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Cellular Metabolism in High-Throughput In Vitro Reporter Gene Assays and Implications for the Quantitative In Vitro-In Vivo Extrapolation.
Chemical Research in Toxicology ( IF 3.7 ) Pub Date : 2020-03-31 , DOI: 10.1021/acs.chemrestox.0c00037 Fabian C Fischer 1 , Cedric Abele 1 , Luise Henneberger 1 , Nils Klüver 2 , Maria König 1 , Marie Mühlenbrink 1 , Rita Schlichting 1 , Beate I Escher 1, 3
Chemical Research in Toxicology ( IF 3.7 ) Pub Date : 2020-03-31 , DOI: 10.1021/acs.chemrestox.0c00037 Fabian C Fischer 1 , Cedric Abele 1 , Luise Henneberger 1 , Nils Klüver 2 , Maria König 1 , Marie Mühlenbrink 1 , Rita Schlichting 1 , Beate I Escher 1, 3
Affiliation
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High-throughput in vitro reporter gene assays are increasingly applied to assess the potency of chemicals to alter specific cellular signaling pathways. Genetically modified reporter gene cell lines provide stable readouts of the activation of cellular receptors or transcription factors of interest, but such reporter gene assays have been criticized for not capturing cellular metabolism. We characterized the metabolic activity of the widely applied AREc32 (human breast cancer MCF-7), ARE-bla (human liver cancer HepG2), and GR-bla (human embryonic kidney HEK293) reporter gene cells in the absence and in the presence of benzo[a]pyrene (BaP), an AhR ligand known to upregulate cytochrome P450 in vitro and in vivo. We combined fluorescence microscopy with chemical analysis, real-time PCR, and ethoxyresorufin-O-deethylase activity measurements to track temporal changes in BaP and its metabolites in the cells and surrounding medium over time in relation to the expression and activity of metabolic enzymes. Decreasing BaP concentrations and formation of metabolites agreed with the high basal CYP1 activity of ARE-bla and the strong CYP1A1 mRNA induction in AREc32, whereas BaP concentrations were constant in GR-bla, in which neither metabolites nor CYP1 induction was detected. The study emphasizes that differences in sensitivity between reporter gene assays may be caused not only by different reporter constructs but also by a varying biotransformation rate of the evaluated parent chemical. The basal metabolic capacity of reporter gene cells in the absence of chemicals is not a clear indication because we demonstrated that the metabolic activity can be upregulated by AhR ligands during the assay. The combination of methods presented here is suitable to characterize the metabolic activity of cells in vitro and can improve the interpretation of in vitro reporter gene effect data and extrapolation to in vivo human exposure.
中文翻译：
高通量体外报道基因基因测定中的细胞代谢及其对体内体外定量分析的影响。
高通量体外报道基因测定法越来越多地用于评估化学物质改变特定细胞信号通路的能力。基因修饰的报告基因细胞系可稳定读出感兴趣的细胞受体或转录因子的激活，但这种报告基因测定法因未捕获细胞代谢而受到批评。我们表征了在不存在和存在下，广泛应用的AREc32（人类乳腺癌MCF-7），ARE-bla（人类肝癌HepG2）和GR-bla（人类胚胎肾脏HEK293）报告基因细胞的代谢活性。苯并[ a ] py（BaP），一种在体内和体外上调细胞色素P450的AhR配体。我们将荧光显微镜与化学分析，实时PCR和乙氧基异氟脲-O-脱乙基酶活性测量相结合，以追踪BaP及其在细胞和周围介质中的代谢产物随时间变化的时间变化，以反映代谢酶的表达和活性。降低BaP浓度和形成代谢产物与ARE-bla的高基础CYP1活性和强CYP1A1一致AREc32中的mRNA诱导，而GR-bla中的BaP浓度恒定，其中未检测到代谢物或CYP1诱导。该研究强调，报道基因测定之间灵敏度的差异不仅可能是由于报道基因结构不同，还可能是由于所评估的母体化学物质的生物转化率不同。没有化学物质时，报告基因细胞的基础代谢能力尚不明确，因为我们证明了在测定过程中，AhR配体可以上调代谢活性。此处介绍的方法的组合适用于表征体外细胞的代谢活性，并且可以改善对体外报道基因效应数据的解释和向体内的外推 人体暴露。
更新日期：2020-03-31
中文翻译：
高通量体外报道基因基因测定中的细胞代谢及其对体内体外定量分析的影响。
高通量体外报道基因测定法越来越多地用于评估化学物质改变特定细胞信号通路的能力。基因修饰的报告基因细胞系可稳定读出感兴趣的细胞受体或转录因子的激活，但这种报告基因测定法因未捕获细胞代谢而受到批评。我们表征了在不存在和存在下，广泛应用的AREc32（人类乳腺癌MCF-7），ARE-bla（人类肝癌HepG2）和GR-bla（人类胚胎肾脏HEK293）报告基因细胞的代谢活性。苯并[ a ] py（BaP），一种在体内和体外上调细胞色素P450的AhR配体。我们将荧光显微镜与化学分析，实时PCR和乙氧基异氟脲-O-脱乙基酶活性测量相结合，以追踪BaP及其在细胞和周围介质中的代谢产物随时间变化的时间变化，以反映代谢酶的表达和活性。降低BaP浓度和形成代谢产物与ARE-bla的高基础CYP1活性和强CYP1A1一致AREc32中的mRNA诱导，而GR-bla中的BaP浓度恒定，其中未检测到代谢物或CYP1诱导。该研究强调，报道基因测定之间灵敏度的差异不仅可能是由于报道基因结构不同，还可能是由于所评估的母体化学物质的生物转化率不同。没有化学物质时，报告基因细胞的基础代谢能力尚不明确，因为我们证明了在测定过程中，AhR配体可以上调代谢活性。此处介绍的方法的组合适用于表征体外细胞的代谢活性，并且可以改善对体外报道基因效应数据的解释和向体内的外推 人体暴露。
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