Toxicology and Applied Pharmacology 204 (2005) 320 – 328

www.elsevier.com/locate/ytaap

Review

Intestinal glutathione: determinant of mucosal peroxide transport,

metabolism, and oxidative susceptibility
Tak Yee Aw*
Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center,
1501 Kings Highway, Shreveport, LA 71130-3932, USA

Received 8 October 2004; accepted 15 November 2004

Available online 23 January 2005

Abstract

The intestine is a primary site of nutrient absorption and a critical defense barrier against dietary-derived mutagens, carcinogens, and
oxidants. Accumulation of oxidants like peroxidized lipids in the gut lumen can contribute to impairment of mucosal metabolic pathways,
enterocyte dysfunction independent of cell injury, and development of gut pathologies, such as inflammation and cancer. Despite this
recognition, we know little of the pathways of intestinal transport, metabolism, and luminal disposition of dietary peroxides in vivo or of the
underlying mechanisms of lipid peroxide-induced genesis of intestinal disease processes. This chapter summarizes our current understanding
of the determinants of intestinal absorption and metabolism of peroxidized lipids. I will review experimental evidence from our laboratory
and others (Table 1) supporting the pivotal role that glutathione (GSH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH)
play in mucosal transport and metabolism of lipid hydroperoxides and how reductant availability can be compromised under chronic stress
such as hypoxia, and the influence of GSH on oxidative susceptibility, and redox contribution to genesis of gut disorders. The discussion is
pertinent to understanding dietary lipid peroxides and GSH redox balance in intestinal physiology and pathophysiology and the significance
of luminal GSH in preserving the integrity of the intestinal epithelium.
D 2004 Elsevier Inc. All rights reserved.

Keywords: Intestinal peroxide transport and disposition; Glutathione and intestinal peroxide elimination; Lymphatic peroxide transport; Intestinal apoptosis;
Mitochondrial apoptotic signaling; Oxidative stress and apoptosis; Intestinal redox and proliferation; GSH/GSSG redox and apoptosis; Glutathione and
intestinal redox balance

Contents

Glutathione and intestinal disposition of peroxidized lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321

Lipid peroxides and intestinal pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
GSH and intestinal lipid peroxide absorption, metabolism, and lymphatic transport . . . . . . . . . . . . . . . . . . . . . . 321
Intestinal handling of luminal lipid peroxides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
GSH in intestinal peroxide transport and elimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
NADPH and GSH redox cycle in intestinal hydroperoxide metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324
Intestinal GSH/GSSG redox balance, oxidative susceptibility, and gut pathology . . . . . . . . . . . . . . . . . . . . . . . . . 325
Cellular GSH redox governs intestinal cell transition and oxidative vulnerability . . . . . . . . . . . . . . . . . . . . . . . 325
GSH redox imbalance and development of intestinal disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326
Summary and perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327

* Fax: +1 318 475 4217.

E-mail address: taw@lsuhsc.edu.

