Methotrexate (MTX) chemotherapy is often limited by its severe side effects which include nephrotoxicity. In the continuous search of efficient antioxidants that could ameliorate MTX toxicity, our study aimed to evaluate the efficiency of N-acetyl cysteine (NAC), Trolox methyl ether (Trolox-Me), and curcumin as potent antioxidants using an in vitro model of MTX-induced toxicity.

Human embryonic kidney (HEK293) cells were pre-exposed to different antioxidants for 2 h before MTX treatment. After 24 hours of exposure to MTX, cell viability and morphology were assessed. Activities of antioxidant enzymes and levels of lipid peroxidation were measured by spectrophotometric methods, and protein expression was determined by Western blotting.

Exposure to MTX at concentrations between 1 and 100 μM for 6 and 24 h decreased cell viability in a dose-dependent manner and was correlated with the increase in p53 protein expression. All three antioxidants tested were able to inhibit MTX-induced apoptosis, as revealed by the expression of heat shock proteins (Hsp27, Hsp60, Hsp70, and Hsp90). Pre-treatment of cells with 50 μM of Trolox-Me succeeded to significantly decrease the MTX-induced cell death. The reduction in the activities of glutathione reductase and glutathione S-transferase after MTX treatment was correlated with a reduced level of GSH, and was attenuated by the pre-incubation with Trolox-Me or curcumin. These antioxidants were able to maintain enough GSH for the reactions of conjugation with MTX metabolites in order to decrease its toxicity.

In conclusion, the pre-treatment with curcumin, Trolox-Me, or NAC was significantly effective in blocking MTX toxicity at the concentration investigated in vitro on kidney cells. The results of our study encourage further clinical assessments in order to use these antioxidants in dietary prevention of renal side effects of MTX.