Chemical and Immunochemical Detection of 8-Halogenated Deoxyguanosines at Early Stage Inflammation

Abstract

Myeloperoxidase (MPO) generates reactive halogenating species that can modify DNA. The aim of this study was to investigate the formation of 8-halogenated 2-deoxyguanosines (8-halo-dGs) during inflammatory events. 8-Bromo-2-dG (8-BrdG) and 8-chloro-2-dG (8-CldG) were generated by treatment of MPO with hydrogen peroxide at physiological concentrations of Cl ؊ and Br ؊ . The formation of 8-halo-dGs with other oxidative stress biomarkers in lipopolysaccharide-treated rats was assessed by liquid chromatography tandem mass spectrometry and immunohistochemistry using a novel monoclonal antibody (mAb8B3) to 8-BrdG-conjugated keyhole limpet hemocyanin. The antibody recognized both 8-BrdG and 8-CldG. In the liver of lipopolysaccharide-treated rats, immunostaining for 8-halodGs, halogenated tyrosines, and MPO were increased at 8 h, whereas those of 8-oxo-2-dG (8-OxodG) and 3-nitrotyrosine were increased at 24 h. Urinary excretion of both 8-CldG and 8-BrdG was also observed earlier than those of 8-OxodG and modified tyrosines (3-nitrotyrosine, 3-chlorotyrosine, and 3-bromotyrosine). Moreover, the levels of the 8-halo-dGs in urine from human diabetic patients were 8-fold higher than in healthy subjects (n ‫؍‬ 10, healthy and diabetic, p < 0.0001), whereas there was a moderate difference in 8-OxodG between the two groups (p < 0.001). Interestingly, positive mAb8B3 antibody staining was observed in liver tissue from hepatocellular carcinoma patients but not in liver tissue from human cirrhosis patients. These data suggest that 8-halo-dGs may be potential biomarkers of early inflammation.Oxidative damage is implicated in the pathogenesis of many diseases including atherosclerosis and cancer (1-4). Involvement of myeloperoxidase (MPO), 2 a heme protein secreted by activated leukocytes such as neutrophils and monocytic cells, is a potential cause of oxidative damage during inflammation. At sites of inflammation, activated leukocytes play a major role in host defense against microorganisms using oxidants such as HOCl and HOBr. However, excessive oxidant release can also induce damage; DNA bases (5-8) and proteins and lipids (9 -12), for instance, are putative targets of oxidants. At plasma halide concentrations (100 mM Cl Ϫ , 20 -100 M Br Ϫ ) (13), MPO converts HOCl to HOBr using Br Ϫ as a halide exchange (14). HOBr is also generated by eosinophil peroxidase using H 2 O 2 and Br Ϫ , such as by the MPO-H 2 O 2 -Cl Ϫ system. Because MPO catalyzes halogenation, oxidation, and nitration (15-17), MPO and its product may be important general markers for evaluating oxidative damage to the body during inflammation. Halogenation is a key reaction for the formation of an MPO activity marker because the presence of halogen in the marker molecule directly indicates a contribution of the halogenating species at the site of oxidative damage. Moreover, no other halogenating pathways have been reported except for MPO and eosinophil peroxidase-derived damage. Therefore, halogenated products have the potential to be specific markers of inflammatio...

Structural Basis for Promutagenicity of 8-Halogenated Guanine

“…Interestingly, 8-chloro-dGTP was efficiently hydrolyzed by hMTH1 (11), which hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP. In addition, haloG was excreted to urine three times faster than oxoG in lipopolysaccharide-treated rats (10), suggesting the existence of an efficient DNA repair enzyme for haloG in cells.…”

“…Despite ϳ1000-fold lower physiological concentration of Br Ϫ (100 M) relative to that of Cl Ϫ (100 m⌴), the concentration of BrG is ϳ5-fold higher than that of 8-chloroguanine under inflammatory conditions (10). The urine concentration of haloG was one tenth of that of 8-oxoguanine (oxoG) in healthy subjects, and half of that in diabetic patients, suggesting haloG as a potentially important lesion (10). Interestingly, 8-chloro-dGTP was efficiently hydrolyzed by hMTH1 (11), which hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP.…”

“…Whereas reactive halogen species, potent oxidants that kill pathogens, play an important role in the host defense mechanism, they can also damage DNA especially under inflammatory conditions to generate various halogenated DNA lesions (5-7), with 8-halogenated G (haloG) 2 such as 8-chloroguanine and 8-bromoguanine (BrG) being the major adducts (8 -10). Recently, haloG has been detected in DNA of rat liver and in the urine of patients with hepatocellular carcinoma (10). Despite ϳ1000-fold lower physiological concentration of Br Ϫ (100 M) relative to that of Cl Ϫ (100 m⌴), the concentration of BrG is ϳ5-fold higher than that of 8-chloroguanine under inflammatory conditions (10).…”

“…Recently, haloG has been detected in DNA of rat liver and in the urine of patients with hepatocellular carcinoma (10). Despite ϳ1000-fold lower physiological concentration of Br Ϫ (100 M) relative to that of Cl Ϫ (100 m⌴), the concentration of BrG is ϳ5-fold higher than that of 8-chloroguanine under inflammatory conditions (10). The urine concentration of haloG was one tenth of that of 8-oxoguanine (oxoG) in healthy subjects, and half of that in diabetic patients, suggesting haloG as a potentially important lesion (10).…”

Inflammation-induced DNA damage and damage-induced inflammation: a vicious cycle

“…1F). PARP-1 is also involved in caspase-1 mediated [73] or caspase-independent [74] apoptosis and autophagy 207 [75]. PARP-1 catalyses the addition of poly (ADP-ribose) (PAR) chains to its substrate proteins and facilitates DNA repair.…”