0041-008X/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.taap.2004.11.016
 T.Y. Aw / Toxicology and Applied Pharmacology 204 (2005) 320–328
Glutathione and intestinal disposition of peroxidized
lipids
Lipid peroxides and intestinal pathology
Lipid peroxides are a major source of dietary oxidants
of mutagenic or carcinogenic potential that are of nutri-
tional and toxicological importance (Ames, 1983; Kinlen,
1983). By virtue of their ability to generate oxyradicals,
lipid peroxides are capable of initiating degenerative
processes and promote digestive system disorders, such
as inflammation and cancer (Grisham and Granger, 1998;
Parks et al., 1983). Dietary intake of highly unsaturated
fats is an important contributor to lipid peroxides in the
intestinal lumen, and an estimated daily intake of 1.4
mmol lipid peroxides per day could occur in humans
consuming an average of 84 g of fat daily (Wolff and
Nourooz-Zadeh, 1996). In animal studies, luminal peroxide
contents of 0.4 Amol have been detected after experimental
duodenal infusion of peroxidized lipids (Aw et al., 1992);
levels that have been shown to alter intestinal cell
responses in vitro (0.2–10 AM; Gotoh et al., 2002; Wang
et al., 2000). Early studies have demonstrated that the
toxicity of dietary polyunsaturated oils correlated directly
with their peroxide contents and that excessive consump-
tion of lipid peroxides was cytotoxic in vivo (Andrews et
al., 1960; Kaneda et al., 1955; Kimura et al., 1984).
Although luminal toxic concentrations have not been
demonstrated in humans, the intake of polysaturated fats
can elevate peroxide contents to levels that impact intestinal
integrity by induction of tissue oxidative stress and redox
imbalance. In rats, administration of hydroperoxy and
hydroxy fatty acids stimulated DNA synthesis and induced
ornithine decarboxylase activity consistent with enhanced
cellular proliferation (Bull et al., 1984). Moreover, mucosal
hypertrophy of the colon developed in rats given peroxi-
dized ethyl linoleate (Hara et al., 1996). These results
underscore the deleterious impact that lipid peroxides have
on normal intestinal cell turnover processes and highlight
their tumorigenic potential. Indeed, the increased incidence
of colon cancer with high intake of dietary fats (Carroll and
Khor, 1975; Correa et al., 1982; Reddy, 1983) is consistent
with the paradigm that dietary fat is a risk factor for colon
cancer development. Despite this recognition, the disposi-
tion of luminal peroxidized lipids by the small intestine
remains poorly understood.
GSH and intestinal lipid peroxide absorption, metabolism,
and lymphatic transport
Intestinal handling of luminal lipid peroxides
The overall processing of luminal lipid peroxides by the
small intestine can be considered to occur in three phases. The
respective absorptive, metabolic, and transport phases involve
the uptake of peroxides from the lumen, the intracellular
catabolism of the absorbed peroxides, and finally the release of
321
peroxides or their metabolic products into lymph in association
with lipoproteins. Early studies in the 1960s and 1970s have
investigated the absorption and lymphatic transport of fatty
acid hydroperoxides (Bergen and Draper, 1970; Bunyan et al.,
1968; Glavind and Sylven, 1970; Nagatsugawa and Kaneda,
1983), but results from these studies are equivocal regarding
whether luminal peroxidized lipids are, in fact, absorbed and
processed by the small intestine. The difficulty is due, in part,
to different experimental models that have been employed to
study intestinal hydroperoxide disposition under in vitro and in
vivo conditions. An experimental model of some utility for
addressing the issue of intestinal handling of lipid peroxides is
that of the everted intestinal sac. First described in 1954 by
Wilson and Wiseman, the intestinal everted sac provided a
convenient in vitro method for quantifying transport of
nutrients when precautions were taken to maintain mucosal
blood flow and oxygen delivery through minimizing villus
vasoconstriction (Pritchard and Porteous, 1977; Wilson and
Wiseman, 1954). The subsequent establishment of the lymph
fistula rat model (see below) offers better quantitative
measures of the absorption and metabolism of peroxidized
lipids without the issue of tissue oxygenation associated with
the everted sac model.
Evidence for transport of dietary lipid peroxides into the
circulation is equally controversial. A few studies docu-
mented the presence of lipid peroxides in the lymph
(Nagatsugawa and Kaneda, 1983), isolated chylomicrons
(Staprans et al., 1994), blood plasma (Ursini et al., 1998),
and tissues (Bunyan et al., 1968; Kaneda et al., 1955) after
administration of lipid peroxide containing diet. Others were
unable to document the presence of lipid peroxides in lymph
(Andrews et al., 1960; Bergen and Draper, 1970; Glavind
and Sylven, 1970; Glavind et al., 1971); rather, evidence
supports the absorption of degradation products of lipid
peroxides, such as hydroxyl fatty acids, conjugated dienes,
and aldehydes (Andrews et al., 1960; Bergen and Draper,
1970; Glavind and Sylven, 1970; Glavind et al., 1971;
Kanazawa and Ashida, 1998; Kanazawa et al., 1985; Orada
et al., 1987). The reasons for the different conflicting
observations are unclear; notably, the factors that affect lipid
peroxide absorption, metabolism, and lymphatic transport
were not evaluated in these studies. The presence of anti-
oxidant defenses in mesenteric lymph offers one possible
mechanism that accounts for the reduced presence of
lymphatic lipid peroxides observed after intragastric admin-
istration of vegetable oil containing triglyceride hydro-
peroxides (Mohr et al., 1999). Additionally, the appearance
of hydroxyl compounds that are reduction products of lipid
peroxides in mesenteric lymph points to a role for mucosal
glutathione (GSH) and GSH peroxidases in enterocyte
metabolism of luminal lipid peroxides. Thus, differences
in mucosal GSH status under the various dietary and
experimental conditions (see below) could conceivably
explain the lack of general agreement in the literature on
how luminal peroxides are handled by the small intestine
(Table 1).
322 T.Y. Aw / Toxicology and Applied Pharmacology 204 (2005) 320–328

