Metabolomics analyses of cancer tissue from patients with colorectal cancer.

The alteration of metabolism is essential for the initiation and progression of numerous types of cancer, including colorectal cancer (CRC). Metabolomics has been used to study CRC. At present, the reprogramming of the metabolism in CRC remains to be fully elucidated. In the present study, comprehensive untargeted metabolomics analysis was performed on the paired CRC tissues and adjacent normal tissues from patients with CRC (n=35) using ultra­high­performance liquid chromatography­mass spectrometry. Subsequently, bioinformatic analysis was performed on the differentially expressed metabolites. The changes in these differential metabolites were compared among groups of patients based on sex, anatomical tumor location, grade of tumor differentiation and stage of disease. A total of 927 metabolites were detected in the tissue samples, and 24 metabolites in the CRC tissue were significantly different compared with the adjacent normal tissue. The present study revealed that the levels of three amino acid metabolites were increased in the CRC tissue, specifically, N­α­acetyl­Îµ­(2­propenal)­Lys, cyclo(Glu­Glu) and cyclo(Phe­Glu). The metabolites with decreased levels in the CRC tissue included quinaldic acid (also referred to as quinoline­2­carboxilic acid), 17α­ and 17ß­estradiol, which are associated with tumor suppression activities, as well as other metabolites such as, anhydro­ß­glucose, Asp­Arg, lysophosphatidylcholine, lysophosphatidylethanolamine (lysoPE), lysophosphatidylinositol, carnitine, 5'­deoxy­5'­(methylthio) adenosine, 2'­deoxyinosine­5'­monophosphate and thiamine monophosphate. There was no difference in the levels of the differential metabolites between male and female patients. The differentiation of CRC also showed no impact on the levels of the differential metabolites. The levels of lysoPE were increased in the right side of the colon compared with the left side of the colon and rectum. Analysis of the different tumor stages indicated that 2­aminobenzenesulfonic acid, P­sulfanilic acid and quinoline­4­carboxylic acid were decreased in stage I CRC tissue compared with stage II, III and IV CRC tissue. The levels of N­α­acetyl­Îµ­(2­propenal)­Lys, methylcysteine and 5'­deoxy­5'­(methylthio) adenosine varied at different stages of tumorigenesis. These differential metabolites were implicated in multiple metabolism pathways, including carbohydrate, amino acid, lipid, nucleotide and hormone. In conclusion, the present study demonstrated that CRC tumors had altered metabolites compared with normal tissue. The data from the metabolic profile of CRC tissues in the present study provided supportive evidence to understand tumorigenesis.

Increased NOX1 and DUOX2 expression in the colonic mucosa of patients with chronic functional constipation.

To determine whether oxidative stress and inflammation are associated with constipation by examining the expression of the main producers of reactive oxygen species, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, and pro-inflammatory cytokines in the colon of patients with chronic functional constipation. The colonic biopsies were collected from 32 patients with chronic functional constipation and 30 healthy subjects who underwent colonoscopy. Colonic mucosal histology was observed. Interleukin (IL)-1ß, IL-6, IL-8 messenger RNA (mRNA), and 4 members of NADPH oxidase (NOX1, NOX2, DUOX2, and NOX4) protein and mRNA were assessed by immunohistochemistry, western blotting, and reverse transcription polymerase chain reaction. The tissues from both patients and healthy subjects showed normal histological structure without increase of inflammatory cells. NOX1 protein and mRNA levels were significantly increased compared to controls (P < .05). DUOX2 protein, but not mRNA, was increased by 2-fold compared to controls (P < .05). The levels of NOX2 and NOX4 protein and mRNA demonstrated no significant difference between patients and control subjects. The levels of IL-1ß and IL-6 mRNA were significantly higher in constipation patients (P < .05), while IL-8 mRNA level was no different between the 2 groups. NADPH oxidase and pro-inflammatory cytokine might be involved in the pathogeneses of chronic functional constipation.

The beginning of GPX2 and 30 years later.

We published the first paper to characterize GPX2 (aka GSHPx-GI) as a selenoenzyme with glutathione peroxidase activity in 1993. Among the four Se-GPX isozymes, GPX1-4, GPX1 and GPX2 are closely related in terms of structure, substrate specificities, and subcellular localization. What sets them apart are distinct patterns of gene regulation, tissue distribution and response to selenium. While we identified the digestive tract epithelium as the main site of GPX2 expression, later work has shown GPX2 is found more widely in epithelial tissues with concentration of expression in stem cell and proliferative compartments. GPX2 expression is regulated over a wide range of levels by many pathways, including NRF2, WNT, p53, RARE and this often results in attaching undue significance to GPX2 as GPX2 is only a part of a system of hydroperoxidase activities, including GPX1, peroxiredoxins and catalase. These other activities may play equal or greater roles, particularly in cell lines cultured without selenium supplementation and often with very low GPX2 levels. This could be assessed by examining levels of mRNA and protein among these various peroxidases at the outset of studies. As an example, it was found that GPX1 responds to the absence of GPX2 in mouse ileum and colon epithelium with higher expression. As such, both Gpx1 and Gpx2 had to be knocked out in mice to produce ileocolitis. However, we note that the actual role of GPX1 and GPX2 in relation to peroxiredoxin function is unclear. There may be an interdependence that requires only low amounts of GPX1 and/or GPX2 in a supporting role to maintain proper peroxiredoxin function. GPX2 levels may be prognostic for cancer progression in colon, breast, prostate and liver, however, there is no consistent trend for higher or lower levels to be favorable.

