Hypoxia/Reoxygenation-Induced Mutations in Mammalian Cells Detected by the Flow Cytometry Mutation Assay and Characterized by Mutant Spectrum

Abstract

Under hypoxic conditions, cells are more resistant to cell killing by ionizing radiation by a factor of 2.5 to 3, potentially compromising the efficacy of radiotherapy. It has been shown recently that hypoxic conditions alone are sufficient to generate mutations in vitro and in vivo, likely due to the creation of reactive oxygen species (ROS) and a decrease in mismatch and homologous recombination DNA repair activity. These factors are known precursors to the onset of genetic instability and poor prognosis. We have previously characterized the flow cytometry mutation assay and its sensitivity to detect significant mutant fractions induced by genotoxic agents that are not detected by other mammalian assays. Here we measure the mutant fraction induced by hypoxia. CHO A L cells cultured at <0.1% O 2 for 24 h generated a significant mutant fraction of 120 × 10 −5 and had growth kinetics and survival characteristics similar to those obtained with other mutagens. We investigated the role of ROS by treating cells with the radical scavenger DMSO, which significantly reduced hypoxia toxicity and mutagenesis. Single cells were sorted from the mutant population, and the resulting clonal populations were stained for five antigens encoded by genes found along chromosome 11 to generate mutant spectra. The mutations were primarily large deletions, similar to those in background mutants, but the frequency was higher. We have demonstrated that hypoxic conditions alone are sufficient to generate mutations in mammalian cells in culture and that the spectrum of mutations is similar to background mutations.

Quercetin and naringenin reduce abnormal development of mouse embryos produced by hydroxyurea

“…Dimethyl sulfoxide, which was used as a vehicle for the flavonoids at a concentration 0.1%, protected embryonic development against hydroxyurea‐induced damage, contributing to the protection exerted by flavonoids. Recently it has been reported that DMSO has cyto‐ and embryoprotective [52,53] activity mediated mainly by antioxidant mechanisms, by reducing the production of reactive oxygen [54] . It is likely that DMSO acts by this mechanism against the teratogenic effect of hydroxyurea.…”

Transient Receptor Potential Ankyrin 1 Activation within the Cardiac Myocyte Limits Ischemia–reperfusion Injury in Rodents

“…The pouch creates an anaerobic environment where the oxygen level within the pouch is less than 0.1%. 17 The cells were then removed from the anaerobic gas pouches and reoxygenated within the cell culture incubator for an additional 4 hours.…”

Role of Hypoxic Stress in Regulating Tumor Immunogenicity, Resistance and Plasticity

“…Indeed, several studies have suggested that hypoxia can induce DNA damage, alter cell cycle checkpoints and/or the sensing and repair of DNA damage, and consequently favor genetic instability ( Figure 1 ) [ 131 , 132 , 133 ]. In this regard, several teams have documented an increase in the rate of DNA mutations in cells exposed to in vitro or in vivo hypoxic conditions, mostly using reporter assays [ 134 , 135 , 136 ]. The origins of these hypoxia-induced DNA mutations are probably multiple, emerging from hypoxia-mediated oncogene amplification, induction of DNA damages or DNA replication stress, deregulation of DNA damage checkpoint signaling, interference with DNA repair or escape from cell death [ 132 ].…”