Table 1 GSH intake and biliary GSH output are the major sources of
Summary of literature evidence for glutathione as a determinant of mucosal luminal GSH. The human diet varies considerably in GSH
peroxide transport, metabolism, and oxidative susceptibility
levels (Jones et al., 1992; Wierzbicka et al., 1989), and ~50%
Studies Literature references of total hepatic GSH efflux goes into bile (Aw, 1994).
Intestinal peroxide handling Furthermore, oral GSH administration was shown to prevent
Peroxide absorption/lymphatic Bergen and Draper (1970), Bunyan et al.
severe degeneration of jejunal and colonic epithelial cells
transport (1968), Glavind and Sylven (1970),
Glavind et al. (1971), Kanazawa and induced by chronic GSH deficiency (Martensson et al.,
Ashida (1998), Mohr et al. (1999), 1990), consistent with its physiological role in preservation of
Nagatsugawa and Kaneda (1983), Orada the integrity of the intestinal epithelium.
et al. (1987), Staprans et al. (1994), Evidence to date implicates GSH as a key determinant in
Ursini et al. (1998)
the elimination of peroxides by the intestine (Aw and
GSH in peroxide absorption Aw (1994), Aw and Williams (1992),
and lymphatic transport Aw et al. (1992), Kowalski et al. (1990) Williams, 1992; Aw et al., 1992; Kowalski et al., 1990).
NADPH and GSH redox Using our conscious lymph fistula rat model, we have
cycle in mucosal provided novel insights into the driving force for lipid
peroxide metabolism peroxide absorption and removal by the small intestine (Aw et
Control conditions Aw and Rhoads (1994), Tribble and
al., 1992). Conceptually, this model predicts that at a given
Jones (1990), Williams and Aw (1994),
Wingler et al. (2000) peroxide concentration, the amount of peroxide (designated
Pathophysiological stress Chu et al. (1993), Iwakiri et al. (1995), as ROOH in Fig. 1) recovered from the intestinal lumen and
LeGrand and Aw (1996, 1997, 1998), lymph is governed by the mucosal GSH status. During GSH
Reddy and Tappel (1974), Vilas et al. sufficiency, increased intracellular peroxide metabolism
(1976)
drives mucosal ROOH uptake that results in decreased
Intestinal GSH/GSSG redox and oxidative susceptibility luminal ROOH retention and attenuated ROOH output into
Redox and cell transitions: intestinal lymph (Fig. 1A). This mechanism of GSH-depend-
Cellular models ent bmetabolic trappingQ highly favors peroxide catabolism
Proliferation Gotoh et al. (2002) and offers an efficient mode for rapid peroxide disposal in the
Growth arrest/differentiation Nkabyo et al. (2002), Noda et al. (2001)
small intestine. Conversely, during GSH deficiency,
Apoptosis Cai et al. (2004), Wang et al. (2000)
Susceptibility in nonintestinal Ekshyyan and Aw (2004), Pias and Aw decreased intracellular peroxide catabolism impairs mucosal
cells (2002a, 2002b), Pias et al. (2003) ROOH absorption, increases luminal ROOH retention, and
Redox and intestinal turnover: Iwakiri et al. (2001), Tsunada et al. promotes lymphatic ROOH transport (Fig. 1A). Thus, the
animal models (2003a, 2003b) capacity for and the efficiency of peroxide catabolism within
Redox and intestinal disorders Keshavarzian et al. (1992),
enterocytes largely determine peroxide elimination from the
Simmons et al. (1992),
Tsunada et al. (2003a, 2003b, 2000c) intestinal lumen. Experimentally, the lymph fistula rat model
allows for the simultaneous quantification of peroxide
recovery in intestinal lumen and lymph, and thereby provides
GSH in intestinal peroxide transport and elimination insights into the intestine’s capacity for peroxide metabolism
in vivo (Aw et al., 1992). As illustrated in the schematic
Glutathione (GSH) is a naturally occurring cellular profiles in Fig. 1B based on experimentally generated results,
reductant and antioxidant (Kaplowitz et al., 1985; Kauffman but without showing actual quantitative data, we found that
et al., 1977) that functions to detoxify reactive oxygen increased intestinal peroxide absorption as well as reduced
metabolites of endogenous or exogenous origins. GSH is luminal and lymph recoveries of infused lipid peroxides
present in high concentrations in tissues (Aw et al., 1992; correlate with higher mucosal GSH concentrations (Aw and
Kaplowitz et al., 1985; Shan et al., 1990), and normal tissue Williams, 1992; Aw et al., 1992; LeGrand and Aw, 2001),
GSH homeostasis is maintained by de novo synthesis from suggestive of an overall decrease in peroxide absorption and
cysteine, from regeneration of glutathione disulfide, GSSG, metabolism in association with reduced GSH levels. These
and from GSH uptake of exogenous sources via Na+- results underscore the quantitative importance of mucosal
dependent transport systems. Cellular GSH transport has GSH in the removal of luminal lipid peroxides.
been linked to cytoprotection against peroxide-induced Exogenous GSH supplements either from the diet or bile
damage in a variety of cell types, including enterocytes increases mucosal GSH concentrations and promotes metab-
(Lash et al., 1986), type II alveolar cells (Hagen et al., 1986), olism of lipid peroxides, thereby increasing luminal peroxide
renal proximal tubule cells (Hagen et al., 1988), and uptake into enterocytes (Aw and Williams, 1992; Kowalski et
endothelial cells (Andreoli et al., 1986). Moreover, GSH al., 1990). That biliary GSH is a major contributor to the
has been shown to be transported intact into the small luminal GSH pool (Aw, 1994) is evidenced by studies
intestine in vivo to supplement the intracellular GSH pool showing that diversion of biliary GSH significantly decreased
(Aw and Williams, 1992; Hagen and Jones, 1987; Hagen et intestinal peroxide metabolism that results in elevated
al., 1990; Lash et al., 1986), suggesting that luminal GSH can peroxide recoveries in lumen and lymph (Aw, 1994; LeGrand
contribute to maintaining mucosal thiol balance. Dietary and Aw, 2001). Using a similar schematic profile as that
 T.Y. Aw / Toxicology and Applied Pharmacology 204 (2005) 320–328 323

