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Xenobiotica-metabolizing enzyme induction potential of chemicals in animal studies: NanoString nCounter gene expression and peptide group-specific immunoaffinity as accelerated and economical substitutions for enzyme activity determinations?

  • Toxicokinetics and Metabolism
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Abstract

Xenobiotica-metabolizing enzyme (XME) induction is a relevant biological/biochemical process vital to understanding the toxicological profile of xenobiotics. Early recognition of XME induction potential of compounds under development is therefore important, yet its determination by traditional XME activity measurements is time consuming and cost intensive. A proof-of-principle study was therefore designed due to the advent of faster and less cost-intensive methods for determination of enzyme protein and transcript levels to determine whether two such methods may substitute for traditional measurement of XME activity determinations. The results of the study show that determination of enzyme protein levels by peptide group-specific immunoaffinity enrichment/MS and/or determination of gene expression by NanoString nCounter may serve as substitutes for traditional evaluation methodology and/or as an early predictor of potential changes in liver enzymes. In this study, changes of XME activity by the known standard XME inducers phenobarbital, beta-naphthoflavone and Aroclor 1254 were demonstrated by these two methods. To investigate the applicability of these methods to demonstrate XME-inducing activity of an unknown, TS was also examined and found to be an XME inducer. More specifically, TS was found to be a phenobarbital-type inducer (likely mediated by CAR rather than PXR as nuclear receptor), but not due to Ah receptor-mediated or antioxidant response element-mediated beta-naphthoflavone-type induction. The results for TS were confirmed via enzymatic activity measurements. The results of the present study demonstrate the potential applicability of NanoString nCounter mRNA quantitation and peptide group-specific immunoaffinity enrichment/MS protein quantitation for predicting compounds under development to be inducers of liver XME activity.

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References

  • Amacher DE, Schomaker SJ, Burkhardt JE (1998) The relationship among microsomal enzyme induction, liver weight and histological change in rat toxicology studies. Food Chem Toxicol 36:831–839

    CAS  PubMed  Google Scholar 

  • Barter RA, Klaassen CD (1994) Reduction of thyroid hormone levels and alteration of thyroid function by four representative UDP-glucuronosyltransferase inducers in rats. Toxicol Appl Pharmacol 128(1):9–17

    CAS  PubMed  Google Scholar 

  • Bock KW, Bock-Hennig BS (2010) UDP-glucuronosyltransferases (UGTs): from purification of Ah-receptor-inducible UGT1A6 to coordinate regulation of subsets of CYPs, UGTs, and ABC transporters by nuclear receptors. Drug Metab Rev 42(1):6–13. https://doi.org/10.3109/03602530903205492

    Article  CAS  PubMed  Google Scholar 

  • Bock KW, Josting D, Lilienblum W, Pfeil H (1979) Purification of rat-liver microsomal UDP-glucuronyltransferase. Separation of two enzyme forms inducible by 3-methylcholanthrene or phenobarbital. Eur J Biochem 98(1):19–26

    CAS  PubMed  Google Scholar 

  • Bock KW, Gschaidmeier H, Heel H, Lehmköster T, Münzel PA, Raschko F, Bock-Hennig B (1998) AH receptor-controlled transcriptional regulation and function of rat and human UDP-glucuronosyltransferase isoforms. Adv Enzyme Regul 38:207–222

    CAS  PubMed  Google Scholar 

  • Bohonowych JE, Denison MS (2007) Persistent binding of ligands to the aryl hydrocarbon receptor. Toxicol Sci 98(1):99–109. https://doi.org/10.1093/toxsci/kfm085(Published online 2007 Apr 12)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Brodie MJ, Mintzer S, Pack AM, Gidal BE, Vecht CJ, Schmidt D (2013) Enzyme induction with antiepileptic drugs: cause for concern? Epilepsia 54(1):11–27. https://doi.org/10.1111/j.1528-1167.2012.03671.x

    Article  CAS  PubMed  Google Scholar 

  • Burke MD, Thompson S, Weaver RJ, Wolf CR, Mayer RT (1994) Cytochrome P450 specificities of alkoxyresorufin O-dealkylation in human and rat liver. Biochem Pharmacol 48(5):923–936

    CAS  PubMed  Google Scholar 

  • Cooney GF, Mochon M, Kaiser B, Dunn SP, Goldsmith B (1995) Effects of carbamazepine on cyclosporine metabolism in pediatric renal transplant recipients. Pharmacotherapy 15:353–356

    CAS  PubMed  Google Scholar 

  • Corcos L, Lagadic-Gossmann D (2001) Gene induction by phenobarbital: an update on an old question that receives key novel answers. Pharmacol Toxicol 89(3):113–122

