title: An untargeted multi-technique metabolomics approach to studying intracellular metabolites of HepG2 cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.
abstract: BACKGROUND
  In vitro cell systems together with omics methods represent promising alternatives to conventional animal models for toxicity testing. Transcriptomic and proteomic approaches have been widely applied in vitro but relatively few studies have used metabolomics. Therefore, the goal of the present study was to develop an untargeted methodology for performing reproducible metabolomics on in vitro systems. The human liver cell line HepG2, and the well-known hepatotoxic and non-genotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were used as the in vitro model system and model toxicant, respectively.
  
  
  RESULTS
  The study focused on the analysis of intracellular metabolites using NMR, LC-MS and GC-MS, with emphasis on the reproducibility and repeatability of the data. State of the art pre-processing and alignment tools and multivariate statistics were used to detect significantly altered levels of metabolites after exposing HepG2 cells to TCDD. Several metabolites identified using databases, literature and LC-nanomate-Orbitrap analysis were affected by the treatment. The observed changes in metabolite levels are discussed in relation to the reported effects of TCDD.
  
  
  CONCLUSIONS
  Untargeted profiling of the polar and apolar metabolites of in vitro cultured HepG2 cells is a valid approach to studying the effects of TCDD on the cell metabolome. The approach described in this research demonstrates that highly reproducible experiments and correct normalization of the datasets are essential for obtaining reliable results. The effects of TCDD on HepG2 cells reported herein are in agreement with previous studies and serve to validate the procedures used in the present work.
authors: RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen, The Netherlands. Ainhoa.ruiz@wur.nl
authors: Ainhoa
authors: A
authors: Ruiz-Aracama
authors: Ad
authors: A
authors: Peijnenburg
authors: Jos
authors: J
authors: Kleinjans
authors: Danyel
authors: D
authors: Jennen
authors: Joost
authors: J
authors: van Delft
authors: Caroline
authors: C
authors: Hellfrisch
authors: Arjen
authors: A
authors: Lommen
references: Hum Mol Genet. 2004 May 1;13(9):905-21
references: 15028668
references: Biochem Pharmacol. 2000 Oct 15;60(8):1129-42
references: 11007951
references: Toxicol Appl Pharmacol. 2008 May 15;229(1):10-9
references: 18295293
references: Toxicol Appl Pharmacol. 2008 Jun 15;229(3):281-9
references: 18336851
references: Drug Metab Dispos. 2003 Aug;31(8):1035-42
references: 12867492
references: Biochem Biophys Res Commun. 2006 Jun 23;345(1):181-7
references: 16677602
references: Toxicol Appl Pharmacol. 1981 Jul;59(3):489-99
references: 7268772
references: J Inorg Biochem. 1995 Oct;60(1):31-43
references: 7595470
references: Annu Rev Pharmacol Toxicol. 1982;22:517-54
references: 6282188
references: Biochem J. 1991 Jun 1;276 ( Pt 2):273-87
references: 1646595
references: Lipids. 1991 Jul;26(7):521-5
references: 1682781
references: Annu Rev Pharmacol Toxicol. 2008;48:653-83
references: 18184107
references: J Comp Physiol B. 2005 May;175(4):221-30
references: 15900503
references: Toxicol Appl Pharmacol. 1984 Apr;73(2):311-22
references: 6710531
references: Proc Natl Acad Sci U S A. 2002 Aug 20;99(17):11482-6
references: 12177411
references: NMR Biomed. 2000 May;13(3):129-53
references: 10861994
references: Pharmacol Toxicol. 1990 May;66(5):399-408
references: 2371248
references: Annu Rev Pharmacol Toxicol. 1995;35:307-40
references: 7598497
references: Anal Chem. 2009 Apr 15;81(8):3079-86
references: 19301908
references: Biochem Pharmacol. 1982 Nov 1;31(21):3367-71
references: 6184059
references: Comp Biochem Physiol C Toxicol Pharmacol. 2003 Sep;136(1):29-45
references: 14522597
references: Toxicol Lett. 2002 Feb 28;127(1-3):161-8
references: 12052654
