Open Full Page

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 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- 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 80jC. 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 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 80jC 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). The phage particles were then and subsequent breeding of heterozygous female progeny to homozygous preadsorbed to G1250 E. coli, and the bacteria were plated on TB1 agar B6 Gpx1/2-DKO males was done to incorporate the ELIZ shuttle vector plates. The plates were incubated for 48 hours at 24jC (selective conditions) transgene into the B6 Gpx1/2-DKO colony. We have maintained a WT B6 or overnight at 37jC (nonselective conditions). The cII mutant frequency ELIZ Big Blue line as controls. We then generated B6.129 ELIZ-transgenic was expressed as the ratio of the number of plaques formed on plates (Tg) mice by crossing B6 Gpx1/2-DKO ELIZ-Tg males with 129S1/Sv females incubated under selective conditions to the number of plaques formed (The Jackson Laboratory; Bar Harbor, ME). Heterozygous B6.129 ELIZ-Tg under nonselective conditions. We screened a minimum of 3 105 rescued progeny were back-crossed to the original B6.129Gpx1/2-DKO colony to phages for each experimental condition. establish a B6.129 ELIZ-Tg colony. B6.129 ELIZ-Tg mice carrying at least cII mutation spectrum analysis. Plaques containing putative mutants one WT Gpx1 or Gpx2 allele, non-DKO ELIZ-Tg, were used as controls of cII were verified after being replated and incubated under selective for B6.129Gpx1/2-DKO mice. We observed similar pattern and severity of conditions. The verified plaques were amplified by PCR using select-cII disease in Gpx1/2-DKO mice of the same genetic background regardless of sequencing primers (Stratagene) and purified with QIAquick PCR whether the mice were carrying the kLIZ gene or not. All colonies harbor purification kits (Qiagen, Valencia, CA). The PCR products were sequenced H. hepaticus, which can cause colitis but not hepatitis in B6 and B6.129 by using a BigDye terminator cycle sequencing kit on an ABI automated mice (20).3 Animal care and treatment was approved by the City of Hope DNA sequencer. At least 100 mutants were analyzed for each of five B6 Research Animal Care Committee (Duarte, CA). Gpx1/2-DKO and five B6 WT 8-month-old ilea. Pathology and tumor studies. We assessed animal health by routine weighing and visual inspection for diarrhea (wet tail), perianal alopecia, and perianal ulceration (14). Terminal ileum and distal colon sections flanking Results the tissues for DNA isolation for cII analysis were processed for histology We have reported previously that B6.129Gpx1/2-DKO mice analysis. Tissues were fixed overnight in phosphate-buffered formalin and are prone to ileocolitis under conventional housing conditions, then processed for paraffin embedment and thin sectioning. Sections were routinely stained with H&E for general pathology surveys. Immunohisto- and their ileocolitis occurs around weaning (3-4 weeks of age). chemistry was done with rabbit polyclonal anti-myeloperoxidase anti- The inflammation is most severe in the distal ileum, and by 6 to bodies (100, Lab Vision, Fremont, CA) and biotinylated preabsorbed goat 9months of age, 25% of these mice have gross tumors mostly in anti-rabbit IgG (Abcam, Inc., Cambridge, MA) to stain neutrophils and the ileum and a few in the colon (14, 17). B6.129non-DKO mice monocytes (17), rat monoclonal anti-macrophage antibody (20 Ag/mL with at least one WT allele of Gpx1 or Gpx2 rarely had inflam- F4/80, CI:A3-1, Abcam) and biotinylated goat anti-rat IgG (Vector mation and none had tumors. Genetic background has a profound Laboratories, Inc., Burlingame, CA), and a bromodeoxyuridine (BrdUrd) effect on both inflammation (16) and tumor susceptibility (25), immunohistochemistry system (Calbiochem, San Diego, CA) to detect with B6 being resistant to inflammation but sensitive to chemical- proliferating cells (17) and counterstained with hematoxylin. Pathology and induced colon carcinogenesis. We back-crossed our original colony inflammation were scored by a 14-point system as described previously (21). of B6.129Gpx1/2-DKO to B6 mice and investigated whether B6 Tumors were generally sufficiently advanced at 5 to 9months of age to be detected by naked eye after dissecting up the intestine longitudinally (17). Gpx1/2-DKO mice are also more resistant to inflammation and All putative tumorous lesions were verified and graded by consultation with inflammation-associated cancer than B6.129Gpx1/2-DKO mice. As expected, under the same housing conditions, which contain H. hepaticus, these B6 Gpx1/2-DKO mice are more resistant to inflammation than B6.129Gpx1/2-DKO mice, which were evaluated, 3 Unpublished data. concurrently. Most importantly, no B6 Gpx1/2-DKO mice were too

