UC Berkeley

The increased presence of chemical contaminants in the environment is an undeniable concern to human health and ecosystems. Historically, by relying heavily upon costly and laborious animal-based toxicity assays, the field of toxicology has often neglected examinations of the cellular and molecular mechanisms of toxicity for the majority of compounds - information that, if available, would strengthen risk assessment analyses. With its unique genetic tools and a high degree of conservation with more complex organisms, the model eukaryote Saccharomyces cerevisiae is an appealing organism in which to conduct functional inquiries into the modes of chemical toxicity. In this series of studies, the yeast deletion mutant collection was screened to identify strains exhibiting altered growth in the presence of various environmental contaminants. This technique, known as functional profiling or functional genomics, yielded (1) novel insights into chemical toxicity; (2) pathways and mechanisms deserving of further study; and (3) candidate human toxicant susceptibility or resistance genes.

Functional profiling determined the toxic mechanism of the dieldrin organochlorinated pesticide in yeast. Exposure to dieldrin has been linked to Parkinson's and Alzheimer's diseases, endocrine disruption, and cancer, but the cellular and molecular mechanisms of toxicity behind these effects remain largely unknown. A functional genomics approach in the model eukaryote Saccharomyces cerevisiae demonstrated that dieldrin altered leucine availability. This model was supported by multiple lines of congruent evidence: (1) mutants defective in amino acid signaling or transport were sensitive to dieldrin, which was reversed by the addition of exogenous leucine; (2) dieldrin sensitivity of wild-type or mutant strains was dependent upon leucine concentration in the media; (3) overexpression of proteins that increased intracellular leucine conferred resistance to dieldrin; (4) leucine uptake was inhibited in the presence of dieldrin; and (5) dieldrin induced the amino acid starvation response. Additionally, it was shown that appropriate negative regulation of the Ras/protein kinase A (PKA) pathway, along with an intact pyruvate dehydrogenase complex, was required for dieldrin tolerance. A model connecting leucine uptake, Ras/PKA signaling, and pyruvate dehydrogenase was hypothesized. Many yeast dieldrin tolerance genes described have orthologs that may modulate dieldrin toxicity in humans.

Yeast functional profiling was also conducted with toxaphene, an environmentally persistent mixture of chlorinated terpenes previously utilized as an insecticide. Toxaphene exposure has been previously associated with various cancers and diseases such as amyotrophic lateral sclerosis, but the cellular and molecular mechanisms responsible for these toxic effects have not been well established. In this section, a functional approach in the model eukaryote Saccharomyces cerevisiae demonstrated that toxaphene affected yeast mutants defective in (1) processes associated with transcription elongation and (2) nutrient utilization. Synergistic growth defects were observed upon exposure to both toxaphene and the known transcription elongation inhibitor mycophenolic acid (MPA). However, unlike MPA, toxaphene did not deplete nucleotides and additionally had no detectable effect on transcription elongation. It was concluded that toxaphene likely affects a process closely associated with transcription elongation, such as mRNA processing, mRNA nuclear export, or transcription-coupled nucleotide excision repair. Future studies are required to pinpoint the exact mechanism, and again, many of the yeast genes identified in this chapter have human homologs, warranting further investigations into the potentially conserved mechanisms of toxaphene toxicity.

A functional screen was devised to identify yeast cellular processes and pathways affected by dimethyl sulfoxide (DMSO), a solvent frequently utilized in toxicological and pharmaceutical investigations. As such, it is important to establish the cellular and molecular targets of DMSO in order to differentiate its intrinsic effects from those elicited by a compound of interest. A genome-wide functional screen in Saccharomyces cerevisiae identified deletion mutants exhibiting sensitivity to 1% DMSO, a concentration standard to yeast chemical profiling studies. Mutants defective in Golgi/ER transport were found to be sensitive to DMSO, including those lacking components of the conserved oligomeric Golgi (COG) complex. Moreover, strains deleted for members of the SWR1 histone exchange complex were hypersensitive to DMSO, with additional chromatin remodeling mutants displaying a range of growth defects. DNA repair genes were also identified as important for DMSO tolerance. Finally, it was demonstrated that overexpression of histone H2A.Z, which replaces chromatin-associated histone H2A in a SWR1-catalyzed reaction, conferred resistance to DMSO. Once again, many yeast DMSO tolerance genes described have homologs in more complex organisms, and the data provided is applicable to future investigations into the cellular and molecular mechanisms of DMSO toxicity.

Finally, a framework for semi-automated functional yeast screening of toxicants is described. Pool growths, chemical exposures, and DNA extraction of barcodes described above were conducted manually, which limited throughput. Additionally, identification of mutants with altered growth in a toxicant required hybridizations to costly microarrays. To transition to a more high-throughput environment, a liquid handler was utilized in conjunction with purpose-built software to perform five and fifteen generations screens of the homozygous diploid yeast deletion collection with various emerging environmental contaminants. Genomic DNA was extracted from the pools with robotics, and the barcodes uniquely identifying each deletion strain were amplified by PCR using primers indexed for sequencing on Illumina machinery. Future studies are needed to analyze the sequencing results and identify strains significantly sensitive or resistant to each of the compounds.