Identification and Expression Profiles of Six Transcripts Encoding Carboxylesterase Protein in Vitis flexuosa Infected with Pathogens.

PubMed

Islam, Md Zaherul; Yun, Hae Keun

2016-08-01

Plants protect themselves from pathogen attacks via several mechanisms, including hypersensitive cell death. Recognition of pathogen attack by the plant resistance gene triggers expression of carboxylesterase genes associated with hypersensitive response. We identified six transcripts of carboxylesterase genes, Vitis flexuosa carboxylesterase 5585 (VfCXE5585), VfCXE12827, VfCXE13132, VfCXE17159, VfCXE18231, and VfCXE47674, which showed different expression patterns upon transcriptome analysis of V. flexuosa inoculated with Elsinoe ampelina. The lengths of genes ranged from 1,098 to 1,629 bp, and their encoded proteins consisted of 309 to 335 amino acids. The predicted amino acid sequences showed hydrolase like domains in all six transcripts and contained two conserved motifs, GXSXG of serine hydrolase characteristics and HGGGF related to the carboxylesterase family. The deduced amino acid sequence also contained a potential catalytic triad consisted of serine, aspartic acid and histidine. Of the six transcripts, VfCXE12827 showed upregulated expression against E. ampelina at all time points. Three genes (VfCXE5585, VfCXE12827, and VfCXE13132) showed upregulation, while others (VfCXE17159, VfCXE18231, and VfCXE47674) were down regulated in grapevines infected with Botrytis cinerea. All transcripts showed upregulated expression against Rhizobium vitis at early and later time points except VfCXE12827, and were downregulated for up to 48 hours post inoculation (hpi) after upregulation at 1 hpi in response to R. vitis infection. All tested genes showed high and differential expression in response to pathogens, indicating that they all may play a role in defense pathways during pathogen infection in grapevines.

Identification and Expression Profiles of Six Transcripts Encoding Carboxylesterase Protein in Vitis flexuosa Infected with Pathogens

PubMed Central

Islam, Md. Zaherul; Yun, Hae Keun

2016-01-01

Plants protect themselves from pathogen attacks via several mechanisms, including hypersensitive cell death. Recognition of pathogen attack by the plant resistance gene triggers expression of carboxylesterase genes associated with hypersensitive response. We identified six transcripts of carboxylesterase genes, Vitis flexuosa carboxylesterase 5585 (VfCXE5585), VfCXE12827, VfCXE13132, VfCXE17159, VfCXE18231, and VfCXE47674, which showed different expression patterns upon transcriptome analysis of V. flexuosa inoculated with Elsinoe ampelina. The lengths of genes ranged from 1,098 to 1,629 bp, and their encoded proteins consisted of 309 to 335 amino acids. The predicted amino acid sequences showed hydrolase like domains in all six transcripts and contained two conserved motifs, GXSXG of serine hydrolase characteristics and HGGGF related to the carboxylesterase family. The deduced amino acid sequence also contained a potential catalytic triad consisted of serine, aspartic acid and histidine. Of the six transcripts, VfCXE12827 showed upregulated expression against E. ampelina at all time points. Three genes (VfCXE5585, VfCXE12827, and VfCXE13132) showed upregulation, while others (VfCXE17159, VfCXE18231, and VfCXE47674) were down regulated in grapevines infected with Botrytis cinerea. All transcripts showed upregulated expression against Rhizobium vitis at early and later time points except VfCXE12827, and were downregulated for up to 48 hours post inoculation (hpi) after upregulation at 1 hpi in response to R. vitis infection. All tested genes showed high and differential expression in response to pathogens, indicating that they all may play a role in defense pathways during pathogen infection in grapevines. PMID:27493610

Evaluating mice lacking serum carboxylesterase as a behavioral model for nerve agent intoxication.

PubMed

Dunn, Emily N; Ferrara-Bowens, Teresa M; Chachich, Mark E; Honnold, Cary L; Rothwell, Cristin C; Hoard-Fruchey, Heidi M; Lesyna, Catherine A; Johnson, Erik A; Cerasoli, Douglas M; McDonough, John H; Cadieux, C Linn

2018-06-07

Mice and other rodents are typically utilized for chemical warfare nerve agent research. Rodents have large amounts of carboxylesterase in their blood, while humans do not. Carboxylesterase nonspecifically binds to and detoxifies nerve agent. The presence of this natural bioscavenger makes mice and other rodents poor models for studies identifying therapeutics to treat humans exposed to nerve agents. To obviate this problem, a serum carboxylesterase knockout (Es1 KO) mouse was created. In this study, Es1 KO and wild type (WT) mice were assessed for differences in gene expression, nerve agent (soman; GD) median lethal dose (MLD) values, and behavior prior to and following nerve agent exposure. No expression differences were detected between Es1 KO and WT mice in more than 34 000 mouse genes tested. There was a significant difference between Es1 KO and WT mice in MLD values, as the MLD for GD-exposed WT mice was significantly higher than the MLD for GD-exposed Es1 KO mice. Behavioral assessments of Es1 KO and WT mice included an open field test, a zero maze, a Barnes maze, and a sucrose preference test (SPT). While sex differences were observed in various measures of these tests, overall, Es1 KO mice behaved similarly to WT mice. The two genotypes also showed virtually identical neuropathological changes following GD exposure. Es1 KO mice appear to have an enhanced susceptibility to GD toxicity while retaining all other behavioral and physiological responses to this nerve agent, making the Es1 KO mouse a more human-like model for nerve agent research.

Functional characterization of carboxylesterase gene mutations involved in Aphis gossypii resistance to organophosphate insecticides.

PubMed

Gong, Y-H; Ai, G-M; Li, M; Shi, X-Y; Diao, Q-Y; Gao, X-W

2017-12-01

Carboxylesterases (CarEs) play an important role in detoxifying insecticides in insects. Over-expression and structural modification of CarEs have been implicated in the development of organophosphate (OP) insecticide resistance in insects. A previous study identified four nonsynonymous mutations (resulting in four amino acid residue substitutions) in the open reading frame of the carboxylesterase gene of resistant cotton aphids compared to the omethoate susceptible strain, which has possibly influenced the development of resistance to omethoate (a systemic OP insecticide). The current study further characterized the function of these mutations, both alone and in combination, in the hydrolysis of OP insecticides. The metabolism results suggest that the combination of four mutations, mainly existing in the laboratory-selected OP-resistant cotton aphid population, increased the OP hydrolase activity (approximately twofold) at the cost of detectable carboxylesterase activity. The functional studies of single or multiple mutations suggest the positive effect of H104R, A128V and T333P on the acquisition of OP hydrolase activity, especially the combination of H104R with A128V or T333P. K484R substitution decreased both the OP hydrolase activity and the CarE activity, indicating that this mutation primarily drives the negative effect on the acquisition of OP hydrolase activity amongst these four mutations in the resistant strain. The modelling and docking results are basically consistent with the metabolic results, which strongly suggest that the structural gene modification is the molecular basis for the OP resistance in this laboratory-selected cotton aphid strain. © 2017 The Royal Entomological Society.

Simultaneous bioremediation and biodetection of mercury ion through surface display of carboxylesterase E2 from Pseudomonas aeruginosa PA1.

PubMed

Yin, Kun; Lv, Min; Wang, Qiaoning; Wu, Yixuan; Liao, Chunyang; Zhang, Weiwei; Chen, Lingxin

2016-10-15

Mercury is a toxic heavy metal and presents significant threats to organisms and natural ecosystems. Recently, the mercury remediation as well as its detection by environmental-friendly biotechnology has received increasing attention. In this study, carboxylesterase E2 from mercury-resistant strain Pseudomonas aeruginosa PA1 has been successfully displayed on the outer membrane of Escherichia coli Top10 bacteria to simultaneously adsorb and detect mercury ion (Hg(2+)). The transmission electron microscopy analysis shows that Hg(2+) can be absorbed by carboxylesterase E2 and accumulated on the outer membrane of surface-displayed E. coli bacteria. The adsorption of Hg(2+) followed a physicochemical, equilibrated and saturatable mechanism, which well fits the traditional Langmuir adsorption model. The surface-displayed system can be regenerated through regulating pH values. As its activity can be inhibited by Hg(2+), carboxylesterase E2 has been used to detect the concentration of Hg(2+) in water samples. The developed surface display system will be of great potential in the simultaneous bioremediation and biodetection of environmental mercury pollution. Copyright © 2016 Elsevier Ltd. All rights reserved.

Analysis of the Inhibition of Mammalian Carboxylesterases by Novel Fluorobenzoins and Fluorobenzils

PubMed Central

Hicks, Latorya D.; Hyatt, Janice L; Moak, Teri; Edwards, Carol C.; Tsurkan, Lyudmila; Wierdl, Monika; Ferreira, Antonio; Wadkins, Randy M.; Potter, Philip M.

2007-01-01

We have synthesized and assessed the ability of symmetrical fluorobenzoins and fluorobenzils to inhibit mammalian carboxylesterases (CE). The majority of the latter were excellent inhibitors of CEs however unexpectedly, the fluorobenzoins were very good enzyme inhibitors. Positive correlations were seen with the charge on the hydroxyl carbon atom, the carbonyl oxygen, and the Hammett constants for the derived Ki values with the fluorobenzoins. PMID:17399985

Further kinetic and molecular characterization of an extremely heat-stable carboxylesterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

PubMed Central

Sobek, H; Görisch, H

1989-01-01

The carboxylesterase (serine esterase, EC 3.1.1.1) from Sulfolobus acidocaldarius was purified 940-fold to homogeneity by an improved purification procedure with a yield of 57%. In the presence of alcohols the enzyme catalyses the transfer of the substrate acyl group to alcohols in parallel to hydrolysis. The results show the existence of an alcohol-binding site and a competitive partitioning of the acyl-enzyme intermediate between water and alcohols. Aniline acts also as a nucleophilic acceptor for the acyl group. On the basis of titration with diethyl p-nitrophenyl phosphate, a number of four active centres is determined for the tetrameric carboxylesterase. The sequence of 20 amino acid residues at the esterase N-terminus and the amino acid composition are reported. PMID:2508625

Comparison of in vitro methods for carboxylesterase activity determination in immortalized cells representative of the intestine, liver and kidney.

PubMed

Lamego, Joana; Ferreira, Pedro; Alves, Márcia; Matias, Ana; Simplício, Ana Luisa

2015-08-01

Herein we compare the fluorimetric determination of total and specific carboxylesterase activity in immortalized human derived living cells and in cell lysates. The cell lines chosen are representative of metabolism occurring in the intestine (Caco-2 and HT-29), kidney (HEK-293T) and liver (Hep G2). Caco-2 and HT-29, as cells prone to differentiation, were tested along the differentiation time. For evaluation of both methods when distinguishing activity of different carboxylesterases, HEK-293T transfected with the human carboxylestarase-2 (hCES2) were also tested. Application to Caco-2 or HT-29 cells demonstrated higher activity detected in cell lysates than in cell monolayers. The difference is most striking when comparing the methods at different stages of Caco-2 and HT-29 cell maturation, highlighting substrate accessibility as a limiting step in the in vivo hydrolysis rates (possibly limited by plasma and Endoplasmic Reticulum membrane permeability) with increasing relevance as the cells differentiate. Application to Hep G2 or to hCES2 transfected and non-transfected HEK-293T cells, demonstrated a tendency for higher sensitivity in living cell suspensions than that obtained with the cell lysates which indicates the importance of cell environment in the maintenance of enzyme activity. However, quantification of hCES2 activity relative to total esterase, or to total carboxylesterase activity, was not significantly different in any case. The results herein presented help to clarify which method is best suited for evaluation of carboxylesterase activity in vitro depending on the final goal of the study. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characteristics of carboxylesterase genes and their expression-level between acaricide-susceptible and resistant Tetranychus cinnabarinus (Boisduval).

