Browsing by Subject "Cytochrom P450"

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Monitoring, mechanisms and management of insecticide resistance and insecticide mode of action in coleopteran pests of winter oilseed rape with special reference to neonicotinoid insecticides under laboratory and applied aspects
(2014) Zimmer, Christoph Thomas; Zebitz, Claus P. W.
Winter oilseed rape, Brassica napus L., has become a vital part of cereal-based crop rotations in Europe. It is attacked by numerous insect pests and their control relies on the intensive use of insecticides (compared to other broad acre crops). The exclusive and continuous use of pyrethroid insecticides for almost twenty years led to an enormous selection pressure and facilitated the development of resistance in oilseed rape pests in Europe. Unsurprising three out of the five major pests of the order Coleoptera are reported to be pyrethroid resistant at present: the pollen beetle, Meligethes aeneus F.; the cabbage stem flea beetle, Psylliodes chrysocephala L. and the cabbage seed weevil, Ceutorhynchus assimilis PAYK.. An adult vial bioassay, which is based on insecticide coated glass vials, was used to monitor the spread and strength of pyrethroid resistance and to determine cross-resistance pattern in pollen beetle and cabbage stem flea beetle. Furthermore, baseline susceptibility towards lambda-cyhalothrin (a widely used pyrethroid) was also established for the cabbage seed weevil. The vial bioassay methodology was adapted to thiacloprid, a neonicotinoid insecticide, to determine baseline susceptibility and to provide a methodology to allow long-term susceptibility monitoring of pollen beetle and cabbage seed weevil. Thiacloprid monitoring revealed that pollen beetle and cabbage seed weevil populations collected across Europe in 2009-2012 and 2012 respectively were highly susceptible to this insecticide class. Metabolism studies using native microsomal preparations as the enzyme source and deltamethrin as substrate revealed metabolism of deltamethrin with 4-OH-deltamethrin being the major metabolite. Metabolite formation in vitro was correlated with the observed pyrethroid resistance level in vivo and was suppressible by PBO. A degenerate PCR approach was used to identify partial P450 gene sequences from pollen beetle. qRT-PCR screening covering a range of pollen beetle populations differing in levels of pyrethroid resistance identified a single P450, CYP6BQ23, as significantly and highly overexpressed (up to ~900-fold) in resistant strains compared to susceptible strains. The expression of CYP6BQ23 was significantly correlated with both the level of resistance and with the rate of deltamethrin metabolism in microsomal preparations of these populations. Recombinant expression of this P450 in an insect cell line demonstrated that it is capable of hydroxylating deltamethrin and tau-fluvalinate. The turnover of these pyrethroids by CYP6BQ23 is in line with the observed moderate cross-resistant phenotype. Molecular modeling suggested a better fit of deltamethrin into the active site of CYP6BQ23 compared to tau-fluvalinate also supporting the biochemical results. The occurrence of target-site resistance was investigated by single nucleotide polymorphism (SNP) analysis of the para-locus encoding the voltage-gated sodium channel (VGSC) in insects. To achieve this goal a partial fragment (domain IIS4-6) encoding an important region of the pyrethroid binding site was PCR amplified and screened for non-synonymous SNPs. One SNP was identified causing a leucine to phenylalanine substitution at amino acid residue number 1014 (Musca domestica L. numbering), well known as knock down resistance (kdr) conferring an absolute cross-resistance to pyrethroids and DDT in various insect species. Sequencing of the very same gene region in the cabbage stem flea beetle also revealed the presence of the L1014F kdr mutation in pyrethroid resistant flea beetle populations, thus explaining the strong cross-resistance pattern observed in vitro. Most mechanistic studies of resistance have focused on elucidating the contribution of particular genes/gene families to pyrethroid resistance. To generate a comprehensive sequence resource and to elucidate global changes in gene regulation related to insecticide resistance in pollen beetle a de novo transcriptome was assembled from sequence pools generated by next-generation sequencing. RNA-sequencing of three pyrethroid resistant and one highly susceptible reference population allowed a global gene expression analysis by short read mapping against the generated transcriptome, as well as a SNP analysis. The implications of these results for resistance management in coleopteran pests in winter oilseed rape and opportunities for future work are discussed.
Variabilität und Induzierbarkeit von Cytochrom P450 Monooxygenasen in humanen Leberproben und Hepatozyten : Untersuchungen mittels LC-MS/MS Cocktail-Assay und RNA-Interferenz
(2010) Feidt, Diana M.; Graeve, Lutz
The variability of the expression and function of the P450 enzymes (CYPs) is a cause for having different intensities and lengths of effects as well as side effects when patients are given the same dosage of medication. Up to this day, one cannot allover explain this inter individual variability of expression and activity of the enzymes. In general, there are several factors that may affect the variability, including biological factors (such us age, gender, hormonal status), environmental factors (such as nutrition, smoking, medication) as well as genetic factors like polymorphisms In order to solve this problem and to analyze relatively easy and fast activity differences, we developed an LC-MS/MS based P450 activity cocktail assay to quantify and detect simultaneously the seven most important CYPs as judged by their roles in the metabolism of clinically used drugs. The assay was established for use in in human hepatocytes as well as in recombinant and microsomal enzymes. We used the newly developed model-substrate cocktail assay to analyze the time-dependent induction of seven drug metabolizing cytochrome P450 activities as response to treatment of primary human hepatocytes with different statins. The strongest induction was observed for amodiaquine N-desalkylation of CYP2C8, which was induced up to 20-fold by atorvastatin and approximately 10-fold by simvastatin and lovastatin. Enzymes CYP3A4, CYP2B6 and 2C9 showed lower, but also significant induction after treatment with atorvastatin and simvastatin (4-11-fold). lovastatin and rosuvastatin demonstrated minor effects. Quantitative RT-PCR confirmed corresponding changes on the mRNA level with even more dramatic induction up to almost 100-fold. These data suggest a broader inducing effect of statins on cytochrome P450 expression and activity than previously known, thus further emphasizing their drug-drug interaction potential, especially for CYP2C8. Based on correlation analysis with P450 enzyme activity to their specific protein amount in human liver samples (liverbank IKP) were different functional results observed. Enzymes like CYP3A4 with atorvastatin hydroxylation or CYP1A2 with formation of acetaminophen showed very good correlations. Others like i.e. CYP2C9 with specific substrate diclofenac correlated much lower. Besides biological and environmental factors could these differences based on variability of the enzymes NADPH:P450 oxidoreductase (POR), cytochrome b5 or the two progesterone receptor-membrane components PGRMC1 and PGRMC2. After all, they would take active part in the metabolism of the xenobiotica as possible electron donors of the CYP enzymes. In order to analyze what kind of influence these proteins have on CYP enzyme activity, we developed a lentiviral based RNA-interference (RNAi) method in human hepatocytes and determined P450 activity with cocktail-assay after knocking down these genes. For the P450 reductase, we achieved a successful gene silencing of about 85% on mRNA level. The expression of cytochrome b5 was reduced by 51%, PGRMC1 about 30%. So far, it has not been possible to prove a significant knock down of PGRMC2. After silencing of reductase, an average light decline of about 10-30% of P450 enzyme activities was observed after 4 days. After a period of 7 days, a rest activity of CYP3A4 of only about 5% was detected. For both other potential electron donators cytochrome b5 and PGRMC1 was a reduced activity of 85% and 75% determined. These first results indicate a clear interaction of the enzymes POR, cytochrome b5 and PGRMC1 with the drug metabolizing enzymes.