Direct antioxidant properties of methotrexate: Inhibition of malondialdehyde-acetaldehyde-protein adduct formation and superoxide scavenging
Matthew C. Zimmerman,
Dahn L. Clemens,
Michael J. Duryee,
Cleofes Sarmiento,
Andrew Chiou,
Carlos D. Hunter,
Jun Tian,
Lynell W. Klassen,
James R. O’Dell,
Geoffrey M. Thiele,
Ted R. Mikuls,
Daniel R. Anderson
Affiliations
Matthew C. Zimmerman
Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, 982650 Nebraska Medical Center, Omaha, NE 68198-2265, United States
Dahn L. Clemens
Department of Internal Medicine, University of Nebraska Medical Center, 982650 Nebraska Medical Center, Omaha, NE 68198-2265, United States
Michael J. Duryee
Department of Internal Medicine, University of Nebraska Medical Center, 982650 Nebraska Medical Center, Omaha, NE 68198-2265, United States
Cleofes Sarmiento
Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, 982650 Nebraska Medical Center, Omaha, NE 68198-2265, United States
Andrew Chiou
Department of Internal Medicine, University of Nebraska Medical Center, 982650 Nebraska Medical Center, Omaha, NE 68198-2265, United States
Carlos D. Hunter
Department of Internal Medicine, University of Nebraska Medical Center, 982650 Nebraska Medical Center, Omaha, NE 68198-2265, United States
Jun Tian
Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, 982650 Nebraska Medical Center, Omaha, NE 68198-2265, United States
Lynell W. Klassen
Department of Internal Medicine, University of Nebraska Medical Center, 982650 Nebraska Medical Center, Omaha, NE 68198-2265, United States
James R. O’Dell
Department of Internal Medicine, University of Nebraska Medical Center, 982650 Nebraska Medical Center, Omaha, NE 68198-2265, United States
Geoffrey M. Thiele
Department of Internal Medicine, University of Nebraska Medical Center, 982650 Nebraska Medical Center, Omaha, NE 68198-2265, United States
Ted R. Mikuls
Department of Internal Medicine, University of Nebraska Medical Center, 982650 Nebraska Medical Center, Omaha, NE 68198-2265, United States
Daniel R. Anderson
Department of Internal Medicine, University of Nebraska Medical Center, 982650 Nebraska Medical Center, Omaha, NE 68198-2265, United States
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.
