Detoxification of ivermectin by ATP binding cassette transporter C4 and cytochrome P450 monooxygenase 6CJ1 in the human body louse,Pediculus humanus humanus

We previously observed that ivermectin‐induced detoxification genes, including ATP binding cassette transporter C4 (PhABCC4) and cytochrome P450 6CJ1 (CYP6CJ1) were identified from body lice following a brief exposure to a sublethal dose of ivermectin using a non‐invasive induction assay. In this current study, the functional properties of PhABCC4 and CYP6CJ1 were investigated after expression in either X. laevis oocytes or using a baculovirus expression system, respectively. Efflux of [³H]‐9‐(2‐phosphonomethoxyethyl) adenine ([³H]‐PMEA), a known ABCC4 substrate in humans, was detected from PhABCC4 cRNA‐injected oocytes by liquid scintillation spectrophotometric analysis and PhABCC4 expression in oocytes was confirmed using ABC transporter inhibitors. Efflux was also determined to be ATP‐dependent. Using a variety of insecticides in a competition assay, only co‐injection of ivermectin and dichlorodiphenyltrichloroethane led to decreased efflux of [³H]‐PMEA. PhABCC4‐expressing oocytes also directly effluxed [³H]‐ivermectin, which increased over time. In addition, ivermectin appeared to be oxidatively metabolized and/or sequestered, although at low levels, following functional expression of CYP6CJ1 along with cytochrome P450 reductase in Sf9 cells. Our study suggests that PhABCC4 and perhaps CYP6CJ1 are involved in the Phase III and Phase I xenobiotic metabolism of ivermectin, respectively, and may play an important role in the evolution of ivermectin resistance in lice and other insects as field selection occurs.     

Molecular characterization,expression pattern and metabolic activity of flavin‐dependent monooxygenases in Spodoptera exigua

Enhanced detoxification is one of the important mechanisms for insecticide resistance. Most research in this field to date has focused on the role of cytochrome P450s. Our previous work revealed that flavin‐dependent monooxygenases (FMOs) were involved in metabolic resistance of Spodoptera exigua. In the present study we investigated the molecular characteristics, expression patterns and oxidative activities of SeFMO on insecticides. Three FMO genes, which encode proteins with the typical FMO motifs, were cloned from S. exigua. The oxidative activities of eukaryotically expressed SeFMO enzymes were verified with the model substrate of FMO. Importantly, the SeFMOs had significantly higher oxidative activities on metaflumizone and lambda‐cyhalothrin than on model substrates and other insecticides tested. The three SeFMOs were mainly expressed in the midgut, fat body and Malpighian tubules. The tissues responsible for xenobiotic metabolism and their expression characteristics were similar to those of P450s acting as detoxification genes. The study also revealed that the expression of SeFMOs could be induced by insecticide exposure, and that SeFMOs were over‐expressed in a metaflumizone‐resistant strain of S. exigua. These results suggest that SeFMOs are important insecticide detoxifying enzymes, and that over‐expression of FMO genes may be one of the mechanisms for metabolic resistance in S. exigua.     

Collaborative contribution of six cytochrome P450 monooxygenase genes to fenpropathrin resistance in Tetranychus cinnabarinus (Boisduval)

Cytochrome P450 monooxygenases (P450s), as an important family of detoxification enzymes, participate in the metabolism of agrochemicals in almost all agricultural pests and play important roles in the development of insecticide resistance. Two P450 genes (CYP389B1 and CYP392A26) were identified and their expression patterns were investigated in our previous study. In this study, four more P450 gene sequences (CYP391A1, CYP384A1, CYP392D11 and CYP392A28) from the Clan 2, Clan 3 and Clan 4 families were identified and characterized. Quantitative PCR analysis showed that these four P450 genes were highly expressed in a fenpropathrin‐resistant (FeR) strain of Tetranychus cinnabarinus. In addition, their expressions were much more sensitive to fenpropathrin induction in the FeR strain than the susceptible strain. Gene‐silencing experiments via double‐stranded RNA feeding were carried out. The results showed that mRNA levels of these six P450 genes were reduced in the FeR strain and the activities of P450s were decreased. Consequently mite susceptibilities to fenpropathrin were increased. Interestingly, silencing all six P450 genes simultaneously had an even greater effect on resistance than silencing them individually. This study increases our understanding of the molecular mechanisms of insecticide detoxification, suggesting that the overexpression of these six P450 genes might play important roles in fenpropathrin resistance in T. cinnabarinus collaboratively.     