Cancer Res 2006; 66: (20). October 15, 2006 9846 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2006 American Association for Cancer Research. Mutations in Gpx1/2 Knockout Mice ill and required euthanasia before 50 days of age, when 15% to 20% pathology/inflammation scores in B6.129over B6 Gpx1/2-KO of B6.129Gpx1/2-DKO mice did. B6 Gpx1/2-DKO mice began to mice are not contributed by subinflammatory responses, such as have ileocolitis later, with peak pathology at 6 to 8 weeks of age apoptosis (data not shown) and proliferation; rather, the higher (2-4 weeks later than B6.129mice), and the pathology was milder scores in B6.129Gpx1/2-DKO mice are contributed by a higher than that in B6.129Gpx1/2-DKO mice. Figure 1 shows the typical number of inflammatory cells and inflammation foci. This may be ileal pathology of a 28-day-old B6.129and B6 Gpx1/2-DKO mouse a major reason we only detected one tenth of the tumor frequency after staining with anti-myeloperoxidase antibodies. At this young originally found in B6.129Gpx1/2-DKO mice (25%) in the B6 Gpx1/ age, there were few mature macrophages detectable by immuno- 2-DKO mice (2.5%) analyzed between 6-9months of age. histochemistry with anti-F4/80 antibodies in either genetic back- Because inflammation can enhance tumor susceptibility, we ground (data not shown). Although the 28-day-old B6 Gpx1/2-DKO hypothesized that inflammation causes gene mutations, which lead ileum still had a high level of apoptotic cells, crypt distortion, and to cancer. To test this hypothesis, we bred B6 Gpx1/2-DKO mice disorganized gland architecture, it rarely had epithelium erosion or with B6 ELIZ-Tg mice carrying a tandem array of E genomes (kLIZ) crypt abscesses. At this young age, B6.129Gpx1/2-DKO ileum had containing mutational reporter genes. These mice contain the a much higher number of inflammatory cells than age-matched transcriptionally silent cII reporter gene, which we used as a B6 (Figs. 1 and 2). We also compared the levels of macrophages, target for mutation analysis. Five to nine each of B6 WT and B6 monocytes, and neutrophils in 8-month-old Gpx1/2-DKO ileum Gpx1/2-DKO mice carrying ELIZ-transgenes were used to study and found that B6.129Gpx1/2-DKO mice had significantly higher cII mutation frequencies and mutation spectra. The mutation numbers of anti-F4/80-stained macrophage cells than B6, although analysis was done on tissue samples free of macroscopic tumors. they had similarly high levels of anti-myeloperoxidase-stained We have isolated mucosal epithelial cells from intestine for this monocytes and a low number of neutrophils (Figs. 1 and 2). This analysis to minimize the contribution by inflammatory cells lower level of inflammation also leads to lower pathology/ (Supplementary Fig. S1). inflammation scores in the B6 Gpx1/2-DKO ileum compared with The 8-month-old mice from both non-DKO control and Gpx1/2- B6.129between 21 to 120 days of age as shown in Fig. 3 A. DKO groups of either B6 or B6.129mice had elevated mutant Because the inflammation scores quantify the morphologic frequencies over the 28-day-old mice (Table 1). These age- changes, including crypt distortion, which is partially attributed accumulated differences were about 3- to 4-fold for the colon to hyperproliferation, we also analyzed cell proliferation in the and ileum samples (P < 0.05, t test) but only f1.7-fold for the liver crypts of ileal and colonic epithelium. As shown in Fig. 3B, B6 and samples (P = 0.06). Comparing the 28-day-old mice, mutant B6.129Gpx1/2-DKO ileal epithelia have 2.5- and 2.4-fold, respec- frequencies in the ileum or the colon of B6 Gpx1/2-DKO mice tively, higher numbers of proliferating cells than their age-matched were about 2- to 3-fold higher than those in B6 WT mice; but in non-DKO controls. Similarly, B6 and B6.129Gpx1/2-DKO colonic the liver, B6 Gpx1/2-DKO mice had only a slight (1.5-fold) increase epithelia have 1.9-fold higher number of proliferating cells than in mutant frequency. In the 8-month-old mice, the mutant frequen- their non-DKO controls (data not shown). Thus, the higher cies in B6 Gpx1/2-DKO ileum were over 4-fold higher than B6 WT

Figure 1. Immunohistochemistry of Gpx1/ 2-DKO mouse ileum on B6.129 and B6 genetic backgrounds. A, has B6.129 ileum. B, has B6 ileum. A and B, left, 28-day-old mice stained with anti-myeloperoxidase (a-MPO) antibodies. Although, both ilea have distorted gland architecture, only B6.129 mice have evident crypt abscess as shown by the exfoliated neutrophils (arrows). Middle, 8-month-old ileum stained with anti-myeloperoxidase antibodies. B6.129 mice have neutrophils located in the abscessed crypt and monocytes located in submucosa (arrows), and B6 mice had fewer monocytes and neutrophils. Right, 8-month-old ilea stained with anti-macrophage (a-M$) antibodies. Only a few macrophage cells are detected in the submucosa of older ileum of both B6 and B6.129 mice. Original magnification, 100.