Fig. 1. Panel A represents a working model for absorption and lymphatic transport of luminal lipid peroxides in intestinal cells with GSH-sufficient or GSH-
deficient status. The abbreviations are GSH, GSSG: reduced glutathione and glutathione disulfide; S, D: GSH-sufficient and -deficient status, respectively;
ROOH, ROH: lipid peroxide and hydroxide, respectively; [Lo], [Hi]: low and high peroxide concentrations, respectively. Panel B represents profiles of
intestinal peroxide absorption, luminal peroxide accumulation, and lymphatic peroxide transport as a function of mucosal GSH concentrations. The
enhancement of intracellular lipid peroxide metabolism will accelerate peroxide uptake from the gut lumen resulting in decrease luminal accumulation and
reduce peroxide output into lymph secondary to cellular detoxication. Redrawn from LeGrand and Aw (2001).

Fig. 2. (A) Exogenous GSH enhances intestinal elimination of lipid peroxides. Supplementation of luminal GSH increases mucosal GSH concentration that
enhances luminal peroxide absorption and peroxide metabolism and reduces lymphatic peroxide transport. Dotted arrow 1 shows the direction of change in
intestinal peroxide uptake (upper panel) and lymphatic transport (lower panel) with GSH supplementation. Blockade of intestinal uptake of GSH from the gut
lumen decreases mucosal GSH concentration that impairs luminal peroxide update and peroxide metabolism and enhances lymphatic peroxide output. Dotted
arrow 2 shows the direction of change in peroxide absorption (upper panel) and lymphatic transport (lower panel) when luminal GSH uptake is prevented. The
profiles are based on experimentally generated data from Aw and Williams (1992). Redrawn from LeGrand and Aw, 2001. (B) Diversion of biliary GSH
decreases intestinal elimination of lipid peroxides. Diversion of biliary GSH in bile fistula animals decreases mucosal GSH that compromises intestinal
peroxide uptake and peroxide metabolism, and enhances lymphatic transport. The dashed line (1: bile fistula) shows the profiles for peroxide absorption (upper
panel) and lymphatic transport (lower panel) in bile fistula animals. Exogenous GSH supply in bile fistula animals restores luminal peroxide elimination and
decreases peroxide output into lymph. The solid line 2 represents the profiles for peroxide absorption (upper panel) and lymphatic transport (lower panel) in
GSH-supplemented bile fistula animals, and the dotted arrow 3 shows the direction of change in these parameters following GSH supplementation. The profiles
of peroxide absorption and lymphatic transport are based on experimentally generated data from Aw (1994). Redrawn from LeGrand and Aw (2001).
324 T.Y. Aw / Toxicology and Applied Pharmacology 204 (2005) 320–328
shown in Fig. 1B that is based on experimentally generated
results (Aw and Williams, 1992), the effect of exogenous
GSH supplementation on intestinal absorption, intramucosal
metabolism, and lymphatic output of lipid peroxides can be
summarized in Fig. 2A. The results show that elevating
luminal GSH leads to an increased mucosal GSH that
promotes luminal peroxide uptake, enhances cellular per-
oxide metabolism, and decreases lymphatic peroxide trans-
port (dotted Arrow 1). Conversely, blockade of luminal GSH
uptake or utilization leads to decreased mucosal GSH that
attenuates luminal peroxide uptake, compromises cellular
peroxide metabolism, and promotes lymphatic output (dotted
arrow 2). The result on the effect of bilary GSH diversion on
intestinal handling of lipid peroxides is summarized in the
schematic model illustrated in Fig. 2B. Again, for simplicity,
the pattern of changes as based on experimental results (Aw,
1994) is shown without presentation of the actual quantitative
data. The data show that biliary diversion of GSH via a bile
fistula resulted in decreased mucosal GSH (dashed line,
2:bile fistula) as compared to controls. The decreased
mucosal GSH in the combined bile fisula and lymph fistula
rats leads to reduced luminal peroxide removal, compromised
cellular peroxide metabolism, and elevated lymphatic output.
Notably, supplementation of these animals with exogenous
GSH restores mucosal GSH content (solid line, 2:+GSH) that
