Malathion Detoxification Research Articles

Unveiling candidate genes for metabolic resistance to malathion in Aedes albopictus through RNA sequencing-based transcriptome profiling.

Aedes albopictus, also known as the Asian tiger mosquito, is indigenous to the tropical forests of Southeast Asia. Ae. albopictus is expanding across the globe at alarming rates, raising concern over the transmission of mosquito-borne diseases, such as dengue, West Nile fever, yellow fever, and chikungunya fever. Since Ae. albopictus was reported in Houston (Harris County, Texas) in 1985, this species has rapidly expanded to at least 32 states across the United States. Public health efforts aimed at controlling Ae. albopictus, including surveillance and adulticide spraying operations, occur regularly in Harris County. Despite rotation of insecticides to mitigate the development of resistance, multiple mosquito species including Culex quinquefasciatus and Aedes aegypti in Harris County show organophosphate and pyrethroid resistance. Aedes albopictus shows relatively low resistance levels as compared to Ae. aegypti, but kdr-mutation and the expression of detoxification genes have been reported in Ae. albopictus populations elsewhere. To identify potential candidate detoxification genes contributing to metabolic resistance, we used RNA sequencing of field-collected malathion-resistant and malathion-susceptible, and laboratory-maintained susceptible colonies of Ae. albopictus by comparing the relative expression of transcripts from three major detoxification superfamilies involved in malathion resistance due to metabolic detoxification. Between these groups, we identified 12 candidate malathion resistance genes and among these, most genes correlated with metabolic detoxification of malathion, including four P450 and one alpha esterase. Our results reveal the metabolic detoxification and potential cuticular-based resistance mechanisms associated with malathion resistance in Ae. albopictus in Harris County, Texas.

Overexpression of an Integument Esterase Gene LbEST-inte4 Infers the Malathion Detoxification in Liposcelis bostrychophila (Psocoptera: Liposcelididae).

Liposcelis bostrychophila, commonly known as booklouse, is an important stored-product pest worldwide. Studies have demonstrated that booklices have developed resistance to several insecticides. In this study, an integument esterase gene, LbEST-inte4, with upregulated expression, was characterized in L. bostrychophila. Knockdown of LbEST-inte4 resulted in a substantial increase in the booklice susceptibility to malathion. Overexpression of LbEST-inte4 in Drosophila melanogaster significantly enhanced its malathion tolerance. Molecular modeling and docking analysis suggested potential interactions between LbEST-inte4 and malathion. When overexpressed LbEST-inte4 in Sf9 cells, a notable elevation in esterase activity and malathion tolerance was observed. HPLC analysis indicated that the LbEST-inte4 enzyme could effectively degrade malathion. Taken together, the upregulated LbEST-inte4 appears to contribute to malathion tolerance in L. bostrychophila by facilitating the depletion of malathion. This study elucidates the molecular mechanism underlying malathion detoxification and provides the foundations for the development of effective prevention and control measures against psocids.

Functional analysis of an overexpressed glutathione S-transferase BdGSTd5 involved in malathion and malaoxon detoxification in Bactrocera dorsalis

Glutathione S-transferases (GSTs) are one of the three detoxification enzyme families. The constitutive and inducible overexpression of GSTs genes plays an important role in insecticide resistance. Previous study showed that malathion resistance was polygenic, and elevated GSTs activity was one of the important factor participating in malathion resistance of Bactrocera dorsalis (Hendel), a serious economic pest worldwide. BdGSTd5 overexpression was inducible upon exposure to malathion. However, the involvement of BdGSTd5 in malathion resistance has not been clarified. In this study, we found that BdGSTd5 sequence harbored the conserved region of delta class GSTs, which were overexpressed in malathion resistant strain of B. dorsalis compared to malathion susceptible strain. The highest mRNA expression level of BdGSTd5 was found in 1-day-old adult, and the levels decreased with aging. The dsBdGSTd5 injection effectively silenced (73.4% reduction) the expression of BdGSTd5 and caused significant increase in susceptibility to malathion with a cumulative mortality increasing of 13.5% at 72 h post malathion treatment (p < 0.05). Cytotoxicity assay demonstrated that BdGSTd5 was capable of malathion detoxification. Molecular docking analysis further indicated the interactive potential of BdGSTd5 with malathion and its toxic oxide malaoxon. The recombinant BdGSTd5 exhibited glutathione-conjugating activity toward 1-chloro-2, 4-dinitrobenzene and malathion and malaoxon metabolic capacity with significant reduction (p < 0.05) of the peak areas by 90.0% and 73.1%, respectively. Taken together, the overexpressed BdGSTd5 contributes to malathion metabolism and resistance, which detoxify the malathion in B. dorsalis via directly depleting malathion and malaoxon.