PubMed

Wei, Peng; Shi, Li; Shen, Guangmao; Xu, Zhifeng; Liu, Jialu; Pan, Yu; He, Lin

2016-07-01

Carboxylesterases (CarEs) play important roles in metabolism and detoxification of dietary and environmental xenobiotics in insects and mites. On the basis of the Tetranychuscinnabarinus transcriptome dataset, 23 CarE genes (6 genes are full sequence and 17 genes are partial sequence) were identified. Synergist bioassay showed that CarEs were involved in acaricide detoxification and resistance in fenpropathrin- (FeR) and cyflumetofen-resistant (CyR) strains. In order to further reveal the relationship between CarE gene's expression and acaricide-resistance in T. cinnabarinus, we profiled their expression in susceptible (SS) and resistant strains (FeR, and CyR). There were 8 and 4 over-expressed carboxylesterase genes in FeR and CyR, respectively, from which the over-expressions were detected at mRNA level, but not DNA level. Pesticide induction experiment elucidated that 4 of 8 and 2 of 4 up-regulated genes were inducible with significance in FeR and CyR strains, respectively, but they could not be induced in SS strain, which indicated that these genes became more enhanced and effective to withstand the pesticides' stress in resistant T. cinnabarinus. Most expression-changed and all inducible genes possess the Abhydrolase_3 motif, which is a catalytic domain for hydrolyzing. As a whole, these findings in current study provide clues for further elucidating the function and regulation mechanism of these carboxylesterase genes in T. cinnabarinus' resistance formation. Copyright © 2015 Elsevier B.V. All rights reserved.

Slow-binding inhibition of carboxylesterase and other serine hydrolases by chlorodifluoroacetaldehyde.

PubMed

Yin, H; Jones, J P; Anders, M W

1993-01-01

The chlorofluorocarbon substitute 1,2-dichloro-1,1-difluoroethane (HCFC-132b) undergoes oxidative metabolism in rats to give a range of metabolites, including chlorodifluoroacetaldehyde [Harris and Anders (1991) Chem. Res. Toxicol. 4, 180]. The present experiments were undertaken after studies to characterize an unidentified metabolite of HCFC-132b revealed that chlorodifluoroacetaldehyde was toxic in vivo: rats given chlorodifluoroacetaldehyde died showing signs of cholinergic stimulation. Because some fluoroketones are known inhibitors of hydrolases, including acetylcholinesterase, the inhibitory effects of chlorodifluoroacetaldehyde on acetylcholinesterase (electric eel and human erythrocyte), on pseudocholinesterase (horse serum), on carboxylesterase (pig liver), and on alpha-chymotrypsin (bovine pancreas) were studied. In aqueous solution, the ratio chlorodifluoroacetaldehyde:chlorodifluroacetaldehyde hydrate, as determined by 1H nuclear magnetic resonance spectroscopy, was 1:157. Chlorodifluoroacetaldehyde was a slow-binding inhibitor of both acetylcholinesterases, of pseudocholinesterase, and of carboxylesterase; the Ki values, corrected for the aldehyde:hydrate ratio, were 150 nM, 1.7 nM, 3.7 nM, and 23 pM, respectively, as determined by final velocity of the progress curves; the kon values were 9.1 x 10(4), 1.1 x 10(5), 3.2 x 10(4), and 9.2 x 10(5) M-1 min-1, respectively. Chlorodifluoroacetaldehyde did not inhibit alpha-chymotrypsin. Acetaldehyde and trichloroacetaldehyde were classical competitive inhibitors of acetylcholinesterase. These results show that hydrochlorofluorocarbon metabolites may exert significant biological effects.

Gene cloning, expression, and characterization of a new carboxylesterase from Serratia sp. SES-01: comparison with Escherichia coli BioHe enzyme.

PubMed

Kwon, Min-A; Kim, Hyun Suk; Oh, Joon Young; Song, Bong Keun; Song, Jae Kwang

2009-02-01

The carboxylesterase-encoding gene (bioHs) of a newly isolated strain, Serratia sp. SES-01, was cloned from the genomic DNA library by detecting formation of transparent halo around the colony on LB-tributyrin agar plates. The amino acid sequence of BioHs was highly similar to the members of the BioH enzyme family involved in the biotin biosynthetic pathway; it showed the highest similarity (91%) with that of Serratia proteamaculans. To compare BioHs with other BioH enzymes, the relatively well-known bioHe gene of E. coli was cloned with PCR. After we achieved high-level expression of soluble BioHs and BioHe through the exploration of different culture conditions, the purified BioHs and BioHe enzymes were characterized in terms of specificity, activity, and stability. BioHe was generally more robust to a change in temperature and pH and an addition of organic solvents than BioHs. The two enzymes exhibited a strong preference for carboxylesterase rather than for thioesterase and were optimal at relatively low temperatures (20-40 degrees ) and alkaline pHs (7.5-9.0). The results in this study strongly suggested that both the BioHs and BioHe enzymes would be potential candidates for use as a carboxylesterase in many industrial applications.

Organophosphorus Flame Retardants Inhibit Specific Liver Carboxylesterases and Cause Serum Hypertriglyceridemia

PubMed Central

2015-01-01

Humans are prevalently exposed to organophosphorus flame retardants (OPFRs) contained in consumer products and electronics, though their toxicological effects and mechanisms remain poorly understood. We show here that OPFRs inhibit specific liver carboxylesterases (Ces) and cause altered hepatic lipid metabolism. Ablation of the OPFR target Ces1g has been previously linked to dyslipidemia in mice. Consistent with OPFR inhibition of Ces1g, we also observe OPFR-induced serum hypertriglyceridemia in mice. Our findings suggest novel toxicities that may arise from OPFR exposure and highlight the utility of chemoproteomic and metabolomic platforms in the toxicological characterization of environmental chemicals. PMID:24597639

Molecular identification of tuliposide B-converting enzyme: a lactone-forming carboxylesterase from the pollen of tulip.

PubMed

Nomura, Taiji; Murase, Tatsunori; Ogita, Shinjiro; Kato, Yasuo

2015-07-01

6-Tuliposides A (PosA) and B (PosB), which are the major secondary metabolites in tulip (Tulipa gesneriana), are enzymatically converted to the antimicrobial lactonized aglycons, tulipalins A (PaA) and B (PaB), respectively. We recently identified a PosA-converting enzyme (TCEA) as the first reported member of the lactone-forming carboxylesterases. Herein, we describe the identification of another lactone-forming carboxylesterase, PosB-converting enzyme (TCEB), which preferentially reacts with PosB to give PaB. This enzyme was isolated from tulip pollen, which showed high PosB-converting activity. Purified TCEB exhibited greater activity towards PosB than PosA, which was contrary to that of the TCEA. Novel cDNA (TgTCEB1) encoding the TCEB was isolated from tulip pollen. TgTCEB1 belonged to the carboxylesterase family and was approximately 50% identical to the TgTCEA polypeptides. Functional characterization of the recombinant enzyme verified that TgTCEB1 catalyzed the conversion of PosB to PaB with an activity comparable with the native TCEB. RT-qPCR analysis of each part of plant revealed that TgTCEB1 transcripts were limited almost exclusively to the pollen. Furthermore, the immunostaining of the anther cross-section using anti-TgTCEB1 polyclonal antibody verified that TgTCEB1 was specifically expressed in the pollen grains, but not in the anther cells. N-terminal transit peptide of TgTCEB1 was shown to function as plastid-targeted signal. Taken together, these results indicate that mature TgTCEB1 is specifically localized in plastids of pollen grains. Interestingly, PosB, the substrate of TgTCEB1, accumulated on the pollen surface, but not in the intracellular spaces of pollen grains. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Role of geraniol against lead acetate-mediated hepatic damage and their interaction with liver carboxylesterase activity in rats.

PubMed

Ozkaya, Ahmet; Sahin, Zafer; Kuzu, Muslum; Saglam, Yavuz Selim; Ozkaraca, Mustafa; Uckun, Mirac; Yologlu, Ertan; Comakli, Veysel; Demirdag, Ramazan; Yologlu, Semra

2018-02-01

In this study, the effect of geraniol (50 mg/kg for 30 d), a natural antioxidant and repellent/antifeedant monoterpene, in a rat model of lead acetate-induced (500 ppm for 30 d) liver damage was evaluated. Hepatic malondialdehyde increased in the lead acetate group. Reduced glutathione unchanged, but glutathione S-transferase, glutathione reductase, as well as carboxylesterase activities decreased in geraniol, lead acetate and geraniol + lead acetate groups. 8-OhDG immunoreactivity, mononuclear cell infiltrations and hepatic lead concentration were lower in the geraniol + lead acetate group than the lead acetate group. Serum aspartate aminotransferase and alanine aminotransferase activities increased in the Pb acetate group. In conclusion, lead acetate causes oxidative and toxic damage in the liver and this effect can reduce with geraniol treatment. However, we first observed that lead acetate, as well as geraniol, can affect liver carboxylesterase activity.

Inhibition of carboxylesterase activity of THP1 monocytes/macrophages and recombinant human carboxylesterase 1 by oxysterols and fatty acids

PubMed Central

Crow, J. Allen; Herring, Katye L.; Xie, Shuqi; Borazjani, Abdolsamad; Potter, Philip M.; Ross, Matthew K.

2009-01-01

Summary Two major isoforms of human carboxylesterases (CEs) are found in metabolically active tissues, CES1 and CES2. These hydrolytic enzymes are involved in xenobiotic and endobiotic metabolism. CES1 is abundantly expressed in human liver and monocytes/macrophages, including the THP1 cell line; CES2 is expressed in liver but not in monocytes/macrophages. The cholesteryl ester hydrolysis activity in human macrophages has been attributed to CES1. Here, we report the direct inhibitory effects of several endogenous oxysterols and fatty acids on the CE activity of THP1 monocytes/macrophages and recombinant human CES1 and CES2. Using THP1 whole-cell lysates we found: (1) 27-hydroxycholesterol (27-HC) is a potent inhibitor of carboxylesterase activity (IC50=33 nM); (2) 24(S),25-epoxycholesterol had moderate inhibitory activity (IC50=8.1 μM); and (3) cholesterol, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 25-hydroxycholesterol each had little inhibitory activity. 27-HC was a partially noncompetitive inhibitor of recombinant CES1 (Kiapp=10 nM) and impaired intracellular CES1 activity following treatment of intact THP1 cells. In contrast, recombinant CES2 activity was not inhibited by 27-HC, suggesting isoform-selective inhibition by 27-HC. Furthermore, unsaturated fatty acids were better inhibitors of CES1 activity than saturated fatty acids, while CES2 activity was unaffected by any fatty acid. Arachidonic acid (AA) was the most potent fatty acid inhibitor of recombinant CES1 and acted by a noncompetitive mechanism (Kiapp=1.7 μM); when not complexed to albumin, exogenous AA penetrated intact THP1 cells and inhibited CES1. Inhibition results are discussed in light of recent structural models for CES1 that describe ligand binding sites separate from the active site. In addition, oxysterol-mediated inhibition of CES1 activity was demonstrated by pretreatment of human liver homogenates or intact THP1 cells with exogenous 27-HC, which

Characterization and optimization of carboxylesterase-catalyzed esterification between capric acid and glycerol for the production of 1-monocaprin in reversed micellar system.

PubMed

Park, Kyung Min; Kwon, Oh Taek; Ahn, Seon Min; Lee, JaeHwan; Chang, Pahn-Shick

2010-02-28

Calotropis procera R. Br. carboxylesterase (EC 3.1.1.1) solubilized in reversed micellar glycerol droplets containing a very small amount of water (less than 5ppm) and stabilized by a surfactant effectively catalyzed the esterification between glycerol and capric acid to produce 1-monocaprin. Reaction variables including surfactant types, organic solvent media, reaction time, G-value ([glycerol]/[capric acid]), R-value ([water]/[surfactant]), pH, temperature, and types of metal ion inhibitors on the carboxylesterase-catalyzed esterification were characterized and optimized to efficiently produce 1-monocaprin. Bis(2-ethylhexyl) sodium sulfosuccinate (AOT) and isooctane were the most effective surfactant and organic solvent medium, respectively, for 1-monocaprin formation in reversed micelles. The optimum G- and R-values were 3.0 and 0.05, respectively, and the optimum pH and temperature were determined to be 10.0 and 60 degrees C, respectively. K(m,app.) and V(max,app.) were calculated from a Hanes-Woolf plot, and the values were 9.64 mM and 2.45 microM/min mg protein, respectively. Among various metal ions, Cu(2+) and Fe(2+) severely inhibited carboxylesterase-catalyzed esterification activity (less than 6.0% of relative activity). Copyright 2009 Elsevier B.V. All rights reserved.

