regulation of RFC was studied For
mRNA expression, total RNA was isolated from the
upper 1 cm of jejunal tissue from both groups of rats
RT-PCR analysis was performed with the use of
gene-specific primers corresponding to a sequence in the
open reading frame of rat RFC and b-actin (as an
internal control); products of 489 and 588 bp for RFC
and b-actin, respectively, were obtained on
electropho-resis using a 1.2% agarose gel From densitometric
analysis, it was deduced that the expression of mRNA
coding for RFC was three-fold lower during chronic
ethanol feeding (Fig 4A,B) Thus, ethanol imparts its
effect through transcriptional regulation of RFC at the
primary absorptive site of folic acid, i.e the small
intestine
Expression of the RFC protein in BBM of the
intestine
The effect of chronic alcoholism on the level of
expres-sion of the RFC protein at the BBM surface was
studied by western blot analysis Analysis of purified
BBMVs was performed to identify RFC using polyclonal antibodies raised against a specific region of rat RFC; reactivity was found at approximately
65 kDa Moreover, there was no cross-reaction of RFC antibodies against any protein in the vesicular preparations used Antisera against the leucine amino-peptidase (LAP) showed reactivity at 80 kDa, which served as an internal control LAP is a membrane-bound aminopeptidase whose activity has been found
to be unaffected by chronic ethanol feeding [18,19] The expression of the RFC protein was observed to be 2.3-fold higher in BBMVs from the control group rela-tive to those from the chronic ethanol-fed group (Fig 5A,B) When studied along the crypt–villus axis
in BBMVs isolated from different cell types from the two groups of rats, maximum RFC expression in the control group was observed in the villus tip membrane, followed by the mid-villus and then the crypt base (Fig 6A,B) In comparison with the villus tip of the control group, there was a two- and 2.5-fold lower RFC expression in the mid-villus and crypt base BBMVs, respectively However, in the ethanol-fed group, RFC expression was observed to be at a maxi-mum at the villus tip, with equal expression in the mid-villus and crypt base, which was three-fold less than that at the villus tip Notably, ethanol feeding reduced the expression of RFC protein in membranes isolated from cells along the entire crypt–villus axis (Fig 6A,B)
1 2
***
0.6
B A
0.4
0.2
0
588 489
Fig 4 RT-PCR analysis of RFC and b-actin (internal control) in jeju-nal tissues: (A) resolved on 1.2% agarose gel electrophoresis; (B) densitometric analysis representing relative change in RFC mRNA expression Data shown are the mean of eight separate sets of experiments ***P < 0.001 versus control Lanes 1, 2, control; lanes 3–5, ethanol; lane 6, negative control.
Trang 5RFC distribution and localization across the
intestinal vertical axis
The distribution pattern of RFC protein was
deter-mined by immunohistochemical localization In control
rats, localization of RFC was mainly seen along the
epithelial cells of the villus lining; stronger expression
was localized along the tip epithelial cells and towards the enterocyte brush border, and positive cells were visible up to the base of the villi, i.e at the villus–crypt junction However, there was a gradual decrease in intensity from villus to crypt cells (Fig 7A) In etha-nol-fed rats, there was a marked decrease in the inten-sity of positively stained cells; only a few cells along the tip of the villi and mid-villus showed positivity (Fig 7B) No staining was detected in the sections incubated with only secondary antibody Furthermore, RFC protein was not detected in the lamina propria, muscularis mucosa, submucosa, muscularis externa or smooth muscle cells of the small intestine (data not shown)
Histochemical assessment of jejunal sections After visualizing the slides under a light microscope,
no changes in intestinal architecture were observed
in the intestinal tissues from control (Fig 8A) and ethanol-fed (Fig 8B) rats However, ethanol-fed rats showed mucodepletion and an increase in intra-epithelial lymphocytes of the intra-epithelial cells of the villus lining There was no evidence of any haemor-rhagic mucosal lesions in the intestines of ethanol-fed rats
Estimation of serum and red blood cell (RBC) folate levels
As this study dealt with folate malabsorption during alcoholism, it was important to determine the folate levels at the end of ethanol treatment The results showed that a significant (P < 0.001) decrease (32%)
in serum folate levels occurred in the chronic ethanol-fed group; the mean serum folate levels were 49.64 ± 5.29 and 33.71 ± 4.95 lgÆL)1 in control and ethanol-fed rats, respectively In addition, the RBC folate concentration showed a 34% decrease (P < 0.001) in chronic ethanol-fed rats, with mean values of 950 ± 29.84 and 624 ± 49.73 lgÆL)1 in con-trol and ethanol-fed rats, respectively
DNA methylation profile of jejunal tissue DNA from highly proliferating jejunal tissue was iso-lated, and methylation was studied using the amount
of labelled S-adenosyl methionine (SAM) incorpo-rated into DNA (Fig 9) The amount of SAM incorporated into DNA is inversely proportional to the degree of methylation It was observed that DNA from the ethanol-fed group incorporated eight-fold more SAM relative to that from the control
80kDa
~65kDa
Control 0
1 2 3 4 5
Ethanol
***
A
B
Fig 5 (A) Western blot analysis of intestinal BBMVs using
anti-RFC (65 kDa) and anti-LAP (80 kDa) IgG (B) Densitometric analysis
representing the relative change in RFC protein levels Data shown
are the mean of eight separate sets of experiments Lane 1,
con-trol; lane 2, ethanol ***P < 0.001 versus control.
