High-Throughput and Combinatorial Gene Expression on a Chip for Metabolism-Induced Toxicology Screening

Differential expression of various drug-metabolizing enzymes in the human liver may cause deviations of pharmacokinetic profiles, resulting in inter-individual variability of drug toxicity and/or efficacy. Here we present the “Transfected Enzyme and Metabolism Chip” (TeamChip), which predicts potential metabolism-induced drug or drug-candidate toxicity. The TeamChip is prepared by delivering genes into miniaturized three-dimensional cellular microarrays on a micropillar chip using recombinant adenoviruses in a complementary microwell chip. The device enables users to manipulate the expression of individual and multiple human metabolizing-enzyme genes (such as CYP3A4, CYP2D6, CYP2C9, CYP1A2, CYP2E1, and UGT1A4) in THLE-2 cell microarrays. To identify specific enzymes involved in drug detoxification, we created 84 combinations of metabolic-gene expressions in a combinatorial fashion on a single microarray. Thus, the TeamChip platform can provide critical information necessary for evaluating metabolism-induced toxicity in a high-throughput manner.

[1]  Elmar Heinzle,et al.  3D organotypic cultures of human HepaRG cells: a tool for in vitro toxicity studies. , 2013, Toxicological sciences : an official journal of the Society of Toxicology.

[2]  S. Hansen,et al.  Development of an in vitro assay for the investigation of metabolism-induced drug hepatotoxicity , 2007, Cell Biology and Toxicology.

[3]  C. Gorman,et al.  Site-specific gene targeting for gene expression in eukaryotes. , 2000, Current opinion in biotechnology.

[4]  U. Breyer,et al.  Metabolism of trifluoperazine, fluphenazine, prochlorperazine and perphenazine in rats: in vitro and urinary metabolites. , 1974, Biochemical pharmacology.

[5]  F. Noor,et al.  3D organotypic HepaRG cultures as in vitro model for acute and repeated dose toxicity studies. , 2014, Toxicology in vitro : an international journal published in association with BIBRA.

[6]  T. Fehm,et al.  Activity Levels of Tamoxifen Metabolites at the Estrogen Receptor and the Impact of Genetic Polymorphisms of Phase I and II Enzymes on Their Concentration Levels in Plasma , 2011, Clinical pharmacology and therapeutics.

[7]  E. Bjornsson,et al.  Oral medications with significant hepatic metabolism at higher risk for hepatic adverse events , 2010, Hepatology.

[8]  A. Pfeifer,et al.  Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Tina Hernandez-Boussard,et al.  Doxorubicin pathways: pharmacodynamics and adverse effects , 2011, Pharmacogenetics and genomics.

[10]  Anne E Carpenter,et al.  Microarrays of lentiviruses for gene function screens in immortalized and primary cells , 2006, Nature Methods.

[11]  M. Eichelbaum,et al.  The influence of CYP2B6, CYP2C9 and CYP2D6 genotypes on the formation of the potent antioestrogen Z-4-hydroxy-tamoxifen in human liver. , 2002, British journal of clinical pharmacology.

[12]  Leming Shi,et al.  Similarities and Differences in the Expression of Drug-Metabolizing Enzymes between Human Hepatic Cell Lines and Primary Human Hepatocytes , 2011, Drug Metabolism And Disposition.

[13]  J. Doehmer,et al.  Heterologous Co-Expression of Human Cytochrome P450 1A2 and Polymorphic Forms of N-Acetyltransferase 2 for Studies on Aromatic Amines in V79 Chinese Hamster Cells , 2005, Alternatives to laboratory animals : ATLA.

[14]  J. G. Kenna,et al.  Cell based approaches for evaluation of drug-induced liver injury. , 2010, Toxicology.

[15]  K. Tilmant,et al.  Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins , 2012, Cell Biology and Toxicology.

[16]  B. Andrews,et al.  New Technologies and Screening Strategies for Hepatotoxicity: Use of In Vitro Models , 2005, Toxicologic pathology.

[17]  Jun-chao Wu,et al.  High efficiency adenovirus-mediated expression of truncated N-terminal huntingtin fragment (htt552) in primary rat astrocytes. , 2009, Acta biochimica et biophysica Sinica.

[18]  D. Sabatini,et al.  Microarrays of cells expressing defined cDNAs , 2001, Nature.

[19]  Feng Li,et al.  Supplementary Table 7. , 2014 .

[20]  E. Coccia,et al.  RNAseq Analyses Identify Tumor Necrosis Factor-Mediated Inflammation as a Major Abnormality in ALS Spinal Cord , 2009, Nature Cell Biology.

[21]  Marie M. Ahlström,et al.  Characterization of THLE-Cytochrome P450 (P450) Cell Lines: Gene Expression Background and Relationship to P450-Enzyme Activity , 2012, Drug Metabolism and Disposition.

[22]  Lee Hoong-Chien,et al.  Supplementary Table 4 , 2015 .

[23]  H. Berman,et al.  Metabolic engineering with recombinant adenoviruses. , 1999, Annual review of nutrition.

[24]  W. Trager,et al.  Oxidation of acetaminophen to its toxic quinone imine and nontoxic catechol metabolites by baculovirus-expressed and purified human cytochromes P450 2E1 and 2A6. , 1998, Chemical research in toxicology.

[25]  Varpu Marjomäki,et al.  Supplementary Table 6 , 2012 .

[26]  J. Dordick,et al.  High-throughput human metabolism and toxicity analysis. , 2006, Current opinion in biotechnology.

[27]  Mario Schelhaas,et al.  Supplementary Table 5 , 2012 .

[28]  C. W. Fisher,et al.  Metabolism of the antiandrogenic drug (Flutamide) by human CYP1A2. , 1997, Drug metabolism and disposition: the biological fate of chemicals.

