Metabolic Detoxification: Mechanism of Insect Resistance to Plant Psoralens

172

138

One of the challenges faced in malarial control is the acquisition of insecticide resistance that has developed in mosquitoes that are vectors for this disease. Anopheles gambiae, which has been the major mosquito vector of the malaria parasite Plasmodium falciparum in Africa, has over the years developed resistance to insecticides including dieldrin, 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), and pyrethroids. Previous microarray studies using fragments of 230 An. gambiae genes identified five P450 loci, including CYP4C27, CYP4H15, CYP6Z1, CYP6Z2, and CYP12F1, that showed significantly higher expression in the DDT-resistant ZAN/U strain compared with the DDT-susceptible Kisumu strain. To predict whether either of the CYP6Z1 and CYP6Z2 proteins might potentially metabolize DDT, we generated and compared molecular models of these two proteins with and without DDT docked in their catalytic sites. This comparison indicated that, although these two CYP6Z proteins share high sequence identity, their metabolic profiles were likely to differ dramatically from the larger catalytic site of CYP6Z1, potentially involved in DDT metabolism, and the more constrained catalytic site of CYP6Z2, not likely to metabolize DDT. Heterologous expressions of these proteins have corroborated these predictions: only CYP6Z1 is capable of metabolizing DDT. Overlays of these models indicate that slight differences in the backbone of SRS1 and variations of side chains in SRS2 and SRS4 account for the significant differences in their catalytic site volumes and DDT-metabolic capacities. These data identify CYP6Z1 as one important target for inhibitor design aimed at inactivating insecticide-metabolizing P450s in natural populations of this malarial mosquito.cytochrome P450 monooxygenases ͉ insecticides ͉ plant allelochemicals I n 2004, the World Health Organization reported that up to 2.7 million people die of malaria every year with 80-90% of these deaths occurring in Africa (ref. 1 and www.africanfront.com/ AIDS1.php). Many prevention and treatment strategies have been developed to tackle this life-threatening disease from the side of the mosquito vector and that of the human host (2, 3). These range from antimalarial drugs to indoor spraying of insecticides and use of bed nets treated with pyrethroid insecticides. Although these practices have helped reduce human mortality, various issues have emerged with mosquito vectors developing insecticide resistance and parasites developing drug resistance. Both of these significantly reduce the efficacy of current malarial prevention and treatment practices (2, 4-8).The development of insecticide resistance in mosquito vectors is illustrated by Anopheles gambiae, which has been the major mosquito vector of the malaria parasite Plasmodium falciparum in Africa (4, 6). Throughout the years, people have reported Anopheles resistance (albeit low-level resistance) to various insecticides including dieldrin (a cyclodiene-type insecticide), 1,1-bis(pchlorophenyl)-2,2,2-trichloroethane (DDT), and pe...

Section: Docking Of Prospective Substrates Into the Cyp6z1 And Cyp6z2mentioning

confidence: 99%

Comparative molecular modeling of Anopheles gambiae CYP6Z1, a mosquito P450 capable of metabolizing DDT

Chiu

Wen

Rupasinghe

et al. 2008

172

138

“…One such furanocoumarin, xanthotoxin (8-methoxypsoralen), is highly toxic to generalist insect herbivores (7)(8)(9) yet is tolerated at high levels by specialist herbivores that selectively feed on furanocoumarin-containing plants. This tolerance in at least two species, the black swallowtail (10,11) and the parsnip webworm Depressaria pastinacella (Lepidoptera: Oecophoridae) (12), is due to the ability of larvae to detoxify xanthotoxin rapidly by midgut cytochrome P-450 activities. In P. polyxenes, this P450 activity appears to be autoregulated in that xanthotoxin induces the P-450 activity responsible for its own metabolism 7-fold over the endogenous activity present in larvae (13) and is associated with the appearance of a protein of -55 kDa in midgut microsomes (14).…”

mentioning

confidence: 99%

“…Onemilliliter reaction mixtures contained 0.1 M sodium phosphate (pH 7.8), 0.3 mM NADP, 3.0 mM glucose 6-phosphate, 0.5 unit of glucose-6phosphate dehydrogenase, 10 (19). RNA was blotted onto a nylon membrane and sequentially probed with the insert of clone CYP6BIvl at both high (40% formamide; 42°C) and low (30%o formamide; 25°C) stringency, and with a 0.8-kb fragment of a Drosophila melanogaster actin clone (20) at high stringency.…”

mentioning

confidence: 99%

A host-inducible cytochrome P-450 from a host-specific caterpillar: molecular cloning and evolution.