Potential key genes involved in metabolic resistance to malathion in the southern house mosquito, Culex quinquefasciatus, and functional validation of CYP325BC1 and CYP9M12 as candidate genes using RNA interference

BackgroundMetabolic detoxification is one of the major mechanisms contributing to the development of resistance in mosquitoes, including the southern house mosquito, Culex quinquefasciatus. The three major detoxification supergene families, cytochrome P450s, glutathione S-transferases and general esterases, have been demonstrated to play an important role in metabolic resistance. In this study, we performed differential gene expression analysis based on high-throughput transcriptome sequencing on samples from four experimental groups to give insight into key genes involved in metabolic resistance to malathion in Cx. quinquefasciatus. We conducted a whole transcriptome analysis of field captured wild Cx. quinquefasciatus from Harris County (WI), Texas and a malathion susceptible laboratory-maintained Sebring colony (CO) to investigate metabolic insecticide resistance. Field captured mosquitoes were also phenotypically classified into the malathion resistant and malathion susceptible groups following a mortality response measure conducted using a Centers for Disease Control and Prevention (CDC) bottle assay. The live (MR) and dead (MS) specimens from the bottle assay, along with an unselected WI sample and a CO sample were processed for total RNA extraction and subjected to whole-transcriptome sequencing.ResultsWe demonstrated that the genes coding for detoxification enzymes, particularly cytochrome P450s, were highly up-regulated in the MR group compared to the MS group with similar up-regulation observed in the WI group compared to the CO group. A total of 1,438 genes were differentially expressed in comparison between MR and MS group, including 614 up-regulated genes and 824 down-regulated genes. Additionally, 1,871 genes were differentially expressed in comparison between WI and CO group, including 1,083 up-regulated genes and 788 down-regulated genes. Further analysis on differentially expressed genes from three major detoxification supergene families in both comparisons resulted in 16 detoxification genes as candidates potentially associated with metabolic resistance to malathion. Knockdown of CYP325BC1 and CYP9M12 using RNA interference on the laboratory-maintained Sebring strain significantly increased the mortality of Cx. quinquefasciatus after exposure to malathion.ConclusionWe generated substantial transcriptomic evidence on metabolic detoxification of malathion in Cx. quinquefasciatus. We also validated the functional roles of two candidate P450 genes identified through DGE analysis. Our results are the first to demonstrate that knockdown of CYP325BC1 and CYP9M12 both significantly increased malathion susceptibility in Cx. quinquefasciatus, indicating involvement of these two genes in metabolic resistance to malathion.

Evaluations of two glutathione S-transferase epsilon genes for their contributions to metabolism of three selected insecticides in Locusta migratoria

The insect-specific epsilon class of glutathione S-transferases (GSTEs) plays important roles in insecticide detoxification in insects. In our previous work, five GSTEs were identified in Locusta migratoria, and two recombinant GSTEs, rLmGSTE1 and rLmGSTE4, showed high catalytic activity when 1-chloro-2,4-dinitrobenzene (CDNB) was used as a substrate. In this work, we further investigated whether these two GSTEs could metabolize three insecticides including malathion, deltamethrin and DDT. Using ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC/MS) method, we found that rLmGSTE4, but not rLmGSTE1, can metabolize malathion and DDT. Malathion bioassays of L.migratoria after the expression of LmGSTE4 was suppressed by RNA interference (RNAi) showed increased insect mortality from 33.8% to 68.9%. However, no changes in mortality were observed in deltamethrin- or DDT-treated L.migratoria after the expression of LmGSTE4 was suppressed by RNAi. Our results provided direct evidences that LmGSTE4 participates in malathion detoxification in L.migratoria. These findings are important for understanding the mechanisms of insecticide resistance in L.migratoria and developing new strategies for managing the insect populations in the field.

Identification of two ABCC transporters involved in malathion detoxification in the red flour beetle, Tribolium castaneum.

