title: Genomic phenotyping by barcode sequencing broadly distinguishes between alkylating agents, oxidizing agents, and non-genotoxic agents, and reveals a role for aromatic amino acids in cellular recovery after quinone exposure.
abstract: Toxicity screening of compounds provides a means to identify compounds harmful for human health and the environment. Here, we further develop the technique of genomic phenotyping to improve throughput while maintaining specificity. We exposed cells to eight different compounds that rely on different modes of action: four genotoxic alkylating (methyl methanesulfonate (MMS), N-Methyl-N-nitrosourea (MNU), N,N'-bis(2-chloroethyl)-N-nitroso-urea (BCNU), N-ethylnitrosourea (ENU)), two oxidizing (2-methylnaphthalene-1,4-dione (menadione, MEN), benzene-1,4-diol (hydroquinone, HYQ)), and two non-genotoxic (methyl carbamate (MC) and dimethyl sulfoxide (DMSO)) compounds. A library of S. cerevisiae 4,852 deletion strains, each identifiable by a unique genetic 'barcode', were grown in competition; at different time points the ratio between the strains was assessed by quantitative high throughput 'barcode' sequencing. The method was validated by comparison to previous genomic phenotyping studies and 90% of the strains identified as MMS-sensitive here were also identified as MMS-sensitive in a much lower throughput solid agar screen. The data provide profiles of proteins and pathways needed for recovery after both genotoxic and non-genotoxic compounds. In addition, a novel role for aromatic amino acids in the recovery after treatment with oxidizing agents was suggested. The role of aromatic acids was further validated; the quinone subgroup of oxidizing agents were extremely toxic in cells where tryptophan biosynthesis was compromised.
authors.affiliation: Biological Engineering Department, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America ; Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America ; Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden.
authors.forename: J Peter
authors.initials: JP
authors.lastname: Svensson
authors.forename: Laia
authors.initials: L
authors.lastname: Quirós Pesudo
authors.forename: Siobhan K
authors.initials: SK
authors.lastname: McRee
authors.forename: Yeyejide
authors.initials: Y
authors.lastname: Adeleye
authors.forename: Paul
authors.initials: P
authors.lastname: Carmichael
authors.forename: Leona D
authors.initials: LD
authors.lastname: Samson
mesh_terms: D000477:Alkylating Agents / Q000494:pharmacology; D024322:Amino Acids, Aromatic / Q000235:genetics / Q000378:metabolism; D053898:Biosynthetic Pathways / Q000187:drug effects / Q000235:genetics; D002219:Carbamates / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005800:Genes, Fungal / Q000235:genetics; D023281:Genomics / Q000379:methods*; D008826:Microbial Sensitivity Tests / Q000379:methods; D009154:Mutation / Q000187:drug effects / Q000235:genetics; D016877:Oxidants / Q000494:pharmacology; D010641:Phenotype; D011809:Quinones / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D018675:Toxicity Tests / Q000379:methods; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism; D015262:Xenobiotics / Q000494:pharmacology*
chemical_list: D000477:Alkylating Agents; D024322:Amino Acids, Aromatic; D002219:Carbamates; D004271:DNA, Fungal; D016877:Oxidants; D011809:Quinones; D015262:Xenobiotics; D014364:Tryptophan; C036868:methyl carbamate; D004121:Dimethyl Sulfoxide
references.citation: PLoS One. 2012;7(5):e37368
references.pmid: 22615993
references.citation: Yeast. 2006 Jul 30;23(10):741-50
references.pmid: 16862604
references.citation: Free Radic Biol Med. 2010 Sep 15;49(6):919-62
references.pmid: 20493944
references.citation: J Photochem Photobiol B. 2007 Sep 25;88(2-3):83-9
references.pmid: 17604179
references.citation: BMC Syst Biol. 2011 Oct 06;5:157
references.pmid: 21978764
references.citation: Mol Cell Biol. 2010 May;30(10):2449-59
references.pmid: 20308323
references.citation: Pharmacol Ther. 2010 Aug;127(2):156-64
references.pmid: 20546776
references.citation: Nat Rev Cancer. 2012 Jan 12;12(2):104-20
references.pmid: 22237395
references.citation: J Cell Biol. 2003 Jun 23;161(6):1117-31
references.pmid: 12810702
references.citation: Microbiol Rev. 1991 Sep;55(3):349-70
references.pmid: 1943992
references.citation: Genome Res. 2009 Oct;19(10):1836-42
references.pmid: 19622793
references.citation: J Clin Invest. 2003 Jan;111(2):163-9
references.pmid: 12531868
references.citation: Genome Biol. 2010;11(6):R60
references.pmid: 20537132
references.citation: Science. 2008 Apr 18;320(5874):362-5
references.pmid: 18420932
references.citation: Lab Chip. 2013 Jul 21;13(14):2754-63
references.pmid: 23661198
references.citation: Nat Protoc. 2007;2(11):2958-74
references.pmid: 18007632
references.citation: J Biochem Mol Toxicol. 2005;19(2):119-28
references.pmid: 15849717
references.citation: Mol Cell Biol. 1993 Aug;13(8):5010-9
references.pmid: 7687745
references.citation: Int J Biochem Cell Biol. 2007;39(1):44-84
references.pmid: 16978905
references.citation: Genetics. 2002 Jun;161(2):563-74
references.pmid: 12072454
references.citation: Mutat Res. 2012 Feb 18;742(1-2):11-25
references.pmid: 22138618
references.citation: Mol Cell. 2004 Oct 8;16(1):117-25
references.pmid: 15469827
references.citation: Mol Cancer Res. 2002 Dec;1(2):103-12
references.pmid: 12496357
references.citation: Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):16934-9
references.pmid: 12482937
references.citation: Chem Res Toxicol. 1999 Jan;12(1):1-18
references.pmid: 9894013
references.citation: Curr Opin Chem Biol. 2011 Feb;15(1):66-78
references.pmid: 21093351
references.citation: J Clin Invest. 2003 Mar;111(5):583-93
references.pmid: 12618510
references.citation: Genome Res. 2003 Nov;13(11):2498-504
references.pmid: 14597658
references.citation: Ageing Res Rev. 2013 Jan;12(1):376-90
references.pmid: 23123177
references.citation: J Am Chem Soc. 2006 Apr 26;128(16):5506-15
references.pmid: 16620124
references.citation: Eukaryot Cell. 2009 May;8(5):768-78
references.pmid: 19304952
references.citation: Genetics. 2011 Jan;187(1):299-317
references.pmid: 20944018
references.citation: Nature. 2002 Jul 25;418(6896):387-91
references.pmid: 12140549
references.citation: Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):14863-8
references.pmid: 9843981
references.citation: Mol Biol Cell. 2000 Dec;11(12):4241-57
references.pmid: 11102521
references.citation: J Am Chem Soc. 2004 Sep 29;126(38):12158-61
references.pmid: 15382952
references.citation: Front Biosci. 2000 Jul 01;5:D629-38
references.pmid: 10877994
references.citation: Nat Genet. 2001 Dec;29(4):426-34
references.pmid: 11726929
references.citation: Eur J Biochem. 2003 Aug;270(15):3189-95
references.pmid: 12869194
references.citation: Cell. 2006 Aug 11;126(3):611-25
references.pmid: 16901791
references.citation: Nature. 1999 Nov 25;402(6760):413-8
references.pmid: 10586881
references.citation: Mol Syst Biol. 2005;1:2005.0001
references.pmid: 16729036