www.aacrjournals.org 9847 Cancer Res 2006; 66: (20). October 15, 2006

comprised 41% (median, 31-47%) of all mutations in the WT ileum but only 19% (median, 15-30%) in B6 Gpx1/2-DKO ileum (P < 0.001, t test). Surprisingly, the relative contribution of G:T transversions was decreased in the B6 Gpx1/2-DKO mice, although their absolute frequency was increased. The relative frequency of small deletions was significantly increased in the B6 Gpx1/2-DKO mice compared with that in the WT mice [f3-fold increase, from 6.6% (median, 3.3-10.3%) to 15% (median, 6.2-35.2%) of all mutations; P = 0.04, t test]. Additionally, mutations at A:T bp and G:C transversions were also more common in the B6 Gpx1/2-DKO mice. The complete mutational spectra of individual ileum in five each of B6 WT and B6 Gpx1/2-DKO mice are shown in the Appendix (Supplementary Figs. S2 and S3). Mutational hotspots, defined as the occurrence of more than five mutations at one particular nucleotide position, occurred in one or more B6 WT and B6 Gpx1/ Figure 2. Inflammatory cells in the ilea of B6.129 Gpx1/2-DKO mice and B6 2-DKO mouse. Many of the hotspots in B6 Gpx1/2-DKO (13 of 28) Gpx1/2-DKO mice. Columns, mean number of inflammatory cells in 28-day-old and B6 WT (5 of 31) mice are unique to a single mouse. The higher (28 d) and 8-month-old (8mo) ileum of B6.129 control, B6.129, and B6 number of unique hotspots in B6 Gpx1/2-DKO mice may come Gpx1/2-DKO mice; bars, SD. WT B6 mice were not included due to scarcity of inflammatory cells. The macrophages were detected with anti-F4/80 from clonal expansion of cells carrying an initial mutation through antibodies, when anti-myeloperoxidase antibodies detected monocytes in rapid cell proliferation and thus better fixation. Nonunique hot- submucosal and neutrophils in the crypt abscess. Three to eight ilea were spots are likely derived from an intrinsic mutability of a particular counted in each group. *, significant difference from others in the same type of cells, except that monocytes in 8-month-old B6 and B6.129 are not different DNA sequence. In the WT mice, the hotspots were largely G:C>A:T from each other; P < 0.05, t test. transitions at CpG dinucleotide sequences. Strikingly, 23 of the mice (P < 0.04, t test). In the mildly affected B6 Gpx1/2-DKO colon, we observed a f2-fold increase of mutant frequencies than in B6 WT mice. As expected, no significant differences in mutant frequencies were observed in the uninvolved B6 Gpx1/2-DKO liver and in B6 WT mice. To test whether gene mutations are correlated with cancer incidence, we have also analyzed cII mutations in B6.129Gpx1/ 2-DKO and non-DKO ELIZ-Tg mouse ileum. We used the age- matched B6.129non-DKO mice as controls because they were close relatives of the B6.129Gpx1/2-DKO mice and rarely had inflammation or pathology and did not have tumors. Three to four each of B6.129non-DKO and Gpx1/2-DKO ilea were analyzed for the mutant frequencies. As shown in Table 1, at 8 months of age, the susceptible B6.129Gpx1/2-DKO mice had similar, f4-fold higher, mutant frequencies as the B6 Gpx1/2-DKO mice compared with their controls. Although the mutant frequency, 38.9 10 5, in the B6.129Gpx1/2-DKO ileum was higher than the age-matched B6 ileum (25.54 10 5), the difference was not statistically significant (P = 0.09, t test). To compare mutational spectra, we sequenced DNA isolated from the cII mutants obtained from 8-month-old B6 WT and B6 Gpx1/2-DKO ileum. From each of five mice per group, we sequenced >100 mutants and confirmed a mutated cII gene in over 96% of the plaques. Because the cII transgene in the Big Blue rodents is not transcribed (26), there is no ‘‘strand-dependent mutagenesis,’’ a phenomenon caused by transcription-coupled Figure 3. Ileal inflammation scores and number of proliferatingepithelial cells in B6.129 and B6 mice. A, ileal pathology or inflammation scores analyzed at DNA repair in mammalian endogenous genes (27), in this system. different ages in B6 Gpx1/2-DKO (.), B6.129 Gpx1/2-DKO (w ), and B6.129 Therefore, we have combined the strand mirror counterparts of non-DKO (D) mice. B6 non-DKO ileum has even lower scores than B6.129 all transitions (e.g., G:C>A:T and C:G>T:A) and transversions (e.g., non-DKO mice and thus is not included. The criteria for the scoringinclude inflammation foci, crypt distortion, abundance of apoptotic cells, neutrophils, G:C>T:A and C:G>A:T) when comparing the mutational spectra. monocytes, and lymphocytes, and degranulation of Paneth cells or mucin As shown in Fig. 4, although the mutational spectrum of the B6 depletion in goblet cells as described previously (21). The differences at 21 and 41 days of age are statistically significant (P V 0.007, t test). B, number of Gpx1/2-DKO mice was generally similar to that of the B6 WT proliferatingcells labeled with BrdUrd, detected by immunohistochemistry. mice, there were some important differences. Although the Two hours before euthanasia, mice were injected with BrdUrd, which was absolute frequency of all types of mutations were increased in detected with an anti-BrdUrd antibody immunohistochemically as we have described previously (17). Each group has 5 to 7 mice at 50 days of age. The B6 the B6 Gpx1/2-DKO mice (Fig. 4A), their relative contribution was and B6.129 Gpx1/2-DKO ileum has 2.5- and 2.4-fold significantly higher number different (Fig. 4B). For example, transition mutations at CpG sites of proliferatingcells than their controls. P < 0.05, t test.

