title: Systematic study of mitochondrial toxicity of environmental chemicals using quantitative high throughput screening.
abstract: A goal of the Tox21 program is to transit toxicity testing from traditional in vivo models to in vitro assays that assess how chemicals affect cellular responses and toxicity pathways. A critical contribution of the NIH Chemical Genomics center (NCGC) to the Tox21 program is the implementation of a quantitative high throughput screening (qHTS) approach, using cell- and biochemical-based assays to generate toxicological profiles for thousands of environmental compounds. Here, we evaluated the effect of chemical compounds on mitochondrial membrane potential in HepG2 cells by screening a library of 1,408 compounds provided by the National Toxicology Program (NTP) in a qHTS platform. Compounds were screened over 14 concentrations, and results showed that 91 and 88 compounds disrupted mitochondrial membrane potential after treatment for 1 or 5 h, respectively. Seventy-six compounds active at both time points were clustered by structural similarity, producing 11 clusters and 23 singletons. Thirty-eight compounds covering most of the active chemical space were more extensively evaluated. Thirty-six of the 38 compounds were confirmed to disrupt mitochondrial membrane potential using a fluorescence plate reader, and 35 were confirmed using a high content imaging approach. Among the 38 compounds, 4 and 6 induced LDH release, a measure of cytotoxicity, at 1 or 5 h, respectively. Compounds were further assessed for mechanism of action (MOA) by measuring changes in oxygen consumption rate, which enabled the identification of 20 compounds as uncouplers. This comprehensive approach allows for the evaluation of thousands of environmental chemicals for mitochondrial toxicity and identification of possible MOAs.
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health , Bethesda, Maryland 20892, United States.
authors.forename: Matias S
authors.initials: MS
authors.lastname: Attene-Ramos
authors.forename: Ruili
authors.initials: R
authors.lastname: Huang
authors.forename: Srilatha
authors.initials: S
authors.lastname: Sakamuru
authors.forename: Kristine L
authors.initials: KL
authors.lastname: Witt
authors.forename: Gyda C
authors.initials: GC
authors.lastname: Beeson
authors.forename: Louie
authors.initials: L
authors.lastname: Shou
authors.forename: Rick G
authors.initials: RG
authors.lastname: Schnellmann
authors.forename: Craig C
authors.initials: CC
authors.lastname: Beeson
authors.forename: Raymond R
authors.initials: RR
authors.lastname: Tice
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Austin
authors.forename: Menghang
authors.initials: M
authors.lastname: Xia
mesh_terms: D004305:Dose-Response Relationship, Drug; D004785:Environmental Pollutants / Q000633:toxicity*; D056945:Hep G2 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D051336:Mitochondrial Membranes / Q000187:drug effects*; D015394:Molecular Structure; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
chemical_list: D004785:Environmental Pollutants
references.citation: J Bioenerg Biomembr. 2012 Aug;44(4):421-37
references.pmid: 22689143
references.citation: Int Rev Cytol. 1992;141:217-32
references.pmid: 1452432
references.citation: Nature. 1961 Jul 8;191:144-8
references.pmid: 13771349
references.citation: Proc Natl Acad Sci U S A. 2006 Aug 1;103(31):11473-8
references.pmid: 16864780
references.citation: Physiol Genomics. 2012 May 1;44(9):495-503
references.pmid: 22433785
references.citation: Environ Health Perspect. 2008 Mar;116(3):284-91
references.pmid: 18335092
references.citation: J Exp Med. 1995 May 1;181(5):1661-72
references.pmid: 7722446
references.citation: J Neurosci Res. 2010 Mar;88(4):877-86
references.pmid: 19813266
references.citation: Cell Calcium. 2006 Aug;40(2):155-64
references.pmid: 16759697
references.citation: Curr Drug Targets. 2011 Jun;12(6):774-82
references.pmid: 21275886
references.citation: J Appl Bacteriol. 1974 Mar;37(1):117-31
references.pmid: 4846735
references.citation: Biochem J. 1968 Apr;107(3):367-75
references.pmid: 4172100
references.citation: Anal Biochem. 2010 Sep 1;404(1):75-81
references.pmid: 20465991
references.citation: J Biomol Screen. 1999;4(2):67-73
references.pmid: 10838414
references.citation: J Biol Chem. 1962 Aug;237:2670-7
references.pmid: 13925019
references.citation: Nucleic Acids Res. 2012 Jan;40(Database issue):D400-12
references.pmid: 22140110
references.citation: J Pharmacol Exp Ther. 2008 May;325(2):536-43
references.pmid: 18267976
references.citation: Trends Pharmacol Sci. 2008 Jul;29(7):361-6
references.pmid: 18501972
references.citation: Exp Cell Res. 1961 Mar;23:228-37
references.pmid: 13762256
references.citation: Nature. 2003 Sep 11;425(6954):191-6
references.pmid: 12939617
references.citation: Apoptosis. 2007 May;12(5):913-22
references.pmid: 17453160
references.citation: Antioxid Redox Signal. 2002 Oct;4(5):769-81
references.pmid: 12470504
references.citation: J Appl Bacteriol. 1971 Sep;34(3):579-91
references.pmid: 4945521
references.citation: Annu Rev Pharmacol Toxicol. 2000;40:353-88
references.pmid: 10836141
references.citation: Adv Drug Deliv Rev. 2009 Nov 30;61(14):1234-49
references.pmid: 19733603
references.citation: J Biomol Screen. 2002 Aug;7(4):383-9
references.pmid: 12230893
references.citation: Physiol Rev. 1980 Jul;60(3):825-63
references.pmid: 6248908
references.citation: Eur J Cancer. 1999 Oct;35(10):1517-25
references.pmid: 10673981
references.citation: Chem Biol Interact. 1987;62(2):179-89
references.pmid: 3594640
references.citation: Biol Chem. 1999 Oct;380(10):1157-66
references.pmid: 10595578
references.citation: J Bioenerg Biomembr. 1999 Dec;31(6):581-90
references.pmid: 10682916
references.citation: Eur J Biochem. 1970 Jan;12(1):117-25
references.pmid: 5434277
references.citation: Toxicol Sci. 2009 Nov;112(1):153-63
references.pmid: 19502547
references.citation: Adv Enzymol Relat Subj Biochem. 1956;17:65-134
references.pmid: 13313307
references.citation: Toxicol Sci. 2007 Jun;97(2):539-47
references.pmid: 17361016
references.citation: Curr Protoc Toxicol. 2011 Aug;Chapter 2:Unit2.20
references.pmid: 21818751
references.citation: Biochem Pharmacol. 1971 Apr;20(4):737-48
references.pmid: 4328324
references.citation: Proc Natl Acad Sci U S A. 1968 Feb;59(2):484-90
references.pmid: 5238978
references.citation: Biochim Biophys Acta. 2006 Jan;1757(1):21-30
references.pmid: 16375850
references.citation: Toxicol In Vitro. 2007 Aug;21(5):902-11
references.pmid: 17346924
references.citation: FEBS Lett. 2003 Jul 10;546(2-3):355-8
references.pmid: 12832068
references.citation: Curr Drug Saf. 2009 Jan;4(1):34-54
references.pmid: 19149524
references.citation: Toxicol In Vitro. 2012 Apr;26(3):511-25
references.pmid: 22261204
references.citation: Drug Discov Today. 2007 Sep;12(17-18):777-85
references.pmid: 17826691
references.citation: J Toxicol Environ Health B Crit Rev. 2010 Feb;13(2-4):51-138
references.pmid: 20574894
references.citation: FEBS Lett. 1986 Jun 9;201(2):267-70
references.pmid: 3086126