Differential expression of cytochrome P450 genes between bromadiolone-resistant and anticoagulant-susceptible Norway rats: a possible role for pharmacokinetics in bromadiolone resistance

BACKGROUND: Anticoagulant resistance in Norway rats, Rattus norvegicus (Berk.), has been suggested to be conferred by mutations in the VKORC1 gene, encoding the target protein of anticoagulant rodenticides. Other factors, e.g. pharmacokinetics, may also contribute to resistance, however. To examine the involvement of pharmacokinetics in bromadiolone resistance in male and female rats, liver expression profiles of seven cytochrome P450 genes from a Danish bromadiolone-resistant rat strain (with an Y139C-VKORC1 mutation) were compared with profiles from an anticoagulant-susceptible strain. RESULTS: In the presence of bromadiolone, the Cyp2e1, Cyp2c13, Cyp3a2 and Cyp3a3 genes were significantly overexpressed, while Cyp2c12 expression was suppressed in resistant female rats compared with susceptible females. Relative to susceptible males, resistant males showed significant overexpression of the Cyp2a1, Cyp2e1, Cyp3a2 and Cyp3a3 genes. On exposure to bromadiolone, females had higher Cyp2e1 expression than males, which possibly explains why female rats are generally more tolerant to anticoagulants than male rats. CONCLUSION: Results suggest that bromadiolone resistance in a Danish strain of Norway rats involves enhanced anticoagulant metabolism catalysed by cytochrome P450-2e1, -3a2 and -3a3. This pharmacokinetically based bromadiolone resistance is to some extent sex differentiated, as female resistance furthermore seems to involve overexpression of cytochrome P450-2c13 and suppression of P450-2c12, whereas male resistance appears to involve P450-2a1 overexpression.     

Involvement of hepatic xenobiotic related genes in bromadiolone resistance in wild Norway rats, Rattus norvegicus (Berk.)

To examine the role of xenobiotic relevant genes in bromadiolone resistance in wild Norway rats (Rattus norvegicus) we compared the constitutive liver gene expression and expression upon bromadiolone administration in bromadiolone resistant and anticoagulant susceptible female rats using a LNA microarray and quantitative PCR. Resistant rats showed significantly higher constitutive expression of the cytochrome P450 genes Cyp2c13 and Cyp3a2 and lower expression of Cyp2e1 and Gpox1 compared to the susceptible rats. The Cyp1a2, Cyp2c13, Cyp2e1, Cyp3a2 and Cyp3a3 genes were significantly higher expressed in resistant than susceptible rats upon bromadiolone exposure. To establish how bromadiolone affected xenobiotic gene expression in the two strains we compared bromadiolone expression profiles to saline profiles of both strains. Bromadiolone mediated significant up-regulation of Cyp2e1 and Cyp3a3 expression in the resistant rats whereas the rodenticide conferred down-regulation of Cyp2e1, Cyp3a3 and Gpox1 and induction of Cyp2c12 expression in susceptible rats. Cyp2c13 and Cyp3a2 expression were markedly suppressed in both strains upon treatment. This suggests that xenobiotic relevant enzymes play a role in bromadiolone resistance in the Norway rat. A high constitutive expression of Cyp2c13 and Cyp3a2 and induction of Cyp1a2, Cyp2e1 and Cyp3a3 expression during bromadiolone exposure may increase the resistance to bromadiolone presumably by facilitating increased detoxification and decreased liver injury.     