Purification of a chymotrypsin-like enzyme present on adult Schistosoma mansoni worms from infected mice and its characterization as a host carboxylesterase.

PubMed

Igetei, Joseph E; Liddell, Susan; El-Faham, Marwa; Doenhoff, Michael J

2016-04-01

A serine protease-like enzyme found in detergent extracts of Schistosoma mansoni adult worms perfused from infected mice has been purified from mouse blood and further characterized. The enzyme is approximately 85 kDa and hydrolyses N-acetyl-DL-phenylalanine β-naphthyl-ester, a chromogenic substrate for chymotrypsin-like enzymes. The enzyme from S. mansoni worms appears to be antigenically and enzymatically similar to a molecule that is present in normal mouse blood and so is seemingly host-derived. The enzyme was partially purified by depleting normal mouse serum of albumin using sodium chloride and cold ethanol, followed by repeated rounds of purification by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified material was subjected to tandem mass spectrometry and its derived peptides found to belong to mouse carboxylesterase 1C. Its ability to hydrolyse α- or β-naphthyl acetates, which are general esterase substrates, has been confirmed. A similar carboxylesterase was purified and characterized from rat blood. Additional evidence to support identification of the enzyme as a carboxylesterase has been provided. Possible roles of the enzyme in the mouse host-parasite relationship could be to ease the passage of worms through the host's blood vessels and/or in immune evasion.

Molecular characterization of the amplified carboxylesterase gene associated with organophosphorus insecticide resistance in the brown planthopper, Nilaparvata lugens.

PubMed

Small, G J; Hemingway, J

2000-12-01

Widespread resistance to organophosphorus insecticides (OPs) in Nilaparvata lugens is associated with elevation of carboxylesterase activity. A cDNA encoding a carboxylesterase, Nl-EST1, has been isolated from an OP-resistant Sri Lankan strain of N. lugens. The full-length cDNA codes for a 547-amino acid protein with high homology to other esterases/lipases. Nl-EST1 has an N-terminal hydrophobic signal peptide sequence of 24 amino acids which suggests that the mature protein is secreted from cells expressing it. The nucleotide sequence of the homologue of Nl-EST1 in an OP-susceptible, low esterase Sri Lankan strain of N. lugens is identical to Nl-EST1. Southern analysis of genomic DNA from the Sri Lankan OP-resistant and susceptible strains suggests that Nl-EST1 is amplified in the resistant strain. Therefore, resistance to OPs in the Sri Lankan strain is through amplification of a gene identical to that found in the susceptible strain.

Elevated carboxylesterase activity contributes to the lambda-cyhalothrin insensitivity in quercetin fed Helicoverpa armigera (Hübner).

PubMed

Chen, Chengyu; Liu, Ying; Shi, Xueyan; Desneux, Nicolas; Han, Peng; Gao, Xiwu

2017-01-01

Quercetin as one of the key plant secondary metabolite flavonol is ubiquitous in terrestrial plants. In this study, the decrease in sensitivity to lambda-cyhalothrin was observed in quercetin-fed Helicoverpa armigera larvae. In order to figure out the mechanisms underlying the decreased sensitivity of H. armigera larvae to lambda-cyhalothrin by quercetin induction, the changes in carboxylesterase activity and in-vitro hydrolytic metabolic capacity to lambda-cyhalothrin were examined. The LC50 value of quercetin-fed H. armigera larvae to lambda-cyhalothrin showed 2.41-fold higher than that of the control. S, S, S-Tributyl phosphorotrithioate (DEF) treatment showed a synergism effect on lambda-cyhalothrin toxicity to quercetin-fed H. armigera. Moreover, the activity of carboxylesterase was significantly higher in quercetin-fed H. armigera larvae after fed on quercetin for 48 h. The in-vitro hydrolytic metabolic capacity to lambda-cyhalothrin in quercetin-fed H. armigera larvae midgut was 289.82 nmol 3-PBA/mg protein/min, which is significant higher than that in the control group (149.60 nmol 3-PBA/mg protein/min). The elevated CarE enzyme activity and corresponding increased hydrolytic metabolic capacity to lambda-cyhalothrin in quercetin-fed H. armigera contributed to the enhanced tolerance to lambda-cyhalothrin.

Elevated carboxylesterase activity contributes to the lambda-cyhalothrin insensitivity in quercetin fed Helicoverpa armigera (Hübner)

PubMed Central

Chen, Chengyu; Liu, Ying; Desneux, Nicolas; Han, Peng; Gao, Xiwu

2017-01-01

Quercetin as one of the key plant secondary metabolite flavonol is ubiquitous in terrestrial plants. In this study, the decrease in sensitivity to lambda-cyhalothrin was observed in quercetin-fed Helicoverpa armigera larvae. In order to figure out the mechanisms underlying the decreased sensitivity of H. armigera larvae to lambda-cyhalothrin by quercetin induction, the changes in carboxylesterase activity and in-vitro hydrolytic metabolic capacity to lambda-cyhalothrin were examined. The LC50 value of quercetin-fed H. armigera larvae to lambda-cyhalothrin showed 2.41-fold higher than that of the control. S, S, S-Tributyl phosphorotrithioate (DEF) treatment showed a synergism effect on lambda-cyhalothrin toxicity to quercetin-fed H. armigera. Moreover, the activity of carboxylesterase was significantly higher in quercetin-fed H. armigera larvae after fed on quercetin for 48 h. The in-vitro hydrolytic metabolic capacity to lambda-cyhalothrin in quercetin-fed H. armigera larvae midgut was 289.82 nmol 3-PBA/mg protein/min, which is significant higher than that in the control group (149.60 nmol 3-PBA/mg protein/min). The elevated CarE enzyme activity and corresponding increased hydrolytic metabolic capacity to lambda-cyhalothrin in quercetin-fed H. armigera contributed to the enhanced tolerance to lambda-cyhalothrin. PMID:28817718

Influence of sulfur oxidation state and steric bulk upon trifluoromethyl ketone (TFK) binding kinetics to carboxylesterases and fatty acid amide hydrolase (FAAH)

PubMed Central

Wheelock, Craig E.; Nishi, Kosuke; Ying, Andy; Jones, Paul D.; Colvin, Michael E.; Olmstead, Marilyn M.; Hammock, Bruce D.

2009-01-01

Carboxylesterases metabolize numerous exogenous and endogenous ester-containing compounds including the chemotherapeutic agent CPT-11, anti-influenza viral agent oseltamivir and many agrochemicals. Trifluoromethyl ketone (TFK)-containing compounds with a sulfur atom beta to the ketone moiety are some of the most potent carboxylesterase and amidase inhibitors identified to date. This study examined the effects of alkyl chain length (i.e., steric effects) and sulfur oxidation state upon TFK inhibitor potency (IC50) and binding kinetics (ki). The selective carboxylesterase inhibitor benzil was used as a non-TFK containing control. These effects were examined using two commercial esterases (porcine and rabbit liver esterase) and two human recombinant esterases (hCE-1 and hCE-2) as well as human recombinant fatty acid amide hydrolase (FAAH). In addition, the inhibition mechanism was examined using a combination of 1H NMR, X-ray crystallography and ab initio calculations. Overall, the data show that while sulfur oxidation state profoundly affects both inhibitor potency and binding kinetics, the steric effects dominate and override the contributions of sulfur oxidation. In addition, the data suggest that inclusion of a sulfur atom beta to the ketone contributes an increase (~5-fold) in inhibitor potency due to effects upon ketone hydration and/or intramolecular hydrogen bond formation. These results provide further information on the nature of the TFK binding interaction and will be useful in increasing our understanding of this basic biochemical process. PMID:18023188

Demonstration of carboxylesterase in cytology samples of human nasal respiratory epithelium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodgers, D.A.; Nikula, K.J.; Avila, K.

1995-12-01

The epithelial lining of the nasal airways is a target for responses induced by a variety of toxicant exposures. The high metabolic capacity of this tissue has been suggested to play a role in both protection of the airways through detoxication of certain toxicants, as well as in activation of other compounds to more toxic metabolites. Specifically, nasal carboxylesterase (CE) has been shown to mediate the toxicity of inhaled esters and acrylates by converting them to more toxic acid and alcohol metabolites which can be cytotoxic and/or carcinogenic to the nasal mucosa. Due to difficulties in extrapolating rodent models tomore » human, new paradigms using human cells and tissues are essential to understanding and evaluating the metabolic processes in human nasal epithelium.« less

Molecular Dynamics Investigation of the Substrate Binding Mechanism in Carboxylesterase

DOE PAGES

Chen, Qi; Luan, Zheng-Jiao; Cheng, Xiaolin; ...

2015-02-25

A recombinant carboxylesterase, cloned from Pseudomonas putida and designated as rPPE, is capable of catalyzing the bioresolution of racemic 2-acetoxy-2-(2 -chlorophenyl)acetate (rac-AcO-CPA) with excellent (S)-enantioselectivity. Semi-rational design of the enzyme showed that the W187H variant could increase the activity by ~100-fold compared to the wild type (WT) enzyme. In this study, we performed all-atom molecular dynamics (MD) simulations of both apo-rPPE and rPPE in complex with (S)-AcO-CPA to gain insights into the origin of the increased catalysis in the W187H mutant. Moreover, our results show differential binding of (S)-AcO-CPA in the WT and W187H enzymes, especially the interactions of themore » substrate with the two active site residues Ser159 and His286. The replacement of Trp187 by His leads to considerable structural rearrangement in the active site of W187H. Unlike in the WT rPPE, the cap domain in the W187 mutant shows an open conformation in the simulations of both apo and substrate-bound enzymes. This open conformation exposes the catalytic triad to the solvent through a water accessible channel, which may facilitate the entry of the substrate and/or the exit of the product. Binding free energy calculations confirmed that the substrate binds more strongly in W187H than in WT. Based on these computational results, furthermore, we predicted that the mutations W187Y and D287G might also be able to increase the substrate binding, thus improve the enzyme s catalytic efficiency. Experimental binding and kinetic assays on W187Y and D287G show improved catalytic efficiency over WT, but not W187H. Contrary to our prediction, W187Y shows slightly decreased substrate binding coupled with a 100 fold increase in turn-over rate, while in D287G the substrate binding is 8 times stronger but with a slightly reduced turn-over rate. Finally, our work provides important molecular-level insights into the binding of the (S)-AcO-CPA substrate to carboxylesterase r

Molecular Dynamics Investigation of the Substrate Binding Mechanism in Carboxylesterase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Qi; Luan, Zheng-Jiao; Cheng, Xiaolin

A recombinant carboxylesterase, cloned from Pseudomonas putida and designated as rPPE, is capable of catalyzing the bioresolution of racemic 2-acetoxy-2-(2 -chlorophenyl)acetate (rac-AcO-CPA) with excellent (S)-enantioselectivity. Semi-rational design of the enzyme showed that the W187H variant could increase the activity by ~100-fold compared to the wild type (WT) enzyme. In this study, we performed all-atom molecular dynamics (MD) simulations of both apo-rPPE and rPPE in complex with (S)-AcO-CPA to gain insights into the origin of the increased catalysis in the W187H mutant. Moreover, our results show differential binding of (S)-AcO-CPA in the WT and W187H enzymes, especially the interactions of themore » substrate with the two active site residues Ser159 and His286. The replacement of Trp187 by His leads to considerable structural rearrangement in the active site of W187H. Unlike in the WT rPPE, the cap domain in the W187 mutant shows an open conformation in the simulations of both apo and substrate-bound enzymes. This open conformation exposes the catalytic triad to the solvent through a water accessible channel, which may facilitate the entry of the substrate and/or the exit of the product. Binding free energy calculations confirmed that the substrate binds more strongly in W187H than in WT. Based on these computational results, furthermore, we predicted that the mutations W187Y and D287G might also be able to increase the substrate binding, thus improve the enzyme s catalytic efficiency. Experimental binding and kinetic assays on W187Y and D287G show improved catalytic efficiency over WT, but not W187H. Contrary to our prediction, W187Y shows slightly decreased substrate binding coupled with a 100 fold increase in turn-over rate, while in D287G the substrate binding is 8 times stronger but with a slightly reduced turn-over rate. Finally, our work provides important molecular-level insights into the binding of the (S)-AcO-CPA substrate to carboxylesterase r

Association of a carboxylesterase 1 polymorphism with appetite reduction in children and adolescents with attention-deficit/hyperactivity disorder treated with methylphenidate.

