Alcohol, intestinal bacterial growth, intestinal permeability to
endotoxin, and medical consequences: Summary of a symposium
Vishnudutt Purohita,*, J. Christian Bodeb, Christiane Bodeb, David A. Brennerc,
Mashkoor A. Choudhryd, Frank Hamiltone, Y. James Kangf, Ali Keshavarziang,
Radhakrishna Raoh, R. Balfour Sartori, Christine Swansonj, Jerrold R. Turnerk
aDivision of Metabolism and Health Effects, National Institute on Alcohol Abuse and Alcoholism,
National Institutes of Health, 5635 Fishers Lane, Room 2035, MSC 9304, Bethesda, MD 20892-9304, USA
bHohenheim University (140), Department of Physiology of Nutrition, Stuttgart, Germany
cUniversity of California San Diego Health Sciences, La Jolla, CA, USA
dCenter for Surgical Research, University of Alabama at Birmingham, Birmingham, AL, USA
eDivision of Digestive Diseases and Nutrition, National Institute of Diabetes, Digestive, and Kidney Diseases,
National Institutes of Health, Bethesda, MD, USA
fDepartments of Medicine and Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY, USA
gDivision of Digestive Diseases and Nutrition, Department of Medicine,
Rush University Medical Center, Chicago, IL, USA
hDepartment of Physiology, University of Tennessee Health Science Center, Memphis, TN, USA
iDepartment of Medicine, University of North Carolina at Chapel Hill, Biomolecular Building, Chapel Hill, NC, USA
jOffice of Dietary Supplements, National Institutes of Health, Bethesda, MD, USA
kDepartment of Pathology, The University of Chicago, Chicago, IL, USA
Received 4 December 2007; received in revised form 11 March 2008; accepted 27 March 2008
This report is a summary of the symposium on Alcohol, Intestinal Bacterial Growth, Intestinal Permeability to Endotoxin, and Medical
Consequences, organized by National Institute on Alcohol Abuse and Alcoholism, Office of Dietary Supplements, and National Institute of
Diabetes and Digestive and Kidney Diseases of National Institutes of Health in Rockville, Maryland, October 11, 2006. Alcohol exposure
can promote the growth of Gram-negative bacteria in the intestine, which may result in accumulation of endotoxin. In addition, alcohol
metabolism by Gram-negative bacteria and intestinal epithelial cells can result in accumulation of acetaldehyde, which in turn can increase
intestinal permeability to endotoxin by increasing tyrosine phosphorylation of tight junction and adherens junction proteins. Alcohol-
induced generation of nitric oxide may also contribute to increased permeability to endotoxin by reacting with tubulin, which may cause
damage to microtubule cytoskeleton and subsequent disruption of intestinal barrier function. Increased intestinal permeability can lead to
increased transfer of endotoxin from the intestine to the liver and general circulation where endotoxin may trigger inflammatory changes in
the liver and other organs. Alcohol may also increase intestinal permeability to peptidoglycan, which can initiate inflammatory response in
liver and other organs. In addition, acute alcohol exposure may potentiate the effect of burn injury on intestinal bacterial growth and per-
meability. Decreasing the number of Gram-negative bacteria in the intestine can result in decreased production of endotoxin as well as
acetaldehyde which is expected to decrease intestinal permeability to endotoxin. In addition, intestinal permeability may be preserved
by administering epidermal growth factor, L-glutamine, oats supplementation, or zinc, thereby preventing the transfer of endotoxin to
the general circulation. Thus reducing the number of intestinal Gram-negative bacteria and preserving intestinal permeability to endotoxin
may attenuate alcoholic liver and other organ injuries. ? 2008 Elsevier Inc. All rights reserved.
Keywords: Alcohol; Bacterial growth; Intestinal permeability; Endotoxin; Acetaldehyde; Nitric oxide
Chronic alcohol consumption is associated with the de-
velopment of various medical disorders such as alcoholic
liver diseases (ALD), pancreatitis, cardiomyopathy, acute
respiratory distress syndrome, and brain injury in some
* Corresponding author. Division of Basic Neuroscience & Behavioral
Research, National Institute on Drug Abuse, National Institutes of Health,
6001 Executive Boulevard, Room 4274, MSC 9555, Bethesda, MD 20892-
9555, USA. Tel.: þ1-301-594-5753; fax: þ1-301-594-6043.
E-mail address: firstname.lastname@example.org (V. Purohit).
0741-8329/08/$ e see front matter ? 2008 Elsevier Inc. All rights reserved.
Alcohol 42 (2008) 349e361
individuals. Endotoxin appears to play a central role in the
initiation of alcohol-induced tissue/organ damage and its
role is most convincing for liver injury. Endotoxin is a lipo-
polysaccharide (LPS) derived from the cell wall of Gram-
negative bacteria residing in the intestine. The connection
between alcohol, endotoxin and liver injury became appar-
ent when researchers observed elevated plasma endotoxin
levels in patients affected with ALD (Bode et al., 1987;
Fukui et al., 1991). This connection was further confirmed
when administration of LPS caused the progression of fatty
liver into necroinflammatory changes in a rat model of al-
coholic liver injury (Bhagwandeen et al., 1987; Pennington
et al., 1997). In addition, elimination of intestinal bacteria
or suppression of intestinal Gram-negative bacteria attenu-
ated alcoholic liver injury in rats by reducing plasma endo-
toxin levels (Adachi et al., 1995; Nanji et al., 1994).
Normally, only a trace amount of endotoxin is absorbed
from the intestine through the intestinal epithelial lining
to the portal vein that carries it to the liver where it is
cleared by Kupffer cells. However, excess amount of endo-
toxin reaching to the liver can activate Kupffer cells to ini-
tiate liver inflammation (Thurman, 1998; Wheeler et al.,
2001). Furthermore, excess amount of endotoxin in the gen-
eral circulation may trigger injury to other organs. Elevated
levels of plasma endotoxin in response to alcohol ingestion
may result from (1) excessive production of endotoxin in
the intestine through overgrowth of Gram-negative bacte-
ria; (2) increased permeability of the intestine to endotoxin;
and (3) delayed clearance of endotoxin by Kupffer cells.