Use of isogenic strains indicates CYP9M10 is linked to permethrin resistance in Culex pipiens quinquefasciatus

Previous studies on a strain of Culex pipiens quinquefasciatus from Saudi Arabia indicated permethrin resistance was a result of cytochrome P450 mediated detoxification and kdr. The P450 detoxification was found to be larval specific and associated with a fitness cost in certain environments. The P450 responsible for resistance (and the fitness cost) has not been identified, but recently two candidate P450s (CYP4H34 and CYP9M10) have been found. We measured cytochrome P450 and cytochrome b₅ content as well as the expression levels of CYP4H34 and CYP9M10 in a susceptible (SLAB) and two isogenic strains (isolated by repeated backcrossing and selection) of mosquito (ISOP450 and ISOJPAL) resistant to permethrin. Cytochrome P450 protein levels of the resistant strains were significantly higher (1.5‐fold) than SLAB, but were not significantly different from one another. Expression of CYP4H34 in the larvae and adults of the resistant (ISOP450 and ISOJPAL) and susceptible (SLAB) strains were not statistically different. CYP9M10 was found to be significantly over‐expressed in larvae of both permethrin‐resistant isogenic strains (1800‐fold in ISOP450 and 870‐fold in ISOJPAL) when compared to SLAB. Partial sequence analysis of CYP9M10 revealed eight polymorphic sites that distinguished the susceptible allele from the resistant allele. We conclude that CYP9M10 is linked to permethrin resistance in these strains of C. p. quinquefasciatus, and is likely to be the P450 gene responsible for resistance in these strains.     

Cytochrome P450s CYP6D3 and CYP6D1 are part of a P450 gene cluster on autosome 1 in the house fly

The P450 monooxygenases of insects are important in the metabolism of numerous endogenous and exogenous compounds. However, identity of the P450 isoform(s) involved in these reactions is rarely known. A critical first step in the identification of important P450s is the cloning and sequencing of their genes. Toward this goal we report the genomic sequence of a new cytochrome P450, termed CYP6D3, from the house fly, Musca domestica. CYP6D3 is part of a P450 gene cluster located on chromosome 1 and is located upstream of a related gene, CYP6D1. The similar genetic structures of CYP6D3 and CYP6D1 (5 exons and 4 introns of similar length) suggest one of these genes may have been the result of a duplication event. The CYP6D3 deduced amino acid sequence indicates a protein with 518 amino acids and a molecular weight of 59.3 kDa. The CYP6D3 protein is most similar to house fly CYP6D1 (78%) and Cyp6D2 (56%) from Drosophila melanogaster. The deduced amino acid sequences of CYP6D3 and CYP6D1 are identical at the Helix I and heme binding regions.     

Complications of Psychotropic and Pain Medications in an Ultrarapid Metabolizer Patient at the Upper 1% of Cytochrome P450 (CYP450) Function Quantified by Combinatorial CYP450 Genotyping

A 44-year-old Caucasian woman presented with a history of empirical treatment with 20 pain and psychotropic medications, as well as dual comorbidity of intractable pain and depression. A multiple gain-of-function profile in the CYP450 family of cytochrome P450 (CYP450) drug metabolism isoenzymes was discovered. The patient was a homozygote of suprafunctional alleles for both CYP2D6 (^*35/^*35) and CYP2C19 (^*17/^*17) genes and functional alleles for CYP2C9 (^*1/^*1), which account for aggregate drug metabolism function at the upper 1% of the population. The patient improved clinically with discontinuation of psychotropics and pain medications that were substrates of CYP2D6 and/or CYP2C19, suggesting that much of her symptomatology was drug induced. Combinatorial genotyping of CYP450 genes is diagnostically useful in individuals with histories of multiple side effects or drug resistance, which could be avoided by genetically informed therapeutics in behavioral health.     