ABC transporters have been suggested to be involved in insecticide detoxification in different insect species mainly based on the indirect observation of transcriptional upregulation of ABC gene expression in response to insecticide exposure. Previous studies performed by us and others in the red flour beetle, Tribolium castaneum, have analyzed the function of TcABCA-C and TcABCG-H genes using RNA interference (RNAi) and demonstrated that specific TcABCA and TcABCC genes are involved in the elimination of the pyrethroid tefluthrin and the benzoylurea diflubenzuron, because gene silencing increased the beetle's susceptibility to the insecticides. In this study, we focused on the potential functions of TcABCA-C genes in detoxification of the pyrethroid cyfluthrin (CF), the organophosphate malathion (MAL) and the diacylhdyazine tebufenozide (TBF). Analysis of transcript levels of selected TcABCA-C genes in response to treatment with these three chemically unrelated insecticides revealed that some genes were particularly upregulated after insecticide treatment. In addition, the ABC inhibitor verapamil synergized significantly the toxicity of MAL but only negligibly CF and TBF toxicities. Finally, silencing of two TcABCC genes by RNAi revealed a significant increase in susceptibility to MAL. In contrast, we did not observe a significant increase in insecticide-induced mortalities when knocking down TcABC genes in larvae treated with CF or TBF, although they were upregulated in response to insecticide treatment. Our results suggest that two pleiotropic ABCC transporters expressed in metabolic and excretory tissues contribute to the elimination of MAL.

Functional Characterization of an α-Esterase Gene Associated with Malathion Detoxification in Bradysia odoriphaga

Carboxylesterases (CarEs) are a multigene superfamily of metabolic enzymes involved in metabolic detoxification of xenobiotics. In this study, an α-esterase gene (BoαE1) was identified from Bradysia odoriphaga. Phylogenetic analysis classified BoαE1 into the α-esterase clade. Developmental expression analysis indicated that BoαE1 was significantly expressed in the second to fourth larval stages. Tissue-specific expression analysis indicated that BoαE1 was highly expressed in the larval midgut. After exposure to LC30 of malathion, the CarE activity of B. odoriphaga was induced and the transcriptional level of BoαE1 was significantly up-regulated. Silencing of BoαE1 significantly increased the susceptibility of B. odoriphaga larvae to malathion. Inhibition assays in vitro indicated that malathion significantly inhibited BoαE1 activity. GC-MS assay showed that BoαE1 possesses hydrolase activity toward malathion and participates in the detoxification of malathion. These results strongly suggest that BoαE1 plays a crucial role in detoxification of malathion in B. odoriphaga.

Characterization of Esterase Genes Involving Malathion Detoxification and Establishment of an RNA Interference Method in Liposcelis bostrychophila.

Esterases (ESTs) play important roles in metabolizing various physiologically endogenous and exogenous compounds, and various environmental xenobiotics in insects. The psocid, Liposcelis bostrychophila is a major pest of stored products worldwide and rapidly develops resistance to commonly insecticides. However, the involvement of ESTs in insecticide metabolization and the application of RNAi approach in psocids have not been well elucidated. In this study, we characterized four LbEST genes and investigated the transcriptional levels of these genes at different developmental stages and under different insecticides exposures to assess their potential roles in response to insecticides. The four LbESTs contain a catalytic triad (Ser-His-Glu) linked to an oxyanion hole and acyl pocket involved in substrate stabilization during its hydrolysis. Synergism observed with the esterase-inhibitor DEF suggests the involvement of esterases in malathion detoxification. LbESTs were expressed during the whole of developmental stages, but predominant abundance in the first nymphal instar and adult stage. The mRNA level of three LbEST genes (except for LbEST4) was induced (1.29- to 5.60 fold) in response to malathion or deltamethrin exposures, indicating that these esterases are involved in the detoxification process. Silencing of LbEST1, LbEST2 or LbEST3 through dsRNA feeding led to a higher mortality of psocids upon the malathion treatment compared to controls (1.83 to 2.69-fold), demonstrating that these esterase genes play roles in malathion detoxification in L. bostrychophila. Our study provides new evidence for understanding of the function and regulation mechanism of esterases in L. bostrychophila in insecticide detoxification. The current study also suggests that the present RNAi method could be applied for gene functional studies in psocids.

