Subunits Rip1p and Cox9p of the respiratory chain contribute to diclofenac-induced mitochondrial dysfunction.

The widely used drug diclofenac can cause serious heart, liver and kidney injury, which may be related to its ability to cause mitochondrial dysfunction. Using Saccharomyces cerevisiae as a model system, we studied the mechanisms of diclofenac toxicity and the role of mitochondria therein. We found that diclofenac reduced cell growth and viability and increased levels of reactive oxygen species (ROS). Strains increasingly relying on respiration for their energy production showed enhanced sensitivity to diclofenac. Furthermore, oxygen consumption was inhibited by diclofenac, suggesting that the drug inhibits respiration. To identify the site of respiratory inhibition, we investigated the effects of deletion of respiratory chain subunits on diclofenac toxicity. Whereas deletion of most subunits had no effect, loss of either Rip1p of complex III or Cox9p of complex IV resulted in enhanced resistance to diclofenac. In these deletion strains, diclofenac did not increase ROS formation as severely as in the wild-type. Our data are consistent with a mechanism of toxicity in which diclofenac inhibits respiration by interfering with Rip1p and Cox9p in the respiratory chain, resulting in ROS production that causes cell death.

Involvement of the pleiotropic drug resistance response, protein kinase C signaling, and altered zinc homeostasis in resistance of Saccharomyces cerevisiae to diclofenac.

Diclofenac is a widely used analgesic drug that can cause serious adverse drug reactions. We used Saccharomyces cerevisiae as a model eukaryote with which to elucidate the molecular mechanisms of diclofenac toxicity and resistance. Although most yeast cells died during the initial diclofenac treatment, some survived and started growing again. Microarray analysis of the adapted cells identified three major processes involved in diclofenac detoxification and tolerance. In particular, pleiotropic drug resistance (PDR) genes and genes under the control of Rlm1p, a transcription factor in the protein kinase C (PKC) pathway, were upregulated in diclofenac-adapted cells. We tested if these processes or pathways were directly involved in diclofenac toxicity or resistance. Of the pleiotropic drug resistance gene products, the multidrug transporter Pdr5p was crucially important for diclofenac tolerance. Furthermore, deletion of components of the cell wall stress-responsive PKC pathway increased diclofenac toxicity, whereas incubation of cells with the cell wall stressor calcofluor white before the addition of diclofenac decreased its toxicity. Also, diclofenac induced flocculation, which might trigger the cell wall alterations. Genes involved in ribosome biogenesis and rRNA processing were downregulated, as were zinc-responsive genes. Paradoxically, deletion of the zinc-responsive transcription factor Zap1p or addition of the zinc chelator 1,10-phenanthroline significantly increased diclofenac toxicity, establishing a regulatory role for zinc in diclofenac resistance. In conclusion, we have identified three new pathways involved in diclofenac tolerance in yeast, namely, Pdr5p as the main contributor to the PDR response, cell wall signaling via the PKC pathway, and zinc homeostasis, regulated by Zap1p.

Efficient screening of cytochrome P450 BM3 mutants for their metabolic activity and diversity toward a wide set of drug-like molecules in chemical space.

In the present study, the diversity of a library of drug-metabolizing bacterial cytochrome P450 (P450) BM3 mutants was evaluated by a liquid chromatography-mass spectrometry (LC-MS)-based screening method. A strategy was designed to identify a minimal set of BM3 mutants that displays differences in regio- and stereoselectivities and is suitable to metabolize a large fraction of drug chemistry space. We first screened the activities of six structurally diverse BM3 mutants toward a library of 43 marketed drugs (encompassing a wide range of human P450 phenotypes, cLogP values, charges, and molecular weights) using a rapid LC-MS method with an automated method development and data-processing system. Significant differences in metabolic activity were found for the mutants tested and based on this drug library screen; nine structurally diverse probe drugs were selected that were subsequently used to study the metabolism of a library of 14 BM3 mutants in more detail. Using this alternative screening strategy, we were able to select a minimal set of BM3 mutants with high metabolic activities and diversity with respect to substrate specificity and regiospecificity that could produce both human relevant and BM3 unique drug metabolites. This panel of four mutants (M02, MT35, MT38, and MT43) was capable of producing P450-mediated metabolites for 41 of the 43 drugs tested while metabolizing 77% of the drugs by more than 20%. We observed this as the first step in our approach to use of bacterial P450 enzymes as general reagents for lead diversification in the drug development process and the biosynthesis of drug(-like) metabolites.

The effect of acetaminophen on ubiquitin homeostasis in Saccharomyces cerevisiae.