promoted luminal peroxide uptake, increased cellular per-
oxide detoxication, and reduced peroxide output into lymph
(dotted arrow 3). Collectively, these studies using the lymph
fistula rat model without or with bile fistula provide
reasonable quantitative measures of the absorption, metabo-
lism, and lymphatic transport of peroxidized lipids in the
small intestine under various conditions of mucosal GSH
sufficiency or GSH deficiency.
While current evidence support mucosal GSH and intra-
cellular metabolism as the major driving force governing
intestinal lipid peroxide uptake, the bioavailability of
peroxidized fatty acids could be influenced by their physical
chemical properties given that lipid uptake from a micellar
phase is a generally accepted paradigm for intestinal transport
of free fatty acids into enterocytes (Johnston and Borgstrom,
1964). The formation of micelles has been shown to be a
mechanism for overcoming the resistance of the unstirred
water barrier to facilitate rapid transfer of fatty acids to the
intestinal cell membrane. At present, the extent to which
oxidative modifications of lipids influence their luminal
solubility, their degree of penetration of plasma membrane,
and their ability to participate in bile acid mixed micelles are
unknown nor is it known whether micellar formation is
necessary for and critical to mucosal uptake of peroxidized
lipids. Future investigations into the effects of carbon chain
lengths, degree of unsaturation, and extent of oxidation of
polyunsaturated fatty acids on their absorption characteristics
should provide additional insights into how oxidative
modifications of fatty acids influence the mucosal handling
of varied luminal lipids of different fatty acid composition
and degree of peroxidation.
NADPH and GSH redox cycle in intestinal hydroperoxide
metabolism
The GSH redox cycle is a key intracellular detoxication
mechanism for peroxide elimination. Indeed, studies by
Wingler et al. (2000) demonstrated that intestinal GSH
peroxidase plays a key role in attenuating transport of lipid
peroxides and thereby prevent against oxidative damage in a
CaCo-2 cell culture model. GSH peroxidase and GSSG
reductase function as an enzyme pair in the reduction of
peroxides with concomitant oxidation of GSH and the
regeneration of GSH with reducing equivalents donated from
nicotinamide adenine dinucleotide phosphate (NADPH). The
supply of NADPH is functionally coupled to the pentose
phosphate pathway (Eggleston and Krebs, 1974) and is
regulated by glucose availability and glucose 6-phosphate
dehydrogenase activity (Bousignone and Flora, 1972). Given
that enterocyte GSH synthetic rate is low (Williams and Aw,
1994) as compared to hepatocytes (Shan et al., 1990), GSSG
reduction is quantitatively an important mechanism for
intestinal GSH maintenance under high rates of lipid peroxide
reduction (Aw and Rhoads, 1994). Glucose stimulation of
enterocyte NADPH supply (Aw and Rhoads, 1994) suggests
that GSH redox cycle activity is dependent on exogenous
glucose availability for NADPH production. The contention
that constant glucose supply is critical in maintaining
NADPH generation and GSH redox cycle function in vivo
is borne out by studies in the lymph fistula rat wherein
removal of glucose from the lipid peroxide infusate in GSH-
sufficient animals results in elevated recoveries of luminal
and lymph peroxide contents, reminiscent of the GSH-
deficient state (Aw and Williams, 1992; Aw et al., 1992).
This responsiveness of the gut to glucose suggests that
nutrient availability is an important contributing factor in the
intestinal catabolism of luminal peroxides and raises the
interesting possibility of regulation of mucosal peroxide
disposition by glucose of luminal or plasma origin. Moreover,
because of the difference in quantitative NADPH supply in
the presence of glucose between the intestine and liver (0.5–
1.0 nmol min 1 per 106 cells and 5–8 nmol min 1 per 106
cells, respectively; Aw and Rhoads, 1994; Tribble and Jones,
1990), a greater sensitivity of the small intestine to peroxide-
induced injury is expected.
Interestingly, studies from our laboratory and others show
that intestinal GSH peroxidase and GSSG reductase activities
