The involvement of cytochrome P450 monooxygenases in methanol elimination in Drosophila melanogaster larvae

Methanol is one of the most common short-chain alcohols in fermenting fruits, the natural food of the fruit fly, Drosophila melanogaster. The larvae cope continuously with methanol at various concentrations in order to survive and develop. In the present article, we found toxicities of dietary methanol and formaldehyde were enhanced by piperonyl butoxide, but not by 3-amino-1, 2, 4-triazole, 4-methylpyrazole, diethylmeleate, and triphenyl phosphate, when assessing by the combination index method. These results reveal that cytochrome P450 monooxygenases (CYPs), rather than catalases, alcohol dehydrogenases, glutathione S-transferases, and esterases, participate in methanol metabolism. Moreover, methanol exposure dramatically increased CYP activity. The ratios of the CYP activities in treated larvae to those in control reached, respectively, up to 3.0-, 3.9-, and 2.7-fold, at methanol concentrations of 22.6, 27.9, and 34.5 mg/g diet. In addition, methanol exposure greatly up-regulated the mRNA expression level of five Cyp genes, which were Cyp304a1, Cyp9f2, Cyp28a5, Cyp4d2, and Cyp4e2. Their resulting proteins were suggested as the candidate enzymes for methanol metabolism in D. melanogaster larvae.     

Metabolism of Methoxychlor by the P450‐Monooxygenase CYP6G1 Involved in Insecticide Resistance of Drosophila melanogaster after Expression in Cell Cultures of Nicotiana tabacum

Cytochrome P450 monooxygenase CYP6G1 of Drosophila melanogaster was heterologously expressed in a cell suspension culture of Nicotiana tabacum. This in vitro system was used to study the capability of CYP6G1 to metabolize the insecticide methoxychlor (=1,1,1‐trichloro‐2,2‐bis(4‐methoxyphenyl)ethane, 1 ) against the background of endogenous enzymes of the corresponding non‐transgenic culture. The Cyp6g1‐transgenic cell culture metabolized 96% of applied methoxychlor (45.8 μg per assay) within 24 h by demethylation and hydroxylation mainly to trishydroxy and catechol methoxychlor (16 and 17%, resp.). About 34% of the metabolism and the distinct formation of trishydroxy and catechol methoxychlor were due to foreign enzyme CYP6G1. Furthermore, methoxychlor metabolism was inhibited by 43% after simultaneous addition of piperonyl butoxide (458 μg), whereas inhibition in the non‐transgenic culture amounted to 92%. Additionally, the rate of glycosylation was reduced in both cultures. These results were supported by the inhibition of the metabolism of the insecticide imidacloprid ( 6 ; 20 μg, 24 h) in the Cyp6g1‐transgenic culture by 82% in the presence of piperonyl butoxide (200 μg). Due to CYP6G1 being responsible for imidacloprid resistance of Drosophila or being involved in DDT resistance, it is likely that CYP6G1 conveys resistance to methoxychlor ( 1 ). Furthermore, treating Drosophila with piperonyl butoxide could weaken the observed resistance phenomena.     

细胞色素P450介导的果蝇抗药性研究进展

细胞色素P450 (cytochrome P450, CYP450)超基因家族是由一些数量多而功能复杂的血红蛋白酶基因所组成,该代谢酶系作为一种几乎地球上所有需氧生物都存在的重要生存策略,可以调控多种内源物质及外源化合物的代谢,参与了众多重要的生命过程,代谢解毒作用是该酶系重要功能之一。细胞色素P450的代谢解毒作用受药物影响,机体通过改变基因表达量,实现增强代谢解毒,加快机体对于有害物质的代谢,从而使得机体对有害环境产生一定的适应性,进而使得机体产生耐药性或抗药性。本研究说明果蝇细胞色素P450介导的杀虫剂类药物代谢机制及代谢抗性的特点等方面的研究,对明确果蝇的抗药性机制研究具有参考意义。     

A comparison of Drosophila melanogaster detoxification gene induction responses for six insecticides, caffeine and phenobarbital

