Article Figures & Data
Figures
- Fig. 1.
Structure of the pan-PAD inhibitor, Cl-amidine.
- Fig. 2.
(A) Outline of the AOM/DSS mouse model of colitis used in this study. (B) Effects of Cl-amidine on the colon histology score in the acute AOM/DSS colitis model. Six mice from each group described in Materials and Methods were euthanized on day 14, and colons were measured and harvested. Then the histology score was determined. Weight was recorded every 48 hours during the 14-day experiment. Values represent the mean ± S.E. of the mean. Representative H&E-stained colons are shown for each group. *Significant difference from the AOM + DSS group (P < 0.01).
- Fig. 3.
iNOS levels are reduced in the colons of mice treated with Cl-amidine. Six mice from each of the indicated groups were euthanized on day 14, and colons were harvested from each animal and stained with iNOS as described in Materials and Methods. (A) Immunoreactivity score (IRS) for each group. Values represent the mean ± S.E. *Significant difference from the AOM + DSS only group (P < 0.01). (B) Representative sections of indicated group. Positive staining is brown colored. (400× magnification.)
- Fig. 4.
Cl-amidine attenuates the activation of macrophages and protects from DNA damage in target epithelial cells in vitro. (A) iNOS and Cox-2 induction after treatment of ANA-1 mouse macrophages with IFN-γ. Numbers below each blot represent the GAPDH-adjusted density of each band, with the control (0 hour, no treatment) being a baseline of 1.0. The observation that for both markers (iNOS and Cox-2) density is lower in unstimulated cells exposed to Cl-amidine (0 hour, +10 µg/ml Cl-amidine, 5th lane) suggests Cl-amidine inhibits basal activity of macrophages. Accordingly, it also inhibits the activation of macrophages. (B) An oxidative burst in ANA-1 mouse macrophages is attenuated by pretreatment with Cl-amidine (10 µg/ml). Chemiluminescence was measured as described in Materials and Methods. Results were compared with no Cl-amidine control (± S.E.). (C) In the presence of an oxidative burst, target epithelial cells (HCT116 colon cancer cells) pretreated with 10 µg/ml Cl-amidine are protected from DNA damage. Results are represented as the mean Comet tail moment ± S.E., scoring 50 comets/treatment group. Representative images of Comets in each treatment group are shown above each bar graph. *Significant difference from the untreated (No Cl-amidine) macrophages that were cocultured for 4 hours (P < 0.01).
- Fig. 5.
Cl-amidine induces antioxidant enzymes (catalase, GPx1, SOD1) in IFN-γ–stimulated ANA-1 mouse macrophages and in vivo. (A) ANA-1 mouse macrophage cells were pretreated with 0–10 µg/ml Cl-amidine for 12 hours and then cells were stimulated with IFN-γ for 8 hours. Antioxidant enzymes of interest were suppressed in activated cells pretreated with 10 µg/ml (lane 4). (B–D) Mice from each of the indicated groups were euthanized on day 14, and colons were harvested from each animal and stained with catalase (B), GPx1 (C), and SOD1 (D) as described in Materials and Methods. Immunoreactivity scores (IRS) are shown for each group. Values represent the mean ± S.E. Significance is compared with the AOM + DSS only group: *P < 0.05; **P < 0.01; ***P < 0.005. Representative sections of each group were taken at 400× magnification, and positive staining is brown colored.
- Fig. 6.
Cl-amidine attenuates the activation of white blood cells and protects from DNA damage in target epithelial cells in vivo. Mice were injected with AOM (10 mg/kg), then 1 week later they were given either water ad libitum or 2% DSS in the drinking water for 14 days as described in Materials and Methods and in Fig. 2. (A) Protein lysates from scraped mucosa of the colon (4 mice per group; lysates were combined) were examined for iNOS and GAPDH (internal control). Mice consuming 2% DSS had activation of only iNOS in both CD45+ and CD45− cells. Mice consuming Cl-amidine + 2% DSS had iNOS attenuated in both cell types. (B) After column separation of inflammatory cells from mucosal cells, we examined an oxidative burst of CD45+ inflammatory cells (4 mice per group). Mice consuming Cl-amidine + DSS exhibit CD45+ inflammatory cells with attenuated activity compared with mice on 2% DSS only. Chemiluminescence was measured as described in Materials and Methods and expressed as mean (± S.E.) relative light units (RLU) per 1 × 106 cells. *Significant difference from the AOM + DSS–only group (P < 0.05). (C) Mucosal epithelial cells were examined for DNA damage by Comet analysis. Results are presented as the mean (± S.E.) tail moment from 200 Comets taken from 4 mice per group. *Significant difference from the AOM + DSS–only group (P < 0.01). Representative Comets for each group are shown.
In this issue
Antioxidant/Anti-DNA Damage Consequences of PAD Inhibition
