Skip to main content
NCBI home page
Environmental Health Perspectives logoLink to Environmental Health Perspectives
. 2004 Mar;112(4):428–438. doi: 10.1289/ehp.6677

Clofibrate-induced gene expression changes in rat liver: a cross-laboratory analysis using membrane cDNA arrays.

Valerie A Baker 1, Helen M Harries 1, Jeff F Waring 1, Colette M Duggan 1, Hong A Ni 1, Robert A Jolly 1, Lawrence W Yoon 1, Angus T De Souza 1, Judith E Schmid 1, Roger H Brown 1, Roger G Ulrich 1, John C Rockett 1
PMCID: PMC1241896  PMID: 15033592

Abstract

Microarrays have the potential to significantly impact our ability to identify toxic hazards by the identification of mechanistically relevant markers of toxicity. To be useful for risk assessment, however, microarray data must be challenged to determine reliability and interlaboratory reproducibility. As part of a series of studies conducted by the International Life Sciences Institute Health and Environmental Science Institute Technical Committee on the Application of Genomics to Mechanism-Based Risk Assessment, the biological response in rats to the hepatotoxin clofibrate was investigated. Animals were treated with high (250 mg/kg/day) or low (25 mg/kg/day) doses for 1, 3, or 7 days in two laboratories. Clinical chemistry parameters were measured, livers removed for histopathological assessment, and gene expression analysis was conducted using cDNA arrays. Expression changes in genes involved in fatty acid metabolism (e.g., acyl-CoA oxidase), cell proliferation (e.g., topoisomerase II-Alpha), and fatty acid oxidation (e.g., cytochrome P450 4A1), consistent with the mechanism of clofibrate hepatotoxicity, were detected. Observed differences in gene expression levels correlated with the level of biological response induced in the two in vivo studies. Generally, there was a high level of concordance between the gene expression profiles generated from pooled and individual RNA samples. Quantitative real-time polymerase chain reaction was used to confirm modulations for a number of peroxisome proliferator marker genes. Though the results indicate some variability in the quantitative nature of the microarray data, this appears due largely to differences in experimental and data analysis procedures used within each laboratory. In summary, this study demonstrates the potential for gene expression profiling to identify toxic hazards by the identification of mechanistically relevant markers of toxicity.