Serum pepsinogen II levels are doubled with Helicobacter pylori infection in an asymptomatic population of 40,383 Chinese subjects.

ABSTRACT: Pepsinogen (PG) I and II are crucial in the gastric digestive processes. This study is to examine the relationship of serum PGI, PGII, and PGI/PGII ratio with Helicobacter pylori (Hp) infection, age, sex, and body mass index (BMI) in subjects in Beijing, China.A total of 40,383 asymptomatic subjects, who underwent medical examination in Beijing Rehabilitation Hospital, were included in this study. Serum PG levels were measured using chemoluminescence techniques. The age, sex, and BMI data were collected, and Hp infection was identified with 13C-urea breath test. Statistical analysis was conducted with Python, Pandas and Seaborn software.Asymptomatic subjects with Hp infection (Hp+) had a significantly higher level of PGI in the serum (111 ng/mL [median]) than those without Hp infection (Hp-) (94 ng/mL, P < .001). The asymptomatic Hp+ subjects had 2-fold higher PGII levels (7.2 ng/mL) than Hp- subjects (3.2 ng/mL, P < .001). These changes produced significantly lower PGI/II ratio in Hp+ patients than in Hp- subjects (16:30, P < .001). The serum PGI and PGII levels were higher in males than in females (PGI: 104 ng/mL vs 95 ng/mL, PGII: 4.3 ng/mL vs 3.7 ng/mL, both P < .001), PGI/II ratio of males is at 95% of that in females (P < .001). PGI and PGII levels gradually increased in older people (P < .001), whereas the PGI/II ratio decreased significantly with age (P < .001). The levels of the two serum PGs were decreased and the ratio increased when BMI were higher than 28 kg/cm2 (P < .05).The levels of serum PGI, especial PGII, were increased by Hp infection, and also influenced by age, sex, and BMI. Therefore, these influencing factors should be considered during clinical practice.

Role of the microbiota in ileitis of a mouse model of inflammatory bowel disease-Glutathione peroxide isoenzymes 1 and 2-double knockout mice on a C57BL background.

C57Bl6 (B6) mice devoid of glutathione peroxidases 1 and 2 (Gpx1/2-DKO) develop ileitis after weaning. We previously showed germ-free Gpx1/2-DKO mice of mixed B6.129 background did not develop ileocolitis. Here, we examine the composition of the ileitis provoking microbiota in B6 Gpx1/2-DKO mice. DNA was isolated from the ileum fecal stream and subjected to high-throughput sequencing of the V3 and V4 regions of the 16S rRNA gene to determine the abundance of operational taxonomic units (OTUs). We analyzed the role of bacteria by comparing the microbiomes of the DKO and pathology-free non-DKO mice. Mice were treated with metronidazole, streptomycin, and vancomycin to alter pathology and correlate the OTU abundances with pathology levels. Principal component analysis based on Jaccard distance of abundance showed 3 distinct outcomes relative to the source Gpx1/2-DKO microbiome. Association analyses of pathology and abundance of OTUs served to rule out 7-11 of 24 OTUs for involvement in the ileitis. Collections of OTUs were identified that appeared to be linked to ileitis in this animal model and would be classified as commensals. In Gpx1/2-DKO mice, host oxidant generation from NOX1 and DUOX2 in response to commensals may compromise the ileum epithelial barrier, a role generally ascribed to oxidants generated from mitochondria, NOX2 and endoplasmic reticulum stress in response to presumptive pathogens in IBD. Elevated oxidant levels may contribute to epithelial cell shedding, which is strongly associated with progress toward inflammation in Gpx1/2-DKO mice and predictive of relapse in IBD by allowing leakage of microbial components into the submucosa.

Control of doxorubicin-induced, reactive oxygen-related apoptosis by glutathione peroxidase 1 in cardiac fibroblasts.

