Down-regulation of reduced folate carrier may result in folate malabsorption across intestinal brush border membrane during experimental alcoholism

Down-regulation of reduced folate carrier may result infolate malabsorption across intestinal brush bordermembrane during experimental alcoholismAbid Hamid1, Nissar Ahmad Wani1, Satyavati Rana2, Kim Vaiphei3, Akhtar Mahmood4 andJyotdeep 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: [email protected]

(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.

FEBS Journal 274 (2007) 6317–6328 ª 2007 The Authors Journal compilation ª 2007 FEBS 6317

The 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 folates 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 malabsorption 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.

Intestinal folate malabsorption in alcoholism A. Hamid et al.

6318 FEBS Journal 274 (2007) 6317–6328 ª 2007 The Authors Journal compilation ª 2007 FEBS

[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

A B

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

1.5 months 2 months 3 months

Control

Ethanol

***

*** ***

V(p

mo

l/30

sec/

mg

pro

tein

)

Fig. 2. [3H]Folic acid transport in intestinal BBMVs at different

intervals during the course of ethanol administration. An incubation

buffer of pH 5.5 and a [3H]folic acid concentration of 0.5 lM

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

60Control

Ethanol

***

0 0.5 1 1.5[S] (µM)

***

**

***

**

V(p

mo

l/30

sec/

mg

pro

tein

)

Fig. 3. [3H]Folic acid uptake in intestinal BBMVs as a function of

substrate concentration (inset Lineweaver–Burk plot). Uptake was

measured by varying the [3H]folic acid concentration from 0.125 to

1.50 lM 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.

A. Hamid et al. Intestinal folate malabsorption in alcoholism


3 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

Control Ethanol

***

0.6B

A

0.4

0.2

0

Mea

n re

lativ

e R

FC

leve

ls (

RF

C/β

-act

in)

3 4 5 6588

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.



RFC 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 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

1 2

~65kDa

Control0

1

2

3

4

5

Ethanol

***

Mea

n re

lativ

e R

FC

pro

tein

le

vels

(R

FC

/LA

P)

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

1 2 3 4 5

Villus tip

Mid villus

Crypt base

Control

1.00

###

### ### ###*****

**0.75

0.50

0.25

0Ethanol

6

Mea

n r

elat

ive

RF

C p

rote

in

leve

ls (

RF

C/L

AP

)

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.



group. 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

A B

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.

A B

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

20SA

M u

tiliz

ed(µ

Mx1

03/µ

g D

NA

)

0Ethanol

***

Fig. 9. [3H]-labelled SAM incorporated (lMÆlg)1 jejunal DNA) as an

index of jejunal DNA methylation profile. Values are means ± stan-

dard deviation (n ¼ 8). ***P < 0.001 versus control.



(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 Km and 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-transcriptional 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



reduced 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¢,7,9-3H]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 ± 1 �C)and humidity (45–55%) and with a 12 h ⁄ 12 h dark ⁄ lightcycle 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 Institu-

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 37 �C 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



d,l-dithiothreitol in NaCl ⁄Pi, and kept at 37 �C 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 ⁄Pi washings 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 4 �C 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 MgCl2 was 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-villus 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 4 �C 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 ⁄Pi at

4 �C 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 60 �C. 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 37 �C 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 95 �C. The denaturation, annealing



and elongation steps were carried out for 1 min at 94 �C,1 min at 68 �C and 1 min at 72 �C, respectively, for 35

cycles. The final extension step was carried out for 10 min

at 72 �C. 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

image software (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 4 �C (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-3H]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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Down-regulation of reduced folate carrier may result in folate malabsorption across intestinal brush border membrane during experimental alcoholism