    CAS  PubMed  Google Scholar 

  • Correia MA (1995) In de Montellano PRO (eds) Cytochrome P450: structure, mechanism, and biochemistry, 2nd edn, Plenum Press, New York, pp 607–630

  • Crofton KM (2008) Thyroid disrupting chemicals: mechanisms and mixtures. Int J Androl 31:209–223

    CAS  PubMed  Google Scholar 

  • Dhakshinamoorthy S, Long DJ 2nd, Jaiswal AK (2000) Antioxidant regulation of genes encoding enzymes that detoxify xenobiotics and carcinogens. Curr Top Cell Regul 36:201–216

    CAS  PubMed  Google Scholar 

  • Dunn TJ, Lindahl R, Pitot HC (1988) Differential gene expression in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Noncoordinate regulation of a TCDD-induced aldehyde dehydrogenase and cytochrome P-450c in the rat. J Biol Chem 263(22):10878–10886

    CAS  PubMed  Google Scholar 

  • Ejiri N, Katayama K, Kiyosawa N, Baba Y, Doi K (2005a) Microarray analysis on CYPs expression in pregnant rats after treatment with pregnenolone-16alpha-carbonitrile and phenobarbital. Exp Mol Pathol 78(1):71–77

    CAS  PubMed  Google Scholar 

  • Ejiri N, Katayama K, Kiyosawa N, Baba Y, Doi K (2005b) Microarray analysis on Phase II drug metabolizing enzymes expression in pregnant rats after treatment with pregnenolone-16alpha-carbonitrile or phenobarbital. Exp Mol Pathol 79(3):272–277

    CAS  PubMed  Google Scholar 

  • Elovaara E, Stockmann-Juvala H, Mikkola J, Gelboin HV (2007) Interactive effects of methyl tertiary-butyl ether (MTBE) and tertiary-amyl methyl ether (TAME), ethanol and some drugs: Triglyceridemia, liver toxicity and induction of CYP (2E1, 2B1) and phase II enzymes in female Wistar rats. Environ Toxicol Pharmacol 23:64–72

    CAS  PubMed  Google Scholar 

  • Ennulat D, Walker D, Clemo F, Magid-Slav M, Ledieu D, Graham M, Botts S, Boone L (2010) Effects of hepatic drug-metabolizing enzyme induction on clinical pathology parameters in animals and man. Toxicol Pathol 38:810–828

    CAS  PubMed  Google Scholar 

  • Freeman DJ, Laupacis A, Keown PA, Stiller CR, Carruthers SG (1984) Evaluation of cyclosporin-phenytoin interaction with observations on cyclosporin metabolites. Br J Clin Pharmacol 18:887–893

    CAS  PubMed  PubMed Central  Google Scholar 

  • Hall AP, Elcombe CR, Foster JR, Harada T, Kaufmann W, Knippel A, Küttler K, Malarkey DE, Maronpot RR, Nishikawa A, Nolte T, Schulte A, Strauss V, York MJ (2012) Liver hypertrophy: a review of adaptive (adverse and non-adverse) changes–conclusions from the 3rd International ESTP Expert Workshop. Toxicol Pathol 40:971–994

    CAS  PubMed  Google Scholar 

  • Hamadeh HK, Bushel PR, Jayadev S, Martin K, DiSorbo O, Sieber S, Bennett L, Tennant R, Stoll R, Barrett JC, Blanchard K, Paules RS, Afshari CA (2002) Gene expression analysis reveals chemical-specific profiles. Toxicol Sci 67(2):219–231

    CAS  PubMed  Google Scholar 

  • Hardwick JP, Gonzalez FJ, Kasper CB (1983) Transcriptional regulation of rat liver epoxide hydratase, NADPH-Cytochrome P-450 oxidoreductase, and cytochrome P- 450b genes by phenobarbital. J Biol Chem 258(13):8081–8085

    CAS  PubMed  Google Scholar 

  • Higgins LG, Hayes JD (2011) Mechanisms of induction of cytosolic and microsomal glutathione transferase (GST) genes by xenobiotics and pro-inflammatory agents. Drug Metab Rev 43(2):92–137

    CAS  PubMed  Google Scholar 

  • Hood A, Klaassen CD (2000) Differential effects of microsomal enzyme inducers on in vitro thyroxine (T(4)) and triiodothyronine (T(3)) glucuronidation. Toxicol Sci 55:78–84

    CAS  PubMed  Google Scholar 

  • Huang HC, Nguyen T, Pickett CB (2000) Regulation of the antioxidant response element by protein kinase C-mediated phosphorylation of NF-E2-related factor 2. Proc Natl Acad Sci USA 97(23):12475–12480. https://doi.org/10.1073/pnas.220418997(Published online 2000 Oct 17)