Reactive oxygen formation plays a mechanistic role in the cardiotoxicity of doxorubicin, a chemotherapeutic agent that remains an important component of treatment programs for breast cancer and hematopoietic malignancies. To examine the role of doxorubicin-induced reactive oxygen species (ROS) in drug-related cardiac apoptosis, murine embryonic fibroblast cell lines were derived from the hearts of glutathione peroxidase 1 (Gpx-1) knockout mice. Cells from homozygous Gpx-1 knockout mice and parental animals were propagated with (Se+) and without (Se-) 100 nM sodium selenite. Activity levels of the peroxide detoxifying selenoprotein glutathione peroxidase (GSHPx) were marginally detectable (<1.6 nmol/min/mg) in fibroblasts from homozygous knockout animals whether or not cells were supplemented with selenium. GSHPx activity in Se- cells from parental murine fibroblasts was also <1.6 nmol/min/mg, whereas GSHPx levels in Se+ parental murine fibroblasts were 12.9 ± 2.7 nmol/min/mg (mean ± SE; P < 0.05). Catalase, superoxide dismutase, glutathione reductase, glutathione S-transferase, glucose 6-phosphate dehydrogenase, and reduced glutathione activities did not differ amongst the four cell lines. Reactive oxygen production increased from 908 ± 122 (arbitrary units) for untreated control cells to 1668 ± 54 following exposure to 1 µM doxorubicin for 24 h in parental fibroblasts not supplemented with selenium (P < 0.03); reactive oxygen formation in doxorubicin-treated parental fibroblasts propagated in selenium was 996 ± 69 (P = not significant compared to untreated control cells). Reactive oxygen levels in homozygous Gpx-1 knockout fibroblasts, irrespective of selenium supplementation status, were increased and equivalent to that in selenium deficient wild type fibroblasts. When cardiac fibroblasts were exposed to doxorubicin (0.05 µM) for 96 h and examined for cell cycle alterations by flow cytometry, and apoptosis by TUNEL assay, marked G2 arrest and TUNEL positivity were observed in knockout fibroblasts in the presence or absence of supplemental selenium, and in parental fibroblasts propagated without selenium. Parental fibroblasts propagated with selenium and exposed to the same concentration of doxorubicin demonstrated modest TUNEL positivity and substantially diminished amounts of low molecular weight DNA. These results were replicated in cardiac fibroblasts exposed to doxorubicin (1-2 µM) for 2 h (to mimic clinical drug dosing schedules) and examined 96 h following initiation of drug exposure. Doxorubicin uptake in cardiac fibroblasts was similar irrespective of the mRNA expression level or activity of GSHPx. These experiments suggest that the intracellular levels of doxorubicin-induced reactive oxygen species (ROS) are modulated by GSHPx and play an important role in doxorubicin-related apoptosis and altered cell cycle progression in murine cardiac fibroblasts.

Dexamethasone and Tofacitinib suppress NADPH oxidase expression and alleviate very-early-onset ileocolitis in mice deficient in GSH peroxidase 1 and 2.

C57BL6/J (B6) mice lacking Se-dependent GSH peroxidase 1 and 2 (GPx1/2-DKO) develop mild to moderate ileocolitis around weaning. These DKO mice have a disease resembling human very-early-onset inflammatory bowel disease (VEOIBD), which is associated with mutations in NADPH oxidase genes. Drugs including dexamethasone (Dex), Tofacitinib (Tofa; a Janus kinase/JAK inhibitor) and anti-TNF antibody are effective to treat adult, but not pediatric IBD. AIMS: To test the efficacy of hydrophobic Dex and hydrophilic Dex phosphate (Dex phos), Tofa, anti-Tnf Ab, Noxa1ds-TAT and gp91ds-TAT peptides (inhibiting NOX1 and NOX2 assembly respectively), antioxidant MJ33 and ML090, and pifithrin-α (p53 inhibitor) on alleviation of gut inflammation in DKO weanlings. MAIN METHODS: All treatments began on 22-day-old GPx1/2-DKO mice. The mouse intestine pathology was compared between the drug- and vehicle-treated groups after six or thirteen days of treatment. KEY FINDINGS: Among all drugs tested, Dex, Dex phos and Tofa were the strongest to suppress ileocolitis in the DKO weanlings. Dex, Dex phos and Tofa inhibited crypt apoptosis and increased crypt density. Dex or Dex phos alone also inhibited cell proliferation, exfoliation and crypt abscess in the ileum. Dex, but not Tofa, retarded mouse growth. Both Dex and Tofa inhibited ileum Nox1, Nox4 and Duox2, but not Nox2 gene expression. Noxa1ds-TAT and gp91ds-TAT peptides as well as MJ33 had subtle effect on suppressing pathology, while others had negligible effect. SIGNIFICANCE: These findings suggest that NADPH oxidases can be novel drug targets for pediatric IBD therapy, and Tofa may be considered for treating VEOIBD.

5-Hydroxytryptamine (5-HT)-exacerbated DSS-induced colitis is associated with elevated NADPH oxidase expression in the colon.

