Direct antioxidant properties of methotrexate: Inhibition of malondialdehyde-acetaldehyde-protein adduct formation and superoxide scavenging

Methotrexate (MTX) is an immunosuppressant commonly used for the treatment of autoimmune diseases. Recent observations have shown that patients treated with MTX also exhibit a reduced risk for the development of cardiovascular disease (CVD). Although MTX reduces systemic inflammation and tissue damage, the mechanisms by which MTX exerts these beneficial effects are not entirely known. We have previously demonstrated that protein adducts formed by the interaction of malondialdehyde (MDA) and acetaldehyde (AA), known as MAA-protein adducts, are present in diseased tissues of individuals with rheumatoid arthritis (RA) or CVD. In previously reported studies, MAA-adducts were shown to be highly immunogenic, supporting the concept that MAA-adducts not only serve as markers of oxidative stress but may have a direct role in the pathogenesis of inflammatory diseases. Because MAA-adducts are commonly detected in diseased tissues and are proposed to mitigate disease progression in both RA and CVD, we tested the hypothesis that MTX inhibits the generation of MAA-protein adducts by scavenging reactive oxygen species. Using a cell free system, we found that MTX reduces MAA-adduct formation by approximately 6-fold, and scavenges free radicals produced during MAA-adduct formation. Further investigation revealed that MTX directly scavenges superoxide, but not hydrogen peroxide. Additionally, using the Nrf2/ARE luciferase reporter cell line, which responds to intracellular redox changes, we observed that MTX inhibits the activation of Nrf2 in cells treated with MDA and AA. These studies define previously unrecognized mechanisms by which MTX can reduce inflammation and subsequent tissue damage, namely, scavenging free radicals, reducing oxidative stress, and inhibiting MAA-adduct formation.
Graphical abstract The production of reactive oxygen species (ROS) can result in lipid peroxidation. Lipid peroxidation can, in turn, result in the formation of malondialdehyde (MDA), which can spontaneously breakdown forming acetaldehyde (AA). Malondialdehyde and acetaldehyde can interact to form the stable hybrid malondialdehyde-acetaldehyde adduct (MAA) on proteins and other macromolecules. Binding of MAA-adducted proteins to cells can lead to expression of pro-inflammatory cytokines, resulting in inflammation. MAA-adducted proteins are also themselves immunogenic and can initiate an inflammatory response. Importantly, methotrexate can scavenge free radicals. This may ameliorate the formation of lipid peroxides and the resulting formation of MDA and AA. Additionally, methotrexate can directly inhibit the formation of MAA-protein adducts. Thus, methotrexate can scavenge free radicals and inhibit the formation of MAA-adducts. These are previously undescribed mechanisms by which methotrexate may reduce inflammation and the tissue damage associated with chronic inflammatory diseases.fx1
Highlights • MTX is commonly used to treat RA and is being tested in CVD patients. • MDA and AA are produced during lipidperoxidation and can interact to form MAA-adducts. • MAA-adducts are found in atheromas and in diseased synovial tissue of RA patients. • MTX scavenges the free radical O2− and prevents the formation of MAA-adducts. • Scavenging O2− may be a mechanism by which MTX reduces inflammation and disease.