Chemical and immunochemical detection of 8-halogenated deoxyguanosines at early stage inflammation.
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-halo-dGs, 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.
Asahi
Takashi
T
Laboratory of Food and Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Kondo
Hajime
H
Masuda
Mitsuharu
M
Nishino
Hoyoku
H
Aratani
Yasuaki
Y
Naito
Yuji
Y
Yoshikawa
Toshikazu
T
Hisaka
Shinsuke
S
Kato
Yoji
Y
Osawa
Toshihiko
T
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000737:chemistry*; D005260:Female; D006219:Halogens / Q000737:chemistry; D007249:Inflammation / Q000473:pathology*; D008070:Lipopolysaccharides / Q000737:chemistry; D008099:Liver / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL; D009195:Peroxidase / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry
D000911:Antibodies, Monoclonal; D006219:Halogens; D008070:Lipopolysaccharides; C002744:3-nitrotyrosine; D014443:Tyrosine; D000080242:8-Hydroxy-2'-Deoxyguanosine; D009195:Peroxidase; D003849:Deoxyguanosine