80kDa
~65kDa
Villus tip
Mid villus
Crypt base
Control
1.00
###
***
**
**
0.75
0.50
0.25
0
Ethanol
6
A
B
Fig 6 (A) Western blot analysis of BBMVs isolated from the
intes-tinal villus tip, mid-villus and crypt base cells using anti-RFC
(65 kDa) and anti-LAP (80 kDa) IgG (B) Densitometric analysis
rep-resenting the relative change in RFC protein levels Data shown are
the mean of four separate sets of experiments Lanes 1, 4, villus
tip; lanes 2, 5, mid-villus; lanes 3, 6, crypt base (lanes
1–3, control; lanes 4–6, ethanol) **P < 0.01, ***P < 0.001 versus
the respective control ###P < 0.001 versus villus tip of respective
group.
Trang 6group Such results indicate a decrease in the
degree of methylation of DNA in chronic
ethanol-fed rats
Discussion
Chronic alcoholism is often associated with folate defi-ciency, which is mainly a result of malabsorption of folate across the intestinal membrane [12,20] In a rat model of experimental alcoholism, we examined the mechanism of the regulation of folate transport medi-ated by RFC, the major folate transporter protein in the intestine It was observed that a significant concen-tration of blood alcohol was maintained when deter-mined 24 h after the last dose of ethanol of 1 gÆkg)1 body weight per day at the end of a 3 month course Such a dose was chosen according to earlier studies [21], which suggested that the ethanol concentration of jejunal tissue should not exceed 6% in animal experi-ments in order to be relevant to the human intestine
In the present study, 1 gÆkg)1 body weight of ethanol
Fig 7 Immunohistochemical analysis of rat jejunal sections exposed to anti-RFC IgGs, showing relative localization and distribution pattern of RFC protein (as depicted by brown counterstaining of haematoxylin) along the intestinal absorptive axis Figures (· 450) shown are representative of each group: (A) control; (B) ethanol.
Fig 8 Haematoxylin–eosin staining of jeju-nal sections, showing no change in intesti-nal architecture after chronic ethanol ingestion Figures (· 450) shown are repre-sentative of each group: (A) control; (B) eth-anol.
Control
80
60
40
20
3/µg DNA)
0
Ethanol
***
Fig 9 [ 3 H]-labelled SAM incorporated (l M Ælg)1jejunal DNA) as an
index of jejunal DNA methylation profile Values are means ±
stan-dard deviation (n ¼ 8) ***P < 0.001 versus control.