Cancer Res 2006; 66: (20). October 15, 2006 9848 www.aacrjournals.org

Table 1. cII mutant frequencies in Gpx1/2-DKO and control mice

Age Ileum, 105 (n) Colon, 105 (n) Liver, 105 (n)

B6* WT Gpx1/2-DKO WT Gpx1/2-DKO WT Gpx1/2-DKO

c c c c 28 d 1.92 F 0.40 (4)* 4.00 F 1.10 (4) 1.33 F 0.66 (4) 4.33 F 1.63 (4) 1.82 F 0.24 (4) 2.70 F 0.69(4) c c c c 8 mo 6.01 F 1.31 (5) 25.54 F 10.33 (9) 5.55 F 2.14 (5) 12.97 F 2.71 (6) 3.22 F 0.80 (4) 4.40 F 2.18 (8)

B6.129* Non-DKO Gpx1/2-DKO

c c 28 d 1.38 F 0.05 (3) 4.00 F 0.28 (3) c c 8 mo 9.03 F 1.68 (4) 38.94 F 15.5 (4)

NOTE: The dagger indicates that that difference between the control and Gpx1/2-DKO groups was statistically significant (P V 0.04, t test). The liver does not have significant increase mutant frequency between 8-month-old WT and Gpx1/2-DKO mice (P = 0.17, t test) and between 28-day-old and 8-month-old Gpx1/2-DKO mice (P = 0.06, t test). *Mutant frequencies are mean F SD analyzed from the number of mice (n). Control for B6 Gpx1/2-DKO mice were WT B6, and control for B6.129 Gpx1/2-DKO mice were their disease-free cousins having at least one WT Gpx allele.