Full Text

The Full Text of this article is available as a PDF (440.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aberg F., Appelkvist E. L. Clofibrate and di(2-ethylhexyl)phthalate increase ubiquinone contents without affecting cholesterol levels. Acta Biochim Pol. 1994;41(3):321–329. [PubMed] [Google Scholar]
  2. Amacher D. E., Beck R., Schomaker S. J., Kenny C. V. Hepatic microsomal enzyme induction, beta-oxidation, and cell proliferation following administration of clofibrate, gemfibrozil, or bezafibrate in the CD rat. Toxicol Appl Pharmacol. 1997 Jan;142(1):143–150. doi: 10.1006/taap.1996.8007. [DOI] [PubMed] [Google Scholar]
  3. Ashby J., Brady A., Elcombe C. R., Elliott B. M., Ishmael J., Odum J., Tugwood J. D., Kettle S., Purchase I. F. Mechanistically-based human hazard assessment of peroxisome proliferator-induced hepatocarcinogenesis. Hum Exp Toxicol. 1994 Nov;13 (Suppl 2):S1–117. doi: 10.1177/096032719401300201. [DOI] [PubMed] [Google Scholar]
  4. Bartosiewicz M. J., Jenkins D., Penn S., Emery J., Buckpitt A. Unique gene expression patterns in liver and kidney associated with exposure to chemical toxicants. J Pharmacol Exp Ther. 2001 Jun;297(3):895–905. [PubMed] [Google Scholar]
  5. Bartosiewicz M., Penn S., Buckpitt A. Applications of gene arrays in environmental toxicology: fingerprints of gene regulation associated with cadmium chloride, benzo(a)pyrene, and trichloroethylene. Environ Health Perspect. 2001 Jan;109(1):71–74. doi: 10.1289/ehp.0110971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  7. Bulera S. J., Eddy S. M., Ferguson E., Jatkoe T. A., Reindel J. F., Bleavins M. R., De La Iglesia F. A. RNA expression in the early characterization of hepatotoxicants in Wistar rats by high-density DNA microarrays. Hepatology. 2001 May;33(5):1239–1258. doi: 10.1053/jhep.2001.23560. [DOI] [PubMed] [Google Scholar]
  8. Burczynski M. E., McMillian M., Ciervo J., Li L., Parker J. B., Dunn R. T., 2nd, Hicken S., Farr S., Johnson M. D. Toxicogenomics-based discrimination of toxic mechanism in HepG2 human hepatoma cells. Toxicol Sci. 2000 Dec;58(2):399–415. doi: 10.1093/toxsci/58.2.399. [DOI] [PubMed] [Google Scholar]
  9. Corton J. C., Anderson S. P., Stauber A. Central role of peroxisome proliferator-activated receptors in the actions of peroxisome proliferators. Annu Rev Pharmacol Toxicol. 2000;40:491–518. doi: 10.1146/annurev.pharmtox.40.1.491. [DOI] [PubMed] [Google Scholar]
  10. Díez-Fernández C., Sanz N., Alvarez A. M., Wolf A., Cascales M. The effect of non-genotoxic carcinogens, phenobarbital and clofibrate, on the relationship between reactive oxygen species, antioxidant enzyme expression and apoptosis. Carcinogenesis. 1998 Oct;19(10):1715–1722. doi: 10.1093/carcin/19.10.1715. [DOI] [PubMed] [Google Scholar]
  11. Fielden M. R., Zacharewski T. R. Challenges and limitations of gene expression profiling in mechanistic and predictive toxicology. Toxicol Sci. 2001 Mar;60(1):6–10. doi: 10.1093/toxsci/60.1.6. [DOI] [PubMed] [Google Scholar]
  12. Gerhold D., Lu M., Xu J., Austin C., Caskey C. T., Rushmore T. Monitoring expression of genes involved in drug metabolism and toxicology using DNA microarrays. Physiol Genomics. 2001 Apr 27;5(4):161–170. doi: 10.1152/physiolgenomics.2001.5.4.161. [DOI] [PubMed] [Google Scholar]
  13. Hamadeh Hisham K., Bushel Pierre R., Jayadev Supriya, DiSorbo Olimpia, Bennett Lee, Li Leping, Tennant Raymond, Stoll Raymond, Barrett J. Carl, Paules Richard S. Prediction of compound signature using high density gene expression profiling. Toxicol Sci. 2002 Jun;67(2):232–240. doi: 10.1093/toxsci/67.2.232. [DOI] [PubMed] [Google Scholar]
  14. Hamadeh Hisham K., Bushel Pierre R., Jayadev Supriya, Martin Karla, DiSorbo Olimpia, Sieber Stella, Bennett Lee, Tennant Raymond, Stoll Raymond, Barrett J. Carl. Gene expression analysis reveals chemical-specific profiles. Toxicol Sci. 2002 Jun;67(2):219–231. doi: 10.1093/toxsci/67.2.219. [DOI] [PubMed] [Google Scholar]
  15. Harries H. M., Fletcher S. T., Duggan C. M., Baker V. A. The use of genomics technology to investigate gene expression changes in cultured human liver cells. Toxicol In Vitro. 2001 Aug-Oct;15(4-5):399–405. doi: 10.1016/s0887-2333(01)00043-1. [DOI] [PubMed] [Google Scholar]
  16. Hertz R., Bar-Tana J. Peroxisome proliferator-activated receptor (PPAR) alpha activation and its consequences in humans. Toxicol Lett. 1998 Dec 28;102-103:85–90. doi: 10.1016/s0378-4274(98)00290-2. [DOI] [PubMed] [Google Scholar]