Trang 7(20% solution) per day produced nontoxic blood
alco-hol concentrations, and rats showed no significant
his-tological alterations in the intestinal mucosa and no
clinical signs of intoxication [22]
A significant decrease in folic acid uptake by
BBMVs in the chronic ethanol-fed group, which
appeared even after 1.5 months of treatment, suggests
that ethanol feeding has a profound malabsorptive
effect on folate uptake, which may be of biological
sig-nificance The decrease was associated with an increase
in Kmand a decrease in Vmax, suggesting that both the
affinity of the transporter and the number of
trans-porter sites on BBMVs are reduced after chronic
etha-nol ingestion The increase in Km may also suggest
that an alternative route of folate transport is
opera-tional after chronic ethanol feeding These
observa-tions confirmed an earlier study which was carried out
at toxic blood alcohol levels in the micropig model of
chronic alcoholism [20] In order to evaluate the
mech-anism of reduced folate uptake, the expression profile
of RFC was of prime importance, as RFC is believed
to be a major folate carrier responsible for intestinal
folate absorption [15,23], although recently a
proton-coupled folate transporter has been found to play an
essential role in folate absorption in the intestine [24]
The decreased Vmax value of intestinal folate uptake
observed in chronic ethanol-fed rats was found to be
associated with a marked decrease in the intestinal
mRNA level of RFC In the present study, only jejunal
tissue was used for expression studies, as earlier
inves-tigations [25] have established that the jejunum is the
preferred site of absorption of exogenous folate The
finding that transcripts were reduced by more than
three-fold, whereas transport, as Vmax, was reduced by
less than two-fold, might suggest that chronic ethanol
ingestion in rats has differential effects on the
tran-scriptional and post-trantran-scriptional regulation of RFC,
or on the stability of the RFC mRNA and protein
Alternatively, another route of folate transport may be
up-regulated in alcoholic conditions In this regard, the
proton-coupled folate transporter may be suggested to
play an important role in intestinal folic acid
trans-port; however, its mechanism and specificity in
alcoholism need to be evaluated independently
Fur-thermore, western blot analysis of BBMVs revealed
that the down-regulation of RFC at the protein level
paralleled that of mRNA analysis The decreased RFC
protein molecules in BBMVs may reflect either greater
turnover or reduced synthesis of transporter molecules
during alcoholism In addition, RFC was less
promi-nently expressed at the basolateral surface; moreover,
down-regulation was evident at the basolateral
mem-brane during alcoholism (A Hamid et al., unpublished
data) Earlier studies carried out in models of dietary folate deficiency support our findings that transcrip-tional regulatory mechanisms operate in the folate transport system via RFC [17,26]
The role of RFC regulation across the crypt–villus axis during alcoholism was evaluated It was observed that the apical membrane folate transport activity was greatest in differentiated upper villus cells, followed by differentiating mid-villus cells, and lowest in proliferat-ing cells, and the proportional distribution of the RFC protein was found along the entire crypt–villus axis These results were in accordance with earlier studies [27], where a similar RFC distribution was shown to exist across the crypt–villus axis Importantly, chronic ethanol feeding decreased RFC protein expression along the entire crypt–villus axis In addition, the higher level of RFC protein in villus tip cells suggests that a larger number of folate transporters are expressed at the villus tip and that the redistribution
of RFC occurs with the maturation of intestinal stem cells Such findings correlate with the observed higher rate of folate uptake in villus tip cells relative to crypt base cells A similar distribution has been reported previously for biotin uptake [28]
Consistent with immunoblot analysis, immunohisto-chemical staining revealed RFC localization along the entire crypt–villus axis; moreover, staining was signifi-cantly more intense in epithelial cells lining the villus tip and decreased towards the crypt–villus junction in the control group A stronger expression was observed towards the enterocyte BBM In the chronic ethanol-fed group, RFC was evident at the villus tip, decreased significantly in the mid-villus and was hardly notice-able in the crypt base However, only a few positive cells along the villus tip and mid-villus could be seen during immunohistochemical staining Thus, chronic ethanol feeding imparts its effect more strongly in pro-liferating and differentiating cells in the context of RFC recruitment in the intestine Such a condition is detrimental