Suppressing the growth of intestinal Gram-negative bacte-
ria and/or restoring/preserving intestinal integrity and
thereby blocking the transfer of endotoxin from the intes-
tine to general circulation may be logical steps in prevent-
ing or attenuating alcohol-associated tissue/organ injuries.
Therefore, understanding the underlying mechanisms by
which alcohol promotes intestinal bacterial growth and
increases intestinal permeability to endotoxin may help
design strategies for the prevention or treatment of alcohol/
endotoxin-associated medical disorders.
The National Institute on Alcohol Abuse and Alcohol-
ism in collaboration with Office of Dietary Supplements
and National Institute of Diabetes and Digestive and
Kidney Diseases of National Institutes of Health organized
a symposium on Alcohol, Intestinal Bacterial Growth, In-
testinal Permeability to Endotoxin, and Medical Conse-
quences in Rockville, Maryland, October 11, 2006. The
following topics were discussed by nine speakers: (1) Role
of Endotoxin in Alcoholic and Non-Alcoholic Liver Injury
(David A. Brenner); (2) Alcohol, Immune System, and
Intestinal Bacterial Growth (Mashkoor A. Choudhry); (3)
Probiotics, Intestinal Bacterial Overgrowth, and Peptido-
glycan (R. Balfour Sartor); (4) The Leaky Epithelial Barrier
in Intestinal Diseases (Jerrold RTurner); (5) Alcohol, Intes-
tinal Permeability, and Endotoxemia (J. Christian Bode and
Christiane Bode); (6) Effects of Acetaldehyde and
(Radhakrishna Rao); (7) Role of Zinc in Preserving Intesti-
nal Integrity in Alcohol-Intoxicated Mice (James Kang);
and (8) Effects of Nitric Oxide and Oats Supplementation
on Alcohol-Induced Leaky Gut (Ali Keshavarzian). The
following is a summary of the symposium.
Alcohol and intestinal bacterial growth
The intestine of normal human subjects is a habitat for
various types of bacteria. Overgrowth of Gram-negative
bacteria can result in increased production of endotoxin that
can escape into portal circulation leading to increased
plasma levels of endotoxin. Only a few studies are available
in literature investigating relationship between alcohol
consumption and bacterial growth in the small intestine.
Increased number of microorganisms, including Gram-
negative coliform bacteria, was reported in the proximal
part of small intestine of chronic alcoholics; however, no
correlation was found between the number or types of mi-
croorganisms in the jejunum and the type or degree of liver
disease in the alcoholics (Bode et al., 1984). Bacterial over
growth was also reported in the duodenum of alcoholics
(Hauge et al., 1997). In another study, the incidence of in-
testinal bacterial overgrowth was three times higher in alco-
holics with ALD than that in control subjects with no
history of alcohol abuse (Bode et al., 1993). These studies
suggest that chronic alcohol abuse can promote bacterial
growth in the intestine; however, further studies are
required to answer the following questions. Does alcohol
affect bacterial growth in a dose-dependent manner? How
does alcohol affect bacterial growth at a mechanistic level?
Is bacterial growth associated with increased amount of
endotoxin production? What types of bacteria are affected
Suppression of bacterial growth by probiotics
Suppressing the growth of Gram-negative bacteria in the
intestine may reduce the amount of endotoxin, which in
turn may attenuate endotoxin-associated organ damage. In
this regard, use of probiotic bacteria, otherwise known as
beneficial bacteria, provides a viable option. The most nu-
merous probiotic bacteria normally inhabiting the small in-
testine are species of Lactobacilli whereas in the colon the
majority is mainly Bifidobacteria. These beneficial bacteria
have been shown to attenuate the growth of Gram-negative
bacteria. Feeding of Lactobacillus acidophilus strain NP51
was found to reduce the number of Escherichia coli
O157:H7 in the fecal samples of beef cattle (Peterson
et al., 2007; Younts-Dahl et al., 2005). Lactobacillus
strains, LAP5 and LF33, obtained from swine and poultry,
respectively, were shown to inhibit the growth of E. coli
and Salmonella typhimurium in an in vitro culture system
(Tsai et al., 2005). Enteral administration of Lactobacillus
R2LC attenuated bacteremia and endotoxemia associated
350V. Purohit et al. / Alcohol 42 (2008) 349e361
with intra-abdominal infection in rats (Thorlacius et al.,
2003). Bifidobacterium animalis MB5 and Lactobacillus
rhamnosus GG were shown to protect intestinal Caco-2
cells from the inflammation-associated response induced
by E. coli K88 (Roselli et al., 2006). Finally, in an intragas-
tric infusion model of alcoholic liver injury, feeding of rats
with Lactobacillus GG reduced plasma levels of endotoxin
and severity of liver injury (Nanji et al., 1994). Whether
probiotic treatment can attenuate ALD in humans needs
The leaky epithelial barrier in intestinal diseases
Intestinal diseases are associated with leaky epithelial
barrier and increased intestinal permeability to toxic agents,
such as endotoxin. This may result in increased transfer of
endotoxin from the intestine into liver and general circula-
tion, which in turn may initiate injury to liver and other
organs. Therefore, an intact intestinal barrier primarily
formed by the epithelium is critical to normal physiological
function and prevention of diseases. The space between
adjacent epithelial cells (the paracellular space) is sealed
primarily by tight junctions, which control the barrier func-
tion of an intact intestinal epithelium. Studies showing an
association of thin actin-like filaments within the apical cy-
toplasm of small intestinal epithelial cells with tight junc-
tions suggest that tight junctions are intimately related to
the perijunctional cytoskeleton (Hull & Staehelin, 1979;
Madara, 1987). Severing of actin filaments by fungal-
derived cytochalasins disrupted tight junction barrier func-
tion and structure (Bentzel et al., 1980). Further studies
showed that cytochalasin caused morphological condensa-
tion of perijunctional actin (Madara et al., 1987), suggest-
ing that cytochalasin-induced condensation and associated
contraction of perijunctional actin may have been responsi-
ble for tight junction disruption. Exposure of intestinal ep-
ithelial monolayer to actin-depolymerizing drugs removed
one transmembrane proteindoccludindfrom the tight
junction by endocytosis at the precise time that barrier
function was disrupted (Shen & Turner, 2005). Thus,
occludin endocytosis may be an important marker of tight
junction regulation mediated by the cytoskeleton.