A comparative study on degradation of complex malathion organophosphate using of Escherichia coli IES-02 and a novel carboxylesterase

Malathion organophosphates considered as the major constituent of herbicides, pesticides and insecticides. Extensively used in agricultural, horticultures and for numerous household applications contributes to precedence organic pollutants leading antagonistic effects on human health and environment. Therefore detoxification of malathion from contaminated site is of general interest. Simultaneously it is very emerging to isolated novel indigenous microbial strains from contaminated site with a record of pesticide application. In this study Escherichia coli IES-02 isolated from malathion contaminant effluent and the strain showed maximum efficiency in malathion degradation that utilized it as the sole source of carbon. Carboxylesterase (33.0, 30.0, 28.0 kDa) were purified (1685.71 U/mg) from Escherichia coli IES-02 showed significant results in malathion degradation approximately 81% within 20 min as compared with Escherichia coli IES-02 cells within 4 h (99.0 to 95.0%) into monocarboxylic acid and diacid derivatives. The generation time of Escherichia coli was also observed at 60 min with 0.1 ppm, 68 min with 0.5 ppm, 74.5 min with 2.0 ppm and 91.37 min with 50 ppm of malathion. The degradation rate and transformation metabolites were estimated by Gas Chromatography-Mass Spectrometry respectively. Malathion metabolites pathway proposed in this study which revealed the potential application against lethal environmental pollution.

Divergent molecular evolution in glutathione S-transferase conferring malathion resistance in the oriental fruit fly, Bactrocera dorsalis (Hendel).

Insect glutathione S-transferases (GSTs) are important in insecticide detoxification and Insect-specific GSTs, Epsilon and Delta, have largely expanded in insects. In this study, we functionally expressed and characterized an epsilon class GST gene (BdGSTe8), predominant in the adult Malpighian tubules of Bactrocera dorsalis. This gene may be associated with malathion resistance based on transcriptional studies of resistant and susceptible strains. RNA interference-mediated knockdown of this gene significantly recovered malathion susceptibility in the adults of a malathion-resistant strain, and overexpression of BdGSTe8 enhanced resistance in transgenic Drosophila. Analysis of BdGSTe8 polymorphism showed that several point mutations may be associated with metabolic resistance to malathion. A cytotoxicity assay in Escherichia coli indicated that both of the recombinant BdGSTe8 proteins may play a functional role in protecting cells from toxicity. The allele of BdGSTe8-B conferred higher levels of malathion detoxification capability. Liquid chromatography and ultra-performance liquid chromatography-tandem mass spectrometry analysis showed that the BdGSTe8-A allele did not metabolize malathion directly. However, the BdGSTe8-B allele was involved in the direct metabolism of malathion, which was caused by a mutation in V128A. Further analysis of the sequence suggests that BdGSTe8 evolved rapidly. It maybe play the role of a backup gene and could become a new gene in the future in order to retain the ability of detoxification of malathion, which was driven by positive selection. These results suggest that divergent molecular evolution in BdGSTe8 has played a role in metabolic resistance to malathion in B.dorsalis.

Two delta class glutathione S-transferases involved in the detoxification of malathion in Bactrocera dorsalis (Hendel).

The oriental fruit fly Bactrocera dorsalis (Hendel), a widespread agricultural pest, has evolved resistance to many insecticides, including organophosphorus compounds. Glutathione S-transferases (GSTs) are involved in xenobiotic detoxification and insecticide resistance in many insects. However, the role of delta class GSTs in detoxifying malathion in B. dorsalis is unknown. Here, we evaluated the roles of two delta class GSTs in malathion detoxification in this species. Two delta class GSTs genes, BdGSTd1 and BdGSTd10, were characterized in B. dorsalis. They were highly expressed in 5-day-old adults, as well as in midgut and Malpighian tubules. Upon malathion exposure, the two genes were upregulated by 2.63- and 2.85-fold, respectively. Injection of double-stranded RNA targeting BdGSTd1 or BdGSTd10 significantly reduced their mRNA levels in adults and also significantly increased adult susceptibility to malathion. The expression of these two GSTs in Escherichia coli helped the host to endure malathion stress at a concentration of 10µg mL-1 according to a Cell Counting Kit-8 assay. High-performance liquid chromatography analyses indicated that malathion could be significantly depleted by the two delta GSTs. The role of BdGSTd10 in malathion sequestration was also discussed. BdGSTd1 and BdGSTd10 play important roles in the detoxification of malathion in B. dorsalis. © 2019 Society of Chemical Industry.

