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Validation of in vitro assays to measure cytotoxicity in 3D cell cultures
Abstracts / Toxicology Letters 229S (2014) S40–S252
P-2.161 Validation of in vitro assays to measure cytotoxicity in 3D cell cultures Terry Riss 1,∗ , Michael Valley 1 , Kevin Kupcho 1 , Chad Zimprich 1 , Donna Leippe 1 , Andrew Niles 1 , Jolanta Vidugiriene 1 , James Cali 1 , Jens Kelm 2 , Wolfgang Moritz 2 , Dan Lazar 1 Promega Corporation, Madison, WI, USA, 2 InSphero AG, Zurich, Switzerland
1
The use of 3D cell cultures continues to emerge as improved in vitro models that are more physiologically relevant and more predictive of cytotoxic events in whole animals. Cells cultured in 3D models often acquire large in vivo-like structures compared to the thickness of a 2D monolayer of cells grown on standard plastic plates. Multicellular 3D culture systems containing more than one cell type and exhibiting formation of a complex extracellular matrix provide a challenge for assay chemistries originally designed for measuring events from monolayers of cells. There is an unmet need for guidelines for design and verification of assays effective for larger 3D microtissues. Critical factors to consider for each model system include effective penetration of detection reagents and/or complete lysis of cells using combinations of detergent and physical disruption. We will present the results of experiments designed to improve performance of assays for 3D spheroids. We have improved the lytic capacity of assay reagents by optimizing the detergent concentration and the parameters for physical disruption necessary to extract the desired markers. Improved protocols and reagents will be described to measure ATP as a cell viability marker, caspase-3/7 activity as an apoptosis marker, glutathione as a marker of oxidative stress and HIF-1 promoter driven expression of luciferase as a hypoxia marker. http://dx.doi.org/10.1016/j.toxlet.2014.06.508 P7: Carcinogenesis P-3.1 Impact of p53 on the metabolic activation of the carcinogenic air pollutant 3-nitrobenzanthrone and its human metabolite 3-aminobenzanthrone in human cells Ann-Christin Baranski 1 , Annette M. Krais 1 , Laura U.E. Wohak 1 , Osman Sozeri 1 , Heinz H. Schmeiser 2 , David H. Phillips 1 , Volker M. Arlt 1,∗ King’s College London, London, UK, 2 German Cancer Research Center (DKFZ), Heidelberg, Germany
1
3-Nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen linked to diesel exposure. We previously found that the cellular TP53 status can influence the metabolism of environmental carcinogens such as benzo[a]pyrene. To further investigate the role of TP53 in the bioactivation of 3-NBA and its human metabolite 3-aminobenzanthrone (3-ABA) a panel of isogenic colorectal HCT116 cells was used, differing only with respect to their endogenous TP53 status. HCT116 cells having TP53(+/+), TP53(+/−) or TP53(−/−) were treated with 1 M 3-NBA or 10 M 3-ABA up to 48 h. Treatment with 3-NBA resulted in DNA adduct levels, as analysed by 32 P-postlabelling, up to ∼800 adducts per 108 nucleotides but the cellular TP53 status had no influence on DNA adduct formation. In contrast, DNA adduct levels induced by 3-ABA were up to 15 times lower in TP53(+/−) and TP53(−/−) relative to TP53(+/+) cells indicating that 3-ABA
S145
metabolism depends on wild-type p53 function. No change in NQO1 and CYP1A1 mRNA expression, determined by qRT-PCR, was observed in 3-NBA-treated cells, cytosolic NQO1 being considered the most efficient nitroreductase activating 3-NBA. After 3-ABA treatment CYP1A1 mRNA expression was up to 5-fold lower in TP53(+/−) and TP53(−/−) relative to TP53(+/+) cells, indicating that loss of wild-type p53 function is linked to lower CYP1A1mediated 3-ABA-DNA adduct formation. Collectively, this study demonstrates a link between p53 and the expression of CYP1A1 by some environmental pollutants and reveals a novel role for p53 in xenobiotic metabolism. http://dx.doi.org/10.1016/j.toxlet.2014.06.510 P-3.2 Impact of P53 on DNA damage and metabolic activation of 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine (PHIP) in TRP53(+/+), TRP53(+/−) and TRP53(−/−) mice Annette M. Krais 1 , Joost P.M. Melis 2,3 , Ewoud N. Speksnijder 3 , Marie Stiborova 4 , Rajinder Singh 5 , Goncalo Gamboa da Costa 6 , Mirjam Luijten 2 , David H. Phillips 1 , Volker M. Arlt 1,∗ King’s College London, London, UK, 2 National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands, 3 Leiden University Medical Center, Leiden, The Netherlands, 4 Charles University, Prague, Czech Republic, 5 University of Leicester, Leicester, UK, 6 National Center for Toxicological Research (NCTR), Jefferson, USA 1
