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

Abstract

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

“…Previously, we reported that yeast cell growth is inhibited at diclofenac concentrations of 50 M and higher (43). Upon incubation with 100 M diclofenac, wild-type yeast cells could hardly grow at all, and after 3 h, only 10% of the cells were viable.…”

“…At this point, diclofenac was added at various concentrations up to 100 M. Controls were treated with equal amounts of DMSO (maximum, 0.1%). High-performance liquid chromatographic (HPLC) analysis of the medium or cellular lysates revealed no degradation products of diclofenac in wild-type (WT) cells after incubation for 24 h (43).…”

“…Furthermore, the pharmacological targets of diclofenac do not exist in yeast, thereby simplifying the test system, whereas many of the mechanisms underlying toxicity and resistance to chemicals and other environmental stresses are conserved (32). For example, both in yeast and in mammalian cells, diclofenac toxicity is related to mitochondrial dysfunction and elevated production of reactive oxygen species (ROS) (15,30,43). The availability of a well-annotated genome sequence makes yeast an ideal model system for genome-wide studies.…”

“…Previously, we have shown in Saccharomyces cerevisiae that subunits Rip1p and Cox9p of the mitochondrial respiratory chain are diclofenac targets and that metabolism of diclofenac by cytochrome P450s increases its toxicity (43,44). Yeast is an excellent eukaryotic model organism for toxicological research (20,45).…”

Mitochondrial dysfunction as a mechanism of drug-induced hepatotoxicity: current understanding and future perspectives

“…In addition, diclofenac caused a decrease in ATP levels in human hepatocytes [104]. While detailed mechanistic studies on molecular targets of diclofenac within the respiratory chain are scarce, studies in saccharomyces cerevisiae indicate that diclofenac inhibits respiration by interfering with Rip1p and Cox9p in the respiratory chain, resulting in ROS production that causes cell death [105]. Analysis of molecular mediators involved in mitochondrial MPT pore opening indicated that diclofenac induced early activation of Bax and Bak in immortalized human hepatocytes (HC-04), and caused mitochondrial translocation of Bax [106].…”