are minimally modified under conditions of peroxide
challenge (Reddy and Tappel, 1974; Vilas et al., 1976),
chronic hypoxia (LeGrand and Aw, 1996), and diabetes
(Iwakiri et al., 1995), suggesting that the redox enzymes per
se are generally not rate limiting in overall lipid peroxide
catabolism and removal in the intestine. Notwithstanding, a
role for the specific intestinal isoform of GSH peroxidase
(GSHPX-GI, Chu et al., 1993) has not been vigorously
evaluated, but given its catalytic rate at one-tenth that of the
classical GSH peroxidase, GSHPX-GI is unlikely to signifi-
cantly influence peroxide elimination. Rather, literature
 T.Y. Aw / Toxicology and Applied Pharmacology 204 (2005) 320–328
evidence suggests that it is the efficiency of lipid peroxide
absorption from the lumen (Aw et al., 1992) and the
availability of cellular reductants (GSH and NADPH) (Aw
and Rhoads, 1994; Aw and Williams, 1992; Kowalski et al.,
1990) that largely dictate the kinetics and extent of intra-
cellular peroxide detoxication by the intestine.
It is apparent that factors that alter intestinal GSH
concentrations, the supply of NADPH, or the function of
the GSH redox cycle are expected to impact metabolic
processing of dietary lipid peroxides. We have demonstrated
that chronic oxygen deficiency has a profound effect on
intestinal handling of luminal peroxides due in part to a
generalized hypoxia-induced decrease in the GSH-to-GSSG
ratio in the small intestine (LeGrand and Aw, 1996, 1997). In
this state of tissue oxidative stress, GSH-dependent metab-
olism of lipid peroxides is compromised, resulting in
decreased luminal peroxide removal and elevated abluminal
peroxide recovery (LeGrand and Aw, 1998). Notably, the
NADPH production rate in hypoxic enterocytes is 50% lower
than that in normoxic cells (0.3 versus 0.7 nmol min 1 per
106 cells), indicating reduced glucose flux through the
pentose phosphate shunt. This decreased ability of the
hypoxic intestine to maintain GSH redox homeostasis and
redox cycle activity is likely to favor tissue oxidative
vulnerability and development of gut disorders.
Intestinal GSH/GSSG redox balance, oxidative
susceptibility, and gut pathology
Cellular GSH redox governs intestinal cell transition and
oxidative vulnerability
One important impact of an altered cellular GSH/GSSG
status is the induction of cell transitions (Fig. 3). An arrested
Fig. 3. Cellular responses to oxidation–reduction (redox) status. Curves I,
II, and III represent the responses of terminally differentiated, mitotically
competent, and transformed cell types, respectively. Redrawn from Aw
(2001).
325
and quiescent state typifies fully differentiated tissues like
liver, kidney, brain, and intestine. Shifting cellular control
checkpoints toward either GSH/GSSG reduction or oxida-
tion can elicit phase transition of a cell from a quiescent
state to that of a proliferative, growth arrested, apoptotic, or
necrotic state (Aw, 1999; Flores and McCord, 1997).
Depending on cell types, three cellular responses are
possible (Fig. 3). Terminally quiescent or differentiated
cells such as hepatocytes or neurons generally exhibit a
biological constraint to proliferate (Curve I) and increased
oxidative stress induces cell apoptosis. Mitotic-competent
enterocytes can maintain a genetic program to proliferate in
response to mitogenic stimuli (Curve II), wherein cells
overcome the G0/G1 regulatory checkpoint to enter the S
phase. Transformed cells possessing altered regulatory cell
cycle barriers respond to oxidative challenge with greater
proliferation or apoptosis (Curve III). Thus, depending on
the degree and duration of the oxidant challenges or redox
shifts, distinct cell transitions or oxidative susceptibility will
be elicited by different cell types.
The fundamental concept that changes in GSH/GSSG
that modulate intestinal cell proliferation, growth arrest,
and apoptosis comes from studies with cultured intestinal
cells. We found that treatment of CaCo-2 cells with low
concentrations of lipid peroxides (1–5 AM) promotes pro-
liferative activity that corresponds directly to the induction
of mild cellular GSH/GSSG imbalance (Gotoh et al.,
2002). As the peroxide induces a progressive oxidized
