Analytical-grade reagents and reference substances were obtained from Aldrich (Milwaukee, MN, USA). Phenobarbital (PB) was purchased from Abbott Laboratories (Mexico City, Mexico). Beta-naphthoflavone (β-NF), resorufin, 7-ethoxyresorufin (ER), methoxyresorufin (MR), benzyloxiresorufin (BR), pentoxyresorufin (BR), NADPH, BP, 2,2-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+), thiobarbituric acid (TBA), corn oil, and Giemsa stain were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Microsomes of Baculovirus expression systems from rat CYP1A1-expressing insect cells (Supersomes) were purchased from BD-Gentest (Woburn, MA, USA).
7 ethoxyresorufin
Manufactured by Merck Group
Sourced in United States, Germany
7-ethoxyresorufin is a fluorescent dye used in analytical applications as a substrate for enzyme activity assays. It is a sensitive indicator of various enzyme activities, particularly those involving cytochrome P450 enzymes.
Automatically generated - may contain errors
Lab products found in correlation
Resorufin, by Merck Group (8 mentions)
Nadph, by Merck Group (7 mentions)
Coumarin, by Merck Group (5 mentions)
Methoxyresorufin, by Merck Group (4 mentions)
7 pentoxyresorufin, by Merck Group (4 mentions)
1 chloro 2 4 dinitrobenzene, by Merck Group (4 mentions)
Glutathione reductase, by Merck Group (4 mentions)
Cytochrome c, by Merck Group (4 mentions)
Benzo a pyrene, by Merck Group (4 mentions)
5 5 dithiobis 2 nitrobenzoic acid, by Merck Group (3 mentions)
Cumene hydroperoxide, by Merck Group (3 mentions)
Dodecyl maltoside, by Merck Group (3 mentions)
2 7 dichlorofluorescein diacetate dcf da, by Thermo Fisher Scientific (3 mentions)
Coenzyme q2, by Merck Group (3 mentions)
Antimycin a, by Merck Group (3 mentions)
Chlorzoxazone, by Merck Group (3 mentions)
7 methoxyresorufin, by Merck Group (3 mentions)
Glucose 6 phosphate (g6p), by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Benzyloxiresorufin, by Merck Group (2 mentions)
33 protocols using 7 ethoxyresorufin
1
In Vitro Characterization of CYP1A1
2
Liver and Kidney Function Assessment in Wistar Rats
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Eight-week old white Wistar rats (180-200 g) were obtained from the Korle Bu Teaching Hospital. Grower mash (15%-16% protein, 3%-5% crude fiber, 1% calcium, 0.4% phosphorus, 0.65% lysine, 0.4% salt; with energy content of 2800 kcal/kg), which was used to feed the rats was from Kosher Feedmills, Accra, Ghana. Clean tap water was given as the source of drinking water, which was changed daily. The study was conducted in accordance with the guidelines provided by the Noguchi Memorial Institute for Medical Research of the University of Ghana, an internationally approved research facility in Ghana.
The following assay kits: alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), total bilirubin, direct or conjugated bilirubin, total serum protein, serum creatinine, and blood urea nitrogen (BUN) were obtained from Randox Chemicals Co (Antrim, UK). Nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), resorufin, 7-ethoxyresorufin, 7-pentoxyresorufin, zoxazolamine (2-amino-5-chlorobenzoxazole), bovine serum albumin (BSA), and perchloric acid were obtained from Sigma Chemical Co (St Louis, MO, USA). Sodium dithionite and dithiothreitol were from Fluka Garantie (St Loius, MO, USA), and p-nitrophenol was from BDH Laboratory (Radnor, PA, USA).
All other chemicals were obtained in their pure forms that were commercially available.
The following assay kits: alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), total bilirubin, direct or conjugated bilirubin, total serum protein, serum creatinine, and blood urea nitrogen (BUN) were obtained from Randox Chemicals Co (Antrim, UK). Nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), resorufin, 7-ethoxyresorufin, 7-pentoxyresorufin, zoxazolamine (2-amino-5-chlorobenzoxazole), bovine serum albumin (BSA), and perchloric acid were obtained from Sigma Chemical Co (St Louis, MO, USA). Sodium dithionite and dithiothreitol were from Fluka Garantie (St Loius, MO, USA), and p-nitrophenol was from BDH Laboratory (Radnor, PA, USA).
All other chemicals were obtained in their pure forms that were commercially available.
3
Quantifying Hepatic Enzyme Activities
4
Region-Specific EROD Activity Assay
5
Cytochrome P450 Enzyme Assay Protocol
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Beta-naphtoflavone (β-NF), 7-ethoxyresorufin (EROD), methoxyresorufin (MROD), benzyloxiresorufin (BROD), penthoxyresorufin (PROD), resorufin and benzo[a]pyrene (BP) were purchased from Sigma (St. Louis MO, USA). Culture media were obtained from BD Difco (Trenton, NJ, USA). The Escherichia coli DH5α recombinant and Salmonella typhimurium strains were kindly provided by Dr. Peter Guengerich (Vanderbilt University, Nashville, TN, USA).
