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Department of Medical Oncology and Therapeutics Research and * Department of Biostatistics, City of Hope Comprehensive Cancer Center, Duarte, CA 91010-3000
2To whom correspondence should be addressed. E-mail: fchu{at}coh.org.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • selenium • glutathione peroxidase • ileocolitis • targeted mutation
Selenium (Se)3 is an essential micronutrient. Selenium deficiency results in several symptoms associated with increased oxidative stress. The classic example is liver necrosis in rats deficient in Se, vitamin E, and sulfur amino acids (1,2). One of the best known symptoms resulting from Se deficiency is muscle weakness; weakened cardiac muscle is highly susceptible to cardiomyopathy induced by Coxsackie virus in Keshan disease (3) or by HIV infection (4,5); and weakened cardiac and skeletal muscles occur in farm animals with white muscle disease (6). The weakened muscles may result from a lack of antioxidant activity provided by Gpx1 and selenoprotein W. Se is also essential for male fertility because the selenoprotein, phospholipid hydroperoxide glutathione peroxidase, is both an abundant antioxidant enzyme and a structural protein in mature spermatozoa (7). More recently, Se deficiency was implicated as a factor increasing cancer risk in esophagus, stomach, colon, and prostate from large-scale clinical trials (8–11). Increases in oxidative stress lead to increased cancer risk.
Se deficiency is not equivalent to Se null, since mice with a disrupted selenocysteine tRNA gene, Trsp, and thus not able to synthesize any of 
25 selenoproteins, die at 6.5 d after implantation (12,13). Se-sufficient Gpx1–/–Gpx2–/– mice are sensitive to luminal microflora and prone to ileocolitis beginning around weaning (14–16). Gpx1–/–Gpx2–/– mice have inflammatory bowel disease (IBD) symptoms including growth retardation, loose stools, perianal ulceration, and hypothermia. Histological pathology manifested in Gpx1–/–Gpx2–/– mice routinely includes the prevalence of apoptotic figures in the crypt epithelium of ileum and colon with distortion of the crypt and mucin depletion in goblet cells or degranulation of Paneth cells, even in the absence of immune infiltration other than by intraepithelial lymphocytes or scattered monocytes. This pathology resembles the gastrointestinal (GI) pathology observed in graft versus host disease (17). Additionally, Gpx1–/–Gpx2–/– mouse ileum and colon also exhibit a predominantly acute inflammation by neutrophils causing epithelial erosion and crypt abscesses (15). Although slow growth is a feature shared by Se-adequate Gpx1–/–Gpx2–/– mice and Se-deficient wild-type animals, Se deficiency does not produce ileocolitis (18–20). We questioned whether a low level of Gpx1 or Gpx2 could prevent the ileocolitis observed in Gpx1–/–Gpx2–/– mice.
We hypothesized that Gpx2 provides a better protection of intestinal mucosa from inflammation than Gpx1, even in Se-deficient mice, based on the following observations. Mice expressing 1 wild-type Gpx1 allele (Gpx1+/–Gpx2–/–) had a low incidence of IBD symptoms both while growing and as adults. Mice expressing 1 wild-type Gpx2 allele (Gpx1–/–Gpx2+/–) seldom had any IBD symptoms while growing and had none as adults (14). The stronger protection afforded by Gpx2 may be due to its crypt localization, whereas Gpx1 is more abundant in the villus (21–24). Both stem epithelial cells and rapidly dividing epithelial cells are located in the crypt, and they may be more vulnerable to oxidative stress than the mature epithelial cells in the villus. Finally, Gpx2 gene expression, at least at the mRNA level, is reportedly more resistant to Se deficiency than Gpx1 gene expression in human hepatoma HepG2 and colon Caco-2 cancer cells (25,26). The residual Gpx2 in Se-deficient Caco-2 cells may be sufficient to reduce lipid hydroperoxides (27).
The goal of the following work was to determine whether Gpx2 activity was protective when Se levels were low. These studies had 2 components: first, to illustrate that Se depletion worsens symptoms in the Gpx+/–Gpx2–/– mice to the same extent as Se-sufficient Gpx1–/–Gpx2–/– mice; and second, to demonstrate that Gpx1–/–Gpx2+/– mice are resistant to pathology under the same conditions.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Diets.
