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Cellular signaling and toxicity

Cellular_signaling_and_toxicity

People involved:
Staff: J. Chris Vos, Nico P. E. Vermeulen
PhD Students: Galvin Vredenburg, Angelina HuseinovicShalenie Sewradj, Stefan Dekker, Katarzyna Lazarska

 

Research activities:
The yeast Saccharomyces cerevisiae has been developed as an alternative eukaryotic model organism to study toxicity in response to drug exposure. To incorporate bioactivation, we expressed bacterial cytochroom P450 BM3 in yeast, notably a mutant with a high activity towards a variety of drugs. To correlate intracellular generation of metabolites with toxicity, yeast strains with and without expressed P450 BM3 are tested. Furthermore, we have introduced human glutathione-S-transferase and sulfotransferases in yeast to investigate phase II detoxification reactions.

When incubated with diclofenac, yeast strains expressing P450 BM3 grow significantly slower and show a higher level of reactive oxygen species (ROS), indicating P450-related toxicity. The interplay between drug metabolizing P450s, drug transporters and phase II enzymes is important for drug efficacy and safety and was subject in a collaborative project aiming at alternatives for animal testing (ASAT). For example, we could demonstrate that human GSTs are able to counteract the toxic response to clozapine metabolism by cytochroom P450 in recombinant yeast cells.

Also, the mechanism of toxicity of drugs, such as paracetamol and diclofenac, are tested in the absence of bioactivation using the genetic tractability of yeast to identify genes important for toxicity. We found that diclofenac itself reduced cell growth, viability and O2 consumption, accompanied by increased levels of ROS. In strains lacking mitochondrial DNA and therefore deficient in respiration [petites, rho0] no diclofenac toxicity was observed. The effect of deletion of individual subunits of the respiratory chain identified Rip1p (complex III) and Cox9p (complex IV) as main determinants of diclofenac toxicity. Adaptation of yeast cells to diclofenac was studied using microarrays, which showed the involvement of the multiple drug resistance response, especially the ABC transporter Pdr5p, the protein kinase C pathway and Zinc homeostasis.

In collaboration with Fred van Leeuwen (Netherlands Cancer Institute), we have screened a gene deletion library for yeast strains that are resistant to paracetamol. Interestingly, genes were found that are linked to the DNA damage response. We are currently characterizing these strains further with a special emphasis on genomic instability in a project that aims to also look at the role of human counterparts in tumor cells. This translational project is supported by AIMMS and is a collaboration with the department of Genetics (Jan Kooter, FALW) and the VUmc.


Key publications:

Activation of the anticancer drugs cyclophosphamide and ifosfamide by cytochrome P450 BM3 mutants.
Vredenburg G, den Braver-Sewradj S, van Vugt-Lussenburg BM, Vermeulen NP, Commandeur JN, Vos JC.
Toxicol Lett. 2014 Nov 6;232(1):182-192.

Reconstitution of the interplay between cytochrome P450 and human glutathione S-transferases in clozapine metabolism in yeast.
Vredenburg G, Vassell KP, Commandeur JN, Vermeulen NP, Vos JC.
Toxicol Lett. 2013 Oct 9;222(3):247-56. Epub 2013 Aug 2.

Yeast as a humanized model organism for biotransformation-related toxicity.
van Leeuwen JS, Vermeulen NP, Chris Vos J.
Curr Drug Metab. 2012 Dec;13(10):1464-75. Review.

Subunits Rip1p and Cox9p of the respiratory chain contribute to diclofenac-induced mitochondrial dysfunction.
van Leeuwen JS, Orij R, Luttik MA, Smits GJ, Vermeulen NP, Vos JC.
Microbiology. 2011 Mar;157(Pt 3):685-94. Epub 2010 Dec 9.