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

Abstract

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.

Keywords: Electron Paramagnetic Resonance (EPR) Spectroscopy; Malondialdehyde-Acetaldehyde (MAA) Adducts; Methotrexate; Superoxide.

Graphical abstract

Fig. 1

Formation of MAA-protein adducts in vitro is attenuated by MTX. MTX (2 μM) was added to 2 mM Malondialdehyde (MDA), 1 mM Acetaldehyde (AA), and 1 mg Albumin (Alb) and examined flourmetrically (398 nm/460 nm) for the formation of the MAA-adduct at 24, 48, and 72 h. MTX significantly inhibited the formation of the MAA -adduct over the 72 h incubation period. #P < 0.001 vs. ALB + Vehicle at the respective time-point; *P < 0.001 vs. ALB + MDA + AA at the respective time-point. a.u. = arbitrary units. N = 4 for each condition and time point.

Fig. 2

MTX decreases levels of free radicals generated during MAA formation. Representative EPR spectra obtained 24–72 h after initiating cell-free reactions of albumin (ALB, 1 mg) + malondialdehyde (MDA, 2 mM) + acetaldehyde (AA, 1 mM) in the presence or absence of MTX (2 µM). Control samples contained only albumin + vehicle. To detect levels of free radicals, all reactions were incubated with the EPR spectroscopy spin probe, CMH (200 µM), for the final 30 min of reaction time. a.u. = arbitrary units. N = 4 for each condition and time point.

Fig. 3

MTX directly scavenges superoxide generated by hypoxanthine + xanthine oxidase, but does not inhibit xanthine oxidase activity. A) Representative EPR spectra obtained from cell-free reactions containing hypoxanthine (HX, 50 µM) + xanthine oxidase (XO, 10 mU/ml) in the presence or absence of MTX (2 µM). All reactions were incubated for 30 min with the superoxide-sensitive EPR spectroscopy spin probe, CMH (200 µM). To ensure CMH was not pre-oxidized, a control reaction of CMH alone was utilized. B) Summary data of EPR spectra amplitude showing MTX scavenging of superoxide in a dose-dependent manner. Confirming the detection of superoxide generated by the HX+XO reaction, superoxide dismutase (SOD, 400 U) abolished the EPR spectra amplitude N = 3-12. C) Percent change in uric acid levels produced over 30 min in cell-free reactions containing HX (50 µM) + XO (10 mU/ml) ± MTX (2 µM) N = 9-21. *P < 0.05 vs. CMH; ‡ P < 0.05 vs. CMH + HX + XO. a.u. = arbitrary units for each condition and time point.

Fig. 4

MTX does not directly scavenge H₂O₂. A) Summary EPR spectra amplitude data obtained from cell-free reactions containing H₂O₂ (10 µM) and various concentrations of MTX (2–10 mM). All samples were made with the KDD buffer supplemented with AAP, HRP, and DTPA (i.e. KDD (+) buffer) and contained the CMH spin probe (200 µM). *P < 0.05 vs. control (i.e. black bar, without H₂O₂). a.u. = arbitrary units. N = 3 for each condition and time point.

Fig. 5

MTX inhibits cellular redox signaling induced by MAA-adduct formation. NRF2/ARE cells were incubated for 24 h in the presence or absence of MTX with the following; ALB + Vehicle, MDA + AA, ALB + MDA + AA, modified MAA-ALB (25 µg/ml). MTX was evaluated for preventing ROS production as a result of MAA formation. N = 5 for each experiment. (A) MTX significantly attenuates the metabolites of MAA from inducing ROS production in a NRF2/ARE Luciferase Reporter cell line. The metabolites MDA and AA increased ROS production compared to the ALB + Vehicle control regardless of the addition of ALB to the culture. Previously modified MAA-ALB produce no ROS demonstrating that it is the metabolites inducing the response. Interesting was the ability of MTX to significantly inhibit NRF2 activation. * P < 0.001 significantly decreased with MTX. **P < 0.001 significantly increased compared to ALB + Vehicle. ***P < 0.001 significantly decreased compared to MDA + AA and ALB + MDA + AA. (B) Cell viability by LDH demonstrating the metabolites of MAA, MTX, and modified MAA are not effecting cell survival. N = 5 for each condition.