Methotrexate (MTX) is a chemotherapeutic agent that can
cause a range of toxic side effects including gastrointestinal damage,
hepatotoxicity, myelosuppression, and nephrotoxicity and has potentially
complex interactions with the gut microbiome. Following untargeted UPLCqtof-MS analysis of urine and fecal samples from male Sprague−Dawley rats
administered at either 0, 10, 40, or 100 mg/kg of MTX, dose-dependent
changes in the endogenous metabolite profiles were detected. Semiquantitative
targeted UPLC-MS detected MTX excreted in urine as well as MTX and two
metabolites, 2,4-diamino-N-10-methylpteroic acid (DAMPA) and 7-hydroxyMTX, in the feces. DAMPA is produced by the bacterial enzyme
carboxypeptidase glutamate 2 (CPDG2) in the gut. Microbiota profiling
(16S rRNA gene amplicon sequencing) of fecal samples showed an increase in
the relative abundance of Firmicutes over the Bacteroidetes at low doses of
MTX but the reverse at high doses. Firmicutes relative abundance was positively correlated with DAMPA excretion in feces at 48 h,
which were both lower at 100 mg/kg compared to that seen at 40 mg/kg. Overall, chronic exposure to MTX appears to induce
community and functionality changes in the intestinal microbiota, inducing downstream perturbations in CPDG2 activity, and thus
may delay MTX detoxication to DAMPA. This reduction in metabolic clearance might be associated with increased gastrointestinal
toxicity.