PubMed

Bruxel, E M; Salatino-Oliveira, A; Genro, J P; Zeni, C P; Polanczyk, G V; Chazan, R; Rohde, L A; Hutz, M H

2013-10-01

Carboxylesterase 1 is the enzyme involved in methylphenidate (MPH) metabolism. The aim of this study was to evaluate the association between a -75 T>G polymorphism and appetite reduction in children with attention-deficit/hyperactivity disorder (ADHD). A sample of 213 children with ADHD was investigated. The primary outcome was appetite reduction measured by the Barkley Stimulant Side Effect Rating Scale applied at baseline, at 1 and 3 months of treatment. MPH doses were augmented until no further clinical improvement or significant adverse events occurred. The G allele presented a trend for association with appetite reduction scores (P=0.05). A significant interaction between the G allele and treatment over time for appetite reduction scores was also observed (P=0.03). The G allele carriers presented a higher risk for appetite reduction worsening when compared with T allele homozygotes (odds ratio=3.47, P=0.01). The present results suggest an influence of carboxylesterase 1 -75 T>G polymorphism on the worsening of appetite reduction with MPH treatment in youths with ADHD.

Recommended nomenclature for five mammalian carboxylesterase gene families: human, mouse, and rat genes and proteins.

PubMed

Holmes, Roger S; Wright, Matthew W; Laulederkind, Stanley J F; Cox, Laura A; Hosokawa, Masakiyo; Imai, Teruko; Ishibashi, Shun; Lehner, Richard; Miyazaki, Masao; Perkins, Everett J; Potter, Phillip M; Redinbo, Matthew R; Robert, Jacques; Satoh, Tetsuo; Yamashita, Tetsuro; Yan, Bingfan; Yokoi, Tsuyoshi; Zechner, Rudolf; Maltais, Lois J

2010-10-01

Mammalian carboxylesterase (CES or Ces) genes encode enzymes that participate in xenobiotic, drug, and lipid metabolism in the body and are members of at least five gene families. Tandem duplications have added more genes for some families, particularly for mouse and rat genomes, which has caused confusion in naming rodent Ces genes. This article describes a new nomenclature system for human, mouse, and rat carboxylesterase genes that identifies homolog gene families and allocates a unique name for each gene. The guidelines of human, mouse, and rat gene nomenclature committees were followed and "CES" (human) and "Ces" (mouse and rat) root symbols were used followed by the family number (e.g., human CES1). Where multiple genes were identified for a family or where a clash occurred with an existing gene name, a letter was added (e.g., human CES4A; mouse and rat Ces1a) that reflected gene relatedness among rodent species (e.g., mouse and rat Ces1a). Pseudogenes were named by adding "P" and a number to the human gene name (e.g., human CES1P1) or by using a new letter followed by ps for mouse and rat Ces pseudogenes (e.g., Ces2d-ps). Gene transcript isoforms were named by adding the GenBank accession ID to the gene symbol (e.g., human CES1_AB119995 or mouse Ces1e_BC019208). This nomenclature improves our understanding of human, mouse, and rat CES/Ces gene families and facilitates research into the structure, function, and evolution of these gene families. It also serves as a model for naming CES genes from other mammalian species.

A novel lactone-forming carboxylesterase: molecular identification of a tuliposide A-converting enzyme in tulip.

PubMed

Nomura, Taiji; Ogita, Shinjiro; Kato, Yasuo

2012-06-01

Tuliposides, the glucose esters of 4-hydroxy-2-methylenebutanoate and 3,4-dihydroxy-2-methylenebutanoate, are major secondary metabolites in tulip (Tulipa gesneriana). Their lactonized aglycons, tulipalins, function as defensive chemicals due to their biological activities. We recently found that tuliposide-converting enzyme (TCE) purified from tulip bulbs catalyzed the conversion of tuliposides to tulipalins, but the possibility of the presence of several TCE isozymes was raised: TCE in tissues other than bulbs is different from bulb TCE. Here, to prove this hypothesis, TCE was purified from petals, which have the second highest TCE activity after bulbs. The purified enzyme, like the bulb enzyme, preferentially accepted tuliposides as substrates, with 6-tuliposide A the best substrate, which allowed naming the enzyme tuliposide A-converting enzyme (TCEA), but specific activity and molecular mass differed between the petal and bulb enzymes. After peptide sequencing, a novel cDNA (TgTCEA) encoding petal TCEA was isolated, and the functional characterization of the recombinant enzyme verified that TgTCEA catalyzes the conversion of 6-tuliposide A to tulipalin A. TgTCEA was transcribed in all tulip tissues but not in bulbs, indicating the presence of a bulb-specific TgTCEA, as suggested by the distinct enzymatic characters between the petal and bulb enzymes. Plastidial localization of TgTCEA enzyme was revealed, which allowed proposing a cytological mechanism of TgTCE-mediated tulipalin formation in the tulip defensive strategy. Site-directed mutagenesis of TgTCEA suggested that the oxyanion hole and catalytic triad characteristic of typical carboxylesterases are essential for the catalytic process of TgTCEA enzyme. To our knowledge, TgTCEA is the first identified member of the lactone-forming carboxylesterases, specifically catalyzing intramolecular transesterification.

Nuclear receptor-mediated regulation of carboxylesterase expression and activity.

PubMed

Staudinger, Jeff L; Xu, Chenshu; Cui, Yue J; Klaassen, Curtis D

2010-03-01

Emerging evidence demonstrates that several nuclear receptor (NR) family members regulate drug-inducible expression and activity of several important carboxylesterase (CES) enzymes in mammalian liver and intestine. Numerous clinically prescribed anticancer prodrugs, carbamate and pyrethroid insecticides, environmental toxicants and procarcinogens are substrates for CES enzymes. Moreover, a key strategy used in rational drug design frequently utilizes an ester linkage methodology to selectively target a prodrug, or to improve the water solubility of a novel compound. This review summarizes the current state of knowledge regarding NR-mediated regulation of CES enzymes in mammals and highlights their importance in drug metabolism, drug-drug interactions and toxicology. New knowledge regarding the transcriptional regulation of CES enzymes by NR proteins pregnane x receptor (NR1I2) and constitutive androstane receptor (NR1I3) has recently come to light through the use of knockout and transgenic mouse models. Novel insights regarding the species-specific cross-regulation of glucocorticoid receptor (NR3C1) and PPAR-alpha (NR1C1) signaling and CES gene expression are discussed. Elucidation of the role of NR-mediated regulation of CES enzymes in liver and intestine will have a significant impact on rational drug design and the development of novel prodrugs, especially for patients on combination therapy.

Carboxylesterases from the seeds of an underutilized legume, Mucuna pruriens; isolation, purification and characterization.

PubMed

Chandrashekharaiah, K S; Swamy, N Ramachandra; Murthy, K R Siddalinga

2011-12-01

Two carboxylesterases (ME-III and ME-IV) have been purified to apparent homogeneity from the seeds of Mucuna pruriens employing ammonium sulfate fractionation, cation exchange chromatography on CM-cellulose, gel-permeation chromatography on Sephadex G-100 and preparative PAGE. The homogeneity of the purified preparations was confirmed by polyacrylamide gel electrophoresis (PAGE), gel-electrofocussing and SDS-PAGE. The molecular weights determined by gel-permeation chromatography on Sephadex G-200 were 20.89 kDa (ME-III) and 31.62 kDa (ME-IV). The molecular weights determined by SDS-PAGE both in the presence and absence of 2-mercaptoethanol were 21 kDa (ME-III) and 30.2 kDa (ME-IV) respectively, suggesting a monomeric structure for both the enzymes. The enzymes were found to have Stokes radius of 2.4 nm (ME-III) and 2.7 nm (ME-IV). The isoelectric pH values of the enzymes, ME-III and ME-IV, were 6.8 and 7.4, respectively. ME-III and ME-IV were classified as carboxylesterases employing PAGE in conjunction with substrate and inhibitor specificity. The K(m) of ME-III and ME-IV with 1-naphthyl acetate as substrate was 0.1 and 0.166 mM while with 1-naphthyl propionate as substrate the K(m) was 0.052 and 0.0454 mM, respectively. As the carbon chain length of the acyl group increased, the affinity of the substrate to the enzyme increased indicating hydrophobic nature of the acyl group binding site. The enzymes exhibited an optimum temperature of 45°C (ME-III) and 37°C (ME-IV), an optimum pH of 7.0 (ME-III) and 7.5 (ME-IV) and both the enzymes (ME-III and ME-IV) were stable up to 120 min at 35°C. Both the enzymes were inhibited by organophosphates (dichlorvos and phosphamidon), but resistant towards carbamates (carbaryl and eserine sulfate) and sulphydryl inhibitors (p-chloromercuricbenzoate, PCMB). Copyright © 2011 Elsevier Ltd. All rights reserved.

The Leu-Arg-Glu (LRE) adhesion motif in proteins of the neuromuscular junction with special reference to proteins of the carboxylesterase/cholinesterase family.

PubMed

Johnson, Glynis; Moore, Samuel W

2013-09-01

Short linear motifs confer evolutionary flexibility on proteins as they can be added with relative ease allowing the acquisition of new functions. Such motifs may mediate a variety of signalling functions. The adhesion-mediating Leu-Arg-Glu (LRE) motif is enriched in laminin beta 2, and has been observed in other proteins, including members of the carboxylesterase/cholinesterase family. It acts as a stop signal for growing axons in the developing neuromuscular junction, binding to the voltage-gated calcium channel. In this bioinformatic analysis, we have investigated the presence of the motif in proteins of the neuromuscular junction, and have also examined its structural position and potential for ligand interaction, as well as phylogenetic conservation, in the carboxylesterase/cholinesterase family. The motif was observed to occur with a significantly higher frequency than expected in the UniProt/Swiss-Prot database, as well as in four individual species (human, mouse, Caenorhabditis elegans and Drosophila melanogaster). Examination of its presence in neuromuscular junction proteins showed it to be enriched in certain proteins of the synaptic basement membrane, including laminin, agrin, acetylcholinesterase and tenascin. A highly significant enrichment was observed in cytoskeletal proteins, particularly intermediate filament proteins and members of the spectrin family. In the carboxylesterase/cholinesterase family, the motif was observed in four conserved positions in the protein structure. It is present in the majority of mammalian acetylcholinesterases, as well as acetylcholinesterases from electric fish and a number of invertebrates. In insects, it is present in the ace-2, rather than in the synaptic ace-1, enzyme. It is also observed in the cholinesterase-like adhesion molecules (neuroligins, neurotactin and glutactin). It is never seen in butyrylcholinesterases, which do not mediate cell adhesion. In conclusion, the significant enrichment of the motif in

Hydrolysis of pyrethroids by human and rat tissues: Examination of intestinal, liver and serum carboxylesterases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crow, J. Allen; Borazjani, Abdolsamad; Potter, Philip M.

2007-05-15

Hydrolytic metabolism of pyrethroid insecticides in humans is one of the major catabolic pathways that clear these compounds from the body. Rodent models are often used to determine the disposition and clearance rates of these esterified compounds. In this study the distribution and activities of esterases that catalyze pyrethroid metabolism have been investigated in vitro using several human and rat tissues, including small intestine, liver and serum. The major esterase in human intestine is carboxylesterase 2 (hCE2). We found that the pyrethroid trans-permethrin is effectively hydrolyzed by a sample of pooled human intestinal microsomes (5 individuals), while deltamethrin and bioresmethrinmore » are not. This result correlates well with the substrate specificity of recombinant hCE2 enzyme. In contrast, a sample of pooled rat intestinal microsomes (5 animals) hydrolyze trans-permethrin 4.5-fold slower than the sample of human intestinal microsomes. Furthermore, it is demonstrated that pooled samples of cytosol from human or rat liver are {approx} 2-fold less hydrolytically active (normalized per mg protein) than the corresponding microsomal fraction toward pyrethroid substrates; however, the cytosolic fractions do have significant amounts ({approx} 40%) of the total esteratic activity. Moreover, a 6-fold interindividual variation in carboxylesterase 1 protein expression in human hepatic cytosols was observed. Human serum was shown to lack pyrethroid hydrolytic activity, but rat serum has hydrolytic activity that is attributed to a single CE isozyme. We purified the serum CE enzyme to homogeneity to determine its contribution to pyrethroid metabolism in the rat. Both trans-permethrin and bioresmethrin were effectively cleaved by this serum CE, but deltamethrin, esfenvalerate, alpha-cypermethrin and cis-permethrin were slowly hydrolyzed. Lastly, two model lipase enzymes were examined for their ability to hydrolyze pyrethroids. However, no hydrolysis products could

Age-Dependent Human Hepatic Carboxylesterase 1 (Ces1) ...