Modifications of metabolic pathways are important in insecticide resistance evolution. Mutations leading to changes in expression levels or substrate specificities of cytochrome P450 (P450), glutathione-S-transferase (GST) and esterase genes have been linked to many cases of resistance with the responsible enzyme shown to utilize the insecticide as a substrate. Many studies show that the substrates of enzymes are capable of inducing the expression of those enzymes. We investigated if this was the case for insecticides and the enzymes responsible for their metabolism. The induction responses for P450s, GSTs and esterases to six different insecticides were investigated using a custom designed microarray in Drosophila melanogaster. Even though these gene families can all contribute to insecticide resistance, their induction responses when exposed to insecticides are minimal. The insecticides spinosad, diazinon, nitenpyram, lufenuron and dicyclanil did not induce any P450, GST or esterase gene expression after a short exposure to high lethal concentrations of insecticide. DDT elicited the low-level induction of one GST and one P450. These results are in contrast to induction responses we observed for the natural plant compound caffeine and the barbituate drug phenobarbital, both of which highly induced a number of P450 and GST genes under the same short exposure regime. Our results indicate that, under the insecticide exposure conditions we used, constitutive over-expression of metabolic genes play more of a role in insect survival than induction of members of these gene families.     

No evidence of mitochondrial genetic variation for sperm competition within a population of Drosophila melanogaster

Recent studies have advocated a role for mitochondrial DNA (mtDNA) in sperm competition. This is controversial because earlier theory and empirical work suggested that mitochondrial genetic variation for fitness is low. Yet, such studies dealt only with females and did not consider that variation that is neutral when expressed in females, might be non-neutral in males as, in most species, mtDNA is never selected in males. We measured male ability to compete for fertilizations, at young and late ages, across 25 cytoplasms expressed in three different nuclear genetic backgrounds, within a population of Drosophila melanogaster. We found no cytoplasmic (thus no mtDNA) genetic variation for either male offence or offensive sperm competitiveness. This contrasts with previous findings demonstrating cytoplasmic genetic variation for female fitness and female ageing across these same lines. Taken together, this suggests that mitochondrial genes do not contribute to variation in sperm competition at the within-population level.     

Seasonal fluctuation in susceptibility to insecticides within natural populations of Drosophila melanogaster. II. Features of genetic variation in susceptibility to organophosphate insecticides within natural populations of D. melanogaster

To elucidate genetic variation in susceptibility to organophosphate insecticides within natural populations of Drosophila melanogaster, we conducted an analysis of variance for mortality data sets of isofemale lines (10-286 lines) used in the previous studies. Susceptibility of isofemale lines to the three organophosphate insecticides was continuously distributed within each natural population, ranging from susceptible to resistant. Analysis of variance showed highly significant variation among isofemale lines in susceptibility to each insecticide for each natural population. Significant genetic variances in susceptibility to the three chemicals were estimated for the Katsunuma population; 0.0529-0.2722 for malathion, 0.0492-0.1603 for prothiophos, and 0.0469-0.1696 for fenitrothion. Contrary to the consistent seasonal tendency towards an increase in mean susceptibility in the fall, reported in the previous study, genetic variances in susceptibility to the three organophosphates did not change significantly in 1997 but tended to increase by 2- to 5-times in 1998. We tested whether both the observed situations, maintenance and increase in genetic variance in organophosphate resistance, can be generated under circumstances in which the levels of resistance to the three organophosphates tended to decrease, by conducting a simulation analysis, based on the hypothesis that resistant genotypes have lower fitnesses than susceptible ones under the density-independent condition. The simulation analysis generally explained the pattern in the mean susceptibility and genetic variances in susceptibility to the three organophosphates, observed in the Katsunuma population of D. melanogaster. It was suggested that the differences in the frequencies of resistance genes in the summer population could affect the patterns in genetic variance in organophosphate resistance in the fall population.     