For maintaining our Gpx1–/–Gpx2–/– mouse colony, we fed the breeders a diet containing 9% fat (5020, Mouse Diet 9F, Purina Mills Inc.) and weaned pups a diet containing 5% fat (5001, Laboratory Mouse Diet) until they were selected for breeding. To study the effect of Gpx1 and Gpx2 in Se-deficient mice, we fed mice with an Se-deficient diet (TD 92087, Table 1, Harlan Teklad). This Se-deficient diet was formulated to have <0.01 mg of Se/kg, when the regular diet has 0.2 mg of Se/kg. Based on the studies by Burk (28), 4 wk of Se depletion beginning at weaning results in severe Se deficiency. Se depletion of mice was carried out for 4–5 wk in accordance with Burk’s methods (28). In study 1, we fed 31- to 51-d-old mice with the Se-deficient diet for 4 wk. Because Se-deficient Gpx1+/–Gpx2–/– mice did not develop ileocolitis as we have observed in Se-adequate Gpx1–/–Gpx2–/– mice, in study 2 we fed 18- to 22-d-old weanlings the Se-deficient diet for 4 wk. This followed our standard dietary regimen of feeding diet with 5% fat at weaning. In study 3, we determined whether more severe ileocolitis would develop if the Se-deficient diet was introduced earlier. Pregnant dams were fed the Se-deficient diet before parturition (–1 to –13 d), and both the dam and the pups were fed the same diet to weaning. The weaned pups were fed the diet until 26–31 d postparturition, unless their health was too poor to continue. This part of the study departed from our standard practice of feeding dams with pups a diet with 9% fat. In all 3 studies, age-matched Se-adequate mice were used as controls. To eliminate the potential effect of the genotype of fostering dams on the pups, we analyzed Gpx1+/–Gpx2–/– pups born of dams from all 3 heterozygous genotypes: Gpx1+/–Gpx2–/–, Gpx1–/–Gpx2+/–, and Gpx1+/–Gpx2+/–.
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Histological analysis. Intestinal samples were processed for histological examination as previously described (29). The scoring for pathology or inflammation is based on a 14-point system, which includes inflammation as shown by lymphocytes or neutrophil infiltration (0–3 points), degranulation in ileal Paneth cells or mucin depletion in colonic goblet cells (0–2 points), reactive epithelium such as crypt distortion (0–3 points), inflammatory foci (0–3 points), and apoptotic figures (0–3 points) (29). Neutrophil infiltration produces crypt abscesses, where the crypts are enlarged and filled with neutrophils and exfoliated epithelial cells, or erosion of the epithelium, which produces denuded stretches where the submucosa is directly exposed to the lumen. The scoring places equal weight on crypt abscesses and erosion, because these are epiphenomena of infiltration. However, the number of abscesses, extent of the eroded area, or multiple eroded areas are counted as a separate component of the scoring system (inflammatory foci). Immunohistochemistry was performed on the paraffin sections to confirm the extent of neutrophil and monocyte infiltration using a rabbit polyclonal anti-myeloperoxidase (MPO) antibody (Dako) and apoptosis using a TdT-FragEL DNA Fragmentation Detection kit (TUNEL, Oncogene) as previously described (29).
Enzyme assays. We analyzed plasma and intestinal Gpx activity and liver glutathione S-transferase (GST) to determine the extent of Se depletion (28,30). To determine epithelial Gpx activity from the ileum, we used the everted-sac method to isolate epithelial cells as previously described except that 1 mmol/L dithiothreitol was added to preserve Gpx activity (31). The Gpx assay used 60 mmol/L H2O2 and 3 mmol/L glutathione as substrates in a Tris-HCl buffer, pH 7.3, as previously described (31). The GST assay used 1-chloro-2,4-dinitrobenzene as the substrate as described (32). The protein concentration was determined by bicinchoninic acid assay (Pierce) with bovine serum albumin as the standard. The Gpx specific activity determined from solid tissue is expressed as units of µmol NADPH consumed x min–1 x mg protein–1. Plasma Gpx activity is expressed as units of kU/L(µmol x min–1 x L–1).