  17. Ito N., Hasegawa R., Imaida K., Masui T., Takahashi S., Shirai T. Pathological markers for non-genotoxic agent-associated carcinogenesis. Toxicol Lett. 1992 Dec;64-65 Spec No:613–620. doi: 10.1016/0378-4274(92)90239-g. [DOI] [PubMed] [Google Scholar]
  18. Karbowska J., Kochan Z., Zelewski L., Swierczynski J. Tissue-specific effect of clofibrate on rat lipogenic enzyme gene expression. Eur J Pharmacol. 1999 Apr 16;370(3):329–336. doi: 10.1016/s0014-2999(99)00129-6. [DOI] [PubMed] [Google Scholar]
  19. Kim S. C., Hong J. T., Jang S. J., Kang W. S., Yoo H. S., Yun Y. P. Formation of 8-oxodeoxyguanosine in liver DNA and hepatic injury by peroxisome proliferator clofibrate and perfluorodecanoic acid in rats. J Toxicol Sci. 1998 May;23(2):113–119. doi: 10.2131/jts.23.2_113. [DOI] [PubMed] [Google Scholar]
  20. Kovacs W., Stangl H., Völkl A., Schad A., Dariush Fahimi H., Baumgart E. Localization of mRNAs encoding peroxisomal proteins in cell culture by non-radioactive in situ hybridization. Comparison of rat and human hepatoma cells and their responses to two divergent hypolipidemic drugs. Histochem Cell Biol. 2001 Jun;115(6):499–508. doi: 10.1007/s004180100277. [DOI] [PubMed] [Google Scholar]
  21. Lake B. G., Gray T. J., Foster J. R., Stubberfield C. R., Gangolli S. D. Comparative studies on di-(2-ethylhexyl) phthalate-induced hepatic peroxisome proliferation in the rat and hamster. Toxicol Appl Pharmacol. 1984 Jan;72(1):46–60. doi: 10.1016/0041-008x(84)90248-5. [DOI] [PubMed] [Google Scholar]
  22. Lake B. G., Rijcken W. R., Gray T. J., Foster J. R., Gangolli S. D. Comparative studies of the hepatic effects of di- and mono-n-octyl phthalates, di-(2-ethylhexyl) phthalate and clofibrate in the rat. Acta Pharmacol Toxicol (Copenh) 1984 Mar;54(3):167–176. doi: 10.1111/j.1600-0773.1984.tb01913.x. [DOI] [PubMed] [Google Scholar]
  23. Latruffe N., Cherkaoui Malki M., Nicolas-Frances V., Jannin B., Clemencet M. C., Hansmannel F., Passilly-Degrace P., Berlot J. P. Peroxisome-proliferator-activated receptors as physiological sensors of fatty acid metabolism: molecular regulation in peroxisomes. Biochem Soc Trans. 2001 May;29(Pt 2):305–309. doi: 10.1042/0300-5127:0290305. [DOI] [PubMed] [Google Scholar]
  24. Lee S. S., Pineau T., Drago J., Lee E. J., Owens J. W., Kroetz D. L., Fernandez-Salguero P. M., Westphal H., Gonzalez F. J. Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. Mol Cell Biol. 1995 Jun;15(6):3012–3022. doi: 10.1128/mcb.15.6.3012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lindauer M., Beier K., Völkl A., Fahimi H. D. Zonal heterogeneity of peroxisomal enzymes in rat liver: differential induction by three divergent hypolipidemic drugs. Hepatology. 1994 Aug;20(2):475–486. [PubMed] [Google Scholar]
  26. Lindquist P. J., Svensson L. T., Alexson S. E. Molecular cloning of the peroxisome proliferator-induced 46-kDa cytosolic acyl-CoA thioesterase from mouse and rat liver--recombinant expression in Escherichia coli, tissue expression, and nutritional regulation. Eur J Biochem. 1998 Feb 1;251(3):631–640. doi: 10.1046/j.1432-1327.1998.2510631.x. [DOI] [PubMed] [Google Scholar]
  27. Louet J. F., Chatelain F., Decaux J. F., Park E. A., Kohl C., Pineau T., Girard J., Pegorier J. P. Long-chain fatty acids regulate liver carnitine palmitoyltransferase I gene (L-CPT I) expression through a peroxisome-proliferator-activated receptor alpha (PPARalpha)-independent pathway. Biochem J. 2001 Feb 15;354(Pt 1):189–197. doi: 10.1042/0264-6021:3540189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lundgren B., DePierre J. W. Proliferation of peroxisomes and induction of cytosolic and microsomal epoxide hydrolases in different strains of mice and rats after dietary treatment with clofibrate. Xenobiotica. 1989 Aug;19(8):867–881. doi: 10.3109/00498258909043147. [DOI] [PubMed] [Google Scholar]
  29. Mannaerts G. P., Van Veldhoven P. P., Casteels M. Peroxisomal lipid degradation via beta- and alpha-oxidation in mammals. Cell Biochem Biophys. 2000;32(SPRING):73–87. doi: 10.1385/cbb:32:1-3:73. [DOI] [PubMed] [Google Scholar]
  30. Nuwaysir E. F., Bittner M., Trent J., Barrett J. C., Afshari C. A. Microarrays and toxicology: the advent of toxicogenomics. Mol Carcinog. 1999 Mar;24(3):153–159. doi: 10.1002/(sici)1098-2744(199903)24:3<153::aid-mc1>3.0.co;2-p. [DOI] [PubMed] [Google Scholar]
