Genome-Wide Functional and Stress Response Profiling Reveals Toxic Mechanism and Genes Required for Tolerance to Benzo[a]pyrene in S. cerevisiae

O’Connor, Sean Timothy Francis; Lan, Jiaqi; North, Matthew; Loguinov, Alex; Zhang, Luoping; Smith, Martyn T.; Gu, April Z.; Vulpe, Chris D.

doi:10.3389/fgene.2012.00316

Cited by 28 publications

(65 citation statements)

References 77 publications

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“…27,28,40 On the basis of the function and nature of the proteins selected in this study that are essential in yeast cellular stress responses (Table S1, Supporting Information), the extent of up-regulation reflecting yeast cellular stress responses is quantified as follows. 27,28,40 …”

Section: Methodsmentioning

confidence: 99%

“…23 The yeast genome is fully characterized, and the primary biological functions are conserved, which makes it a suitable model for toxicology. 24–27 The stress response ensemble-based toxicogenomics assay with GFP-fused yeast cell library has been recently proposed and demonstrated by our group. 27,28 The selected stress response genes are present and highly conserved in most cell types of metazoans and are activated at significantly lower toxicant concentrations than those causing overt cellular injury, 29,30 which make them suitable to reveal potential toxicity mechanisms comprehensively.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Proteomics analysis of 148 proteins involved in known cellular response pathways in yeast cells was performed using GFP-tagged yeast reporters’ library. 27,28 In addition, toxicity in human lung epithelial cells was also conducted, using gene expression analysis of 12 key biomarkers via RT q-PCR Although 4-MCHM has been considered moderate for acute toxicity based on limited data, our molecular assays provide more subcytotoxic mechanistic toxicity information on 4-MCHM and its metabolites mixture, to help better understand its potential chronic effects. The results provide useful information for guiding further 4-MCHM toxicity study, public health protection strategy, and regulation formation.…”

Section: ■ Introductionmentioning

confidence: 99%

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Toxicity Assessment of 4-Methyl-1-cyclohexanemethanol and Its Metabolites in Response to a Recent Chemical Spill in West Virginia, USA

Lan

Gao

et al. 2015

Environ. Sci. Technol.

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The large-scale chemical spill on January 9, 2014 from coal processing and cleaning storage tanks of Freedom Industries in Charleston affected the drinking water supply to 300,000 people in Charleston, West Virginia metropolitan, while the short-term and long-term health impacts remain largely unknown and need to be assessed and monitored. There is a lack of publically available toxicological information for the main contaminant 4-methyl-1-cyclohexanemethanol (4-MCHM). Particularly, little is known about 4-MCHM metabolites and their toxicity. This study reports timely and original results of the mechanistic toxicity assessment of 4-MCHM and its metabolites via a newly developed quantitative toxicogenomics approach, employing proteomics analysis in yeast cells and transcriptional analysis in human cells. These results suggested that, although 4-MCHM is considered only moderately toxic based on the previous limited acute toxicity evaluation, 4-MCHM metabolites were likely more toxic than 4-MCHM in both yeast and human cells, with different toxicity profiles and potential mechanisms. In the yeast library, 4-MCHM mainly induced chemical stress related to transmembrane transport and transporter activity, while 4-MCHM metabolites of S9 mainly induced oxidative stress related to antioxidant activity and oxidoreductase activity. With human A549 cells, 4-MCHM mainly induced DNA damage-related biomarkers, which indicates that 4-MCHM is related to genotoxicity due to its DNA damage effect on human cells and therefore warrants further chronic carcinogenesis evaluation.

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Section: Methodsmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

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Toxicity Assessment of 4-Methyl-1-cyclohexanemethanol and Its Metabolites in Response to a Recent Chemical Spill in West Virginia, USA

Lan

Gao

et al. 2015

Environ. Sci. Technol.

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“…We and others have published data on genes involved in resistance to toxic metabolites of arsenic [33], benzene [34], and benzo[a]pyrene [35], formaldehyde [36, 37] as well as mechanisms of toxicity associated with these chemicals. In follow up studies, we validated the roles of human orthologs of some of the yeast genes associated with benzene [38–40] and arsenic [41, 42] toxicity in mammalian cell lines using RNAi-based in vitro knockdown approaches.…”

Section: Functional Genomics In Yeastmentioning

confidence: 99%

Functional genomic screening approaches in mechanistic toxicology and potential future applications of CRISPR-Cas9