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed during the cooking of foods and is regarded as a potential human carcinogen. PhIP is metabolically activated by cytochrome P450 (CYP) enzymes, mainly CYP1A2. Recent findings in human cells have indicated that the cellular TP53 status can influence the metabolism of environmental carcinogens suggesting a new role for TP53 in CYP1A-mediated carcinogen bioactivation. In order to investigate the potential role of p53 in the in vivo metabolism of PhIP, Trp53(+/+), Trp53(+/−) and Trp53(−/−) mice were treated with a single oral dose of 50 mg/kg body weight PhIP and tissues were collected after 24 h. DNA adduct formation by PhIP (dG-C8-PhIP) was analysed by liquid chromatography-tandem mass spectrometry. In the liver 65.9 ± 27.6, 42.9 ± 19.7 and 31.0 ± 4.4 adducts per 108 nucleotides were detected in Trp53(+/+), Trp53(+/−) and Trp53(−/−) mice, respectively. Similarly, a ∼2-fold lower DNA binding was also observed in the colon of Trp53(−/−) relative to Trp53(+/+) mice. Cyp1a1 and Cyp1a2 protein levels, determined by Western blotting, were lower in the livers of PhIP-treated Trp53(−/−) than in Trp53(+/+) mice. Hepatic microsomes were also analysed for specific Cyp1a1 and Cyp1a2 activities by monitoring ethoxyresorufin O-deethylation (EROD) [Cyp1a1/2] and methoxyresorufin O-deethylation (MROD) [Cyp1a2] which were both ∼2.5 times lower in Trp53(−/−) than in Trp53(+/+) mice. Cyp1a protein levels/activities in the livers correlated with the DNA adduct formation by PhIP in this tissue in each mouse line. Collectively, our results indicate that p53 can affect the expression of Cyp1a in vivo after PhIP treatment. http://dx.doi.org/10.1016/j.toxlet.2014.06.511
P-2.161 Validation of in vitro assays to measure cytotoxicity in 3D cell cultures Terry Riss 1,∗ , Michael Valley 1 , Kevin Kupcho 1 , Chad Zimprich 1 , Donna Leippe 1 , Andrew Niles 1 , Jolanta Vidugiriene 1 , James Cali 1 , Jens Kelm 2 , Wolfgang Moritz 2 , Dan Lazar 1 Promega Corporation, Madison, WI, USA, 2 InSphero AG, Zurich, Switzerland
1
The use of 3D cell cultures continues to emerge as improved in vitro models that are more physiologically relevant and more predictive of cytotoxic events in whole animals. Cells cultured in 3D models often acquire large in vivo-like structures compared to the thickness of a 2D monolayer of cells grown on standard plastic plates. Multicellular 3D culture systems containing more than one cell type and exhibiting formation of a complex extracellular matrix provide a challenge for assay chemistries originally designed for measuring events from monolayers of cells. There is an unmet need for guidelines for design and verification of assays effective for larger 3D microtissues. Critical factors to consider for each model system include effective penetration of detection reagents and/or complete lysis of cells using combinations of detergent and physical disruption. We will present the results of experiments designed to improve performance of assays for 3D spheroids. We have improved the lytic capacity of assay reagents by optimizing the detergent concentration and the parameters for physical disruption necessary to extract the desired markers. Improved protocols and reagents will be described to measure ATP as a cell viability marker, caspase-3/7 activity as an apoptosis marker, glutathione as a marker of oxidative stress and HIF-1 promoter driven expression of luciferase as a hypoxia marker. http://dx.doi.org/10.1016/j.toxlet.2014.06.508 P7: Carcinogenesis P-3.1 Impact of p53 on the metabolic activation of the carcinogenic air pollutant 3-nitrobenzanthrone and its human metabolite 3-aminobenzanthrone in human cells Ann-Christin Baranski 1 , Annette M. Krais 1 , Laura U.E. Wohak 1 , Osman Sozeri 1 , Heinz H. Schmeiser 2 , David H. Phillips 1 , Volker M. Arlt 1,∗ King’s College London, London, UK, 2 German Cancer Research Center (DKFZ), Heidelberg, Germany