Evidence suggests that intestinal epithelial barrier func-
tion is regulated by physiological stimuli. Using isolated
segments of hamster small intestine, it was shown that that
triggers contraction of perijunctional actomyosin, thereby
increasing intestinal tight junction permeability and enhanc-
ingabsorption ofnutrients (Madara& Pappenheimer,1987).
The role of Naþ-glucose transport in the regulation of
epithelial tight junction permeability was further confirmed
using Caco-2 intestinal epithelial cells transfected with the
intestinal Naþ-glucose cotransporter, wherein the activation
of the cotransporter increased and inactivation decreased
the permeability of the epithelial cells (Turner et al.,
1997). Further research showed that the phosphporylation
of myosin light-chain (MLC) catalyzed by myosin light-
chain kinase (MLCK) was necessary for Naþ-glucose
cotransport-induced tight junction permeability regulation
because pharmacological inhibition of MLCK prevented
both MLC phosphorylation and regulation of tight junction
barrier function in cultured intestinal epithelial cells (Turner
et al., 1997).
Intestinal epithelial barrier function can be compromised
in intestinal disease. This was initially recognized in pa-
tients with Crohn’s disease (Hollander, 1988; Ukabam
et al., 1983), but has subsequently been reported in patients
or experimental models of a spectrum of inflammatory,
immune-mediated, and infectious
(Clayburgh et al., 2004). Tumor necrosis factor-a (TNF-
a) appears to play an important role in regulating intestinal
epithelial barrier function since TNF-a-neutralizing anti-
bodies restored barrier function in Crohn’s disease patients
(Suenaert et al., 2002). Exposure of cultured epithelial
monolayers to TNF-a in vitro caused epithelial barrier loss
(Marano et al., 1998; Mullin et al., 1992). In another study,
exposure of cultured epithelial monolayers to TNF-a in-
creased MLC phosphorylation, which was associated with
impaired barrier function (Zolotarevsky et al., 2002). In this
study, specific inhibition of MLCK prevented MLC phos-
phorylation and restored barrier function in TNF-a-treated
monolayers (Zolotarevsky et al., 2002). Thus, TNF-a ap-
pears to increase MLC phosphorylation by increasing the
expression of MLCK (Ma et al., 2005; Wang et al.,
2005a). In inflammatory bowel disease patients the extent
of increased MLCK expression and MLC phosphorylation
correlates directly with the magnitude of active inflamma-
tion, suggesting a relationship between MLCK expression
and disease severity (Blair et al., 2006).
Alcohol and intestinal permeability
Several researchers have investigated the effect of alco-
hol on intestinal permeability to various molecules includ-
ing endotoxin. In rats, chronic alcohol feeding by gavage
increased permeability of the intestinal mucosa to macro-
molecules such as hemoglobin (Bungert, 1973) and horse-
radish peroxidase (Worthington et al., 1978). Intestinal
permeability assessed by the lactulose/mannitol (L/M) ratio
was significantly increased in rats administered ethanol by
intragastic infusion (Mathurin et al., 2000). An ethanol-
induced increase in intestinal permeability was also re-
ported in human subjects when smaller molecules were
used as a permeability probe. For example, an increased
absorption of51Cr ethylene diamine tetraacetic acid was
found in subjects chronically abusing alcohol (Bjarnason
et al., 1984) and an increase in absorption of polyethylene
glycol (PEG) 400 was observed after the oral administra-
tion of alcohol to healthy volunteers (Robinson et al.,
1981). To determine whether alcohol can increase intestinal
permeability to macromolecules, permeability to PEG with
different molecular masses (Mr 400, Mr 1,500, Mr 4,000,
351V. Purohit et al. / Alcohol 42 (2008) 349e361
and Mr 10,000) was measured in recently drinking alco-
holics with different stages of ALD (Parlesak et al.,
2000). The permeability to PEG Mr 400 was found to be
unchanged when compared to healthy controls, whereas
the permeability to PEG Mr 1,500 and Mr 4,000 were dis-
tinctly enhanced and the prevalence of increased permeabil-
ity to PEG 10,000 was more than 10-fold higher in
alcoholics (Parlesak et al., 2000). Direct evidence for
increased gut permeability to endotoxin by alcohol was ob-
tained in a rat study where intragastric administration of
LPS (5 mg/kg) to alcohol-fed rats significantly increased
portal vein endotoxin level after 2 h (Mathurin et al.,
2000). No such increases were observed in pair-fed
controls. In another rat study, plasma endotoxin levels in
long-term ethanol-fed rats were higher than those in control
rats after intragastric administration of high-dose endotoxin
(20 mg/kg) (Tamai et al., 2002). Furthermore, intestinal
permeability to fluorescein isothiocyanate-labeled dextran
with an average molecular weight of 4,000 D (FD4) was in-
creased by long-term ethanol administration (Tamai et al.,
2002). Similar results were obtained in a mice study where
animals were administered 1 mg/kg bacterial LPS by intra-
gastirc gavage 1 h after administration of alcohol by gavage
(Lambert et al., 2003). Ethanol alone, but not LPS alone,
significantly increased plasma endotoxin level, and etha-
nol þ LPS caused further significant increase in plasma en-
dotoxin levels (Lambert et al., 2003). These studies clearly
indicate that alcohol can increase intestinal permeability to
various macromolecules including endotoxin.
Alcohol and intestinal mucosal damage
Acute administration of alcohol to rodents or dogs at
concentrations corresponding to those found in commonly
available alcoholic beverages ($4%, vol/vol) leads to
mucosal damage in the upper small intestine with a loss
of epithelium at the tips of the villi, hemorrhagic erosions
and even hemorrhage in the lamina propria (Bode et al.,
2001). Similar lesions were observed in volunteers 2e3 h
after ingestion of alcohol (15e20%, vol/vol) and in recently
drinking alcohol abusing subjects (Bode et al., 2001). In
rats, administration of 20% ethanol by gavage for 4 h re-
sulted in hemorrhagic erosions of the proximal small intes-
tine with epithelial cell loss (Tamai et al., 2000). Similarly,
administration of 20% ethanol to rats for 14 days by gavage
resulted in significant mucosal disruption of ileum as evi-
denced by subepithelial edema of villous tips, cellular infil-
tration of the villi and exfoliation (Napolitano et al., 1995).