to the cell and represents the severe patho-physiological condition in alcoholics, not only with respect to body folate homeostasis, but also because crypt cells form the intestinal stem cells and require regulated RFC expression for the sustained supply of folate to meet the burden of the high proliferation and turnover of these cells Importantly, there was no change in the villus architecture during ethanol inges-tion, suggesting that the observed reduced folate uptake is a specific effect of ethanol, rather than a secondary effect caused by a general loss of intestinal epithelial architecture Furthermore, the significant decrease in serum and RBC folate levels in the etha-nol-fed group in this study was an expected finding, as
Trang 8reduced intestinal folate uptake associated with
decreased expression of RFC will influence body folate
homeostasis These results may explain indirectly the
observations in a recent study [8], where chronic
alco-hol ingestion for 4 weeks in rats was found to be
asso-ciated with hyperhomocysteinaemia and lower levels of
SAM The low folate levels result in low SAM levels
which, in turn, may influence DNA methylation, as
reflected by the observed hypomethylated jejunal DNA
in alcohol-fed rats Our study is in agreement with that
of Choi et al [29], who observed hypomethylation of
colonic mucosal DNA in rats after chronic ethanol
ingestion, although no systemic folate reduction was
observed, by contrast with our study Such a
discrep-ancy may be attributed to the different methods
employed for ethanol administration and the
restric-tion of the study to 4 weeks only, in comparison with
3 months in our investigation Regardless of how
chronic ethanol ingestion produces genomic DNA
hypomethylation of jejunal tissue in rats, it may have
implications regarding the mechanism(s) by which
chronic alcohol exposure increases the risk of different
cancers in humans
Taken together, the results show that chronic
etha-nol ingestion leads to decreased intestinal BBM folic
acid uptake and reduced jejunal mRNA levels encoded
by RFC, resulting in low RFC protein levels and
recruitment along the entire BBM of the crypt–villus
axis The decreased transport efficiency of intestinal
BBM is reflected in reduced serum and RBC folate
lev-els, which may result in the observed hypomethylation
of jejunal DNA
Experimental procedures
Chemicals
Radiolabelled [3¢,5¢,7,9-3
H]folic acid, potassium salt (specific activity, 24.0 CiÆmmol)1) and
S-adenosyl-[methyl-3H]methionine (specific activity, 70.0 CiÆmmol)1)
were purchased from Amersham Pharmacia Biotech (Kwai
Chung, Hong Kong) d-[U-14C]Glucose (specific activity,
140 mCiÆmmol)1) was provided by Bhabha Atomic
Research Centre, Mumbai, India Prokaryotic CpG DNA
methyl transferase was obtained from New England Biolabs
(Beverly, MA, USA) A Moloney murine leukaemia virus
reverse transcriptase kit (RevertAidTM M-MuLV RT) was
purchased from MBI Fermentas Life Sciences (Rockville,
MD, USA) RNAlater (RNA stabilization solution) and
diethylpyrocarbonate were obtained from Ambion, Inc
(Austin, TX, USA) and Amresco (Solon, OH, USA)
respectively Methotrexate, bovine serum albumin and
d,l-dithiothreitol or Cleland’s reagent were purchased from
Sigma-Aldrich Co (St Louis, MO, USA) Cellulose nitrate membrane filters (0.45 lm) were obtained from Millipore Corporation (Bedford, MA, USA)
Animals Young adult male albino rats (Wistar strain), weighing 100–150 g, were obtained from the Postgraduate Institute
of Medical Education and Research’s Central Animal House (Chandigarh, India) The rats were housed in clean wire mesh cages with controlled temperature (23 ± 1C) and humidity (45–55%) and with a 12 h⁄ 12 h dark ⁄ light cycle throughout the study The rats were randomized into two groups of eight animals each, such that the mean body weights and range of body weights for each group of ani-mals were similar The rats in group I were given 1 gÆkg)1 body weight of ethanol (20% solution) per day for
3 months, and those in group II received an isocaloric amount of sucrose (36% solution) orally by Ryle’s tube daily for 3 months Such a dose does not produce a toxic blood alcohol concentration [21] and is therefore relevant
to human studies The rats were fed a commercially avail-able pellet diet (Ashirwad Industries, Chandigarh, India) containing 2 mgÆkg)1 folic acid and water ad libitum The body weights of the rats were recorded twice weekly Animals from both groups were killed under anaesthesia using sodium pentothal, and blood was drawn from the tail vein for alcohol and folate estimations Starting from the ligament of Treitz, two-thirds of the small intestine was removed, flushed with ice-cold saline and processed for the isolation of cells
The protocol of the study was approved by the tional Animal Ethical Committee (IAEC) and the Institu-tional Biosafety Committee (IBC)
