Abstract
Acetaminophen (APAP) overdose is the leading cause of acute liver failure. Yet the mechanisms underlying adaptive tolerance toward APAP-induced liver injury are not fully understood. To better understand molecular mechanisms contributing to adaptive tolerance to APAP is an underpinning foundation for APAP-related precision medicine. In the current study, the mRNA and microRNA (miRNA) expression profiles derived from next generation sequencing data for APAP-treated (5 and 10 mM) HepaRG cells and controls were analyzed systematically. Putative miRNAs targeting key dysregulated genes involved in APAP hepatotoxicity were selected using in silico prediction algorithms, un-biased gene ontology, and network analyses. Luciferase reporter assays, RNA electrophoresis mobility shift assays, and miRNA pull-down assays were performed to investigate the role of miRNAs affecting the expression of dysregulated genes. Levels of selected miRNAs were measured in serum samples obtained from children with APAP overdose (58.6–559.4 mg/kg) and from healthy controls. As results, 2758 differentially expressed genes and 47 miRNAs were identified. Four of these miRNAs (hsa-miR-224-5p, hsa-miR-320a, hsa-miR-449a, and hsa-miR-877-5p) suppressed drug metabolizing enzyme (DME) levels involved in APAP-induced liver injury by downregulating HNF1A, HNF4A and NR1I2 expression. Exogenous transfection of these miRNAs into HepaRG cells effectively rescued them from APAP toxicity, as indicated by decreased alanine aminotransferase levels. Importantly, hsa-miR-320a and hsa-miR-877-5p levels were significantly elevated in serum samples obtained from children with APAP overdose compared to health controls. Collectively, these data indicate that hsa-miR-224-5p, hsa-miR-320a, hsa-miR-449a, and hsa-miR-877-5p suppress DME expression involved in APAP-induced hepatotoxicity and they contribute to an adaptive response in hepatocytes.
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References
Adjei AA, Gaedigk A, Simon SD, Weinshilboum RM, Leeder JS (2008) Interindividual variability in acetaminophen sulfation by human fetal liver: implications for pharmacogenetic investigations of drug-induced birth defects. Birth Defects Res A Clin Mol Teratol 82(3):155–165. doi:10.1002/bdra.20535
Baraniskin A, Kuhnhenn J, Schlegel U et al (2012) Identification of microRNAs in the cerebrospinal fluid as biomarker for the diagnosis of glioma. Neuro Oncol 14(1):29–33. doi:10.1093/neuonc/nor169
Chen X, Ba Y, Ma L et al (2008) Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res 18(10):997–1006. doi:10.1038/cr.2008.282
Court MH, Duan SX, von Moltke LL et al (2001) Interindividual variability in acetaminophen glucuronidation by human liver microsomes: identification of relevant acetaminophen UDP-glucuronosyltransferase isoforms. J Pharmacol Exp Ther 299(3):998–1006
Davidson DG, Eastham WN (1966) Acute liver necrosis following overdose of paracetamol. Br Med J 2(5512):497–499
Eakins R, Walsh J, Randle L et al (2015) Adaptation to acetaminophen exposure elicits major changes in expression and distribution of the hepatic proteome. Sci Rep 5:16423. doi:10.1038/srep16423
Friedlander MR, Chen W, Adamidi C et al (2008) Discovering microRNAs from deep sequencing data using miRDeep. Nat Biotechnol 26(4):407–415. doi:10.1038/nbt1394
Jetten MJ, Ruiz-Aracama A, Coonen ML et al (2015) Interindividual variation in gene expression responses and metabolite formation in acetaminophen-exposed primary human hepatocytes. Arch Toxicol. doi:10.1007/s00204-015-1545-2
Jetten MJ, Ruiz-Aracama A, Coonen ML et al (2016) Interindividual variation in gene expression responses and metabolite formation in acetaminophen-exposed primary human hepatocytes. Arch Toxicol 90(5):1103–1115. doi:10.1007/s00204-015-1545-2
Jin Y, Yu D, Tolleson WH et al (2016) MicroRNA hsa-miR-25-3p suppresses the expression and drug induction of CYP2B6 in human hepatocytes. Biochem Pharmacol 113:88–96. doi:10.1016/j.bcp.2016.06.007