EPA Pesticide Factsheets

Human hepatic carboxylesterase 1 and 2 (CES1 and CES2) are important for ester- and amide- bond containing pharmaceutical and environmental chemical disposition. Despite concern regarding juvenile sensitivity to such compounds, CES1 and CES2 ontogeny has not been well characterized. To define human hepatic microsomal and cytosolic CES1 and CES2 expression during early postnatal life, microsomal and cytosolic fractions were prepared using liver samples from subjects without liver disease [N=165, 1d-18 yrs]. Proteins were fractionated, detected and quantitated by western blotting. Median microsomal CES1 was lower among samples from subjects < 3 weeks of age (N=36) compared to the rest of the population (N=126; 6.27 vs 17.5 pmoles/mg microsomal protein, respectively; p<0.001; Kruskal Wallis test). Cytosolic CES1 increased sequentially with expression being lowest among samples from individuals between birth and 3 weeks of age (N=36), markedly greater among those from ages 3 weeks to 6 years (N=90), and then modestly greater still among those over 6 years of age (N=36; median values = 4.7, 15.8, and 16.6 pmoles/mg cytosolic protein, respectively; p values <0.001 and 0.05, respectively, Kruskal Wallis test). Microsomal CES2 also increased sequentially across the same three age groups with median values of 1.8, 2.9, and 4.2 pmoles/mg microsomal protein, respectively (p<0.001, both), whereas for cytosolic CES2, only the youngest age group differed from the two older g

A Novel Lactone-Forming Carboxylesterase: Molecular Identification of a Tuliposide A-Converting Enzyme in Tulip1[W

PubMed Central

Nomura, Taiji; Ogita, Shinjiro; Kato, Yasuo

2012-01-01

Tuliposides, the glucose esters of 4-hydroxy-2-methylenebutanoate and 3,4-dihydroxy-2-methylenebutanoate, are major secondary metabolites in tulip (Tulipa gesneriana). Their lactonized aglycons, tulipalins, function as defensive chemicals due to their biological activities. We recently found that tuliposide-converting enzyme (TCE) purified from tulip bulbs catalyzed the conversion of tuliposides to tulipalins, but the possibility of the presence of several TCE isozymes was raised: TCE in tissues other than bulbs is different from bulb TCE. Here, to prove this hypothesis, TCE was purified from petals, which have the second highest TCE activity after bulbs. The purified enzyme, like the bulb enzyme, preferentially accepted tuliposides as substrates, with 6-tuliposide A the best substrate, which allowed naming the enzyme tuliposide A-converting enzyme (TCEA), but specific activity and molecular mass differed between the petal and bulb enzymes. After peptide sequencing, a novel cDNA (TgTCEA) encoding petal TCEA was isolated, and the functional characterization of the recombinant enzyme verified that TgTCEA catalyzes the conversion of 6-tuliposide A to tulipalin A. TgTCEA was transcribed in all tulip tissues but not in bulbs, indicating the presence of a bulb-specific TgTCEA, as suggested by the distinct enzymatic characters between the petal and bulb enzymes. Plastidial localization of TgTCEA enzyme was revealed, which allowed proposing a cytological mechanism of TgTCE-mediated tulipalin formation in the tulip defensive strategy. Site-directed mutagenesis of TgTCEA suggested that the oxyanion hole and catalytic triad characteristic of typical carboxylesterases are essential for the catalytic process of TgTCEA enzyme. To our knowledge, TgTCEA is the first identified member of the lactone-forming carboxylesterases, specifically catalyzing intramolecular transesterification. PMID:22474185

Biotransformation Capacity of Carboxylesterase in Skin and Keratinocytes for the Penta-Ethyl Ester Prodrug of DTPA.

PubMed

Fu, Jing; Sadgrove, Matthew; Marson, Lesley; Jay, Michael

2016-08-01

The penta-ethyl ester prodrug of the chelating agent diethylene triamine pentaacetic acid (DTPA), referred to as C2E5, effectively accelerated clearance of americium after transdermal delivery. Carboxylesterases (CESs) play important roles in facilitating C2E5 hydrolysis. However, whether CESs in human skin hydrolyze C2E5 remains unknown. We evaluated the gene and protein expression of CESs in distinctive human epidermal cell lines: HEKa, HEKn, HaCaT, and A431. The substrates p-nitrophenyl acetate (pNPA) and 4-nitrophenyl valerate (4-NPV) were used to access esterase and CES activity. C2E5 hydrolysis was measured by radiometric high-performance liquid chromatography after incubation of [(14)C]C2E5 with supernatant fractions after centrifugation at 9000g (S9) prepared from skin cell lines. CES-specific inhibitors were used to access metabolism in human skin S9 fractions with analysis by liquid chromatography-tandem mass spectrometry. We identified the human carboxylesterase 1 and 2 (CES1 and CES2) bands in a Western blot. The gene expression of these enzymes was supported by a real-time polymerase chain reaction (qPCR). pNPA and 4-NPV assays demonstrated esterase and CES activity in all the cell lines that were comparable to human skin S9 fractions. The prodrug C2E5 was hydrolyzed by skin S9 fractions, resulting in a primary metabolite, C2E4. In human skin S9 fractions, inhibition of C2E5 hydrolysis was greatest with a pan-CES inhibitor (benzil). CES1 inhibition (troglitazone) was greater than CES2 (loperamide), suggesting a primary metabolic role for CES1. These results indicate that human keratinocyte cell lines are useful for the evaluation of human cutaneous metabolism and absorption of ester-based prodrugs. However, keratinocytes from skin provide a small contribution to the overall metabolism of C2E5. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Identification and Characterization of Carboxylesterases from Brachypodium distachyon Deacetylating Trichothecene Mycotoxins

PubMed Central

Schmeitzl, Clemens; Varga, Elisabeth; Warth, Benedikt; Kugler, Karl G.; Malachová, Alexandra; Michlmayr, Herbert; Wiesenberger, Gerlinde; Mayer, Klaus F. X.; Mewes, Hans-Werner; Krska, Rudolf; Schuhmacher, Rainer; Berthiller, Franz; Adam, Gerhard

2015-01-01

Increasing frequencies of 3-acetyl-deoxynivalenol (3-ADON)-producing strains of Fusarium graminearum (3-ADON chemotype) have been reported in North America and Asia. 3-ADON is nearly nontoxic at the level of the ribosomal target and has to be deacetylated to cause inhibition of protein biosynthesis. Plant cells can efficiently remove the acetyl groups of 3-ADON, but the underlying genes are yet unknown. We therefore performed a study of the family of candidate carboxylesterases (CXE) genes of the monocot model plant Brachypodium distachyon. We report the identification and characterization of the first plant enzymes responsible for deacetylation of trichothecene toxins. The product of the BdCXE29 gene efficiently deacetylates T-2 toxin to HT-2 toxin, NX-2 to NX-3, both 3-ADON and 15-acetyl-deoxynivalenol (15-ADON) into deoxynivalenol and, to a lesser degree, also fusarenon X into nivalenol. The BdCXE52 esterase showed lower activity than BdCXE29 when expressed in yeast and accepts 3-ADON, NX-2, 15-ADON and, to a limited extent, fusarenon X as substrates. Expression of these Brachypodium genes in yeast increases the toxicity of 3-ADON, suggesting that highly similar genes existing in crop plants may act as susceptibility factors in Fusarium head blight disease. PMID:26712789

Inhibition of human carboxylesterases hCE1 and hiCE by cholinesterase inhibitors.

PubMed

Tsurkan, Lyudmila G; Hatfield, M Jason; Edwards, Carol C; Hyatt, Janice L; Potter, Philip M

2013-03-25

Carboxylesterases (CEs) are ubiquitously expressed proteins that are responsible for the detoxification of xenobiotics. They tend to be expressed in tissues likely to be exposed to such agents (e.g., lung and gut epithelia, liver) and can hydrolyze numerous agents, including many clinically used drugs. Due to the considerable structural similarity between cholinesterases (ChE) and CEs, we have assessed the ability of a series of ChE inhibitors to modulate the activity of the human liver (hCE1) and the human intestinal CE (hiCE) isoforms. We observed inhibition of hCE1 and hiCE by carbamate-containing small molecules, including those used for the treatment of Alzheimer's disease. For example, rivastigmine resulted in greater than 95% inhibition of hiCE that was irreversible under the conditions used. Hence, the administration of esterified drugs, in combination with these carbamates, may inadvertently result in decreased hydrolysis of the former, thereby limiting their efficacy. Therefore drug:drug interactions should be carefully evaluated in individuals receiving ChE inhibitors. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Inhibition of human carboxylesterases hCE1 and hiCE by cholinesterase inhibitors

PubMed Central

Tsurkan, Lyudmila G.; Hatfield, M. Jason; Edwards, Carol C.; Hyatt, Janice L.; Potter, Philip M.

2012-01-01

Carboxylesterases (CEs) are ubiquitously expressed proteins that are responsible for the detoxification of xenobiotics. They tend to be expressed in tissues likely to be exposed to such agents (e.g., lung and gut epithelia, liver) and can hydrolyze numerous agents, including many clinically used drugs. Due to the considerable structural similarity between cholinesterases (ChE) and CEs, we have assessed the ability of a series of ChE inhibitors to modulate the activity of the human liver (hCE1) and the human intestinal CE (hiCE) isoforms, We observed inhibition of hCE1 and hiCE by carbamate-containing small molecules, including those used for the treatment of Alzheimer’s disease. For example, rivastigmine resulted in greater than 95% inhibition of hiCE that was irreversible under the conditions used. Hence, the administration of esterified drugs, in combination with these carbamates, may inadvertently result in decreased hydrolysis of the former, thereby limiting their efficacy. Therefore drug:drug interactions should be carefully evaluated in individuals receiving ChE inhibitors. PMID:23123248

The in vitro metabolism of irinotecan (CPT-11) by carboxylesterase and β-glucuronidase in human colorectal tumours

PubMed Central

Tobin, Peter; Clarke, Stephen; Seale, J Paul; Lee, Soon; Solomon, Michael; Aulds, Sally; Crawford, Michael; Gallagher, James; Eyers, Tony; Rivory, Laurent

2006-01-01

Aims Irinotecan (CPT-11) is a prodrug that is used to treat metastatic colorectal cancer. It is activated to the topoisomerase poison SN-38 by carboxylesterases. SN-38 is metabolized to its inactive glucuronide, SN-38 glucuronide. The aim of this study was to determine, the reactivation of SN-38 from SN-38 glucuronide by β-glucuronidase may represent a significant pathway of SN-38 formation. Methods The production of SN-38 from irinotecan and SN-38 glucuronide (2.4, 9.6 and 19.2 µm) was measured in homogenates of human colorectal tumour, and matched normal colon mucosa from 21 patients). Results The rate of conversion of irinotecan (9.6 µM) was lower in tumour tissue than matched normal colon mucosa samples (0.30 ± 0.14 pmol min−1 mg−1 protein and 0.77 ± 0.59 pmol min−1 mg−1 protein, respectively; P < 0.005). In contrast, no significant difference was observed in β-glucuronidase activity between tumour and matched normal colon samples (4.56 ± 6.9 pmol min−1 mg−1 protein and 3.62 ± 2.95 pmol min−1 mg−1 protein, respectively, using 9.6 µM SN-38 glucuronide; P > 0.05). β-Glucuronidase activity in tumour correlated to that observed in matched normal tissue (r2 > 0.23, P < 0.05), whereas this was not the case for carboxylesterase activity. At equal concentrations of irinotecan and SN-38 glucuronide, the rate of β-glucuronidase-mediated SN-38 production was higher than that formed from irinotecan in both tumour and normal tissue (P < 0.05). However, at concentrations that reflect the relative plasma concentrations observed in patients, the rate of SN-38 production via these two pathways was comparable. Conclusions Tumour β-glucuronidase may play a significant role in the exposure of tumours to SN-38 in vivo. PMID:16842384

Design, synthesis, and structure-activity relationship study of glycyrrhetinic acid derivatives as potent and selective inhibitors against human carboxylesterase 2.

