Abstract
The US Environmental Protection Agency (EPA) launched the Transform Tox Testing Challenge in 2016 with the goal of developing practical methods that can be integrated into conventional high-throughput screening (HTS) assays to better predict the toxicity of parent compounds and their metabolites in vivo. In response to this need and to retrofit existing HTS assays for assessing metabolism-induced toxicity of compounds, we have developed a 384-pillar plate that is complementary to traditional 384-well plates and ideally suited for culturing human cells in three dimensions at a microscale. Briefly, human embryonic kidney (HEK) 293 cells in a mixture of alginate and Matrigel were printed on the 384-pillar plates using a microarray spotter, which were coupled with 384-well plates containing nine model compounds provided by the EPA, five representative Phase I and II drug metabolizing enzymes (DMEs), and one no enzyme control. Viability and membrane integrity of HEK 293 cells were measured with the calcein AM and CellTiter-Glo® kit to determine the IC50 values of the nine parent compounds and DME-generated metabolites. The Z′ factors and the coefficient of variation measured were above 0.6 and below 14%, respectively, indicating that the assays established on the 384-pillar plate are robust and reproducible. Out of nine compounds tested, six compounds showed augmented toxicity with DMEs and one compound showed detoxification with a Phase II DME. This result indicates that the 384-pillar plate platform can be used to measure metabolism-induced toxicity of compounds in high-throughput with individual DMEs. As xenobiotics metabolism is a complex process with a variety of DMEs involved, the predictivity of our approach could be further improved with mixtures of DMEs.
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References
Asha S, Vidyavathi M (2010) Role of human liver microsomes in in vitro metabolism of drugs—a review. Appl Biochem Biotechnol 160(6):1699–1722. https://doi.org/10.1007/s12010-009-8689-6
Cho TM, Rose RL, Hodgson E (2006) In vitro metabolism of naphthalene by human liver microsomal cytochrome P450 enzymes. Drug Metab Dispos 34(1):176–183. https://doi.org/10.1124/dmd.105.005785
Cook D, Brown D, Alexander R, March R, Morgan P, Satterthwalite G, Pangalos MN (2014) Lessons learned from the fate of AstraZeneca’s drug pipeline: a five-dimensional framework. Nat Rev Drug Discov 13(6):419–431. https://doi.org/10.1038/nrd4309
Deeni YY, Ibbotson SH, Woods JA, Wolf CR, Smith G (2013) Cytochrome P450 CYP1B1 interacts with 8-methoxypsoralen (8-MOP) and influences psoralen-ultraviolet A (PUVA) sensitivity. PLoS One. 8(9):e75494. https://doi.org/10.1371/journal.pone.0075494
Duan X, Shen G, Yang H, Lambert G, Wei F, Zhang JJ (2016) Measurement of human CYP1A2 induction by inhalation exposure to benzo(a)pyrene based on in vivo isotope breath method. Environ Pollut 208(Pt B):506–511. https://doi.org/10.1016/j.envpol.2015.10.023
Ekhart C, Doodeman VD, Rodenhuis S, Smits PH, Beijnen JH, Huitema AD (2008) Influence of polymorphisms of drug metabolizing enzymes (CYP2B6, CYP2C9, CYP2C19, CYP3A4, CYP3A5, GSTA1, GSTP1, ALDH1A1 and ALDH3A1) on the pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide. Pharmacogenet Genom 18(6):515–523. https://doi.org/10.1097/FPC.0b013e3282fc9766
Gautier JC, Lecoeur S, Cosme J, Perret A, Urban P, Beaune P, Pompon D (1996) Contribution of human cytochrome P450 to benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol metabolism, as predicted from heterologous expression in yeast. Pharmacogenetics 6(6):489–499
Genter MB, Marlowe J, Kevin Kerzee J, Dragin N, Puga A, Dalton TP, Nebert DW (2006) Naphthalene toxicity in mice and aryl hydrocarbon receptor-mediated CYPs. Biochem Biophys Res Commun 348(1):120–123. https://doi.org/10.1016/j.bbrc.2006.07.025
Gundert-Remy U, Bernauer U, Blömeke B, Döring B, Fabian E, Goebel C, Hessel S, Jäckh C, Lampen A, Oesch F, Petzinger E, Völkel W, Roos PH (2014) Extrahepatic metabolism at the body’s internal-external interfaces. Drug Metab Rev 46(3):291–324. https://doi.org/10.3109/03602532.2014.900565
Gupta RC (ed) (2012) Veterinary toxicology: basic and clinical principles. Academic Press, Amsterdam
