Induction of xenobiotic-metabolizing enzymes in hepatocytes by beta-naphthoflavone: Time-dependent changes in activities, protein and mRNA levels

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In the present study, time-dependency of the induction effect of a selective inducer on the activity, protein and mRNA levels of cytochromes P450 1A1/2 (CYP1A1/2), NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferases (GSTA), in primary culture of rat hepatocytes was tested and evaluated. To show the differences in responses of tested enzymes, the common aryl hydrocarbon receptor (AhR) ligand agonist, beta-naphthoflavone (BNF), was used. Induction of CYP1A1/2 by BNF was detected at all time intervals and at all levels (i.e., mRNA, protein, enzyme activity). Different responses of NQO1 and GSTA upon BNF treatment were observed. Our results demonstrate that the responses of different xenobiotic-metabolizing enzymes to the inducer vary in time and depend on the measured parameter. For these reasons, an induction study featuring only one-time interval treatment and/ or one parameter testing could produce misleading information.

1. H. Fujimura, N. Murakami, S. Miwa, C. Aruga and W. Toriumi, The suitability of rat hepatoma cell line H4IIE for evaluating the potentials of compounds to induce CYP3A23 expression, Exp. Toxicol. Pathol. 64 (2012) 527-533;

2. D. B. Buckley and C. D. Klaassen, Induction of mouse UDP-glucuronosyltransferase mRNA expression in liver and intestine by activators of aryl-hydrocarbon receptor, constitutive androstane receptor, pregnane X receptor, peroxisome proliferator-activated receptor alpha, and nuclear factor erythroid 2-related factor 2, Drug Metab. Dispos. 37 (2009) 847-856;

3. Food and Drug Administration, Guidance for Industry: Drug Interaction Studies - Study Design, Data Analysis, and Implications for Dosing and Labeling, Draft Guidance, FDA, Center for Drug Evaluation and Research, Rockville 2006;; last access date October 3, 2017

4. L. Richert, G. Tuschl, C. Abadie, N. Blanchard, D. Pekthong, G. Mantion, J. C. Wr andebe S. O. Mueller, Use of mRNA expression to detect the induction of drug metabolising enzymes in rat and human hepatocytes, Toxicol. Appl. Pharmacol. 235 (2009) 86-96; J. Skorkovská 1.019, V. Hanušová, V. Tománková, E. Anzenbacherová, B. Lišková, P. Anzenbacher

5. P. Matoušková, H. Bártíková, I. Boušová, B. Szotáková, J. Martin, and L. Skálová, Effect of defined green tea extract in various dosage schemes on drug-metabolizing enzymes in mice in vivo, J. Funct. Foods 10 (2014) 327-335;

6. O. H. Elshenawy and A. O. El-Kadi, Modulation of aryl hydrocarbon receptor regulated genes by acute administration of trimethylarsine oxide in the lung, kidney and heart of C57BL/6 mice, Xenobiotica 45 (2015) 930-943;

7. O. Hankinson, The aryl hydrocarbon receptor complex, Annu. Rev. Pharmacol. 35 (1995) 307-340;

8. H. M. Korashy and A. O. El-Kadi, The role of aryl hydrocarbon receptor in the pathogenesis of cardiovascular diseases, Drug Metab. Rev. 38 ( 2006) 4 11-450;

9. C. Abadie-Viollon, H. Martin, N. Blanchard, D. Pekthong, P. Bachellier, G. Mantion, B. Heyd, F. Schuler, P. Coassolo, E. Alexandre and L. Richert, Follow-up to the pre-validation of a harmonised protocol for assessment of CYP induction responses in freshly isolated and cryopreserved human hepatocytes with respect to culture format, treatment, positive reference inducers and incubation conditions, Toxicol. In Vitro 24 (2010) 346-356;

10. R. Glockner, A. Lieder and A. Lupp, Determination of CYP activity in precision-cut liver slices: whether to use intact slices or slice homogenate, Anal. Bioanal. Chem. 392 (2008) 1167-1172;

11. M. Monshouwer, G. A. Van’t Klooster, S. M. Nijmeijer, R. F. Witkamp and A. S. van Miert, Characterization of cytochrome P450 isoenzymes in primary cultures of pig hepatocytes, Toxicol. In Vitro 12 (1998) 715-723.

12. M. N. Berry, G. J. Barritt and A. M. Edwards, Isolated Hepatocytes: Preparation, Properties and Application, Elsevier, Amsterdam 1991, Vol. 21.