references: Biochem Pharmacol. 2003 Oct 1;66(7):1231-44
references: 14505802
references: Toxicol Appl Pharmacol. 2004 Apr 1;196(1):156-68
references: 15050417
references: Lipids Health Dis. 2008 Oct 17;7:38
references: 18925970
references: Bull Environ Contam Toxicol. 1985 Jun;34(6):787-96
references: 4016287
references: J Biosci Bioeng. 2009 Oct;108(4):277-81
references: 19716514
references: Am J Physiol. 1982 Nov;243(5):C212-21
references: 6814260
references: Toxicol Appl Pharmacol. 2002 Jan 1;178(1):15-21
references: 11781075
references: Toxicol Sci. 2005 Jun;85(2):1048-63
references: 15800033
references: Mutat Res. 1998 Jun 18;402(1-2):185-202
references: 9675276
references: Nat Med. 2007 Sep;13(9):1070-7
references: 17767167
references: Curr Med Chem. 2009;16(36):4814-27
references: 19929785
references: Chem Biol Interact. 1978 Dec;23(3):315-30
references: 719812
references: Toxicol Appl Pharmacol. 2005 Aug 22;207(1):1-24
references: 16054898
references: Chem Res Toxicol. 2008 Jan;21(1):9-27
references: 18171018
references: Toxicol Appl Pharmacol. 2004 May 1;196(3):410-21
references: 15094312
references: Neurochem Res. 2003 Jun;28(6):941-53
references: 12718449
references: Int J Mol Med. 2002 Nov;10(5):535-9
references: 12373287
references: Trends Genet. 2009 Jan;25(1):39-48
references: 19027981
references: J Cell Sci. 1998 Nov;111 ( Pt 22):3311-22
references: 9788873
references: Magn Reson Chem. 2009 Dec;47 Suppl 1:S2-11
references: 19504464
references: OMICS. 2009 Apr;13(2):115-25
references: 19245359
references: Curr Opin Chem Biol. 2006 Aug;10(4):309-15
references: 16815732
references: Clin Chem. 2006 Jul;52(7):1395-405
references: 16709621
references: Bull Environ Contam Toxicol. 1983 Dec;31(6):649-57
references: 6661579
references: Biochem Biophys Res Commun. 1998 Dec 9;253(1):44-8
references: 9875217
references: Eur J Pediatr. 1995 Nov;154(11):871-7
references: 8582396
references: Toxicol Sci. 2010 May;115(1):66-79
references: 20106945
references: Nat Protoc. 2007;2(4):778-91
references: 17446877
references: Life Sci. 1995;56(26):2319-30
references: 7791519
references: Toxicol Sci. 2009 Nov;112(1):229-44
references: 19684285
references: Carcinogenesis. 2004 Jul;25(7):1265-76
references: 14963013
references: Toxicol Lett. 2008 Apr 21;178(1):20-8
references: 18358643
references: Physiol Rev. 1992 Jan;72(1):101-63
references: 1731369
references: Toxicol Appl Pharmacol. 2005 Feb 1;202(3):215-28
references: 15667827
references: Neurotoxicology. 2008 Jan;29(1):1-12
references: 18023877
references: J Pharmacol Exp Ther. 1989 Jan;248(1):62-6
references: 2913289
references: Xenobiotica. 1999 Nov;29(11):1181-9
references: 10598751
references: Toxicology. 2002 Feb 28;171(2-3):117-25
references: 11836018
references: Toxicol Sci. 2006 Dec;94(2):398-416
references: 16960034
references: Lipids. 1988 Sep;23(9):904-6
references: 3185127
references: Anal Chim Acta. 2007 Feb 12;584(1):43-9
references: 17386583
references: Mutat Res. 2005 Aug 4;575(1-2):17-33
references: 15924884
references: J Biotechnol. 2000 Jan 28;77(1):103-14
references: 10674217
references: Am J Physiol. 1989 Oct;257(4 Pt 1):L163-73
references: 2572174
references: Annu Rev Pharmacol Toxicol. 1990;30:603-31
references: 2188580
references: Toxicol Appl Pharmacol. 1985 Nov;81(2):356-61
references: 4060161
references: Altern Lab Anim. 2002 Dec;30 Suppl 2:129-31
references: 12513664
references: Toxicol Lett. 1990 Oct;53(3):315-25
references: 2237938
references: J Proteome Res. 2010 Jan;9(1):413-9
references: 19894772
references: Toxicol Lett. 2009 Dec 1;191(1):79-87
references: 19686823
references: Arch Toxicol. 1999 Aug;73(6):323-36
references: 10447560
references: Cardiovasc Drugs Ther. 1991 Feb;5 Suppl 1:11-5
references: 2031869
references: Arch Toxicol. 2009 Dec;83(12):1037-42
references: 19908029
references: Toxicol Appl Pharmacol. 2010 Mar 1;243(2):154-66
references: 19932708
references: Toxicol Lett. 2001 Mar 31;120(1-3):1-7
references: 11323156