Charles G. Knutson

Infection-induced colitis in mice causes dynamic and tissue-specific changes in stress response and DNA damage leading to colon cancer

Helicobacter hepaticus-infected Rag2-/- mice emulate many aspects of human inflammatory bowel disease, including the development of colitis and colon cancer. To elucidate mechanisms of inflammation-induced carcinogenesis, we undertook a comprehensive analysis of histopathology, molecular damage, and gene expression changes during disease progression in these mice. Infected mice developed severe colitis and hepatitis by 10 wk post-infection, progressing into colon carcinoma by 20 wk post-infection, with pronounced pathology in the cecum and proximal colon marked by infiltration of neutrophils and macrophages. Transcriptional profiling revealed decreased expression of DNA repair and oxidative stress response genes in colon, but not in liver. Mass spectrometric analysis revealed higher levels of DNA and RNA damage products in liver compared to colon and infection-induced increases in 5-chlorocytosine in DNA and RNA and hypoxanthine in DNA. Paradoxically, infection was associated with decreased levels of DNA etheno adducts. Levels of nucleic acid damage from the same chemical class were strongly correlated in both liver and colon. The results support a model of inflammation-mediated carcinogenesis involving infiltration of phagocytes and generation of reactive species that cause local molecular damage leading to cell dysfunction, mutation, and cell death. There are strong correlations among histopathology, phagocyte infiltration, and damage chemistry that suggest a major role for neutrophils in inflammation-associated cancer progression. Further, paradoxical changes in nucleic acid damage were observed in tissue- and chemistry-specific patterns. The results also reveal features of cell stress response that point to microbial pathophysiology and mechanisms of cell senescence as important mechanistic links to cancer.

Chemical and cytokine features of innate immunity characterize serum and tissue profiles in inflammatory bowel disease

Significance Our study investigates chemical damage associated with chronic inflammation and relates these macromolecular damage products to inflammatory bowel disease activity. Using mice as a model system, we show that chronic inflammatory responses that are common to mice and humans produce similar types and quantities of damage products in both species. Additional analysis of signaling molecules in the serum and tissue of diseased samples highlights the role of the innate immune response in the overall pathology of inflammatory bowel disease. Inflammatory bowel disease (IBD) arises from inappropriate activation of the mucosal immune system resulting in a state of chronic inflammation with causal links to colon cancer. Helicobacter hepaticus-infected Rag2−/− mice emulate many aspects of human IBD, and our recent work using this experimental model highlights the importance of neutrophils in the pathology of colitis. To define molecular mechanisms linking colitis to the identity of disease biomarkers, we performed a translational comparison of protein expression and protein damage products in tissues of mice and human IBD patients. Analysis in inflamed mouse colons identified the neutrophil- and macrophage-derived damage products 3-chlorotyrosine (Cl-Tyr) and 3-nitrotyrosine, both of which increased with disease duration. Analysis also revealed higher Cl-Tyr levels in colon relative to serum in patients with ulcerative colitis and Crohn disease. The DNA chlorination damage product, 5-chloro-2′-deoxycytidine, was quantified in diseased human colon samples and found to be present at levels similar to those in inflamed mouse colons. Multivariate analysis of these markers, together with serum proteins and cytokines, revealed a general signature of activated innate immunity in human IBD. Signatures in ulcerative colitis sera were strongly suggestive of neutrophil activity, and those in Crohn disease and mouse sera were suggestive of both macrophage and neutrophil activity. These data point to innate immunity as a major determinant of serum and tissue profiles and provide insight into IBD disease processes.

Site-specific and redox-controlled S-nitrosation of thioredoxin

Protein S-nitrosation on cysteine residues has emerged as an important posttranslational modification in mammalian cells. Previous studies have suggested a primary role for thioredoxin (Trx) in controlling protein S-nitrosation reactions. Human Trx contains five conserved Cys, including two redox-active catalytic Cys (Cys32 and Cys35) and three non-active-site Cys (Cys62, Cys69, and Cys73), all of which have been reported as targets of S-nitrosation. Prior reports have studied thermodynamic end points of nitrosation reactions; however, the kinetics of Trx nitrosation has not previously been investigated. Using the transnitrosation agent, S-nitrosoglutathione, a kinetic analysis of the selectivity and redox dependence of Trx nitrosation at physiologically relevant concentrations and times was performed, utilizing a mass spectrometry-based method for the direct analysis of the nitrosated Trx. Reduced Trx (rTrx) was nitrosated 2.7-times faster than oxidized Trx (oTrx), and rTrx was nitrosated selectively on Cys62, whereas oTrx was nitrosated only on Cys73. These sites of nitrosation were confirmed at the peptide level using a novel modification of the biotin-switch technique called the reductive switch. These results suggest separate signaling pathways for Trx-SNO under different cellular redox states.