PubMed

Zou, Li-Wei; Li, Yao-Guang; Wang, Ping; Zhou, Kun; Hou, Jie; Jin, Qiang; Hao, Da-Cheng; Ge, Guang-Bo; Yang, Ling

2016-04-13

Human carboxylesterase 2 (hCE2), one of the major carboxylesterases in the human intestine and various tumour tissues, plays important roles in the oral bioavailability and treatment outcomes of ester- or amide-containing drugs or prodrugs, such as anticancer agents CPT-11 (irinotecan) and LY2334737 (gemcitabine). In this study, 18β-glycyrrhetinic acid (GA), the most abundant pentacyclic triterpenoid from natural source, was selected as a reference compound for the development of potent and specific inhibitors against hCE2. Simple semi-synthetic modulation on GA was performed to obtain a series of GA derivatives. Structure-activity relationship analysis brought novel insights into the structure modification of GA. Converting the 11-oxo-12-ene of GA to 12-diene moiety, and C-3 hydroxyl and C-30 carboxyl group to 3-O-β-carboxypropionyl and ethyl ester respectively, led to a significant enhancement of the inhibitory effect on hCE2 and the selectivity over hCE1. These exciting findings inspired us to design and synthesize the more potent compound 15 (IC50 0.02 μM) as a novel and highly selective inhibitor against hCE2, which was 3463-fold more potent than the parent compound GA and demonstrated excellent selectivity (>1000-fold over hCE1). The molecular docking study of compound 15 and the active site of hCE1 and hCE2 demonstrated that the potent and selective inhibition of compound 15 toward hCE2 could partially be attributed to its relatively stronger interactions with hCE2 than with hCE1. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Comparison of cholin- and carboxylesterase enzyme inhibition and visible effects in the zebra fish embryo bioassay under short-term paraoxon-methyl exposure.

PubMed

Küster, E; Altenburger, R

2006-01-01

The acute zebra fish embryo test (Danio rerio Hamilton-Buchanan, 1822) is an accepted bioassay to assess the toxicity of waste water that may be used for the replacement of testing with adult fish. It is also suggested for chemical hazard characterization and assessment, although only a few groups of substances have yet been studied. Specifically acting substances such as neurotoxic insecticides pose a potentially hazard for non-target fish. To establish whether the proposed zebra fish embryo test protocol and the inhibition of cholinesterases (acetylcholinesterase EC 3.1.1.7, propionylcholinesterase EC 3.1.1.8) and carboxylesterase (EC 3.1.1.1) enzymes can be used in a similar fashion for hazard characterization and risk assessment of chemicals and environmental samples, two types of experiments were conducted. Visual effects of exposure to the organophosphate metabolite paraoxon-methyl after 24 and 48 h in the zebra fish embryo test system were analysed with the use of an inverse microscope (rate of mortality, developmental disturbances, heart rate and others). The inhibition to cholinesterases and carboxylesterase was also measured. Enzyme inhibition as a biomarker of exposure was about 70 times more sensitive than the effects in the zebra fish embryo test with an IC50 below 1.2 micromol compared with an EC50 of 91 micromol. The dose-response relationships showed different curve characteristics with a linear increase of enzyme inhibition compared with a sigmoidal curve for the overt effects. Significant overt effects could only be seen at concentrations at which already 80% of the activities of the different esterases were inhibited.

Magnetic-Fe/Fe(3)O(4)-nanoparticle-bound SN38 as carboxylesterase-cleavable prodrug for the delivery to tumors within monocytes/macrophages.

PubMed

Wang, Hongwang; Shrestha, Tej B; Basel, Matthew T; Dani, Raj Kumar; Seo, Gwi-Moon; Balivada, Sivasai; Pyle, Marla M; Prock, Heidy; Koper, Olga B; Thapa, Prem S; Moore, David; Li, Ping; Chikan, Viktor; Troyer, Deryl L; Bossmann, Stefan H

2012-01-01

The targeted delivery of therapeutics to the tumor site is highly desirable in cancer treatment, because it is capable of minimizing collateral damage. Herein, we report the synthesis of a nanoplatform, which is composed of a 15 ± 1 nm diameter core/shell Fe/Fe(3)O(4) magnetic nanoparticles (MNPs) and the topoisomerase I blocker SN38 bound to the surface of the MNPs via a carboxylesterase cleavable linker. This nanoplatform demonstrated high heating ability (SAR = 522 ± 40 W/g) in an AC-magnetic field. For the purpose of targeted delivery, this nanoplatform was loaded into tumor-homing double-stable RAW264.7 cells (mouse monocyte/macrophage-like cells (Mo/Ma)), which have been engineered to express intracellular carboxylesterase (InCE) upon addition of doxycycline by a Tet-On Advanced system. The nanoplatform was taken up efficiently by these tumor-homing cells. They showed low toxicity even at high nanoplatform concentration. SN38 was released successfully by switching on the Tet-On Advanced system. We have demonstrated that this nanoplatform can be potentially used for thermochemotherapy. We will be able to achieve the following goals: (1) Specifically deliver the SN38 prodrug and magnetic nanoparticles to the cancer site as the payload of tumor-homing double-stable RAW264.7 cells; (2) Release of chemotherapeutic SN38 at the cancer site by means of the self-containing Tet-On Advanced system; (3) Provide localized magnetic hyperthermia to enhance the cancer treatment, both by killing cancer cells through magnetic heating and by activating the immune system.

Magnetic-Fe/Fe3O4-nanoparticle-bound SN38 as carboxylesterase-cleavable prodrug for the delivery to tumors within monocytes/macrophages

PubMed Central

Shrestha, Tej B; Basel, Matthew T; Dani, Raj Kumar; Seo, Gwi-Moon; Balivada, Sivasai; Pyle, Marla M; Prock, Heidy; Koper, Olga B; Thapa, Prem S; Moore, David; Li, Ping; Chikan, Viktor

2012-01-01

Summary The targeted delivery of therapeutics to the tumor site is highly desirable in cancer treatment, because it is capable of minimizing collateral damage. Herein, we report the synthesis of a nanoplatform, which is composed of a 15 ± 1 nm diameter core/shell Fe/Fe3O4 magnetic nanoparticles (MNPs) and the topoisomerase I blocker SN38 bound to the surface of the MNPs via a carboxylesterase cleavable linker. This nanoplatform demonstrated high heating ability (SAR = 522 ± 40 W/g) in an AC-magnetic field. For the purpose of targeted delivery, this nanoplatform was loaded into tumor-homing double-stable RAW264.7 cells (mouse monocyte/macrophage-like cells (Mo/Ma)), which have been engineered to express intracellular carboxylesterase (InCE) upon addition of doxycycline by a Tet-On Advanced system. The nanoplatform was taken up efficiently by these tumor-homing cells. They showed low toxicity even at high nanoplatform concentration. SN38 was released successfully by switching on the Tet-On Advanced system. We have demonstrated that this nanoplatform can be potentially used for thermochemotherapy. We will be able to achieve the following goals: (1) Specifically deliver the SN38 prodrug and magnetic nanoparticles to the cancer site as the payload of tumor-homing double-stable RAW264.7 cells; (2) Release of chemotherapeutic SN38 at the cancer site by means of the self-containing Tet-On Advanced system; (3) Provide localized magnetic hyperthermia to enhance the cancer treatment, both by killing cancer cells through magnetic heating and by activating the immune system. PMID:23016149

Gastrointestinal Degradation of Fumonisin B₁ by Carboxylesterase FumD Prevents Fumonisin Induced Alteration of Sphingolipid Metabolism in Turkey and Swine.

PubMed

Masching, Sabine; Naehrer, Karin; Schwartz-Zimmermann, Heidi-Elisabeth; Sărăndan, Mihai; Schaumberger, Simone; Dohnal, Ilse; Nagl, Veronika; Schatzmayr, Dian

2016-03-21

The mycotoxin fumonisin B₁ (FB₁) is a frequent contaminant of feed and causes various adverse health effects in domestic animals. Hence, effective strategies are needed to prevent the impact of fumonisins on livestock productivity. Here we evaluated the capability of the fumonisin carboxylesterase FumD to degrade FB₁ to its less toxic metabolite hydrolyzed FB₁ (HFB₁) in the gastrointestinal tract of turkeys and pigs. First, an ex vivo pig model was used to examine the activity of FumD under digestive conditions. Within 2 h of incubation with FumD, FB₁ was completely degraded to HFB₁ in the duodenum and jejunum, respectively. To test the efficacy of the commercial application of FumD (FUMzyme) in vivo, female turkeys (n = 5) received either basal feed (CON), fumonisin-contaminated feed (15 mg/kg FB₁+FB₂; FB) or fumonisin-contaminated feed supplemented with FUMzyme (15 U/kg; FB+FUMzyme) for 14 days ad libitum. Addition of FUMzyme resulted in significantly decreased levels of FB₁ in excreta, whereas HFB₁ concentrations were significantly increased. Compared to the FB group (0.24 ± 0.02), the mean serum sphinganine-to-sphingosine (Sa/So) ratio was significantly reduced in the FB+FUMzyme group (0.19 ± 0.02), thus resembling values of the CON group (0.16 ± 0.02). Similarly, exposure of piglets (n = 10) to 2 mg/kg FB₁+FB₂ for 42 days caused significantly elevated serum Sa/So ratios (0.39 ± 0.15) compared to the CON group (0.14 ± 0.01). Supplementation with FUMzyme (60 U/kg) resulted in gastrointestinal degradation of FB₁ and unaffected Sa/So ratios (0.16 ± 0.02). Thus, the carboxylesterase FumD represents an effective strategy to detoxify FB₁ in the digestive tract of turkeys and pigs.

Gene expression analysis and enzyme assay reveal a potential role of the carboxylesterase gene CpCE-1 from Cydia pomonella in detoxification of insecticides.

PubMed

Yang, Xue-Qing

2016-05-01

Carboxylesterases (CarEs) are responsible for metabolism of xenobiotics including insecticides in insects. Understanding the expression patterns of a such detoxifying gene and effect of insecticides on its enzyme activity are important to clarify the function of this gene relevant to insecticides-detoxifying process, but little information is available in the codling moth Cydia pomonella (L.). In this study, we investigated the expression profiles of CarE gene CpCE-1 at different developmental stages and in different tissues of C. pomonella, as well as the larvae exposed to chlorpyrifos-ethyl and lambda-cyhalothrin by using absolute real-time quantitative PCR (absolute RT-qPCR). Results indicated that CpCE-1 expression was significantly altered during C. pomonella development stages, and this expression differed between sexes, with a higher transcript in females than males. Meanwhile, CpCE-1 is overexpressed in cuticle, midgut and head than silk gland, fat body and Malpighian tubules. Exposure of third instar larvae to a non-lethal dosage of chlorpyrifos-ethyl and lambda-cyhalothrin resulted in induction of CpCE-1 transcript. The total carboxylesterase enzyme activity was inhibited by chlorpyrifos-ethyl in vivo; in contrast, the activity of Escherichia coli produced recombinant CpCE-1 was significantly inhibited by both lambda-cyhalothrin and chlorpyrifos-ethyl in vitro. These results suggested that CpCE-1 in C. pomonella is potentially involved in the development and in detoxification of chlorpyrifos-ethyl and lambda-cyhalothrin.

Sacubitril Is Selectively Activated by Carboxylesterase 1 (CES1) in the Liver and the Activation Is Affected by CES1 Genetic Variation

PubMed Central

Shi, Jian; Wang, Xinwen; Nguyen, Jenny; Wu, Audrey H.; Bleske, Barry E.

