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Crypt Organoid Culture as an in Vitro Model in Drug Metabolism and Cytotoxicity Studies
Wenqi Lu1, Eva Rettenmeier1, Miles Paszek1
1Laboratory of Environmental Toxicology, Department of Pharmacology, University of California, San Diego, La Jolla, California (W.L., E.R., M.P., M-F.Y., R.H.T., S.C.); and Laboratory of Molecular Pharmacology, CHU de Quebec Research Centre and Faculty of Pharmacy, Laval University, Québec (Québec), Canada (J.T., O.B.).
Abstract:
The gastrointestinal tract is enriched with xenobiotic processing proteins that play important roles in xenobiotic bioactivation, metabolism, and detoxification. The application of genetically modified mouse models has been instrumental in characterizing the function of xenobiotic processing genes (XPG) and their proteins in drug metabolism. Here, we report the utilization of three-dimensional crypt organoid cultures from these animal models to study intestinal drug metabolism and toxicity. With the successful culturing of crypt organoids, we profiled the abundance of Phase I and Phase II XPG expression, drug transporter gene expression, and xenobiotic nuclear receptor (XNR) gene expression. Functions of XNRs were examined by treating crypt cells with XNR prototypical agonists. Real-time quantitative polymerase chain reaction demonstrated that the representative downstream target genes were induced. These findings were validated from cultures developed from XNR-null mice. In crypt cultures isolated from Pxr mice, pregnenolone 16α-carbonitrile failed to induce Cyp3a11 gene expression; similarly, WY14643 failed to induce Cyp4a10 in the Pparα crypts. Crypt cultures from control (Ugt1 ) and intestinal epithelial cell (IEC) specific Ugt1 null mice (Ugt1 ) were treated with camptothecin-11, an anticancer prodrug with severe intestinal toxicity that originates from insufficient UGT1A1-dependent glucuronidation of its active metabolite SN-38. In the absence of Ugt1 gene expression, Ugt1 crypt cultures exhibit very limited production of SN-38 glucuronide, concordant with increased apoptosis in comparison with Ugt1 crypt cultures. This study suggests crypt organoid cultures as an effective in vitro model for studying intestinal drug metabolism and toxicity.
Insights
Three-dimensional (3D) intestinal crypt organoid cultures from genetically modified mice offer a novel in vitro model. This approach effectively studies xenobiotic processing, drug metabolism, and toxicity within the gastrointestinal tract.
Area of Science:
- Pharmacology
- Gastroenterology
- Toxicology
Background:
- The gastrointestinal tract possesses numerous xenobiotic processing proteins crucial for drug metabolism and detoxification.
- Genetically modified mouse models have advanced understanding of xenobiotic processing genes (XPG) and their roles in drug metabolism.
Purpose of the Study:
- To establish and utilize 3D intestinal crypt organoid cultures from genetically modified mice as an in vitro model for studying intestinal drug metabolism and toxicity.
- To investigate the expression of Phase I/II XPG, drug transporters, and xenobiotic nuclear receptors (XNRs) in these organoid cultures.
- To validate the function of XNRs and the impact of specific gene deficiencies on drug metabolism and toxicity.
Main Methods:
- Culturing of 3D intestinal crypt organoids from genetically modified mice.
- Profiling of xenobiotic processing gene (XPG) and drug transporter gene expression via real-time quantitative polymerase chain reaction (RT-qPCR).
- Functional assays involving treatment with XNR agonists and anticancer prodrugs in organoid cultures from wild-type and knockout mice.
Main Results:
- Organoid cultures successfully profiled XPG, drug transporter, and XNR gene expression.
- Treatment with XNR agonists induced downstream target genes, validated in XNR-null mice.
- Organoids from UGT1A1-deficient mice showed impaired glucuronidation of SN-38 and increased apoptosis when exposed to camptothecin-11.
Conclusions:
- 3D intestinal crypt organoid cultures serve as a powerful and effective in vitro model for investigating intestinal drug metabolism and toxicity.
- This model system allows for the study of genetic variations and their impact on xenobiotic processing and drug response.
- The findings highlight the utility of organoids in preclinical drug development and safety assessment.