In mice, acute ethanol exposure (single oral dose of 6 g/kg)
caused severe injury to the mucosal lining of ileum of the
small intestine (Lambert et al., 2003). This included
breaches in the epithelial layer of the villi, submucosal
blebbing, and ulceration of the villi (Lambert et al.,
2003). The mucosal damage caused by alcohol might result
in an impaired intestinal barrier function, enabling toxins of
gut-inhabiting bacteria such as endotoxins to enter the
systemic circulation and to contribute to liver injury after
Role of acetaldehyde in increasing
Acetaldehyde is the first and most toxic metabolite of
ethanol metabolism. In the gastrointestinal tract, acetalde-
hyde can be generated from ethanol through mucosal and/
or bacterial alcohol dehydrogenase (Seitz & Oneta, 1998).
During chronic ethanol administration, high concentration
of acetaldehyde can be detected in the mucosa of large in-
testine. In rats, colonic luminal concentrations of acetalde-
hyde were significantly increased after ethanol intake
(132.6 6 31.6 mmol/l vs. 20.8 6 1.4 mmol/l) and antibiotic
treatment attenuated this increase to some extent (86.2 6
10.9 mmol/l) (Ferrier et al., 2006), indicating a role of bac-
teria in the generation of acetaldehyde. Other investigators
have reported further higher concentrations of acetaldehyde
ranging from 0.12 to 3 mM in rat colonic lumen after
ethanol administration (Rao et al., 2004). Accumulation
of acetaldehyde in the colonic lumen could be due to low
efficiency of bacterial aldehyde dehydrogenase to metabo-
lize acetaldehyde in the colon (Nosova et al., 1998).
Increasing evidence suggest that acetaldehyde can in-
crease intestinal permeability to endotoxin (Ferrier et al.,
2006; Rao, 1998; Rao et al., 2004). Acetaldehyde in the
concentration of 99e760 micromolar increased paracellular
permeability in a human colonic epithelial (Caco-2) cell
monolayer (Rao, 1998). The paracellular permeability
was assessed by measuring transepithelial electrical resis-
tance, sodium chloride dilution potential, and unidirectional
flux of labeled mannitol. However, ethanol up to 5% con-
centration failed to increase paracellular permeability, sug-
gesting that ethanol metabolism to acetaldehyde is required
for the action. This was confirmed by another study where
ethanol up to 3% showed no significant effect on paracellu-
lar permeability in Caco-2 cell monolayers (Ma et al.,
1999). This study showed that ethanol increased paracellu-
lar permeability at 7.5% concentration. Subsequent studies
by Banan et al. (1999, 2000) showed that ethanol increased
paracellular permeability at doses 2.5% and above by re-
ducing the cell viability. The importance of ethanol metab-
olism into acetaldehyde was further confirmed when
acetaldehyde, and not ethanol, was shown to increase mu-
cosal permeability in proximal rat colonic strips mounted
to Ussing chambers in vitro (Ferrier et al., 2006). Further
mechanistic studies showed that acetaldehyde-induced in-
crease in paracellular permeability is associated with redis-
tribution of tight junction proteins (occludin and ZO-1) and
adherens junction (E-cadherin and b-catenin) proteins from
the intercellular junctions into the intracellular compart-
ments (Atkinson & Rao, 2001; Seth et al., 2004; Sheth
et al., 2004).
Researchers further investigated the role of protein tyro-
sine phosphorylation in acetaldehyde-induced disruption of
352 V. Purohit et al. / Alcohol 42 (2008) 349e361
epithelial tight junctions in the Caco-2 cell monolayer
(Atkinson & Rao, 2001). Acetaldehyde increased tyrosine
phosphorylation of ZO-1, E-cadherin, and beta-catenin,
which was associated with a significant reduction in protein
tyrosine phosphatase (PTP) activity without affecting tyro-
sine kinase activity. Treatment with acetaldehyde resulted
in a 97% loss of PTP1B activity and a partial reduction
of PTP1C and PTP1D activities. These results suggest that
acetaldehyde inhibits PTP to increase protein tyrosine phos-
phorylation of tight junction and adherens junction pro-
teins, which may result in disruption of the tight junctions
and adherens junctions via redistribution of their proteins.
Since interactions between E-cadherin, b-catenin, and
PTP1B are crucial for the organization of adherens junc-
tions and epithelial cellecell adhesion and subsequent
maintenance of intestinal epithelial barrier, researchers
further investigated the effect of acetaldehyde on the inter-
actions between E-cadherin, b-catenin, and PTP1B in
Caco-2 cell monolayers (Sheth et al., 2007). In this study,
investigators demonstrated that acetaldehyde induced dis-
ruption of interactions between E-cadherin, b-catenin, and
suggesting that this may be a mechanism whereby acetalde-
hyde impairs intestinal permeability.
Role of nitric oxide in increasing intestinal permeability
The role of nitric oxide (NO) in intestinal barrier
dysfunction was recognized when researchers observed in-
creased expression of inducible nitric oxide synthase
(iNOS) in concert with intestinal barrier dysfunction in in-
flamed human colonic mucosa (Kimura et al., 1998; Singer
et al., 1996). In human Caco-2 monolayers, ethanol
(2.5%e15%) significantly increased expression of iNOS
that was associated with increased nitration of tubulin and
disruption of barrier function as measured by apical-to-
basolateral flux of fluroscent marker (Banan et al., 1999).
