Differences in Gene Expression Profiles Reflecting Differences in Drug Sensitivity to Acetaminophen in Normal and Transformed Hepatic Cell Lines In vitro

  • Jeong, Youn-Kyoung (Division of Genetic Toxicology, National Institute of Toxicological Research, Korea Food and Drug Administration) ;
  • Kang, Jin-Seok (Department of Biomedical Laboratory Science, Namseoul University) ;
  • Kim, Joo-Whan (Division of Genetic Toxicology, National Institute of Toxicological Research, Korea Food and Drug Administration) ;
  • Suh, Soo-Kyung (Division of Genetic Toxicology, National Institute of Toxicological Research, Korea Food and Drug Administration) ;
  • Lee, Michael (Department of Biology, College of Natural Sciences, Incheon University) ;
  • Kim, Seung-Hee (Division of Genetic Toxicology, National Institute of Toxicological Research, Korea Food and Drug Administration) ;
  • Lee, Sang-Kook (Department of Pharmacognosy, College of Pharmacy, Ewha Womans University) ;
  • Park, Sue-Nie (Division of Genetic Toxicology, National Institute of Toxicological Research, Korea Food and Drug Administration)
  • Published : 2009.03.31

Abstract

Acetaminophen (APAP) overdose is known to cause severe hepatotoxicity mainly through the depletion of glutathione. In this study, we compared the cytotoxic effects of APAP on both a normal murine hepatic cell line, BNL CL.2, and its SV40-transformed cell line, BNL SV A.8. Gene expression profiles for APAP-treated cells were also obtained using microarray and analyzed to identify differences in genes or profiles that may explain the differences of susceptibility to APAP in these cell lines. These two cell lines exhibited different susceptibilities to APAP (0-$5,000{\mu}M$); BNL SV A.8 cells were more susceptible to APAP treatment compared to BNL CL.2 cells. A dose of $625{\mu}M$ APAP, which produced significant differences in cytotoxicity in these cell lines, was tested. Microarray analysis was performed to identify significant differentially expressed genes (DEGs) irrespective of APAP treatment. Genes up-regulated in BNL SV A.8 cells were associated with immune response, defense response, and apoptosis, while down-regulated genes were associated with catalytic activity, cell adhesion and the cytochrome P450 family. Consistent with the cytotoxicity data, no significant DEGs were found in BNL CL.2 cells after treatment with $625{\mu}M$ APAP, while cell cycle arrest and apoptosis-related genes were up-regulated in BNL SV A.8 cells. Based on the significant fold-changes in their expression, a genes were selected and their expressions were confirmed by quantitative real-time RT-PCR; there was a high correlation between them. These results suggest that gene expression profiles may provide a useful method for evaluating drug sensitivity of cell lines and eliciting the underlying molecular mechanism. We further compared the genes identified from our current in vitro studies to the genes previously identified in our lab as regulated by APAP in both C57BL/6 and ICR mice in vivo. We found that a few genes are regulated in a similar pattern both in vivo and in vitro. These genes might be useful to develop as in vitro biomarkers for predicting in vivo hepatotoxicity. Based on our results, we suggest that gene expression profiles may provide useful information for elucidating the underlying molecular mechanisms of drug susceptibility and for evaluating drug sensitivity in vitro for extrapolation to in vivo.

