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Application of cDNA Microarray Technology to In Vitro Toxicology and the Selection of Genes for a Real-Time RT-PCR-Based Screen for Oxidative Stress in Hep-G2 Cells

Aventis Pharmaceuticals, Raleigh, North Carolina, USA, kevin.morgan{at}aventis.com

Hong Ni

GlaxoSmithKline, Inc, Research Triangle Park, North Carolina, USA

H. Roger Brown

GlaxoSmithKline, Inc, Research Triangle Park, North Carolina, USA

Lawrence Yoon

GlaxoSmithKline, Inc, Research Triangle Park, North Carolina, USA

Charles W. Qualls, JR

GlaxoSmithKline, Inc, Research Triangle Park, North Carolina, USA

Lynn M. Crosby

US Environmental Protection Agency, Research Triangle Park, North Carolina, USA

Randall Reynolds

GlaxoSmithKline, Inc, Research Triangle Park, North Carolina, USA

Betty Gaskill

GlaxoSmithKline, Inc, Research Triangle Park, North Carolina, USA

Steven P. Anderson

GlaxoSmithKline, Inc, Research Triangle Park, North Carolina, USA

Thomas B. Kepler

Santa Fe Institute, Santa Fe, New Mexico, USA

Tracy Brainard

GlaxoSmithKline, Inc, Research Triangle Park, North Carolina, USA

Nik Liv

GlaxoSmithKline, Inc, Research Triangle Park, North Carolina, USA

Marilyn Easton

GlaxoSmithKline, Inc, Research Triangle Park, North Carolina, USA

Christine Merrill

GlaxoSmithKline, Inc, Research Triangle Park, North Carolina, USA

Don Creech

GlaxoSmithKline, Inc, Research Triangle Park, North Carolina, USA

Dirk Sprenger

GlaxoSmithKline, Inc, Research Triangle Park, North Carolina, USA

Gary Conner

GlaxoSmithKline, Inc, Research Triangle Park, North Carolina, USA

Paul R. Johnson

GlaxoSmithKline, Inc, Research Triangle Park, North Carolina, USA

Tony Fox

GlaxoSmithKline, Inc, Research Triangle Park, North Carolina, USA

Maureen Sartor

GlaxoSmithKline, Inc, Research Triangle Park, North Carolina, USA

Erika Richard

GlaxoSmithKline, Inc, Research Triangle Park, North Carolina, USA

Sabu Kuruvilla

GlaxoSmithKline, Inc, Research Triangle Park, North Carolina, USA

Warren Casey

GlaxoSmithKline, Inc, Research Triangle Park, North Carolina, USA

Gina Benavides

GlaxoSmithKline, Inc, Research Triangle Park, North Carolina, USA

Large-scale analysis of gene expression using cDNA microarrays promises the rapid detection of the mode of toxicity for drugs and other chemicals. cDNA microarrays were used to examine chemically induced alterations of gene expression in HepG2 cells exposed to a diverse group of toxicants at an equitoxic exposure concentration. The treatments were ouabain (43 µM), lauryl sulfate (260 µ M), dimethylsulfoxide (1.28 M), cycloheximide (62.5 µM), tolbutamide (12.8 mM), sodium fluoride (3 mM), diethyl maleate (1.25 mM), buthionine sulfoximine (30 mM), potassium bromate (2.5 mM), sodium selenite (30 µM), alloxan (130 mM), adriamycin (40 µM), hydrogen peroxide (4 mM), and heat stress (45•C x 30 minutes). Patterns of gene expression were correlated with morphologic and biochemical indicators of toxicity. Gene expression responses were characteristically different for each treatment. Patterns of expression were consistent with cell cycle arrest, DNA damage, diminished protein synthesis, and oxidative stress. Based upon these results, we concluded that gene expression changes provide auseful indicator of oxidative stress, as assessed by the GSH:GSSG ratio. Under the conditions of this cell culture test system, oxidative stress upregulated 5 genes, HMOX1, p21waf1/cip1, GCLM, GR, TXNR1 while downregulating CYP1A1 and TOPO2A. Primers and probes for these genes were incorporated into the design of a 7-gene plate for RT-PCR. The plate design permitted statistical analysis and allowed clear discrimination between chemicals inducing oxidative vs nonoxidative stress. A simple oxidative stress score (0—1), based on the responses by the 7 genes (including p-value) on the RT-PCR plate, was correlated with the GSH:GSSG ratio using linear regression and ranking (Pearson product) procedures. These analyses yielded correlation coefficients of 0.74 and 0.87, respectively, for the treatments tested (when 1 outlier was excluded), indicating a good correlation between the biochemical and transcriptional measures of oxidative stress. We conclude that it is essential to measure the mechanism of interest directly in the test system being used when assessing gene expression as a tool for toxicology.

