Methotrexate-induced decrease in embryonic 5-methyl-tetrahydrofolate is irreversible with leucovorin supplementation

Folate is a nutrient crucial for rapidly growing tissues, including developing embryos and cancer cells. Folate participates in the biosynthesis of nucleic acids, proteins, amino acids, S-adenosylmethionine, many neurotransmitters, and some vitamins. The intracellular folate pool consists of different folate adducts, which carry one-carbon units at three different oxidative states and participate in distinct biochemical reactions. Therefore, the content and dynamics of folate adducts will affect the homeostasis of the metabolites generated in these folate-mediated reactions. Currently, the knowledge on the level of each individual folate adduct in developing embryos is limited. With an improved high-performance liquid chromatography protocol, we found that tetrahydrofolate (THF), the backbone of one-carbon carrier, gradually increased and became dominant in developing zebrafish embryos. 5-methyl-tetrahydrofolate (5-CH3-THF) was abundant in unfertilized eggs but decreased rapidly when embryos started to proliferate and differentiate. 10-formyltetrahydrofolate at first increased after fertilization, and then dropped dramatically before reaching a sustained level at later stages. Dihydrofolate (DHF) slightly decreased initially and remained low throughout embryogenesis. Exposure to methotrexate significantly decreased 5-CH3-THF levels and increased DHF pools, besides causing brain ventricle anomaly. Rescuing with leucovorin partly reversed the abnormal phenotype. Unexpectedly, the level of 5-CH3-THF remained low even when leucovorin was added for rescue. Our results show that different folate adducts fluctuated significantly and differentially in concert with the physiological requirement specific for the corresponding developmental stages. Furthermore, methotrexate lowered the level of 5-CH3-THF in developing embryos, which could not be reversed with folate supplementation and might be more substantial to cellular methylation potential and epigenetic control than to nucleotide synthesis.