Humanized mouse lines and their application for prediction of human drug metabolism and toxicological risk assessment

Cytochrome P450s (P450s) are important enzymes involved in the metabolism of xenobiotics, particularly clinically used drugs, and are also responsible for metabolic activation of chemical carcinogens and toxins. Many xenobiotics can activate nuclear receptors that in turn induce the expression of genes encoding xenobiotic metabolizing enzymes and drug transporters. Marked species differences in the expression and regulation of cytochromes P450 and xenobiotic nuclear receptors exist. Thus, obtaining reliable rodent models to accurately reflect human drug and carcinogen metabolism is severely limited. Humanized transgenic mice were developed in an effort to create more reliable in vivo systems to study and predict human responses to xenobiotics. Human P450s or human xenobiotic-activated nuclear receptors were introduced directly or replaced the corresponding mouse gene, thus creating "humanized" transgenic mice. Mice expressing human CYP1A1/CYP1A2, CYP2E1, CYP2D6, CYP3A4, CY3A7, pregnane X receptor, and peroxisome proliferator-activated receptor alpha were generated and characterized. These humanized mouse models offer a broad utility in the evaluation and prediction of toxicological risk that may aid in the development of safer drugs.     

Protective effect of fenofibrate against ischemia‐/reperfusion‐induced cardiac arrhythmias in isolated rat hearts

Fenofibrate is a peroxisome proliferator‐activated receptor (PPAR)‐α activator that lowers triglycerides and influences cytochrome P‐450 (CYP‐450) epoxygenase‐dependent arachidonic acid (AA) metabolism. CYP‐450 epoxygenase metabolizes AA to epoxyeicosatrienoic acids (EETs). EETs have coronary dilating and cardiac and renal protective properties. Fibrates possess similar properties due to their CYP‐450 epoxygenase‐inducing properties that lead to increase in endogenous EET production. In the current investigations, fenofibrate (100 mg/kg, orally) for 2 weeks decreased ischemia‐/reperfusion (I/R)‐induced premature ventricular contractions (PVCs), ventricular tachycardia (VT), and ventricular fibrillation (VF) in the isolated rat hearts. Fenofibrate caused marked inhibition of the reperfusion‐induced cardiac arrhythmias. The incidence of reperfusion‐induced VF decreased from 80% in the control vehicle‐treated animals to 33% in the fenofibrate‐treated animals (P < 0.001). PVCs were also significantly (P < 0.01) decreased from 223.2 ± 51 in control vehicle‐treated animals to 136.8 ± 22 in fenofibrate‐treated animals. Total duration of reperfusion‐induced VT decreased from 29.2 ± 6.3 s in control, vehicle‐treated animals to 4.8 ± 1.3 s in fenofibrate‐treated animals, P < 0.001. Heart rate and perfusion pressure were not significantly affected by fenofibrate pretreatment. Diltiazem, a clinically used anti‐arrhythmic agent, produced complete protection against I/R‐induced cardiac arrhythmias in this model reducing the incidence of VF from 80% in control, vehicle‐treated animals to 10% in diltiazem‐treated hearts. These findings indicate that fenofibrate suppresses arrhythmias in isolated rat hearts subjected to I/R‐induced injury.     

