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DNA photoproducts released by repair in biological fluids as biomarkers of the genotoxicity of UV radiation

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Abstract

UV-induced formation of photoproducts in DNA is a major initiating event of skin cancer. Consequently, many analytical tools have been developed for their quantification in DNA. In the present work, we extended our previous liquid chromatography–mass spectrometry method to the quantification of the short DNA fragments containing photoproducts that are released from cells by the repair machinery. We designed a robust protocol including a solid-phase extraction step (SPE), an enzymatic treatment aimed at releasing individual photoproducts, and a liquid chromatography method combining on-line SPE and ultra-high-performance liquid chromatography for optimal specificity and sensitivity. We also added relevant internal standards for a better accuracy. The method was validated for linearity, repeatability, and reproducibility. The limits of detection and quantification were found to be in the fmol range. The proof of concept of the use of excreted DNA repair products as biomarkers of the genotoxicity of UV was obtained first in in vitro studies using cultured HaCat cells and ex vivo on human skin explants. Further evidence was obtained from the detection of pyrimidine dimers in the urine of human volunteers collected after recreational exposure in summer.

Graphical abstract

An assay was designed to quantify the DNA photoproducts released from cells within short fragments by the DNA repair machinery. These oligonucleotides were isolated by solid-phase extraction and enzymatically hydrolyzed. The photoproducts were then quantified by on-line SPE combined with UHPLC-MS/MS with isotopic dilution.

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Authors and Affiliations

  1. Univ. Grenoble Alpes, CEA, CNRS, IRIG, SyMMES, F-38000, Grenoble, France

    Noémie Reynaud, Laura Belz, David Béal & Thierry Douki

  2. Service Recherche Pharmaco-Clinique, Département Recherche Appliquée, Centre R&D Pierre Fabre, 31000, Toulouse, France

    Daniel Bacqueville, Hélène Duplan & Camille Géniès

  3. Centre de Recherche sur la Peau, Pierre Fabre Dermo-Cosmétique, 31000, Toulouse, France

    Emmanuel Questel & Gwendal Josse

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  1. Noémie Reynaud
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Reynaud, N., Belz, L., Béal, D. et al. DNA photoproducts released by repair in biological fluids as biomarkers of the genotoxicity of UV radiation. Anal Bioanal Chem 414, 7705–7720 (2022). https://doi.org/10.1007/s00216-022-04302-1

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