De novo sequencing, assembly and analysis of the genome of the laboratory strain Saccharomyces cerevisiae CEN.PK113-7D, a model for modern industrial biotechnology
Nijkamp, J.F. ; Broek, M. van den; Datema, E. ; Kok, S. de; Bosman, L. ; Luttik, M.A. ; Daran-Lapujade, P. ; Vongsangnak, W. ; Nielsen, J. ; Heijne, W.H.M. ; Klaassen, P. ; Paddon, C.J. ; Platt, D. ; Kotter, P. ; Ham, R.C.H.J. van; Reinders, M.J.T. ; Pronk, J.T. ; Ridder, D. de; Daran, J.M. - \ 2012
Microbial Cell Factories 11 (2012). - ISSN 1475-2859
l-arabinose - alcoholic fermentation - biotin-prototrophy - chemostat cultures - gene prediction - yeast genome - glucose - evolutionary - protein - xylose
Saccharomyces cerevisiae CEN.PK 113-7D is widely used for metabolic engineering and systems biology research in industry and academia. We sequenced, assembled, annotated and analyzed its genome. Single-nucleotide variations (SNV), insertions/deletions (indels) and differences in genome organization compared to the reference strain S. cerevisiae S288C were analyzed. In addition to a few large deletions and duplications, nearly 3000 indels were identified in the CEN.PK113-7D genome relative to S288C. These differences were overrepresented in genes whose functions are related to transcriptional regulation and chromatin remodelling. Some of these variations were caused by unstable tandem repeats, suggesting an innate evolvability of the corresponding genes. Besides a previously characterized mutation in adenylate cyclase, the CEN. PK113-7D genome sequence revealed a significant enrichment of non-synonymous mutations in genes encoding for components of the cAMP signalling pathway. Some phenotypic characteristics of the CEN. PK113-7D strains were explained by the presence of additional specific metabolic genes relative to S288C. In particular, the presence of the BIO1 and BIO6 genes correlated with a biotin prototrophy of CEN. PK113-7D. Furthermore, the copy number, chromosomal location and sequences of the MAL loci were resolved. The assembled sequence reveals that CEN. PK113-7D has a mosaic genome that combines characteristics of laboratory strains and wild-industrial strains.
Systems toxicology: applications of toxicogenomics, transcriptomics, proteomics and metabolomics in toxicology
Heijne, W.H.M. ; Kienhuis, A.S. ; Ommen, B. van; Stierum, R. ; Groten, J.P. - \ 2005
Expert Review of Proteomics 2 (2005)5. - ISSN 1478-9450 - p. 767 - 780.
gene-expression patterns - 2-dimensional gel-electrophoresis - laser desorption/ionization-time - magnetic-resonance spectroscopy - complementary-dna microarray - ionization mass-spectrometry - coded affinity tags - in-vitro - rat hepatocytes - induced hepatotoxic
Toxicogenomics can facilitate the identification and characterization of toxicity, as illustrated in this review. Toxicogenomics, the application of the functional genomics technologies (transcriptomics, proteomics and metabolomics) in toxicology enables the study of adverse effects of xenobiotic substances in relation to structure and activity of the genome. The advantages and limitations of the different technologies are evaluated, and the prospects for integration of the technologies into a systems biology or systems toxicology approach are discussed. Applications of toxicogenomics in various laboratories around the world show that the crucial steps and sequence of events at the molecular level can be studied to provide detailed insights into mechanisms of toxic action. Toxicogenomics allowed for more sensitive and earlier detection of adverse effects in (animal) toxicity studies. Furthermore, the effects of exposure to mixtures could be studied in more detail. This review argues that in the (near) future, human health risk assessment will truly benefit from toxicogenomics (systems toxicology).
Toxicogenomic analysis of gene expression changes in rat liver after a 28-day oral benzene exposure
Heijne, W.H.M. ; Jonker, D. ; Stierum, R.H. ; Ommen, B. van; Groten, J.P. - \ 2005
Mutation Research. Fundamental and Molecular Mechanisms of Mutagenesis 575 (2005)1-2. - ISSN 0027-5107 - p. 85 - 101.