RNA analysis. Total RNA was isolated from snap-frozen ileal segments (0.6 cm) using an RNeasy mini kit (Qiagen) as we described previously (31). We used real-time PCR to quantify Gpx1, Gpx2, and ß-actin mRNA levels; the mRNA levels of Gpx1 and Gpx2 are normalized with that of ß-actin (33,34). The primer set for Gpx1 mRNA is mGpx1_265F (5'-GACTGGTGGTGCTCGGTTTC-3') and mGpx1_357R (5'-GTCGGACGTACTTGAGGGAATT-3') to generate a 93-bp product. The primer set for Gpx2 mRNA is mGpx2_275F (5'-CCAGCTCAATGAGCTGCAATG-3') and mGpx2_409R (5'-CCCCCAGGTCGGACATACTT-3') to generate a 135-bp product. The primer set for ß-actin is mActB1F (5'-GCTCCTCCTGAGCGCAAGT-3') and mActB1R (5'-TCATCGTACTCCTGCTTGCTGAT-3') to generate a 101-bp product. We used a real-time PCR thermocycler (PTC-200 DNA Engine Cycler with CFD-3200 Opticon Detector, MJ Research) to detect fluorescent products stained with SYBR green (Roche) in reaction mixtures previously described (34). The annealing and denaturing temperatures were 58 and 94°C, and the product was monitored at 75°C for 36 cycles for all genes. We used an RNA sample isolated from an Se-sufficient Gpx1+/–Gpx2+/– mouse ileum to generate standard curves to determine Gpx1 and Gpx2 mRNA levels. The standard curve was a plot of the threshold cycle (Ct) against the log of the amount of DNA added; the linear correlation was >98%. A linear regression analysis of the standard plot was used to calculate the relative amount of DNA in experimental samples. The Gpx1 and Gpx2 mRNA levels determined from real-time PCR were normalized against levels of ß-actin mRNA.
Statistical analysis. Results are means ± SD. The sample sizes are indicated in the text and figure legends. The significance of differences among sample means (P < 0.05) was tested as follows: a Wilcoxon rank sum test was used to compare inflammation/pathology subscores and totals; paired tests between genotypes for Gpx activity at each time point and within genotype for time of Se depletion were done by t test; the effect of Se depletion on mRNA levels within each genotype was done by t test; Fisher’s exact test was used to evaluate significance of differences in numbers of mice in symptom categories between genotypes and between Se-sufficient and Se-deficient groups.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Study 1: Se depletion of young adult mice. The plasma Gpx activity of Se-deficient adult mice used in the assessment of intestinal pathology (63- to 83-d-old) was 0.29 ± 0.15 kU x L–1 vs. 4.2 ± 0.16 kU x L–1 in age-matched Se-sufficient mice (P < 0.001, t test). Liver GST specific activity did not differ between groups (data not shown). Because GST activity is induced in severe Se depletion (28), these results suggested that the Se-deficient adult mice were only mildly Se depleted. A similar absence of pathology was observed in the ileum and colon of the mildly Se-depleted adult Gpx1+/–Gpx2–/– and Gpx1–/–Gpx2+/– mice as in age-matched Se-sufficient Gpx1+/–Gpx2–/– and Gpx1–/–Gpx2+/– mice analyzed at the same time.
Study 2: Se depletion on weanling mice.
Gpx activity in the ileal epithelium from the same mice that were scored for pathology decreased to a similar level after Se depletion in both Gpx1+/–Gpx2–/– and Gpx1–/–Gpx2+/– mice, about 16 mU x mg protein–1 after the background activity found in Gpx1–/–Gpx2–/– mice was subtracted. After mice were fed the Se-deficient diet for 4 wk, their activity levels were 6% of those in Se-sufficient Gpx1+/–Gpx2–/– mice (252 ± 70 mU x mg–1) and 12% of the activity of Se-sufficient Gpx1–/–Gpx2+/– mice (144 ± 32 mU x mg–1) (P < 0.01, Se-deficient vs. Se-sufficient for both genotypes, n 
4, t test). Gpx activities were 20–23% of Se-sufficient levels by d 13 of Se depletion in mice of both genotypes (P < 0.01, Se deficient vs. Se sufficient for both genotypes, n 4, t test).
