Assessment of Biomarkers in Acetaminophen-Induced Hepatic Toxicity by siRNA

  • Kang, Jin-Seok (Department of Biomedical Laboratory Science, Namseoul University) ;
  • Yum, Young-Na (National Institute of Food and Drug Safety Evaluation, Korea Food and Drug Administration) ;
  • Kim, Joo-Hwan (National Institute of Food and Drug Safety Evaluation, Korea Food and Drug Administration) ;
  • Park, Sue-Nie (National Institute of Food and Drug Safety Evaluation, Korea Food and Drug Administration)
  • Published : 2009.10.31


We investigated global gene expression from both mouse liver and mouse hepatic cell lines treated with acetaminophen (APAP) in order to compare in vivo and in vitro profiles and to assess the feasibility of the two systems. During our analyses of gene expression profiles, we picked up several down-regulated genes, such as the cytochrome P450 family 51 (Cyp51), sulfotransferase family cytosolic 1C member 2 (Sult1c2), 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (Hmgcs1), and several genes that were up-regulated by APAP, such as growth arrest and DNA-damage-inducible 45 alpha (Gadd45a), transformation related protein 53 inducible nuclear protein 1 (Trp53inp1) and zinc finger protein 688 (Zfp688). For validation of gene function, synthesized short interfering RNAs (siRNAs) for these genes were transfected in a mouse hepatic cell line, BNL CL.2, for investigation of cell viability and mRNA expression level. We found that siRNA transfection of these genes induced down-regulation of respective mRNA expression and decreased cell viability. siRNA transfection for Cyp51 and others induced morphological alterations, such as membrane thickening and nuclear condensation. Taken together, siRNA transfection of these six genes decreased cell viability and induced alteration in cellular morphology, along with effective inhibition of respective mRNA, suggesting that these genes could be associated with APAP-induced toxicity. Furthermore, these genes may be used in the investigation of hepatotoxicity, for better understanding of its mechanism.


