ABSTRACT
Myeloperoxidase (MPO) is involved in the development of many chronic inflammatory diseases, in addition to its key role in innate immune defenses. This is attributed to the excessive production of hypochlorous acid (HOCl) by MPO at inflammatory sites, which causes tissue damage. This has sparked wide interest in the development of therapeutic approaches to prevent HOCl-induced cellular damage including supplementation with thiocyanate (SCN-) as an alternative substrate for MPO. In this study, we used an enzymatic system composed of glucose oxidase (GO), glucose, and MPO in the absence and presence of SCN-, to investigate the effects of generating a continuous flux of oxidants on macrophage cell function. Our studies show the generation of hydrogen peroxide (H2O2) by glucose and GO results in a dose- and time-dependent decrease in metabolic activity and cell viability, and the activation of stress-related signaling pathways. Interestingly, these damaging effects were attenuated by the addition of MPO to form HOCl. Supplementation with SCN-, which favors the formation of hypothiocyanous acid, could reverse this effect. Addition of MPO also resulted in upregulation of the antioxidant gene, NAD(P)H:quinone acceptor oxidoreductase 1. This study provides new insights into the role of MPO in the modulation of macrophage function, which may be relevant to inflammatory pathologies.
ABSTRACT
The exposure of RNA and DNA nucleobases to the oxidant hypochlorous acid (HOCl) results in the generation of different stable chlorinated products. These chlorinated nucleobases are formed in vivo, particularly in chronic inflammatory pathologies, which are characterized by the overproduction of HOCl by myeloperoxidase. As such, chlorinated nucleosides are used as biomarkers of inflammation. However, these compounds have also attracted attention as potential chemotherapeutic agents with 8-chloro-adenosine (8ClA), for example, currently in clinical trials for the treatment of hematological cancers, including chronic lymphocytic leukemia. 8ClA has mainly RNA-directed effects in malignant cells, with exposure resulting in ATP depletion and apoptotic cell death. Whether 8ClA has significant reactivity with nonmalignant cells has not been widely studied. Here we show that prolonged incubation of J774A.1 macrophage-like cells with 8ClA results in the perturbation of cellular metabolism and apoptotic cell death. These effects are associated with an accumulation of 8-chloroadenosine triphosphate (8Cl-ATP), an effect not seen in experiments utilizing other chlorinated nucleosides. Exposure of the macrophages to 8ClA did not significantly change basal mitochondrial respiration or glycolysis but resulted in an increase in maximal mitochondrial respiration as well as spare respiratory capacity within these cells. Additionally, 8ClA exposure also altered the mRNA expression of a range of antioxidant and DNA damage repair genes in the macrophages in a manner consistent with a reduction in the capacity of the cells to cope with oxidative stress and repair DNA damage. Taken together, these results provide new insight into pathways by which the production of HOCl during chronic inflammation could perturb immune cell function and may also have implications for the use of 8ClA as a chemotherapeutic drug.