31 hotspots in WT mice but only 9of the 28 hotspots in the colonic epithelium, but not in the unaffected liver, of B6 and B6.129 Gpx1/2-DKO mice were of this type. The B6 Gpx1/2-DKO mice had Gpx1/2-DKO mice compared with their control mice. It is unlikely other types of dominant mutational events, including hotspots of that the increase in mutant frequencies is contributed by nonepi- G:C>C:G transversions (at positions 31, 86, 211, and 224), G:C>T:A thelial cells, such as infiltrating inflammatory cells. First, for DNA transversions (at positions 106, 167, and 190), and a hotspot of isolation, we separated intestinal epithelial cells from lamina T:A>C:G transitions (at position 53). B6 Gpx1/2-DKO mice had propria and submucosa, where the inflammatory cells reside frequent occurrence of small deletions at two very large mutational (Fig. 1; Supplementary Fig. S1). Second, the number of inflamma- hotspots; one at a mononucleotide G repeat, between positions tory cells present among epithelial cells is very small especially in 179to 184, and the other at a mononucleotide A repeat, between the B6 mice. Third, B6 Gpx1/2-DKO ileum had significantly lower nucleotides 241 to 246, the latter occurring in every DKO mouse. levels of inflammatory cells than B6.129Gpx1/2-DKO mice (Fig. 3 A) We did not analyze mutation spectra of B6.129mice due to their but still accumulated a 2-fold and a 4-fold higher mutation load, lack of uniform genetic background. For example, 129mice are respectively, than control mice at 28 days and 8 months of age. defective in the error-prone DNA polymerase iota, which may be Fourth, B6.129Gpx1/2-DKO mice at 28 days of age had as many involved in replication, bypassing some of the DNA lesions inflammatory cells as 8-month-old B6.129Gpx1/2-DKO mice but produced under oxidative stress.3 Analyzing mice with mixed yet had lower mutant frequencies. Lastly, intestinal epithelial cells genetic background would have complicated data interpretation. have similar, if not higher, levels of mutant frequencies than bone marrow cells and spleen (30, 31), sources of inflammatory cells and lymphocytes. Thus, the increased mutant frequencies found in Discussion cells isolated from B6 and B6.129Gpx1/2-DKO intestinal and ROS play a significant role in the pathogenesis of diseases of the colonic mucosa are most likely reflecting cumulative mutations gastrointestinal tract, including IBD (4, 7, 9). B6.129 Gpx1/2-DKO present in the epithelium rather than in the inflammatory cells. mice, as an IBD model, are highly susceptible to ileocolitis beginning A large variation of mutant frequency was detected in the ileum around weaning (13–15, 17). GPX enzymes in the gastrointestinal among both 8-month-old B6 and B6.129Gpx1/2-DKO mice; this is tract play a critical protective role in the detoxification of ROS likely due to differences in the disease history and severity. produced during inflammation in response to bacterial colonization Oxidative stress not only induces DNA damage, as will be (13). We considered the possibility that inflammation and increased discussed later, but also increases cell proliferation and apoptosis. levels of ROS in B6.129Gpx1/2-DKO mice may produce an elevated We have found that both B6 and B6.129Gpx1/2-DKO ileal epithelia load of mutations, which can increase the risk for malignant have f2.5-fold higher number of proliferating cells than the age- transformation. Thus far, there are only few reports showing a direct matched non-DKO controls (Fig. 3B). Because cell proliferation relationship between inflammation and DNA mutation load in would increase the probability of mutation accumulation (32), specific tissues. UC patients have an increased load of p53 mutations the higher level of cell proliferation in Gpx1/2-DKO intestinal in their affected tissues within areas of active inflammation (28). epithelium can also contribute to a higher mutation load with Oxygen radical overload diseases, such as Haemochromatosis and more efficient fixation. Wilson disease, are characterized by accumulation of iron and It is interesting that genetic background plays a significant role copper, respectively, in the liver. These conditions produce enhanced in the severity of inflammation and cancer incidence in the Gpx1/ oxidative stress, increase p53 mutations in premalignant tissue, and 2-DKO model, but B6 and B6.129Gpx1/2-DKO ilea have similar increase the risk for liver cancer (29). levels of gene mutations and cell proliferation. Although we In this study, using a mouse model for IBD, we have found detected higher mutant frequencies in the ileum of B6.129mice significantly higher mutant frequencies in the affected ileal and compared with the age-matched B6 mice in the 8-month-old www.aacrjournals.org 9849 Cancer Res 2006; 66: (20). October 15, 2006

