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

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Urothelial Carcinoma

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1655))

Abstract

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.

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Acknowledgment

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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Authors and Affiliations

  1. Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Rama IV Road, Bangkok, 10330, Thailand

    Patcharawalai Whongsiri, Suchittra Phoyen & Chanchai Boonla

Authors
  1. Patcharawalai Whongsiri
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  2. Suchittra Phoyen
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  3. Chanchai Boonla
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Corresponding author

Correspondence to Chanchai Boonla .

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Editors and Affiliations

  1. Department of Urology, Heinrich Heine University, Düsseldorf, Germany

    Wolfgang A Schulz

  2. Department of Urology, Heinrich Heine University, Düsseldorf, Germany

    Michèle J. Hoffmann

  3. Department of Urology, Heinrich Heine University, Düsseldorf, Germany

    Günter Niegisch

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Whongsiri, P., Phoyen, S., Boonla, C. (2018). Oxidative Stress in Urothelial Carcinogenesis: Measurements of Protein Carbonylation and Intracellular Production of Reactive Oxygen Species. In: Schulz, W., Hoffmann, M., Niegisch, G. (eds) Urothelial Carcinoma. Methods in Molecular Biology, vol 1655. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7234-0_9

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  • DOI: https://doi.org/10.1007/978-1-4939-7234-0_9

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7233-3

  • Online ISBN: 978-1-4939-7234-0

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