Shen¹,

McHale²,

Smith³

et al. 2015

Mutation Research/Reviews in Mutation Research

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Characterizing variability in the extent and nature of responses to environmental exposures is a critical aspect of human health risk assessment. Chemical toxicants act by many different mechanisms, however, and the genes involved in adverse outcome pathways (AOPs) and AOP networks are not yet characterized. Functional genomic approaches can reveal both toxicity pathways and susceptibility genes, through knockdown or knockout of all non-essential genes in a cell of interest, and identification of genes associated with a toxicity phenotype following toxicant exposure. Screening approaches in yeast and human near-haploid leukemic KBM7 cells, have identified roles for genes and pathways involved in response to many toxicants but are limited by partial homology among yeast and human genes and limited relevance to normal diploid cells. RNA interference (RNAi) suppresses mRNA expression level but is limited by off-target effects (OTEs) and incomplete knockdown. The recently developed gene editing approach called clustered regularly interspaced short palindrome repeats-associated nuclease (CRISPR)-Cas9, can precisely knock-out most regions of the genome at the DNA level with fewer OTEs than RNAi, in multiple human cell types, thus overcoming the limitations of the other approaches. It has been used to identify genes involved in the response to chemical and microbial toxicants in several human cell types and could readily be extended to the systematic screening of large numbers of environmental chemicals. CRISPR-Cas9 can also repress and activate gene expression, including that of non-coding RNA, with near-saturation, thus offering the potential to more fully characterize AOPs and AOP networks. Finally, CRISPR-Cas9 can generate complex animal models in which to conduct preclinical toxicity testing at the level of individual genotypes or haplotypes. Therefore, CRISPR-Cas9 is a powerful and flexible functional genomic screening approach that can be harnessed to provide unprecedented mechanistic insight in the field of modern toxicology.

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“…The protein expression levels based on GFP signals were calculated using the method previously described (39,40). The raw data from the OD 600 and GFP measurements were corrected by considering the background signals in control with medium only, and the protein expression per cell unit for each measurement was calculated by normalizing the GFP data to the cell number (OD 600 ), with P equal to the GFP measurement over the OD 600 .…”

Section: Methodsmentioning

confidence: 99%

Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of the WHI2 Gene Identified through Inverse Metabolic Engineering

Chen

Stabryla

Wei

2016

Appl Environ Microbiol

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bDevelopment of acetic acid-resistant Saccharomyces cerevisiae is important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge due to limited information on effective genetic perturbation targets for improving acetic acid resistance in the yeast. This study employed a genomic-library-based inverse metabolic engineering approach to successfully identify a novel gene target, WHI2 (encoding a cytoplasmatic globular scaffold protein), which elicited improved acetic acid resistance in S. cerevisiae. Overexpression of WHI2 significantly improved glucose and/or xylose fermentation under acetic acid stress in engineered yeast. The WHI2-overexpressing strain had 5-times-higher specific ethanol productivity than the control in glucose fermentation with acetic acid. Analysis of the expression of WHI2 gene products (including protein and transcript) determined that acetic acid induced endogenous expression of Whi2 in S. cerevisiae. Meanwhile, the whi2⌬ mutant strain had substantially higher susceptibility to acetic acid than the wild type, suggesting the important role of Whi2 in the acetic acid response in S. cerevisiae. Additionally, overexpression of WHI2 and of a cognate phosphatase gene, PSR1, had a synergistic effect in improving acetic acid resistance, suggesting that Whi2 might function in combination with Psr1 to elicit the acetic acid resistance mechanism. These results improve our understanding of the yeast response to acetic acid stress and provide a new strategy to breed acetic acid-resistant yeast strains for renewable biofuel production. Lignocellulosic biomass from nonfood stocks such as agricultural and forestry residues has been identified as the prime source for production of renewable biofuels to substitute for conventional fossil fuels in the face of growing demand for energy and rising concerns about greenhouse gas emissions (1-4). Bioconversion of plant cell wall materials by microbial fermentation is typically preceded by harsh (physico)chemical hydrolysis designed to release sugars; this hydrolysis treatment also generates by-products that are toxic to fermenting microorganisms (5, 6). Since hemicellulose and lignin in the plant cell wall are ubiquitously acetylated (7,8), the typical acidic pretreatment of lignocellulosic biomass generates substantial amounts of acetic acid (with concentrations ranging from 1 g/liter to 15 g/liter) in the resulting hydrolysates (9, 10). Acetic acid severely inhibits cell growth and fermentation activity in Saccharomyces cerevisiae (5, 6, 11-13), the predominant microorganism used in industrial fermentation (14, 15). Therefore, improvement in S. cerevisiae resistance to acetic acid is highly desired and critical for achieving efficient and economically viable bioconversion of cellulosic sugars to biofuels.The toxic effects of acetic acid in S. cerevisiae have been intensively characterized, and toxicity mechanisms have been proposed (10,11,(16)(17)(18)(19)(20). When the external pH is lower than the...

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Genome-Wide Functional and Stress Response Profiling Reveals Toxic Mechanism and Genes Required for Tolerance to Benzo[a]pyrene in S. cerevisiae

Cited by 28 publications

References 77 publications

Toxicity Assessment of 4-Methyl-1-cyclohexanemethanol and Its Metabolites in Response to a Recent Chemical Spill in West Virginia, USA

Toxicity Assessment of 4-Methyl-1-cyclohexanemethanol and Its Metabolites in Response to a Recent Chemical Spill in West Virginia, USA

Functional genomic screening approaches in mechanistic toxicology and potential future applications of CRISPR-Cas9

Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of the WHI2 Gene Identified through Inverse Metabolic Engineering

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