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HepG2-1A2 C2 and C7: Lentivirus vector-mediated stable and functional overexpression of cytochrome P450 1A2 in human hepatoblastoma cells
Toxicology Letters 319 (2020) 155–159
Contents lists available at ScienceDirect
Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
HepG2-1A2 C2 and C7: Lentivirus vector-mediated stable and functional overexpression of cytochrome P450 1A2 in human hepatoblastoma cells
T
Susanne Steinbrechta, Nadine Pfeifera, Natalie Herzoga, Nadine Katzenbergera, Christian Schulzb, Sarah Kammerera,*,1, Jan-Heiner Küppera,1 a
Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, Universitätsplatz 1, 01968 Senftenberg, Germany Project Group Pz-Syn of the Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses IZI-BB, Am Mühlenberg 13, 14476 Potsdam, located at the Brandenburg University of Technology Cottbus-Senftenberg, Germany
b
A R T I C LE I N FO
A B S T R A C T
Keywords: HepG2 CYP1A2 Cytochrome P450 Liver metabolism
Novel HepG2 cell clones 1A2 C2 and 1A2 C7 were independently generated by lentiviral transduction to functionally overexpress cytochrome P450 1A2 (CYP1A2). We found similar and stable CYP1A2 transcript and protein levels in both cell clones leading to specific enzyme activities of about 370 pmol paracetamol x min-1 x mg-1 protein analyzed by phenacetin conversion. Both clones showed dramatically increased sensitivity to the hepatotoxic compound aflatoxin B1 (EC50 < 100 nM) when compared to parental HepG2 cells (EC50 ∼5 μM). Thus, newly established cell lines are an appropriate tool to study metabolism and toxicity of substances depending on conversion by CYP1A2.
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1. Resource Table
Name of cell line construct
HepG2-1A2 C2, HepG2-1A2 C7
Institution
Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany Natalie Herzog (C2), Nadine Katzenberger (C7) Sarah Kammerer, [email protected] Apr, 2014 (C7); Oct, 2015 (C2) human hepatoblastoma cells (HepG2) HepG2 cells with stable overexpression of CYP1A2 Cell clone CYP1A2 Identity and purity of cell line confirmed
Person who created resource Contact person and email Date archived/stock date Origin Type of resource Sub-type Transfected or deleted genes Authentication
https://content.iospress.com/articles/journalof-cellular-biotechnology/jcb189013 Steinbrecht, S., Kammerer, S., Küpper, J.-H. HepG2 cells with recombinant cytochrome P450 enzyme overexpression: Their use and limitation as in vitro liver model. Journal of Cellular Biotechnology., DOI: 10.3233/JCB189013; https://content.iospress.com/articles/journalof-cellular-biotechnology/jcb179012 Kammerer, S., Küpper, J.-H. Expert Opinion: Human Hepatocyte Systems for in vitro Toxicology Analysis. Journal of Cellular Biotechnology., DOI: 10.3233/JCB-179012
Information in public databases
2. Resource Details Due to their unlimited availability and easy handling, human hepatoblastoma HepG2 cells are widely used for in vitro toxicology studies as alternative to primary human hepatocytes. HepG2 cells were derived from an epithelial hepatoblastoma of a 15-year-old Caucasian male (López-Terrada et al., 2009). However, compared to primary human hepatocytes, they have the disadvantage of limited biotransformation activity due to their low or even absent expression of cytochrome P450
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Corresponding author at: Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, Universitätsplatz 1, 01968 Senftenberg, Germany. E-mail address: [email protected] (S. Kammerer). 1 both authors shared the function as principal investigator https://doi.org/10.1016/j.toxlet.2019.11.006 Received 13 August 2019; Received in revised form 9 October 2019; Accepted 5 November 2019 Available online 06 November 2019 0378-4274/ © 2019 Elsevier B.V. All rights reserved.
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(Fig. 1A, B). Both cell clones showed significantly increased levels of CYP1A2 mRNA and protein expression when compared to parental HepG2 cells, as determined by qRT-PCR and western blot (Fig. 1C, D). In addition to expression analyses at mRNA and protein levels, CYP1A2 functionality was tested by enzyme activity assays. As first test system, we used the P450-Glo™ CYP1A2 induction/inhibition assay. While only low CYP1A2 activity could be measured in the parental HepG2 cells, it was increased by a factor of 700-800 in the HepG2-1A2 cell clones (Fig. 2A). In addition, specific enzyme activity of CYP1A2 was determined by conversion of the standard substrate phenacetin and HPLC-based measurement of its CYP1A2-dependent product paracetamol (Huang et al., 2012). Recombinant expression of CYP1A2 led to an enzyme activity of 363-373 pmol paracetamol x min-1 x mg-1 total cellular protein in both CYP-expressing HepG2 cell clones (Fig. 2B). In contrast, parental HepG2 cells lacked significant CYP1A2 activity. Inhibition studies involving 1-aminobenzotriazole (1-ABT), a pan-specific inactivator of CYPs involved in xenobiotic metabolism (de Montellano, 2018), showed that the high conversion rates of phenacetin to paracetamol are reversible (Fig. 2B) and thus were indeed associated with functional integration of recombinant CYP1A2 cDNA into HepG2 cells. It is well known that CYP1A2 is a major player in the biotransformation of aflatoxin B1 (AFB1) resulting in the production of genotoxic metabolites (Bbosa et al., 2013). When measuring EC50
oxidoreductases (CYPs) (Rodríguez-Antona et al., 2002; Westerink and Schoonen, 2007). Strategies to overcome the lack of CYP activities in HepG2 include, amongst other methods, usage of chemical agonists for CYP induction which, however, do not lift CYP1A2 enzyme activity to the levels of primary human hepatocytes (Westerink and Schoonen, 2007). Alternatively, the genetic engineering of these cells with e.g. lentiviral expression vectors coding for human CYPs can be applied. In the latter case, the virally encoded integrase is used for the efficient integration of recombinant DNA sequences into the mammalian genome to obtain HepG2 cells with a stable CYP overexpression and physiologically relevant enzyme activity levels (Steinbrecht et al., 2019a). CYP1A2 is one of the most abundant CYPs in the human liver that metabolizes about 20 % of clinically used drugs and several important endogenous compounds (Shu-Feng et al., 2009; Wang and Zhou, 2009). Here, we report the generation and characterization of two new genetically engineered HepG2 cell clones (HepG2-1A2 C2 and C7) with stable overexpression of human CYP1A2. The cell lines were generated by transduction of a lentivirus-based expression vector encoding human CYP1A2 cDNA, followed by single clone selection with antibiotic zeocin. HepG2-1A2 cell clones 2 and 7 both showed a cobblestone-like morphology typical for hepatocytes and have population doubling times that were not significantly different from the parental HepG2 cells