Keywords

References

  1. Sun, Y., Oberley, L. W., Elwell, J. H. & Sierra-Rivera, E. Antioxidant enzyme activities in normal and transformed mouse liver cells. Int J Cancer 44:1028-1033 (1989)
  2. Mitchell, J. R., Jollow, D. J., Potter, W. Z., Gillette, J. R. & Brodie, B. B. Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione. J Pharmacol Exp Ther 187:211-217 (1973)
  3. Pumford, N. R., Halmes, N. C. & Hinson, J. A. Covalent binding of xenobiotics to specific proteins in the liver. Drug Metab Rev 29:39-57 (1997) https://doi.org/10.3109/03602539709037572
  4. Ray, S. D. & Jena, N. A hepatotoxic dose of acetaminophen modulates expression of BCL-2, BCL-X (L), and BCL-X(S) during apoptotic and necrotic death of mouse liver cells in vivo. Arch Toxicol 73:594-606 (2000) https://doi.org/10.1007/s002040050013
  5. Gibson, J. D., Pumford, N. R., Samokyszyn, V. M. & Hinson, J. A. Mechanism of acetaminophen-induced hepatotoxicity: covalent binding versus oxidative stress. Chem Res Toxicol 9:580-585 (1996) https://doi.org/10.1021/tx950153d
  6. Adamson, G. M. & Harman, A.W. Oxidative stress in cultured hepatocytes exposed to acetaminophen. Biochem Pharmacol 45:2289-2294 (1993) https://doi.org/10.1016/0006-2952(93)90201-7
  7. Burcham, P. C. & Harman, A. W. Acetaminophen toxicity results in site-specific mitochondrial damage in isolated mouse hepatocytes. J Biol Chem 266:5049-5054 (1991)
  8. Brown, P. O. & Botstein, D. Exploring the new world of the genome with DNA microarrays. Nat Genet 21: 33-37 (1999) https://doi.org/10.1038/4462
  9. Young, R. R. Genetix toxicology: Web resources. Toxicology 173:103-121 (2002) https://doi.org/10.1016/S0300-483X(02)00026-4
  10. Kim, J. H. et al. Toxicogenomics study on TK6 human lymphoblast cells treated with mitomycin C. Mol Cell Toxicol 3:165-171 (2007)
  11. Kim, J. Y. et al. Identification of potential biomarkers of genotoxicity and carcinogenicity in L5178Y mouse lymphoma cells by cDNA microarray analysis. Environ Mol Mutagen 45:80-89 (2005) https://doi.org/10.1002/em.20077
  12. Lee, E. M. et al. Genetic toxicity test of o-Nitrotoluene by ames, micronucleus, comet assays and microarray analysis. Mol Cell Toxicol 3:107-112 (2007)
  13. Lee, M. et al. cDNA microarray gene expression profiling of hydroxyurea, paclitaxel, and p-anisidine, genotoxic compounds with differing tumorigenicity results. Environ Mol Mutagen 42:91-97 (2003) https://doi.org/10.1002/em.10177
  14. Lee, W. S. et al. Genetic toxicity test of 8-hydroxyquinoline by ames, micronucleus, comet assays and microarray analysis. Mol Cell Toxicol 3:90-97 (2007)
  15. Heinloth, A. N. et al. Gene expression profiling of rat livers reveals indicators of potential adverse effects. Toxicol Sci 80:193-202 (2004) https://doi.org/10.1093/toxsci/kfh145
  16. Reilly, T. P. et al. Expression profiling of acetaminophen liver toxicity in mice using microarray technology. Biochem Biophys Res Commun 282: 321-328 (2001) https://doi.org/10.1006/bbrc.2001.4576
  17. Suh, S. K. et al. Gene expression profiling of acetaminophen induced hepatotoxicity in mice. Mol Cell Toxicol 2:246-243 (2006)
  18. Arnaiz, S. L., Llesuy, S., Cutrin, J. C. & Boveris, A. Oxidative stress by acute acetaminophen administration in mouse liver. Free Radic Biol Med 19:303-310 (1995) https://doi.org/10.1016/0891-5849(95)00023-Q
  19. Reid, A. B., Kurten, R. C., McCullough, S. S., Brock, R. W. & Hinson, J. A. Mechanisms of acetaminopheninduced hepatotoxicity: role of oxidative stress and mitochondrial permeability transition in freshly isolated mouse hepatocytes. J Pharmacol Exp Ther 312: 509-516 (2005) https://doi.org/10.1124/jpet.104.075945
  20. Vasiliou, V., Buetler, T., Eaton, D. L. & Nebert, D.W. Comparison of oxidative stress response parameters in newborn mouse liver versus simian virus 40 (SV40)-transformed hepatocyte cell lines. Biochem Pharmacol 59:703-712 (2000) https://doi.org/10.1016/S0006-2952(99)00360-3
