Oxidative Stress in Urothelial Carcinogenesis: Measurements of Protein Carbonylation and Intracellular Production of Reactive Oxygen Species

  • Patcharawalai Whongsiri
  • Suchittra Phoyen
  • Chanchai BoonlaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1655)


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.

Key words

Bladder cancer Oxidative stress Protein carbonyl DNPH ROS DCFH 



This work was supported by Thailand Research Fund and Chulalongkorn University (RSA5680019), and Ratchadapiseksompotch Fund, Faculty of Medicine, Chulalongkorn University (RA57/118).


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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Patcharawalai Whongsiri
    • 1
  • Suchittra Phoyen
    • 1
  • Chanchai Boonla
    • 1
    Email author
  1. 1.Department of Biochemistry, Faculty of MedicineChulalongkorn UniversityBangkokThailand

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