Fig. 1. Characterization of HepG2-1A2 C2 and C7. (A) Phase contrast microscopy of HepG2 and CYP1A2-overexpressing clones C2 and C7. Images were obtained by using the microscope CKX41 and 20x objective (scale bar 200 μm). (B) Doubling times of exponentially growing cultures of HepG2, HepG2-1A2 C2 and C7. Results are shown as mean ± SEM of two independent experiments; n.s.: not significant. C) mRNA expression levels in HepG2-1A2 C2 and C7 cells relative to parental HepG2 cells. Data represent the mean ± SEM; n = 5; *p < 0.05, ****p < 0.0001, each in comparison to the control cell line HepG2. (D) Western blot analysis of non-modified HepG2 cells and CYP1A2-expressing HepG2 cell clones C2 and C7. Equal protein amounts (20 μg) of HepG2, HepG2-1A2 C2 and C7, and 10 μg of microsomes as positive control were used for western blotting. Detection of GAPDH served as loading control. Note that microsomal protein extracts do not contain GAPDH. Top: A representative blot is shown. Bottom: Quantitative Western blot data are shown as CYP1A2/GAPDH ratio of luminescent band intensities as mean ± SEM; n = 3; **p < 0.01, ***p < 0.001, each in comparison to the control cell line HepG2. 156
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Fig. 2. Functional analysis of CYP1A2 in HepG2 and CYP1A2-overexpressing cell clones. (A) Relative enzyme activity of CYP1A2 in HepG2-1A2 cell clones C2 and C7 relative to parental HepG2 cells measured by the P450Glo™ CYP1A2 induction/inhibition assay. Results are shown as mean ± SEM; n = 4; ****p < 0.0001 in comparison to the control cell line HepG2. Please note that the bar of HepG2 is not visible due to the comparatively low values. (B) Determination of enzyme activity in HepG2 cells and CYP1A2-overexpressing cell clones C2 and C7 by conversion of phenacetin to paracetamol. Paracetamol concentration in the cell culture supernatant after 1 h phenacetin treatment of the cells was measured by HPLC and related to the total amount of protein. For inhibition studies, cells were incubated with 1 mM 1-aminobenzotriazole (1-ABT) 4 h before treatment with phenacetin. Data represent mean values ± SEM; n = 3; ****p < 0.0001 in comparison to the control cell line HepG2; + +++p < 0.0001 in comparison to previous treatment with 1-ABT. (C) Relative cell viability and calculated EC50 values of HepG2 cell clones C2 and C7 in comparison to HepG2 cells after treatment with different concentrations of aflatoxin B1 as evaluated by CellTiter-Glo®2.0 ATP assay. Results are shown as mean ± SEM of two independent experiments. Statistical analysis is given in comparison to the control cell line HepG2.
toxicological and metabolic studies.
values, CYP1A2-overexpressing HepG2 cell clones showed a remarkable 70- (clone C2; p < 0.0001) and 130-fold (clone C7; p < 0.0001) higher sensitivity towards AFB1 compared to parental HepG2 cells (Fig. 2C). Experiments shown here were performed over a range of 20 passages and results did not differ between younger and older passages. Therefore, we assume a very high stability of CYP1A2 expression in both cell clones even under long-term culture conditions. When studying new drugs, it will become necessary to analyze additional CYP isoforms known to be involved in human drug metabolism. This might be realized using freshly isolated primary hepatocytes, primary-like hepatocytes such as upcyte® and HepaFH3 cells (Herzog et al., 2016; Ramachandran et al., 2015) or panels of HepG2 cells with overexpression of single or multiple CYP enzymes (Wu et al., 2017). Our newly generated CYP1A2-overexpressing HepG2 clones C2 and C7 display high stability of CYP1A2 expression as well as functional activity which makes them valuable lab resources for studies on CYP1A2dependent metabolism. Both clones represent a well characterized, robust and easy-to-handle in vitro tool for different kinds of
3. Verification and Authentication The results of DNA fingerprinting analysis to indicate cell authenticity revealed an identical STR pattern of the CYP1A2-expressing cell clones to those of their parental HepG2 cells (Data available with the authors). The mycoplasma test by luminescence was negative for both HepG2-1A2 clones and parental HepG2 cells (Supplementary material, Table S1). 4. Materials and Methods 4.1. Cell Culture Human hepatoblastoma (HepG2) cells (HB-8065; ATCC, Manassas, USA) were cultivated in Dulbecco’s minimal essential medium (DMEM) (Biochrom AG, Berlin, Germany) supplemented with 10 % fetal bovine serum (Biochrom AG) and 2 mM L-glutamine (PAA Laboratories GmbH, 157
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to the manufacturer´s protocol. Total RNA was treated with DNase I (Thermo Fisher Scientific) to remove genomic DNA, and RNA integrity was controlled by 1 % agarose gel electrophoresis. Isolated RNA was used for synthesis of cDNA according to manufacturer´s instruction using oligo(dT)18 primer and RevertAid Reverse Transcriptase (Thermo Fisher Scientific). Resulting cDNA was diluted 1:10 and qRT-PCR was performed for CYP1A2 (forward primer: 5`-CTGGAGACCTTCCGACA CTC-3`; reverse primer: 5`-AGGGCTTGTTAATGGCAGTG-3`) and reference genes GAPDH (glyceraldehyde-3-phosphate dehydrogenase; forward primer: 5`-TGCACCACCAACTGCTTAGC-3`; reverse primer: 5`GGCATGGACTGTGGTCATGAG-3`) and RPL-P0 (Ribosomal Protein Lateral Stalk Subunit P0; forward primer: 5`-AAATGTTTCATTGTGGG AGC-3`; reverse primer: 5`-ATATGAGGCAGCAGTTTCTC-3`; all primers from BioTez, Berlin, Germany). Primers, Maxima Probe qPCR Master Mix (Thermo Fisher Scientific), Evagreen (Biotium, Inc., Fremont, California, USA) and cDNA were mixed accordingly in a 10 μl reaction volume per sample and the following settings on a C1000 Touch™ Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, USA) were used: initial denaturation at 95 °C for 3 min; 50 cycles of 95 °C for 10 s, 62 °C for 10 s and 72 °C for 30 s; final elongation at 72 °C for 30 s. Size of PCR products was controlled by a 3 % agarose gel electrophoresis. Relative expression levels were calculated from the threshold cycle (CT) values according to the 2-ΔΔCT method.
