8-Chloroadenosine Alters the Metabolic Profile and Downregulates Antioxidant and DNA Damage Repair Pathways in Macrophages.

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.

A Role for Chlorinated Nucleosides in the Perturbation of Macrophage Function and Promotion of Inflammation.

During inflammation, myeloperoxidase released from activated phagocytes generates the highly reactive oxidant hypochlorous acid (HOCl). This oxidant plays an important role in the immune response but can also promote tissue damage and has been strongly linked with the development of numerous inflammatory diseases. HOCl reacts with cellular DNA forming chlorinated nucleobases, which induce strand breaks, mutations, and cross-links. Although it has been shown that chlorinated nucleosides are present within inflammatory pathologies and diseased tissue, whether or not these species are biomarkers formed as a byproduct of chronic inflammation or play a role in the disease progression has not been ascertained. In this study, we show that exposure of J774A.1 macrophage-like cells to chlorinated ribose and deoxyribose nucleosides results in the incorporation of 5-chloro-cytidine (5ClC), 8-chloro-adenosine (8ClA), and 8-chloro-guanosine (8ClG) into the cellular RNA and 5-chloro-deoxycytidine (5CldC) but not 8-chloro-deoxyguanosine (8CldG) or 8-chloro-deoxyadenosine (8CldA) into cellular DNA. Evidence was obtained for the clearance of 5ClC from the RNA, with a loss of 8ClA and 8ClG observed to a lesser extent, whereas an increase in the level of 5CldC in DNA was seen on further incubation of treated cells in the absence of chlorinated nucleosides. Importantly, exposure of the macrophages to chlorinated nucleosides, particularly 8ClG and 5ClC, resulted in the increased expression of interleukin-1ß, and other pro-inflammatory cytokines and chemokines. With 5ClC, this inflammatory response was associated with the increased nuclear translocation of the NF-κB subunit, p65, rather than inflammasome activation. This alteration in gene expression appeared to be unrelated to the extent of incorporation of the chlorinated nucleosides into RNA or DNA and was not associated with any significant changes in cell viability or proliferation. Taken together, these results highlight a potential biological role for chlorinated nucleosides to promote inflammatory disease, in addition to their utility as biomarkers.