Tables 1—15, referenced in this paper, are not printed in this issue of Toxicologic Pathology. They are available as downloadable text files at http://taylorandfrancis.metapress.com/openurl.asp?genre=journal&issn=0192-6233. To access them, click on the issue link for 30(4), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org.

Key Words: Oxidative stress • GSH:GSSG • gene expression • cDNA arrays • RT-PCR • toxicity screen • Hep-G2 cells • transcriptomics.

References

• Gille G., Sigler K. (1995). Oxidative stress and living cells. Folia Microbiol 40: 131—152.[CrossRef]
• Hauptmann N., Cadenas E. (1997). The oxygen paradox: Biochemistry of active oxygen. In: Oxidative Stress and the Molecular Biology of Antioxidant Defenses, Scandalios JG (ed). Cold Spring Harbor Laboratory Press, New York, pp 1—20.
• Cadenas E., Packer L. (1996). In: Handbook of Antioxidants. Antioxidants in Health and Disease, Packer L, Fuchs J (eds). Marcel Dekker, Inc, New York, 1—602.
• Scandalios JG (1997). Oxidative Stress and the Molecular Biology of Antioxidant Defenses. Cold Spring Harbor Monograph Series, Cold Spring Harbor Press, NewYork.
• Morel Y., Barouki R. (1998). Down-regulation of cytochrome P4501A1 gene promotor by oxidative stress. J Biol Chem 273: 26969—26976.[Abstract/Free Full Text]
• Polyak K., Xia Y., Zweier JL, Kinzler KW, Vogelstein B. (1997). A model for p53-induced apoptosis. Nature 389: 300—305.[CrossRef][Medline] [Order article via Infotrieve]
• Granville DJC, Carthy CM, Hunt Dwc, McManue BM (1998). Apoptosis: Molecular aspects of cell death and disease. Lab Invest 78: 893—913.[Web of Science][Medline] [Order article via Infotrieve]
• Esposito F., Cuccorillo F., Russo L., Casella F., Russo T., Cimino F. (1998). A new p21 waf1/cip1 isoform is an early event of cell response to oxidative stress. Cell Death Diff 5: 940—945.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Armstrong DE (1998). Free Radical and Antioxidant Protocols. Vol. 108: Humana Press, Totawa, New Jersey.
• Bains JS, Shaw CA ( 1997). Neurodegenerative disorders in humans: The role of glutathione in oxidative stress-mediated neuronal death. Brain Res Rev 25: 335—358.[CrossRef][Medline] [Order article via Infotrieve]
• Gurwitz D. (1998). Oxidative stress in Parkinson's disease: Clues for new therapeutic directions. Mol Med Today: 371(4).
• Klaassen CD, ed. (1996). Casarett & Doull's Toxicology, The Basic Science of Poisons. 5th ed, McGraw-Hill, New York.
• Primiano TS, Thomas R., Kensler, TW (1997). Redox regulation of genes that protect against carcinogens. Biochem Physiol 118B: 487—497.
• Dalton TP, Shertzer HG, Puga A. ( 1999). Regulation of gene expression by reactive oxygen. Annu Rev Pharmacol Toxicol 39: 67—101.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Sun Y., Oberley W. (1996). Redox regulation of transcriptional activators. Free Rad Biol Med 21: 335—348.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Palmer HJ, Paulson KE (1997). Reactive oxygen species and antioxidants in signal transduction and gene expression. Nutr Rev 55: 353—361.[Web of Science][Medline] [Order article via Infotrieve]
• Abraham NG (1998). Quantitation of heme oxygenase (HO-1) copies in human tissues by competitive RT/PCR, In: Free Radical and Antioxidant Protocols, Armstrong D (ed). Humana Press, Totawa, New Jersey, pp 199— 210.
• Burczynski ME, McMillian M., Ciervo J., Li L., Parker JB, Dunn RTI, Hicken S., Farr S., Johnson MD (2000). Toxicogenomics-based discrimination of toxic mechanism in HepG2 human hepatoma cells. Toxicol Sci 58: 399— 415.[Abstract/Free Full Text]