Cis-acting mutation and duplication: History of molecular evolution in a P450 haplotype responsible for insecticide resistance in Culex quinquefasciatus

A cytochrome P450 gene, Cyp9m10, is more than 200-fold overexpressed in a pyrethroid resistant strain of Culex quinquefasciatus, JPal-per. The haplotype of this strain contains two copies of Cyp9m10 resulted from recent tandem duplication. In this study, we discovered and isolated a Cyp9m10 haplotype closely related to this duplicated Cyp9m10 haplotype from JHB, a strain used for the recent genome project for this mosquito species. The isolated haplotype (JHB-NIID-B haplotype) shared the same insertion of a transposable element upstream of the coding region with JPal-per strain but not duplicated. The JHB-NIID-B haplotype was considered to have diverged from the JPal-per lineage just before the duplication event. Cyp9m10 was moderately overexpressed in larvae with the JHB-NIID-B haplotype. The overexpressions in JHB-NIID-B and JPal-per haplotypes were developmentally regulated in similar pattern indicating both haplotypes share a common cis-acting mutation responsible for the overexpressions. The isolated moderately overexpressed haplotype conferred resistance, however, its efficacy was relatively small. We hypothesized that the first cis-acting mutation modified the consequence of the subsequent duplication in JPal-per lineage to confer stronger phenotypic effect than that if it occurred before the first cis-acting mutation.     

Functional characterization of the Tetranychus urticae CYP392A11, a cytochrome P450 that hydroxylates the METI acaricides cyenopyrafen and fenpyroximate

Cyenopyrafen is a Mitochondrial Electron Transport Inhibitor (METI) acaricide with a novel mode of action at complex II, which has been recently developed for the control of the spider mite Tetranychus urticae, a pest of eminent importance globally. However, some populations of T. urticae are cross-resistant to this molecule, and cyenopyrafen resistance can be readily selected in the lab. The cytochrome P450s genes CYP392A11 and CYP392A12 have been strongly associated with the phenotype. We expressed the CYP392A11 and the CYP392A12 genes with T. urticae cytochrome P450 reductase (CPR) in Escherichia coli. CYP392A12 was expressed predominately as an inactive form, witnessed by a peak at P420, despite optimization efforts on expression conditions. However, expression of CYP392A11 produced a functional enzyme, with high activity and preference for the substrates Luciferin-ME EGE and ethoxycoumarin. CYP392A11 catalyses the conversion of cyenopyrafen to a hydroxylated analogue (k_cat = 2.37 pmol/min/pmol P450), as well as the hydroxylation of fenpyroximate (k_cat = 1.85 pmol/min/pmol P450). In addition, transgenic expression of CYP392A11 in Drosophila melanogaster, in conjunction with TuCPR, confers significant levels of fenpyroximate resistance.The overexpression of CYP392A11 in multi-resistant T. urticae strains, not previously exposed to cyenopyrafen, which had been indicated by microarray studies, was confirmed by qPCR, and it was correlated with significant levels of cyenopyrafen and fenpyroximate cross-resistance. The implications of our findings for insecticide resistance management strategies are discussed.     

Homology modeling of cytochrome P450scc and the mutations for optimal amperometric sensor

A new homology model of bovine cytochrome P450scc is obtained starting from the recently determined crystal structure of mammalian cytochrome P450 2B4. The new emerging structure appears compatible with recent diffraction patterns of bovine P450scc microcrystals as obtained at the Microfocus Beamline of the European Synchrotron Radiation in Grenoble and here reported for the first time. The same atomic structure is utilized thereby to predict the mutations needed for modifying redox potential. A comprehensive comparison is finally carried out with the previous model present in the RCSB Protein DataBank also in terms of the alternative mutations being predicted for the same functional modification. The implication of these studies for optimal sensor construction is discussed.     

