Studien zum Metabolismus von Pestiziden und Xenobiotika durch humane Cytochrom-P450-Monooxygenasen in transgenen Tabakzellkulturen
type: CreativeWork
description: Detailed data on the metabolism of pesticides in plants is required for registration, therefore several in vitro systems have been developed to generate and identify a broad spectrum of possible metabolites. Therefore transgenic plant cell cultures with the human isoenzymes CYP1A1 and CYP1A2 were established for an oxidative metabolic profiling of pesticides. The cDNA of human P450 (CYP1A1 and CYP1A2) was introduced into tobacco cells (N. tabacum L.) by Agrobacterium-mediated transformation. For the screening of the transgenic calli an in vivo 7?Ethoxycoumarin O-de-ethylation (ECOD)-assay was established, which was performed with intact plant cells. The ECOD activity was measured as the concentration of non-metabolised 7?ethoxycoumarin remaining in the media. The developing transgenic calli or transgenic suspension cultures were characterised regarding their ECOD-activity. Expression of the P450 genes was further examined by PCR and western blotting. For metabolism studies in the transgenic tobacco cell cultures 14C-labelled atrazine, metamitron and dimethoat were used as model substances. The herbicide atrazine was metabolised by oxidative N-Dealkylation. The conversion rate was considerably higher in the transformed cell cultures (CYP1A1 and CYP1A2) than in non-transformed cell culture. However, CYP1A2 exhibited a higher conversion rate than CYP1A1. In a time-course study the enzyme CYP1A2 catalysed predominately N-deethylation whereas the enzyme CYP1A1 catalysed predominately the N-deisopropylation. In experiments with a constant amount of CYP1A2 plant cell culture the originally used concentration of atrazine was raised up to a 20fold quantity. In these large-scale experiments the high activity of the transgenic clones was demonstrated. Metabolism studies with the herbicide metamitron demonstrated that the arylhydroxylation and the previous desamination both can be catalysed by the isoenzymes CYP1A1 and CYP1A2. The conversion rate was considerably higher in the …
abstract: Detailed data on the metabolism of pesticides in plants is required for registration, therefore several in vitro systems have been developed to generate and identify a broad spectrum of possible metabolites. Therefore transgenic plant cell cultures with the human isoenzymes CYP1A1 and CYP1A2 were established for an oxidative metabolic profiling of pesticides. The cDNA of human P450 (CYP1A1 and CYP1A2) was introduced into tobacco cells (N. tabacum L.) by Agrobacterium-mediated transformation. For the screening of the transgenic calli an in vivo 7?Ethoxycoumarin O-de-ethylation (ECOD)-assay was established, which was performed with intact plant cells. The ECOD activity was measured as the concentration of non-metabolised 7?ethoxycoumarin remaining in the media. The developing transgenic calli or transgenic suspension cultures were characterised regarding their ECOD-activity. Expression of the P450 genes was further examined by PCR and western blotting. For metabolism studies in the transgenic tobacco cell cultures 14C-labelled atrazine, metamitron and dimethoat were used as model substances. The herbicide atrazine was metabolised by oxidative N-Dealkylation. The conversion rate was considerably higher in the transformed cell cultures (CYP1A1 and CYP1A2) than in non-transformed cell culture. However, CYP1A2 exhibited a higher conversion rate than CYP1A1. In a time-course study the enzyme CYP1A2 catalysed predominately N-deethylation whereas the enzyme CYP1A1 catalysed predominately the N-deisopropylation. In experiments with a constant amount of CYP1A2 plant cell culture the originally used concentration of atrazine was raised up to a 20fold quantity. In these large-scale experiments the high activity of the transgenic clones was demonstrated. Metabolism studies with the herbicide metamitron demonstrated that the arylhydroxylation and the previous desamination both can be catalysed by the isoenzymes CYP1A1 and CYP1A2. The conversion rate was considerably higher in the …
author: Bode, Maren
language: ger
keywords: info:eu-repo/classification/ddc/570, Tabak, Transgene Pflanzen, Zellkultur, Pestizid, Xenobiotikum, Oxidativer Stoffwechsel, Cytochrom P-450, Biowissenschaften, Biologie, Humane CYP1A1 und CYP1A2, transgene Pflanzenzellkulturen, Biotransformation, ECOD, Atrazin, Metamitron, Dimethoat
date published: 2004
date modified: 2026-07-30T19:43:19Z
provider: RWTH Aachen University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Publikationsserver der RWTH Aachen University