• Collins FS (1999). Overview of DNA chip technology. Nature Gen 21: 1—60.[CrossRef][Web of Science]
• Amundson SA, Bittner M., Chen Y., Trent J., Meltzer P., Fornace AJ (1999). Fluorescent cDNA microarray hybridization reveals complexity and heterogeneity of cellular stress responses. Oncogene 18: 3666—3672.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Waring JF, Ulrich RG (2000). The impact of genomics-based technologies on drug safety evaluation. Annu Rev Pharmacol Toxicol 40: 335— 352.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Taniguchi M., Miura K., Iwao H., Yamanaka S. (2001). Quantitative assessment of DNA microarrays—comparison with Northern blot analysis. Geneomics 71: 34—39.[CrossRef]
• Crosby LM, Benavides G., Yoon L., Hyder K., DeAngelo AB, Morgan KT (2000). Morphologic analysis correlates with gene expression changes in cultured F344 rat mesothelial cells. Toxicol Appl Pharmacol 169: 205—221.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Lewin B. (1997). Genes. 6th ed. Oxford University Press, New york.
• Velculescu VE, Zhang L., Zhou W., Vogelstein J., Basrai MA, Bassett Jr DE, Hieter P., Vogelstein B., Kinzler KW (1997). Characterization of the yeast transcriptome. Cell 88: 243—251.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Farr S., Dunn RTI (1999). Concise review: Gene expression applied to toxicology. Toxicol Sci 50: 1—9.[Free Full Text]
• Pennie WD (2000). Use of cDNA microarrays to probe and understand the toxicological consequences of altered gene expression. Toxicol Letts 112—113: 473—477.[CrossRef]
• Marshall NJ, Goodwin CJ, Holt SJ (1995). A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function. Growth Reg 5: 69—84.[Web of Science][Medline] [Order article via Infotrieve]
• Atcc (1998). American Type Culture Collection (ATCC) Number: CRL-10741 Organism: Homo sapiens (human), HepG2/C3A. ATCC.
• Reed DJ, Babson JR, Beatty PW, Brodie AE, Ellis WW, Potter DW (1980). High-performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide, and related thiols and disulfides. Anal Biochem 106: 55—62.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Kepler TB, Crosby LM, Morgan KT (2002). Normalization and analysis of DNA microarray data by self-consistency and local regression. In press: Genome Biology.
• Taniguchi N, ed. (1989). Glutathione Centennial: Molecular Perspectives and Clinical Implications. Academic Press, New York.
• Chenchik A. (1997). Atlas Human cDNA Expression Array 1. Clontechniques. 12.
• Crosby LM (2000). Studies on mechanisms of potassium bromate toxicity and carcinogenicity in the male F344 rat. Ph.D Dissertation. In Department of Toxicology. North Carolina State University, Raleigh.
• Smith TW, Antman EM, Friedmann PL, Blatt CM, Marsh JD (1984). Digitasis glycosides: Mechanisms and manifestations oftoxicity. Prog Cardiovasc Dis 26: 495—540.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Anderson RC (1998). Carnitine palmitoyltransferase: A viable target for the treatment of NIDDM? Curr Pharm Des 4: 1—15.[Web of Science][Medline] [Order article via Infotrieve]
• Schiaffonati L., Tiberio L. (1997). Gene expression in liver after toxic injury: Analysis of heat shock response and oxidative stress-inducible genes. Liver 17: 183—191.[Web of Science][Medline] [Order article via Infotrieve]