Stereoselective sulfoxidation of the pesticide methiocarb by flavin-containing monooxygenase and cytochrome P450-dependent monooxygenases of rat liver microsomes. Anticholinesterase activity of the two sulfoxide enantiomers

Evidence based on thermal lability and enzyme inhibition data suggests that the sulfoxidation of methiocarb (an N-methylcarbamate insecticide) by rat liver microsomes is catalyzed by flavin-containing monooxygenase(s) (FMO) and by cytochrome(s) P450 (P450). In control rats, the relative proportion is ca. 50% P450:50% FMO. Stereoselective formation of methiocarb sulfoxide from the corresponding sulfide has also been examined to compare the enantioselectivity of the two different enzyme systems. Only the FMO-dependent sulfoxidation presents a high stereoselectivity with an enantiomeric excess of 88% in favor of the (A)-enantiomer. Pretreatment of rats with different P450 inducers such as phenobarbital, 3-methylcholanthrene, dexamethasone, and pyrazole did not affect, or decreased, the rate of methiocarb sulfoxidation. Stereoselectivity of the reaction was modified, mainly because of changes in the relative involvement of FMO and P450 in sulfoxidase activity in pretreated animals. The acetylcholinesterase inhibition properties of methiocarb and its main metabolites were also investigated. Racemic methiocarb sulfoxide was slightly less inhibitory (Ki = 0.216 μM^?1· min^?1) than methiocarb, but a 10-fold difference was observed between the bimolecular rate constants found for the two sulfoxides produced (0.054 and 0.502 μM^?1·min^?1 for the (A) and (B) enantiomers, respectively). © 1995 John Wiley & Sons, Inc.     

Continuing studies on chromosomal polymorphism in a Korean natural population of Drosophila melanogaster

Investigations on the chromosomal inversion polymorphism were conducted on a Korean (Taenung) natural population of D. melanogaster during the period 1978 to 1992. A total of 66 different endemic and cosmopolitan inversions were found on both major chromosome pairs II and III. Some of them proved to be rare cosmopolitan types (2LKA, 2LNS, 2LF, 2RCy, 3LM, 3RKI, and 3RK), while others were endemics. The distribution of breakpoints for endemic and rare cosmopolitan inversions are not random along the two autosome arms.With respect to frequency changes, the 15-year survey revealed that five of the cosmopolitan types (2Lt, 2RNS, 3LP, 3RC, and 3RMo) exhibit cyclical frequency changes, whereas gene arrangement 3RP shows relatively stable frequencies. Tests for correlations between gene arrangement frequencies and several climatic variables gave no clear evidence for such relationships. Only one correlation coefficient out of 64 was statistically significant. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cross-linking of human cytochrome P450 2B6 to NADPH-cytochrome P450 reductase: Identification of a potential site of interaction

The site(s) of interaction between human cytochrome P450 2B6 and NADPH-cytochrome P450 reductase (P450 reductase) have yet to be identified. To investigate this, the cross-linking agent 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) was used to covalently link P450 2B6-P450 reductase. Following digestion with trypsin, the cross-linked peptides were identified by reconstituting the peptides in ¹⁸O-water based on the principle that the cross-linked peptides would be expected to incorporate twice as many ¹⁸O atoms as the non-cross-linked peptides. Subsequent mass spectrometric analyses of the resulting peptides led to the identification of one cross-linked peptide candidate. De novo sequencing of the peptide indicated that it is a complex between residues in the C-helix of the P450 (based upon solved X-ray crystal structures of P450 2B4) and the connecting domain of the P450 reductase. To confirm this experimentally, the P450 2B6 peptide identified through the cross-linking studies was synthesized and peptide competition studies were performed. In the presence of the synthetic peptide, P450 catalytic activity was decreased by up to 60% when compared to competition studies performed using a nonsense peptide. Taken together, these studies indicate that residues in the C-helix of P450 2B6 play a major role in the interaction with the P450 reductase.     