2016-01-01

Sacubitril was recently approved by the Food and Drug Administration for use in combination with valsartan for the treatment of patients with heart failure with reduced ejection fraction. As a prodrug, sacubitril must be metabolized (hydrolyzed) to its active metabolite sacubitrilat (LBQ657) to exert its intended therapeutic effects. Thus, understanding the determinants of sacubitril activation will lead to the improvement of sacubitril pharmacotherapy. The objective of this study was to identify the enzyme(s) responsible for the activation of sacubitril, and determine the impact of genetic variation on sacubitril activation. First, an incubation study of sacubitril with human plasma and the S9 fractions of human liver, intestine, and kidney was conducted. Sacubitril was found to be activated by human liver S9 fractions only. Moreover, sacubitril activation was significantly inhibited by the carboxylesterase 1 (CES1) inhibitor bis-(p-nitrophenyl) phosphate in human liver S9. Further incubation studies with recombinant human CES1 and carboxylesterase 2 confirmed that sacubitril is a selective CES1 substrate. The in vitro study of cell lines transfected with wild-type CES1 and the CES1 variant G143E (rs71647871) demonstrated that G143E is a loss-of-function variant for sacubitril activation. Importantly, sacubitril activation was significantly impaired in human livers carrying the G143E variant. In conclusion, sacubitril is selectively activated by CES1 in human liver. The CES1 genetic variant G143E can significantly impair sacubitril activation. Therefore, CES1 genetic variants appear to be an important contributing factor to interindividual variability in sacubitril activation, and have the potential to serve as biomarkers to optimize sacubitril pharmacotherapy. PMID:26817948

Gastrointestinal Degradation of Fumonisin B1 by Carboxylesterase FumD Prevents Fumonisin Induced Alteration of Sphingolipid Metabolism in Turkey and Swine

PubMed Central

Masching, Sabine; Naehrer, Karin; Schwartz-Zimmermann, Heidi-Elisabeth; Sărăndan, Mihai; Schaumberger, Simone; Dohnal, Ilse; Nagl, Veronika; Schatzmayr, Dian

2016-01-01

The mycotoxin fumonisin B1 (FB1) is a frequent contaminant of feed and causes various adverse health effects in domestic animals. Hence, effective strategies are needed to prevent the impact of fumonisins on livestock productivity. Here we evaluated the capability of the fumonisin carboxylesterase FumD to degrade FB1 to its less toxic metabolite hydrolyzed FB1 (HFB1) in the gastrointestinal tract of turkeys and pigs. First, an ex vivo pig model was used to examine the activity of FumD under digestive conditions. Within 2 h of incubation with FumD, FB1 was completely degraded to HFB1 in the duodenum and jejunum, respectively. To test the efficacy of the commercial application of FumD (FUMzyme) in vivo, female turkeys (n = 5) received either basal feed (CON), fumonisin-contaminated feed (15 mg/kg FB1+FB2; FB) or fumonisin-contaminated feed supplemented with FUMzyme (15 U/kg; FB+FUMzyme) for 14 days ad libitum. Addition of FUMzyme resulted in significantly decreased levels of FB1 in excreta, whereas HFB1 concentrations were significantly increased. Compared to the FB group (0.24 ± 0.02), the mean serum sphinganine-to-sphingosine (Sa/So) ratio was significantly reduced in the FB+FUMzyme group (0.19 ± 0.02), thus resembling values of the CON group (0.16 ± 0.02). Similarly, exposure of piglets (n = 10) to 2 mg/kg FB1+FB2 for 42 days caused significantly elevated serum Sa/So ratios (0.39 ± 0.15) compared to the CON group (0.14 ± 0.01). Supplementation with FUMzyme (60 U/kg) resulted in gastrointestinal degradation of FB1 and unaffected Sa/So ratios (0.16 ± 0.02). Thus, the carboxylesterase FumD represents an effective strategy to detoxify FB1 in the digestive tract of turkeys and pigs. PMID:27007395

Structural Insights into Drug Processing by Human Carboxylesterase 1: Tamoxifen, Mevastatin, and Inhibition by Benzil

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fleming, Christopher D.; Bencharit, Sompop; Edwards, Carol C.

2010-07-19

Human carboxylesterase 1 (hCE1) exhibits broad substrate specificity and is involved in xenobiotic processing and endobiotic metabolism. We present and analyze crystal structures of hCE1 in complexes with the cholesterol-lowering drug mevastatin, the breast cancer drug tamoxifen, the fatty acyl ethyl ester (FAEE) analogue ethyl acetate, and the novel hCE1 inhibitor benzil. We find that mevastatin does not appear to be a substrate for hCE1, and instead acts as a partially non-competitive inhibitor of the enzyme. Similarly, we show that tamoxifen is a low micromolar, partially non-competitive inhibitor of hCE1. Further, we describe the structural basis for the inhibition ofmore » hCE1 by the nanomolar-affinity dione benzil, which acts by forming both covalent and non-covalent complexes with the enzyme. Our results provide detailed insights into the catalytic and non-catalytic processing of small molecules by hCE1, and suggest that the efficacy of clinical drugs may be modulated by targeted hCE1 inhibitors.« less

Structural insights into drug processing by human carboxylesterase 1: tamoxifen, mevastatin, and inhibition by benzil.

PubMed

Fleming, Christopher D; Bencharit, Sompop; Edwards, Carol C; Hyatt, Janice L; Tsurkan, Lyudmila; Bai, Feng; Fraga, Charles; Morton, Christopher L; Howard-Williams, Escher L; Potter, Philip M; Redinbo, Matthew R

2005-09-09

Human carboxylesterase 1 (hCE1) exhibits broad substrate specificity and is involved in xenobiotic processing and endobiotic metabolism. We present and analyze crystal structures of hCE1 in complexes with the cholesterol-lowering drug mevastatin, the breast cancer drug tamoxifen, the fatty acyl ethyl ester (FAEE) analogue ethyl acetate, and the novel hCE1 inhibitor benzil. We find that mevastatin does not appear to be a substrate for hCE1, and instead acts as a partially non-competitive inhibitor of the enzyme. Similarly, we show that tamoxifen is a low micromolar, partially non-competitive inhibitor of hCE1. Further, we describe the structural basis for the inhibition of hCE1 by the nanomolar-affinity dione benzil, which acts by forming both covalent and non-covalent complexes with the enzyme. Our results provide detailed insights into the catalytic and non-catalytic processing of small molecules by hCE1, and suggest that the efficacy of clinical drugs may be modulated by targeted hCE1 inhibitors.

Population Pharmacokinetics of Methylphenidate in Healthy Adults Emphasizing Novel and Known Effects of Several Carboxylesterase 1 (CES1) Variants

PubMed Central

Stage, C; Bergmann, TK; Ferrero‐Milliani, L; Bjerre, D; Thomsen, R; Dalhoff, KP; Rasmussen, HB; Jürgens, G

2016-01-01

The aim of this study was to identify demographic and genetic factors that significantly affect methylphenidate (MPH) pharmacokinetics (PK), and may help explain interindividual variability and further increase the safety of MPH. d‐MPH plasma concentrations, demographic covariates, and carboxylesterase 1 (CES1) genotypes were gathered from 122 healthy adults and analyzed using nonlinear mixed effects modeling. The structural model that best described the data was a two‐compartment disposition model with absorption transit compartments. Novel effects of rs115629050 and CES1 diplotypes, as well as previously reported effects of rs71647871 and body weight, were included in the final model. Assessment of the independent and combined effect of CES1 covariates identified several specific risk factors that may result in severely increased d‐MPH plasma exposure. PMID:27754602

Sacubitril Is Selectively Activated by Carboxylesterase 1 (CES1) in the Liver and the Activation Is Affected by CES1 Genetic Variation.

PubMed

Shi, Jian; Wang, Xinwen; Nguyen, Jenny; Wu, Audrey H; Bleske, Barry E; Zhu, Hao-Jie

2016-04-01

Sacubitril was recently approved by the Food and Drug Administration for use in combination with valsartan for the treatment of patients with heart failure with reduced ejection fraction. As a prodrug, sacubitril must be metabolized (hydrolyzed) to its active metabolite sacubitrilat (LBQ657) to exert its intended therapeutic effects. Thus, understanding the determinants of sacubitril activation will lead to the improvement of sacubitril pharmacotherapy. The objective of this study was to identify the enzyme(s) responsible for the activation of sacubitril, and determine the impact of genetic variation on sacubitril activation. First, an incubation study of sacubitril with human plasma and the S9 fractions of human liver, intestine, and kidney was conducted. Sacubitril was found to be activated by human liver S9 fractions only. Moreover, sacubitril activation was significantly inhibited by the carboxylesterase 1 (CES1) inhibitor bis-(p-nitrophenyl) phosphate in human liver S9. Further incubation studies with recombinant human CES1 and carboxylesterase 2 confirmed that sacubitril is a selective CES1 substrate. The in vitro study of cell lines transfected with wild-type CES1 and the CES1 variant G143E (rs71647871) demonstrated that G143E is a loss-of-function variant for sacubitril activation. Importantly, sacubitril activation was significantly impaired in human livers carrying the G143E variant. In conclusion, sacubitril is selectively activated by CES1 in human liver. The CES1 genetic variant G143E can significantly impair sacubitril activation. Therefore, CES1 genetic variants appear to be an important contributing factor to interindividual variability in sacubitril activation, and have the potential to serve as biomarkers to optimize sacubitril pharmacotherapy. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Identification and characterization of novel benzil (diphenylethane-1,2-dione) analogues as inhibitors of mammalian carboxylesterases.

PubMed

Wadkins, Randy M; Hyatt, Janice L; Wei, Xin; Yoon, Kyoung Jin P; Wierdl, Monika; Edwards, Carol C; Morton, Christopher L; Obenauer, John C; Damodaran, Komath; Beroza, Paul; Danks, Mary K; Potter, Philip M

2005-04-21

Carboxylesterases (CE) are ubiquitous enzymes responsible for the metabolism of xenobiotics. Because the structural and amino acid homology among esterases of different classes, the identification of selective inhibitors of these proteins has proved problematic. Using Telik's target-related affinity profiling (TRAP) technology, we have identified a class of compounds based on benzil (1,2-diphenylethane-1,2-dione) that are potent CE inhibitors, with K(i) values in the low nanomolar range. Benzil and 30 analogues demonstrated selective inhibition of CEs, with no inhibitory activity toward human acetylcholinesterase or butyrylcholinesterase. Analysis of structurally related compounds indicated that the ethane-1,2-dione moiety was essential for enzyme inhibition and that potency was dependent on the presence of, and substitution within, the benzene ring. 3D-QSAR analyses of these benzil analogues for three different mammalian CEs demonstrated excellent correlations of observed versus predicted K(i) (r(2) > 0.91), with cross-validation coefficients (q(2)) of 0.9. Overall, these results suggest that selective inhibitors of CEs with potential for use in clinical applications can be designed.

Two alternative modes for optimizing nylon-6 byproduct hydrolytic activity from a carboxylesterase with a β-lactamase fold: X-ray crystallographic analysis of directly evolved 6-aminohexanoate-dimer hydrolase

PubMed Central

Ohki, Taku; Shibata, Naoki; Higuchi, Yoshiki; Kawashima, Yasuyuki; Takeo, Masahiro; Kato, Dai-ichiro; Negoro, Seiji

2009-01-01

Promiscuous 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity originally obtained in a carboxylesterase with a β-lactamase fold was enhanced about 80-fold by directed evolution using error-prone PCR and DNA shuffling. Kinetic studies of the mutant enzyme (Hyb-S4M94) demonstrated that the enzyme had acquired an increased affinity (Km = 15 mM) and turnover (kcat = 3.1 s−1) for Ald, and that a catalytic center suitable for nylon-6 byproduct hydrolysis had been generated. Construction of various mutant enzymes revealed that the enhanced activity in the newly evolved enzyme is due to the substitutions R187S/F264C/D370Y. Crystal structures of Hyb-S4M94 with bound substrate suggested that catalytic function for Ald was improved by hydrogen-bonding/hydrophobic interactions between the Ald—COOH and Tyr370, a hydrogen-bonding network from Ser187 to , and interaction between and Gln27-Oɛ derived from another subunit in the homo-dimeric structure. In wild-type Ald-hydrolase (NylB), Ald-hydrolytic activity is thought to be optimized by the substitutions G181D/H266N, which improve an electrostatic interaction with (Kawashima et al., FEBS J 2009; 276:2547–2556). We propose here that there exist at least two alternative modes for optimizing the Ald-hydrolytic activity of a carboxylesterase with a β-lactamase fold. PMID:19521995

Interaction of organophosphorus compounds with carboxylesterases in the rat.