Functional characterization of BdB1, a well-conserved carboxylesterase among tephritid fruit flies associated with malathion resistance in Bactrocera dorsalis (Hendel)

There are many evidences that insect carboxylesterase possess important physiological roles in xenobiotic metabolism and are implicated in the detoxification of organophosphate (OP) insecticides. Despite the ongoing resistance development in the oriental fruit fly, Bactrocera dorsalis (Hendel), the molecular basis of carboxylesterase and its ability to confer OP resistance remain largely obscure. This study was initiated to provide a better understanding of carboxylesterase-mediated resistance mechanism in a tephritid pest fly. Here, we narrow this research gap by demonstrating a well-conserved esterase B1 gene, BdB1, mediates malathion resistance development via gene upregulation with the use of a laboratory selected malathion-resistant strain (MR) of B. dorsalis. No sequence mutation of BdB1 was detected between MR and the susceptible strain (MS) of B. dorsalis. BdB1 is predominantly expressed in the midgut, a key insect tissue for detoxification. As compared with transcripts in MS, BdB1 was significantly more abundant in multiple tissues in the MR. RNA interference (RNAi)-mediated knockdown of BdB1 significantly increased malathion susceptibility. Furthermore, heterologous expression along with cytotoxicity assay revealed BdB1 could probably have the function of malathion detoxification.

Combinatorial evolution of phosphotriesterase toward a robust malathion degrader by hierarchical iteration mutagenesis.

Malathion is one of the most widely used organophosphorus pesticides in the United States and developing countries. Herein, we enhanced the degradation rate of malathion starting with a phosphotriesterase PoOPHM2 while also considering thermostability. In the first step, iterative saturation mutagenesis at residues lining the binding pocket (CASTing) was employed to optimize the enzyme active site for substrate binding and activity. Hot spots for enhancing activity were then discovered through epPCR-based random mutagenesis, and these beneficial mutations were then recombined by DNA shuffling. Finally, guided by in silico energy calculations (FoldX), thermostability of the variant was improved. The mutations extend from the core region to the enzyme surface during the evolutionary pathway. After screening <9,000 mutants, the best variant PoOPHM9 showed 25-fold higher activity than wild-type PoOPHM2 , with a thermostability (T50 (15) ) of 67.6°C. Thus, PoOPHM9 appears to be an efficient and robust candidate for malathion detoxification. Biotechnol. Bioeng. 2016;113: 2350-2357. © 2016 Wiley Periodicals, Inc.

Functional characterization of an α-esterase gene involving malathion detoxification in Bactrocera dorsalis (Hendel)

Extensive use of insecticides in many orchards has prompted resistance development in the oriental fruit fly, Bactrocera dorsalis (Hendel). In this study, a laboratory selected strain of B. dorsalis (MR) with a 21-fold higher resistance to malathion was used to examine the resistance mechanisms to this organophosphate insecticide. Carboxylesterase (CarE) was found to be involved in malathion resistance in B. dorsalis from the synergism bioassay by CarE-specific inhibitor triphenylphosphate (TPP). Molecular studies further identified a previously uncharacterized α-esterase gene, BdCarE2, that may function in the development of malathion resistance in B. dorsalis via gene upregulation. This gene is predominantly expressed in the Malpighian tubules, a key insect tissue for detoxification. The transcript levels of BdCarE2 were also compared between the MR and a malathion-susceptible (MS) strain of B. dorsalis, and it was significantly more abundant in the MR strain. No sequence mutation or gene copy changes were detected between the two strains. Functional studies using RNA interference (RNAi)-mediated knockdown of BdCarE2 significantly increased the malathion susceptibility in the adult files. Furthermore, heterologous expression of BdCarE2 combined with cytotoxicity assay in Sf9 cells demonstrated that BdCarE2 could probably detoxify malathion. Taken together, the current study bring new molecular evidence supporting the involvement of CarE-mediated metabolism in resistance development against malathion in B. dorsalis and also provide bases on functional analysis of insect α-esterase associated with insecticide resistance.

