Uric acid effects on glutathione metabolism estimated by induction of glutamate-cysteine ligase, glutathione reductase and glutathione synthetase in mouse J744A.1 macrophage cell line

Abstract

Introduction: Elevated plasma levels of uric acid (UA) are considered an independent risk factor for hypertension, diabetes, cardiovascular disease, endothelial and vascular damage, obesity, and metabolic syndrome. Even physiological concentrations of soluble UA have been proved to induce gene expression of macrophage-secreted inflammatory cytokines and stimulate production of reactive oxygen species in mature adipocytes. UA is also described as a powerful endogenous plasma antioxidant, which reveals a paradox of duality for this parameter. Aim: The aim of this study was to investigate the effect of UA on expression of antioxidant defense related enzymes in cultured J744A.1 macrophage cell line. Materials and methods: Mouse macrophage J744A.1 cells were treated with uric acid at increasing concentrations of 200 to 800 μM. Changes in expression levels of genes related to the metabolism of glutathione – glutamate-cysteine ligase, catalytic subunit (GCLc), glutathione peroxidase 1 (GPx1), glutathione reductase (GR) and glutathione synthetase (GS) were analyzed. Gene expression levels were calculated using the 2-ΔΔCt method. Results: When UA is applied in concentrations of 200 µM and 400 µM, cell viability did not change significantly. Higher, pathophysiological concentrations of 600 μM, 800 μM UA, and 1000 μM of UA caused significant decrease in cell viability to 95.81% (p<0.01), 76.22% (p<0.001), and 18.01% (p<0.001), respectively. UA treatment in concentrations of 200 μM, 400 μM, 500 μM, and 800 μM induced significant transcription levels of glutathione reductase – 8.14 (p<0.05), 7.15 (p<0.01), 22.07 (p<0.001), and 27.77 (p<0.01), respectively, and of glutathione synthetase – 13.71 (p<0.01), 13.05 (p<0.05), 18 (p<0.01), and 48.60 (p<0.01) folds, respectively. GCLc and GPx1 genes were transcriptionally activated by higher (500 μM and 800 μM) concentrations of UA. For these UA concentrations the measured levels of mRNA were 7.51 (p<0.05) and 12 fold (p<0.05) higher than the non-treated control for GCLc and 1.90 (p<0.05) and 1.93 (p<0.01) for GPx1. Significant difference in the GCLc expression was found between the 200 μM and 500 μM (p<0.05) and 800 μM (p<0.01) treated cells. mRNA levels were significantly different between 400 μM and 800 μM (p<0.05) for both GCLc and GR genes. Very strong correlation was found between GCLc and GR (0.974, p=0.005) and GS (0.935, p=0.020) expression and between GS and GR (0.886, p=0.045) expression levels. Conclusions: It appears that 500 μM and pathophysiological concentrations (800 μM) of UA induce antioxidant cell response in J744A.1 macrophages proved by the indicative elevation GCL, GPx1, GR, and GS transcription. GR and GS can be stimulated even by lower concentrations (200 μM and 400 μM) indicating that glutathione metabolism in macrophages is tightly regulated in order to keep adequate GSH levels.