• Mankovitz R., Kisilevsky R., Florian M. (1978). Chinese hamster cell lines resistant to the cytotoxic action of fluoride. Can J Genet Cytol 20: 71—84.[Web of Science][Medline] [Order article via Infotrieve]
• Shen H., Yang C., Ong C. (1999). Sodium selenite-induced oxidative stress and apoptosis in human hepatoma HepG2 cells. Int J Cancer 81: 820—828.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Zou J., Salminen WF, Roberts SM, Voellmy R. (1998). Correlation between glutathione oxidation and trimerization of heat shock factor 1, an early step in stress induction of the Hsp response. Cell Str Chaper 3: 130—141.[CrossRef]
• Rockett JC, Dix DJ (1999). Application of DNA arrays to toxicology. Environ Health Perspect 107: 681—685.[Web of Science][Medline] [Order article via Infotrieve]
• Nuwaysir EF, Bittner M., Trent J., Barrett JC, Afshan CA (1999). Microarrays and toxicology: The advent of toxicogenomics. Mol Carcinogen 24: 153— 159f.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Stevens JL, Lui H., Halleck M., Bowes RC, Chen QM, Water vdB (2000). Toxicology Linking gene expression to mechanisms of toxicity. Toxicol Letts 112—113: 479—486.[CrossRef]
• Steiner S., Anderson NL (2000). Expression profiles in toxicology— Potentials and limitations. Toxicol Letts 112—113: 467—471.[CrossRef]
• Corton CJ, Stauber AJ (2000). Toxicological highlight: Toward construction of a transcript profile database predictive of chemical toxicity. Toxicol Sci 58: 217—219.[Abstract/Free Full Text]
• Haffner SM (2000). Clinical relevance of the oxidative stress concept. Metabolism 49: 30—34.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Sen CK, Sies H, Baeuerle PA, eds. ( 2000). Antioxidant and Redox Regulation of Genes. Academic Press: San Diego, CA.
• Eaton DL, Klaassen CD (1996). Principles of toxicology. In: Casarett and Doull's Toxicology, The Basic Science of Poisons, Klaassen CD (ed). McGraw-Hill, New York.
• Faustman EM, Omenn GS (1996). Risk assessment. In: CasarettandDoull's Toxicology, The Basic Science of Poisons, Klaassen CD (ed). McGraw-Hill, Health Professions Division, New York, pp 75—88.
• Gregus Z., Klaassen CD (1996). Mechanisms of toxicity. In: Casarett and Doull's Toxicology, The Basic Science of Poisons, Klaassen CD (ed). McGraw-Hill, New York.
• Bolon B., Dorman DC, Bonnefoi MS, Randall HW, Morgan KT (1993). Histopathologic approaches to chemical toxicity using primary cultures of dissociated neural cells grown in chamber slides. Toxicol Pathol 21: 465— 479.[Abstract/Free Full Text]
• Bouma M-ER, E., Verthier N., LaBarre C., Feldmann G. (1989). Further cellular investigation of the human hepatoblastoma-derived cell line HEPG2: Morphology and immunocyto-chemical studies of hepatic-secreted proteins. In Vitro Cell Devel Biol 25: 267—275.[Web of Science][Medline] [Order article via Infotrieve]
• Neuman MG, Koren G., Tiribelli C. (1993). In vitro assessment of the ethanol-induced hepatotoxicity on HepG2 cell line. Biochem Biophys Res Comm 197: 932—941.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Salminen WFJ, Voellmy R., Roberts SM (1996). Induction of hsp 70 in HepG2 cells in response to hepatotoxicants. Toxicol Appl Pharmacol 141: 117—123.[Web of Science][Medline] [Order article via Infotrieve]
• Shimada H., Hirai K-I., Simamura E., Pan J. (1998). Mitochondrial NADHquinone oxidoreductase of the outer membrane is responsible for paraquat cytotoxicity in rat livers. Arch Biochem Biophys 351: 75—81.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Ueno M., Masutani H., Araj RJ, Yamauchi A., Hirota K., Sakai T., Inamoto T., Yamaoka Y., Yodoi J., Nikaido T. (1999). Thioredoxin-dependent redox regulation of p53-mediated p21 activation. J Biol Chem 274: 35809— 35815.[Abstract/Free Full Text]
