title: Determination of the CYP1A-inducing potential of single substances, mixtures and extracts of samples in the micro-EROD assay with H4IIE cells.
abstract: This protocol describes a quantitative and robust 96-well-plate-reader-based assay for the measurement of ethoxyresorufin-O-deethylase (EROD) activity using the rat hepatoma cell line H4IIE. The assay can be used to determine the cytochrome P450 subfamily 1A (CYP1A)-inducing potential of single substances, as well as of mixtures and extracts of samples. It is based on the aryl hydrocarbon receptor (AhR)-mediated induction of cytochrome P450 enzymes (subfamily 1A) in cells after exposure to dioxins and dioxin-like compounds. One enzymatic reaction catalyzed by CYP1A is the deethylation of the exogenous substrate 7-ethoxyresorufin to the fluorescent product resorufin, which is measured as EROD activity in the assay. The CYP1A-inducing potential of a sample can be reliably quantified by comparing the EROD activity with the concentration-response curve of the standard substance 2,3,7,8-tetrachlorodibenzo-p-dioxin, which can be detected at concentrations down to the picogram per liter range. A researcher familiar with the procedure can process up to 160 samples with four wells each within 3 d. The series described uses four plates with three concentrations per sample, which can be easily scaled to accommodate different sample sizes.
authors.affiliation: Department of Ecosystem Analysis, Institute for Environmental Research, Aachen Biology and Biotechnology (ABBt), Rheinisch-Westfälische Technische Hochschule Aachen (RWTH) Aachen University, Aachen, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schiwy
authors.affiliation: Department of Ecosystem Analysis, Institute for Environmental Research, Aachen Biology and Biotechnology (ABBt), Rheinisch-Westfälische Technische Hochschule Aachen (RWTH) Aachen University, Aachen, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Brinkmann
authors.affiliation: Food and Veterinary Institute Braunschweig/Hannover, Lower Saxony State Office for Consumer Protection and Food Safety (LAVES), Braunschweig, Germany.
authors.forename: Ines
authors.initials: I
authors.lastname: Thiem
authors.affiliation: Food and Veterinary Institute Braunschweig/Hannover, Lower Saxony State Office for Consumer Protection and Food Safety (LAVES), Braunschweig, Germany.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Guder
authors.affiliation: Department of Ecosystem Analysis, Institute for Environmental Research, Aachen Biology and Biotechnology (ABBt), Rheinisch-Westfälische Technische Hochschule Aachen (RWTH) Aachen University, Aachen, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Winkens
authors.affiliation: Department of Ecosystem Analysis, Institute for Environmental Research, Aachen Biology and Biotechnology (ABBt), Rheinisch-Westfälische Technische Hochschule Aachen (RWTH) Aachen University, Aachen, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Eichbaum
authors.affiliation: Department of Ecosystem Analysis, Institute for Environmental Research, Aachen Biology and Biotechnology (ABBt), Rheinisch-Westfälische Technische Hochschule Aachen (RWTH) Aachen University, Aachen, Germany.
authors.forename: Leonie
authors.initials: L
authors.lastname: Nüßer
authors.affiliation: Department of Ecosystem Analysis, Institute for Environmental Research, Aachen Biology and Biotechnology (ABBt), Rheinisch-Westfälische Technische Hochschule Aachen (RWTH) Aachen University, Aachen, Germany.
authors.forename: Beat
authors.initials: B
authors.lastname: Thalmann
authors.affiliation: Federal Institute of Hydrology (BFG), Department G3: Biochemistry, Ecotoxicology, Koblenz, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Buchinger
authors.affiliation: Federal Institute of Hydrology (BFG), Department G3: Biochemistry, Ecotoxicology, Koblenz, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Reifferscheid
authors.affiliation: Department of Ecosystem Analysis, Institute for Environmental Research, Aachen Biology and Biotechnology (ABBt), Rheinisch-Westfälische Technische Hochschule Aachen (RWTH) Aachen University, Aachen, Germany.
authors.forename: Thomas-Benjamin
authors.initials: TB
authors.lastname: Seiler
authors.affiliation: Food and Veterinary Institute Braunschweig/Hannover, Lower Saxony State Office for Consumer Protection and Food Safety (LAVES), Braunschweig, Germany.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Thoms
authors.affiliation: Department of Ecosystem Analysis, Institute for Environmental Research, Aachen Biology and Biotechnology (ABBt), Rheinisch-Westfälische Technische Hochschule Aachen (RWTH) Aachen University, Aachen, Germany.|College of Resources and Environmental Science, Chongqing University, Chongqing, China.|College of Environmental Science and Engineering and State Key Laboratory of Pollution Control and Resource Reuse, Tongji University, Shanghai, China.|State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, China.
authors.forename: Henner
authors.initials: H
authors.lastname: Hollert
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis* / Q000235:genetics; D004147:Dioxins / Q000633:toxicity*; D005453:Fluorescence; D005470:Fluorometry / Q000379:methods; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology*; D010078:Oxazines / Q000378:metabolism; D051381:Rats; D015533:Transcriptional Activation
chemical_list: D004147:Dioxins; D010078:Oxazines; C007649:ethoxyresorufin; C014180:resorufin; D019363:Cytochrome P-450 CYP1A1