The role of nitric oxide in mediating the effect of ethanol
on intestinal barrier function was further confirmed in an-
other Coca-2 monolayer study (Banan et al., 2000). In this
study, ethanol exposure (1%, 2.5%, and 15%) increased ex-
pression of iNOS and increased production of NO and su-
peroxide. These findings were associated with nitration
and oxidation of tubulin, decreased levels of stable poly-
merized tubulin, increased levels of disassembled tubulin,
and damage to microtubule cytoskeleton. In addition,
ethanol significantly disrupted epithelial barrier function
as determined by measuring apical-to-basolateral flux of
a fluorescent marker. The effects of ethanol were mimicked
by peroxynitrite (a product of the reaction of NO with
superoxide anions). Pretreatment with iNOS inhibitor
(L-N6-1-iminoethyl-lysine), peroxynitrite scavengers (urate
or L-cysteine) or superoxide anion scavenger (superoxide
dismutase) attenuated the damage caused by ethanol. These
results suggest that NO and superoxide anion generated in
response to ethanol exposure may react with each other to
form peroxynitrite which in turn may cause damage to mi-
crotubule cytoskeleton and subsequently disrupt intestinal
barrier function. It was further demonstrated that NF-
kappaB is required for oxidant-induced iNOS upregulation
and for the consequent nitration and oxidation of cytoskel-
eton (Banan et al., 2004). These researchers further showed
that epidermal growth factor (EGF) may protect intestinal
barrier function by stabilizing cytoskeleton via down
regulating the activity of iNOS (Banan et al., 2003).
Combined effect of alcohol and burn injury on intestinal
bacterial growth and barrier function
Researchers have investigated the combined effect of
alcohol and burn injury on intestinal bacterial growth and
barrier function. The reason for this two-hit insult is that al-
cohol intoxication is recognized as a major problem in post-
burn injury pathogenesis. According to an estimate nearly
one million people are affected with burn injuries every
year within the United States and nearly half of these in-
juries occur under the influence of ethanol intoxication
(Choudhry & Chaudry, 2006; Messingham et al., 2002).
The effects of acute ethanol intoxication combined with
burn injury were determined on intestinal bacterial growth,
intestinal permeability, intestinal immune defense, and bac-
terial translocation (Choudhry et al., 2002; Kavanaugh
et al., 2005; Li et al., 2006). Ethanol was administered to
rats by gavage (5 ml of 20% ethanol) 4 h prior to burn in-
jury. An analysis of various parameters on days 1 and 2 af-
ter injury revealed that the combined insult of ethanol and
burn injury significantly increased intestinal permeability
and the number of bacteria in mesenteric lymph nodes. Fur-
thermore, the combined insult caused a significant decrease
in intestinal T cell functions and their numbers in intestinal
mucosa (Choudhry et al., 2002; Kavanaugh et al., 2005).
Additionally, the finding of Kavanaugh et al. (2005) showed
that the combined insult significantly increases bacterial
growth in the small intestine on day 2 after injury. The eth-
anol alone group exhibited an increase in intestinal perme-
ability on day 1 and not on day 2 (Choudhry et al., 2002;
Kavanaugh et al., 2005; Li et al., 2006). No change in other
parameters was noted in the ethanol alone group. In con-
trast, as compared to sham, rats receiving burn injury alone
did not exhibit a significant difference in intestinal perme-
ability and intestinal T cell function on day 1 after injury.
However, on day 2, the burn alone group exhibited a ten-
dency of an increase in bacterial growth (though not signif-
icant), intestinal permeability and number of bacteria in
the mesenteric lymph nodes, and a decrease in intestinal
T cell functions. Nonetheless, the changes in these above
parameters were more severe in the group of rats that has
undergone a combined insult of alcohol and burn injury
(Choudhry et al., 2002; Kavanaugh et al., 2005). These re-
sults suggest that acute alcohol intoxication potentiated the
effect of burn injury on all of the parameters listed above
including intestinal bacterial growth and permeability.
353V. Purohit et al. / Alcohol 42 (2008) 349e361
Preservation of intestinal epithelial barrier function
Preservation of intestinal epithelial barrier function may
prevent the diffusion of colonic luminal endotoxin and
other toxic products into portal vein, which in turn may at-
tenuate endotoxin-induced organ/tissue injury. Several
researchers have investigated the role of the following fac-
tors in the preservation of intestinal epithelial barrier func-
tion disrupted by alcohol.
Role of epidermal growth factor
Epidermal growth factor is known to promote growth and
differentiation of gastrointestinal mucosa and provide pro-
tection against injurious agents. The role of EGF in the pro-
tection of epithelial barrier function from acetaldehyde was
evaluated in Caco-2 intestinal epithelial cell monolayer
(Sheth et al., 2004). Epidermal growth factor administration
significantly reduced acetaldehyde-induced increases in
paracellular permeability to inulin and LPS in a time and
dose-dependent manner. Epidermal growth factor prevented
acetaldehyde-induced reorganization of occludin, zonula
occludens-1 (ZO-1), E-cadherin, and b-catenin from the
cellular junctions to the intracellular compartments. In addi-
actin cytoskeleton and the interaction of occludin, ZO-1,
E-cadherin, andb-catenin with the actincytoskeleton. These
results indicate that EGF can preserve intestinal paracellular
permeability by attenuating acetaldehyde-induced disrup-
tion of tight junctions and adherens junctions and by
preventing acetaldehyde-induced redistribution of actin cy-
toskeleton and its interaction with occludin, ZO-1, E-
cadherin, and b-catenin. Further investigation revealed that
EGF protects the tight junction barrier function from acetal-
dehyde by PLCg1, calcium, PKCbI, and PKC3-dependent
mechanisms (Suzuki et al., 2007). Epidermal growth
factor-mediated prevention of acetaldehyde-induced in-
by shRNA. EGF induces membrane translocation of PKC3
location of PKC3 and PKCbI was required for prevention of
acetaldehyde-induced increase in permeability. A similar
protective role of EGF on acetaldehyde-induced disruption
of tight junctions and adherens junctions was also demon-
strated in human colonic mucosa (Basuroy et al., 2005).
Role of L-glutamine
Glutamine, a nonessential amino acid, has been shown
to improve intestinal barrier function in experimental
biliary obstruction (White et al., 2005) and in patients with
systemic infections (De-Souza & Greene, 2005). Role of
L-glutamine in the protection of intestinal epithelium from
acetaldehyde-induced disruption of barrier function was
evaluated in Caco-2 cell monolayer (Seth et al., 2004).
L-Glutamine reduced the acetaldehyde-induced decrease
in transepithelilal electrical resistance and increase in per-
meability to inulin and LPS in a time and dose-dependent
manner. L-glutamine reduced the acetaldehyde-induced re-
distribution of occludin, ZO-1, E-cadherin, and b-catenin
from the intercellular junctions to the intracellular compart-
ments. L-glutamine reduced acetaldehyde-induced dissocia-
tion of occludin, ZO-1, E-cadherin, and b-catenin from the
actin cytoskeleton. These researchers further demonstrated
that L-glutamine protects the intestinal barrier function by
an EGF receptor-dependent mechanism. A similar protec-
tive role of L-glutamine on acetaldehyde-induced disruption
of tight junctions and adherens junctions was also demon-
strated in human colonic mucosa (Basuroy et al., 2005).