AIM: This study investigated the impact of 5-hydroxytryptamine (5-HT) on the expression of NOXs in dextran sulfate sodium (DSS)-induced colitis in mice. METHODS: C57BL/6J (B6) mice at 6 to 8 weeks of age were treated with 5-HT, DSS, or DSS + 5-HT. After 6-day treatment, the severity of colitis, infiltration of leukocytes, and messenger RNA (mRNA) and/or protein levels of Nox1, Nox2, Nox4, and Duox2 were analyzed in the colon by real-time quantitative polymerase chain reaction (qPCR), immunohistochemistry (IHC), and Western blot analysis. The direct effect of 5-HT on NOX gene and protein expression in HT-29 colon cancer cells and in U-937 macrophage cells were determined by qPCR and Western blot analysis. RESULTS: Mice treated with 5-HT alone did not develop colitis, while those treated with 1.0% DSS or DSS + 5-HT had mild and severe colitis, respectively. All treated mice had more myeloperoxidase-positive cells in the colon compared with untreated control mice. Mice treated with 5-HT or DSS alone had increased Nox2 and Nox4 mRNA and protein levels in the colon determined by qPCR, IHC, and Western blot analysis. These two Nox expressions were even higher in mice treated with DSS + 5-HT, while the expression levels of epithelium-localized Nox1 and Duox2 tended to decrease. Additionally, mice treated with 5-HT alone had elevated Nox1 and Duox2 expression as shown by qPCR and IHC. However, these gene expressions were diminished in DSS + 5-HT-treated mice likely due to erosion of epithelium. Furthermore, 5-HT induced NOX1 and DUOX2 gene and protein expression in HT-29 colon cancer epithelial cells, whereas induced NOX2 and NOX4 gene and protein expression in U-937 cells. CONCLUSION: As 5-HT induced NOX1 and DUOX2 gene and protein expression in colon epithelial and HT-29 cells, NOX2 and NOX4 in the infiltrating leukocyte in mouse colon and in U-937 cells, the exacerbate colitis induced by combined 5-HT and DSS treatment might be relevant to increased NOX expression in mice colons.

Mice Deficient in Cyp4a14 Have An Increased Number of Goblet Cells and Attenuated Dextran Sulfate Sodium-Induced Colitis.

BACKGROUND/AIMS: Cyp4a14 is a member of cytochrome P450 (Cyp450) enzyme superfamily that possesses NADPH monooxygenase activity, which catalyzes omega-hydroxylation of medium-chain fatty acids and arachidonic acid. Study suggests that down-regulation of Cyp4a14 has an anti-inflammatory response in intestine. The present study was to test the function of Cyp4a14 in dextran sulfate sodium (DSS)-induced colitis. METHODS: Female Cyp4a14-knockout (KO) and wild-type (WT) mice were treated with DSS for 6 days to induce colitis. The colon of mice was histologically observed by hematoxylin and eosin (H&E) and periodic acid Schiff (PAS) staining. The serum malondialdehyde (MDA), an endogenous indicator of oxidative stress, was chemically measured. Proinflammatory and NADPH oxidase genes were examined by quantitative polymerase chain reaction (qPCR). RESULTS: Cyp4a14-KO mice had a significantly higher number of goblet cells in the colon and were more resistant to DSS-induced colitis compared with the WT mice. The DSS-treated KO mice had lower levels of MDA. Consistent with the milder inflammatory pathological changes, DSS-treated KO mice had lower levels of IL-1ß, IL-6 and TNF-α mRNA in the liver and the colon. Moreover, the colon of DSS-treated Cyp4a14-KO and WT mice had higher mRNA levels of two members of NADPH oxidases, Nox2 and Nox4, suggesting that both Nox2 and Nox4 are inflammatory markers. By contrast, DSS-treated WT and KO mice had drastically decreased epithelium-localized Nox1 and dual oxidase (Duox) 2 mRNA levels, coinciding with the erosion of the mucosa induced by DSS. CONCLUSION: These results suggests a hypothesis that the increased goblet cell in the colon of Cyp4a14-KO mice provides protection from mucosal injury and Cyp4a14-increased oxidative stress exacerbates DSS-induced colitis. Therefore, Cyp4a14 may represent a potential target for treating colitis.

Expression of inositol-requiring enzyme 1ß is downregulated in colorectal cancer.

The endoplasmic reticulum stress inositol-requiring enzyme (IRE) 1α/X-box binding protein (XBP) 1 signaling pathway is involved in the tumorigenesis of breast and prostate cancer. Mucin 2 (MUC2) protects colon tissues from the formation of tumors. In human colorectal cancer (CRC) the role of IRE1α, and its analogue, IRE1ß, has yet to be elucidated. In the present study, the expression levels of IRE1α, IRE1ß, un-spliced XBP1u, spliced XBP1s and MUC2 in surgically resected cancerous and adjacent non-cancerous tissues from patients with CRC were investigated. The IRE1α, IRE1ß, XBP1u, XBP1s and MUC2 mRNA expression levels were determined using reverse transcription-quantitative polymerase chain reaction, and the protein expression levels were detected using immunohistochemistry and western blotting. The association between the expression levels of IRE1α, IRE1ß and MUC2 and the clinicopathological features of patients with CRC was subsequently analyzed. The mRNA expression levels of IRE1ß and MUC2 were decreased by ~2.1 and ~4.5-fold in CRC tissues, respectively, as compared with the adjacent normal tissues. The protein expression levels of IRE1ß and MUC2 were decreased by ~8.0 and ~2.0-fold in the CRC tissues, respectively. IRE1ß mRNA expression levels were positively correlated with MUC2 mRNA expression levels. IRE1ß expression levels were revealed to be significantly associated with lymph node metastasis, tumor stage and histological differentiation. However, IRE1α, XBP1u and XBP1s mRNA and IRE1α protein expression levels were not observed to significantly differ between cancerous tissues and the adjacent normal tissues. The results indicated that the expression of IRE1ß, but not IRE1α, may protect colon tissue from developing CRC by inducing MUC2 expression. Therefore, decreased IRE1ß expression levels may be associated with the development of CRC through the inhibition of MUC2 expression.

