Research Article Mutation Accumulation in the Intestine and Colon of Mice Deficient in Two Intracellular Glutathione Peroxidases Dong-Hyun Lee,1 R. Steven Esworthy,2 Christy Chu,2 Gerd P. Pfeifer,1 and Fong-Fong Chu2 1Department of Biology and 2Department of Radiation Biology, City of Hope Cancer Center, Duarte, California Abstract Inflammatory diseases are often associated with significant oxidative stress that can damage cells and tissues. A link between Mice deficient in two glutathione peroxidases (GPX), Gpx1 and inflammation and cancer is well established (4, 6, 7). An increased Gpx2, [Gpx1/2-double knockout (DKO) mice] are prone to incidence of cancer is seen in patients with chronic gastritis, ileocolitis on a mixed C57BL/6 and 129S1/SvJ (B6.129) genetic chronic pancreatitis, and inflammatory bowel disease (IBD; refs. 4, background. We reported previously that f25% of B6.129 7–9). IBD includes Crohn’s disease (CD) and ulcerative colitis (UC); Gpx1/2-DKO mice develop ileocolonic tumors by 6 to 9 each affects approximately one in a thousand people in Western months of age, when their non-DKO littermates [having at countries. Although CD and UC may have different genetic origins least one wild-type (WT) Gpx1 or Gpx2 allele] rarely have and affect different areas of the gastrointestinal tract, both inflammation and none have tumors. Because genetic back- diseases result in an increased risk of cancer compared with the ground affects tumor susceptibility, we have generated a B6 age-matched general population (10, 11). Clearly, chronic inflam- Gpx1/2-DKO colony and discovered that these mice have mation contributes to carcinogenesis in the gastrointestinal tract; fewer inflammatory cells, milder ileocolitis, and low mortality, however, the mechanisms are not understood. and only 2.5% of B6 mice developed tumors. The mutant Inflammation is caused by the recruitment of inflammatory frequency of a cII reporter gene was about 2- to 3-fold higher cells, including neutrophils, monocytes, and eosinophils (7), which in 28-day-old Gpx1/2-DKO and 4-fold higher in 8-month-old release ROS. Chronic inflammation can be a tumor promoter, Gpx1/2-DKO ileal mucosa than in controls in both genetic which may affect signal transduction mechanisms, influence cell backgrounds. In contrast, mutant frequencies in the unaffected proliferation, and modulate apoptosis. However, it is also possible B6 liver were not significantly different between WT and Gpx1/ that inflammation acts as a tumor initiator by inducing DNA 2-DKO mice. The mutant frequency of 8-month-old B6.129 À damage and gene mutations in the affected tissue. Such a proposal, Gpx1/2-DKO ileum was 38.94 F 15.5 5, which was not signifi- À in which inflammation, DNA damage, increased mutations, and cantly higher than the age-matched B6 ileum, 25.54 F 10.33 5. tumorigenesis are directly linked in consecutive steps, has been The mutation spectra analysis has shown that B6 Gpx1/2-DKO put forward (12). ileum had a 3-fold increase in small nucleotide deletions at Selenium-dependent glutathione peroxidases (GPX) represent mononucleotide repeats over control B6, which are a signa- a major selenoprotein-containing gene family in mammals. The ture mutation associated with oxidative stress. Unexpectedly, GPXs include four selenium-dependent hydroperoxide-reducing B6 Gpx1/2-DKO mice had fewer C to T transitions at CpG isozymes: (a) the ubiquitous GPX1, (b) the epithelium-specific GPX dinucleotides than the WT B6 (18.0% versus 40.1%; P < 0.001). (GPX2), (c) the secreted plasma GPX (GPX-P), and (d)the Our results suggest that inflammation drives gene mutations, monomeric phospholipid hydroperoxide GPX (PHGPX), which are which leads to neoplastic transformation of intestinal epithe- encoded by the Gpx1, Gpx2, Gpx3, and Gpx4 genes, respectively lium in the B6.129 Gpx1/2-DKO mice but rarely in the B6 (13). Among these GPXs, GPX1 and GPX2 are the major H2O2- Gpx1/2-DKO mice. (Cancer Res 2006; 66(20): 9845-51) reducing GPX activities in the gastrointestinal epithelium. The GPX1 and GPX2 isozymes have very similar properties, such as Introduction substrate specificity and cytosolic localization. They both reduce Oxidative stress occurs when the generation of reactive oxygen H2O2 and fatty acid hydroperoxides very efficiently but reduce lipid species (ROS) in a system exceeds the antioxidant capacity to hydroperoxides poorly. Unlike the ubiquitous GPX1, GPX2 is neutralize and eliminate them (1–4). This imbalance can result expressed mainly in epithelium, most highly in the gastrointestinal from a lack of available antioxidants or from an overabundance of epithelium. We found that, although homozygous mice deficient in ROS from environmental or internal sources. An excess of ROS either the wild-type (WT) Gpx1 or Gpx2 alleles appeared to be can damage cellular macromolecules, such as lipids, proteins, normal under standard housing conditions, homozygous Gpx1 and and nucleic