Gpx1 and Gpx2 mRNA levels in Gpx1+/–Gpx2–/– and Gpx1–/–Gpx2+/– mice, respectively, were decreased in mice fed Se-deficient diet for 4 wk. The Gpx1 mRNA levels in the Se-deficient mice were 25% of control levels and the Gpx2 mRNA levels in the Se-deficient mice were 18% of control levels (P < 0.05, Se deficient vs. Se sufficient for both mRNAs, n 3, t test)
Unlike the adult Se-deficient Gpx1+/–Gpx2–/– mice, which did not have pathology, these juvenile Se-deficient Gpx1+/–Gpx2–/– mice exhibited significantly more pathology in the ileum or colon than age-matched controls analyzed concurrently, and 2 mice had pathology in both areas (Table 2). These mice had frank inflammation as shown by neutrophil or monocyte infiltration in the ilea and colons: 17% (2 of 12) of mice had inflamed ilea, and 33% (4 of 12) of mice had inflamed colons (Fig. 2), involving 33% of the mice in all. Two animals in this set exhibited focal erosion of the colon epithelium, which is often observed in Gpx1–/–Gpx2–/– mice. On the contrary, the juvenile Se-deficient Gpx1–/–Gpx2+/– mice did not have symptoms or pathology. The mean pathology score in the ileum of Se-deficient Gpx1–/–Gpx2+/– mice was 0; the mean score in the colon was 0.5, range 0–1, significantly less than the scores for Se-deficient Gpx1+/–Gpx2–/– mice (P < 0.0001, Wilcoxon’s rank-sum test, n = 12). Se-sufficient Gpx1+/–Gpx2–/– mice had no inflammation but still manifested mild pathology (focal mucin depletion and apoptosis) (Fig. 2).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The pathology of the Se-deficient Gpx1+/–Gpx2–/– juvenile mice (Se depletion in utero or at weaning) included focal neutrophil infiltration producing erosion of the epithelium of the colon and scattered crypt abscesses. Monocyte infiltration of the submucosa was also prevalent in the colons of Se-depleted Gpx1+/–Gpx2–/– mice. However, this monocyte infiltration did not produce the severe pathology observed in many Gpx1–/–Gpx2–/– mice. External signs developed in Gpx1+/–Gpx2–/– mice that were subjected to Se depletion before birth and were clearly associated with anal and rectal pathology. These pathological features, which are common in Se-sufficient Gpx1–/–Gpx2–/– mice, were absent from the Se-depleted Gpx1–/–Gpx2+/– mice in this study. Although Gpx1 is not as effective as Gpx2, there were indications that Gpx1 was important for prevention of frank inflammation, especially in the ileum. The relative resistance of the ileum in Se-depleted Gpx1+/–Gpx2–/– mice to inflammation was notable, whereas other pathology worsened in the ileum.
Whereas the colon pathology occurred in the Se-depleted Gpx1+/–Gpx2–/– mice at the same time as in Gpx1–/–Gpx2–/– mice, the ileal pathology tended to be delayed and did not reach the same level of incidence found in the colon. In Gpx1–/–Gpx2–/– mice, the ileum is inflamed by 21–28 d. We did not see this until 46–50 d of age in Se-depleted Gpx1+/–Gpx2–/– mice. In Gpx1–/–Gpx2–/– mice, the pathology is triggered by a combination of microbial load and diminished Gpx activity levels (16,29). Because the ileum has a much lower microbial load than the colon does, the mild Se depletion may have left some resistance to a microbial trigger, whereas in the colon the microbial assault would be relentless. There was a marked difference between ileal and colonic responses during microbial loading in Gpx1–/–Gpx2–/– mice in an earlier study. When germ-free Gpx1–/–Gpx2–/– mice were exposed to microflora by contact with soiled bedding from conventional colonies, the colons responded with severe uniform inflammation in 6–7 d (16), whereas the ilea only had sporadic and sparse inflammation at this time (unpublished observations). Depletion of Gpx activity was complete in this case, but the colonization of the colon was probably much more rapid than in the ileum, conceivably producing marked differences in the inflammatory response between these 2 tissues.