Pasching, Austria) at 37 °C and 5 % CO2 in a humidified incubator. The cells were routinely passaged by trypsinization with 0.05 % (w/v) Trypsin/0.02 % (w/v) EDTA (Biochrom AG). Medium for CYP1A2overexpressing HepG2 clones was supplemented with 500 μg x ml-1 zeocin (Thermo Fisher Scientific Inc., Waltham, USA). 4.2. Construction of plasmids Plasmids were constructed using the Invitrogen™ Gateway® Technology from Thermo Fisher Scientific according to the manufacturer´s protocol. In brief, an entry vector (GeneCopoeia™, Rockville, USA) that already contained the coding sequence for CYP1A2 (NCBI reference sequence: BC067428) flanked by attachment sites (attL) was used to recombine the CYP1A2 cDNA sequences into the attR-containing destination vector pLenti4/V5-DEST (Thermo Fischer Scientific) using the LR recombination reaction. Consequently, the CYP1A2 sequence in the resulting lentivirus expression vector was controlled by the human cytomegalovirus (CMV) promoter. The resulting vector plasmid pLenti4/V5-DEST-CYP1A2 was then used to generate recombinant lentivirus particles. 4.3. Lentivirus packaging For recombinant lentivirus generation, the ViraPower™ Lentiviral Expression System from Thermo Fisher Scientific was used according to the manufacturer´s protocol. Virus production was based on helper cell line 293FT (Thermo Fisher Scientific; R70007) which was routinely cultivated in Dulbecco’s MEM supplemented with 10 % fetal bovine serum, 0.1 mM non-essential amino acids, 4 mM L-glutamine, 1 mM sodium pyruvate and 500 μg/ml Geneticin® (all from Biochrom AG) at 37 °C and 5 % CO2. The cells were cotransfected with vector plasmid pLenti4/V5-DEST-CYP1A2 along with helper plasmids pLP1, pLP2, pLP/VSVG (Thermo Fisher Scientific) via Lipofectamine™ 2000 (Thermo Fisher Scientific). Next day, the medium was replaced with fresh culture medium and after an additional incubation period of 4872 h, recombinant lentivirus was collected from the supernatant. Virus solution was 20 fold concentrated by a filtration system of Sartorius (Vivaspin® 20) and used directly for HepG2 infection.
4.7. Western blot The general procedure of Western Blot analysis was adopted from our previous publication (Steinbrecht et al., 2019b) with following modifications: For CYP1A2-overexpressing and parental HepG2 cells, 20 μg total protein and 10 μg total protein of microsomes (pooled from 50 different individual donors; Thermo Fisher Scientific) were applied per lane for SDS-PAGE. For the detection of CYP1A2 and the reference gene GAPDH, anti-CYP1A2 monoclonal antibody (Thermo Fisher Scientific; dilution 1:2000) and anti-GAPDH monoclonal antibody (Antikoerper-online GmbH, Aachen, Germany; dilution 1:5000) were used as primary antibodies. 4.8. Determination of enzyme activity by P450-Glo™ and CellTiter-Glo® 2.0 assay
4.4. Lentiviral infection of HepG2 cells Cells were seeded at 3 × 104 cells/well into 96-well plates (Sarstedt AG & Co. KG) 24 h before start of analysis with the P450-Glo™ CYP1A2 induction/inhibition assay (Promega, Madison, WI, USA) that was used according to the manufacturer’s protocol. In brief, cells were incubated with 50 μl CYP1A2 substrate diluted in PBS for 60 min at 37 °C. Then, 25 μl of supernatants were transferred into a white-walled 96-well plate (Sarstedt AG & Co. KG), and an equal volume of detection reagent was added followed by incubation for 20 min at room temperature in the dark. Thereafter, luminescence signals were measured with FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany). In addition, the cells and the 25 μl substrate solution remaining in the wells were mixed with 25 μl ATP reagent solution of the CellTiter-Glo®2.0 assay (Promega). Thus, the ATP level could be determined according to the manufacturer's instructions to allow normalization to the seeded cell number.
HepG2 cells were grown to 60 % confluence in 24-well plates (Sarstedt AG & Co. KG, Nümbrecht, Germany). Then, concentrated recombinant lentivirus solution and 6 μg x ml-1 hexadimethrine bromide (Sigma-Aldrich, St. Louis, USA) were added. After 24 h, infection medium was replaced with fresh HepG2 medium (see above) followed by start of selection pressure (500 μg x ml-1 zeocin) 48 hours post infection. Within 3 – 4 weeks, zeocin-resistant cell colonies were picked and further expanded in selection medium to obtain single cell-derived HepG2 clones. 4.5. Determination of population doubling times 4 × 104 cells/well were seeded into 6-well plates (Sarstedt AG & Co. KG) and cultured under standard conditions without antibiotics. Cells were counted daily using a Neubauer hemocytometer and population doubling times (PDT) were determined using the formula PDT = (t2t1)*log2/log(q2/q1), with t1-t2 = time interval of constant growth rate in hours, q1 = cell number per cm2 at t1, and q2 = cell number per cm2 at t2.
4.9. Determination of enzyme activity by phenacetin conversion Cells were seeded at 2 × 106 cells per well into 6-well plates (Sarstedt AG & Co. KG) and cultured for 48 h. Cells were first washed with Krebs-Henseleit Buffer (KHB; completed with 25 mM NaHCO3, 25 mM HEPES, 2 mM CaCl2 x 2H2O; pH 7.4; all from Sigma-Aldrich except HEPES from Carl Roth GmbH + Co. KG, Karlsruhe, Germany). Then, cells were incubated with 360 μM of CYP1A2 substrate phenacetin (Sigma-Aldrich) diluted in KHB (500 μl per well) for 1 h in a humidified incubator at 5 % CO2 and 37 °C. For inhibition studies, 1 mM
4.6. Quantitative reverse transcriptase-polymerase chain reaction (qRTPCR) Total RNA was extracted from cell pellets (1-4 × 106 cells) using the innuPREP RNA Mini Kit (Analytik Jena AG, Jena, Germany) according 158
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one-way ANOVA with Turkey’s or Dunnett’s multiple comparison test using the respective control was performed. Results were considered as statistically significant if p < 0.05.
1-aminobenzotriazole (1-ABT; Sigma-Aldrich) was added to the cell medium 4 h prior to phenacetin treatment. After phenacetin treatment with or without 1-ABT, supernatants were collected, centrifuged at 13,000 x g for 15 min and metabolite formation was measured using VWR-Hitachi Elite LaChrom series HPLC (VWR International GmbH, Darmstadt, Germany) with photodiode array L-2455 (column: Phenomenex Kinetex C18, 4.6 × 150 mm, 5 μm particle size, 100 A°; security guard CK18, 4.6 × 2 mm). HPLC method: injection volume 20 μl; flow rate 1.0 ml x min-1; column temperature 40 °C; mobile phase A (10 mM KH2PO4; pH 3); mobile phase B (acetonitrile); HPLC gradient profile: 0-5 min eluent B 5 %, 15 min eluent B 40 %, 16 min eluent B 90 %, 20 min eluent B 90 %. In general, eluted substances were detected in the wavelength range 210–400 nm by the detector. The metabolite paracetamol was identified by comparison to an authentic standard (Dr. Ehrenstorfer GmbH, Augsburg, Germany) based on retention time and UV absorption spectrum. Quantification of paracetamol was performed by using a calibration curve at the wavelength of its absorption maximum of 245 nm. To determine enzyme activity in relation to total cellular protein, cells were trypsinized, pelleted by centrifugation, resuspended in RIPA buffer (50 mM Tris, 150 mM NaCl, 0.5 % DOC, 1 % NP-40, 0.5 % SDS, 1 mM PMSF, pH 8) and incubated on ice for 15 min followed by centrifugation (10,000 x g, 10 min, 4 °C). Protein concentrations of lysate supernatants were analyzed with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) according to manufacturer’s protocol.
