A Two-Way Interaction between Methotrexate and the Gut Microbiota of Male Sprague–Dawley Rats

Abstract

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 UPLC-qtof-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-hydroxy-MTX, 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.

Methotrexate impacts conserved pathways in diverse human gut bacteria leading to decreased host immune activation

“…MTX can directly bind DHFR from multiple bacteria (Bolin et al, 1982), and DHFR overexpression rescues the growth of a sensitive E. coli strain (Kopytek et al, 2000). More recently, a high-throughput screen of human gut bacterial isolates identified multiple MTX sensitive strains (Maier et al, 2018), experiments in rats and mice revealed shifts in the gut microbiota in response to intraperitoneal MTX (Letertre et al, 2020;Zhou et al, 2018), and clinical studies suggest that some of the differences between the microbiomes of RA patients relative to healthy controls (Scher et al, 2013) are reversed during treatment (Zhang et al, 2015). However, it remains unclear whether the observed changes to the gut microbiota are a direct result of MTX or reflective of concomitant treatment with other medications and dietary supplements, improvement of the disease process, reduction of inflammation, and/or other confounding factors that may be present in observational studies.…”

Microbiota and Drug Response in Inflammatory Bowel Disease

“…In the gut, both MTX-PG1 and 7-OH-MTX are transported into the cells where they are metabolized by folylpolyglutamate synthetase (FPGS) into MTX polyglutamates (MTX-PGs), which are not able to get out of cells because of their low affinity for folate transporters. MTX-PGs are subsequently reconverted by human γ-glutamyl hydrolase or bacterial glutamate carboxypeptidase II to MTX-PG1, which is again able to leave the cells [ 113 , 114 , 115 ].…”

“…These two products are inactive, so they contribute to the detoxification of the organism from MTX, which is fundamental especially in patients with delayed drug clearance and/or MTX-related adverse effects. Despite Pseudomonas (Proteobacteria), other bacteria able to synthesize glutamate carboxypeptidase II might be Prevotellaceae (Bacteroidetes) and Anaeroplasmataceae (Tenericutes); indeed, studies showed that the presence of DAMPA in the feces is positively associated with these bacterial families [ 114 ].…”

“…High doses of MTX increment Peptostreptococcaceae (Firmicutes) and Porphyromonadaceae (Bacteroidetes) levels and reduce Clostridium, Eubacterium, Ruminococcus (Firmicutes) and Bifidobacterium (Actinobacteria) levels; indeed, high concentrations of this drug can inhibit the growth of almost a half of the representative gut bacteria. Additionally, long-term exposure to MTX or the use of high doses have deleterious effects on the GM, as they induce some perturbations leading to an altered glutamate carboxypeptidase II activity, which reduces MTX conversion to the nontoxic DAMPA and thus increases MTX-related gastrointestinal toxicity [ 114 ]. MTX is able to inhibit the growth of the IBD-associated pathogenic microorganism Mycobacterium avium subspecies paratuberculosis (Actinobacteria), so its therapeutic actions may not only be related to the antiproliferative or anti-inflammatory effects, but also to the treatment of MAP infections; however, it is still unknown if the eradication of MAP could be beneficial in IBD [ 100 ].…”

Opportunities and Challenges for Gut Microbiota in Acute Leukemia

“…The relative abundance of the Firmicutes was positively correlated with the 48-h fecal excretion of 2,4-diamino-N-10-methylpteroic acid (DAMPA), a metabolite of MTX. Long-term exposure to MTX can alter the composition and function of the microbiome, which in turn affects its ability to detoxify MTX ( 69 ). MTX largely alters the human gut microbiota.…”