1
3-Nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen linked to diesel exposure. We previously found that the cellular TP53 status can influence the metabolism of environmental carcinogens such as benzo[a]pyrene. To further investigate the role of TP53 in the bioactivation of 3-NBA and its human metabolite 3-aminobenzanthrone (3-ABA) a panel of isogenic colorectal HCT116 cells was used, differing only with respect to their endogenous TP53 status. HCT116 cells having TP53(+/+), TP53(+/−) or TP53(−/−) were treated with 1 M 3-NBA or 10 M 3-ABA up to 48 h. Treatment with 3-NBA resulted in DNA adduct levels, as analysed by 32 P-postlabelling, up to ∼800 adducts per 108 nucleotides but the cellular TP53 status had no influence on DNA adduct formation. In contrast, DNA adduct levels induced by 3-ABA were up to 15 times lower in TP53(+/−) and TP53(−/−) relative to TP53(+/+) cells indicating that 3-ABA
S145
metabolism depends on wild-type p53 function. No change in NQO1 and CYP1A1 mRNA expression, determined by qRT-PCR, was observed in 3-NBA-treated cells, cytosolic NQO1 being considered the most efficient nitroreductase activating 3-NBA. After 3-ABA treatment CYP1A1 mRNA expression was up to 5-fold lower in TP53(+/−) and TP53(−/−) relative to TP53(+/+) cells, indicating that loss of wild-type p53 function is linked to lower CYP1A1mediated 3-ABA-DNA adduct formation. Collectively, this study demonstrates a link between p53 and the expression of CYP1A1 by some environmental pollutants and reveals a novel role for p53 in xenobiotic metabolism. http://dx.doi.org/10.1016/j.toxlet.2014.06.510 P-3.2 Impact of P53 on DNA damage and metabolic activation of 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine (PHIP) in TRP53(+/+), TRP53(+/−) and TRP53(−/−) mice Annette M. Krais 1 , Joost P.M. Melis 2,3 , Ewoud N. Speksnijder 3 , Marie Stiborova 4 , Rajinder Singh 5 , Goncalo Gamboa da Costa 6 , Mirjam Luijten 2 , David H. Phillips 1 , Volker M. Arlt 1,∗ King’s College London, London, UK, 2 National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands, 3 Leiden University Medical Center, Leiden, The Netherlands, 4 Charles University, Prague, Czech Republic, 5 University of Leicester, Leicester, UK, 6 National Center for Toxicological Research (NCTR), Jefferson, USA 1
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed during the cooking of foods and is regarded as a potential human carcinogen. PhIP is metabolically activated by cytochrome P450 (CYP) enzymes, mainly CYP1A2. Recent findings in human cells have indicated that the cellular TP53 status can influence the metabolism of environmental carcinogens suggesting a new role for TP53 in CYP1A-mediated carcinogen bioactivation. In order to investigate the potential role of p53 in the in vivo metabolism of PhIP, Trp53(+/+), Trp53(+/−) and Trp53(−/−) mice were treated with a single oral dose of 50 mg/kg body weight PhIP and tissues were collected after 24 h. DNA adduct formation by PhIP (dG-C8-PhIP) was analysed by liquid chromatography-tandem mass spectrometry. In the liver 65.9 ± 27.6, 42.9 ± 19.7 and 31.0 ± 4.4 adducts per 108 nucleotides were detected in Trp53(+/+), Trp53(+/−) and Trp53(−/−) mice, respectively. Similarly, a ∼2-fold lower DNA binding was also observed in the colon of Trp53(−/−) relative to Trp53(+/+) mice. Cyp1a1 and Cyp1a2 protein levels, determined by Western blotting, were lower in the livers of PhIP-treated Trp53(−/−) than in Trp53(+/+) mice. Hepatic microsomes were also analysed for specific Cyp1a1 and Cyp1a2 activities by monitoring ethoxyresorufin O-deethylation (EROD) [Cyp1a1/2] and methoxyresorufin O-deethylation (MROD) [Cyp1a2] which were both ∼2.5 times lower in Trp53(−/−) than in Trp53(+/+) mice. Cyp1a protein levels/activities in the livers correlated with the DNA adduct formation by PhIP in this tissue in each mouse line. Collectively, our results indicate that p53 can affect the expression of Cyp1a in vivo after PhIP treatment. http://dx.doi.org/10.1016/j.toxlet.2014.06.511