Declaration of Competing Interest The authors report no declarations of interest. Acknowledgements This work was supported by the European Regional Development Fund (ERDF, Brandenburg, Germany; project “PERsonalisierte Medizin durch FUNCTIONomics in Berlin-Brandenburg: DrugMetabolisierungsmodul für Wirkstofftests an Patientenzellen”, project number: 85002925; and project “Entwicklung eines physiologisch relevanten Testsystems zur In-vitro-Erfassung von Hepatotoxizität im Hochdurchsatz”, project number: 85009748). We thank Kai-Uwe Schmidtke, Institute of Biotechnology, BTU Cottbus-Senftenberg for support with HPLC measurements. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.toxlet.2019.11.006. References
4.10. Cell treatment with aflatoxin B1 Bbosa, G.S., Kitya, D., Odda, J., Ogwal-Okeng, J., 2013. Aflatoxins metabolism, effects on epigenetic mechanisms and their role in carcinogenesis. Health 5, 14–34. https://doi. org/10.4236/health.2013.510A1003. de Montellano, P.R.O., 2018. 1-Aminobenzotriazole: A Mechanism-Based Cytochrome P450 Inhibitor and Probe of Cytochrome P450 Biology. Med Chem (Los Angeles) 8, 038–065. https://doi.org/10.4172/2161-0444.1000495. Herzog, N., Hansen, M., Miethbauer, S., Schmidtke, K.U., Anderer, U., Lupp, A., Sperling, S., Seehofer, D., Damm, G., Scheibner, K., Kupper, J.H., 2016. Primary-like human hepatocytes genetically engineered to obtain proliferation competence display hepatic differentiation characteristics in monolayer and organotypical spheroid cultures. Cell biology international 40, 341–353. https://doi.org/10.1002/cbin.10574. Huang, Q., Deshmukh, R.S., Ericksen, S.S., Tu, Y., Szklarz, G.D., 2012. Preferred Binding Orientations of Phenacetin in CYP1A1 and CYP1A2 Are Associated with IsoformSelective Metabolism. Drug Metabolism and Disposition 40, 2324–2331. https://doi. org/10.1124/dmd.112.047308. López-Terrada, D., Cheung, S.W., Finegold, M.J., Knowles, B.B., 2009. Hep G2 is a hepatoblastoma-derived cell line. Human Pathology 40, 1512–1515. https://doi.org/ 10.1016/j.humpath.2009.07.003. Ramachandran, S.D., Vivares, A., Klieber, S., Hewitt, N.J., Muenst, B., Heinz, S., Walles, H., Braspenning, J., 2015. Applicability of second-generation upcyte(R) human hepatocytes for use in CYP inhibition and induction studies. Pharmacology research & perspectives 3, e00161. https://doi.org/10.1002/prp2.161. Rodríguez-Antona, C., Donato, M.T., Boobis, A., Edwards, R.J., Watts, P.S., Castell, J.V., Gómez-Lechón, M.J., 2002. Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells. Xenobiotica 32, 505–520. https://doi.org/10.1080/ 00498250210128675. Shu-Feng, Z., Li-Ping, Y., Ming Qian, W., Wei, D., Eli, C., 2009. Insights into the Structure, Function, and Regulation of Human Cytochrome P450 1A2. Current Drug Metabolism 10, 713–729. https://doi.org/10.2174/138920009789895552. Steinbrecht, S., Kammerer, S., Küpper, J.-H., 2019a. HepG2 cells with recombinant cytochrome P450 enzyme overexpression: Their use and limitation as in vitro liver model. Journal of Cellular Biotechnology 5, 55–64. https://doi.org/10.3233/JCB189013. Steinbrecht, S., König, R., Schmidtke, K.-U., Herzog, N., Scheibner, K., Krüger-Genge, A., Jung, F., Kammerer, S., Küpper, J.-H., 2019b. Metabolic activity testing can underestimate acute drug cytotoxicity as revealed by HepG2 cell clones overexpressing cytochrome P450 2C19 and 3A4. Toxicology 412, 37–47. https://doi.org/10.1016/j. tox.2018.11.008. Wang, B., Zhou, S.F., 2009. Synthetic and Natural Compounds that Interact with Human Cytochrome P450 1A2 and Implications in Drug Development. Current Medicinal Chemistry 16, 4066–4218. https://doi.org/10.2174/092986709789378198. Westerink, W.M.A., Schoonen, W.G.E.J., 2007. Cytochrome P450 enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells. Toxicology in Vitro 21, 1581–1591. https://doi.org/10.1016/j.tiv.2007.05.014. Wu, Y., Chitranshi, P., Loukotková, L., Gamboa da Costa, G., Beland, F.A., Zhang, J., Fang, J.-L., 2017. Cytochrome P450-mediated metabolism of triclosan attenuates its cytotoxicity in hepatic cells. Archives of toxicology 91, 2405–2423. https://doi.org/10. 1007/s00204-016-1893-6.
CYP1A2-overexpressing and parental HepG2 cells (104 cells/well) were seeded into 96-well plates (Sarstedt AG & Co. KG). The next day, cells were treated with eight different concentrations of aflatoxin B1 (AFB1) from Aspergillus flavus (Sigma-Aldrich) at a range of 0 – 5 μM or with 0.1% DMSO (solvent control) in HepG2 standard medium. Cells were cultured for 72 h with daily medium change including the respective AFB1 or DMSO concentration. Cell viability was determined using the CellTiter-Glo®2.0 assay (Promega) according to the manufacturer´s instructions, and EC50 values were calculated. 4.11. Mycoplasma testing MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, Basel, Switzerland) was used according to the manufacturer´s protocol. In short, cells were cultivated for three days without medium change. Thereafter, 100 μl cell culture supernatant were transferred to a 96-well plate (Sarstedt AG & Co. KG), supplemented with 100 μl MycoAlert™ PLUS reagent, and the luminescence signals were measured with a FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany) resulting in reading A. Then, 100 μl of the MycoAlert™ PLUS substrate were applied and the luminescence signal was measured again (reading B). A ratio of reading B : reading A < 1 indicates absence of mycoplasma infection. 4.12. DNA fingerprinting analysis For checking authenticity of cell clones and parental HepG2 cells, we used the service CellCheck™ (9) from IDEXX BioAnalytics. This was based on short tandem repeat (STR) analysis using the Promega CELL ID™ System (8 STR markers + amelogenin). 4.13. Statistical analysis ImageJ (https://imagej.nih.gov/ij/index.html, version 1.52q) was used to determine band intensities for western blot quantification. GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA) was used for the statistical and non‐linear regression analysis to determine EC50 (half maximal effective concentration) values. To compare groups, 159
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
HepG2-1A2 C2 and C7: Lentivirus vector-mediated stable and functional overexpression of cytochrome P450 1A2 in human hepatoblastoma cells
T
Susanne Steinbrechta, Nadine Pfeifera, Natalie Herzoga, Nadine Katzenbergera, Christian Schulzb, Sarah Kammerera,*,1, Jan-Heiner Küppera,1 a
Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, Universitätsplatz 1, 01968 Senftenberg, Germany Project Group Pz-Syn of the Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses IZI-BB, Am Mühlenberg 13, 14476 Potsdam, located at the Brandenburg University of Technology Cottbus-Senftenberg, Germany
b
A R T I C LE I N FO
A B S T R A C T
Keywords: HepG2 CYP1A2 Cytochrome P450 Liver metabolism
Novel HepG2 cell clones 1A2 C2 and 1A2 C7 were independently generated by lentiviral transduction to functionally overexpress cytochrome P450 1A2 (CYP1A2). We found similar and stable CYP1A2 transcript and protein levels in both cell clones leading to specific enzyme activities of about 370 pmol paracetamol x min-1 x mg-1 protein analyzed by phenacetin conversion. Both clones showed dramatically increased sensitivity to the hepatotoxic compound aflatoxin B1 (EC50 < 100 nM) when compared to parental HepG2 cells (EC50 ∼5 μM). Thus, newly established cell lines are an appropriate tool to study metabolism and toxicity of substances depending on conversion by CYP1A2.
