Studien zum Metabolismus von Pestiziden und Xenobiotika durch humane Cytochrom-P450-Monooxygenasen in transgenen Tabakzellkulturen
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 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 using a CYP1A2 cell culture the transformation of metamitron could be demonstrated, starting with the formation of the metabolite desaminometamitron, followed by 4?hydroxydesaminometamitron and ended with the glucosid of the later. In experiments with a constant amount of CYP1A2 plant cell culture the originally used concentration of metamitron was raised up to a 20fold quantity. In these large-scale experiments the relative metabolic profile had changed slightly. This was due to the slower glucosylation compared to P450-oxidation. Therefore a high P450 activity of the transgenic clones was demonstrated. The third substance used was the insecticide dimethoate with three potential P450 reaction centres present in this molecule. The three reactions are the desulfuration, N-demethylation and O-demethylation. Nevertheless, there was no evidence that dimethoat was a suitable substrate for the two P450 isoenzymes. Only a slight increase in the conversion rate of dimethoate could be found by the CYP1A2 enzyme which was attributed to the O-demethylation. Finally the metabolism of dimethoate was tested in liver microsomes of rats, which were prior induced with Aroclor 1254 to increase the P450 enzyme activity. On the basis of the obtained results it is obvious, that dimethoate is not metabolised by P450-isoenzymes in rats. Plant cell cultures in which the appropriate P450 cDNAs are introduced, appear to be suitable in vitro systems in order to produce large quantities of primary oxidised pesticide metabolites.
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
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