Spinosad resistance in female Musca domestica L. from a field-derived population

BACKGROUND: Bait-formulated spinosad is currently being introduced for housefly (Musca domestica L.) control around the world. Spinosad resistance was evaluated in a multiresistant field population and strains derived from this by selection with insecticides. Constitutive and spinosad-induced expression levels of three cytochrome P450 genes, CYP6A1, CYP6D1 and CYP6D3, previously reported to be involved in insecticide resistance, were examined. RESULTS: In 2004 a baseline for spinosad toxicity of Danish houseflies where all field populations were considered to be susceptible was established. In the present study, females of a multiresistant field population 791a were, however, 27-fold spinosad resistant at LC₅₀, whereas 791a male houseflies were susceptible. Strain 791a was selected with spinosad, thiamethoxam, fipronil and imidacloprid, resulting in four strains with individual characteristics. Selection of 791a with spinosad did not alter spinosad resistance in either males or females, but counterselected against resistance to the insecticides thiamethoxam and imidacloprid targeting nicotinic acetylcholine receptors. A synergist study with piperonyl butoxide, as well as gene expression studies of CYP6A1, CYP6D1 and CYP6D3, indicated a partial involvement of cytochrome P450 genes in spinosad resistance. CONCLUSION: This study reports female-linked spinosad resistance in Danish houseflies. Negative cross-resistance was observed between spinosad and neonicotinoids in one multiresistant housefly strain. Spinosad resistance involved alterations of cytochrome P450 gene expression. Copyright © 2011 Society of Chemical Industry     

Regulation of cytochrome P450 expression in Drosophila: Genomic insights

Genomic tools such as the availability of the Drosophila genome sequence, the relative ease of stable transformation, and DNA microarrays have made the fruit fly a powerful model in insecticide toxicology research. We have used transgenic promoter-GFP constructs to document the detailed pattern of induced Cyp6a2 gene expression in larval and adult Drosophila tissues. We also compared various insecticides and xenobiotics for their ability to induce this cytochrome P450 gene, and show that the pattern of Cyp6a2 inducibility is comparable to that of vertebrate CYP2B genes, and different from that of vertebrate CYP1A genes, suggesting a degree of evolutionary conservation for the “phenobarbital-type” induction mechanism. Our results are compared to the increasingly diverse reports on P450 induction that can be gleaned from whole genome or from “detox” microarray experiments in Drosophila. These suggest that only a third of the genomic repertoire of CYP genes is inducible by xenobiotics, and that there are distinct subsets of inducers/induced genes, suggesting multiple xenobiotic transduction mechanisms. A relationship between induction and resistance is not supported by expression data from the literature. The relative abundance of expression data now available is in contrast to the paucity of studies on functional expression of P450 enzymes, and this remains a challenge for our understanding of the toxicokinetic aspects of insecticide action.     

Resistance evolution in Drosophila: the case of CYP6G1

The massive use of DDT as an insecticide between 1940 and 1970 has resulted in the emergence of a resistant population of insects. One of the main metabolic mechanisms developed by resistant insects involves detoxification enzymes such as cytochrome P450s. These enzymes can metabolise the insecticide to render it less toxic and facilitate its elimination from the organism. The P450 Cyp6g1 was identified as the major factor responsible for DDT resistance in Drosophila melanogaster field populations. In this article, we review the data available for this gene since it was associated with resistance in 2002. The knowledge gained on Cyp6g1 allows a better understanding of the evolution of insecticide resistance mechanisms and highlights the major role of transposable elements in evolutionary processes. © 2016 Society of Chemical Industry     

Low expression of nicotinic acetylcholine receptor subunit Mdα2 in neonicotinoid‐resistant strains of Musca domestica L.

BACKGROUND: Neonicotinoid action as well as resistance involves interaction with nicotinic acetylcholine receptors (nAChRs). In the housefly, neonicotinoid resistance also involves cytochrome P450, as indicated by bioassay with synergist as well as altered expression. In bioassay, synergism was only partial and indicated possible target‐site resistance. The nAChR α2 subunit is important in neonicotinoid toxicity to insects, and gene expression of the Mdα2 subunit was investigated in field populations and laboratory strains of neonicotinoid‐resistant and insecticide‐susceptible houseflies, Musca domestica L. The genomic sequence covering exon III–VII of Mdα2 was analysed for mutations. RESULTS: Gene expression profiling of Mdα2 revealed notable differences between neonicotinoid‐resistant and insecticide‐susceptible houseflies. On average, the neonicotinoid‐resistant field population 766b and the imidacloprid selected strain 791imi had 60% lower copy numbers of Mdα2 compared with the susceptible reference strain. Sequencing of exon III–VII of the Mdα2, encoding acetylcholine binding‐site regions and three out of four transmembrane domains, did not reveal any mutations explaining the increased neonicotinoid tolerance in the strains examined. CONCLUSION: Previous discoveries and the results of this study suggest that the neonicotinoid resistance mechanism in Danish houseflies involves both cytochrome P450 monooxygenase‐mediated detoxification and reduced expression of the nAChR subunit α2. Copyright © 2010 Society of Chemical Industry     