Link to related literature (direct URL links and full references)
1. Resource Table
Name of cell line construct
HepG2-1A2 C2, HepG2-1A2 C7
Institution
Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany Natalie Herzog (C2), Nadine Katzenberger (C7) Sarah Kammerer, [email protected] Apr, 2014 (C7); Oct, 2015 (C2) human hepatoblastoma cells (HepG2) HepG2 cells with stable overexpression of CYP1A2 Cell clone CYP1A2 Identity and purity of cell line confirmed
Person who created resource Contact person and email Date archived/stock date Origin Type of resource Sub-type Transfected or deleted genes Authentication
https://content.iospress.com/articles/journalof-cellular-biotechnology/jcb189013 Steinbrecht, S., Kammerer, S., Küpper, J.-H. HepG2 cells with recombinant cytochrome P450 enzyme overexpression: Their use and limitation as in vitro liver model. Journal of Cellular Biotechnology., DOI: 10.3233/JCB189013; https://content.iospress.com/articles/journalof-cellular-biotechnology/jcb179012 Kammerer, S., Küpper, J.-H. Expert Opinion: Human Hepatocyte Systems for in vitro Toxicology Analysis. Journal of Cellular Biotechnology., DOI: 10.3233/JCB-179012
Information in public databases
2. Resource Details Due to their unlimited availability and easy handling, human hepatoblastoma HepG2 cells are widely used for in vitro toxicology studies as alternative to primary human hepatocytes. HepG2 cells were derived from an epithelial hepatoblastoma of a 15-year-old Caucasian male (López-Terrada et al., 2009). However, compared to primary human hepatocytes, they have the disadvantage of limited biotransformation activity due to their low or even absent expression of cytochrome P450
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Corresponding author at: Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, Universitätsplatz 1, 01968 Senftenberg, Germany. E-mail address: [email protected] (S. Kammerer). 1 both authors shared the function as principal investigator https://doi.org/10.1016/j.toxlet.2019.11.006 Received 13 August 2019; Received in revised form 9 October 2019; Accepted 5 November 2019 Available online 06 November 2019 0378-4274/ © 2019 Elsevier B.V. All rights reserved.
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(Fig. 1A, B). Both cell clones showed significantly increased levels of CYP1A2 mRNA and protein expression when compared to parental HepG2 cells, as determined by qRT-PCR and western blot (Fig. 1C, D). In addition to expression analyses at mRNA and protein levels, CYP1A2 functionality was tested by enzyme activity assays. As first test system, we used the P450-Glo™ CYP1A2 induction/inhibition assay. While only low CYP1A2 activity could be measured in the parental HepG2 cells, it was increased by a factor of 700-800 in the HepG2-1A2 cell clones (Fig. 2A). In addition, specific enzyme activity of CYP1A2 was determined by conversion of the standard substrate phenacetin and HPLC-based measurement of its CYP1A2-dependent product paracetamol (Huang et al., 2012). Recombinant expression of CYP1A2 led to an enzyme activity of 363-373 pmol paracetamol x min-1 x mg-1 total cellular protein in both CYP-expressing HepG2 cell clones (Fig. 2B). In contrast, parental HepG2 cells lacked significant CYP1A2 activity. Inhibition studies involving 1-aminobenzotriazole (1-ABT), a pan-specific inactivator of CYPs involved in xenobiotic metabolism (de Montellano, 2018), showed that the high conversion rates of phenacetin to paracetamol are reversible (Fig. 2B) and thus were indeed associated with functional integration of recombinant CYP1A2 cDNA into HepG2 cells. It is well known that CYP1A2 is a major player in the biotransformation of aflatoxin B1 (AFB1) resulting in the production of genotoxic metabolites (Bbosa et al., 2013). When measuring EC50
oxidoreductases (CYPs) (Rodríguez-Antona et al., 2002; Westerink and Schoonen, 2007). Strategies to overcome the lack of CYP activities in HepG2 include, amongst other methods, usage of chemical agonists for CYP induction which, however, do not lift CYP1A2 enzyme activity to the levels of primary human hepatocytes (Westerink and Schoonen, 2007). Alternatively, the genetic engineering of these cells with e.g. lentiviral expression vectors coding for human CYPs can be applied. In the latter case, the virally encoded integrase is used for the efficient integration of recombinant DNA sequences into the mammalian genome to obtain HepG2 cells with a stable CYP overexpression and physiologically relevant enzyme activity levels (Steinbrecht et al., 2019a). CYP1A2 is one of the most abundant CYPs in the human liver that metabolizes about 20 % of clinically used drugs and several important endogenous compounds (Shu-Feng et al., 2009; Wang and Zhou, 2009). Here, we report the generation and characterization of two new genetically engineered HepG2 cell clones (HepG2-1A2 C2 and C7) with stable overexpression of human CYP1A2. The cell lines were generated by transduction of a lentivirus-based expression vector encoding human CYP1A2 cDNA, followed by single clone selection with antibiotic zeocin. HepG2-1A2 cell clones 2 and 7 both showed a cobblestone-like morphology typical for hepatocytes and have population doubling times that were not significantly different from the parental HepG2 cells