Role of oats supplementation
Chronic administration of ethanol (8 g/kg/day) to rats for
10 weeks by gavage induced gut leakiness as assessed by
urinary excretion of lactulose and mannitol, which was
associated with increased levels of blood endotoxin as well
as liver injury. Oat supplementation ameliorated all of these
changes (Keshavarzian et al., 2001). Studies are required to
understand the mechanisms by which oat supplement pre-
serves intestinal permeability.
Role of zinc
Zinc is an essential trace element involved in many
physiological functions. Zinc deficiency has been reported
in patients with inflammatory bowl disease (Hendricks &
Walker, 1988; Solomons et al., 1977), which is associated
with increased intestinal permeability. On the other hand,
zinc supplementation has been found to preserve intestinal
permeability in patients with Crohn’s disease (Sturniolo
et al., 2001) and in rats with experimental colitis (Sturniolo
et al., 2002). Ethanol appears to impair the absorption of
zinc from the intestine. In rats, chronic ethanol ingestion
significantly impaired zinc absorption in the ileum, which
is the most active area of zinc uptake in the intestine
(Antonson & Vanderhoof, 1983). Similarly in mice, acute
alcohol treatment decreased zinc concentration in the ileum
(Lambert et al., 2004).
Using a mouse model of acute alcohol toxicity mimick-
ing human binge drinking (Carson & Pruett, 1996), re-
could preserve intestinal permeability that is increased by
alcohol consumption. Preserving intestinal permeability
may block the transfer of endotoxin to the circulation and
prevent alcoholic liver injury. Mice were treated with three
intragastric doses of zinc sulfate at 5 mg zinc element/kg
each dosing, with an interval of 12 h, prior to acute ethanol
challenge with a single oral dose of 6 g/kg ethanol. Alcohol
caused severe damage to the small intestine as determined
by morphological and permeability changes. Serum endo-
toxin levels were significantly higher in alcohol-treated
mice compared with control animals. Mice treated with eth-
anol and subsequently loaded with exogenous LPS showed
a further increase in serum endotoxin levels compared with
mice exposed to ethanol alone. These findings demonstrate
that ethanol increased the transfer of endogenous as well as
354V. Purohit et al. / Alcohol 42 (2008) 349e361
exogenous endotoxin from the intestine to the general cir-
culation. Increased serum endotoxin levels were associated
with liver injury as detected by an elevation in serum ala-
nine aminotransferase activity, fatty liver, and hepatic de-
generative necrotic foci along with a significant increase
in hepatic TNF-a levels (Lambert et al., 2003, 2004). Zinc
supplementation attenuated ethanol-induced increases in
serum endotoxin levels, serum alanine aminotransferase ac-
tivity, and hepatic TNF-a levels. These changes were asso-
ciated with prevention of ethanol-induced liver injury
(Lambert et al., 2003, 2004). These results suggest that zinc
brought these changes, at least in part, by preventing etha-
nol-induced transfer of endotoxin from intestine to circula-
tion which could be ascribed to preservation of intestinal
morphology and permeability. The mechanism by which
zinc preserves intestinal morphology and permeability is
a subject of investigation.
Alcohol and plasma endotoxin levels
Alcohol-induced increases in intestinal bacterial growth
and intestinal permeability to endotoxin are expected to re-
sult in elevated plasma endotoxin levels. An association be-
tween alcohol consumption and plasma endotoxin levels
has been investigated in humans as well as in animals.
Plasma endotoxin levels were significantly elevated in pa-
tients affected with different stages of ALDdfatty liver,
hepatitis, and cirrhosis when compared with healthy control
subjects (Fukui et al., 1991; Parlesak et al., 2000; Scha ¨fer
et al., 2002). Amount of alcohol appeared to make signifi-
cant impact on endotoxin levels and liver injury because in
these studies healthy control subjects consumed less than
20 g/day of alcohol compared to more than 60 g/d of alco-
hol by ALD patients. Furthermore, plasma endotoxin levels
were also significantly elevated in actively drinking alco-
holics without evidence of liver disease (Bode et al.,
1987; Nolan, 1989), suggesting that alcohol itself can in-
crease endotoxin levels presumably via increasing intestinal
permeability to endotoxin. Endotoxemia was reversible in
the majority of patients with alcoholic fatty liver and in
about 50% of patients with mild alcoholic hepatitis within
1 week following the cessation of alcohol intake (Fukui
et al., 1991), further suggesting a direct role of alcohol in
increasing plasma endotoxin levels. Liver disease itself
may increase plasma endotoxin levels since they were sig-
nificantly elevated in nonalcoholic cirrhotic patients (Bode
et al., 1987; Fukui et al., 1991). However, plasma endotoxin
levels were significantly higher in patients with alcoholic
cirrhosis than in patients with nonalcoholic cirrhosis (Bode
et al., 1987; Fukui et al., 1991), thus implicating alcohol in
increasing endotoxin levels.
The relationship between alcohol consumption and
plasma endotoxin levels has been shown in animals with al-
coholic liver injury. Serum endotoxin levels were elevated
in rats with alcoholic liver injury developed after ethanol
administration for 10 weeks by gavage (Keshavarzian
et al., 2001). Similarly, plasma endotoxin levels were sig-
nificantly elevated in rats with alcoholic liver injury devel-
oped in response to intragastric infusion of ethanol for 3e4
weeks (Adachi et al., 1995; Nanji et al., 1994). In mice,
plasma endotoxin levels were significantly elevated 1.5 h
after intragastric administration of 6-g/kg ethanol by ga-
vage, and this was associated with significant small intesti-
nal injury (Lambert et al., 2003). These mice developed
significant liver injury 6 h after ethanol administration, sug-
gesting that alcohol is the causal factor for endotoxemia.
Both human and animal studies suggest that alcohol medi-
ates the transfer of endotoxin from intestine to the liver that
results in the elevation of plasma enotoxin levels.
