Abstract
Endogenous stress represents a major source of genome instability, but is in essence difficult to apprehend. Incorporation of labeled radionuclides into DNA constitutes a tractable model to analyze cellular responses to endogenous attacks. Here we show that incorporation of [3H]thymidine into CHO cells generates oxidative-induced mutagenesis, but, with a peak at low doses. Proteomic analysis showed that the cellular response differs between low and high levels of endogenous stress. In particular, these results confirmed the involvement of proteins implicated in redox homeostasis and DNA damage signaling pathways. Induced-mutagenesis was abolished by the anti-oxidant N-acetyl cysteine and plateaued, at high doses, upon exposure to L-buthionine sulfoximine, which represses cellular detoxification. The [3H]thymidine-induced mutation spectrum revealed mostly base substitutions, exhibiting a signature specific for low doses (GC > CG and AT > CG). Consistently, the enzymatic activity of the base excision repair protein APE-1 is induced at only medium or high doses. Collectively, the data reveal that a threshold of endogenous stress must be reached to trigger cellular detoxification and DNA repair programs; below this threshold, the consequences of endogenous stress escape cellular surveillance, leading to high levels of mutagenesis. Therefore, low doses of endogenous local stress can jeopardize genome integrity more efficiently than higher doses.
Highlights
The DNA most of the energy is locally deposited into the nucleus by low-energy compounds, such as tritium[1,2]
CHO-K1 hamster cells lines were cultured in the presence of different specific activities of labeled [3H]thymidine[1]. These cell lines were chosen because the impact of [3H] thymidine incorporation on DNA double strand break repair has been extensively analyzed in this background[1] and mutagenesis can be measured at different loci
Radioactivity incorporated into the DNA was counted in trichloroacetic acid (TCA) precipitates 20 hours after exposure to [3H]thymidine, i.e., when incorporation plateaued and greater than 95% of cells contained labeled nucleotides
Summary
The DNA most of the energy is locally deposited into the nucleus by low-energy compounds, such as tritium[1,2]. In a previous study using labeled nucleoside [3H] thymidine, which is incorporated into DNA, we established a model to analyze the genetic impact of endogenous self-irradiation of DNA by a low energy-emitting source, such as tritium. We have demonstrated that low doses of incorporated [3H]thymidine, which are barely toxic, induce DNA double strand breaks and stimulate homologous recombination (HR) in a linear dose-response manner, reaching a plateau between 1 and 2 Gy/nucleus (>70% viability). External γ-rays behave differently, and 4 to 6 Gy (>10% viability) are required to detect rare HR events[1] These findings highlights the differences between external and internal (endogenous) exposure to radiation. Given that radiation generates ROS through water radiolysis, incorporation of labeled radionuclides constitutes a model for the production of local endogenous oxidative stress. When a threshold is achieved, the cell response induces both detoxification of ROS and induction of DNA repair enzymes specialized in oxidative DNA damage, decreasing the mutagenic outcome
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