Estimation of blood alcohol levels Alcohol was estimated from whole blood drawn from rats
24 h after the last dose of ethanol at the end of the treat-ment period using the alcohol dehydrogenase method [30]
Isolation of intestinal epithelial cells The intestinal epithelial cells were isolated following the method of Weiser [31] with modifications The upper two-thirds of the small intestine was cut and flushed two to three times with 0.9% saline One end of the intestine was tied with a thread and filled with rinsing buffer containing
1 mm d,l-dithiothreitol in normal saline The rinsing buffer was then replaced with a solution consisting of 1.5 mm KCl, 96 mm NaCl, 27 mm sodium citrate, 8 mm KH2PO4
and 8 mm Na2HPO4, and kept at 37C for 15 min in a beaker containing NaCl⁄ Pi The intestine was then filled with a solution containing 1.5 mm EDTA and 0.5 mm
Trang 9d,l-dithiothreitol in NaCl⁄ Pi, and kept at 37C in a shaker
at 100 r.p.m for 30 min; the solution was then collected for
the isolation of total enterocytes Furthermore, small
intes-tinal epithelial cells enriched in enterocytes of different
ori-gins along the crypt–villus axis were also isolated In this
case, different cell fractions were collected after filling the
intestine for different time intervals Fractions 1–3 were
col-lected at 4, 2 and 2 min intervals, fractions 4–6 at 3, 4 and
5 min intervals, and fractions 7–9 at 7, 10 and 15 min
inter-vals Each consecutive three fractions were pooled and
represented the villus tip, mid-villus and crypt base cells,
respectively The collected cells were centrifuged at 800 g
for 15 min The pellet contents were mixed with a Pasteur
pipette and centrifuged at 800 g for 10 min after the addition
of 5 mL of cold NaCl⁄ Pi Two more NaCl⁄ Piwashings were
performed These cells were then used for BBM isolation
Preparation of BBMVs from isolated intestinal
epithelial cells
BBMVs were prepared from isolated total intestinal cells
from control and ethanol-fed rats at different time intervals
during the course of treatment at 4C by the method of
Kessler et al [32] with some modifications The final pellet
containing cells was homogenized by adding 2 mm
Tris)50 mm mannitol buffer, and 10 mm MgCl2was added
to the homogenate followed by intermittent shaking for
10 min The contents were centrifuged at 3000 g for 15 min
and the supernatant was then run at 27 000 g for 30 min
The pellet thus obtained was suspended in a small amount
of loading buffer containing 280 mm mannitol and 20 mm
Hepes–Tris, pH 7.4, and centrifuged at 27 000 g for
30 min The final pellet obtained was suspended in loading
buffer so as to obtain a protein concentration of
approxi-mately 5 mgÆmL)1 These BBMVs were used to study
[3H]folic acid uptake at 1.5, 2 and 3 months of ethanol
treatment Experiments to determine kinetic constants and
western blot analysis were carried out using BBMV
prepa-rations from rats fed ethanol for 3 months
BBMVs were also isolated from cells representing the
vil-lus tip, mid-vilvil-lus and crypt base from rats sacrificed at the
end of treatment The respective cell fractions from two
animals were pooled for this purpose to obtain sufficient
BBMVs These BBMVs were used to determine [3H]folic
acid uptake across the crypt–villus axis and to analyse the
RFC protein levels in different cell types
Assessment of morphological purity of
membrane vesicles by transmission electron
microscopy
The final BBMV preparations obtained were suspended in
NaCl⁄ Pi and centrifuged at 27 000 g for 30 min Vesicular
suspensions were fixed at 4C in 3% buffered
glutaralde-hyde for 5–6 h and centrifuged at 10 000 g for 10 min Suspensions were gently rinsed twice with 0.2 m NaCl⁄ Piat
4C and postfixed for 1 h at 4 C with 1% buffered osmium tetroxide After dehydration in 70%, 90% and absolute ethanol for 2 h, 20 min and 1 h, respectively, the suspensions were treated with propylene oxide at room tem-perature The preparations were embedded in epoxy resin TAAB-812 (TAAB Laboratories, Aldermaston, UK) and polymerized for 24 h at 60C Semi-thin sections were placed on microslides, stained with 0.5% alkaline toluidine blue and examined under a light microscope to verify the areas of intensity Ultrathin sections (60 nm) were cut, placed on metal grids, stained on ultracut E (Reichert-Jung, Nuslock, Germany) and double stained with uranyl acetate and lead citrate The microslides were then examined under
a Zeiss EM-906 transmission electron microscope (Carl Zeiss, Dresden, Germany)
