Untersuchungen zur metabolismus-bedingten Insektizid-Resistenz von Drosophila melanogaster als Beispiel für den Einsatz Cytochrom P450-transgener Zellsuspensionskulturen von Nicotiana tabacum zur Aufklärung der enzymatischen Kapazität des Ursprungsorganismus

With the worldwide use of insecticides, the number of resistant insect species increases resulting in decreased efficacy of pesticides and threat of human welfare by crop failures and disease transmission. Environmental concentrations of some insecticides are so high that even populations of non-target organisms are selected for resistance. Thus, Cyp6g1-overexpressing strains of Drosophila melanogaster Meig. were selected; they are resistant to a wide range of diverse insecticides, including DDT (dichlorodiphenyltrichloroethane) and imidacloprid. Up to now however, no evidence has been available that the cytochrome P450 monooxygenase CYP6G1 is able to metabolise insecticides. This should be studied regarding imidacloprid, DDT, and methoxychlor by heterologous expression of Cyp6g1 in cell suspension cultures of Nicotiana tabacum L. as convenient in vitro system. The required vectors with cDNA of Cyp6g1 and vectors with cDNA of Cyp71u4v2 (barley) and Cyp3a4 (human) for additional studies, were constructed, tobacco cells were transformed and studied by molecular biology. It was demonstrated, that CYP6G1 is capable of metabolising the (14C-labelled) insecticides. Cyp6g1-transgenic culture 6L converted 400 µg imidacloprid within 48 h to 60% of 4-hydroxyimidacloprid, 20% of 5-hydroxyimidacloprid, 7% of olefinic imidacloprid and minor amounts of 4,5-dihydroxyimidacloprid with an absolute metabolic rate of 86% (no detectable turnover in the non-transgenic culture LS). In supplementary studies, the metabolic pathway of imidacloprid derived from CYP6G1 was elucidated, and it was shown that the appearance of the olefinic metabolite was traced back to the influence of the acidic media. After simultaneous application of P450 inhibitor piperonyl butoxide (200 µg), the CYP6G1-dependent metabolism of imidacloprid (20 µg, 24 h) was inhibited by 82%. DDT (20 µg) was only converted under anaerobic conditions in a closed system within 48 h by reductive dechlorination to 16% DDD (dichlorodiphenyldichloroethane) (in one parallel) besides low amounts of possible further metabolites with an absolute metabolic rate of 37%. However, this result showed only a limited reproducibility. In the LS culture only 2% DDD were detected in the closed system with an absolute metabolic rate of 11%. Methoxychlor (45.8 µg) was converted to 17% of catechol methoxychlor, 16% of trishydroxymethoxychlor, 8% of bishydroxymethoxychlor, and 6% of monohydroxymethoxychlor in the 6L cell culture within 24 h (absolute metabolic rate: 96%). In contrast to studies with imidacloprid and DDT, LS culture showed a high absolute metabolic rate of 62% regarding methoxychlor. Nevertheless, 34% of the metabolic rate and especially the increased formation of trishydroxymethoxychlor and catechol methoxychlor could be traced back to the enzymatic activity of CYP6G1. Similarly, the absolute metabolic rate of methoxychlor in 6L and LS culture was inhibited by 43% and 92%, respectively, after simultaneous application of piperonyl butoxide (458 µg). Due to this treatment, the glycoside fraction decreased in favour of primary metabolites. The metabolic capacity of CYP6G1 leads to detoxification of different insecticides towards Drosophila. Thus, the gap between the supposed resistance gene Cyp6g1 and the observed resistance phenomenon was closed by the evidence that CYP6G1 is at least able to metabolise three insecticides. It should be mentioned that CYP6G1 is presumably not alone responsible for the resistance towards DDT but confers probably resistance towards methoxychlor. Using 4-n-nonylphenol, a substrate of CYP71U4v2, which was shown to be overexpressed in barley during pathogen defence, was finally identified. Cyp71u4v2-transgenic culture formed the main metabolites 4’-hydroxy-4-n-nonylphenol and 5’-hydroxy-4-n-nonylphenol with an absolute metabolic rate of 93%. In the non-transgenic culture, a high metabolic rate of 60% was also detected. However, the pattern of side chain hydroxylated metabolites with the main metabolite 8’-hydroxy-4-n-nonylphenol differed obviously from that of the transgenic culture – thus clearly proving the enzymatic activity of CYP71U4v2. The turnover of the degradation product of nonylphenol polyethoxylates formerly used in pesticide formulations by endogenous enzymes could be quite relevant for barley. The present work showed that the tobacco cell suspension culture is a convenient expression system for P450 genes of diverse origin enabling to elucidate or predict metabolism-based resistance phenomena without the need of expression of appropriate P450 reductase.

Grantor dc:publisher

Hut

Year dc:date

2008

Author dc:creator

• Joußen, Nicole

Contributors dc:contributor

• Schuphan, Ingolf

• info:eu-repo/classification/ddc/570
• Taufliege
• Cytochrom P-450
• Heterologe Genexpression
• Gerste
• Biowissenschaften, Biologie
• Insektizid-Resistenz
• Tabak-Zellsuspensionskulturen
• Metabolismusstudien
• insecticide resistance
• Drosophila melanogaster
• cytochrome P450 monooxygenase
• heterologous expression
• tobacco cell suspension cultures

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-113611%22

OAI record oai:identifier

https://publications.rwth-aachen.de/oai2d?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Apublications.rwth-aachen.de%3A51305