shift in the GSH/GSSG redox state, cells progress from
proliferation to growth arrest (Gotoh et al., 2002; Noda
et al., 2001). When intracellular GSH is compromised, the
redox threshold governing the transition between cell
proliferation and growth arrest is lowered (Noda et al.,
2001) such that proliferating cell populations with low
baseline cellular GSH are selectively vulnerable to oxidiz-
ing events that result in growth arrest or apoptosis. An
interesting aspect is that the cellular redox potential per se
varies with growth status in that the proliferative phenotype
is associated with a more reduced potential than the
differentiated phenotype (Aw, 2003; Nkabyo et al., 2002).
Elicitation of cell apoptosis has been linked to an
intracellular oxidized redox state; notably apoptotic death
is preceded by an early loss of cellular GSH/GSSG redox
balance that was the primary inciting event of the apoptotic
cascade (Wang et al., 2000). Interestingly, we found that in
a nonintestinal cell line, the PC-12 cells, the induction of
GSH/GSSG redox imbalance independently of ROS for-
mation is sufficient to initiate apoptotic signaling (Pias and
Aw, 2002a, 2002b). Moreover, the relative cellular vulner-
ability to apoptosis during peroxide challenge is directly
linked to the cell’s ability to maintain intracellular redox
balance through enhanced NADPH production (Pias and
Aw, 2002a) and the ability to attenuate mitochondrial ROS
production (Pias et al., 2003). While an early loss of
cellular redox balance alone is sufficient to induce cell
apoptosis in intestinal cells (Wang et al., 2000), it remains
326 T.Y. Aw / Toxicology and Applied Pharmacology 204 (2005) 320–328
to be tested as to whether apoptosis can result from a loss
of cellular redox homeostasis per se without an accom-
panying ROS generation.
A recent emerging paradigm from our studies in PC-12
cells is that cell transition states (i.e., proliferative versus
differentiated) can significantly influence their survival
outcome. At a given peroxide load, the transition from a
proliferative to a quiescent or differentiated phenotype
confers protection against oxidative challenge that is largely
associated with significant increases in total cellular GSH,
most notably in the mitochondrial GSH pool (Ekshyyan and
Aw, 2004). Studies are underway to address the issue of
oxidative susceptibility of intestinal cells as they transition
from the proliferative to the differentiated phenotype. Cai
et al. (2004) recently demonstrated that sodium butyrate-
induced terminal differentiation of HT 29 colon carcinoma
cells is associated with apoptotic death that is tightly linked
to caspase activation, suggesting an enhanced vulnerability
with cell differentiation, but a role for redox status in this
process has not been established. Taken together, it is
evident that the cellular redox state serves as a physiological
context for optimization of thiol-disulfide control of signal-
ing pathways and gene activation in cell proliferation and
apoptosis.
Studies in animal models support a paradigm of redox
modulation of oxidative susceptibility of the intestinal
epithelium in vivo. The intestinal mucosa is a labile organ
system whose epithelium turns over every 4–5 days
(Creamer et al., 1961; Miller et al., 1977; Tutton, 1973)
through a regulated balance of enterocyte proliferation and
apoptosis (Iwakiri et al., 2001). We found that rats placed on
chronic lipid peroxide diets exhibited suppressed mucosal
proliferative and apoptotic activity in accordance with
oxidized tissue GSH/GSSG (Tsunada et al., 2003a,
2003b). This state of cytostasis created by chronic peroxide
challenge was ameliorated by supplemental GSH that
restored tissue GSH/GSSG redox balance (Tsunada et al.,
2003a). Additionally, small intestinal cytostasis was
reversed by administration of epidermal growth factor
(Tsunada et al., 2003b). Significantly, in conjunction with
an increase in colonic GSH levels, EGF administration
induced a hyperproliferative response in the colon that
normally exhibits a slow cell turnover (Tsunada et al.,
2003b). Taken together, these results demonstrate the
centrality of cellular GSH/GSSG redox in modulating cell
