The selenoprotein GPX4 is essential for mouse development and protects from radiation and oxidative damage insults

Lipid peroxidation has been implicated in a variety of pathophysiological processes, including inflammation, atherogenesis, neurodegeneration, and the ageing process. Phospholipid hydroperoxide glutathione peroxidase (GPX4) is the only major antioxidant enzyme known to directly reduce phospholipid hydroperoxides within membranes and lipoproteins, acting in conjunction with alpha tocopherol (vitamin E) to inhibit lipid peroxidation. Here we describe the generation and characterization of GPX4-deficient mice by targeted disruption of the murine Gpx4 locus through homologous recombination in embryonic stem cells. Gpx4(-/-) embryos die in utero by midgestation (E7.5) and are associated with a lack of normal structural compartmentalization. Gpx4(+/-) mice display reduced levels of Gpx4 mRNA and protein in various tissues. Interestingly, cell lines derived from Gpx4(+/-) mice are markedly sensitive to inducers of oxidative stress, including gamma-irradiation, paraquat, tert-butylhydroperoxide, and hydrogen peroxide, as compared to cell lines derived from wild-type control littermates. Gpx4(+/-) mice also display reduced survival in response to gamma-irradiation. Our observations establish GPX4 as an essential antioxidant enzyme in mice and suggest that it performs broad functions as a component of the mammalian antioxidant network.     

Embryonic fibroblasts from Gpx4+/- mice: a novel model for studying the role of membrane peroxidation in biological processes

A previous study using mice null for Gpx4 indicates that PHGPx plays a critical role in antioxidant defense and is essential for the survival of the mouse. In the present study, we further analyzed the stress response of MEFs (murine embryonic fibroblasts) derived from mice heterozygous for the Gpx4 gene (Gpx4(+/-) mice). MEFs from Gpx4(+/-) mice have a 50% reduction in PHGPx expression without any changes in the activities of other major antioxidant defense enzymes. Compared to MEFs from Gpx4(+/+) mice, MEFs from Gpx4(+/-) mice were more sensitive to exposure to the oxidizing agent t-butyl hydroperoxide (t-BuOOH), and t-BuOOH exposure induced increased apoptosis in MEFs from Gpx4(+/-) mice. When cultured at low cell density, MEFs from Gpx4(+/-) mice also showed retarded growth under normal culture conditions (20% oxygen) that was reversed by culturing under low oxygen (2% oxygen). In addition, oxidative damage was increased in the MEFs from the Gpx4(+/-) mice, as indicated by increased levels of F(2)-isoprostanes and 8-oxo-2-deoxyguanosine in these cells. Our data demonstrate that MEFs from Gpx4(+/-) mice are more sensitive to oxidative stress because of reduced expression of PHGPx.     

Distinct Alterations in Mitochondrial Mass and Function Characterize Different Models of Apoptosis

Recent studies have shown that reduction in mitochondrial membrane potential (ΔΨ_m) and generation of reactive oxygen species are early events in apoptosis. In this study, we present two different models of apoptotic cell death, Chinese hamster ovary (CHO) cells treated with aphidicolin and dexamethasone-treated 2B4 T-cell hybridoma cells, which display opposing mitochondrial changes. CHO cells arrested at G1/S with aphidicolin have a progressive increase in mitochondria mass and number, assessed by flow cytometry and fluorescent microscopy with mitochondria-specific probes. The increase in mitochondrial mass was not accompanied by a gain in net cellular mitochondrial membrane potential, consistent with an accumulation of relatively depolarized mitochondria. Fluorescent microscopy demonstrated an increased content of low ΔΨ_mmitochondria in aphidicolin-treated CHO cells, but high ΔΨ_mmitochondria were also present and remained stable in number. Mitochondrial mass correlated with decreased clonogenicity of aphidicolin-treated CHO cells. Cycloheximide prevented both the proliferation of mitochondria and subsequent cell death. In contrast, dexamethasone treatment of 2B4 T-cell hybridoma cells caused a decrease in ΔΨ_mwithout mitochondrial proliferation. Cycloheximide and Bcl-2 overexpression inhibited the loss of ΔΨ_m, as well as apoptosis. In both models, cell death was associated with a decrease in mitochondrial potential relative to mitochondrial mass, suggesting that an accumulation of damaged or dysfunctional mitochondria had occurred.