昆虫细胞色素P450基因的表达与调控及P450介导抗性的分子机制

本文综述了在杀虫剂抗性中起重要作用的 P4 50酶系研究的最新进展。内容包括 :细胞色素 P4 50酶系基因及其基因的表达与调控 ,P4 50介导抗性的分子基础。细胞色素 P4 50表达表现出发育期、组织、品系特异性及可诱导性。 P4 50表达的调控机制复杂 ,可能受顺式调控元件 (如 CYP6 B1)或反式作用因子 (如CYP6 A1)或顺式、反式因子的共同调控 (如 CYP6 D1)。调控可能涉及转录增强的转录机制或 m RNA稳定性增加的转录后机制。 P4 50的超量表达是 P4 50酶系介导抗性的主要机制 ,P4 50的氨基酸替换也可能在杀虫剂抗性中起作用。     

Cytochrome P450 monooxygenase-mediated neonicotinoid resistance in the house fly Musca domestica L

Neonicotinoids play an essential role in the control of house flies Musca domestica. The development of neonicotinoid resistance was found in two field populations. 766b was 130- and 140-fold resistant to imidacloprid and 17- and 28-fold resistant to thiamethoxam in males and females, respectively. 791a was 22- and 20-fold resistant to imidacloprid and 9- and 23-fold resistant to thiamethoxam in males and females, respectively. Imidacloprid selection of 791a increased imidacloprid resistance to 75- and 150-fold in males and females, respectively, whereas selection with thiamethoxam had minimum impact. Neonicotinoid resistance was in all cases suppressed by PBO. The cytochrome P450 genes CYP6A1, CYP6D1 and CYP6D3 were constitutively over-expressed in resistant strains and CYP6D1 and CYP6D3 differentially expressed between sexes. The highest level of CYP6A1 expression was observed in both gender of the imidacloprid-selected strain after neonicotinoid exposure. CYP6D1 expression was increased after neonicotinoid exposure in resistant males. CYP6D3 expression was induced in both sexes upon neonicotinoid exposure but significantly higher in females.     

抗阿维菌素小菜蛾的细胞色素P450酶系研究

通过对不同发育时期敏感和抗阿维菌素小菜蛾品系细胞色素P450含量的测定,以及使用不同模式底物对P450单加氧酶活性的比较研究发现:除成虫期外,不同发育时期抗性品系小菜蛾中P450和细胞色素b5的含量都高于敏感品系;抗性品系还原型辅酶Ⅱ(NADPH)-细胞色素P450还原酶活性是敏感品系的1.97倍;同时发现抗性品系中甲氧试卤灵-O-脱甲基酶(MROD)、乙氧试卤灵-O-脱乙基酶(EROD)、乙氧基香豆素-O-脱乙基酶(ECOD)以及对硝基苯甲醚-O-脱甲基酶(PNOD)的活性均明显高于敏感品系,分别为敏感品系的9.41、4.15、1.67和2.94倍。研究结果表明,细胞色素P450含量和单加氧酶活性的增高是小菜蛾对阿维菌素产生抗性的一个重要机制。     