Oxidation and glycolytic cleavage of etheno and propano DNA base adducts.

Non-invasive strategies for the analysis of endogenous DNA damage are of interest for the purpose of monitoring genomic exposure to biologically produced chemicals. We have focused our research on the biological processing of DNA adducts and how this may impact the observed products in biological matrixes. Preliminary research has revealed that pyrimidopurinone DNA adducts are subject to enzymatic oxidation in vitro and in vivo and that base adducts are better substrates for oxidation than the corresponding 2′-deoxynucleosides. We tested the possibility that structurally similar exocyclic base adducts may be good candidates for enzymatic oxidation in vitro. We investigated the in vitro oxidation of several endogenously occurring etheno adducts [1,N2-ε-guanine (1,N2-ε-Gua), N2,3-ε-Gua, heptanone-1,N2-ε-Gua, 1,N6-ε-adenine (1,N6-ε-Ade), and 3,N4-ε-cytosine (3,N4-ε-Cyt)] and their corresponding 2′-deoxynucleosides. Both 1,N2-ε-Gua and heptanone-1,N2-ε-Gua were substrates for enzymatic oxidation in rat liver cytosol; heteronuclear NMR experiments revealed that oxidation occurred on the imidazole ring of each substrate. In contrast, the partially or fully saturated pyrimidopurinone analogues [i.e., 5,6-dihydro-M1G and 1,N2-propanoguanine (PGua)] and their 2′-deoxynucleoside derivatives were not oxidized. The 2′-deoxynucleoside adducts, 1,N2-ε-dG and 1,N6-ε-dA, underwent glycolytic cleavage in rat liver cytosol. Together, these data suggest that multiple exocyclic adducts undergo oxidation and glycolytic cleavage in vitro in rat liver cytosol, in some instances in succession. These multiple pathways of biotransformation produce an array of products. Thus, the biotransformation of exocyclic adducts may lead to an additional class of biomarkers suitable for use in animal and human studies.

One-pot syntheses of malondialdehyde adducts of nucleosides.

Short, “one-pot” syntheses of malondialdehyde adducts of deoxyguanosine, deoxyadenosine, and deoxycytidine are described. These syntheses proceed in improved yield and easier purification than previous syntheses and are well suited for the preparation of isotopically labeled nucleoside adducts for biomarker and metabolic studies.

γ-Glutamyltranspeptidase-Mediated Degradation of Diclofenac-S-acyl-glutathione in Vitro and in Vivo in Rat

Diclofenac, a nonsteroidal antiinflammatory drug, is known to be metabolized to chemically reactive intermediates that transacylate GSH forming diclofenac-S-acyl-glutathione (D-SG) in vivo in rat and in vitro in rat and human hepatocytes. Recently, it was reported that the treatment of rats with diclofenac led to a substantial decrease in the activity of hepatic gamma-glutamyltranspeptidase (gamma-GT), an extracellular canalicular membrane enzyme. Because studies have indicated that D-SG is a chemically reactive transacylating species that is excreted into rat bile, we propose that D-SG formed in the liver may be a substrate for, and potential inhibitor of, hepatic gamma-GT. The present experiments were performed to investigate the ability of D-SG to be a substrate for gamma-GT in vivo in rat and in vitro with commercially available gamma-GT enzyme. We also examined the ability of D-SG to inhibit gamma-GT in vitro. Thus, LC-MS/MS analysis of bile extracts from diclofenac-dosed rats (200 mg/kg, iv) showed the presence of the gamma-GT-mediated D-SG degradation product diclofenac-N-acyl-cysteinylglycine (D- N-CG), where a total of approximately 8 microg was excreted 6 h postadministration. When D-SG (100 microM) was incubated with gamma-GT (1 unit/mL), the GSH adduct was degraded in a linear time-dependent fashion where approximately 94 microM D- N-CG was formed after 20 min of incubation. Dialysis studies showed that inhibition of gamma-GT by D-SG was completely reversible. Further inhibition studies showed that D-SG is a competitive inhibitor of the gamma-GT enzyme. Results from theses studies indicate that D-SG is a substrate for gamma-GT; however, the conjugate may not contribute significantly to the decrease in gamma-GT activity reported to occur in vivo in rat.