Joshi P, Datar A, Yu KN, Kang SY, Lee MY (2018) High-content imaging assays on a miniaturized 3D cell culture platform. Toxicol In Vitro 50:147–159. https://doi.org/10.1016/j.tiv.2018.02.014
Karmaus AL, Filer DL, Martin MT, Houck KA (2016) Evaluation of food-relevant chemicals in the ToxCast high-throughput screening program. Food Chem Toxicol 92:188–196. https://doi.org/10.1016/j.fct.2016.04.012
Kienzler A, Halder M, Worth A (2017) Waiving chronic fish tests: possible use of acute-to-chronic relationships and interspecies correlations. Toxicol Environ Chem 99(7):1129–1151. https://doi.org/10.1080/02772248.2016.1246663
Kwon SJ, Lee DW, Shah DA, Ku B, Jeon SY, Solanki K, Ryan JD, Clark DS, Dordick JS, Lee MY (2014) High-throughput and combinatorial gene expression on a chip for metabolism-induced toxicology screening. Nat Commun 5:3739. https://doi.org/10.1038/ncomms4739
Langouët S, Coles B, Morel F, Becquemont L, Beaune P, Guengerich FP, Ketterer B, Guillouzo A (1995) Inhibition of CYP1A2 and CYP3A4 by oltipraz results in reduction of aflatoxin B1 metabolism in human hepatocytes in primary culture. Cancer Res 1 55(23):5574–5579
Lee MY (ed) (2017) Microarray bioprinting technology: fundamentals and practices. Springer, Berlin
Lee MY, Dordick JS (2006) High-throughput human metabolism and toxicity analysis. Curr Opin Biotechnol 17(6):619–627. https://doi.org/10.1016/j.copbio.2006.09.003
Lee MY, Park CB, Dordick JS, Clark DS (2005) Metabolizing enzyme toxicology assay chip (MetaChip) for high-throughput microscale toxicity analyses. Proc Natl Acad Sci 102(4):983–987. https://doi.org/10.1073/pnas.0406755102
Lee MY, Kumar RA, Sukumaran SM, Hogg MG, Clark DS, Dordick JS (2008) Three-dimensional cellular microarray for high-throughput toxicology assays. Proc Natl Acad Sci 105(1):59–63. https://doi.org/10.1073/pnas.0708756105
Lee MY, Dordick JS, Clark DS (2010) Metabolic enzyme microarray coupled with miniaturized cell-culture array technology for high-throughput toxicity screening. Methods Mol Biol 632:2212–2237. https://doi.org/10.1007/978-1-60761-663-4_14
Lee DW, Lee MY, Ku B, Nam DH (2015) Automatic 3D cell analysis in high-throughput microarray using micropillar and microwell chips. J Biomol Screen 20(9):1178–1184. https://doi.org/10.1177/1087057115597635
Liebler DC, Guengerich FP (2005) Elucidating mechanisms of drug-induced toxicity. Nat Rev Drug Discov 4(5):410–420. https://doi.org/10.1038/nrd1720
Luckert C, Ehlers A, Buhrke T, Seidel A, Lampen A, Hessel S (2013) Polycyclic aromatic hydrocarbons stimulate human CYP3A4 promoter activity via PXR. Toxicol Lett 222(2):180–188. https://doi.org/10.1016/j.toxlet.2013.06.243
Marks BD, Thompson DV, Goossens TA, Trubetskoy OV (2004) High-throughput screening assays for the assessment of CYP2C9*1, CYP2C9*2, and CYP2C9*3 metabolism using fluorogenic Vivid substrates. J Biomol Screen 9(5):439–449
May JE, Xu J, Morse HR, Avent ND, Donaldson C (2009) Toxicity testing: the search for an in vitro alternative to animal testing. Br J Biomed Sci 66(3):160–165
Mironov SL, Ivannikov MV, Johansson M (2005) [Ca2+]i signaling between mitochondria and endoplasmic reticulum in neurons is regulated by microtubules. From mitochondrial permeability transition pore to Ca2+-induced Ca2+ release. J Biol Chem 280(1):715–721. https://doi.org/10.1074/jbc.M409819200
Monamy V (2017) Animal experimentation: a guide to the issues. Cambridge University Press, Cambridge
Raccor BS, Claessens AJ, Dinh JC, Park JR, Hawkins DS, Thomas SS, Makar KW, McCune JS, Totah RA (2012) Potential contribution of cytochrome P450 2B6 to hepatic 4-hydroxycyclophosphamide formation in vitro and in vivo. Drug Metab Dispos 40(1):54–63. https://doi.org/10.1124/dmd.111.039347
Rendic S, Guengerich FP (2012) Contributions of human enzymes in carcinogen metabolism. Chem Res Toxicol 16(7):1316–1383. https://doi.org/10.1021/tx300132k
Richard AM, Judson RS, Houck KA, Grulke CM, Volarath P, Thillainadarajah I, Yang C, Rathman J, Martin MT, Wambaugh JF, Knudsen TB, Kancherla J, Mansouri K, Patlewicz G, Williams AJ, Little SB, Crofton KM, Thomas RS (2016) ToxCast chemical landscape: paving the road to 21st century toxicology. Chem Res Toxicol 29(8):1225–1251. https://doi.org/10.1021/acs.chemrestox.6b00135