Evolutionary plasticity of monooxygenase-mediated resistance

The cytochrome P450 monooxygenases are an important metabolic system involved in the detoxification of xenobiotics, and are thus one of the major mechanisms by which insects evolve insecticide resistance. However, comparatively little is known about the evolutionary constraints of this insecticide resistance mechanism. We investigated the genetic basis of resistance in a strain of house fly (NG98) from Georgia, USA that had evolved 3700-fold resistance to the pyrethroid insecticide permethrin, and compared this to other permethrin resistant strains of house flies from the US and Japan. Resistance in NG98 was due to kdr on autosome 3 and monooxygenase-mediated resistance on autosomes 1, 2, and 5. These results indicate that the genes which evolve to produce monooxygenase-mediated resistance to permethrin are different between different populations, and that the P450 monooxygenases have some degree of plasticity in response to selection. Monooxygenase-mediated resistance appears to evolve using different P450s, and possibly different regulatory signals controlling P450 expression, even in strains selected with the same insecticide.     

Q型烟粉虱细胞色素P450 CYP6DV5基因克隆及其在烟粉虱对噻虫嗪抗性中的作用

烟粉虱Bemisia tabaci已严重危害农作物的正常生长,而新烟碱类杀虫剂噻虫嗪已被广泛用于烟粉虱的防治,但由于常年应用,致使烟粉虱对噻虫嗪产生了严重的抗性,而目前其抗药性分子机制尚不明确.本研究通过荧光定量PCR比较分析烟粉虱噻虫嗪抗性及敏感种群,发现细胞色素P450基因CYP6DV5在噻虫嗪抗性品系中的表达量为...     

Influences of sub-acute exposure to paraquat on cytochrome P450 3A2 expression in rat liver and lungs

This study was designed to investigate the possible effects of paraquat sub-acute poisoning on cytochrome P450 3A2 expression in the liver and lungs. Twenty adult male rats (150-200 g) were exposed either against saline normal as control group or various doses of paraquat (3.5, 7 and 10 mg/kg, s.c.) as test groups for 7 consecutive days. Paraquat-exposed animals showed loss of body weight and elevation in serum levels of alkaline phosphates and alanine aminotransferase in a dose-dependent fashion. Moreover, animals in the test groups demonstrated a significant (P < 0.05) increase of malondialdehyde content in both the liver and lung tissues. Ultimately, by using RT-PCR method it became clear that 7 days exposure to paraquat resulted in a total suppression of cytochrome P450 3A2 at the mRNA level in the lungs. By contrast, a considerable up regulation of the same gene occurred in the liver. This data suggest that paraquat not only affect the lungs as a main target tissue but also up regulates the predominant cytochrome P450 gene in the liver which may induce detoxification processes.     

Molecular analysis of pyrethroid resistance conferred by target insensitivity and increased metabolic detoxification in Plutella xylostella

BACKGROUND: The pyrethroid resistance of the diamondback moth Plutella xylostella (L.) is conferred by increased gene expression of cytochrome P450 to detoxify the insecticide and/or through gene mutation of the sodium channel, which makes the individual insensitive to pyrethroids. However, no information is available about the correlation between the increased metabolic detoxification and the target insensitivity in pyrethroid resistance. RESULTS: Frequencies of pyrethroid‐resistant alleles (L1014F, T929I and M918I) and two resistance‐related mutations (A1101T and P1879S) at the sodium channel and expression levels of the cytochrome P450 gene CYP6BG1 were examined individually using laboratory and field strains of P. xylostella. Real‐time quantitative PCR analysis using the laboratory strains revealed that levels of larval expression of the resistant strain, homozygous for the pyrethroid‐resistant alleles other than the M918I, are significantly higher than those of the susceptible strain. In the field strains, the expression levels in insects having the same resistant alleles as those of the resistant strains varied greatly among individuals. The expression levels were not significantly higher than those in the heterozygotes. CONCLUSION: Significant correlation between the target insensitivity and the increased metabolic detoxification in pyrethroid resistance of P. xylostella was observed in the laboratory but not in the field. Copyright © 2010 Society of Chemical Industry     