Deficiency in Duox2 activity alleviates ileitis in GPx1- and GPx2-knockout mice without affecting apoptosis incidence in the crypt epithelium.

Mice deficient in glutathione peroxidase (GPx)-1 and -2 (GPx1-/-GPx2-/- double knockout or DKO mice) develop very-early-onset (VEO) ileocolitis, suggesting that lack of defense against reactive oxygen species (ROS) renders susceptibility to intestinal inflammation. Two members of ROS-generating NADPH oxidase family, NOX1 and DUOX2, are highly inducible in the intestinal epithelium. Previously, we reported that Nox1 deficiency ameliorated the pathology in DKO mice (Nox1-TKO). The role of Duox2 in ileocolitis of the DKO mice is evaluated here in Duoxa-TKO mice by breeding DKO mice with Duoxa-/- mice (Duoxa-TKO), which do not have Duox2 activity. Similar to Nox1-TKO mice, Duoxa-TKO mice no longer have growth retardation, shortened intestine, exfoliation of crypt epithelium, crypt abscesses and depletion of goblet cells manifested in DKO mice by 35 days of age. Unlike Nox1-TKO mice, Duoxa-TKO mice still have rampant crypt apoptosis, elevated proliferation, partial loss of Paneth cells and diminished crypt density. Treating DKO mice with NOX inhibitors (di-2-thienyliodonium/DTI and thioridazine/THZ) and an antioxidant (mitoquinone/MitoQ) significantly reduced gut pathology. Furthermore, in the inflamed human colon, DUOX protein expression is highly elevated in the apical, lateral and perinuclear membrane along the whole length of gland. Taken together, we conclude that exfoliation of crypt epithelium, but not crypt apoptosis, is a major contributor to inflammation. Both Nox1 and Duox2 induce exfoliation of crypt epithelium, but only Nox1 induces apoptosis. NOX1 and DUOX2 may be potential therapeutic targets for treating ileocolitis in human patients suffering inflammatory bowel disease (IBD).

NADPH oxidase-1 deficiency offers little protection in Salmonella typhimurium-induced typhlitis in mice.

AIM: To test whether Nox1 plays a role in typhlitis induced by Salmonella enterica serovar Typhimurium (S. Tm) in a mouse model. METHODS: Eight-week-old male wild-type (WT) and Nox1 knockout (KO) C57BL6/J (B6) mice were administered metronidazole water for 4 d to make them susceptible to S. Tm infection by the oral route. The mice were given plain water and administered with 4 different doses of S. Tm by oral gavage. The mice were followed for another 4 d. From the time of the metronidazole application, the mice were observed twice daily and weighed daily. The ileum, cecum and colon were removed for sampling at the fourth day post-inoculation. Portions of all three tissues were fixed for histology and placed in RNAlater for mRNA/cDNA preparation and quantitative real-time PCR. The contents of the cecum were recovered for estimation of S. Tm CFU. RESULTS: We found Nox1-knockout (Nox1-KO) mice were not more sensitive to S. Tm colonization and infection than WT B6 mice. This conclusion is based on the following observations: (1) S. Tm-infection induced similar weight loss in Nox1-KO mice compared to WT mice; (2) the same S. Tm CFU was recovered from the cecal content of Nox1-KO and WT mice regardless of the inoculation dose, except the lowest inoculation dose (2 × 106 CFU) for which the Nox1-KO had one-log lower CFU than WT mice; (3) there is no difference in cecal pathology between WT and Nox1-KO groups; and (4) there are no S. Tm infection-induced changes in gene expression levels (IL-1b, TNF-α, and Duox2) between WT and Nox1-KO groups. The Alpi gene expression was more suppressed by S. Tm treatment in WT than the Nox1-KO cecum. CONCLUSION: Nox1 does not protect mice from S. Tm colonization. Nox1-KO provides a very minor protective effect against S. Tm infection. Using NOX1-specific inhibitors for colitis therapy should not increase risks in bacterial infection.

Serotonin-Exacerbated DSS-Induced Colitis Is Associated with Increase in MMP-3 and MMP-9 Expression in the Mouse Colon.