• Hainaut P., Milner J. (1993). Redox modulation of p53 conformation and sequence-specific DNA binding in vitro. Cancer Res 53: 4469—4473.[Abstract/Free Full Text]
• Rainwater R., Parks D., Anderson ME, Tegtmeyer P., Mann K. (1995). Role of cysteine residues in regulation of p53 function. Mol Cell Biol 15: 3892— 3903.[Abstract]
• Morel Y., Barouki R. (1999). Repression of gene expression by oxidative stress. Biochem J 342: 481—496.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Guyton KZ, Gorospe M., Holbrook NJ (1997). Oxidative stress, gene expression, and the aging process. In: Oxidative Stress and the Molecular Biology of Antioxidant Defenses, Scandalios JG (ed). Cold Spring Harbor Laboratory Press, New York, pp 247—272.
• Kamata H., Hirata H. (1999). Redox regulation of cellular signaling. Cell Signal 11: 1—14.[Medline] [Order article via Infotrieve]
• Murray RK, Granner DK, Mayes PA, Rodwell VW (1996). Harper's Biochemistry, 24th ed. Appleton & Lange, stamford, CT.
• Bjornstedt M., Kumar S., Bjorkhem L., Spyrou G., Holmgran A. (1997). Selenium and the thioredoxin and glutaredoxin systems. Biomed Environ Sci 10: 271—279.[Medline] [Order article via Infotrieve]
• Mansur CP (1997). The regulation and function of the p53 tumor suppressor, In: Advances in Dermatology 13: 121—166.
• Lu S. (1999). Regulation of hepatic glutathione synthesis: Current concepts and controversies. FASEB J 13: 1169—1183.[Abstract/Free Full Text]
• McAdams HH, Shapiro L. (1995). Circuit simulation of genetic networks. Science 269: 650.[Abstract/Free Full Text]
• Pawson T., Saxton TM (1999). Signaling networks-do all roads lead to the same genes? Cell 97: 675—678.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Schuster S., Fell DA, Dandekar T. (2000). A general definition of metabolic pathways useful for systemic organization and analysis of complex metabolic networks. Nature Biotechnol 18: 326—332.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Thieffry D., Huerta AM, Pérez-Rueda E., Collado-Vides J. (1998). From specific gene regulation to genomic networks: A global analysis of transcriptional regulation in Escherichia coli. Bioessays 20: 433—440.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Weaver DC, Workman CT, Stormo GD (1999). Modeling regulatory networks with weight matrices. Pac Symp Biocomput, pp 112—123.
• Somogyi R., Sniegoski CA (1996). Modeling the complexity of genetic networks: Understanding multigenic and pleiotropic regulation. In: Complexity. H. Morowitz (ed). John Wiley & Sons, New York, pp 45—63.
• Anderson ME (1998). Glutathione: An overview of biosynthesis and modulation. Chem-Biol Inter 111—112: 1—14.
• Sai KU, Takashi TA, Hasegawa R., Kurokawa Y. (1992). The protective role of glutathione, cysteine and vitamin C against oxidative DNA damage induced in rat kidney by potassium bromate. Jpn J Cancer Res 83: 45—51.[CrossRef][Web of Science]
• Stewart MS, Spallholz JE, Neldner KH, Pence BC (1999). Selenium compounds have disparate abilities to impose oxidative stress and induce apoptosis. Free Rad Biol Med 26: 42—48.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

Toxicologic Pathology, Vol. 30, No. 4, 435-451 (2002)
DOI: 10.1080/01926230290105613


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