Oxidative stress contributes substantially to urothelial carcinogenesis. Its extent can be assessed by measurements of reactive species (mainly reactive oxygen species (ROS)), oxidatively modified damage products, and levels of various antioxidants. We presented herein the methods for the measurement of protein carbonyl content and intracellular production of ROS. Protein carbonyl is the most commonly used indicator of protein oxidation because it is early formed and relatively stable under oxidative stress. Determination of protein carbonyl relies on the derivatization of carbonyl groups (aldehydes: R-CHO and ketones: R-CO-R) with 2,4-dinitrophenylhydrazine (DNPH) under strongly acidic conditions to yield stable dinitrophenyl (DNP) hydrazones. Absorbance of the DNP hydrazones at 370–375 nm is proportional to the content of carbonyl groups. To report the protein carbonyl content, it is usually normalized by total proteins. Detection of intracellular ROS production is based on oxidation of 2′,7′-dichlorofluorescein-diacetate (DCFH-DA) by ROS to produce the highly fluorescent 2′,7′-dichlorofluorescein (DCF). Fluorescent intensity measured at 480 nm excitation and 535 nm emission is directly proportional to the amount of ROS generated.
Bladder cancer Oxidative stress Protein carbonyl DNPH ROS DCFH
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
This work was supported by Thailand Research Fund and Chulalongkorn University (RSA5680019), and Ratchadapiseksompotch Fund, Faculty of Medicine, Chulalongkorn University (RA57/118).
Kawai K, Yamamoto M, Kameyama S, Kawamata H, Rademaker A, Oyasu R (1993) Enhancement of rat urinary bladder tumorigenesis by lipopolysaccharide-induced inflammation. Cancer Res 53(21):5172–5175PubMedGoogle Scholar
Wei M, Wanibuchi H, Morimura K et al (2002) Carcinogenicity of dimethylarsinic acid in male F344 rats and genetic alterations in induced urinary bladder tumors. Carcinogenesis 23(8):1387–1397CrossRefPubMedGoogle Scholar
Brown NS, Streeter EH, Jones A, Harris AL, Bicknell R (2005) Cooperative stimulation of vascular endothelial growth factor expression by hypoxia and reactive oxygen species: the effect of targeting vascular endothelial growth factor and oxidative stress in an orthotopic xenograft model of bladder carcinoma. Br J Cancer 92(9):1696–1701CrossRefPubMedPubMedCentralGoogle Scholar
Akcay T, Saygili I, Andican G, Yalcin V (2003) Increased formation of 8-hydroxy-2′-deoxyguanosine in peripheral blood leukocytes in bladder cancer. Urol Int 71(3):271–274CrossRefPubMedGoogle Scholar
Badjatia N, Satyam A, Singh P, Seth A, Sharma A (2010) Altered antioxidant status and lipid peroxidation in Indian patients with urothelial bladder carcinoma. Urol Oncol 28(4):360–367CrossRefPubMedGoogle Scholar
Opanuraks J, Boonla C, Saelim C et al (2010) Elevated urinary total sialic acid and increased oxidative stress in patients with bladder cancer. Asian Biomed 4(5):703–710Google Scholar
Soini Y, Haapasaari KM, Vaarala MH, Turpeenniemi-Hujanen T, Karja V, Karihtala P (2011) 8-hydroxydeguanosine and nitrotyrosine are prognostic factors in urinary bladder carcinoma. Int J Clin Exp Pathol 4(3):267–275PubMedPubMedCentralGoogle Scholar
Patchsung M, Boonla C, Amnattrakul P, Dissayabutra T, Mutirangura A, Tosukhowong P (2012) Long interspersed nuclear element-1 hypomethylation and oxidative stress: correlation and bladder cancer diagnostic potential. PLoS One 7(5):e37009CrossRefPubMedPubMedCentralGoogle Scholar
Wongpaiboonwattana W, Tosukhowong P, Dissayabutra T, Mutirangura A, Boonla C (2013) Oxidative stress induces hypomethylation of LINE-1 and hypermethylation of the RUNX3 promoter in a bladder cancer cell line. Asian Pac J Cancer Prev 14(6):3773–3778CrossRefPubMedGoogle Scholar
Kloypan C, Srisa-art M, Mutirangura A, Boonla C (2015) LINE-1 hypomethylation induced by reactive oxygen species is mediated via depletion of S-adenosylmethionine. Cell Biochem Funct 33(6):375–385CrossRefPubMedGoogle Scholar
Berlett BS, Stadtman ER (1997) Protein oxidation in aging, disease, and oxidative stress. J Biol Chem 272(33):20313–20316CrossRefPubMedGoogle Scholar
Levine RL, Wehr N, Williams JA, Stadtman ER, Shacter E (2000) Determination of carbonyl groups in oxidized proteins. Methods Mol Biol 99:15–24PubMedGoogle Scholar
Levine RL, Garland D, Oliver CN et al (1990) Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol 186:464–478CrossRefPubMedGoogle Scholar
Dalle-Donne I, Rossi R, Giustarini D, Milzani A, Colombo R (2003) Protein carbonyl groups as biomarkers of oxidative stress. Clin Chim Acta 329(1–2):23–38CrossRefPubMedGoogle Scholar
Bartosz G (2006) Use of spectroscopic probes for detection of reactive oxygen species. Clin Chim Acta 368(1–2):53–76CrossRefPubMedGoogle Scholar
Gomes A, Fernandes E, Lima JL (2005) Fluorescence probes used for detection of reactive oxygen species. J Biochem Biophys Methods 65(2–3):45–80CrossRefPubMedGoogle Scholar
Chen X, Zhong Z, Xu Z, Chen L, Wang Y (2010) 2′,7′-Dichlorodihydrofluorescein as a fluorescent probe for reactive oxygen species measurement: forty years of application and controversy. Free Radic Res 44(6):587–604CrossRefPubMedGoogle Scholar