The epsilon glutathione S-transferases contribute to the malathion resistance in the oriental fruit fly, Bactrocera dorsalis (Hendel)

Epsilon glutathione S-transferases (eGSTs) play important roles in xenobiotics detoxification and insecticides resistance in insects. However, the molecular mechanisms of eGSTs-mediated insecticide resistance remain largely unknown in the Bactrocera dorsalis (Hendel), one of the most notorious pests in the world. Here, we investigated the roles of eight GST genes which belonged to epsilon class (BdGSTe1, BdGSTe2, BdGSTe3, BdGSTe4, BdGSTe5, BdGSTe6, BdGSTe7 and BdGSTe9) in conferring malathion resistance in B. dorsalis. Adult developmental stage-, sex- and tissue-specific expression patterns of the eight eGST genes were analyzed via quantitative reverse transcription PCR. The results showed that BdGSTe2, BdGSTe3, BdGSTe4 and BdGSTe9 were abundant in the midgut, fat body and Malpighian tubules. Notably, BdGSTe2, BdGSTe4 and BdGSTe9 were significantly overexpressed in a malathion-resistant (MR) strain of B. dorsalis compared to the malathion-susceptible (MS) strain. Functional expression and cytotoxicity assays showed significantly higher malathion detoxification capabilities in BdGSTe2-, BdGSTe3-, BdGSTe4- and BdGSTe9-expressing Sf9 cells compared to the parental and green fluorescent protein (GFP)-expressing Sf9 cells. Moreover, malathion susceptibility in MS adults was increased 30%, 14%, and 33% when BdGSTe2, BdGSTe3 and BdGSTe4 mRNA levels were repressed by RNA interference (RNAi)-mediated knockdown, respectively. Taken together, overexpression of the isoforms of eGSTs, including BdGSTe2, BdGSTe4, and particularly, BdGSTe9 plays an important role in the malathion resistant development in B. dorsalis.

Molecular and functional characterization of cDNAs putatively encoding carboxylesterases from the migratory locust, Locusta migratoria.

Carboxylesterases (CarEs) belong to a superfamily of metabolic enzymes encoded by a number of genes and are widely distributed in microbes, plants and animals including insects. These enzymes play important roles in detoxification of insecticides and other xenobiotics, degradation of pheromones, regulation of neurodevelopment, and control of animal development. In this study, we characterized a total of 39 full-length cDNAs putatively encoding different CarEs from the migratory locust, Locusta migratoria, one of the most severe insect pests in many regions of the world, and evaluated the role of four CarE genes in insecticide detoxification. Our phylogenetic analysis grouped the 39 CarEs into five different clades including 20 CarEs in clade A, 3 in D, 13 in E, 1 in F and 2 in I. Four CarE genes (LmCesA3, LmCesA20, LmCesD1, LmCesE1), representing three different clades (A, D and E), were selected for further analyses. The transcripts of the four genes were detectable in all the developmental stages and tissues examined. LmCesA3 and LmCesE1 were mainly expressed in the fat bodies and Malpighian tubules, whereas LmCesA20 and LmCesD1 were predominately expressed in the muscles and hemolymph, respectively. The injection of double-stranded RNA (dsRNA) synthesized from each of the four CarE genes followed by the bioassay with each of four insecticides (chlorpyrifos, malathion, carbaryl and deltamethrin) increased the nymphal mortalities by 37.2 and 28.4% in response to malathion after LmCesA20 and LmCesE1 were silenced, respectively. Thus, we proposed that both LmCesA20 and LmCesE1 played an important role in detoxification of malathion in the locust. These results are expected to help researchers reveal the characteristics of diverse CarEs and assess the risk of insecticide resistance conferred by CarEs in the locust and other insect species.

Inclusion Effect of Alpha-Cyclodextrin on Chemical Degradation of Malathionin Water

Chemical degradation of malathion in water is one of the important environmental processes of malathion, and plays a critical role in detoxification of malathion. Alpha-cyclodextrin (alpha-CD), one of the cyclic oligosaccharides different from beta-cyclodextrin (beta-CD) in glucose units, solubility, and size of cavity, is capable of producing an environmental effect different from those of the latter CD and its derivatives. This work evaluated the effect of alpha-CD on chemical degradation of malathion in water in an attempt to further study the effects of different CDs on hydrolysis of malathion and assess the potential applications of alpha-CD in remediation of malathion contamination. The results showed that alpha-CD had a remarkable facilitative effect on the hydrolysis of malathion in both buffer solutions and natural surface waters. The facilitative effects of alpha-CD were an obverse function of pH value and inverse function of temperature. The rate constant of malathion hydrolysis increased with increasing the concentration of alpha-CD in both types of waters. Urea, a compound widely used in agriculture, decreased the facilitative effect of alpha-CD at 25 degrees C and pH 9.0. The formation of inclusion complexes between alpha-CD and malathion was evidenced by nuclear magnetic resonance studies.

