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.