Kim SN, Seo JY, Jung DW, Lee MY, Jung YS, Kim YC (2007) Induction of hepatic CYP2E1 by a subtoxic dose of acetaminophen in rats: increase in dichloromethane metabolism and carboxyhemoglobin elevation. Drug Metab Dispos 35(10):1754–1758. doi:10.1124/dmd.107.015545
Koturbash I, Tolleson WH, Guo L et al (2015) microRNAs as pharmacogenomic biomarkers for drug efficacy and drug safety assessment. Biomark Med 9(11):1153–1176. doi:10.2217/bmm.15.89
Krauskopf J, Caiment F, Claessen SM et al (2015) Application of high-throughput sequencing to circulating microRNAs reveals novel biomarkers for drug-induced liver injury. Toxicol Sci 143(2):268–276. doi:10.1093/toxsci/kfu232
McGill MR, Yan HM, Ramachandran A, Murray GJ, Rollins DE, Jaeschke H (2011) HepaRG cells: a human model to study mechanisms of acetaminophen hepatotoxicity. Hepatology 53(3):974–982. doi:10.1002/hep.24132
McKillop IH, Schmidt CM, Cahill PA, Sitzmann JV (1999) Altered Gq/G11 guanine nucleotide regulatory protein expression in a rat model of hepatocellular carcinoma: role in mitogenesis. Hepatology 29(2):371–378. doi:10.1002/hep.510290201
Miyakawa K, Albee R, Letzig LG et al (2015) A cytochrome P450-independent mechanism of acetaminophen-induced injury in cultured mouse hepatocytes. J Pharmacol Exp Ther 354(2):230–237. doi:10.1124/jpet.115.223537
Mohri T, Nakajima M, Fukami T, Takamiya M, Aoki Y, Yokoi T (2010) Human CYP2E1 is regulated by miR-378. Biochem Pharmacol 79(7):1045–1052. doi:10.1016/j.bcp.2009.11.015
Morishita K, Mizukawa Y, Kasahara T et al (2006) Gene expression profile in liver of differing ages of rats after single oral administration of acetaminophen. J Toxicol Sci 31(5):491–507
O’Brien PJ, Slaughter MR, Swain A et al (2000) Repeated acetaminophen dosing in rats: adaptation of hepatic antioxidant system. Hum Exp Toxicol 19(5):277–283
Pan YZ, Gao W, Yu AM (2009) MicroRNAs regulate CYP3A4 expression via direct and indirect targeting. Drug Metab Dispos 37(10):2112–2117. doi:10.1124/dmd.109.027680
Rodrigues RM, Heymans A, De Boe V et al (2016) Toxicogenomics-based prediction of acetaminophen-induced liver injury using human hepatic cell systems. Toxicol Lett 240(1):50–59. doi:10.1016/j.toxlet.2015.10.014
Shayiq RM, Roberts DW, Rothstein K et al (1999) Repeat exposure to incremental doses of acetaminophen provides protection against acetaminophen-induced lethality in mice: an explanation for high acetaminophen dosage in humans without hepatic injury. Hepatology 29(2):451–463. doi:10.1002/hep.510290241
Smutny T, Mani S, Pavek P (2013) Post-translational and post-transcriptional modifications of pregnane X receptor (PXR) in regulation of the cytochrome P450 superfamily. Curr Drug Metab 14(10):1059–1069
Snawder JE, Roe AL, Benson RW, Roberts DW (1994) Loss of CYP2E1 and CYP1A2 activity as a function of acetaminophen dose: relation to toxicity. Biochem Biophys Res Commun 203(1):532–539. doi:10.1006/bbrc.1994.2215
Strubelt O, Siegers CP, Volpel M, Younes M (1979) Studies on the mechanism of paracetamol-induced protection against paracetamol hepatotoxicity. Toxicology 12(2):121–133
Subramanian M, Li XL, Hara T, Lal A (2015) A biochemical approach to identify direct microRNA targets. Methods Mol Biol 1206:29–37. doi:10.1007/978-1-4939-1369-5_3
Thorgeirsson SS, Sasame HA, Mitchell JR, Jollow DJ, Potter WZ (1976) Biochemical changes after hepatic injury from toxic doses of acetaminophen or furosemide. Pharmacology 14(3):205–217
Trapnell C, Roberts A, Goff L et al (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7(3):562–578. doi:10.1038/nprot.2012.016
Tsuchiya Y, Nakajima M, Takagi S, Taniya T, Yokoi T (2006) MicroRNA regulates the expression of human cytochrome P450 1B1. Cancer Res 66(18):9090–9098. doi:10.1158/0008-5472.CAN-06-1403