Fig. 1. Characterization of HepG2-1A2 C2 and C7. (A) Phase contrast microscopy of HepG2 and CYP1A2-overexpressing clones C2 and C7. Images were obtained by using the microscope CKX41 and 20x objective (scale bar 200 μm). (B) Doubling times of exponentially growing cultures of HepG2, HepG2-1A2 C2 and C7. Results are shown as mean ± SEM of two independent experiments; n.s.: not significant. C) mRNA expression levels in HepG2-1A2 C2 and C7 cells relative to parental HepG2 cells. Data represent the mean ± SEM; n = 5; *p < 0.05, ****p < 0.0001, each in comparison to the control cell line HepG2. (D) Western blot analysis of non-modified HepG2 cells and CYP1A2-expressing HepG2 cell clones C2 and C7. Equal protein amounts (20 μg) of HepG2, HepG2-1A2 C2 and C7, and 10 μg of microsomes as positive control were used for western blotting. Detection of GAPDH served as loading control. Note that microsomal protein extracts do not contain GAPDH. Top: A representative blot is shown. Bottom: Quantitative Western blot data are shown as CYP1A2/GAPDH ratio of luminescent band intensities as mean ± SEM; n = 3; **p < 0.01, ***p < 0.001, each in comparison to the control cell line HepG2. 156
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Fig. 2. Functional analysis of CYP1A2 in HepG2 and CYP1A2-overexpressing cell clones. (A) Relative enzyme activity of CYP1A2 in HepG2-1A2 cell clones C2 and C7 relative to parental HepG2 cells measured by the P450Glo™ CYP1A2 induction/inhibition assay. Results are shown as mean ± SEM; n = 4; ****p < 0.0001 in comparison to the control cell line HepG2. Please note that the bar of HepG2 is not visible due to the comparatively low values. (B) Determination of enzyme activity in HepG2 cells and CYP1A2-overexpressing cell clones C2 and C7 by conversion of phenacetin to paracetamol. Paracetamol concentration in the cell culture supernatant after 1 h phenacetin treatment of the cells was measured by HPLC and related to the total amount of protein. For inhibition studies, cells were incubated with 1 mM 1-aminobenzotriazole (1-ABT) 4 h before treatment with phenacetin. Data represent mean values ± SEM; n = 3; ****p < 0.0001 in comparison to the control cell line HepG2; + +++p < 0.0001 in comparison to previous treatment with 1-ABT. (C) Relative cell viability and calculated EC50 values of HepG2 cell clones C2 and C7 in comparison to HepG2 cells after treatment with different concentrations of aflatoxin B1 as evaluated by CellTiter-Glo®2.0 ATP assay. Results are shown as mean ± SEM of two independent experiments. Statistical analysis is given in comparison to the control cell line HepG2.
toxicological and metabolic studies.
values, CYP1A2-overexpressing HepG2 cell clones showed a remarkable 70- (clone C2; p < 0.0001) and 130-fold (clone C7; p < 0.0001) higher sensitivity towards AFB1 compared to parental HepG2 cells (Fig. 2C). Experiments shown here were performed over a range of 20 passages and results did not differ between younger and older passages. Therefore, we assume a very high stability of CYP1A2 expression in both cell clones even under long-term culture conditions. When studying new drugs, it will become necessary to analyze additional CYP isoforms known to be involved in human drug metabolism. This might be realized using freshly isolated primary hepatocytes, primary-like hepatocytes such as upcyte® and HepaFH3 cells (Herzog et al., 2016; Ramachandran et al., 2015) or panels of HepG2 cells with overexpression of single or multiple CYP enzymes (Wu et al., 2017). Our newly generated CYP1A2-overexpressing HepG2 clones C2 and C7 display high stability of CYP1A2 expression as well as functional activity which makes them valuable lab resources for studies on CYP1A2dependent metabolism. Both clones represent a well characterized, robust and easy-to-handle in vitro tool for different kinds of
3. Verification and Authentication The results of DNA fingerprinting analysis to indicate cell authenticity revealed an identical STR pattern of the CYP1A2-expressing cell clones to those of their parental HepG2 cells (Data available with the authors). The mycoplasma test by luminescence was negative for both HepG2-1A2 clones and parental HepG2 cells (Supplementary material, Table S1). 4. Materials and Methods 4.1. Cell Culture Human hepatoblastoma (HepG2) cells (HB-8065; ATCC, Manassas, USA) were cultivated in Dulbecco’s minimal essential medium (DMEM) (Biochrom AG, Berlin, Germany) supplemented with 10 % fetal bovine serum (Biochrom AG) and 2 mM L-glutamine (PAA Laboratories GmbH, 157
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to the manufacturer´s protocol. Total RNA was treated with DNase I (Thermo Fisher Scientific) to remove genomic DNA, and RNA integrity was controlled by 1 % agarose gel electrophoresis. Isolated RNA was used for synthesis of cDNA according to manufacturer´s instruction using oligo(dT)18 primer and RevertAid Reverse Transcriptase (Thermo Fisher Scientific). Resulting cDNA was diluted 1:10 and qRT-PCR was performed for CYP1A2 (forward primer: 5`-CTGGAGACCTTCCGACA CTC-3`; reverse primer: 5`-AGGGCTTGTTAATGGCAGTG-3`) and reference genes GAPDH (glyceraldehyde-3-phosphate dehydrogenase; forward primer: 5`-TGCACCACCAACTGCTTAGC-3`; reverse primer: 5`GGCATGGACTGTGGTCATGAG-3`) and RPL-P0 (Ribosomal Protein Lateral Stalk Subunit P0; forward primer: 5`-AAATGTTTCATTGTGGG AGC-3`; reverse primer: 5`-ATATGAGGCAGCAGTTTCTC-3`; all primers from BioTez, Berlin, Germany). Primers, Maxima Probe qPCR Master Mix (Thermo Fisher Scientific), Evagreen (Biotium, Inc., Fremont, California, USA) and cDNA were mixed accordingly in a 10 μl reaction volume per sample and the following settings on a C1000 Touch™ Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, USA) were used: initial denaturation at 95 °C for 3 min; 50 cycles of 95 °C for 10 s, 62 °C for 10 s and 72 °C for 30 s; final elongation at 72 °C for 30 s. Size of PCR products was controlled by a 3 % agarose gel electrophoresis. Relative expression levels were calculated from the threshold cycle (CT) values according to the 2-ΔΔCT method.
