15-lipoxygenase-1 mediated formation of endogenous GSH- and RNA-adducts and its impact on GSH/GSSG homeostasis in monocytes/macrophages

15-lipoxygenase-1 (15-LOX-1) converts arachidonic acid to 15(S)-hydroperoxy-[5Z,8Z,11Z,13E]-eicosatetraenoic acid (15(S)-HpETE), which can decompose to the bifunctional electrophile 4-oxo-2-nonenal (ONE). ONE forms adducts with DNA and proteins, which are potential biomarkers of lipid-peroxidation-mediated oxidative stress. Recently a novel ONE-GSH-adduct was characterized as thiadiazabicyclo-ONE-GSH (TOG). A major objective of the present study was to test whether 15-LOX-1-mediated lipid peroxidation can lead to endogenous TOG formation. The impact of 15-LOX-1 expression on glutathione (GSH) homeostasis was also evaluated. Furthermore, ONE-RNA adducts were also characterized for the first time. 15-LOX-1-mediated formation of TOG was established in RAW267.4 cells that constitutively over-express 15-LOX-1 (R15LO cells). Intracellular TOG levels increased upon treatment with arachidonic acid or calcium ionophore A23187, accompanied by increased lipid peroxidation mediated by 15-LOX-1. The LOX inhibitor cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC) significantly reduced lipid peroxidation and abated TOG formation. Further analysis of membrane lipids suggested translocation of 15-LOX-1 upon calcium mobilization and subsequent oxidation of membrane lipids by 15-LOX-1. The 15-LOX-1-dependent TOG formation was further studied in primary human monocytes. Interleukin-4 (IL-4) treatment differentiated primary human monocytes and induced 15-LOX-1 expression. Arachidonic acid and calcium ionophore A23187 significantly increased TOG levels in IL-4-differentiated primary human monocytes and the increased levels were abolished by CDC. Primary human monocytes had much higher 15-LOX-1 activity than R15LO cells. The increase and decrease of lipid oxidation followed that of TOG levels. In order to study the impact of 15-LOX-1 on GSH/GSH-disulfide (GSSG) homeostasis, a liquid chromatography (LC)/mass spectrometry (MS) method was developed to quantify cellular GSH and GSSG. ONE treatment depleted GSH and compromised the cellular redox state. Interestingly, 15-LOX-1 enhanced the resistance of cells to ONE-induced GSH depletion. Calcium ionophore also depleted GSH. However, 15-LOX-1 exacerbated the effect of calcium ionophore on GSH/GSSG homeostasis. Adducts formed between ONE and the RNA nucleotides guanosine (Guo), adenosine (Ade) and cytidine (Cyd) were characterized to be the heptanone-etheno-adducts. A LC/MS method was developed to quantify these adducts in cells. All three adducts were detected in RAW267.4 cells after ONE treatment. The endogenous levels of these adducts were very low. These results suggest that RNA is highly protected from damage by endogenous ONE