Riss TL, Moravec RA, Niles AL, Duellman S, Benink HA, Worzella TJ, Minor L (2013) Cell viability assays [Updated 2016 Jul 1]. In: Sittampalam GS,Coussens NP, Brimacombe K et al (eds) Assay Guidance Manual [Internet]. Eli Lilly and Company and the National Center for Advancing Translational Sciences, Bethesda, MD. Available from: https://www.ncbi.nlm.nih.gov/books/NBK144065/
Roth AD, Lee MY (2017) Idiosyncratic drug-induced liver injury (IDILI): potential mechanisms and predictive assays. Biomed Res Int 2017:9176937. https://doi.org/10.1155/2017/9176937
Settels E, Bernauer U, Palavinskas R, Klaffke HS, Gundert-Remy U, Appel KE (2008) Human CYP2E1 mediates the formation of glycidamide from acrylamide. Arch Toxicol 82:717–727
Sui Y, Wu Z (2007) Alternative statistical parameter for high-throughput screening assay quality assessment. J Biomol Screen 12(2):229–234. https://doi.org/10.1177/1087057106296498
Tang C, Lin JH, Lu AYH (2005) Metabolism-based drug–drug interactions: what determines individual variability in cytochrome P450 induction? Drug Metab Dispos 33(5):603–613. https://doi.org/10.1124/dmd.104.003236
Trubetskov OV, Gibson JR, Marks BD (2005) Highly miniaturized formats for in vitro drug metabolism assays using vivid fluorescent substrates and recombinant human cytochrome P450 enzymes. J Biomol Screen 10(1):56–66
Tukey RH, Strassburg CP (2000) Human UDP-glucuronosyltransferases: metabolism, expression, and disease. Annu Rev Pharmacol Toxicol 40:581–616. https://doi.org/10.1146/annurev.pharmtox.40.1.581
Vijayakumar TM, Kumar RM, Agrawal A, Dubey GP, Ilango K (2015) Comparative inhibitory potential of selected dietary bioactive polyphenols, phytosterols on CYP3A4 and CYP2D6 with fluorometric high-throughput screening. J Food Sci Technol 52(7):4537–4543. https://doi.org/10.1007/s13197-014-1472-x
Wilson AS, Davis CD, Williams DP, Buckpitt AR, Pirmohamed M, Park BK (1996) Characterisation of the toxic metabolite(s) of naphthalene. Toxicology 114(3):233–242. https://doi.org/10.1016/S0300-483X(96)03515-9
Yang L, Yan C, Zhang F, Jiang B, Gao S, Liang Y, Huang L, Chen W (2018) Effects of ketoconazole on cyclophosphamide metabolism: evaluation of CYP3A4 inhibition effect using the in vitro and in vivo models. Exp Anim 67(1):71–82. https://doi.org/10.1538/expanim.17-0048
Yu KN, Nadanaciva S, Rana P, Lee DW, Ku B, Roth AD, Dordick JS, Will Y, Lee MY (2017) Prediction of metabolism-induced hepatotoxicity on three-dimensional hepatic cell culture and enzyme microarrays. Arch Toxicol 92(3):1295–1310. https://doi.org/10.1007/s00204-017-2126-3
Zamek-Gliszczynski MJ, Hoffmaster KA, Nezasa K-I, Tallman MN, Brouwer KLR (2006) Integration of hepatic drug transporters and phase II metabolizing enzymes: mechanisms of hepatic excretion of sulfate, glucuronide, and glutathione metabolites. Eur J Pharm Sci 27(5):447–486. https://doi.org/10.1016/j.ejps.2005.12.007
Zerilli A, Ratanasavanh D, Lucas D, Goasduff T, Dréano Y, Menard C, Picart D, Berthou F (1997) Both cytochromes P450 2E1 and 3A are involved in the O-hydroxylation of p-nitrophenol, a catalytic activity known to be specific for P450 2E1. Chem Res Toxicol 10(10):1205–1212. https://doi.org/10.1021/tx970048z
Acknowledgements
This study was partially supported by the US Environmental Protection Agency (US EPA Transform Tox Testing Challenge), Medical & Bio Device (MBD) Korea, the Cleveland State University (Faculty Innovation Fund), and the National Institutes of Health (NIEHS R01ES025779). We also acknowledge Dr. Kevin Kuhn at the US EPA and Mr. Rayton Gerald at 3D MicroArray, Inc. for their guidance and help during the Transform Tox Testing Challenge.
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The authors declare a potential conflict of interest as the 384-pillar plates manufactured by their industrial partner, Medical and Bio Device (MBD) Korea, have been used in this study.
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Yu, KN., Kang, SY., Hong, S. et al. High-throughput metabolism-induced toxicity assays demonstrated on a 384-pillar plate. Arch Toxicol 92, 2501–2516 (2018). https://doi.org/10.1007/s00204-018-2249-1
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DOI: https://doi.org/10.1007/s00204-018-2249-1