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Oxidation of cellular thiols by hydroxyethyldisulphide inhibits DNA double-strand-break rejoining in G6PD deficient

I S Ayene1, C J Koch, S W Tuttle

  • 1Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia 19104, USA. ayenei@mail.med.upenn.edu

International Journal of Radiation Biology
|December 1, 2000
PubMed
Abstract

Insights

Oxidizing cellular thiols impairs DNA double-strand-break (DSB) rejoining and cell survival after irradiation. A functional oxidative pentose phosphate pathway is crucial for repairing DSBs when cells encounter mild thiol oxidants.

Area of Science:

  • Cellular biology
  • Radiation biology
  • Biochemistry

Background:

  • Cellular thiols play a critical role in DNA repair mechanisms.
  • The oxidative pentose phosphate pathway generates NADPH, essential for maintaining cellular redox balance and reducing oxidized thiols.
  • Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in this pathway.

Purpose of the Study:

  • To investigate the impact of protein and non-protein thiol oxidation on DNA double-strand-break (DSB) rejoining after irradiation.
  • To determine the relevance of thiol oxidation in the survival of Chinese Hamster Ovary (CHO) cells.

Main Methods:

  • Utilized G6PD-deficient mutant cells to study thiol redox balance.
  • Oxidized cellular thiols using hydroxyethyldisulfide (HEDS).
  • Quantified thiol levels (NPSH, PSH) using HPLC and Ellman's reagent.
  • Assessed cell survival via clonogenic assay and DSB rejoining by Pulse Field Gel Electrophoresis.

Main Results:

  • G6PD-deficient cells showed significantly lower bioreduction of HEDS and depletion of thiols compared to wild-type cells.
  • Irradiation of G6PD-deficient cells in the presence of HEDS resulted in increased cell killing and impaired DSB rejoining.
  • Complementation of G6PD deficiency restored normal thiol status, cell survival, and DSB rejoining.

Conclusions:

  • A functional oxidative pentose phosphate pathway is essential for efficient DNA double-strand-break (DSB) rejoining.
  • Mild thiol oxidation significantly impacts cellular radiosensitivity and DNA repair capacity.
  • NADPH production via G6PD is critical for thiol reduction and subsequent DNA repair post-irradiation.

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