  21. Larson, A. M. et al. Acetaminophen-induced acute liver failure: results of a United States multicenter, prospective study. Hepatology 42:1364-1372 (2005) https://doi.org/10.1002/hep.20948
  22. Cohen, S. D. et al. Selective protein covalent binding and target organ toxicity. Toxicol Appl Pharmacol 143:1-12 (1997) https://doi.org/10.1006/taap.1996.8074
  23. Mirochnitchenko, O. et al. Acetaminophen Toxicity. Opposite effects of two forms of glutathione peroxidase. J Biol Chem 274:10349-10355 (1999)
  24. Canales, R. D. et al. Evaluation of DNA microarray results with quantitative gene expression platforms. Nat Biotechnol 24:1115-1122 (2006) https://doi.org/10.1038/nbt1236
  25. Rico-Bautista, E., lores-Morales, A. & Fernandez-Perez, L. Suppressor of cytokine signaling (SOCS) 2, a protein with multiple functions. Cytokine Growth Factor Rev 17:431-439 (2006) https://doi.org/10.1016/j.cytogfr.2006.09.008
  26. Tanaka, S. et al. Grb7 signal transduction protein mediates metastatic progression of esophageal carcinoma. J Cell Physiol 183:411-415 (2000) https://doi.org/10.1002/(SICI)1097-4652(200006)183:3<411::AID-JCP14>3.0.CO;2-Z
  27. Crabbe, J. C. et al. Elevated alcohol consumption in null mutant mice lacking 5-HT1B serotonin receptors. Nat Genet 14:98-101 (1996) https://doi.org/10.1038/ng0996-98
  28. Baurain, D., Dinant, M., Coosemans, N. & Matagne, R. F. Regulation of the alternative oxidase Aox1 gene in Chlamydomonas reinhardtii. Role of the nitrogen source on the expression of a reporter gene under the control of the Aox1 promoter. Plant Physiol 131:1418- 1430 (2003) https://doi.org/10.1104/pp.013409
  29. Villeneuve, D. J. et al. cDNA microarray analysis of isogenic paclitaxel- and doxorubicin-resistant breast tumor cell lines reveals distinct drug-specific genetic signatures of resistance. Breast Cancer Res Treat 96: 17-39 (2006) https://doi.org/10.1007/s10549-005-9026-6
  30. Liu, S. H. et al. Down-regulation of annexin A10 in hepatocellular carcinoma is associated with vascular invasion, early recurrence, and poor prognosis in synergy with p53 mutation. Am J Pathol 160:1831-1837 (2002) https://doi.org/10.1016/S0002-9440(10)61129-7
  31. Keyse, S. M. & Emslie, E. A. Oxidative stress and heat shock induce a human gene encoding a proteintyrosine phosphatase. Nature 359:644-647 (1992) https://doi.org/10.1038/359644a0
  32. Zimmers, T. A. Growth differentiation factor-15: induction in liver injury through p53 and tumor necrosis factor-independent mechanisms. J Surg Res 130: 45-51 (2006) https://doi.org/10.1016/j.jss.2005.07.036
  33. Koizumi, H., Kartasova, T., Tanaka, H., Ohkawara, A. & Kuroki, T. Differentiation-associated localization of small proline-rich protein in normal and diseased human skin. Br J Dermatol 134:686-692 (1996) https://doi.org/10.1111/j.1365-2133.1996.tb06971.x
  34. Belperio, J. A. et al. The role of the Th2 CC chemokine ligand CCL17 in pulmonary fibrosis. J Immunol 173:4692-4698 (2004) https://doi.org/10.4049/jimmunol.173.7.4692
  35. Turnley, A. M. Role of SOCS2 in growth hormone actions. Trends Endocrinol Metab 16:53-58 (2005) https://doi.org/10.1016/j.tem.2005.01.006
  36. Hirabayashi, S. et al. JAM4, a junctional cell adhesion molecule interacting with a tight junction protein, MAGI-1. Mol Cell Biol 23:4267-4282 (2003) https://doi.org/10.1128/MCB.23.12.4267-4282.2003
  37. Fielden, M. R. & Zacharewski, T. R. Challenges and limitations of gene expression profiling in mechanistic and predictive toxicology. Toxicol Sci 60:6-10 (2001) https://doi.org/10.1093/toxsci/60.1.6
  38. Cabibbo, A., Consalez, G. G., Sardella, M., Sitia, R. & Rubartelli, A. Changes in gene expression during growth arrest of hepG2 hepatoma cells induced by reducing agents or TGBb1. Oncogene 16:2935-2943 (1998) https://doi.org/10.1038/sj.onc.1201825
  39. Kim, B. H. et al. Expression profiling of actaminophen liver toxicity in mice using microarray technology. International symposium of the Korean society of toxicogenomics and toxicoproteomics. P-008, 111 (2004)