Volume 120, Issue 1, Pages 190-199 (January 2001) Nitric oxide–mediated inhibition of DNA repair potentiates oxidative DNA damage in cholangiocytes  Meeta Jaiswal, Nicholas F. LaRusso, Richard A. Shapiro, Timothy R. Billiar, Gregory J. Gores  Gastroenterology  Volume 120, Issue 1, Pages 190-199 (January 2001) DOI: 10.1053/gast.2001.20875 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Expression of iNOS and NO generation after transfection are not affected by inhibitors of guanylyl cyclase or PKG. (A) Cells were transfected with an expression vector for iNOS or the empty plasmid. Twenty-four hours after transfection, iNOS protein expression was assessed by immunoblot analysis. iNOS expression was not affected by the presence of 40 mmol/L ODQ, 0.5 mmol/L KT5823, 50 mmol/L 1400W, 30 mmol/L L-NIL, or 30 mmol/L L-NMMA. (B) Nitrite and nitrate (NO3−/NO2−) levels in the media measured by a chemiluminescence assay 24 hours after transfection. Each column represents the mean ± SEM of 3 different experiments. Gastroenterology 2001 120, 190-199DOI: (10.1053/gast.2001.20875) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 iNOS transfection inhibits global and specific 8-oxodG DNA repair in an NO-dependent manner. Cells were transfected with an expression vector for iNOS or an empty plasmid and incubated in the presence or absence of 50 μmol/L 1400W. Twenty-four hours later, whole-cell extracts were prepared as described in Materials and Methods. (A) Global DNA repair was calculated as a percentage of relative repair incorporation of radiolabeled dGMP into photoactivated methylene blue–damaged plasmid DNA by the transfected H69 and KMBC whole-cell extracts. (B) 8-OxodG specific repair. Specific repair activity is indicated by the recognition and excision of the oligonucleotide at the 8-oxodG lesion giving the 15-bp excised product. The bacterial glycosylase Fpg was added as a positive control. Gastroenterology 2001 120, 190-199DOI: (10.1053/gast.2001.20875) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Inhibition of DNA repair is independent of NO-mediated activation of cGMP/PKG pathway. Cells were transfected with an expression vector for iNOS or an empty plasmid and incubated for 24 hours in the presence of 50 mmol/L 1400W to prevent generation of NO before initiation of experimental treatment. (A) Global repair efficiency was analyzed by in vitro repair incorporation of radiolabeled dGMP into damaged plasmid DNA substrate as described in Figure 1. (B) The cell-permeable cyclic G analogue, 500 mmol/L dibutyryl cGMP, an activator of the PKG pathway, does not result in inhibition of global DNA repair activity. (C) Specific 8-oxodG repair activity in cell extracts was measured as described in Figure 1. NO-mediated inhibition of 8-oxodG specific DNA repair activity also is not prevented by guanylyl cyclase or PKG inhibitors. Gastroenterology 2001 120, 190-199DOI: (10.1053/gast.2001.20875) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 DNA repair is normal during treatment of iNOS-transfected cells incubated with NO scavengers but not peroxynitrite scavengers. Cells were transfected with an iNOS expression vector or an empty plasmid. Twenty-four hours after transfection, whole-cell extracts were obtained, and (A) global DNA repair and (B) 8-oxodG–specific DNA repair were measured as described in Figure 1. The NO scavengers (100 mmol/L carboxy-PTIO, 25 mmol/L heme, and 50 mmol/L PTIO) and the peroxynitrite scavengers (40 mmol/L MnTBAP, 10 mmol/L trolox, and 25 mmol/L ebselen) were included in the media throughout the 24-hour incubation. Gastroenterology 2001 120, 190-199DOI: (10.1053/gast.2001.20875) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 DTT prevents loss of DNA repair as a media adjunct but does not restore global or 8-oxodG–specific DNA repair in whole-cell extracts from iNOS-transfected cells. Cells were transfected with an expression vector for iNOS or an empty plasmid. Twenty-four hours later, whole-cell extracts were prepared, and (A) global and (B) 8- oxodG DNA repair were quantitated as described in Figure 1. When included in the media after transfection, DTT prevented loss of DNA repair capability, presumably by acting as an NO scavenger. However, when added only to cell extracts, DTT did not restore either global or specific DNA repair activity. Gastroenterology 2001 120, 190-199DOI: (10.1053/gast.2001.20875) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 8-OxodG DNA lesions are not repaired in the presence of NO. 8-OxodG lesions were quantitated by the comet assay. The percentage of DNA in 50 comet tails was analyzed using computer analysis of cells from the following experimental groups: (1) untreated cells (A and control); (2) cells treated with 150 mmol/L H2O2 for 1 hour (B and H2O2) and subjected immediately to the comet assay; (3) cells treated with 150 mmol/L H2O2 for 1 hour followed by incubation in H2O2-free media for 24 hours (C and H2O2 after 24 hours); (4) cells treated with 150 mmol/L H2O2 for 1 hour followed by incubation with 0.3 mmol/L SNAP for 24 hours (D and H2O2+ SNAP after 24 hours); (5) cells treated with 0.3 mmol/L SNAP for 24 hours (E); and (6) cells treated with 0.3 mmol/L SNAP plus the NO scavenger 100 mmol/L c-PTIO for 24 hours (F). *P < 0.01 compared with all other treatment as determined using ANOVA. Gastroenterology 2001 120, 190-199DOI: (10.1053/gast.2001.20875) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 Immunohistochemistry for iNOS, 3-nitrotyrosine, and 8-oxodG in human liver specimens. Top panel: The biliary epithelia (arrows) did not contain immunoreactivity for (A) iNOS, (D) 3-nitrotyrosine, or (G) 8-oxodG in normal liver tissue. Middle panels: Intense immunoreactivity was observed in biliary epithelia (arrows) in liver specimens from patients with PSC for (B) iNOS, (E) 3-nitrotyrosine, and (H) 8-oxodG. Bottom panel: Immunoreactivity for (C) iNOS, (F) 3-nitrotyrosine, and (I) 8-oxodG was minimal in the biliary epithelia of patients with alcohol-induced cirrhosis. Gastroenterology 2001 120, 190-199DOI: (10.1053/gast.2001.20875) Copyright © 2001 American Gastroenterological Association Terms and Conditions