    Article  CAS  PubMed  Google Scholar 

  • Jones CR, Lubet RA (1992) Induction of a pleiotropic response by phenobarbital and related compounds. Response in various inbred strains of rats, response in various species and the induction of aldehyde dehydrogenase in Copenhagen rats. Biochem Pharmacol 44(8):1651–1660

    CAS  PubMed  Google Scholar 

  • Kalsotra A, Cui X, Antonovic L, Robida AM, Morgan ET, Strobel HW (2003) Inflammatory prompts produce isoform-specific changes in the expression of leukotriene B(4) omega-hydroxylases in rat liver and kidney. FEBS Lett 555(2):236–242

    CAS  PubMed  Google Scholar 

  • Kalsotra A, Anakk S, Brommer CL, Kikuta Y, Morgan ET, Strobel HW (2007) Catalytic characterization and cytokine mediated regulation of cytochrome P450 4Fs in rat hepatocytes. Arch Biochem Biophys 461(1):104–112 (Epub 2007 Mar 15)

    CAS  PubMed  PubMed Central  Google Scholar 

  • Klaassen CD, Hood AM (2001) Effects of microsomal enzyme inducers on thyroid follicular cell proliferation and thyroid hormone metabolism. Toxicol Pathol 29(1):34–40

    CAS  PubMed  Google Scholar 

  • Lau PP, Strobel HW (1982) Multiple forms of cytochrome P-450 in liver microsomes from ß-naphthoflavone-pretreated rats. J Biol Chem 257:5257–5262

    CAS  PubMed  Google Scholar 

  • Leontaridou M et al (2007) The borderline range of toxicological methods: quantification and implications for evaluating precision. ALTEX Altern Anim Exp 34(4):525–538

    Google Scholar 

  • Liu J, Liu Y, Barter RA, Klaassen CD (1995) Alteration of thyroid homeostasis by UDP-glucuronosyltransferase inducers in rats: a dose-response study. J Pharmacol Exp Ther 273:977–985

    CAS  PubMed  Google Scholar 

  • McLean AE, McLean EK (1966) The effect of diet and 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) on microsomal hydroxylating enzymes and on sensitivity of rats to carbon tetrachloride poisoning. Biochem J 100:564–571

    CAS  PubMed  PubMed Central  Google Scholar 

  • Oesch-Bartlomowicz B, Oesch F (2007) Mechanisms of toxication and detoxication which challenge drug candidates and drugs. In: Taylor JB, Triggle DJ (eds) Comprehensive medicinal chemistry. Elsevier, Amsterdam, pp 193–214

    Google Scholar 

  • Oesch F, Arand M, Strolin-Benedetti M, Castelli MG, Dostert P (1996) Inducing properties of rifampicin and rifabutin for selected enzyme activities of the cytochrome P-450 and UDP-glucuronosyltransferase superfamilies in female rat liver. J Antimicrobial Chemother 37:1111–1119

    CAS  Google Scholar 

  • Remmer H (1958) Die Beschleunigung des Evipanabbaues unter der Wirkung von Barbituraten. Naturwissenschaften 8:189

    Google Scholar 

  • Rushmore TH, Kong AN (2002) Pharmacogenomics, regulation and signaling pathways of phase I and II drug metabolizing enzymes. Curr Drug Metab 3(5):481–490

    CAS  PubMed  Google Scholar 

  • Sabers A (2008) Pharmacokinetic interactions between contraceptives and antiepileptic drugs. Seizure 17(2):141–144. https://doi.org/10.1016/j.seizure.2007.11.012

    Article  PubMed  Google Scholar 

  • Safe S, Bandiera S, Sawyer T, Robertson L, Safe L, Parkinson A, Thomas PE, Ryan DE, Reik LM, Levin W, Denomme MA, Fujita T (1985) PCBs: structure–function relationships and mechanism of action. Environ Health Perspect 60:47–56. https://doi.org/10.1289/ehp.856047

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schuur AG, Bergman A, Brouwer A, Visser TJ (1999) Effects of pentachlorophenol and hydroxylated polychlorinated biphenyls on thyroid hormone conjugation in a rat and a human hepatoma cell line. Toxicol Vitro 13(3):417–425

    CAS  Google Scholar 

  • Strolin-Benedetti M, Brogin G, Bani M, Oesch F, Hengstler JG (1999) Association of cytochrome P450 induction with oxidative stress in vivo as evidenced by 3-hydroxylation of salicylate. Xenobiotica 29:1171–1180

    CAS  PubMed  Google Scholar 

  • Thomas PE, Reik LM, Ryan DE, Levin W (1983) Induction of two immunochemically related rat liver cytochrome P-450 isozymes, cytochrornes P-450c and P-450d, by structurally diverse xenobiotics. J Biol Chem 258:4590–4598