13. K. Lnenickova, E. Prochazkova, L. Skalova, P. Matouskova, H. Bartikova, P. Soucek and B. Szotakova, Catechins variously affect activities of conjugation enzymes in proliferating and differentiated Caco-2 cells, Molecules 21 (2016) e1186 (11 pages);

14. J. J. Cullen, M. M. Hinkhouse, M. Grady, A. W. Gaut, J. Liu, Y. P. Zhang, C. J. Weydert, F. E. Domann and L. W. Oberley, Dicumarol inhibition of NADPH:quinone oxidoreductase induces growth inhibition of pancreatic cancer via a superoxide-mediated mechanism, Cancer Res. 63 (2003) 5513-5520.

15. M. D. Burke and R. T. Mayer, Ethoxyresorufin: direct fluorimetric assay of a microsomal O-dealkylation which is preferentially inducible by 3-methylcholanthrene, Drug Metab. Dispos. 2 (1974) 583-588.

16. A. Untergasser, I. Cutcutache, T. Koressaar, J. Ye, B. C. Faircloth, M. Remm and S. G. Rozen, Primer3 - new capabilities and interfaces, Nucleic Acids Res. 40 (2012) e115 (12 pages);

17. K. J. Livak and T. D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method, Methods 25 (2001) 402-408;

18. Z. Dvorak, P. Maurel, M. J. Vilarem, J. Ulrichova and M. Modriansky, Expression and transcriptional activities of nuclear receptors involved in regulation of drug-metabolizing enzymes are not altered by colchiceine: Focus on PXR, CAR, and GR in primary human hepatocytes, Cell Biol. Toxicol. 23 (2007) 63-73;

19. M. Nishimura, A. Koeda, E. Suzuki, T. Shimizu, Y. Kawano, M. Nakayama and T. Satoh, Effects of prototypical drug-metabolizing enzyme inducers on mRNA expression of housekeeping genes in primary cultures of human and rat hepatocytes, Biochem. Bioph. Res. Commun. 346 (2006) 1033-1039;

20. Y. Korenori, S. Tanigawa, T. Kumamoto, S. Qin, Y. Daikoku, K. Miyamori, M. Nagai and D. X. Hou, Modulation of Nrf2/Keap1 system by Wasabi 6-methylthiohexyl isothiocyanate in ARE-mediated NQO1 expression, Mol. Nutr. Food Res. 57 (2013) 854-864;

21. I. Bousova and L. Skalova, Inhibition and induction of glutathione S-transferases by flavonoids: possible pharmacological and toxicological consequences, Drug Metab. Rev. 44 (2012) 2 67-286;

22. E. P. Chen, L. F. Chen, Y. Ji, G. Y. Tai, Y. H. Wen and H. Ellens, A mechanism-based mathematical model of aryl hydrocarbon receptor-mediated CYP1A induction in rats using beta-naphthoflavone as a tool compound, Drug Metab. Dispos. 38 (2010) 2278-2285;

23. S. Karakurt, Modulatory effects of rutin on the expression of cytochrome P450s and antioxidant enzymes in human hepatoma cells, Acta Pharm. 66 (2016) 491-502;

24. L. G. Higgins and J. D. Hayes, Mechanisms of induction of cytosolic and microsomal glutathione transferase (GST) genes by xenobiotics and pro-inflammatory agents, Drug Metab. Rev. 43 (2011) 92-137;

25. O. H. Elshenawy and A. O. S. El-Kadi, Modulation of aryl hydrocarbon receptor-regulated enzymes by trimethylarsine oxide in C57BL/6 mice: In vivo and in vitro studies, Toxicol. Lett. 238 (2015) 17-31;

26. I. Bousova, J. Hajek, J. Drsata and L. Skalova, Naturally occurring flavonoids as inhibitors of purified cytosolic glutathione S-transferase, Xenobiotica 42 (2012) 872-879;

27. D. Brauze, K. Fijalkiewicz, M. Szaumkessel, K. Kiwerska, K. Bednarek, M. Rydzanicz, J. Richter, R. Grenman and M. Jarmuz-Szymczak, Diversified expression of aryl hydrocarbon receptor dependent genes in human laryngeal squamous cell carcinoma cell lines treated with beta-naphthoflavone, Toxicol. Lett. 231 (2014) 99-107;

28. D. Brauze, M. Widerak, J. Cwykiel, K. Szyfter and W. Baer-Dubowska, The effect of aryl hydrocarbon receptor ligands on the expression of AhR, AhRR, ARNT, Hif1 alpha, CYP1A1 and NQO1 genes in rat liver, Toxicol. Lett. 167 (2006) 212-220;

29. T. Maier, M. Guell and L. Serrano, Correlation of mRNA and protein in complex biological samples, FEBS Lett. 583 (2009) 3966-3973;

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