Scientific Abstract

Proposal No.  IBD-0050
Principal Investigator:  Robert Steven Esworthy, Ph.D.
Applicant Organization:  City of Hope National Medical Center (Duarte, California, U.S.A.)
Project Title:  Barrier breakdown in mice deficient in antioxidant defenses leads to ileal colitis
Period of Award:  February 1, 2003 - May 31, 2004

Pools of the major antioxidant glutathione (GSH) are adversely affected in IBD.  Supplementation with N-acetylcysteine (NAC) to replenish the reduced GSH pool is reported to have a therapeutic effect on rodents with experimental inflammatory bowel disease (IBD).  One function of GSH is to supply reducing equivalents for selenium-dependent glutathione peroxidases (GPX), major hydroperoxide metabolizing activities.  The association of GSH with IBD may fit with our observations that mice deficient in two GPX isoenzymes (GPX-1 and GPX-2; homozygous double knockout mice, GPX-DKO) found in the GI tract develop early and aggressive ilealcolitis.  These observations suggest that hydroperoxides could be a major factor in IBD pathology.

We propose to study GPX-DKO mice based on the premise that GSH depletion in IBD would impair hydroperoxide detoxification.  Significant depletion of GSH would occur in chronic IBD.  As such, our findings that depression of GPX activity induces acute inflammation would be relevant to understanding and treating IBD.

We postulate that oxidative stress causes reactivity, the term we will use for the collective histopathology of apoptosis, hyperproliferation, Paneth cell loss, and mucin depletion.  Since we do not see signs of reactivity in acclimated germ-free mice, we infer that the hydroperoxide production is induced upon exposure to gut microflora.  The link between oxidative stress and immune infiltration is the state of reactivity in the mucosal epithelium that results in penetration of luminal antigens into the submucosa.  We propose to test the premises that reactivity is a direct impact of oxidative stress and that microflora cause oxidative stress in GPX-DKO mice.  The reactivity causes barrier breakdown that leads to acute inflammation in the presence of intestinal microflora.  Relating specific oxidative stress to IBD through reactivity in the GPX-DKO mice constitutes novel findings.

The specific aims of this proposal are:

Aim 1.  To determine under what conditions GPX-DKO mouse ileum and colon have increased oxidative stress.  We will analyze the hydroperoxide levels in germ-free GPX-DKO mice with and without reactivity to confirm that hydroperoxides are generated endogenously and are associated with reactivity.  Using another approach, we will determine if hydroperoxides induce reactivity by using antioxidants to suppress reactivity in GPX-DKO mice, and using pro-oxidants to induce reactivity in Gpx2 gene knockout mice.
 
Aim 2.  To determine whether hydroperoxides and reactivity are associated with barrier breakdown in GPX-DKO mice.  We propose that hydroperoxides can cause barrier breakdown. We will first determine whether GPX-DKO ileal epithelium is more permeable than that from heterozygous control mice under conventional housing.  If so, we will compare permeability in reactive and non-reactive germ-free GPX-DKO mice.  We will determine if antioxidants can improve barrier function in the GPX-DKO mice and pro-oxidants will cause barrier breakdown in non-DKO mice.