Assessment of cytochrome P450 induction in canine intestinal organoid models

Abstract

Understanding cytochrome P450 (CYP) enzymes in the canine intestine is vital for predicting drug metabolism and developing safer oral medications. This study evaluates canine colonoids as a model to assess the expression and induction of essential intestinal CYP enzymes.Canine colonoids were cultured in expansion medium (EM) with Wnt-3A and in differentiation medium (DM) without Wnt-3A. We assessed the mRNA expression of CYP2B11, CYP2C21, CYP3A12, and CYP3A98 using qPCR and examined the effects of rifampicin and phenobarbital as inducers.Our findings show that DM significantly increased the mRNA expression of CYP3A98 and CYP2B11, but not CYP3A12, compared to EM. CYP2C21, not typically expressed in the intestine, remained unexpressed in colonoids. Rifampicin induced CYP3A98, aligning with pregnane x receptor (PXR) regulation, while phenobarbital did not, suggesting no constitutive androstane receptor (CAR) involvement. CYP2B11 did not respond to either inducer, suggesting alternative regulatory pathways in canine colonoids.This study is a pioneering effort to establish conditions for studying P450 expression in canine colonoids, confirming significant CYP3A98 expression in the canine intestine. It demonstrated colonoids can induce CYP activity post drug treatments. Further research is needed to enhance species-specific drug metabolism understanding and validate this model for broader applications.

Keywords: CYP2B11; CYP3A98; CYP450; Dog; colonoid; intestinal organoid.

Figure 1.. Expansion and differentiation medium on canine colonoids.

(A) schematic representation of the canine colonoids cultured in expansion medium (EM) and differentiation medium (DM). The protocol involved initially incubating the organoids in EM for 4 days until they reached maturity. Then, the colonoids were cultured for 4 days in EM or DM. This schematic was created with BioRender.com. (B) Representative phase-contrast microscopy images displaying canine colonoids at Day 8 following EM (Exp) or DM (Diff) culture. Scale bar = 100 μm. (c) Differentiation marker genes including LGR5 (a stem cell marker), MUC2 (a goblet cell marker), Chromogranin A (an enteroendocrine cell marker), and Lysozyme (a Paneth cell marker) were compared between canine colonoids cultured in EM (Exp) or DM (Diff). The error bars represent the standard error of the mean (SEM). (D) Confocal microscopy images demonstrating presence of enteroendocrine cells (Chromogranin A, yellow), goblet cells (SNA, green), apical brush border (F-actin, cyan) and basal nuclei (DAPI, blue) in colonoids grown in DM. Scale bar = 100 μm.

Figure 2.. Major P450 gene expression level in canine colonoids.

Gene expression levels of CYP2B11, CYP3A98, CYP2C21, and CYP3A12 were assessed using quantitative reverse transcription polymerase chain reaction (qPCR). This analysis compared canine colonoids cultured solely in expansion medium for 8 days (Exp) with those initially cultured in expansion medium for 4 days and then in differentiation medium for another 4 days (Diff). The error bars represent the standard error of the mean (SEM).

Figure 3.. P450 induction and activity assay in canine colonoids.

(A) A schematic representation of the drug induction of the canine colonoids. The protocol involved initially incubating the organoids in expansion medium for 4 days until they reached maturity. Then, the colonoids were cultured for 4 days in DM or DM containing 250μM of phenobarbital or 50μM of rifampicin. This schematic was created with BioRender.com. (B) Gene expression levels of CYP2B11 and CYP3A98 were assessed using qPCR following phenobarbital and rifampicin induction assays. The error bars represent the standard error of the mean (SEM). (C) Canine colonoids were cultured for four days in EM before being exposed to DM enriched with 50 μM rifampicin for three days to induce CYP3A enzyme activity. To quantify this activity, the medium was sampled at three time points (0, 12, and 24 hours) following the administration of midazolam. These samples were analyzed to measure the concentration of a principal metabolite, 1'-hydroxymidazolam, using HPLC-MS/MS. The error bars represent the standard deviation (SD).