Identification of promoter polymorphisms in the cytochrome P450 CYP6AY1 linked with insecticide resistance in the brown planthopper,Nilaparvata lugens

Imidacloprid resistance in the brown planthopper, Nilaparvata lugens, is primarily the result of the over‐expression of cytochrome P450 monooxygenases. Here, a field‐collected strain of N. lugens was shown to be highly resistant to both imidacloprid and buprofezin. Insecticide exposure and quantitative real‐time PCR revealed that its resistance was mainly associated with a cytochrome P450 gene, CYP6AY1. CYP6AY1 is known to metabolize imidacloprid but its effect on buprofezin is unclear. In the 5′‐untranslated region of CYP6AY1, a novel alternative splicing was detected. After a 1990‐bp promoter region was cloned, its basal luciferase activity was assessed. Furthermore, genotyping studies identified 12 variations in the promoter region that discriminated between the field‐collected and control strain. Finally, survival bioassays revealed a single nucleotide polymorphism and an insertion‐deletion polymorphism linked to buprofezin and imidacloprid resistance. Mutagenesis of these sites enhanced the promoter activity of CYP6AY1. These results suggest that promoter polymorphisms may affect P450‐mediated multiple insecticide resistance of pests.     

A cluster of CYP6 gene family associated with the major quantitative trait locus is responsible for the pyrethroid resistance in Culex pipiens pallen

The emergence and rapid spread of insecticide resistance in several mosquito species has become a significant obstacle in management of mosquito‐borne diseases, including deltamethrin resistance in Culex pipiens pallens. Previous study identified a major deltamethrin resistance quantitative trait locus (DR‐6) that alone explained 62% of the genetic variance. In this study, the marker L4B1.102 and L4B1.175 associated with the DR‐6 were characterized. We searched for potential candidate genes in the flank region of two markers in the genome sequence and showed that a cluster of CYP6 cytochrome P450 genes (CYP6BB4, CYP6BB3, CYP6CC2, CYP6P14, CYP6BZ2, CYP6AA9, CYP6AA8, CYP6AA7) was in the vicinity of DR‐6. Significant differences in the expression of these P450s in the larval and adult stages were identified in the resistant strains compared with the susceptible strain. For CYP6AA9 and CYP6BB4, the correlation analysis showed a highly positive correlation between relative gene expression quantification and the resistance level in different strains. Knockdown of CYP6BB4 increased the sensitivity of mosquitoes to deltamethrin. We identified that the deltamethrin resistance was in a cluster of CYP6 genes in C. pipiens pallens, and CYP6BB4 may play a significant role in the development of deltamethrin resistance.     

A cytochrome P450 gene plays a role in the recognition of sex pheromones in the tobacco cutworm,Spodoptera litura

Cytochrome P450 (P450 or CYP) genes are involved in fundamental physiological functions, and might be also associated with the olfactory recognition of sex pheromones in beetles and moths. A P450 gene, Spodoptera litura CYP4L4 (SlituCYP4L4), was cloned for the first time from the antennae of S. litura. SlituCYP4L4 was almost exclusively expressed in the adult stage and predominantly expressed in the adult antennae. In situ hybridization showed that SlituCYP4L4 localized mainly at the base of the long sensilla trichoidea, which responds to sex pheromone components. Pretreatment with an S. litura sex pheromone significantly reduced the expression levels of SlituCYP4L4, consistent with other genes involved in sex pheromone recognition. The expression level of SlituCYP4L4 was different in moths collected with different ratios of sex pheromone lures and collected in different geographical locations. After gene knockdown of SlituCYP4L4 in the antennae, the electroantennogram (EAG) responses of male and female moths to (9Z,11E)‐tetradecadienyl acetate or (9Z,12E)‐tetradecadienyl acetate were significantly decreased. In contrast, EAG responses to plant volatiles and sex pheromones of other moth species were not significantly influenced in these moths. SlituCYP4L4 was also expressed in the gustatory tissues and sensilla, which suggests that SlituCYP4L4 may have other functions in the chemosensory system. Our results have shown for the first time the function of a CYP gene with appendage‐specific expression in insect sex pheromone recognition, especially in adult moths.