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Metabolism related toxicity of diclofenac in yeast as model system
Jolanda S van Leeuwen1, Galvin Vredenburg, Sanja Dragovic
1LACDR, Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Abstract:
Diclofenac is a widely used drug that can cause serious hepatotoxicity, which has been linked to metabolism by cytochrome P450s (P450). To investigate the role of oxidative metabolites in diclofenac toxicity, a model for P450-related toxicity was set up in Saccharomyces cerevisiae. We expressed a drug-metabolizing mutant of cytochrome P450 BM3 (BM3 M11) in yeast. Importantly, BM3 M11 yielded similar oxidative metabolite profiles of diclofenac as human P450s. It was found that yeast strains expressing BM3 M11 grew significantly slower when exposed to diclofenac than strains without BM3 M11. Furthermore, the amount of reactive oxygen species (ROS) after incubation with diclofenac was higher in strains expressing BM3 M11 than in strains without this enzyme, confirming that P450 activity increases diclofenac toxicity. Interestingly, 4'- and 5-hydroxydiclofenac had no effect on cell growth or ROS formation in cells expressing BM3 M11, although hydroxydiclofenac-derived quinone imines were identified in these strains by detection of their glutathione conjugates. This suggests that 4'- and 5-hydroxydiclofenac, as well as their quinone imines, are not involved in toxicity in yeast. Rather, the P450-related toxicity of diclofenac is caused by primary metabolites such as arene oxides resulting in hydroxydiclofenac or radical species formed during decarboxylation.
Insights
Diclofenac toxicity in yeast models is linked to cytochrome P450 activity. Primary metabolites, not hydroxydiclofenac, cause reactive oxygen species and reduced cell growth, revealing diclofenac
Area of Science:
- Biochemistry
- Pharmacology
- Yeast Genetics
Background:
- Diclofenac, a common NSAID, can cause severe liver damage (hepatotoxicity).
- Cytochrome P450 enzymes (P450) are implicated in diclofenac metabolism and subsequent toxicity.
- Understanding the specific metabolites responsible for diclofenac toxicity is crucial for risk assessment.
Purpose of the Study:
- To establish a yeast model for investigating P450-mediated diclofenac toxicity.
- To identify the specific diclofenac metabolites responsible for toxicity in a P450-expressing system.
- To differentiate the roles of primary versus secondary metabolites in diclofenac-induced cellular damage.
Main Methods:
- Expression of a drug-metabolizing cytochrome P450 mutant (BM3 M11) in Saccharomyces cerevisiae.
- Analysis of diclofenac oxidative metabolite profiles in yeast, comparing them to human P450s.
- Assessment of cell growth and reactive oxygen species (ROS) production in diclofenac-exposed yeast strains with and without BM3 M11 expression.
Main Results:
- Yeast strains expressing BM3 M11 exhibited significantly reduced growth and increased ROS levels upon diclofenac exposure compared to controls.
- BM3 M11 produced similar diclofenac oxidative metabolite profiles as human P450s.
- 4'- and 5-hydroxydiclofenac and their derived quinone imines did not induce toxicity or ROS formation in the yeast model.
Conclusions:
- Cytochrome P450 activity significantly enhances diclofenac toxicity in a yeast model.
- Primary diclofenac metabolites, such as arene oxides or radical species from decarboxylation, are likely responsible for P450-related toxicity.
- Hydroxydiclofenac and its quinone imines are not the causative agents of diclofenac toxicity in this yeast system, suggesting alternative toxic pathways.