hepatocyte nuclear factor-6 - bone-marrow - messenger-rna - induced hematotoxicity - microarray experiments - inhaled benzene - stem-cells - male-mice - metabolism - toxicity
Benzene is an industrial chemical, component of automobile exhaust and cigarette smoke. After hepatic bioactivation benzene induces bone marrow, blood and hepatic toxicity. Using a toxicogenomics approach this study analysed the effects of benzene at three dose levels on gene expression in the liver after 28 daily doses. NMR based metabolomics was used to assess benzene exposure by identification of characteristic benzene metabolite profiles in urine. The 28-day oral exposure to 200 and 800 mg/kg/day but not 10 mg/kg/day benzene-induced hematotoxicity in male Fisher rats. Additionally these upper dose levels slightly reduced body weight and increased relative liver weights. Changes in hepatic gene expression were identified with oligonucleotide microarrays at all dose levels including the 10 mg/kg/day dose level where no toxicity was detected by other methods. The benzene-induced gene expression changes were related to pathways of biotransformation, glutathione synthesis, fatty acid and cholesterol metabolism and others. Some of the effects on gene expression observed here have previously been observed after induction of acute hepatic necrosis with bromobenzene and acetaminophen. In conclusion, changes in hepatic gene expression were found after treatment with benzene both at the toxic and non-toxic doses. The results from this study show that toxicogenomics identified hepatic effects of benzene exposure possibly related to toxicity. The findings aid to interpret the relevance of hepatic gene expression changes in response to exposure to xenobiotics. In addition, the results have the potential to inform on the mechanisms of response to benzene exposure.
Profiles of metabolites and gene expression in rats with chemically induced hepatic necrosis
Heijne, W.H.M. ; Lamers, R.J.A.N. ; Bladeren, P.J. van; Groten, J.P. ; Nesselrooij, J.H.J. ; Ommen, B. van - \ 2005
Toxicologic Pathology 33 (2005)4. - ISSN 0192-6233 - p. 425 - 433.
pattern-recognition - bromobenzene - mechanism - toxicity - transcriptomics - hepatotoxicity - toxicogenomics - invivo - model
This study investigated whether integrated analysis of transcriptomics and metabolomics data increased the sensitivity of detection and provided new insight in the mechanisms of hepatotoxicity. Metabolite levels in plasma or urine were analyzed in relation to changes in hepatic gene expression in rats that received bromobenzene to induce acute hepatic centrilobular necrosis. Bromobenzene-induced lesions were only observed after treatment with the highest of 3 dose levels. Multivariate statistical analysis showed that metabolite profiles of blood plasma were largely different from controls when the rats were treated with bromobenzene, also at doses that did not elicit histopathological changes. Changes in levels of genes and metabolites were related to the degree of necrosis, providing putative novel markers of hepatotoxicity. Levels of endogenous metabolites like alanine, lactate, tyrosine and dimethylglycine differed in plasma from treated and control rats. The metabolite profiles of urine were found to be reflective of the exposure levels. This integrated analysis of hepatic transcriptomics and plasma metabolomics was able to more sensitively detect changes related to hepatotoxicity and discover novel markers. The relation between gene expression and metabolite levels was explored and additional insight in the role of various biological pathways in bromobenzene-induced hepatic necrosis was obtained, including the involvement of apoptosis and changes in glycolysis and amino acid metabolism.
Bromobenzene-induced hepatotoxicity at the transcriptome level
Heijne, W.H.M. ; Slitt, A.L. ; Bladeren, P.J. van; Groten, J.P. ; Klaassen, C.D. ; Stierum, R.H. ; Ommen, B. van - \ 2004
Toxicological sciences 79 (2004)2. - ISSN 1096-6080 - p. 411 - 422.
glyceraldehyde-3-phosphate dehydrogenase gene - acute-phase response - rat-liver - glutathione - element - identification - expression - induction - binding - acetaminophen
Rats were exposed to three levels of bromobenzene, sampled at 6, 24, and 48 h, and liver gene expression profiles were determined to identify dose and time-related changes. Expression of many genes changed transiently, and dependent on the dose. Few changes were identified after 6 h, but many genes were differentially expressed after 24 h, while after 48 h, only the high dose elicited large effects. Differentially expressed genes were involved in drug metabolism (upregulated GSTs, mEH, NQO1, Mrps, downregulated CYPs, sulfotransferases), oxidative stress (induced HO-1, peroxiredoxin, ferritin), GSH depletion (induced GCS-l, GSTA, GSTM) the acute phase response, and in processes like cholesterol, fatty acid and protein metabolism, and intracellular signaling. Trancriptional regulation via the electrophile and sterol response elements seemed to mediate part of the response to bromobenzene. Recovery of the liver was suggested in response to BB by the altered expression of genes involved in protein synthesis and cytoskeleton rearrangement. Furthermore, after 48 h, rats in the mid dose group showed no toxicity, and gene expression patterns resembled the normal situation. For certain genes (e.g., CYP4A, metallothioneins), intraday variation in expression levels was found, regardless of the treatment. Selected cDNA microarray measurements were confirmed using the specific and sensitive branched DNA signal amplification assay.