6
Fluorescence-based CYP1A Induction Assay
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The induction of CYP1A is measured by the EROD-assay in the presence of NADPH (β-nicotinamide adenine dinucleotide phosphate). CYP1A converts the artificial substrate EROD to resorufin, which can be measured via fluorescence spectroscopy. The assay was done as described in [27 (link)] with minor modifications. Briefly, the cells were seeded in black 96 well plates (100,000 cells/cm2) and incubated for 24 h in culture medium. The cells were then incubated with the CYP1A inducer, beta-naphtaflavone (BNF; 1–100 nM; Sigma-Aldrich) for 24 h. The next day, the culture medium was replaced with 200 µL EROD assay media (DMEM w/o phenol red; Gibco™, Thermo Fisher, 10% FBS, 8 µM 7-Ethoxyresorufin; Sigma-Aldrich). After 30 min of incubation, resorufin fluorescence (Ex: 530 nm/Em: 580 nm) was quantified using a plate reader (Spectramax i3x plate reader, San Jose, CA, USA).
7
Mechanism-based CYP1A Inhibition Assay
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V79 Chinese hamster fibroblast cells were grown in a flat-bottom, 96-well plate for 2 days as described previously59 (link). 1-(1-Propynyl)-pyrene (1-PP) (synthesized by Prof. A. Seidel, Biochemical Institute for Environmental Carcinogens, Großhansdorf) in DMSO was serially diluted in culture medium to final concentrations of 0.1 nM to 10 μM. CYP activity was measured in V79 cell lines with cDNA-directed expression of human CYP1A1, CYP1A2, and CYP1B1. Cells were pre-incubated with 1-PP, a mechanism-based (suicide) inhibitor for CYP1A136 (link)60 (link), for 30 min at 37 °C and rinsed with PBS. Then, 100 μl 1 μM 7-ethoxyresorufin (Sigma-Aldrich, Steinheim, Germany) solved in sodium phosphate buffer (pH 8) was added at 37 °C for 15 min14 (link). The reaction was stopped with ice-cold methanol, and the resorufin thus formed was quantified with excitation/emission wavelengths of 544/590 nm. The percentage of inhibition was determined relative to a medium-only control, and the IC50 was estimated from the plots.
8
CYP1A1 Activity Quantification in Skin and Th17 Cells
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CYP1A1 enzymatic activity was measured in mouse tail epidermal sheet biopsies and in vitro skewed Th17 cells by the EROD assay, in which the 7-ethoxyresorufin (Sigma-Aldrich) is converted to resorufin (Sigma-Aldrich). Tail mouse skin was processed by the mechanical removal of subcutaneous fat tissue and floated on 0.5% w/v trypsin (Sigma-Aldrich) for 1 hour at 37 °C. Epidermal sheets were mechanically separated, and quadruplicate from each epidermal sheet was transferred to a 96-wells plate. EROD reaction was initiated by adding 2 μM 7-ethoxyresorufin in sodium phosphate buffer (50 mM, PH 8.0) to each well. The plate was incubated at 37 oC for 20 minutes, the reaction was stopped by adding fluorescamine (150 μg/ml in acetonitrile), and epidermal sheets biopsies were discarded. resorufin formation was measured against a resorufin standard curve on a plate reader (FLUOstar Omega, BMG LabTech, Ortenberg, Germany) with excitation and emission wavelengths of 535 and 590, respectively. CYP1A1 enzymatic activity was normalized for the skin protein content simultaneously determined by fluorescamine fluorescence of a standard curve of BSA (Sigma-Aldrich) at excitation and emission wavelengths of 390 and 480, respectively. In vitro skewed Th17 cells were assayed similarly, and CYP1A1 enzymatic activity was normalized to the IL-17 secreted in each well.
9
Cytochrome P450 Enzyme Assay Protocol
10
CYP1A1 Activity Assay Protocol
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Effects on the ethoxyresorufin deethylase (EROD) activity of human recombinant CYP1A1 (2.5nM) was assayed by first pre-incubating the enzyme with the compound to be tested for 5 min in Tris-HCl (0.1M, pH 7.4) with EDTA (1mM) at 37°C followed by addition of ethoxyresorufin (0.1 μM) and NADPH (0.4 mM). Formation of resorufin was quantified using a multiwell plate reader with the excitation/emission wavelengths of 535/590 nm) and activity of the enzyme was determined by the rate of resorufin formation. ICZ and FICZ were purchased from Syntastic AB (Stockholm, Sweden). DIM, I3C, 7-ethoxyresorufin, β-Nicotinamide adenine dinucleotide 2′-phosphate (NADPH, N7505) and human recombinant cytochrome P4501A1 with P450 reductase (C3735) was purchased from Sigma-Aldrich.
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