transitions and their oxidative susceptibility. Thus, physio-
logical or pathological factors that induce peroxide gen-
eration, decrease luminal GSH transport, or promote
intestinal GSH oxidation are expected to impact intestinal
turnover characteristics and organ survival.
GSH redox imbalance and development of intestinal
disorders
One deleterious consequence of an altered cellular redox
status in the intestine is the potential for genesis of gut
pathologies. Chronic gut pathological states like inflamma-
tory bowel diseases are associated with chronic inflamma-
tion of the intestine (Fiocchi, 1998; Grisham and Granger,
2000; Powrie, 1995). Damage to the intestinal epithelium
resulting from an acute inflammatory response is typically
viewed as an event secondary to the primary systematic
inflammatory cascade of neutrophil adhering to vascular
endothelial cells, disruption of the endothelial barrier, and
enhanced inflammatory cell infiltration into the intestinal
interstitium. It has been long appreciated that oxidants
mediate inflammatory reactions, and pivotal to such
inflammatory processes, regardless of stimuli, is the
induction of cellular oxidative stress. Previous studies have
demonstrated a link between reactive oxygen species and
the inflammatory response in vivo in inflammatory bowel
disease (Keshavarzian et al., 1992; Simmons et al., 1992),
suggesting a role for GSH/GSSG redox in disease etiology.
Indeed, our recent studies demonstrated that induction
of intestinal GSH/GSSG imbalance contributes to the
development of chronic inflammation of the gut in SCID
mice reconstituted with CD4+CD45RBhigh T-lymphocytes.
It is notable that loss of GSH/GSSG redox balance
temporally preceded tissue hyperplasia, mucosal inflamma-
tion, and symptoms of clinical colitis, indicating that redox
imbalance is a contributor rather than a consequence of
disease progression. Restored tissue redox homeostasis and
attenuated colitis manifestation were associated with admin-
istration of the thiol antioxidant N-acetylcysteine. Recently,
we demonstrated a linkage between the oxidation of
mucosal GSH and intestinal inflammation and diseases
activity in human ulcerative colitis (Tsunada et al., 2003c).
Active disease state was resolved with normalization of
mucosal redox balance by therapeutic intervention with
salazosulphapyridine (Tsunada et al., 2003c), an effective
agent for ulcerative colitis and a free radical scavenger
(Ahnfelt-Ronne et al., 1990).
Summary and perspective
Our current understanding of the physiological deter-
minants of intestinal disposition of luminal lipid peroxides
is limited, despite the recognition that oxidants like
peroxidized lipids can contribute to the pathogenesis of
degenerative processes in the gut. Studies from our
laboratory in the conscious lymph fistula rat underscore
a pivotal role for intestinal GSH in quantitative lipid
peroxide transport and metabolism in vivo wherein luminal
peroxide elimination is found to be rate limited by the
availability of tissue reductants (viz., GSH and NADPH)
rather than the functions of the redox enzymes per se.
Unresolved oxidative stress coupled to the loss of mucosal
redox homeostasis can induce intestinal cell transition to
proliferative or apoptotic phenotypes. Given that dietary
intake of highly unsaturated fats results in luminal
peroxide accumulation and altered tissue redox state, these
 T.Y. Aw / Toxicology and Applied Pharmacology 204 (2005) 320–328
differential cellular responses to GSH/GSSG redox imbal-
ance could contribute to a loss of intestinal integrity and
promote pathogenesis of chronic gut disorders like
inflammatory bowel disease. Evidence that the intestinal
epithelium is capable of transporting luminal GSH to
augment intracellular peroxide catabolism and the notion
that the etiology of chronic gut pathologies may be directly
linked to an early loss of mucosal redox balance suggest
that dietary strategies for intestinal redox maintenance
would be a viable consideration in chronic gut disease
management.
Acknowledgments
Research in the author’s laboratory was supported by
grants from the National Institutes of Health, DK 44510 and
DK 43785.
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