Intestinal mucosal inflammation leads to systemic genotoxicity in mice

Abstract

Inflammatory bowel disease, including ulcerative colitis and Crohn's disease, substantially increases the risk of colorectal cancer. However, mechanisms linking mucosal inflammation to the sequence of dysplasia are incompletely understood. Whereas studies have shown oxidative damage to the colon, this study tests whether genotoxicity is elicited systemically by acute and chronic intestinal inflammation. In this study, genotoxic endpoints were assessed in peripheral leukocytes (DNA single- and double-stranded breaks and oxidative DNA damage) and normochromatic erythrocytes (micronuclei) during chemical or immune-mediated colitis. During three consecutive cycles of intestinal inflammation induced by dextran sulfate sodium administration, genotoxicity to peripheral leukocytes and erythroblasts was detected in both acute and chronic phases of dextran sulfate sodium-induced inflammation. Reactive oxygen species-mediated oxidative stress and DNA damage was confirmed with positive 8-oxoguanine and nitrotyrosine staining in peripheral leukocytes. Levels of DNA damage generally decreased during remission and increased during treatment, correlating with clinical symptoms and systemic inflammatory cytokine levels. In Galphai2(-/-) and interleukin-10(-/-) transgenic mice susceptible to immune-mediated colitis and inflammation-associated adenocarcinoma, similar levels of peripheral leukocyte and erythroblast genotoxicity were also observed. Moreover, this systemic genotoxicity was observed in mice with subclinical inflammation, which was further elevated in those with severe mucosal inflammation. We propose that mucosal inflammation, by eliciting substantial and ongoing systemic DNA damage, contributes early on to genetic instability necessary for progression to inflammatory bowel disease-associated dysplasia and the development of cancer.

Fig. 1. Disease Activity Index (DAI) of DSS Treated vs. Non-Treated Mice

DSS treated mice (n=10) demonstrated significantly higher disease activity indices everyday after Day 4 of Cycle 1 (p<0.001), Day 3 of Cycle 2 (p<0.001), and Day 2 of Cycle 3 (p

Fig. 2. Mean Olive Tail Moments

At least 150 “comets” were scored per mouse in the DSS treated group (n=10) and in the non-treated group (n=10). Data were log transformed before applying statistical tests, and are represented as mean ± SEM. *: p<0.05, **: p<0.01.

Fig. 3. γ-H2AX foci and Micronucleus Induction

A.Percent positive cells for γ-H2AX foci in peripheral leukocytes. Presence of double strand breaks was confirmed by immunofluorescence of γ-H2AX. Positive cells contained >4 distinct nuclear foci. Image caption: Positive and negative cell for nuclear foci, 100X magnification. At least 125 cells were analyzed per sample. Data are represented as mean ± SEM, n=10 per treatment group. **: p<0.01, *: p<0.05 B. Micronucleus induction in peripheral normochromatic erythrocytes. At least 4000 normochromatic erythrocytes were counted and scored for presence of micronuclei. Data are represented as mean ± SEM of micronucleated normochromatic erythrocytes (MN-NCE) per 1000 NCEs. Image caption: MN-NCEs and NCEs, 100X magnification. **: p<0.01, *: p<0.05, by nonparametric one way ANOVA with Dunn’s multiple comparison test. ANOVA of normal linear regression showed effect of treatment, cycle of treatment and interaction of effect of treatment and cycle of treatment to be significant (p<0.01).

Fig. 4. Quantitative Real Time-PCR of Cytokines in Peripheral Blood

Expression levels of cytokines were determined only in DSS treated mice (n=10). Data are represented as mean ± SEM of gene expression divided by Tbp expression. A. Transcript levels of TNF-α divided by Tbp. B. Transcript levels of MCP-1 divided by Tbp. C. Transcript levels of IFN-γ divided by Tbp. D. Transcript levels of TGF-β divided by Tbp. Statistical significance was determined by non-parametric one-way ANOVAs with Dunn’s multiple comparison test. *: p<0.05, **: p<0.01

Fig. 5. Systemic Genotoxicity in Mouse Models of Mucosal Inflammation

Blood was sampled from Gαi2^−/−, IL-10^−/−, and control IL-10^+/+ mice for genotoxicity assays at age 3 months; in addition, IL-10^−/− mice were sampled at age 6 months, when colitis in this genetic background has progressed to greater clinical activity. A. Representative colon histology (hematoxylin and eosin staining at indicated magnifications) from Gαi2 and IL-10 mice, both at age 3 months. B. Alkaline comet assay with and without hOgg1 incubation was carried out in peripheral leukocytes. Error bars are SEM, n=6 per group. *: p<0.05, **: p<0.01 by Student’s unpaired t-test. C. Percent positive cells for γ-H2AX foci in peripheral leukocytes. Error bars are SEM. *:p<0.05 by Student’s unpaired t-test. D. MN-NCEs per 1000 NCEs in peripheral blood. Error bars are SEM. **: p<0.01 by Student’s unpaired t-test.

Fig. 6. 8-oxoguanine and Nitrotyrosine Formation in Peripheral Leukocytes and the Colon

A. Representative images of positive staining for 8-oxoguanine (green, left) and nitrotyrosine (red, right) in leukocytes of DSS-treated wildtype (7 days) and IL-10^−/−mice (6 months). B. Percent positive cells for 8-oxoguanine and nitrotyrosine staining before and after DSS treated mice (7 days), n=6 per group (LEFT) and in IL-10^−/− mice (6 months), n=4 per group (RIGHT). *: p<0.05, **: p<0.01 by Student’s unpaired t-test. C. Representative images of 8-oxoguanine (green) and nitrotyrosine (red) in colon sections of IL-10^−/− mice (6 months) and wildtype mice.