Background. 5-HT enhances dextran sulfate sodium- (DSS-) induced colitis and is involved in inflammatory bowel disease (IBD). Matrix metalloproteinases (MMPs) play roles in the process of intestinal inflammation. Aims. To examine whether 5-HT induces MMPs expression in mouse colon to enhance DSS-induced colitis. Materials and Methods. C57BL/6J (B6) mice were treated with either low-dose (1.0 mg/kg) or high-dose (2.0 mg/kg) 5-HT by enema, low-dose (1.0%) or high-dose (2.5%) DSS, or combined low-dose (1.0%) DSS and (1.0 mg/kg) 5-HT. Mouse colitis was analyzed. MMPs and tissue inhibitors of MMPs (TIMPs) mRNA were measured by real-time quantitative RT-PCR in mouse colon and in human Caco-2 cells and neutrophils. MMP-3 and MMP-9 protein levels were quantified from immunohistochemistry (IHC) images of mouse colons. Results. 5-HT exacerbated DSS-induced colitis, low-dose 5-HT induces both MMP-3 and MMP-9, and high-dose 5-HT only increased MMP-3 mRNA expression in mouse colon. Mouse colon MMP-3 and MMP-9 protein levels were also elevated by 5-HT treatment. The MMP-2, TIMP-1, and TIMP-2 mRNA levels were increased in the inflamed colon. 5-HT induced MMP-3 and MMP-9 mRNA expression in Caco-2 and human neutrophils, respectively, in vitro. Conclusion. 5-HT induced MMP-3 and MMP-9 expression in mouse colon; these elevated MMPs may contribute to DSS-induced colitis.

The Gastric Mucosa from Patients Infected with CagA+ or VacA+ Helicobacter pylori Has a Lower Level of Dual Oxidase-2 Expression than Uninfected or Infected with CagA-/VacA- H. pylori.

BACKGROUND: Helicobacter pylori (H. pylori) is a well-recognized gastroduodenal pathogen and class I carcinogen. Dual oxidase-2 (DUOX2), a member of NADPH oxidase family, has several critical physiological functions, including thyroid hormone biosynthesis and host mucosal defense. AIM: To investigate the effect of H. pylori infection on DUOX2 gene expression in human stomach. MATERIALS AND METHODS: The biopsies were obtained from patients who underwent endoscopic diagnosis. The patient serum was assayed for two virulence factors of H. pylori, CagA IgG and VacA. The inflammation in gastric mucosa was analyzed with histology. Real-time quantitative PCR was used to detect the expression of three members of NADPH oxidase, NOX1, NOX2, and DUOX2, as well as lactoperoxidase (LPO) in the gastric mucosa. NOX2, DUOX2, and myeloperoxidase (MPO) protein levels were quantified by Western blots or immunohistochemistry. RESULTS: The H. pylori-infected gastric mucosa had more severe inflammation than uninfected samples. However, the expression of DUOX2 mRNA and protein was lower in gastric mucosa of patients with H. pylori infection compared to the uninfected. Among the H. pylori-infected patients, those having CagA IgG or VacA in the serum had lower DUOX2 expression levels than those infected with H. pylori without either virulence factor. The NOX2 and MPO levels were higher in those patients infected with H. pylori irrespective of the virulence factors than those uninfected patients. NOX1 and LPO mRNA were undetectable in the gastric mucosa. CONCLUSION: CagA+ or VacA+ H. pylori in the stomach of patients may suppress DUOX2 expression to promote its own survival. Increased NOX2 could not eliminate H. pylori infection.

Nox1 causes ileocolitis in mice deficient in glutathione peroxidase-1 and -2.

We previously reported that mice deficient in two Se-dependent glutathione peroxidases, GPx1 and GPx2, have spontaneous ileocolitis. Disease severity depends on mouse genetic background. Whereas C57BL/6J (B6) GPx1/2-double-knockout (DKO) mice have moderate ileitis and mild colitis, 129S1Svlm/J (129) DKO mice have severe ileocolitis. Because GPx's are antioxidant enzymes, we hypothesized that elevated reactive oxygen species trigger inflammation in these DKO mice. To test whether NADPH oxidase 1 (Nox1) contributes to colitis, we generated B6 triple-KO (TKO) mice to study their phenotype. Because the Nox1 gene is X-linked, we analyzed the effects of Nox1 on male B6 TKO mice and female B6 DKO mice with the Nox1(+/-) (het-TKO) genotype. We found that the male TKO and female het-TKO mice are virtually disease-free when monitored from 8 through 50 days of age. Male TKO and female het-TKO mice have nearly no signs of disease (e.g., lethargy and perianal alopecia) that are often exhibited in the DKO mice; further, the slower growth rate of DKO mice is almost completely eliminated in male TKO and female het-TKO mice. Male TKO and female het-TKO mice no longer have the shortened small intestine present in the DKO mice. Finally, the pathological characteristics of the DKO ileum, including the high level of crypt apoptosis (analyzed by apoptotic figures, TUNEL, and cleaved caspase-3 immunohistochemical staining), high numbers of Ki-67-positive crypt epithelium cells, and elevated levels of monocytes expressing myeloperoxidase, are all significantly decreased in male TKO mice. The attenuated ileal and colonic pathology is also evident in female het-DKO mice. Furthermore, the male DKO ileum has eightfold higher TNF cytokine levels than TKO ileum. Nox1 mRNA is highly elevated in both B6 and 129 DKO ileum compared to wild-type mouse ileum. Taking these results together, we propose that ileocolitis in the DKO mice is caused by Nox1, which is induced by TNF. The milder disease in female het-TKO intestine is probably due to random or imprinted X-chromosome inactivation, which produces mosaic Nox1 expression.