[29]  Liang-Shang Gan,et al.  Effects of prototypical microsomal enzyme inducers on cytochrome P450 expression in cultured human hepatocytes. , 2003, Drug metabolism and disposition: the biological fate of chemicals.

[30]  Martyn T. Smith,et al.  Mechanisms of troglitazone hepatotoxicity. , 2003, Chemical research in toxicology.

[31]  Y. Masubuchi,et al.  Mechanism-based inactivation of CYP2C11 by diclofenac. , 2001, Drug metabolism and disposition: the biological fate of chemicals.

[32]  S. Auriola,et al.  Acetaminophen bioactivation by human cytochrome P450 enzymes and animal microsomes. , 2009, Xenobiotica; the fate of foreign compounds in biological systems.

[33]  E. Whitelaw,et al.  Epigenetic effects on transgene expression. , 2001, Methods in molecular biology.

[34]  D. Grant,et al.  Biotransformation of caffeine, paraxanthine, theophylline, and theobromine by polycyclic aromatic hydrocarbon-inducible cytochrome(s) P-450 in human liver microsomes. , 1987, Drug metabolism and disposition: the biological fate of chemicals.

[35]  Yusuke Nakamura,et al.  Pharmacogenomics of tamoxifen: roles of drug metabolizing enzymes and transporters. , 2012, Drug metabolism and pharmacokinetics.

[36]  E. Hazai,et al.  Reduction of toxic metabolite formation of acetaminophen. , 2002, Biochemical and biophysical research communications.

[37]  J. Nieman,et al.  Bioactivation of Flutamide Metabolites by Human Liver Microsomes , 2008, Drug Metabolism and Disposition.

[38]  Jos H Beijnen,et al.  An update on in vitro test methods in human hepatic drug biotransformation research: pros and cons. , 2003, Toxicology and applied pharmacology.

[39]  Y. Masubuchi,et al.  Diclofenac-induced inactivation of CYP3A4 and its stimulation by quinidine. , 2002, Drug metabolism and disposition: the biological fate of chemicals.

[40]  HighWire Press,et al.  Drug metabolism and disposition : the biological fate of chemicals. , 1973 .

[41]  R. Roeder,et al.  Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. , 1983, Nucleic acids research.

[42]  Y. Masubuchi,et al.  MECHANISM-BASED INACTIVATION OF CYP 2 C 11 BY DICLOFENAC , 2001 .

[43]  M. T. Donato,et al.  HepG2 cells simultaneously expressing five P450 enzymes for the screening of hepatotoxicity: identification of bioactivable drugs and the potential mechanism of toxicity involved , 2013, Archives of Toxicology.

[44]  J. Hayes,et al.  The hepatotoxic metabolite of acetaminophen directly activates the Keap1‐Nrf2 cell defense system , 2008, Hepatology.

[45]  F. Guengerich,et al.  Cytochrome P450s and other enzymes in drug metabolism and toxicity , 2006, The AAPS Journal.

[46]  A. Rettie,et al.  Involvement of human cytochrome P450 2D6 in the bioactivation of acetaminophen. , 2000, Drug metabolism and disposition: the biological fate of chemicals.

[47]  Laura P James,et al.  Acetaminophen-induced hepatotoxicity. , 2003, Drug metabolism and disposition: the biological fate of chemicals.

[48]  Ian D. Wilson,et al.  Managing the challenge of chemically reactive metabolites in drug development , 2011, Nature Reviews Drug Discovery.

[49]  R. Dellinger,et al.  Characterization of tamoxifen and 4-hydroxytamoxifen glucuronidation by human UGT1A4 variants , 2006, Breast Cancer Research.

[50]  J. Castell,et al.  An update on metabolism studies using human hepatocytes in primary culture , 2008 .

[51]  S. Strom,et al.  Transport, metabolism, and hepatotoxicity of flutamide, drug-drug interaction with acetaminophen involving phase I and phase II metabolites. , 2007, Chemical research in toxicology.

[52]  Kei-ichi Minami,et al.  An in vitro drug-induced hepatotoxicity screening system using CYP3A4-expressing and gamma-glutamylcysteine synthetase knockdown cells. , 2010, Toxicology in vitro : an international journal published in association with BIBRA.

[53]  J V Castell,et al.  Cell lines: a tool for in vitro drug metabolism studies. , 2008, Current drug metabolism.

[54]  Michael B H Smith.  Pros and cons …. , 2014, Evidence-based child health : a Cochrane review journal.

[55]  Munir Pirmohamed,et al.  The role of metabolic activation in drug-induced hepatotoxicity. , 2005, Annual review of pharmacology and toxicology.

[56]  J. Schlager,et al.  In vitro cytotoxicity of nanoparticles in mammalian germline stem cells. , 2005, Toxicological sciences : an official journal of the Society of Toxicology.

[57]  Hartmut Jaeschke,et al.  HepaRG cells: A human model to study mechanisms of acetaminophen hepatotoxicity , 2011, Hepatology.

[58]  Y. L. Chen,et al.  Supplementary Figure 8 , 2012 .

[59]  Moo-Yeal Lee,et al.  On-chip, cell-based microarray immunofluorescence assay for high-throughput analysis of target proteins. , 2008, Analytical chemistry.

[60]  O. Pelkonen,et al.  The effect of cigarette smoking on 7-ethoxyresorufin O-deethylase and other monooxygenase activities in human liver: analyses with monoclonal antibodies. , 1986, British journal of clinical pharmacology.

[61]  D W Nebert,et al.  Pharmacogenomics: out of the lab and into the community. , 2001, Trends in Biotechnology.

[62]  M. T. Donato,et al.  Human hepatocytes in primary culture: the choice to investigate drug metabolism in man. , 2004, Current drug metabolism.