    CAS  PubMed  Google Scholar 

  • Viollon-Abadie C, Bigot-Lasserre D, Nicod L, Carmichael N, Richert L (2000) Effects of model inducers on thyroxine UDP-glucuronosyl-transferase activity in vitro in rat and mouse hepatocyte cultures. Toxicol Vitro 14:505–512

    CAS  Google Scholar 

  • Visser TJ, Kaptein E, van Toor H, van Raaij JA, van den Berg KJ, Joe CT, van Engelen JG, Brouwer A (1993) Glucuronidation of thyroid hormone in rat liver: effects of in vivo treatment with microsomal enzyme inducers and in vitro assay conditions. Endocrinology 133:2177–2186

    CAS  PubMed  Google Scholar 

  • Waterman CL, Currie RA, Cottrell LA, Dow J, Wright J, Waterfield CJ, Griffin JL (2010) An integrated functional genomic study of acute phenobarbital exposure in the rat. BMC Genom 6(11):9. https://doi.org/10.1186/1471-2164-11-9

    Article  CAS  Google Scholar 

  • Waxman DJ, Azaroff L (1992) Phenobarbital induction of cytochrome P-450 gene expression. Biochem J 281(Pt 3):577–592

    CAS  PubMed  PubMed Central  Google Scholar 

  • Weiss F, Hammer HS, Klein K, Planatscher HP, Zanger HU, Noren A, Wegler C, Artursson P, Joos TO, Poetz O (2018) Direct quantification of cytochromes P450 and drug transporters—a rapid, targeted mass spectrometry-based immunoassay panel for tissues and cell culture lysates. Drug Metab Dispos 46:387–396. https://doi.org/10.1124/dmd.117.078626(Epub 2018 Jan 17)

    Article  CAS  PubMed  Google Scholar 

  • Whomsley R, Strolin Benedetti M, Espié P, Chen C, Klaassen C, Oesch F, Baltes E (2004) Comparison of induction of glucuronidation of thyroid hormones and bilirubin in rat and human hepatocytes. In: Abstract book, MDO 2004, Mainz, 4–9 July, p 129 (P6–CH–21)

  • Williams GM, Iatropoulos MJ (2002) Alteration of liver cell function and proliferation: differentiation between adaptation and toxicity. Toxicol Pathol 30(1):41–53

    CAS  PubMed  Google Scholar 

  • Wong H, Lehman-McKeeman LD, Grubb MF, Grossman SJ, Bhaskaran VM, Solon EG, Shen HS, Gerson RJ, Car BD, Zhao B, Gemzik B (2005) Increased hepatobiliary clearance of unconjugated thyroxine determines DMP 904-induced alterations in thyroid hormone homeostasis in rats. Toxicol Sci 84:232–242

    CAS  PubMed  Google Scholar 

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Author notes

  1. Brandy Riffle and Franz Oesch contributed equally.

Authors and Affiliations

  1. BASF Corporation, 26 Davis Drive, Research Triangle Park, NC, 27709, USA

    Brandy Riffle, Mao Wang, Anita Samuga & Peifeng Ren

  2. Institute of Toxicology, Johannes Gutenberg University of Mainz, Rheinblick 21, 55263, Wackernheim, Germany

    Franz Oesch

  3. BASF SE, Experimental Toxicology and Ecology, RB/TB, Z470, Carl-Bosch-Strasse 38, 67056, Ludwigshafen am Rhein, Germany

    Annika Heckmanns, Eric Fabian, Bennard van Ravenzwaay & Robert Landsiedel

  4. Natural and Medical Sciences Institute at the University of Tübingen, Markwiesenstrasse 55, 72770, Reutlingen, Germany

    Helen Hammer & Oliver Pötz

  5. SIGNATOPE GmbH, Markwiesenstrasse 55, 72770, Reutlingen, Germany

    Felix Schmidt

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  1. Brandy Riffle
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Correspondence to Robert Landsiedel.

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B.R,, A.H., E.F., M.W., A.S., P.R., B.R., and R.L. are employees of BASF, a chemical company which may use the described methods to develop commercial products;. O.P. is a shareholder of Signatope GmbH. Signatope offers assay development and service using MS-based immunoassay technology; F.S. works at Sigmatope; F.O. and H.H. have no conflict of interest.

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Riffle, B., Oesch, F., Heckmanns, A. et al. Xenobiotica-metabolizing enzyme induction potential of chemicals in animal studies: NanoString nCounter gene expression and peptide group-specific immunoaffinity as accelerated and economical substitutions for enzyme activity determinations?. Arch Toxicol 94, 2663–2682 (2020). https://doi.org/10.1007/s00204-020-02777-4

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