The ability of Se depletion to produce pathology in the ileum and the colon was dependent on the age of the mice. Inflammatory pathology in the colon was common in neonates and nearly absent when Se depletion began in young adult mice. Although the ileal pathology peaked when Se depletion began at 18–22 d, Se depletion that began at 31 d or later did not produce severe symptoms at this site. This suggests that there are other mechanisms to suppress inflammation when the mice reach adulthood or that microbial pathogens have been eliminated.
Compared to Gpx1, we detected only marginal resistance of Gpx2 activity to Se depletion after 2–4 wk. The reason for protection of 1 isoenzyme and not the other may lie in differences in the distribution of the 2 isoenzymes along the crypt to villus axis. Gpx1 is sparsely expressed in the crypt epithelium, whereas Gpx2 is found in both crypt and villus with an increasing gradient of activity from villus to crypt (21,22,24,27,35). Whereas the final Gpx specific activity in the epithelium of Se-depleted Gpx1+/–Gpx2–/– and Gpx1–/–Gpx2+/– mice is similar, because Gpx2 is in the crypt, Gpx activity levels can remain quite high in this compartment in Se-deprived Gpx1–/–Gpx2+/– mice.
Intestinal Gpx2 gene expression is as sensitive to Se deprivation as the Gpx1 gene. Wingler et al. (25) reported that although the Gpx1 mRNA level is sensitive to Se deprivation, Gpx2 mRNA is resistant to Se deprivation in human hepatoma HepG2 and human colon Caco-2 cancer cells. We were unable to document such resistance to nonsense-mediated decay by Gpx2 mRNA at 4 wk of Se depletion in Gpx1–/–Gpx2+/– mouse ileum. It is not clear whether this discrepancy is due to the difference between normal and cancer cells or is species and tissue dependent.
In summary, the ability to induce increased pathology, particularly in the colons of Gpx1+/–Gpx2–/– mice by Se depletion, and clear resistance to almost any pathology in Gpx1–/–Gpx2+/– mice strongly indicates that Gpx2 affords resistance to GI pathology in Se-depleted mice. The presence of Gpx2 seems to be an alternative mechanism of resistance to Se depletion, complementing the redistribution of Se to brain and testis by selenoprotein P (36). Interestingly, the use of Gpx2 does not require sequestering of Se by the tissue but seems to rely on the potency and robustness of Gpx activity and strategic placement of Gpx2 to operate at seemingly trace levels.
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FOOTNOTES |
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3 Abbreviations used: GI, gastrointestinal; Gpx, Se-dependent glutathione peroxidase; Gpx1, the gene encoding the ubiquitous Gpx1; Gpx2, the gene encoding an epithelium-specific Gpx2 or Gpx-GI; Gpx1–/–Gpx2–/–, homozygous targeted mutation of both Gpx1 and Gpx2 genes; GST, glutathione S-transferase; IBD, inflammatory bowel disease; MPO, myeloperoxidase; PCR, polymerase chain reaction; Se, selenium.
Manuscript received 1 September 2004. Initial review completed 20 October 2004. Revision accepted 14 January 2005.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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 A. Singh, T. Rangasamy, R. K. Thimmulappa, H. Lee, W. O. Osburn, R. Brigelius-Flohe, T. W. Kensler, M. Yamamoto, and S. Biswal Glutathione Peroxidase 2, the Major Cigarette Smoke-Inducible Isoform of GPX in Lungs, Is Regulated by Nrf2 Am. J. Respir. Cell Mol. Biol., December 1, 2006; 35(6): 639 - 650. [Abstract] [Full Text] [PDF]
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D.-H. Lee, R. S. Esworthy, C. Chu, G. P. Pfeifer, and F.-F. Chu Mutation accumulation in the intestine and colon of mice deficient in two intracellular glutathione peroxidases. Cancer Res., October 15, 2006; 66(20): 9845 - 9851. [Abstract] [Full Text] [PDF]
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