The Gdac1 locus modifies spontaneous and Salmonella-induced colitis in mice deficient in either Gpx2 or Gpx1 gene.

We previously identified the Gdac1 (Gpx-deficiency-associated colitis 1) locus, which influences the severity of spontaneous colitis in Gpx1- and Gpx2-double-knockout (Gpx1/2-DKO) mice. Congenic Gpx1/2-DKO mice in the 129S1/SvImJ (129) background but carrying the Gdac1(B6) allele have milder spontaneous colitis than 129 Gpx1/2-DKO mice carrying the Gdac1(129) allele. Here, we evaluated the effect of the Gdac1(B6) allele on 129 strain non-DKO mice that had a wild-type (WT) Gpx1 or Gpx2 allele and WT mice. We found that the congenic Gdac1(B6) Gpx2-KO, Gpx1-KO, and WT mice also had better health than the corresponding 129 mice measured by at least one of the parameters including disease signs, colon length, or weight gain. The Gdac1(B6) allele prevented loss of goblet cells and crypt epithelium exfoliation in the Gpx1/2-DKO mice, but did not affect epithelial cell apoptosis or proliferation. Because Gdac1(B6) affects gut dysbiosis in the DKO mice, we then tested its impact on bacteria-induced colitis in non-DKO mice. First, we found both Gpx1-KO and Gpx2-KO mice were susceptible to Salmonella enterica serotype typhimurium (S. Tm)-induced colitis under conditions where WT B6 and 129 mice were resistant. Second, the S. Tm-infected Gdac1(B6) Gpx1-KO mice had stronger inflammatory responses than 129 Gpx1-KO or 129 Gpx2-KO with both Gdac1 alleles and WT mice by having higher mRNA levels of Nod2, Nox2, Tnf, and Cox2. We conclude that the Gdac1 locus affects both spontaneous and S. Tm-induced colitis in 129 non-DKO mice, although in opposite directions.

Deletion of glutathione peroxidase-2 inhibits azoxymethane-induced colon cancer development.

The selenoprotein glutathione peroxidase-2 (GPx2) appears to have a dual role in carcinogenesis. While it protected mice from colon cancer in a model of inflammation-triggered carcinogenesis (azoxymethane and dextran sodium sulfate treatment), it promoted growth of xenografted tumor cells. Therefore, we analyzed the effect of GPx2 in a mouse model mimicking sporadic colorectal cancer (azoxymethane-treatment only). GPx2-knockout (KO) and wild-type (WT) mice were adjusted to an either marginally deficient (-Se), adequate (+Se), or supranutritional (++Se) selenium status and were treated six times with azoxymethane (AOM) to induce tumor development. In the -Se and ++Se groups, the number of tumors was significantly lower in GPx2-KO than in respective WT mice. On the +Se diet, the number of dysplastic crypts was reduced in GPx2-KO mice. This may be explained by more basal and AOM-induced apoptotic cell death in GPx2-KO mice that eliminates damaged or pre-malignant epithelial cells. In WT dysplastic crypts GPx2 was up-regulated in comparison to normal crypts which might be an attempt to suppress apoptosis. In contrast, in the +Se groups tumor numbers were similar in both genotypes but tumor size was larger in GPx2-KO mice. The latter was associated with an inflammatory and tumor-promoting environment as obvious from infiltrated inflammatory cells in the intestinal mucosa of GPx2-KO mice even without any treatment and characterized as low-grade inflammation. In WT mice the number of tumors tended to be lowest in +Se compared to -Se and ++Se feeding indicating that selenium might delay tumorigenesis only in the adequate status. In conclusion, the role of GPx2 and presumably also of selenium depends on the cancer stage and obviously on the involvement of inflammation.

Analysis of candidate colitis genes in the Gdac1 locus of mice deficient in glutathione peroxidase-1 and -2.

