Improving the in vitro ethoxyresorufin-O-deethylase (EROD) assay with RTL-W1 by metabolic normalization and use of 尾-naphthoflavone as the reference substance

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• 作者：Patrick Heinrich ; ^{patrick.heinrich@cos.uni-heidelberg.de" class="auth_mail} ; ^{patrick@patrickheinrich.de" class="auth_mail} ; Ulrike Diehl ; Franziska Fö ; rster ; Thomas Braunbeck
• 关键词：BSA ; Bovine serum albumin ; DMEM ; Dulbecco's Modified Eagle Medium ; DMSO ; Dimethyl sulfoxide ; EROD ; 7-Ethoxyresorufin-O-deethylase ; FBS ; Fetal bovine serum ; MCE ; Metabolic cell equivalent(s) ; MTT ; 3-(4 ; 5-Dimethylthiazol-2-yl)-2 ; 5-diphenyltetrazolium bromide ; thiazolyl blue tetrazolium bromide ; NADPH ; Nicotinamide adenindinucleotide phosphate (reduced) ; RFU ; Relative fluorescence units (arbitrary units) ; RTL-W1 ; Rainbow trout liver Waterloo 1 (cell line) ; SD ; Standard deviation(s) ; SEQ ; Sediment e
• 刊名：Comparative Biochemistry and Physiology, Part C
• 出版年：August 2014
• 年：2014
• 卷：164
• 期：Complete
• 页码：27-34
• 全文大小：1059 K

文摘

The ethoxyresorufin-O-deethylase (EROD) assay is a widely applied method for the evaluation of the dioxin-like activity of single substances and environmental samples. As for most enzyme assays, the specific activity is normally related to total protein contents, the determination of which has clear limitations in high-throughput assays. EROD induction potentials are usually expressed as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents, a substance highly toxic to humans. In order to compensate for these shortcomings, two modifications of the EROD protocol are proposed: (1) EROD activity is normalized to the metabolic activity of the cells as determined by a modified thiazolyl blue tetrazolium (MTT) assay and expressed as metabolic cell equivalents (MCE) based on MTT data rather than to protein contents. Via MCE data, cytotoxicity information can always be reported in parallel to EROD data; with the protocol presented here, MTT and EROD data are collected simultaneously. (2) Among several reference substances tested (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 尾-naphthoflavone and benzo[a]pyrene), 尾-naphthoflavone proved to be the most suitable reference for the routine in vitro EROD assay, although TCDD has generally been preferred for purely scientific reasons.