Vliegenthart AD, Shaffer JM, Clarke JI et al (2015) Comprehensive microRNA profiling in acetaminophen toxicity identifies novel circulating biomarkers for human liver and kidney injury. Sci Rep 5:15501. doi:10.1038/srep15501
Wang Z, Burke PA (2013) The role of microRNAs in hepatocyte nuclear factor-4alpha expression and transactivation. Biochem Biophys Acta 1829(5):436–442. doi:10.1016/j.bbagrm.2012.12.009
Wang K, Zhang S, Marzolf B et al (2009) Circulating microRNAs, potential biomarkers for drug-induced liver injury. Proc Natl Acad Sci USA 106(11):4402–4407. doi:10.1073/pnas.0813371106
Wang Y, Yu D, Tolleson WH et al (2017) A systematic evaluation of microRNAs in regulating human hepatic CYP2E1. Biochem Pharmacol. doi:10.1016/j.bcp.2017.04.020
Ward J, Kanchagar C, Veksler-Lublinsky I et al (2014) Circulating microRNA profiles in human patients with acetaminophen hepatotoxicity or ischemic hepatitis. Proc Natl Acad Sci USA 111(33):12169–12174. doi:10.1073/pnas.1412608111
Yang X, Greenhaw J, Shi Q et al (2012) Identification of urinary microRNA profiles in rats that may diagnose hepatotoxicity. Toxicol Sci 125(2):335–344. doi:10.1093/toxsci/kfr321
Yang X, Salminen WF, Shi Q et al (2015) Potential of extracellular microRNAs as biomarkers of acetaminophen toxicity in children. Toxicol Appl Pharmacol 284(2):180–187. doi:10.1016/j.taap.2015.02.013
Yu X, Dhakal IB, Beggs M et al (2010) Functional genetic variants in the 3′-untranslated region of sulfotransferase isoform 1A1 (SULT1A1) and their effect on enzymatic activity. Toxicol Sci 118(2):391–403. doi:10.1093/toxsci/kfq296
Yu D, Green B, Marrone A et al (2015a) Suppression of CYP2C9 by microRNA hsa-miR-128-3p in human liver cells and association with hepatocellular carcinoma. Sci Rep 5:8534. doi:10.1038/srep08534
Yu D, Green B, Tolleson W et al (2015b) MicroRNA hsa-miR-29a-3p modulates CYP2C19 production in human liver cells. Biochem Pharmacol. doi:10.1061/j.bcp.2015.08.094
Yu D, Tolleson WH, Knox B et al (2015c) Modulation of ALDH5A1 and SLC22A7 by microRNA hsa-miR-29a-3p in human liver cells. Biochem Pharmacol. doi:10.1016/j.bcp.2015.09.020
Zeng L, Chen Y, Wang Y et al (2017) MicroRNA hsa-miR-370-3p suppresses the expression and induction of CYP2D6 by facilitating mRNA degradation. Biochem Pharmacol. doi:10.1016/j.bcp.2017.05.018
Zhang QX, Melnikov Z, Feierman DE (2004) Characterization of the acetaminophen-induced degradation of cytochrome P450-3A4 and the proteolytic pathway. Basic Clin Pharmacol Toxicol 94(4):191–200. doi:10.1111/j.1742-7843.2004.pto940406.x
Zhao L, Pickering G (2011) Paracetamol metabolism and related genetic differences. Drug Metab Rev 43(1):41–52. doi:10.3109/03602532.2010.527984
Acknowledgements
This work was supported by the US FDA, protocol # E0731311. B. N., W. T., R. D. B. and L. J. proposed and organized the study; D. Y., P. G. and B. N. designed the study; D. Y., S. C., Y. J. and J. S. conducted the total RNA extraction; L. W., W. X., H. H., L. S., S. B., S. M., H. F. and T. S. analyzed the gene and miRNA expression data; D. Y., Z. R., Y. W., Y. C., and L. Z. conducted the Reporter assays and RNA EMSA assays; D. Y., V. T., I. P., S. M. and S. B. conducted miRNA pull-down experiment; B. K., J. S. and L. S. provided the technical support; X. Y., K. D., Y. G, L. J. and L. S analyzed the miRNA levels in serum samples. D. Y. and B. N. wrote the manuscript; W. H. T, W. X., L. G., Y. G., W. M., L. J., P. G., S. B., S. M., N. M., I. P. and W. T. contributed to data interpretation and revised the manuscript. All authors reviewed the manuscript.
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Yu, D., Wu, L., Gill, P. et al. Multiple microRNAs function as self-protective modules in acetaminophen-induced hepatotoxicity in humans. Arch Toxicol 92, 845–858 (2018). https://doi.org/10.1007/s00204-017-2090-y
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DOI: https://doi.org/10.1007/s00204-017-2090-y