Toxicogenomics: Applications of new functional genomics technologies in toxicology
Heijne, W.H.M. - \ 2004
Wageningen University. Promotor(en): Peter van Bladeren; John Groten, co-promotor(en): B. van Ommen. - [S.I.] : S.n. - ISBN 9789085041214 - 213
toxicologie - genexpressie - trichloorethyleen - benzeen - toxicogenomica - toxicology - gene expression - trichloroethylene - benzene - toxicogenomics
Toxicogenomics studies toxic effects of substances on organisms in relation to the composition of the genome. It applies the functional genomics technologies transcriptomics, proteomics and metabolomics that determine expression of the genes, proteins and metabolites in a sample. These methods could facilitate toxicological research and eventually, toxicogenomics could improve human health risk assessment. This thesis evaluated applications of toxicogenomics, especially to investigate mechanisms of toxicity, to obtain new toxicity markers and to assess toxicity of mixtures.
In the first studies, protein and gene expression were characterised in livers of rats treated with bromobenzene, well-known to cause liver damage. The metabolite contents of plasma and urine were also measured. Many changes were found related to biotransformation, glutathione metabolism, oxidative stress and to unexpectedly involved processes like cholesterol, fatty acid and protein metabolism. The studies enabled to identify crucial events in the mechanisms of hepatotoxic by bromobenzene.
The specificity of bromobenzene-induced liver gene expression changes was delineated by comparison with published effects of high doses of acetaminophen (paracetamol), that also caused liver necrosis. Subsequently, effects of abundant chemicals benzene and trichloroethylene, not typical liver toxicants, were analysed. Benzene primarily causes hematotoxicity and trichloroethylene nephrotoxicity. However, both compounds induced liver weight. They were also found to modulate liver gene expression. Effects were related to biotransformation, fatty acid metabolism and other processes. Benzene, trichloroethylene, bromobenzene and acetaminohen induced several common and many specific changes in liver gene expression.
Transcriptomics, proteomics and metabolomics were applied to obtain new markers for identification of liver toxicity at early stages and low exposure levels. Potential markers were correlated to the liver damage induced by bromobenzene. Furthermore, characteristic profiles of metabolites in urine enabled specific and robust assessment of exposure to bromobenzene, benzene and trichloroethylene.
A study with mixtures of benzene and trichloroethylene showed for the first time how transcriptomics may facilitate assessment of mixture toxicity. The gene expression changes by trichloroethylene were found to be enhanced by benzene. More importantly, transcriptomics provided insights in molecular mechanisms of joint action.
Transcriptomics and metabolomics proved more sensitive than conventional toxicity parameters. However, it remains to be clarified whether observed effects are adverse. At present, transcriptomics is the most informative of the toxicogenomics methods, while proteomics and metabolomics wil! rapidly become more relevant. Linking the technologies could provide detailed insights in (dynamic) processes within the eell in relation to environmental stimuli.
In conclusion, toxicogenomics methods provided detailed insights in molecular mechanisms of toxicity. They enabled more sensitive and early detection of effects and facilitated assessment of effects of mixtures of compounds. Thus, toxicogenomics methods may be used to improve human health risk assessment.
Toxicogenomics of bromobenzene hepatotoxicity: a combined transcriptomics and proteomics approach
Heijne, W.H.M. ; Stierum, R.H. ; Slijper, M. ; Bladeren, P.J. van; Ommen, B. van - \ 2003
Biochemical Pharmacology 65 (2003)5. - ISSN 0006-2952 - p. 857 - 875.
gene-expression profiles - acute-phase response - rat-liver - lipid-peroxidation - bacillus-subtilis - heme oxygenase-1 - hepatoma-cells - toxicity - glutathione - mechanism
Toxicogenomics is a novel approach integrating the expression analysis of thousands of genes (transcriptomics) or proteins (proteomics) with classical methods in toxicology. Effects at the molecular level are related to pathophysiological changes of the organisms, enabling detailed comparison of mechanisms and early detection and prediction of toxicity. This report addresses the value of the combined use of transcriptomics and proteomics technologies in toxicology. Acute hepatotoxicity was induced in rats by bromobenzene administration resulting in depleted glutathione levels and reduced average body weights, 24 hr after dosage. These physiological symptoms coincided with many changes of hepatic mRNA and protein content. Gene induction confirmed involvement of glutathione-S-transferase isozymes and epoxide hydrolase in bromobenzene metabolism and identified many genes possibly relevant in bromobenzene toxicity. Observed glutathione depletion coincided with induction of the key enzyme in glutathione biosynthesis, ¿-glutamylcysteine synthetase. Oxidative stress was apparent from strong upregulation of heme oxygenase, peroxiredoxin 1 and other genes. Bromobenzene-induced protein degradation was suggested from two-dimensional gel electrophoresis, upregulated mRNA levels for proteasome subunits and lysosomal cathepsin L, whereas also genes were upregulated with a role in protein synthesis. Both protein and gene expression profiles from treated rats were clearly distinct from controls as shown by principal component analysis, and several proteins found to significantly change upon bromobenzene treatment were identified by mass spectrometry. A modest overlap in results from proteomics and transcriptomics was found. This work indicates that transcriptomics and proteomics technologies are complementary to each other and provide new possibilities in molecular toxicology.