Quantitative immunohistochemical determination of 8-hydroxy-2'-deoxyguanosine by a monoclonal antibody N45.1: its application to ferric nitrilotriacetate-induced renal carcinogenesis model.
The DNA base-modified product 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of the most commonly used markers for the evaluation of oxidative DNA damage. A monoclonal antibody specific for 8-OHdG (N45.1) was characterized and applied in quantitative immunohistochemistry. N45.1 recognized both the modified base and deoxyribose structure of 8-OHdG and required a concentration two orders higher of 8-hydroxyguanosine as a competitor in the ELISA. In addition, N45.1 did not cross-react with the original four deoxyribonucleosides, other DNA base-modified products such as 8-hydroxy-2'-deoxyadenosine and O6-methyl-2'-deoxyguanosine, or urine components such as uric acid, creatine, and creatinine. A ferric nitrilotriacetate-induced rat renal carcinogenesis model was used for the evaluation of quantitative immunohistochemistry. The 8-OHdG index of quantitative immunohistochemistry, as analyzed by NIH image freeware, correlated reasonably well with the 8-OHdG amount determined by high-performance liquid chromatography with an electrochemical detector-except for a difference in peak time, which could be attributed to the selection of target location. The present method has advantages over the high-performance liquid chromatography/electrochemical detector, gas chromatography/mass spectrometry, and 32P-postlabeling methods in that it allows localization of 8-OHdG to be specified without the risk of artifactual production of 8-OHdG during the DNA extraction and hydrolytic processes.
Toyokuni
S
S
Department of Pathology, Graduate School of Medicine, Kyoto University, Japan.
Tanaka
T
T
Hattori
Y
Y
Nishiyama
Y
Y
Yoshida
A
A
Uchida
K
K
Hiai
H
H
Ochi
H
H
Osawa
T
T
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002273:Carcinogens; D002851:Chromatography, High Pressure Liquid; D004249:DNA Damage; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis; D005290:Ferric Compounds; D007150:Immunohistochemistry / Q000379:methods; D007668:Kidney / Q000187:drug effects / Q000473:pathology*; D007680:Kidney Neoplasms / Q000139:chemically induced / Q000473:pathology*; D008297:Male; D008954:Models, Biological; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives*; D018384:Oxidative Stress; D051381:Rats; D017208:Rats, Wistar
D000911:Antibodies, Monoclonal; D002273:Carcinogens; D005290:Ferric Compounds; D000080242:8-Hydroxy-2'-Deoxyguanosine; D003849:Deoxyguanosine; D009571:Nitrilotriacetic Acid; C020326:ferric nitrilotriacetate