Pasching, Austria) at 37 °C and 5 % CO2 in a humidified incubator. The cells were routinely passaged by trypsinization with 0.05 % (w/v) Trypsin/0.02 % (w/v) EDTA (Biochrom AG). Medium for CYP1A2overexpressing HepG2 clones was supplemented with 500 μg x ml-1 zeocin (Thermo Fisher Scientific Inc., Waltham, USA). 4.2. Construction of plasmids Plasmids were constructed using the Invitrogen™ Gateway® Technology from Thermo Fisher Scientific according to the manufacturer´s protocol. In brief, an entry vector (GeneCopoeia™, Rockville, USA) that already contained the coding sequence for CYP1A2 (NCBI reference sequence: BC067428) flanked by attachment sites (attL) was used to recombine the CYP1A2 cDNA sequences into the attR-containing destination vector pLenti4/V5-DEST (Thermo Fischer Scientific) using the LR recombination reaction. Consequently, the CYP1A2 sequence in the resulting lentivirus expression vector was controlled by the human cytomegalovirus (CMV) promoter. The resulting vector plasmid pLenti4/V5-DEST-CYP1A2 was then used to generate recombinant lentivirus particles. 4.3. Lentivirus packaging For recombinant lentivirus generation, the ViraPower™ Lentiviral Expression System from Thermo Fisher Scientific was used according to the manufacturer´s protocol. Virus production was based on helper cell line 293FT (Thermo Fisher Scientific; R70007) which was routinely cultivated in Dulbecco’s MEM supplemented with 10 % fetal bovine serum, 0.1 mM non-essential amino acids, 4 mM L-glutamine, 1 mM sodium pyruvate and 500 μg/ml Geneticin® (all from Biochrom AG) at 37 °C and 5 % CO2. The cells were cotransfected with vector plasmid pLenti4/V5-DEST-CYP1A2 along with helper plasmids pLP1, pLP2, pLP/VSVG (Thermo Fisher Scientific) via Lipofectamine™ 2000 (Thermo Fisher Scientific). Next day, the medium was replaced with fresh culture medium and after an additional incubation period of 4872 h, recombinant lentivirus was collected from the supernatant. Virus solution was 20 fold concentrated by a filtration system of Sartorius (Vivaspin® 20) and used directly for HepG2 infection.
4.7. Western blot The general procedure of Western Blot analysis was adopted from our previous publication (Steinbrecht et al., 2019b) with following modifications: For CYP1A2-overexpressing and parental HepG2 cells, 20 μg total protein and 10 μg total protein of microsomes (pooled from 50 different individual donors; Thermo Fisher Scientific) were applied per lane for SDS-PAGE. For the detection of CYP1A2 and the reference gene GAPDH, anti-CYP1A2 monoclonal antibody (Thermo Fisher Scientific; dilution 1:2000) and anti-GAPDH monoclonal antibody (Antikoerper-online GmbH, Aachen, Germany; dilution 1:5000) were used as primary antibodies. 4.8. Determination of enzyme activity by P450-Glo™ and CellTiter-Glo® 2.0 assay
4.4. Lentiviral infection of HepG2 cells Cells were seeded at 3 × 104 cells/well into 96-well plates (Sarstedt AG & Co. KG) 24 h before start of analysis with the P450-Glo™ CYP1A2 induction/inhibition assay (Promega, Madison, WI, USA) that was used according to the manufacturer’s protocol. In brief, cells were incubated with 50 μl CYP1A2 substrate diluted in PBS for 60 min at 37 °C. Then, 25 μl of supernatants were transferred into a white-walled 96-well plate (Sarstedt AG & Co. KG), and an equal volume of detection reagent was added followed by incubation for 20 min at room temperature in the dark. Thereafter, luminescence signals were measured with FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany). In addition, the cells and the 25 μl substrate solution remaining in the wells were mixed with 25 μl ATP reagent solution of the CellTiter-Glo®2.0 assay (Promega). Thus, the ATP level could be determined according to the manufacturer's instructions to allow normalization to the seeded cell number.
HepG2 cells were grown to 60 % confluence in 24-well plates (Sarstedt AG & Co. KG, Nümbrecht, Germany). Then, concentrated recombinant lentivirus solution and 6 μg x ml-1 hexadimethrine bromide (Sigma-Aldrich, St. Louis, USA) were added. After 24 h, infection medium was replaced with fresh HepG2 medium (see above) followed by start of selection pressure (500 μg x ml-1 zeocin) 48 hours post infection. Within 3 – 4 weeks, zeocin-resistant cell colonies were picked and further expanded in selection medium to obtain single cell-derived HepG2 clones. 4.5. Determination of population doubling times 4 × 104 cells/well were seeded into 6-well plates (Sarstedt AG & Co. KG) and cultured under standard conditions without antibiotics. Cells were counted daily using a Neubauer hemocytometer and population doubling times (PDT) were determined using the formula PDT = (t2t1)*log2/log(q2/q1), with t1-t2 = time interval of constant growth rate in hours, q1 = cell number per cm2 at t1, and q2 = cell number per cm2 at t2.
4.9. Determination of enzyme activity by phenacetin conversion Cells were seeded at 2 × 106 cells per well into 6-well plates (Sarstedt AG & Co. KG) and cultured for 48 h. Cells were first washed with Krebs-Henseleit Buffer (KHB; completed with 25 mM NaHCO3, 25 mM HEPES, 2 mM CaCl2 x 2H2O; pH 7.4; all from Sigma-Aldrich except HEPES from Carl Roth GmbH + Co. KG, Karlsruhe, Germany). Then, cells were incubated with 360 μM of CYP1A2 substrate phenacetin (Sigma-Aldrich) diluted in KHB (500 μl per well) for 1 h in a humidified incubator at 5 % CO2 and 37 °C. For inhibition studies, 1 mM
4.6. Quantitative reverse transcriptase-polymerase chain reaction (qRTPCR) Total RNA was extracted from cell pellets (1-4 × 106 cells) using the innuPREP RNA Mini Kit (Analytik Jena AG, Jena, Germany) according 158
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one-way ANOVA with Turkey’s or Dunnett’s multiple comparison test using the respective control was performed. Results were considered as statistically significant if p < 0.05.
1-aminobenzotriazole (1-ABT; Sigma-Aldrich) was added to the cell medium 4 h prior to phenacetin treatment. After phenacetin treatment with or without 1-ABT, supernatants were collected, centrifuged at 13,000 x g for 15 min and metabolite formation was measured using VWR-Hitachi Elite LaChrom series HPLC (VWR International GmbH, Darmstadt, Germany) with photodiode array L-2455 (column: Phenomenex Kinetex C18, 4.6 × 150 mm, 5 μm particle size, 100 A°; security guard CK18, 4.6 × 2 mm). HPLC method: injection volume 20 μl; flow rate 1.0 ml x min-1; column temperature 40 °C; mobile phase A (10 mM KH2PO4; pH 3); mobile phase B (acetonitrile); HPLC gradient profile: 0-5 min eluent B 5 %, 15 min eluent B 40 %, 16 min eluent B 90 %, 20 min eluent B 90 %. In general, eluted substances were detected in the wavelength range 210–400 nm by the detector. The metabolite paracetamol was identified by comparison to an authentic standard (Dr. Ehrenstorfer GmbH, Augsburg, Germany) based on retention time and UV absorption spectrum. Quantification of paracetamol was performed by using a calibration curve at the wavelength of its absorption maximum of 245 nm. To determine enzyme activity in relation to total cellular protein, cells were trypsinized, pelleted by centrifugation, resuspended in RIPA buffer (50 mM Tris, 150 mM NaCl, 0.5 % DOC, 1 % NP-40, 0.5 % SDS, 1 mM PMSF, pH 8) and incubated on ice for 15 min followed by centrifugation (10,000 x g, 10 min, 4 °C). Protein concentrations of lysate supernatants were analyzed with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) according to manufacturer’s protocol.
