Methotrexate induces alterations in one-carbon metabolism and in plasmalogen and phosphatidylcholine levels in CSF of juvenile rats

Treatment for pediatric acute lymphoblastic leukemia (ALL) is associated with neurotoxicity and leads to chemotherapy-related cognitive impairment (CRCI) in 40-70% of survivors. Methotrexate (MTX), a key component of ALL chemotherapy regimens, is a major contributor to CRCI. However, because ALL chemotherapy involves multiple agents, the independent contribution of MTX to the previously observed metabolic changes remains unclear. Using a juvenile rat model (N = 40, evenly split between males and females) designed to isolate MTX-specific effects within a pediatric-relevant context, we administered six intraperitoneal (0.5 mg/kg per dose) and four intrathecal (1 mg/kg per dose) MTX injections and performed CSF metabolomics at the time of the first intrathecal injection and last. Five weeks later, we assessed spatial and visual memory using object placement (OP) and novel object recognition (OR) behavioral tests. MTX-treated rats exhibited spatial and visual memory impairments compared with controls. In accordance with our previous results and MTX's mechanism of action, determinants of one-carbon metabolism were downregulated in the CSF of MTX-treated animals between the timepoints tested. In contrast, transsulfuration pathway metabolites such as cystathionine and cysteine were upregulated. Last, MTX treatment also induced alterations in lipid metabolism, with a significant over-representation of 8 plasmalogens and 9 phosphatidylcholines. These findings support the use of CSF metabolomics to characterize MTX-associated neurotoxicity and provide insight into metabolic pathways that may be associated with CRCI and therefore warrant future mechanistic investigation.