control group (9.03 F 1.68 10 5 versus 6.01 F 1.31 10 5)as (Fig. 4A). CpG transition mutations are the single most important well as in the Gpx1/2-DKO group (38.94 F 15.50 10 5 versus endogenous mutations linked to cancer and inherited disease 25.54 F 10.33 10 5), these increases were not statistically signi- (34–36). The major mechanism for CpG mutagenesis is thought to ficant. This result suggests that, although mutations are driven be the deamination of 5-methylcytosine bases at CpG sequences. by inflammation and may be required for tumor formation, diffe- Other possibilities, including oxidation of 5-methylcytosine, have also rences in genetic background between B6 and B6.129Gpx1/2-DKO been considered (37, 38). Our data suggest that increased production mice are essential to produce malignancy in intestinal epithelium. of ROS and inflammation associated with GPX deficiency does not There may be a maximal mutation load that cells can tolerate. The substantially increase mutagenesis at methylated CpG sequences. concept of maximal preneoplastic increase of mutant frequencies There are at least two possible reasons why the absolute has been suggested by Busuttil et al. (33) when they observed a frequencies of all types of mutations were increased in the B6 similarly high mutant frequency (20 10 5) in 6- and 12-month- Gpx1/2-DKO mice. The first is that enhanced cell proliferation, old B6 Sod1-null mouse liver, which was 4-fold higher than that in relative to non-DKO mice, plays a role in enhanced mutant the control B6 liver. The 4-fold increase of mutation load during frequencies in general, and this would apply to all types of 7 months may be reaching the maximal load in the ileum. Cells mutations. However, another possible explanation is that oxidative with higher mutation load may be subjected to apoptosis, which DNA damage can produce many different types of mutations. For may be more prominent in the ileal and colonic crypt epithelium example, damage of guanine or cytosine can produce all types of of B6.129Gpx1/2-DKO mice (17). Alternatively, it is possible that transitions and transversions observed at G:C bp, and damage B6.129Gpx1/2-DKO ileum do have a slightly higher mutation load at thymine (and less likely adenine) has the potential to produce any than B6 mice, and a 1.5-fold higher mutant frequency can result in type of mutation at A:T bp. It is even possible that the mutations in 10-fold higher tumor incidence. the control mice are in part due to oxidative DNA damage. Analysis of mutation spectra was done to determine the likely One major mutation type induced by oxidative damage is the cause of the mutations. The majority of G:C>A:T transition G:C>T:A transversion, presumably a consequence of the presence mutations in both B6 Gpx1/2-DKO and B6 WT mice occurred of 8-oxodeoxyguanosine (8-oxo-dG). B6 Gpx1/2-DKO mice not at CpG sites but this was much more pronounced for the WT mice only had increased transversion mutation frequencies (Fig. 4A) but also had prominent G:C>T:A hotspots at positions 109and 192 (Supplementary Fig. S3). However, the relative contribution of G:T transversions to the mutational spectrum was decreased in the B6 Gpx-DKO mice (Fig. 4B). Because 8-oxo-dG is induced by oxidative damage and mainly causes G:C>T:A transversions (39), we quantified 8-oxo-dG in ileal genomic DNA of B6 WT or B6 Gpx1/2-DKO mice using high-pressure liquid chromatography (HPLC)–mass spectrometry (MS)/MS (24). However, we observed no significant differences (data not shown). The new hotspots occurring specifically in B6 Gpx1/2-DKO mice were characterized mainly by G:C>C:G transversions and single-base deletions in small mononucleotide repeats (Supplementary Figs. S2 and S3). Four unique hotspots of G:C>C:G transversions (at positions 31, 86, 211, and 224) were observed in B6 Gpx1/2-DKO mice and thus might represent clonal expansions. Even if we exclude the unique hotspots, which can contribute up to 40% of the total mutation load (Supplementary Fig. S3; 93 DKO), B6 Gpx1/2-DKO ileum would still have a z2.5-fold higher mutant frequency than its control ileum. The G:C>C:G transversions can be produced by replicative bypass of oxidation products of 8-oxo-dG, such as guanidinohydantoin and spiroiminodihydantoin (40), types of lesions, which currently cannot be quantified by HPLC/MS/MS analysis. This type of mutation also seems to be unique to B6 Gpx1/2-DKO mice and is not elevated in B6 mice deficient in SOD1 (33). However, increases in single-base deletions seem to be associated with oxidative stress and were also elevated in B6 Sod1-null liver and kidney (33). Deletions in runs of Gs may be induced directly by oxidative DNA damage because guanine Figure 4. Mutational spectra of the cII transgene in B6 Gpx1/2-DKO and WT is the base most susceptible to oxidation. Oxidative stress is known mice. A, absolute frequencies of each type of mutation. The cII mutants were to increase frameshift mutations in mismatch repair proficient sequenced to evaluate mutational spectra. The mutation spectra were compiled bacterial cells (41, 42) and in human cells (43). Oxidant-induced from at least 100 mutant sequences from each mouse ileum. Five each of 8-month-old B6 WT and Gpx1/2-DKO mice were analyzed. The strand mirror strand breaks at mononucleotide repeats may increase the counterparts of all transitions (e.g., G:C to A:T and C:G to T:A) and transversions probability of misalignment or strand slippage during DNA (e.g., G:C to T:A and C:G to A:T) were combined. The mutant frequency for each type of mutation was determined by multiplyingthe percentageof replication (2, 41). In addition, ROS associated with chronic each type of mutation with the average mutant frequencies obtained from the WT inflammation have the capacity to damage the protein components (white columns) and Gpx1/2-DKO (black columns) group. Gray columns, mutant of the mismatch repair system directly, leading to a failure to correct frequencies occurringat CpG sites. B, relative frequencies of each type of mutation. Combined data for each of the five WT and five Gpx1/2-DKO mice single-base mismatches and small insertion/deletion loops that (white and black columns, respectively). Ins, insertion; Del, deletion. occur during DNA replication (44).

Cancer Res 2006; 66: (20). October 15, 2006 9850 www.aacrjournals.org

In summary, we observed an increased mutation load in the Acknowledgments ileum of both B6 and B6.129Gpx1/2-DKO mice compared with Received 3/2/2006; revised 8/3/2006; accepted 8/10/2006. their age-matched control mice. Because B6 Gpx1/2-DKO mice had Grant support: NIH grant CA84469(G.P. Pfeifer), Crohn’s and Colitis Foundation milder ileocolitis and 10-fold lower tumor incidence than B6.129 of America Senior Research Award (F-F. Chu), NIH grants CA119272 and CA114569 (F-F. Chu), and Eli and Edythe L. Broad Foundation BMRP (R.S. Esworthy). Gpx1/2-DKO mice, these data suggest that, although inflammation The costs of publication of this article were defrayed in part by the payment of page is clearly associated with gene mutations, an increase in mutations charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. rarely leads to neoplastic transformation in B6 Gpx1/2-DKO mice We thank Dr. Paul Frankel for statistical analysis, Tina Montgomery and Sofia having a tumor-resistant genetic background. Loera for tissue processing, and Dr. Peiguo Chu for tumor grading.