烟粉虱细胞色素CYP6EM1基因的克隆及对吡虫啉抗性的作用

烟粉虱是一种世界性农业害虫,其防治手段以化学防治为主,新烟碱类杀虫剂吡虫啉常年用于防治烟粉虱,田间烟粉虱已经形成严重的抗药性。本研究通过分析烟粉虱吡虫啉抗性和敏感种群,发现细胞色素CYP6EM1基因在吡虫啉抗性品系中上调了4.7倍,进而克隆了其全长基因,进行了荧光定量PCR分析,发现该基因在吡虫啉抗性烟粉虱3龄若虫期和雄虫成虫期过量表达,并且在抗性成虫胸部和腹部过量表达。最后通过RNA干扰的方法使成虫的CYP6EM1基因表达量下降了54.8%,之后发现当烟粉虱暴露于吡虫啉时死亡率显著升高了39.65%,这表明CYP6EM1与烟粉虱对吡虫啉抗性的形成相关。研究结果对于揭示烟粉虱对吡虫啉产生抗性的机制有帮助,也为烟粉虱抗性水平田间监测及烟粉虱综合治理提供理论依据。     

Molecular analysis of resistance in a deltamethrin-resistant strain of Musca domestica from China

We investigated the molecular basis of resistance in a strain of house fly (BJD) from Beijing, China. This strain showed 567-fold resistance to commonly used deltamethrin. Flies were 64-fold resistant to deltamethrin synergized by piperonyl butoxide (PBO). The 5′-flanking sequence of the cytochrome P450 gene CYP6D1 in BJD strain had a 15-bp insert as in the LPR strain. Two mutations (kdr, super-kdr) in the voltage sensitive sodium channel (VSSC) were also detected in the BJD strain. Our results showed that a combination of resistance alleles for CYP6D1 and VSSC existed in deltamethrin resistant house flies in China.     

Homology modelling of Drosophila cytochrome P450 enzymes associated with insecticide resistance

BACKGROUND: Overexpression of the cytochrome P450 gene Cyp6g1 confers resistance against DDT and a broad range of other insecticides in Drosophila melanogaster Meig. In the absence of crystal structures of CYP6G1 or complexes with its substrates, structural studies rely on homology modelling and ligand docking to understand P450–substrate interactions. RESULTS: Homology models are presented for CYP6G1, a P450 associated with resistance to DDT and neonicotinoids, and two other enzymes associated with insecticide resistance in D. melanogaster, CYP12D1 and CYP6A2. The models are based on a template of the X‐ray structure of the phylogenetically related human CYP3A4, which is known for its broad substrate specificity. The model of CYP6G1 has a much smaller active site cavity than the template. The cavity is also ‘V’‐shaped and is lined with hydrophobic residues, showing high shape and chemical complementarity with the molecular characteristics of DDT. Comparison of the DDT–CYP6G1 complex and a non‐resistant CYP6A2 homology model implies that tight‐fit recognition of this insecticide is important in CYP6G1. The active site can accommodate differently shaped substrates ranging from imidacloprid to malathion but not the pyrethroids permethrin and cyfluthrin. CONCLUSION: The CYP6G1, CYP12D1 and CYP6A2 homology models can provide a structural insight into insecticide resistance in flies overexpressing P450 enzymes with broad substrate specificities. Copyright © 2010 Society of Chemical Industry     

Deletion of Cyp6d4 does not alter toxicity of insecticides to Drosophila melanogaster

Cytochrome P450-dependent monooxygenases are important in the activation and detoxification of numerous insecticides. In this study, a Drosophila melanogaster Cyp6d4 null mutant was used to determine the role of this P450 in insecticide metabolism. This null mutant was generated by imprecise excision of a mobile P element located upstream to the P450 gene Cyp6d4. Comparative analysis between the non-functional mutant and relevant control strains shows that Cyp6d4 does not appear to be involved in the metabolism of chlorfenapyr, cypermethrin, diazinon, imidacloprid, malathion, oxamyl, parathion, or pyrethrum extract, even though these insecticides are known to be activated or detoxified by P450-monooxygenases. No obvious abnormalities in development were seen in the Cyp6d4 null mutant, indicating that Cyp6d4 is not critical for the metabolism of vital endogenous substrates.