Role of endotoxin in alcoholic liver injury
Increasing evidence suggests that gut-derived endotoxin
plays a central role in the initiation and perhaps progression
of alcoholic liver injury. The evidence for the role of endo-
toxin in alcoholic liver injury was primarily obtained from
animal studies. In rats exposed to chronic ethanol in a liquid
diet, administration of endotoxin led to the progression of
liver injury from fatty liver to necroinflammatory changes
(Apte et al., 2005; Bhagwandeen et al., 1987; Pennington
et al., 1997). In an intragstric infusion rat model of alco-
holic liver injury, sterilization of intestine by antibiotics sig-
nificantly attenuated liver injury by reducing plasma levels
of endotoxin (Adachi et al., 1995), and in the same model,
similar results were obtained by simultaneous feeding of
probiotic lactobacillus GG bacteria (competitive inhibitor
of Gram negative bacteria) to the rats (Nanji et al., 1994).
Endotoxin initiates liver injury via activation of hepatic
Kupffer cells because inactivation of Kupffer cells with
gadolinium chloride significantly attenuated liver injury
caused by chronic ethanol exposure (Adachi et al., 1994).
Endotoxin after binding with LPS-binding protein activates
Kupffer cells through two types of receptors, cluster of dif-
ferentiation-14 (CD-14) and Toll-like receptors-4 (TLR-4).
CD-14 is a surface receptor without a cytoplasmic domain
and thus it lacks the ability to transduce LPS-induced cyto-
plasmic signal across a cell membrane. On the other hand,
TLR-4 is a transmembrane protein with a cytoplasmic do-
main, which is capable of transducing LPS-induced cyto-
plasmic signal across a cell membrane. The finding that
alcoholic liver injury is blocked in both CD-14 (Yin
et al., 2001) and TLR-4 deficient mice (Uesugi et al.,
2001) suggests that both of these receptors are required to
initiate liver injury caused by alcohol. Binding of LPS to
these receptors on Kupffer cells initiates a cascade of events
leading to free radical generation, nuclear factor kappaB
activation, and production of inflammatory mediators such
as cytokines (e.g., TNF-a), chemokines, and adhesion mol-
ecules (Nagata et al., 2007; Park et al., 2006; Thakur et al.,
2006; Thurman, 1998; Wheeler et al., 2001). These media-
tors eventually initiate necroinflammatory and fibrotic
changes in the liver.
355 V. Purohit et al. / Alcohol 42 (2008) 349e361
Researchers have discovered that in addition to activating
Kupffer cells, LPS may also activate hepatic stellate cells
liver. Culture-activated human HSCs expressed LPS recep-
tors CD-14 and TLR-4, and stimulation of these cells with
LPS induced nuclear factor kappaB activation and upregu-
lated gene expression of chemokinesdinterleukin (IL)-8
and monocyte chemoattractant protein-1dand adhesion
molecules (intercellular adhesion molecules and vascular
cellular adhesion molecules-1) (Paik et al., 2003). Activated
mouse HSCs expressed TLR-4 and CD-14 and responded to
LPS with an up regulation of extracellular signal-regulated
kinase phosphorylation and IL-6, transforming growth fac-
tor-b1, and monocyte chemoattractant protein-1 secretion
(Brun et al., 2005). In quiescent rat hepatic stellate cells,
LPS stimulated the synthesis of proinflammatory cytokines
TNF-a, IL-6, and IL-1 (Thirunavukkarasu et al., 2005).
These results suggest that LPS may directly activate HSCs,
thereby contributing to liver fibrosis. Endotoxin may also
sue/organ injuries such as pancreatitis (Wang et al., 2005b),
acute respiratory distress syndrome (Ryffel et al., 2005),
and brain injury (Duncan et al., 2006).
Peptidoglycan: intestinal permeability and liver toxicity
In addition to endotoxin, intestinal bacteria may release
many other TLR ligands that activate innate immune cells
such as Kupffer cells. Peptidoglycan and flagellin, which
bind to TLR-2 and TLR-5, respectively, have been impli-
cated in the pathogenesis of inflammation of the liver and
intestine. Peptidoglycan is present in all types of bacteria
residing in the intestinal tract. The Gram-positive bacteria
have a very thick peptidoglycan layer whereas the Gram-
negative bacteria have a relatively thin layer. Limited
information is available on the role of alcohol in intestinal
permeability to peptidoglycan. In a rat study, plasma pepti-
doglycan concentration of portal blood was significantly
increased 24 h after the acute administration of 10 ml/kg
of 20% ethanol (Tabata et al., 2002), suggesting a role of
alcohol in increasing intestinal permeability to peptidogly-
can and possible pathological consequences. In another
study, injection of peptidoglycan increased TNF-a mRNA
expression more in the livers of ethanol-fed mice than in
control mice. In addition, peptidoglycan induced liver in-
flammatory infiltrate in ethanol-fed mice but not in control
mice (Gustot et al., 2006). These studies suggest that alcohol
may not only increase intestinal permeability to peptidogly-
can, but also works synergistically with it to induce liver in-
jury. Experimental jejunal bacterial overgrowth in rats leads
to increased portal blood concentrations of peptidoglycan
that induces hepatic inflammation and steatosis (Lichtman
et al., 1992). Peptidoglycan activates TNF-a production
by Kupffer cells. Further studies are required to confirm
the role of alcohol in increasing intestinal permeability to
peptidoglycan and its medical consequences.
Alcohol, Intestinal Bacterial Growth, Intestinal Permeability to Endotoxin,
and Organ Injury: A Summary
Intestinal nitric oxide
Other organ injury
Portal vein endotoxin
Endotoxin in circulation
Fig. 1. Alcohol, intestinal bacterial growth, intestinal permeability to endotoxin, and organ injury: a summarydAlcohol exposure can promote the growth of
Gram-negative bacteria in the intestine, which may result in accumulation of endotoxin. In addition, alcohol metabolism by Gram-negative bacteria and in-
testinal epithelial cells can result in accumulation of acetaldehyde, which in turn can increase intestinal permeability to endotoxin. Alcohol-induced gener-
ation of nitric oxide may also contribute to increased permeability to endotoxin. Increased intestinal permeability to endotoxin leads to increased transfer of
endotoxin from the intestine to the portal vein which carries endotoxin to the liver where it binds to Kupffer cells and initiates a cascade of events leading to
tumor necrosis factor-a production and liver injury. Endotoxin that escapes to general circulation may induce injury to other organs. A part of tumor necrosis
factor-a produced in the liver may reach to intestine via bile duct or general circulation and further increase intestinal permeability to endotoxin.