  1. Boverhof, D. R. and Zacharewski, T. R. (2006). Toxicogenomics in risk assessment: applications and needs. Toxicol. Sci. 89, 352-360.
  2. Coen, M., Ruepp, S. U., Lindon, J. C., Nicholson, J. K., Pognan, F., Lenz, E. M. and Wilson, I. D. (2004). Integrated application of transcriptomics and metabonomics yields new insight into the toxicity due to paracetamol in the mouse. J. Pharm. Biomed. Anal. 35, 93-105.
  3. Corvi, R., Ahr, H. J., Albertini, S., Blakey, D. H., Clerici, L., Coecke, S., Douglas, G. R., Gribaldo, L., Groten, J. P., Haase, B., Hamernik, K., Hartung, T., Inoue, T., Indans, I., Maurici, D., Orphanides, G., Rembges, D., Sansone, S. A., Snape, J. R., Toda, E., Tong, W., van Delft, J. H., Weis, B. and Schechtman, L. M. (2006). Meeting report: Validation of toxicogenomics-based test systems: ECVAM-ICCVAM/NIC EATM considerations for regulatory use. Environ. Health Perspect. 114, 420-429.
  4. Cover, C., Mansouri, A., Knight, T. R., Bajt, M. L., Lemasters, J. J., Pessayre, D. and Jaeschke, H. (2005). Peroxynitriteinduced mitochondrial and endonuclease-mediated nuclear DNA damage in acetaminophen hepatotoxicity. J Pharmacol Exp. Ther. 315, 879-887.
  5. Dykxhoorn, D. M., Novina, C. D. and Sharp, P. A. (2003). Killing the messenger: short RNAs that silence gene expression. Nat. Rev. Mol. Cell Biol. 4, 457-467.
  6. Fink, M., Acimovic, J., Rezen, T., Tansek, N. and Rozman, D. (2005). Cholesterogenic lanosterol 14alpha-demethylase (CYP51) is an immediate early response gene. Endocrinology 146, 5321-5331.
  7. Fornace, A. J. Jr., Jackman, J., Hollander, M. C., Hoffman- Liebermann, B. and Liebermann, D. A. (1992). Genotoxicstress- response genes and growth-arrest genes. gadd, MyD, and other genes induced by treatments eliciting growth arrest. Ann. N. Y. Acad. Sci. 663, 139-153.
  8. Hamilton, A. J. and Baulcombe, D. C. (1999). A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950-952.
  9. Huang, Q., Jin, X., Gaillard, E. T., Knight, B. L., Pack, F. D., Stoltz, J. H., Jayadev, S. and Blanchard, K. T. (2004). Gene expression profiling reveals multiple toxicity endpoints induced by hepatotoxicants. Mutat. Res. 549, 147-168.
  10. James, L. P., Mayeux, P. R. and Hinson, J. A. (2003). Acetaminophen- induced hepatotoxicity. Drug Metab. Dispos. 31, 1499- 1506.
  11. Kang, J. S., Jeong, Y. K., Suh, S. K., Kim, J. H., Lee, W. S., Lee, E. M., Shin, J. H., Jung, H. K., Kim, S. H. and Park, S. N. (2007). Assessment of feasibility for developing toxicogenomics biomarkers by comparing in vitro and in vivo genomic profiles specific to liver toxicity induced by acetaminophen. Mol. & Cellular Toxicol. 3, 177-184.
  12. Leighton, J. K., Brown, P., Ellis, A., Harlow, P., Harrouk, W., Pine, P. S., Robison, T., Rosario, L. and Thompson, K. (2006). Workgroup report: Review of genomics data based on experience with mock submissions--view of the CDER Pharmacology Toxicology Nonclinical Pharmacogenomics Subcommittee. Environ. Health Perspect. 114, 573-578.
  13. Mattingly, C. J., Rosenstein, M. C., Davis, A. P., Colby, G. T., Forrest, J. N. Jr. and Boyer, J. L. (2006). The comparative toxicogenomics database: a cross-species resource for building chemical-gene interaction networks. Toxicol. Sci. 92, 587-595.
  14. Napirei, M., Basnakian, A. G., Apostolov, E. O. and Mannherz, H. G. (2006). Deoxyribonuclease 1 aggravates acetaminopheninduced liver necrosis in male CD-1 mice. Hepatology 43, 297-305.
  15. Pennie, W., Pettit, S. D. and Lord, P. G. (2004). Toxicogenomics in risk assessment: an overview of an HESI collaborative research program. Environ. Health Perspect. 112, 417- 419.
  16. Reilly, T. P., Bourdi, M., Brady, J. N., Pise-Masison, C. A., Radonovich, M. F., George, J. W. and Pohl, L. R. (2001). Expression profiling of acetaminophen liver toxicity in mice using microarray technology. Biochem. Biophys. Res. Commun 282, 321-328.
  17. Rozman, D., Stromstedt, M., Tsui, L. C., Scherer, S. W. and Waterman, M. R. (1996). Structure and mapping of the human lanosterol 14alpha-demethylase gene (CYP51) encoding the cytochrome P450 involved in cholesterol biosynthesis; comparison of exon/intron organization with other mammalian and fungal CYP genes. Genomics 38, 371-381.
  18. Seliskar, M. and Rozman, D. (2007). Mammalian cytochromes P450--importance of tissue specificity. Biochim. Biophys. Acta. 1770, 458-466.
  19. Stanley, E. L., Hume, R. and Coughtrie, M. W. (2005). Expression profiling of human fetal cytosolic sulfotransferases involved in steroid and thyroid hormone metabolism and in detoxification. . 240, 32-42.
  20. Tomasini, R., Seux, M., Nowak, J., Bontemps, C., Carrier, A., Dagorn, J. C., Pebusque, M. J., Iovanna, J. L. and Dusetti, N. J. (2005). TP53INP1 is a novel p73 target gene that induces cell cycle arrest and cell death by modulating p73 transcriptional activity. Oncogene 24, 8093-8104.
  21. Waters, M. D. and Fostel, J. M. (2004). Toxicogenomics and systems toxicology: aims and prospects. Nat. Rev. Genet. 5, 936-948.
  22. Welch, C. L., Xia, Y. R., Shechter, I., Farese, R., Mehrabian, M., Mehdizadeh, S., Warden, C. H. and Lusis, A. J. (1996). Genetic regulation of cholesterol homeostasis: chromosomal organization of candidate genes. J. Lipid. Res. 37, 1406-1421.