Transport of [3H]folic acid Uptake studies were performed at 37C using incubation buffer containing 100 mm NaCl, 80 mm mannitol, 10 mm Hepes, 10 mm 2-morpholinoethanesulfonic acid, pH 5.5, and 0.5 lm [3H]folic acid, unless otherwise noted Isolated BBMVs (10 lL; 50 lg protein) from control and ethanol-fed rats were added to incubation buffer containing [3H]folic acid of known concentration for different specific assays Reaction was stopped by the addition of ice-cold stop solution containing 280 mm mannitol and 20 mm Hepes–Tris, pH 7.4, followed by rapid vacuum filtration Nonspecific binding to the filters was determined by resid-ual filter counts after filtration of the incubation buffer and labelled substrate without vesicles [33,34] The radioactivity retained by the filters was determined by liquid scintillation counting (Beckman Coulter LS 6500, Beckman Coulter, Fullerton, CA, USA) For the determination of the kinetic constants Km and Vmax, transport of [3H]folic acid was measured by varying the concentration of [3H]folic acid from 0.125 to 1.50 lm in the incubation buffer at pH 5.5
RT-PCR analysis Total RNA from all animals was isolated from the upper
1 cm of jejunal tissues following the method of Chomeczyn-ski and Sacchi [35] cDNA synthesis was carried out from the purified and intact total RNA, according to the manu-facturer’s instructions (MBI Life Sciences) Expression of RFC and b-actin was evaluated using sequence-specific primers corresponding to the sequence in the open reading frame A 20 lL PCR mixture was prepared in 1· PCR buffer consisting of 0.6 U of Taq polymerase, 2 lm of each primer (for both b-actin and RFC) and 200 lm of each dNTP In optimized PCR, the initial denaturation step was carried out for 2 min at 95C The denaturation, annealing
Trang 10and elongation steps were carried out for 1 min at 94C,
1 min at 68C and 1 min at 72 C, respectively, for 35
cycles The final extension step was carried out for 10 min
at 72C The primers designed using primer3 input
(version 0.3.0) were as follows: RFC: forward,
5¢GA-ACGTCCGGCAACCACAG3¢; reverse,
5¢GATGGACTT-GGAGGCCCAG3¢; b-actin: forward,
5¢CACTGTGCCCA-TCTATGAGGG3¢; reverse,
5¢TCCACATCTGCTGGAA-GGTGG3¢ The expected PCR products of 489 and 588 bp
were obtained for RFC and b-actin, respectively, when
electrophoresed on a 1.2% agarose gel The densitometric
analyses of the products were determined using scion
imagesoftware (Scion Image Corporation, Frederick, MD,
USA)
Western blot analysis
For protein expression studies, BBMVs (100–150 lg)
isolated from epithelial cell preparations (either total cells
or different cell fractions) were resolved by 10%
SDS⁄ PAGE and transferred to nitrocellulose membrane
for 4–5 h at 4C (transfer at 25 V and 300 mA) Western
blotting was performed using the procedure described by
Towbin et al [36], employing polyclonal primary
anti-bodies (rabbit anti-rat RFC, 1 : 500 dilution) kindly
provided by H M Said (University of California, Irvine,
USA) These were raised against a specific region of rat
RFC synthetic peptide corresponding to amino acids 495–
512 The polyclonal antibodies against LAP, an intestinal
brush border peptidase, were rabbit anti-rat LAP (1 : 500
dilution) Secondary antibodies were goat anti-rabbit IgG
[horseradish peroxidase (HRP)-labelled] (1 : 2000 dilution)
Blot quantification was carried out using scion image
software
Immunohistochemical analysis
Freshly cut intestinal jejunal sections were cut into 2 cm
pieces and slit open, followed by fixing in a sufficient
amount of 10% formalin [37] using primary antibodies
[rabbit polyclonal anti-rat RFC (1 : 100)] and secondary
antibodies [goat anti-rabbit IgG (HRP-labelled) (1 : 500)]
Haematoxylin was employed for counterstaining
Haematoxylin–eosin staining
Haematoxylin–eosin staining was carried out following
the routine histological method described by Kayser and
Bubenzer [38] The haematoxylin–eosin staining technique
employs haematoxylin, which is a basic dye and stains
acidic components, such as nucleoproteins and
muco-polysaccharides, and eosin, which is an acidic dye and
stains the basic components present in cytoplasmic
proteins
Estimation of folate by microbiological assay Folate estimations were determined by micotitre plate assay using Lactobacillus casei [39] All steps were carried out in aseptic conditions
Genomic DNA methylation studies DNA isolation was performed by the conventional method using a lysis buffer containing proteinase K, as described previously [29] The methylation status of CpG sites in genomic DNA was determined by the in vitro methyl accep-tance capacity of DNA using S-adenosyl-[methyl-3 H]methi-onine as a methyl donor and a prokaryotic CpG DNA methyltransferase [40]
Statistics Each uptake assay was performed three times with eight independent preparations from each group The data were computed as the mean ± standard deviation Group means were compared using Student’s t-test, and analysis of variance was used when necessary The acceptable level of significance was P < 0.05 for each analysis
Acknowledgements
Financial assistance by the Council of Scientific and Industrial Research (CSIR), New Delhi, India is grate-fully acknowledged
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