Cohen

Schuler

Berenbaum

1992

179

113

Cytochrome P-450 monooxygenases (P-450s) play a critical role in the detoxification of natural and synthetic toxins in a wide range of organisms. We have isolated and sequenced cDNA clones encoding a P-450, CYP6BI, from larvae of Papilo polyxenes (Lepidoptera: Papilionidae), the black swallowtail butterfly. This P-450, cloned from a herbivorous insect, is highly inducible by xanthotoxin, a secondary Larvae of the black swallowtail, Papilio polyxenes (Lepidoptera: Papilionidae), feed on only two plant families, the Apiaceae and Rutaceae (5). These plants produce a diversity and an abundance of furanocoumarins, secondary metabolites that occur only rarely in other families (6). One such furanocoumarin, xanthotoxin (8-methoxypsoralen), is highly toxic to generalist insect herbivores (7-9) yet is tolerated at high levels by specialist herbivores that selectively feed on furanocoumarin-containing plants. This tolerance in at least two species, the black swallowtail (10, 11) and the parsnip webworm Depressaria pastinacella (Lepidoptera: Oecophoridae) (12), is due to the ability of larvae to detoxify xanthotoxin rapidly by midgut cytochrome P-450 activities. In P. polyxenes, this P450 activity appears to be autoregulated in that xanthotoxin induces the P-450 activity responsible for its own metabolism 7-fold over the endogenous activity present in larvae (13) and is associated with the appearance of a protein of -55 kDa in midgut microsomes (14).The molecular cloning of cDNAs encoding this xanthotoxin-inducible protein has allowed us to confirm its identity as a newly discovered P-450, designated CYP6BJ. To determine the origin of xanthotoxin-inducible P-450s in the evolution of the subfamily Papilioninae with respect to colonization offuranocoumarin-producing plants, we examined six additional species within this subfamily for their constitutive and xanthotoxin-induced P-450-mediated metabolism of xanthotoxin and screened Northern blots of midgut poly(A)+ RNA for crossreactivity with CYP6BI. Of the additional swallowtails examined, Papilio brevicauda and Papilio cresphontes are specialists on furanocoumarin-producing plants in the Apiaceae and Rutaceae, respectively (5); Papilio glaucus is a broadly polyphagous species that only occasionally encounters furanocoumarins in a few rutaceous hosts (15); Papilio troilus, Battus philenor, and Eurytides marcellus are specialists on families that do not produce furanocoumarins, the Lauraceae, Aristolochiaceae, and Annonaceae, respectively (5). Poly(A)+ RNA from D. pastinacella, which feeds exclusively on two genera of the Apiaceae high in furanocoumarins (16) but is only distantly related to P. polyxenes, was also screened. These experiments were designed to analyze the evolution of P-450 genes with respect to the speciation and host shifts that have occurred in a closely related group of herbivores and to compare P450 genes in distantly related herbivores specialized to feed on the same plant species. Our results suggest that CYP6B1 is an adaptation enabling the black...

“…Enhanced excretion (22) and sequestration (23) of insecticides seem to be responsible for the resistance of some insects. To check for these two mechanisms, larvae were topically treated with [3H]JH III.…”

Section: Resultsmentioning

confidence: 99%

Resistance to juvenile hormone and an insect growth regulator in Drosophila is associated with an altered cytosolic juvenile hormone-binding protein.

Shemshedini

Wilson²

1990

100

The Met mutant ofDrosophila melanogaster is highly resistant to juvenile hormone Im (JH III) GBq) were racemic mixes.[3H]Methoprene (R isomer, 83.9 Ci/mmol) was a generous gift of G. Prestwich (Stony Brook, NY). Each was stored in a stock solution in hexane at -20'C. Purity was monitored periodically by thin-layer chromatography. Breakdown was almost negligible over a 1-year period under these conditions. Concentrations were determined by radioactivity or UV spectroscopy.Flies were raised at 25 ± 1PC on a cornmeal/agar/yeast/ molasses diet supplemented with Tegosept or propionic acid to retard mold growth. Adults were collected from uncrowded cultures following eclosion. Third-instar larvae were selected several hours before pupariation from the walls of culture bottles.Three alleles of Met were examined in this study (Table 1). Each was recovered from separate screens for methopreneresistant mutants following ethyl methanesulfonate mutagenesis of susceptible strains. Met and Met2 were recovered following mutagenesis of the Oregon-RC wild-type strain and Met3 of the yellow vermilion strain. Each has been maintained for several years as homozygotes; none has shown significant change in resistance to methoprene during this time. Two susceptible Met' strains were examined. First Multiple Seven (FM7) is a laboratory balancer strain having a useful semidominant eye mutation (described in ref. 10). FM7 flies are sensitive to methoprene, and this strain has been used as a Met' strain in our previous studies (8,11). For additional comparison, the wild-type Ho-R strain was also examined; these flies have been shown to be sensitive to methoprene (7).Tenebrio molitor were maintained at room temperature on locally purchased chicken feed. Hemolymph was withdrawn with a microcapillary tube from 1-to 2-day pupae.Penetration and Excretion. A quantity of 4 pmol of [3H]JH III (New England Nuclear) was topically applied in one dose in acetone solution to third-instar larvae as described (12). This dose was chosen as a physiological dose because the amount of hormone that penetrated the cuticle could be readily measured by its radioactivity. Treated larvae were held in glass scintillation vials for 1 hr at 250C. They were then rinsed in acetone to remove unpenetrated hormone. Pene-