References induced colon cancer in Rag2-deficient mice. Am J 31. Ono T, Ikehata H, Nakamura S, et al. Age-associated Pathol 2003;162:691–702. increase of spontaneous mutant frequency and molec- 1. Epe B. Role of endogenous oxidative DNA damage in 17. Chu FF, Esworthy RS, Chu PG, et al. Bacteria-induced ular nature of mutation in newborn and old lacZ- carcinogenesis: what can we learn from repair-deficient intestinal cancer in mice with disrupted Gpx1 and Gpx2 transgenic mouse. Mutat Res 2000;447:165–77. mice? Biol Chem 2002;383:467–75. genes. Cancer Res 2004;64:962–8. 32. Busuttil RA, Rubio M, Dolle ME, et al. Mutant 2. Evans MD, Cooke MS. Factors contributing to the 18. Tan M, Li S, Swaroop M, et al. Transcriptional frequencies and spectra depend on growth state and outcome of oxidative damage to nucleic acids. Bio- activation of the human glutathione peroxidase pro- passage number in cells cultured from transgenic lacZ- Essays 2004;26:533–42. moter by p53. J Biol Chem 1999;274:12061–6. plasmid reporter mice. DNA Repair (Amst) 2006;5:52–60. 3. Termini J. Hydroperoxide-induced DNA damage and 19. Yan W, Chen X. GPX2, a direct target of p63, inhibits 33. Busuttil RA, Garcia AM, Cabrera C, et al. Organ- mutations. Mutat Res 2000;450:107–24. oxidative stress-induced apoptosis in a p53-dependent specific increase in mutation accumulation and apo- 4. Hussain SP, Hofseth LJ, Harris CC. Radical causes of manner. J Biol Chem 2006;281:7856–62. ptosis rate in CuZn-superoxide dismutase deficient cancer. Nat Rev Cancer 2003;3:276–85. 20. Ihrig M, Schrenzel MD, Fox JG. Differential suscep- mice. Cancer Res 2005;65:11271–5. 5. Cadet J, Douki T, Gasparutto D, Ravanat JL. Oxidative tibility to hepatic inflammation and proliferation in AXB 34. Jones PA, Baylin SB. The fundamental role of damage to DNA: formation, measurement, and bio- recombinant inbred mice chronically infected with epigenetic events in cancer. Nat Rev Genet 2002;3: chemical features. Mutat Res 2003;531:5–23. Helicobacter hepaticus. Am J Pathol 1999;155:571–82. 415–28. 6. Jackson AL, Loeb LA. The contribution of endogenous 21. Esworthy RS, Yang L, Frankel PH, Chu FF. Epitheli- 35. Gonzalgo ML, Jones PA. Mutagenic and epigenetic sources of DNA damage to the multiple mutations in um-specific glutathione peroxidase, Gpx2, is involved in effects of DNA methylation. Mutat Res 1997;386:107–18. cancer. Mutat Res 2001;477:7–21. the prevention of intestinal inflammation in selenium- 36. Pfeifer GP. p53 mutational spectra and the role of 7. Coussens LM, Werb Z. Inflammation and cancer. deficient mice. J Nutr 2005;135:740–5. methylated CpG sequences. Mutat Res 2000;450:155–66. Nature 2002;420:860–7. 22. Esworthy RS, Swiderek KM, Ho YS, Chu FF. Selenium- 37. Lee DH, O’Connor TR, Pfeifer GP. Oxidative DNA 8. Seril DN, Liao J, Yang GY, Yang CS. Oxidative stress and dependent glutathione peroxidase-GI is a major glutathi- damage induced by copper and hydrogen peroxide ulcerative colitis-associated carcinogenesis: studies in one peroxidase activity in the mucosal epithelium of promotes CG!TT tandem mutations at methylated humans and animal models. Carcinogenesis 2003;24: rodent intestine. Biochim Biophys Acta 1998;1381:213–26. CpG dinucleotides in nucleotide excision repair-defi- 353–62. 23. Pfeifer GP, Chen HH, Komura J, Riggs AD. Chromatin cient cells. Nucleic Acids Res 2002;30:3566–73. 9. Itzkowitz SH, Yio X. Inflammation and cancer IV. structure analysis by ligation-mediated and terminal 38. Yoon JH, Iwai S, O’Connor TR, Pfeifer GP. Human Colorectal cancer in inflammatory bowel disease: the transferase-mediated polymerase chain reaction. Meth- thymine DNA glycosylase (TDG) and methyl-CpG- role of inflammation. Am J Physiol Gastrointest Liver ods Enzymol 1999;304:548–71. binding protein 4 (MBD4) excise thymine glycol (Tg) Physiol 2004;287:G7–17. 