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Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures
Published on: March 28, 2017
Metabolic-induced cytotoxicity of diosbulbin B in CYP3A4-expressing cells
Ji-Zong Jiang1, Bao-Hua Yang1, Li-Li Ji2
1Institute of Chinese MateriaMedica, Shanghai University of Traditional Chinese Medicine; The MOE Key Laboratory for Standardization of Chinese Medicines and The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicine, 1200 Cailun Rood, Shanghai 201203, China.
Abstract:
As a candidate antitumor agent, diosbulbin B (DB) can induce serious liver toxicity and other adverse reactions. DB is mainly metabolized by CYP3A4 in vitro and in vivo, but the cytotoxicity and anti-tumor mechanisms of DB have yet to be clarified. This study aimed to determine whether the cytotoxicity and anti-tumor effects of DB are related to the metabolism-induced activation of CYP3A4 in various cell models, including CYP-free NIH3T3 cells, primary rat hepatocytes, HepG2 and L02 cells of high CYP3A4 expression and wild-type. Results showed that DB did not markedly decrease the viability of NIH3T3 cells. DB metabolites, obtained from the metabolism by mouse liver microsomes, did not elicit cytotoxicity on NIH3T3 cells either. By contrast, DB could induce significant cytotoxicity on primary rat hepatocytes. The DB induced cytotoxicity on HepG2 or L02 cells with high CYP3A4 expression were stronger than those on wild-type cells. As a metabolic biomarker, the metabolite conjugate (M31) of DB with GSH was detected in the incubation system. A higher amount of M31 was generated in the transfected HepG2 and L02 cells than in the wild-type cells at different time points. Ketoconazole, however, could restrain DB induced cytotoxicity on primary rat hepatocytes and in CYP3A4 transfected HepG2 and L02 cells. Therefore, the cytotoxicity of DB was closely related to CYP3A4-metabolized reactive DB metabolites.
Insights
Diosbulbin B (DB) shows antitumor potential but causes liver toxicity. Its cytotoxicity is linked to reactive metabolites generated by CYP3A4 enzyme activity, impacting cell viability.
Area of Science:
- Pharmacology
- Hepatotoxicity
- Drug Metabolism
Background:
- Diosbulbin B (DB) is a potential antitumor agent with known liver toxicity.
- The precise mechanisms of DB's cytotoxicity and antitumor effects remain unclear.
- DB is primarily metabolized by Cytochrome P450 3A4 (CYP3A4).
Purpose of the Study:
- To investigate the relationship between DB's cytotoxicity and its metabolism by CYP3A4.
- To elucidate the role of CYP3A4-mediated metabolism in DB's anti-tumor effects and toxicity.
- To assess DB's effects across various cell models with differing CYP3A4 expression levels.
Main Methods:
- Utilized CYP-free NIH3T3 cells, primary rat hepatocytes, and HepG2/L02 cells (high CYP3A4 expression).
- Assessed DB cytotoxicity and metabolite formation.
- Employed ketoconazole, a CYP3A4 inhibitor, to evaluate its impact on DB-induced cytotoxicity.
- Detected DB-glutathione conjugate (M31) as a metabolic biomarker.
Main Results:
- DB exhibited minimal cytotoxicity in CYP-free NIH3T3 cells.
- DB-induced cytotoxicity was significantly higher in primary rat hepatocytes and HepG2/L02 cells with high CYP3A4 expression compared to wild-type cells.
- Increased levels of the M31 metabolite were observed in cells with higher CYP3A4 activity.
- Ketoconazole treatment attenuated DB-induced cytotoxicity in relevant cell models.
Conclusions:
- DB's cytotoxicity is closely associated with the generation of reactive metabolites via CYP3A4 metabolism.
- CYP3A4-mediated metabolic activation plays a crucial role in the toxic effects of Diosbulbin B.
- Targeting CYP3A4 activity could be a strategy to mitigate DB-induced liver toxicity.
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