Malathion detoxification by human hepatic carboxylesterases and its inhibition by isomalathion and other pesticides

The organophosphorothioate (OPT) pesticide malathion (MAL) in mammals is readily hydrolyzed by mammalian carboxylesterases (CE). The reaction competes with the CYP-catalyzed formation of malaoxon (MOX), the toxic metabolite. Alterations or individual variations in CE activity may result in increased MOX formation, enhancing MAL toxicity. We have characterized the human hepatic CE activity in a panel of 18 human liver microsomes as well as the inhibitory effect of IsoMAL, a major impurity of MAL commercial formulations, parathion (PAR), chlorpyrifos (CPF), and chlorpyrifos-oxon (CPFO). CE activity showed a low level of variation among individuals (4-fold). The reaction consists of two different phases, differing in their affinity for mal (k(m1)=0.25-0.69 microm; K(m2)=10.3-26.8 microM). The relatively low K(m1) values confirmed that human CE efficiently detoxify MAL. IsoMAL was shown to be a potent noncompetitive inhibitor of MAL detoxification (K(i)=0.6 microM), with a higher inhibitory potency than CPF and PAR (K(i)=7.5 microM and 50 microM, respectively). These two latter compounds very likely act as mixed inhibitors. CPFO showed the highest inhibitory potency toward CE-mediated detoxification, being characterized by a K(i)=22 nM. The present results provide useful information for a better understanding of possible interactions between different OPTs and for assessing the potential cumulative risk for exposure to OPT mixtures.

Detoxification of malathion a chemical warfare agent analog using oxygen activation at room temperature and pressure

The organophosphorus insecticide malathion was selected as an analog for the chemical nerve agent, VX. Degradation of 0.44 mM malathion in a 10 mL aqueous solution containing 0.50 g granular zero valent iron (ZVI) under ambient air and pressure was complete after 4 h to the detection limit of GC-FID. The degradation kinetics demonstrate the system to be pseudo-first-order with respect to malathion disappearance with a rate constant of 0.92 h−1. The only non-polar organic intermediates detected were diethyl succinate and malaoxon, of which malaoxon is degraded to below the limit of detection of the GC-FID after 12 h. Electrospray ionization mass spectral analyses show the final reaction products to be low molecular weight carboxylic acids (propionic, oxalic and iminodiacetic acid).

Differential mRNA expression levels and gene sequences of a putative carboxylesterase-like enzyme from two strains of the parasitoid Anisopteromalus calandrae (Hymenoptera: Pteromalidae)

Carboxylesterase-like enzyme cDNAs have been cloned and sequenced from malathion-resistant and susceptible strains of the parasitoid Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae). The cDNAs consist of 1963 nucleotides including a 35 bp untranslated 5′-end, a 1596 bp open reading frame, and a 332 bp untranslated 3′-end. The open reading frame encodes 532 amino acid residues. The predicted protein sequence from these cDNAs includes 2 potential N-glycosylation sites, a carboxylesterase type-B serine active site FGGDSENVTIFGE SAG, and conserved residues Ser 187, Glu 317, and His 432 to function as the catalytic triad. The predicted carboxylesterase-like enzyme sequence is most similar to that of the carboxylesterase from the peach-potato aphid, Myzus persicae with 45% sequence identity. Alignment of the parasitoid carboxylesterase-like enzyme cDNAs revealed that there are two nucleotide differences in the open reading frame between the parasitoid strains, including a silent mutation and a point mutation that presumably causes a gene product difference. A nucleotide thymine at position 658 in the susceptible strain cDNA is replaced by a guanine in the resistant strain cDNA. This substitution leads to an amino acid change from tryptophan (Trp 220) in the susceptible strain to glycine (Gly 220) in the resistant strain. This substitution is genetically linked to resistance but it is not known how or if this amino acid substitution affects detoxification of malathion. Northern blot analyses demonstrated that expression level of the carboxylesterase-like enzyme mRNA in adult A. calandrae is approximately 30-fold higher in the resistant strain relative to that in the susceptible strain. Southern analysis indicated that Pst I or Eco RI restriction sites are different in the two strains. Both a modified gene structure and an increase in expression of carboxylesterase may be responsible for the high level of resistance found in this beneficial wasp.