acids. For example, ROS can directly interact with Gpx2 double knockout mice (Gpx1/2-DKO), with combined nucleic acids, resulting in a variety of modifications, including base disruption of both Gpx1 and Gpx2 genes, are highly susceptible damage, sugar damage, deletions, cross-linked lesions, and DNA to ileocolitis beginning around weaning (14). Similar to other strand breaks (1, 3, 5). mouse IBD models, when these mice are derived into germ-free conditions, they are disease-free (15). With long-term follow-up, the tumor incidence in Gpx1/2-DKO mice raised conventionally is f25% in mice harboring Helicobacter hepaticus (an enterohepatic Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Helicobacter species that can cause colitis in immunodeficient Requests for reprints: Gerd P. Pfeifer, Beckman Research Institute of the City of mice; ref. 16) on a mixed B6.129genetic background (17). Oxidative Hope, 1450 East Duarte Road, Duarte, CA 91010. Phone: 626-301-8853; Fax: 626-358- stress associated with cellular damage is thought to play a key 7704; E-mail: [email protected]. I2006 American Association for Cancer Research. role in the pathogenesis of the colitis itself (8, 9). Lack of the doi:10.1158/0008-5472.CAN-06-0732 antioxidant GPX system in the intestinal mucosa may set up a www.aacrjournals.org 9845 Cancer Res 2006; 66: (20). October 15, 2006 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2006 American Association for Cancer Research. Cancer Research continuous cycle of ROS and inflammation; inflammation and staff pathologists on H&E-stained sections. When harvesting DNA for cII production of ROS by inflammatory cells eventually leads to mutation analysis, we excluded mice with tumors. cancer. DNA isolation. Epithelial cells were isolated from the distal 10 to 12 cm Because inflammatory responses produce DNA-damaging oxi- of the ileum (lower 60% of small intestine, corresponding to the diseased segment in Gpx1/2-DKO mice at the height of pathology and distribution dative species and chronic inflammation in the gastrointestinal of tumors) and total colon (less f0.5 cm flanking segments taken for tract can cause cancer, we hypothesized that inflammation drives histology) by the everted sac method as described previously (22) to recover gene mutations, which can lead to carcinogenesis. To test this the villus and crypt compartments. The cells were rinsed in PBS thrice and hypothesis in a mouse model of IBD, we compared mutation frozen at À80jC. Genomic DNA was isolated using a standard phenol and frequencies and mutation spectra of the cII reporter gene in the chloroform extraction and ethanol precipitation protocol (23). Liver DNA affected intestinal and unaffected liver tissues of Gpx1/2-DKO and was isolated using a NaCl method. Briefly, the excised tissue was control mice on both B6.129and B6 genetic background. Because homogenized in 4 mL cold PBS in a 7 mL Wheaton Dounce tissue grinder GPX1 is up-regulated by p53 activation (18) and GPX2 by p63 (19), (Millville, NJ), washed twice with PBS, and lysed with 4 mL of a solution a homologue of p53, to inhibit oxidative stress–induced apoptosis, containing 0.5 mol/L Tris-HCl (pH 8.0), 20 mmol/L EDTA, 10 mmol/L NaCl, j studying the effect of GPXs in prevention of gene mutations may 1% SDS, and 0.5 mg/mL proteinase K at 37 C overnight. Subsequently, 2 mL saturated NaCl (f6 mol/L) was added to each sample, and the samples provide an insight to the mechanism of action of these antioxidant were incubated at 56jC for 10 minutes. After centrifugation at 5,000 Â g enzymes in IBD-associated tumorigenesis. for 30 minutes, the supernatant containing the DNA was mixed with 2 volumes of cold 100% ethanol, and the DNA was spooled by gently inverting the mix. The DNA was washed thoroughly with 70% ethanol, air Materials and Methods dried, and subsequently dissolved in TE buffer (pH 8.0), and kept at À80jC Animal breeding. We have established the B6.129Gpx1/2-DKO mouse until further analysis. colony as described previously (14). Breeders were maintained on diet 5020 cII mutant frequency analysis. The cII mutant frequency was (Laboratory Rodent Diet, Purina Mills, Inc., Richmond, IN) with 9% fat. examined by using the select-cII mutation detection system for Big Blue At weaning, pups were placed on diet 5001 with 5% fat until recruitment rodents as described previously (24). The assay is based on the ability of the into breeding. To generate a B6 Gpx1/2-DKO colony, the B6.129mice were phage to multiply either lytically or lysogenically in Escherichia coli host back-crossed to B6 for seven generations. B6 Gpx1/2-DKO males were bred cells. Briefly, we recovered the LIZ shuttle vectors from mouse genomic to homozygous B6 Big Blue females (Stratagene, La Jolla, CA) carrying a DNA (5 Ag) and packaged them into viable phage particles using the tandem array of E genomes (kLIZ) containing mutational reporter genes, Transpack packaging extract (Stratagene).