24. Besaratinia A, Synold TW, Xi B, Pfeifer GP. G-to-T from a Tg:G mispair. Nucleic Acids Res 2003;31: 10. Kilgore SP, Sigel JE, Goldblum JR. Hyperplastic-like transversions and small tandem base deletions are the 5399–404. mucosal change in Crohn’s disease: an unusual form of hallmark of mutations induced by ultraviolet a radiation 39. Wood ML, Dizdaroglu M, Gajewski E, Essigmann JM. dysplasia? Mod Pathol 2000;13:797–801. in mammalian cells. Biochemistry 2004;43:8169–77. Mechanistic studies of ionizing radiation and oxidative 11. Rhodes JM, Campbell BJ. Inflammation and colorec- 25. Jacoby RF, Hohman C, Marshall DJ, et al. Genetic mutagenesis: genetic effects of a single 8-hydroxygua- tal cancer: IBD-associated and sporadic cancer com- analysis of colon cancer susceptibility in mice. nine (7-hydro-8-oxoguanine) residue inserted at a pared. Trends Mol Med 2002;8:10–6. Genomics 1994;22:381–7. unique site in a viral genome. Biochemistry 1990;29: 12. Touati E, Michel V, Thiberge JM, et al. Chronic 26. Swiger RR, Cosentino L, Shima N, et al. The cII locus 7024–32. Helicobacter pylori infections induce gastric mutations in the MutaMouse system. Environ Mol Mutagen 1999; 40. Henderson PT, Delaney JC, Muller JG, et al. The in mice. Gastroenterology 2003;124:1408–19. 34:201–7. hydantoin lesions formed from oxidation of 7,8-dihydro- 13. Chu FF, Esworthy RS, Doroshow JH. Role of Se- 27. Hanawalt PC. Transcription-coupled repair and 8-oxoguanine are potent sources of replication errors dependent glutathione peroxidases in gastrointestinal human disease. Science 1994;266:1957–8. in vivo. Biochemistry 2003;42:9257–62. inflammation and cancer. Free Radic Biol Med 2004;36: 28. Hussain SP, Amstad P, Raja K, et al. Increased p53 41. Jackson AL, Chen R, Loeb LA. Induction of micro- 1481–95. mutation load in noncancerous colon tissue from satellite instability by oxidative DNA damage. Proc Natl 14. Esworthy RS, Aranda R, Martin MG, et al. Mice with ulcerative colitis: a cancer-prone chronic inflammatory Acad Sci U S A 1998;95:12468–73. combined disruption of Gpx1 and Gpx2 genes have disease. Cancer Res 2000;60:3333–7. 42. Jackson AL, Loeb LA. Microsatellite instability colitis. Am J Physiol Gastrointest Liver Physiol 2001;281: 29. Hussain SP, Raja K, Amstad PA, et al. Increased p53 induced by hydrogen peroxide in Escherichia coli. Mutat G848–55. mutation load in nontumorous human liver of wilson Res 2000;447:187–98. 15. Esworthy RS, Binder SW, Doroshow JH, Chu FF. disease and hemochromatosis: oxyradical overload 43. Gasche C, Chang CL, Rhees J, et al. Oxidative stress Microflora trigger colitis in mice deficient in selenium- diseases. Proc Natl Acad Sci U S A 2000;97:12770–5. increases frameshift mutations in human colorectal dependent glutathione peroxidase and induce Gpx2 30. Hernandez LG, Heddle JA. A carcinogenic western cancer cells. Cancer Res 2001;61:7444–8. gene expression. Biol Chem 2003;384:597–607. diet does not induce somatic mutations in various 44. Chang CL, Marra G, Chauhan DP, et al. Oxidative 16. Erdman SE, Poutahidis T, Tomczak M, et al. CD4+ target tissues of transgenic C56BL/6 mice. Mutat Res stress inactivates the human DNA mismatch repair CD25+ regulatory T lymphocytes inhibit microbially 2005;570:185–96. system. Am J Physiol Cell Physiol 2002;283:C148–54.

www.aacrjournals.org 9851 Cancer Res 2006; 66: (20). October 15, 2006

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2006 American Association for Cancer Research. Mutation Accumulation in the Intestine and Colon of Mice Deficient in Two Intracellular Glutathione Peroxidases

Dong-Hyun Lee, R. Steven Esworthy, Christy Chu, et al.

Cancer Res 2006;66:9845-9851.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/66/20/9845

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2006/10/18/66.20.9845.DC1

Cited articles This article cites 44 articles, 10 of which you can access for free at: http://cancerres.aacrjournals.org/content/66/20/9845.full#ref-list-1

Citing articles This article has been cited by 1 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/66/20/9845.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/66/20/9845. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.