生活中潜在诱变剂对果蝇存活率及遗传变异的影响

用紫外线、抗氧化剂、化妆品等多种生活中潜在的诱变剂处理野生型黑腹果蝇,观察果蝇的存活率及性状遗传变异情况,进而分析这些诱变剂对于果蝇的影响。实验结果表明,随紫外线照射时间的增加,果蝇生活力降低,子代果蝇突变率增加;随培养基中抗氧化剂浓度的增加,果蝇突变率与死亡率均呈上升趋势;不同化妆品也对果蝇造成了明显伤害,造成亲代个体死亡,后代出现突变型果蝇。     

A tissue specific cytochrome P450 required for the structure and function of Drosophila sensory organs

Cytochrome P450s have generally been acknowledged as broadly tuned detoxifying enzymes. However, emerging evidence argues P450s have an integral role in cell signaling and developmental processes, via their metabolism of retinoic acid, arachidonic acid, steroids, and other cellular ligands. To study the morphogenesis of Drosophila sensory organs, we examined mutants with impaired mechanosensation and discovered one, nompH, encodes the cytochrome P450 CYP303a1. We now report the characterization of nompH, a mutant defective in the function of peripheral chemo- and mechanoreceptor cells, and demonstrate CYP303a1 is essential for the development and structure of external sensory organs which mediate the reception of vital mechanosensory and chemosensory stimuli. Notably this P450 is expressed only in sensory bristles, localizing in the apical region of the socket cell. The wide diversity of the P450 family and the growing number of P450s with developmental phenotypes suggests the exquisite tissue and subcellular specificity of CYP303a1 illustrates an important aspect of P450 function; namely, a strategy to process critical developmental signals in a tissue- and cell-specific manner.     

Establishment of a yeast system that stably expresses human cytochrome P450 reductase: application for the study of drug metabolism of cytochrome P450s in vitro

Cytochrome P450s (CYPs) hold a balance in studying pharmacokinetics, toxico-kinetics, drug metabolism, and drug-drug interactions, which require association with cytochrome P450 reductase (CPR) to achieve optimal activity. A novel system of Saccharomyces cerevisiae useful for expression studies of mammalian microsomal CYPs was established. Human CPR (hCPR) was co-expressed with human CYP3A4 (hCYP3A4) in this system, and two expression plasmids pTpLC and pYeplac195-3A4 containing the cDNA of hCPR and hCYP3A4 were constructed, respectively. The two plasmids were applied first and controlled by phosphoglycerate kinase (PGK) promoter. S. cerevisiae BWG1-7alpha transformed with the expression plasmids produced the respective proteins in the expected molecular sizes reactive with both anti-hCYP3A4 immunoglobulin (Ig) and anti-hCPR Ig. The activity of hCPR in yeast BWG-CPR was 443.2 nmol reduced cytochrome c/min/mg, which was about three times the CPR activity of the microsome prepared from the parental yeast. The protein amount of hCYP3A4 in BWG-CPR/3A4 was 35.53 pmol/mg, and the 6beta-hydroxylation testosterone formation activity of hCYP3A4 expressed was 7.5 nmol/min/nmol CYP, 30 times higher than the activity of hCYP3A4 expressed in the parental yeast, and almost two times the activity of hCYP3A4 from homologous human liver microsome. Meanwhile, BWG-CPR/3A4 retained 100 generations under nonselective culture conditions, indicating this yeast was a mitotically stable transformant. BWG-CPR was further tested daily by the PCR amplification of hCPR of yeast genome, Western blot analysis, and the activity assay of hCPR of yeast microsome. This special expression host for CYPs was validated to be stable and efficient for the expression of CYPs, applying as an effective selection model for the drug metabolism in vitro.     