356 V. Purohit et al. / Alcohol 42 (2008) 349e361
The mechanisms whereby alcohol exposure leads to in-
creased intestinal permeability to endotoxin and subsequent
injury to the liver and other organs have been presented in
Fig. 1. Alcohol exposure can promote the growth of Gram-
negative bacteria in the intestine, which may result in accu-
mulation of endotoxin. In addition, alcohol metabolism by
Gram-negative bacteria and intestinal epithelial cells can
result in accumulation of acetaldehyde, which in turn can
increase intestinal permeability to endotoxin. Alcohol-
induced generation of nitric oxide may also contribute to
increased permeability to endotoxin. Increased intestinal
permeability to endotoxin leads to increased transfer of en-
dotoxin from the intestine to the portal vein which carries
endotoxin to the liver where it binds to Kupffer cells and
initiates a cascade of events leading to TNF-a production
and liver injury. Endotoxin that escapes to general circula-
tion may induce injury to other organs. A part of
TNF-a produced in the liver may reach to intestine via bile
duct or general circulation and further increase intestinal
permeability to endotoxin.
Acetaldehyde may increase intestinal permeability to en-
dotoxin by a following mechanism comprised of multiple
steps (Fig. 2). Acetaldehyde has been shown to decrease
the activity of protein tyrosine phosphatase in the intestinal
epithelial paracellular space. This results in the increased
tyrosine phosphorylation of tight junction proteins (occlu-
din and ZO-1) and adherens junction proteins (E-cadherin
and b-catenin). Increased phosphorylation of these proteins
leads to their redistribution from intercellular junctions to
intracellular compartments and that probably results in
increased intestinal permeability to endotoxin.
Alcohol may also increase intestinal permeability by in-
creasing the production of nitric oxide, via upregulating
iNOS activity, and superoxide (Fig. 3). These radicals can
react with each other to form peroxynitrite, which in turn
can react with tubulin leading to damage to microtubule
cytoskeleton, disruption of barrier function, and increased
In intestinal inflammatory diseases, TNF-a may play an
important role in impairing intestinal barrier function by
upregulating theactivity of
increased phosphorylation of MLC (Fig. 4).
Decreasing plasma endotoxin levels may attenuate alco-
holic liver injury as well as alcohol-associated injury of
other organs. This can be accomplished by reducing the
number of Gram-negative bacteria in the intestine or pre-
serving intestinal permeability to endotoxin (Fig. 5). The
number of Gram-negative bacteria can be reduced by feed-
ing of probiotics such as Lactobacillus GG or Bifidobacte-
ria. This can result in decreased production of endotoxin as
well as acetaldehyde, which is expected to decrease
intestinal permeability to endotoxin. In addition, intestinal
permeability may be preserved by administering EGF, L-
glutamine, oats supplementation, or zinc (which is expected
to prevent the transfer of endotoxin to the general
Alcohol may also increase intestinal permeability to
peptidoglycan, which can initiate inflammatory response
in liver and other organs.
Acute alcohol exposure may potentiate the effect of burn
injury on intestinal bacterial growth and permeability.
? Investigating mechanisms whereby alcohol promotes
intestinal bacterial growth. Is it dose dependent? Is
bacterial growth associated with increased amount of
endotoxin production? What types of bacteria grow
in the presence of alcohol? Does alcohol promote bac-
terial growth directly or indirectly through suppression
of immune system?
? Developing effective and safe strategies to prevent
production of endotoxin in the intestine. This may in-
clude direct killing of Gram-negative bacteria or sup-
pressing their growth by probiotics or prebiotics.
? Developing strategies for accelerating acetaldehyde
metabolism in the intestine. This may include downre-
gulating the activity of alcohol dehydrogenase or upre-
gulating the activity of aldehyde dehydrogenase
through various mechanisms.
Role of Acetaldehyde in Increasing Intestinal
Permeability to Endotoxin: A Proposed Mechanism
Epithelial protein tyrosine
Tyrosine phosphorylation of tight
junction and adherens junction proteins
Redistribution of junctional proteins from
intercellular junctions into intracellular compartments
Disruption of barrier function
Intestinal permeablility to endotoxin
Fig. 2. Role of acetaldehyde in increasing intestinal permeability to endo-
toxin: a proposed mechanismdAcetaldehyde may increase intestinal per-
meability to endotoxin by decreasing the activity of protein tyrosine
phosphatase in the intestinal epithelial paracellular space. This results in
the increased tyrosine phosphorylation of tight junction proteins (occludin
and ZO-1) and adherens junction proteins (E-cadherin and b-catenin). In-
creased phosphorylation of these proteins leads to their redistribution from
intercellular junctions to intracellular compartments and that probably re-
sults in increased intestinal permeability to endotoxin.
357V. Purohit et al. / Alcohol 42 (2008) 349e361
? Further discern the underlying mechanisms by which
acetaldehyde makes intestinal epithelium more perme-
able to endotoxin. For example, is it an effect of acet-
aldehyde itself or an effect of acetaldehyde adducts
such as acetaldehyde-protein adducts or acetaldehy-
? Understand the relative and interactive role of acetal-
dehyde, nitric oxide, and TNF-a in impairing intesti-
nal barrier function.
? Developing safe and effective agents that will preserve
the integrity of intestine and thus prevent the transfer
of endotoxin to portal vein.
? Understanding the underlying mechanisms by which
zinc, oat supplementation, and L-glutamine preserve
intestinal permeability to endotoxin.
? Examining the ability of alcohol to increase intestinal
permeability to other toxic bacterial products such as
peptidoglycan and flagellin.
Studies reported in this manuscript were supported in
part by the National Institutes of Health (R21 AA015979,
R01 AA015731, R01 HL059225, R01 H063760, R01
AA013745, R01 AA012307, and R01 DK055532).
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Alcoholic liver disease
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