Rescuing effect of folates on methotrexate cytotoxicity in human trophoblast cells

Objectives: Methotrexate (MTX)is a folate antagonist that is administered in several conditions such as rheumatoid arthritis. Its use may associate with adverse effects presumably originating from folate deficiency. Although MTX side effects could be decreased by folate supplementation, the current guideline on folate administration is not precisely established, which could result in irreversible damage especially in high-risk groups like women in childbearing-age. Thus, this study aimed to investigate the in vitro rescuing effect of different folates including folic acid (FA), 5-methyltetrahydrofolate (MTHF) and folinic acid (5-Formyltetrahydrofolic acid, FTHF) on MTX-treated trophoblast cells. Methods: HTR-8/SVneo cells were stressed with a minimum effective dose of MTX and simultaneously treated with different concentrations of FA, MTHF or FTHF. The rescuing effect was assessed by MTT viability assay. The evaluation was completed by microscopic monitoring, apoptosis assessment and measuring LINE-1 DNA methylation. Results: The MTT viability assay showed no MTX-rescuing effect of FA, but a significant effect of FTHF or MTHF. Microscopic observations supported the results of the viability assay. Accordingly, apoptosis was reduced in MTHF or FTHF treatments, while FA has no effect on the apoptosis induced by MTX. The LINE-1 methylation was not affected by MTX treatment, and not significantly modified in folate supplemented cultures. Conclusions: Despite the general acceptance of administering FA to prevent adverse events of MTX therapy, our findings suggest that FA may not be optimal, and indicate FTHF or MTHF as a better choice. This study on trophoblast cells suggests that FTHF may be the optimal folate, particularly for women in childbearing-age.