Fig. 4: Cyclophosphamide-induced small intestinal toxicity is due to increased drug metabolism.

a Schematic of cyclophosphamide (CP) activation. b Quantification of the percent change in total cells relative to untreated cells in small and large intestine-derived monolayers treated with 100 µM CP and 100 µM 4-hydroperoxycyclophosphamide (4-HC) for 48 h. n = 6 wells (control) or n = 3 wells (drug treatment). Statistical significance was calculated by a two-way ANOVA followed by Sidak’s multiple comparison test. c Measured CYP3A activity in small and large intestine-derived monolayers. n = 6 wells. Statistical significance was calculated by an unpaired t-test with Welch’s correction. d Detection of 4-hydroxycyclophosphamide (4-OHCP) in the media of small and large intestine-derived monolayers incubated with 100 µM CP for 24 h by LC–MS/MS. Peak area ratio: sample 4-OHCP peak area/4-OHCP internal standard peak area. n = 3 wells. Statistical significance was calculated by an unpaired t-test with Welch’s correction. e Schema for CP in vivo treatment. f Changes in apoptosis in small and large intestines from mice treated with CP or vehicle. Representative images of small and large intestines stained for TUNEL and propidium iodide. Scale bars, 100 µm. Quantification of TUNEL⁺ cells per crypt. Boxplot showing median value, whiskers showing lower 1^st and upper 99^th percentiles. n = 60 crypts from 3 mice. Statistical significance was calculated by a two-way ANOVA followed by Sidak’s multiple comparison test. Error bars mean ± SEM. SI: small intestine; LI: large intestine. * indicates p-values < 0.05; ** indicates p-values < 0.01; *** indicates p-values < 0.001; **** indicates p-values < 0.0001.