Acetaminophen (APAP), although considered a safe drug, is one of the major causes of acute liver failure by overdose, and therapeutic chronic use can cause serious health problems. Although the reactive APAP metabolite N-acetyl-p-benzoquinoneimine (NAPQI) is clearly linked to liver toxicity, toxicity of APAP is also found without drug metabolism of APAP to NAPQI. To get more insight into mechanisms of APAP toxicity, a genome-wide screen in Saccharomyces cerevisiae for APAP-resistant deletion strains was performed. In this screen we identified genes related to the DNA damage response. Next, we investigated the link between genotype and APAP-induced toxicity or resistance by performing a more detailed screen with a library containing mutants of 1522 genes related to nuclear processes, like DNA repair and chromatin remodelling. We identified 233 strains that had an altered growth rate relative to wild type, of which 107 showed increased resistance to APAP and 126 showed increased sensitivity. Gene Ontology analysis identified ubiquitin homeostasis, regulation of transcription of RNA polymerase II genes, and the mitochondria-to-nucleus signalling pathway to be associated with APAP resistance, while histone exchange and modification, and vesicular transport were connected to APAP sensitivity. Indeed, we observed a link between ubiquitin levels and APAP resistance, whereby ubiquitin deficiency conferred resistance to APAP toxicity while ubiquitin overexpression resulted in sensitivity. The toxicity profile of various chemicals, APAP, and its positional isomer AMAP on a series of deletion strains with ubiquitin deficiency showed a unique resistance pattern for APAP. Furthermore, exposure to APAP increased the level of free ubiquitin and influenced the ubiquitination of proteins. Together, these results uncover a role for ubiquitin homeostasis in APAP-induced toxicity.

Yeast as a humanized model organism for biotransformation-related toxicity.

High drug attrition rates due to toxicity, the controversy of experimental animal usage, and the EU REACH regulation demanding toxicity profiles of a high number of chemicals demonstrate the need for new, in vitro toxicity models with high predictivity and throughput. Metabolism by cytochrome P450s (P450s) is one of the main causes of drug toxicity. As some of these enzymes are highly polymorphic leading to large differences is metabolic capacity, isotype-specific test systems are needed. In this review, we will discuss the use of yeast expressing (mammalian) P450s as a powerful, additional model system in drug safety. We will discuss the various cellular model systems for bioactivation-related toxicity and subsequently describe the properties of yeast as a model system, including the endogenous bioactivation enzymes present, the heterologous expression of (mammalian) P450s and the application of yeasts expressing heterologous P450s and/or other biotransformation enzymes in toxicity studies. All major human drug-metabolizing P450s have been successfully expressed in yeast and various mutagenicity tests have been performed with these humanized yeast strains. The few examples of non-mutagenic toxicity studies with these strains and of the combination of P450s with phase II or other human enzymes show the potential of yeast as a model system in metabolism-related toxicity studies. The wide variety of genome-wide screens available in yeast, combined with its well-annotated genome, also facilitate follow-up studies on the genes involved in toxicity. Unless indicated otherwise "yeast" will refer to baker's yeast Saccharomyces cerevisiae.

Differential involvement of mitochondrial dysfunction, cytochrome P450 activity, and active transport in the toxicity of structurally related NSAIDs.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in the treatment of pain and inflammation. However, this group of drugs is associated with serious adverse drug reactions. Previously, we studied the mechanisms underlying toxicity of the NSAID diclofenac using Saccharomycescerevisiae as model system. We identified the involvement of several mitochondrial proteins, a transporter and cytochrome P450 activity in diclofenac toxicity. In this study, we investigated if these processes are also involved in the toxicity of other NSAIDs. We divided the NSAIDs into three classes based on their toxicity mechanisms. Class I consists of diclofenac, indomethacin and ketoprofen. Mitochondrial respiration and reactive oxygen species (ROS) play a major role in the toxicity of this class. Metabolism by cytochrome P450s further increases their toxicity, while ABC-transporters decrease the toxicity. Mitochondria and oxidative metabolism also contribute to toxicity of class II drugs ibuprofen and naproxen, but another cellular target dominates their toxicity. Interestingly, ibuprofen was the only NSAID that was unable to induce upregulation of the multidrug resistance response. The class III NSAIDs sulindac, ketorolac and zomepirac were relatively non-toxic in yeast. In conclusion, we demonstrate the use of yeast to investigate the mechanisms underlying the toxicity of structurally related drugs.

Metabolism related toxicity of diclofenac in yeast as model system.

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.