Abstract 2885: Features of innate immunity dominate serum and tissue protein and cytokine profiles in both mouse and human inflammatory bowel disease.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Inflammatory bowel disease (IBD) is a chronic and relapsing intestinal inflammatory disease that arises through unknown genetic, environmental, and bacterial origins. Ulcerative colitis (UC) and Crohns disease (CD) are the two main forms of IBD, and their incidence is increasing in industrialized countries. Further, IBD is a significant risk factor for the development of colon cancer. Though the specific determinants remain elusive, persistent inflammation is believed to play a significant role in colon cancer carcinogenesis. To better define the molecular mechanisms linking colitis to the identity of disease biomarkers, we performed a translational comparison of protein expression and protein damage products in mouse and human IBD. Helicobacter hepaticus-infected Rag2-/- mice emulate many aspects of human IBD, and our recent work with this model highlights the importance of neutrophils in the pathology of colitis and colon cancer. Analysis of neutrophil- and macrophage-derived damage products revealed accumulation of 3-chlorotyrosine (CTyr) and 3-nitrotyrosine (NTyr) in inflamed mice colons that increased with disease duration. These results were further corroborated in mouse studies by histological evaluation, which demonstrated strong infiltration of neutrophils and macrophages to the site of inflammation. Human studies revealed an increase in CTyr in the colon of UC and CD tissues relative to serum levels. The nucleic acid chlorination damage product, 5-chloro-2′-deoxycytidine (5-Cl-dC), was quantified in human colon and found to be present at similar levels to that of inflamed mice colons. Multivariate analysis of these markers along with serum proteins and cytokines revealed a general signature of activated innate immunity in human IBD. UC sera were strongly suggestive of neutrophil activity while CD and mouse sera were suggestive of macrophage and neutrophil activity. These data point to innate immunity as a major determinant of serum and tissue profiles and provide insight into disease activity in IBD. Citation Format: Charles G. Knutson, Aswin Mangerich, Yu Zeng, Arkadiusz R. Raczynski, Rosa G. Liberman, Pilsoo Kang, Wenjie Ye, Guanyu Gong, Erin Prestwich, Kun Lu, John S. Wishnok, Joshua R. Korzenik, Gerald N. Wogan, James G. Gox, Peter C. Dedon, Steven R. Tannenbaum. Features of innate immunity dominate serum and tissue protein and cytokine profiles in both mouse and human inflammatory bowel disease. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2885. doi:10.1158/1538-7445.AM2013-2885

Abstract 5113: Discovery and application of a biomarker cassette to inflammatory bowel disease

Inflammatory bowel disease (IBD) results from intermittent and severe activation of the mucosal immune system in the gastrointestinal tract to promote a chronic state of inflammation. Crohn9s disease (CD) and ulcerative colitis (UC) are the two major forms of IBD. Infiltration of gut tissue by lymphocytes, neutrophils, and macrophages results in prolonged exposure to chemical agents such as pro-inflammatory cytokines and reactive oxygen/nitrogen species. Chronic exposure to these inflammation products leads to mis-regulated cell signaling, altered protein expression, and chemical damage to lipids, protein, and nucleic acids. IBD is a significant risk factor for colon cancer. IBD exhibits phases of active disease (active inflammation) and remission, which complicate therapeutic intervention. Clinical biomarkers currently in use are not predictive of the transition from remission to active disease. Our laboratory has conducted an unbiased, four-pronged approach to IBD biomarker identification in human serum utilizing proteomic discovery of acute phase proteins, cytokine profiling, quantification of oxidative tyrosine modifications (chlorotyrosine (Cl-Tyr), bromotyrosine (Br-Tyr), and nitrotyrosine (NO-Tyr)–markers of neutrophil activity), and measurement of carbonylated proteins. One hundred and ten human serum samples were analyzed. This network of data was analyzed by orthogonormalized partial least squares (OPLS) analysis to identify covariance in the data set. Variable importance in projection (VIP) scores were determined to identify which variables were most predictive of clinically diagnosed disease score (VIP >1). Using data available from the cytokine and oxidative tyrosine analysis, the data set was stratified to clinically diagnosed active and inactive disease, with the intent of identifying serum markers that were predictive of this transition. Several variables for UC and CD demonstrated strong covariance with disease score. The top VIP scores identified for UC were: Interleukin-8 (IL-8), granulocyte-colony stimulating factor (G-CSF), IL-6, and Cl-Tyr. The top VIP scores identified for CD were: Cl-Tyr, macrophage inflammatory protein-1β (MIP-1β), inflammatory chemokine-10 (IP-10), IL-6, and IL-1 receptor antagonist (IL-1ra). A Total Score was generated for each UC and CD serum sample by normalizing the raw data to a base-10 spread, scaling by the VIP factor, and summing the total. Based on the Total Score the calculated specificity and sensitivity for identifying active UC was 81.8% and 84.2%, respectively, while the specificity and sensitivity for identifying active CD was 87.5% and 75%, respectively. We anticipate that the addition of acute phase and cabonylated protein analysis will improve the Total Score assessment. Results from this study strongly suggest that a cassette of in vivo serum markers may be predictive of the transition from remission to active disease in IBD. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5113. doi:10.1158/1538-7445.AM2011-5113