BACKGROUND: Mice that are deficient for glutathione peroxidases 1 and 2 (GPX) show large variations in the penetrance and severity of colitis in C57BL/6J and 129S1/SvImJ backgrounds. We mapped a locus contributing to this difference to distal chromosome 2 (∼119-133 mbp) and named it glutathione peroxidase-deficiency-associated colitis 1 (Gdac1). The aim of this study was to identify the best gene candidates within the Gdac1 locus contributing to the murine colitis phenotype. METHOD/PRINCIPAL FINDINGS: We refined the boundaries of Gdac1 to 118-125 mbp (95% confidence interval) by increasing sample size and marker density across the interval. The narrowed region contains 128 well-annotated protein coding genes but it excludes Fermt1, a human inflammatory bowel disease candidate that was within the original boundaries of Gdac1. The locus we identified may be the Cdcs3 locus mapped by others studying IL10-knockout mice. Using in silico analysis of the 128 genes, based on published colon expression data, the relevance of pathways to colitis, gene mutations, presence of non-synonymous-single-nucleotide polymorphisms (nsSNPs) and whether the nsSNPs are predicted to have an impact on protein function or expression, we excluded 42 genes. Based on a similar analysis, twenty-five genes from the remaining 86 genes were analyzed for expression-quantitative-trait loci, and another 15 genes were excluded. CONCLUSION/SIGNIFICANCE: Among the remaining 10 genes, we identified Pla2g4f and Duox2 as the most likely colitis gene candidates, because GPX metabolizes PLA2G4F and DUOX2 products. Pla2g4f is a phospholipase A2 that has three potentially significant nsSNP variants and showed expression differences across mouse strains. PLA2G4F produces arachidonic acid, which is a substrate for lipoxygenases and, in turn, for GPXs. DUOX2 produces H(2)O(2) and may control microbial populations. DUOX-1 and -2 control microbial populations in mammalian lung and in the gut of several insects and zebrafish. Dysbiosis is a phenotype that differentiates 129S1/SvImJ from C57BL/6J and may be due to strain differences in DUOX2 activity.

Expression of lactoperoxidase in differentiated mouse colon epithelial cells.

Lactoperoxidase (LPO) is known to be present in secreted fluids, such as milk and saliva. Functionally, LPO teams up with dual oxidases (DUOXs) to generate bactericidal hypothiocyanite in the presence of thiocyanate. DUOX2 is expressed in intestinal epithelium, but there is little information on LPO expression in this tissue. To fill the gap of knowledge, we have analyzed Lpo gene expression and its regulation in mouse intestine. In wild-type (WT) C57BL/6 (B6) mouse intestine, an appreciable level of mouse Lpo gene expression was detected in the colon, but not the ileum. However, in B6 mice deficient in glutathione peroxidase (GPx)-1 and -2, GPx1/2-double-knockout (DKO), which had intestinal pathology, the colon Lpo mRNA levels increased 5- to 12-fold depending on mouse age. The Lpo mRNA levels in WT and DKO 129S1/SvlmJ (129) colon were even higher, 9- and 5-fold, than in B6 DKO colon. Higher levels of Lpo protein and enzymatic activity were also detected in the 129 mouse colon compared to B6 colon. Lpo protein was expressed in the differentiated colon epithelial cells, away from the crypt base, as shown by immunohistochemistry. Similar to human LPO mRNA, mouse Lpo mRNA had multiple spliced forms, although only the full-length variant 1 was translated. Higher methylation was found in the 129 than in the B6 strain, in DKO than in control colon, and in older than in juvenile mice. However, methylation of the Lpo intragenic CpG island was not directly induced by inflammation, because dextran sulfate sodium-induced colitis did not increase DNA methylation in B6 DKO colon. Also, Lpo DNA methylation is not correlated with gene expression.

Glutathione peroxidase-2 and selenium decreased inflammation and tumors in a mouse model of inflammation-associated carcinogenesis whereas sulforaphane effects differed with selenium supply.

Chronic inflammation and selenium deficiency are considered as risk factors for colon cancer. The protective effect of selenium might be mediated by specific selenoproteins, such as glutathione peroxidases (GPx). GPx-1 and -2 double knockout, but not single knockout mice, spontaneously develop ileocolitis and intestinal cancer. Since GPx2 is induced by the chemopreventive sulforaphane (SFN) via the nuclear factor E2-related factor 2 (Nrf2)/Keap1 system, the susceptibility of GPx2-KO and wild-type (WT) mice to azoxymethane and dextran sulfate sodium (AOM/DSS)-induced colon carcinogenesis was tested under different selenium states and SFN applications. WT and GPx2-KO mice were grown on a selenium-poor, -adequate or -supranutritional diet. SFN application started either 1 week before (SFN4) or along with (SFN3) a single AOM application followed by DSS treatment for 1 week. Mice were assessed 3 weeks after AOM for colitis and Nrf2 target gene expression and after 12 weeks for tumorigenesis. NAD(P)H:quinone oxidoreductases, thioredoxin reductases and glutathione-S-transferases were upregulated in the ileum and/or colon by SFN, as was GPx2 in WT mice. Inflammation scores were more severe in GPx2-KO mice and highest in selenium-poor groups. Inflammation was enhanced by SFN4 in both genotypes under selenium restriction but decreased in selenium adequacy. Total tumor numbers were higher in GPx2-KO mice but diminished by increasing selenium in both genotypes. SFN3 reduced inflammation and tumor multiplicity in both Se-adequate genotypes. Tumor size was smaller in Se-poor GPx2-KO mice. It is concluded that GPx2, although supporting tumor growth, inhibits inflammation-mediated tumorigenesis, but the protective effect of selenium does not strictly depend on GPx2 expression. Similarly, SFN requires selenium but not GPx2 for being protective.