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To understand the effects of global Cyp27a1 and Cyp3a4 gene deletions on bile acid synthesis

ABSTRACT: The overall goal of the project is to generate a potential mouse model for Cerebrotendinous Xanthomatosis (CTX), a rare autosomal recessive bile synthesis disorder. Patients with this disorder were found to have mutations in Cyp27a1 gene, which results in deficiency of the enzyme sterol 27-hydroxylase. This loss leads to inhibition of cholesterol being converted into bile acids. Global deletion of Cyp27a1 in mice showed none of the CTX phenotype and do not accumulate bile alcohols. As livers of these mice show tremendous upregulation of Cyp3a4, so we hypothesized that it may have "detoxified’ the intermediates and therefore presented a normal phenotype. For answering this, we made double KO mice with Cyp27a1-/- and Cyp3a cluster (Cyp3a13/57/16/41/44/11/25/59) and found no phenotype till 6 months of age.

ORGANISM(S): Mus musculus

PROVIDER: GSE146521 | GEO | 2020/09/04

REPOSITORIES: GEO

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Project description:In drug development, a system for predicting drug metabolism and drug-induced toxicity is necessary to ensure drug safety. Cytochrome P450 family 3 subfamily A member 4 (CYP3A4) is an important drug-metabolizing enzyme expressed in the liver and small intestine, and predicting CYP3A4-mediated drug metabolism and drug-induced toxicity is essential. We previously developed procedures to differentiate human induced pluripotent stem (iPS) cells into hepatocyte-like cells (HLCs) or intestinal epithelial-like cells (IECs) with a fetal phenotype as well as a highly efficient genome editing technology that could enhance the homologous recombination efficiency at any locus, including CYP3A4. By using human iPS cells and our genome editing technology, we generated CYP3A4-knockout (KO) iPS cell-derived HLCs and IECs for the evaluation of CYP3A4-mediated drug metabolism and drug-induced toxicity. CYP3A4 deficiency did not affect pluripotency and hepatic and intestinal differentiation capacities, and CYP3A4 activity was entirely eradicated by CYP3A4 KO. Off-target effects (e.g., inhibition of bile acid excretion) were hardly observed in CYP3A4-KO cells but were observed in CYP3A4 inhibitor-treated (e.g., ketoconazole) cells. To evaluate whether drug-induced hepatotoxicity and enterotoxicity could be predicted using our model, we exposed CYP3A4-KO HLCs and IECs to acetaminophen, amiodarone, desipramine, leflunomide, tacrine, and tolcapone and confirmed that these cells could predict CYP3A4-mediated toxicity. Finally, we examined whether the therapeutic effects of an anti-hepatitis C virus (HCV) drug metabolized by CYP3A4 would be predicted using our model. CYP3A4-KO HLCs were treated with asunaprevir (antiviral drug metabolized by CYP3A4) after HCV infection, and the anti-viral effect was indeed strengthened by CYP3A4 KO. Conclusion: We succeeded in generating a novel evaluation system for prediction of CYP3A4-mediated drug metabolism and drug-induced toxicity.

Project description:The presence of plastic and microplastic within the oceans as well as in marine flora and fauna have caused a multitude of problems which have been topic of numerous investigations for many years. However, their impact on human health remains largely unknown. Such plastic and microplastic particles have been detected in blood and placenta, underlining their ability to enter the human body. Plastics also contain other compounds, such as plasticizers, antioxidants, or dyes, whose impact on human health is currently being studied. Critical enzymes within the metabolism of endogenous molecules, especially of xenobiotics, are the cytochrome P450 monooxygenases (CYPs). Although their importance in maintaining cellular balance has been confirmed, their interactions with plastics and related products are poorly understood. In this study, the possible relationship between different plastic-related compounds and CYP3A4 as one of the most important CYPs was analyzed using hepatic cells overexpressing this enzyme. Beginning with virtual compound screening and molecular docking of more than 1,000 plastic-related compounds, several candidates were identified to interact with CYP3A4. In a second step, RNA-sequencing was used to study in detail the transcriptome-wide gene expression levels affected by the selected compounds. The results showed that three candidate molecules (2,2'-methylene bis(6-tert-butyl-4-methylphenol, 1,1-bis(3,5-di-tert-butyl-2-hydroxyphenyl)ethane, and 2,2'-methylene bis(6-cyclohexyl-4-methylphenol) had an excellent binding affinity to CYP3A4 in silico as well as cytotoxic effects and interactions with several metabolic pathways in vitro. We identified common pathways influenced by all three selected plastic-related compounds. In particular, the suppression of pathways related to mitosis and ‘DNA-templated DNA replication’. Furthermore, several mis-regulated metabolic and inflammation-related pathways were identified, suggesting the induction of hepatotoxicity at different levels. These findings imply that these compounds may cause problems in the liver, which could subsequently affect the entire organism.

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To understand the effects of global Cyp27a1 and Cyp3a4 gene deletions on bile acid synthesis

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