Declaration of Competing Interest The authors report no declarations of interest. Acknowledgements This work was supported by the European Regional Development Fund (ERDF, Brandenburg, Germany; project “PERsonalisierte Medizin durch FUNCTIONomics in Berlin-Brandenburg: DrugMetabolisierungsmodul für Wirkstofftests an Patientenzellen”, project number: 85002925; and project “Entwicklung eines physiologisch relevanten Testsystems zur In-vitro-Erfassung von Hepatotoxizität im Hochdurchsatz”, project number: 85009748). We thank Kai-Uwe Schmidtke, Institute of Biotechnology, BTU Cottbus-Senftenberg for support with HPLC measurements. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.toxlet.2019.11.006. References
4.10. Cell treatment with aflatoxin B1 Bbosa, G.S., Kitya, D., Odda, J., Ogwal-Okeng, J., 2013. Aflatoxins metabolism, effects on epigenetic mechanisms and their role in carcinogenesis. Health 5, 14–34. https://doi. org/10.4236/health.2013.510A1003. de Montellano, P.R.O., 2018. 1-Aminobenzotriazole: A Mechanism-Based Cytochrome P450 Inhibitor and Probe of Cytochrome P450 Biology. Med Chem (Los Angeles) 8, 038–065. https://doi.org/10.4172/2161-0444.1000495. Herzog, N., Hansen, M., Miethbauer, S., Schmidtke, K.U., Anderer, U., Lupp, A., Sperling, S., Seehofer, D., Damm, G., Scheibner, K., Kupper, J.H., 2016. Primary-like human hepatocytes genetically engineered to obtain proliferation competence display hepatic differentiation characteristics in monolayer and organotypical spheroid cultures. Cell biology international 40, 341–353. https://doi.org/10.1002/cbin.10574. Huang, Q., Deshmukh, R.S., Ericksen, S.S., Tu, Y., Szklarz, G.D., 2012. Preferred Binding Orientations of Phenacetin in CYP1A1 and CYP1A2 Are Associated with IsoformSelective Metabolism. Drug Metabolism and Disposition 40, 2324–2331. https://doi. org/10.1124/dmd.112.047308. López-Terrada, D., Cheung, S.W., Finegold, M.J., Knowles, B.B., 2009. Hep G2 is a hepatoblastoma-derived cell line. Human Pathology 40, 1512–1515. https://doi.org/ 10.1016/j.humpath.2009.07.003. Ramachandran, S.D., Vivares, A., Klieber, S., Hewitt, N.J., Muenst, B., Heinz, S., Walles, H., Braspenning, J., 2015. Applicability of second-generation upcyte(R) human hepatocytes for use in CYP inhibition and induction studies. Pharmacology research & perspectives 3, e00161. https://doi.org/10.1002/prp2.161. Rodríguez-Antona, C., Donato, M.T., Boobis, A., Edwards, R.J., Watts, P.S., Castell, J.V., Gómez-Lechón, M.J., 2002. Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells. Xenobiotica 32, 505–520. https://doi.org/10.1080/ 00498250210128675. Shu-Feng, Z., Li-Ping, Y., Ming Qian, W., Wei, D., Eli, C., 2009. Insights into the Structure, Function, and Regulation of Human Cytochrome P450 1A2. Current Drug Metabolism 10, 713–729. https://doi.org/10.2174/138920009789895552. Steinbrecht, S., Kammerer, S., Küpper, J.-H., 2019a. HepG2 cells with recombinant cytochrome P450 enzyme overexpression: Their use and limitation as in vitro liver model. Journal of Cellular Biotechnology 5, 55–64. https://doi.org/10.3233/JCB189013. Steinbrecht, S., König, R., Schmidtke, K.-U., Herzog, N., Scheibner, K., Krüger-Genge, A., Jung, F., Kammerer, S., Küpper, J.-H., 2019b. Metabolic activity testing can underestimate acute drug cytotoxicity as revealed by HepG2 cell clones overexpressing cytochrome P450 2C19 and 3A4. Toxicology 412, 37–47. https://doi.org/10.1016/j. tox.2018.11.008. Wang, B., Zhou, S.F., 2009. Synthetic and Natural Compounds that Interact with Human Cytochrome P450 1A2 and Implications in Drug Development. Current Medicinal Chemistry 16, 4066–4218. https://doi.org/10.2174/092986709789378198. Westerink, W.M.A., Schoonen, W.G.E.J., 2007. Cytochrome P450 enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells. Toxicology in Vitro 21, 1581–1591. https://doi.org/10.1016/j.tiv.2007.05.014. Wu, Y., Chitranshi, P., Loukotková, L., Gamboa da Costa, G., Beland, F.A., Zhang, J., Fang, J.-L., 2017. Cytochrome P450-mediated metabolism of triclosan attenuates its cytotoxicity in hepatic cells. Archives of toxicology 91, 2405–2423. https://doi.org/10. 1007/s00204-016-1893-6.
CYP1A2-overexpressing and parental HepG2 cells (104 cells/well) were seeded into 96-well plates (Sarstedt AG & Co. KG). The next day, cells were treated with eight different concentrations of aflatoxin B1 (AFB1) from Aspergillus flavus (Sigma-Aldrich) at a range of 0 – 5 μM or with 0.1% DMSO (solvent control) in HepG2 standard medium. Cells were cultured for 72 h with daily medium change including the respective AFB1 or DMSO concentration. Cell viability was determined using the CellTiter-Glo®2.0 assay (Promega) according to the manufacturer´s instructions, and EC50 values were calculated. 4.11. Mycoplasma testing MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, Basel, Switzerland) was used according to the manufacturer´s protocol. In short, cells were cultivated for three days without medium change. Thereafter, 100 μl cell culture supernatant were transferred to a 96-well plate (Sarstedt AG & Co. KG), supplemented with 100 μl MycoAlert™ PLUS reagent, and the luminescence signals were measured with a FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany) resulting in reading A. Then, 100 μl of the MycoAlert™ PLUS substrate were applied and the luminescence signal was measured again (reading B). A ratio of reading B : reading A < 1 indicates absence of mycoplasma infection. 4.12. DNA fingerprinting analysis For checking authenticity of cell clones and parental HepG2 cells, we used the service CellCheck™ (9) from IDEXX BioAnalytics. This was based on short tandem repeat (STR) analysis using the Promega CELL ID™ System (8 STR markers + amelogenin). 4.13. Statistical analysis ImageJ (https://imagej.nih.gov/ij/index.html, version 1.52q) was used to determine band intensities for western blot quantification. GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA) was used for the statistical and non‐linear regression analysis to determine EC50 (half maximal effective concentration) values. To compare groups, 159