  31. Peng Xuejun, Wood Constance L., Blalock Eric M., Chen Kuey Chu, Landfield Philip W., Stromberg Arnold J. Statistical implications of pooling RNA samples for microarray experiments. BMC Bioinformatics. 2003 Jun 24;4:26–26. doi: 10.1186/1471-2105-4-26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pennie William, Pettit Syril D., Lord Peter G. Toxicogenomics in risk assessment: an overview of an HESI collaborative research program. Environ Health Perspect. 2004 Mar;112(4):417–419. doi: 10.1289/ehp.6674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Popp J. A., Cattley R. C., Miller R. T., Marsman D. S. Relationship of peroxisome proliferator-induced cellular effects to hepatocarcinogenesis. Prog Clin Biol Res. 1994;387:193–207. [PubMed] [Google Scholar]
  34. Reddy J. K., Qureshi S. A. Tumorigenicity of the hypolipidaemic peroxisome proliferator ethyl-alpha-p-chlorophenoxyisobutyrate (clofibrate) in rats. Br J Cancer. 1979 Sep;40(3):476–482. doi: 10.1038/bjc.1979.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schoonjans K., Staels B., Auwerx J. Role of the peroxisome proliferator-activated receptor (PPAR) in mediating the effects of fibrates and fatty acids on gene expression. J Lipid Res. 1996 May;37(5):907–925. [PubMed] [Google Scholar]
  36. Simpson A. E. The cytochrome P450 4 (CYP4) family. Gen Pharmacol. 1997 Mar;28(3):351–359. doi: 10.1016/s0306-3623(96)00246-7. [DOI] [PubMed] [Google Scholar]
  37. Singh I. Biochemistry of peroxisomes in health and disease. Mol Cell Biochem. 1997 Feb;167(1-2):1–29. doi: 10.1023/a:1006883229684. [DOI] [PubMed] [Google Scholar]
  38. Small G. M., Burdett K., Connock M. J. A sensitive spectrophotometric assay for peroxisomal acyl-CoA oxidase. Biochem J. 1985 Apr 1;227(1):205–210. doi: 10.1042/bj2270205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Staels B., van Tol A., Andreu T., Auwerx J. Fibrates influence the expression of genes involved in lipoprotein metabolism in a tissue-selective manner in the rat. Arterioscler Thromb. 1992 Mar;12(3):286–294. doi: 10.1161/01.atv.12.3.286. [DOI] [PubMed] [Google Scholar]
  40. Svoboda D. J., Azarnoff D. L. Tumors in male rats fed ethyl chlorophenoxyisobutyrate, a hypolipidemic drug. Cancer Res. 1979 Sep;39(9):3419–3428. [PubMed] [Google Scholar]
  41. Thomas R. S., Rank D. R., Penn S. G., Zastrow G. M., Hayes K. R., Pande K., Glover E., Silander T., Craven M. W., Reddy J. K. Identification of toxicologically predictive gene sets using cDNA microarrays. Mol Pharmacol. 2001 Dec;60(6):1189–1194. doi: 10.1124/mol.60.6.1189. [DOI] [PubMed] [Google Scholar]
  42. Tosh D., Alberti K. G., Agius L. Clofibrate induces carnitine acyltransferases in periportal and perivenous zones of rat liver and does not disturb the acinar zonation of gluconeogenesis. Biochim Biophys Acta. 1989 Sep 15;992(3):245–250. doi: 10.1016/0304-4165(89)90081-0. [DOI] [PubMed] [Google Scholar]
  43. Ulrich Roger G., Rockett John C., Gibson G. Gordon, Pettit Syril D. Overview of an interlaboratory collaboration on evaluating the effects of model hepatotoxicants on hepatic gene expression. Environ Health Perspect. 2004 Mar;112(4):423–427. doi: 10.1289/ehp.6675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Vanden Heuvel J. P. Peroxisome proliferator-activated receptors (PPARS) and carcinogenesis. Toxicol Sci. 1999 Jan;47(1):1–8. doi: 10.1093/toxsci/47.1.1. [DOI] [PubMed] [Google Scholar]
  45. Vu-Dac N., Chopin-Delannoy S., Gervois P., Bonnelye E., Martin G., Fruchart J. C., Laudet V., Staels B. The nuclear receptors peroxisome proliferator-activated receptor alpha and Rev-erbalpha mediate the species-specific regulation of apolipoprotein A-I expression by fibrates. J Biol Chem. 1998 Oct 2;273(40):25713–25720. doi: 10.1074/jbc.273.40.25713. [DOI] [PubMed] [Google Scholar]
  46. Waring J. F., Ciurlionis R., Jolly R. A., Heindel M., Ulrich R. G. Microarray analysis of hepatotoxins in vitro reveals a correlation between gene expression profiles and mechanisms of toxicity. Toxicol Lett. 2001 Mar 31;120(1-3):359–368. doi: 10.1016/s0378-4274(01)00267-3. [DOI] [PubMed] [Google Scholar]
  47. Waring J. F., Jolly R. A., Ciurlionis R., Lum P. Y., Praestgaard J. T., Morfitt D. C., Buratto B., Roberts C., Schadt E., Ulrich R. G. Clustering of hepatotoxins based on mechanism of toxicity using gene expression profiles. Toxicol Appl Pharmacol. 2001 Aug 15;175(1):28–42. doi: 10.1006/taap.2001.9243. [DOI] [PubMed] [Google Scholar]
  48. Yaacob N. S., Norazmi M. N., Kass G. E., Gibson G. G. Use of competitive RT-PCR in the molecular analysis of peroxisome proliferation. Eur J Drug Metab Pharmacokinet. 1997 Oct-Dec;22(4):321–324. doi: 10.1007/BF03190964. [DOI] [PubMed] [Google Scholar]

Articles from Environmental Health Perspectives are provided here courtesy of National Institute of Environmental Health Sciences

RESOURCES