Implications of Altered Glutathione Metabolism in Aspirin-Induced Oxidative Stress and Mitochondrial Dysfunction in HepG2 Cells

Faculty of Pharmacy, Ain Shams University, Egypt
PLoS ONE (Impact Factor: 3.23). 04/2012; 7(4):e36325. DOI: 10.1371/journal.pone.0036325
Source: PubMed


We have previously reported that acetylsalicylic acid (aspirin, ASA) induces cell cycle arrest, oxidative stress and mitochondrial dysfunction in HepG2 cells. In the present study, we have further elucidated that altered glutathione (GSH)-redox metabolism in HepG2 cells play a critical role in ASA-induced cytotoxicity. Using selected doses and time point for ASA toxicity, we have demonstrated that when GSH synthesis is inhibited in HepG2 cells by buthionine sulfoximine (BSO), prior to ASA treatment, cytotoxicity of the drug is augmented. On the other hand, when GSH-depleted cells were treated with N-acetyl cysteine (NAC), cytotoxicity/apoptosis caused by ASA was attenuated with a significant recovery in oxidative stress, GSH homeostasis, DNA fragmentation and some of the mitochondrial functions. NAC treatment, however, had no significant effects on the drug-induced inhibition of mitochondrial aconitase activity and ATP synthesis in GSH-depleted cells. Our results have confirmed that aspirin increases apoptosis by increased reactive oxygen species production, loss of mitochondrial membrane potential and inhibition of mitochondrial respiratory functions. These effects were further amplified when GSH-depleted cells were treated with ASA. We have also shown that some of the effects of aspirin might be associated with reduced GSH homeostasis, as treatment of cells with NAC attenuated the effects of BSO and aspirin. Our results strongly suggest that GSH dependent redox homeostasis in HepG2 cells is critical in preserving mitochondrial functions and preventing oxidative stress associated complications caused by aspirin treatment.

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Available from: Haider Raza, Sep 08, 2014
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    • "J774.2 macrophage cells were cultured to 80% confluence and treated with LPS alone or in combination with ASA with or without NAC as described in Materials and Methods. GST-dependent GSH conjugation (3A–3B) and GSH-Px (3C–3D) activities were measured as described before [13]. Results are expressed as mean +/− SEM of at least three experiments. "
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    ABSTRACT: Bacterial endotoxin lipopolysaccharide (LPS) induces the production of inflammatory cytokines and reactive oxygen species (ROS) under in vivo and in vitro conditions. Acetylsalicylic acid (ASA, aspirin) is a commonly used anti-inflammatory drug. Our aim was to study the effects of N-acetyl cysteine (NAC), an antioxidant precursor of GSH synthesis, on aspirin-sensitized macrophages treated with LPS. We investigated the effects of LPS alone and in conjunction with a sub-toxic concentration of ASA, on metabolic and oxidative stress, apoptosis, and mitochondrial function using J774.2 mouse macrophage cell line. Protection from LPS-induced toxicity by NAC was also studied. LPS alone markedly induced ROS production and oxidative stress in macrophage cells. When ASA was added to LPS-treated macrophages, the increase in oxidative stress was significantly higher than that with LPS alone. Similarly, alteration in glutathione-dependent redox metabolism was also observed in macrophages after treatment with LPS and ASA. The combination of LPS and ASA selectively altered the CYP 3A4, CYP 2E1 and CYP 1A1 catalytic activities. Mitochondrial respiratory complexes and ATP production were also inhibited by LPS-ASA treatment. Furthermore a higher apoptotic cell death was also observed in LPS-ASA treated macrophages. NAC pre-treatment showed protection against oxidative stress induced apoptosis and mitochondrial dysfunction. These effects are presumed, at least in part, to be associated with alterations in NF-κB/Nrf-2 mediated cell signaling. These results suggest that macrophages are more sensitive to LPS when challenged with ASA and that NAC pre-treatment protects the macrophages from these deleterious effects.
    PLoS ONE 07/2014; 9(7):e103379. DOI:10.1371/journal.pone.0103379 · 3.23 Impact Factor
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    • "It has been shown that the proapoptotic induction of oxidative stress induced by NSAIDs such as aspirin is strongly associated with the modulation of cellular redox homeostasis. This is exemplified by the observed increase of aspirin-induced apoptosis in HepG2 cells with GSH depletion and compromised redox balance [174]. In addition to this, studies in S. cerevisiae yeast cells have shown that aspirin-treated MnSOD-deficient yeast cells grown in ethanol medium experienced a very significant decrease in cellular reducing power with respect to wildtype cells, as measured by the NADPH/NADP+ concentration ratio. "
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