Origin and decay of the P element-associated latitudinal cline in Australian Drosophila melanogaster

The latitudinal cline in P transposable element-associated characteristics in eastern Australian populations of Drosophila melanogaster has changed between 1986 and 1991–1994. New collections were made in 1991–1994 from localities along the eastern coast of Australia. P element-associated properties of 256 isofemale lines from 43 localities were evaluated using gonadal dysgenesis and/or singed-weak hypermutability assays. The overall results indicate that both P activity and P susceptibility have declined, with all populations showing a tendency towards a state with little P activity potential but with P repressor function (neutral or ‘Q’). P repressor function is strong in all populations except some of the most southerly. P activity potential peaks at about 27° SLat, and drops off to the south (as in 1983–1986 collections) and to the north (in contrast to 1983–1986 collections); thus the cline is no longer a simple P-to-Q-to-M pattern from north to south, but is now Q-P-Q-M. A mtDNA RFLP that putatively distinguishes North American and European populations varies in frequency among the populations but the frequency does not vary clinally with latitude, ruling out massive introductions from North America and Europe as causing the cline. This revised version was published online in July 2006 with corrections to the Cover Date.     

Expression of a cytochrome P450 gene family in maize

Maize seedlings, like seedlings of many other plants, are rich in cytochrome P450 (P450) enzyme activity. Four P450 genes (CYPzm1–4), isolated from a seedling-specific cDNA library, are characterised by a transient and seedling-specific expression pattern. The maximum steady state mRNA levels are reached at 3 days in root and at 7 days in shoot tissue, respectively. All four genes belong to one gene family and are closely related to the CYP71 family of plant P450 genes, which includes the enzymes of the ripening avocado fruit (CYP71A1) and eggplant hypocotyls (CYP71A2, A3, A4). The expression of these related P450 genes in monocot and dicot plants indicates that these enzymes play a significant role in plants; however, the in vivo enzyme functions are unknown. The divergence of the four members of the maize gene family is sufficiently high to account for different substrate and/or reaction specificity. Although the general expression pattern of the four genes is identical, the maximum steady-state mRNA levels vary in different maize lines. In situ hybridisation reveals the highest mRNA levels in the coleoptile, the first developed leaflets, the ground tissue of the nodular complex, and in the cortex and pith of the region of cell division in the root. The mapping of the maize CYPzm genes shows that, as in animals, P450 genes of the same family can be clustered. The presence of the CYPzm gene cluster in maize argues for generation of distinct plant P450 gene families by gene duplication.     

One oxidant,many pathways: a theoretical perspective of monooxygenation mechanisms by cytochrome P450 enzymes

Density functional theoretical studies of monooxygenation reactivity of the high-valent oxoiron(IV) porphyrin cation-radical compound of cytochrome P450, the so-called Compound I, and of its precursor, the ferric(III)-hydroperoxide species, are described. The degeneracy of the spin states of Compound I, its electron deficiency, and dense orbital manifold lead to two-state and multi-state reactivity scenarios and may thereby create reactivity patterns as though belonging to two or more different oxidants. Most of the controversies in the experimental data are reconciled using Compound I as the sole competent oxidant. Theory finds ferric(III)-hydroperoxide to be a very sluggish oxidant, noncompetitive with Compound I. If and when Compound I is absent, P450 oxidation will logically proceed by another form, but this has to be more reactive than ferric(III)-hydroperoxide. Theoretical studies are conducted to pinpoint such an oxidant for P450.

Effect of acetylcholinesterase oxime-type reactivators K-48 and HI-6 on human liver microsomal cytochromes P450 in vitro

Substances K-48 and HI-6, oxime-type acetylcholinesterase (AChE) reactivators, were tested for their potential to inhibit the activities of human liver microsomal cytochromes P450 (CYP). The compounds were shown to bind to microsomal cytochromes P450 with spectral binding constants of 0.25 ± 0.05 μM (K-48) and 0.54 ± 0.15 μM (HI-6). To find which cytochrome P450 from the human liver microsomal fraction interacts with these compounds, an inhibition of enzyme activities specific for nine individual CYP enzymes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) was studied. The results have shown no prominent inhibition of individual CYP activities with both compounds except the CYP2E1 activity and the HI-6 reactivator. However, the inhibition of this activity was less than 50% which makes the possible drug interactions highly unlikely. Hence, the interaction of K-48 and HI-6 oxime-type AChE reactivators with human liver microsomal CYP enzymes does not seem to be clinically significant and both compounds could be taken in this respect as antidotal drugs with low risk of drug interactions.