PubMed

Jokanović, M; Kosanović, M; Maksimović, M

1996-01-01

Carboxylesterases (CarbE) are involved in detoxication of organophosphorus compounds (OPC) through two mechanisms: hydrolysis of ester bonds in OPC which contain them and binding of OPC at the active site of CarbE which reduces the amount of OPC available for acetylcholinesterase inhibition. This study of the interaction of rat plasma and liver CarbE with dichlorvos, soman and sarin in vitro and in vivo was undertaken in order to contribute to better understanding of the role of CarbE in detoxication of OPC. The results obtained have shown that inhibitory potency (I50) of dichlorvos, sarin and soman towards rat liver CarbE was 0.2 microM, 0.5 microM and 4.5 microM, respectively, for 20-min incubation at 25 degrees C. Second-order rate constants (k(a)) for liver CarbE inhibition were 2.3 x 10(5) M-1 min-1, 6.9 x 10(4) M-1 min-1 and 1.1 x 10(4) M-1 min-1 for dichlorvos, sarin and soman, respectively. The corresponding values for plasma CarbE could not be calculated because of dominant spontaneous reactivation of inhibited CarbE. CarbE inhibited with these OPC in vitro spontaneously reactivate with half-times of 18, 143 and 497 min for sarin, dichlorvos and soman in plasma and 111, 163 and 297 min for sarin, soman and dichlorvos in liver, respectively. These results were also confirmed in experiments in vivo in which rats were subcutaneously treated with 0.5 LD50 of these agents. The half-times of spontaneous reactivation of rat plasma CarbE in vivo were 1.2, 2.0 and 2.7 h for dichlorvos, sarin and soman, respectively. These findings have changed current understanding of the mechanism of interaction of CarbE with OPC and involvement of the enzymes in detoxication of OPC, suggesting an active and important role of the enzymes in metabolic conversions of OPC to their less toxic metabolites.

New catalytic roles for serine esterases: a 19F-NMR study of the interaction of 3,3,3-trifluoro-2,2-dihydroxy-1-phenyl-1-propanone with chicken liver carboxylesterase.

PubMed

Bowles, M R; King, G J; Berndt, M C; Zerner, B

1996-12-05

The reactions of 3,3,3-trifluoro-2,2-dihydroxy-1-phenyl-1-propanone (TDPP) with chicken liver carboxylesterase have shown that this ketone hydrate is not only a potent inhibitor of the enzyme, but also a substrate for a number of enzyme-catalyzed reactions. The kinetics of inhibition are consistent with a mechanism in which the bound hydrate is initially dehydrated in a rate-limiting step catalyzed by the enzyme. Nucleophilic attack by the active-site serine on the parent ketone then produces a hemiketal adduct. However, the slow reactivation (by dialysis) of TDPP-inhibited enzyme indicates that the interaction with this inhibitor is more complex. At equilibrium, a dissociation constant of 2.4 pM was obtained for this interaction. 19F-NMR studies of the enzyme-TDPP complex show that after pre-equilibration, the major adduct is not the hemiketal adduct. It is proposed that this final adduct is a cross-linked adduct formed between TDPP, the active-site serine and the active-site histidine. 19F-NMR studies reveal that chicken liver carboxylesterase catalyses the cleavage of TDPP to yield either fluoride ion or trifluoroacetate, and also the benzilic acid rearrangement of TDPP to alpha-trifluoromethylmandelate. These products have also been identified in model studies of the reaction between TDPP and imidazole.

Acylsugar Acylhydrolases: Carboxylesterase-Catalyzed Hydrolysis of Acylsugars in Tomato Trichomes1[OPEN

PubMed Central

Gilgallon, Karin; Ghosh, Banibrata

2016-01-01

Glandular trichomes of cultivated tomato (Solanum lycopersicum) and many other species throughout the Solanaceae produce and secrete mixtures of sugar esters (acylsugars) on the plant aerial surfaces. In wild and cultivated tomato, these metabolites consist of a sugar backbone, typically glucose or sucrose, and two to five acyl chains esterified to various positions on the sugar core. The aliphatic acyl chains vary in length and branching and are transferred to the sugar by a series of reactions catalyzed by acylsugar acyltransferases. A phenotypic screen of a set of S. lycopersicum M82 × Solanum pennellii LA0716 introgression lines identified a dominant genetic locus on chromosome 5 from the wild relative that affected total acylsugar levels. Genetic mapping revealed that the reduction in acylsugar levels was consistent with the presence and increased expression of two S. pennellii genes (Sopen05g030120 and Sopen05g030130) encoding putative carboxylesterase enzymes of the α/β-hydrolase superfamily. These two enzymes, named ACYLSUGAR ACYLHYDROLASE1 (ASH1) and ASH2, were shown to remove acyl chains from specific positions of certain types of acylsugars in vitro. A survey of related genes in M82 and LA0716 identified another trichome-expressed ASH gene on chromosome 9 (M82, Solyc09g075710; LA0716, Sopen09g030520) encoding a protein with similar activity. Characterization of the in vitro activities of the SpASH enzymes showed reduced activities with acylsugars produced by LA0716, presumably contributing to the high-level production of acylsugars in the presence of highly expressed SpASH genes. PMID:26811191

Purification, Characterization, and Sensitivity to Pesticides of Carboxylesterase From Dendrolimus superans (Lepidoptera: Lasiocampidae)

PubMed Central

Zou, Chuan-shan; Cao, Chuan-wang; Zhang, Guo-cai; Wang, Zhi-ying

2014-01-01

Abstract Through a combination of steps including centrifugation, ammonium sulfate gradient precipitation, sephadex G-25 gel chromatography, diethylaminoethyl cellulose 52 ion-exchange chromatography and hydroxyapatite affinity chromatography, carboxylesterase (CarE, EC3.1.1.1) from sixth instar larch caterpillar moth, Dendrolimus superans (Lepidoptera: Lasiocampidae) larvae was purified and its biochemical properties were compared between crude homogenate and purified CarE. The final purified CarE after hydroxyapatite chromatography had a specific activity of 52.019 μmol/(min·mg protein), 138.348-fold of crude homogenate, and the yield of 2.782%. The molecular weight of the purified CarE was approximately 84.78 kDa by SDS-PAGE. Three pesticides (dichlorvos, lambda-cyhalothrin, and avermectins) showed different inhibition to crude CarE and purified CarE, respectively. In vitro median inhibitory concentration indicated that the sensitivity of CarE (both crude homogenate and final purified CarE) to pesticides was in decreasing order of dichlorvos > avermectins > lambda-cyhalothrin. By the kinetic analysis, the substrates alpha-naphthyl acetate (α-NA) and beta-naphthyl acetate (β-NA) showed lesser affinity to crude extract than purified CarE. The results also indicated that both crude homogenate and purified CarE had more affinity to α-NA than to β-NA, and the Kcat and Vmax values of crude extract were lower than purified CarE using α-NA or β-NA as substrate. PMID:25525114

Effect of environmental contaminants in the Mississippi River Basin on carboxylesterases from four aquatic species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jaiswal, R.; Huang, T.; Obih, P.

1995-12-31

The objectives of this study are to investigate the sensitivity of different classes of esterases in various aquatic species to environmental contaminants and the possible use of these enzymes as biomarkers for monitoring the effects of pollutants. Acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and the non-specific carboxylesterases (CaE) were analyzed in three fish species, Ictiobus bubalus (small mouth buffalo), Ictiobus cyprinellus (big mouth buffalo) and Lepisosteus oculatus (spotted gar) and the green tree frog, Hyla cinerea. These samples were collected from the Devil`s Swamp Site (DSS), an industrial site known to be highly contaminated at the Mississippi River Basin, and Lake Tunica,more » a nonindustrial site. ACHE and BuChE activities in the subcellular fractions of liver and brain were significantly lower in fishes and frogs obtained from DSS when compared to the same species obtained from Tunica swamp site. The greatest decrease was observed with ACHE activity in the liver and brain of Ictiobus bubalus from DSS. CaE activity analyzed with p-nitrophenyl acetate was found to be significantly lower in the liver of all three fish species collected from DSS when compared to the same fish species obtained from the Tunica swamp site.« less

Requirements for mammalian carboxylesterase inhibition by substituted ethane-1,2-diones

PubMed Central

Parkinson, Elizabeth I.; Hatfield, M. Jason; Tsurkan, Lyudmila; Hyatt, Janice L.; Edwards, Carol C.; Hicks, Latorya D.; Yan, Bing; Potter, Philip M.

2011-01-01

Carboxylesterases (CE) are ubiquitous enzymes found in both human and animal tissues and are responsible for the metabolism of xenobiotics. This includes numerous natural products, as well as a many clinically used drugs. Hence the activity of these agents is likely dependent upon the levels and location of CE expression. We have recently identified benzil is a potent inhibitor of mammalian CEs, and in this study, we have assessed the ability of analogues of this compound to inhibit these enzymes. Three different classes of molecules were assayed: One containing different atoms vicinal to the carbonyl carbon atom and the benzene ring [PhXC(O)C(O)XPh, where X = CH2, CHBr, N, S, or O]; a second containing a panel of alkyl 1,2-diones demonstrating increasing alkyl chain length; and a third consisting of a series of 1-phenyl-2-alkyl-1,2-diones. In general, with the former series of molecules, heteroatoms resulted in either loss of inhibitory potency (when X =N), or conversion of the compounds into substrates for the enzymes (when X = S or O). However, the inclusion of a brominated methylene atom resulted in potent CE inhibition. Subsequent analysis with the alkyl diones [RC(O)C(O)R, where R ranged from CH3 to C8H17] and 1-phenyl-2-alkyl-1,2-diones [PhC(O)C(O)R where R ranged from CH3 to C6H13], demonstrated that the potency of enzyme inhibition directly correlated with the hydrophobicity (clogP) of the molecules. We conclude from these studies that that the inhibitory power of these 1,2-dione derivatives depends primarily upon the hydrophobicity of the R group, but also on the electrophilicity of the carbonyl group. PMID:21733699

Requirements for mammalian carboxylesterase inhibition by substituted ethane-1,2-diones.

PubMed

Parkinson, Elizabeth I; Jason Hatfield, M; Tsurkan, Lyudmila; Hyatt, Janice L; Edwards, Carol C; Hicks, Latorya D; Yan, Bing; Potter, Philip M

2011-08-01

Carboxylesterases (CE) are ubiquitous enzymes found in both human and animal tissues and are responsible for the metabolism of xenobiotics. This includes numerous natural products, as well as a many clinically used drugs. Hence, the activity of these agents is likely dependent upon the levels and location of CE expression. We have recently identified benzil is a potent inhibitor of mammalian CEs, and in this study, we have assessed the ability of analogues of this compound to inhibit these enzymes. Three different classes of molecules were assayed: one containing different atoms vicinal to the carbonyl carbon atom and the benzene ring [PhXC(O)C(O)XPh, where X=CH₂, CHBr, N, S, or O]; a second containing a panel of alkyl 1,2-diones demonstrating increasing alkyl chain length; and a third consisting of a series of 1-phenyl-2-alkyl-1,2-diones. In general, with the former series of molecules, heteroatoms resulted in either loss of inhibitory potency (when X=N), or conversion of the compounds into substrates for the enzymes (when X=S or O). However, the inclusion of a brominated methylene atom resulted in potent CE inhibition. Subsequent analysis with the alkyl diones [RC(O)C(O)R, where R ranged from CH₃ to C₈H₁₇] and 1-phenyl-2-alkyl-1,2-diones [PhC(O)C(O)R where R ranged from CH₃ to C₆H₁₃], demonstrated that the potency of enzyme inhibition directly correlated with the hydrophobicity (clogP) of the molecules. We conclude from these studies that that the inhibitory power of these 1,2-dione derivatives depends primarily upon the hydrophobicity of the R group, but also on the electrophilicity of the carbonyl group. Copyright © 2011 Elsevier Ltd. All rights reserved.

Annotation and expression of carboxylesterases in the silkworm, Bombyx mori.

PubMed

Yu, Quan-You; Lu, Cheng; Li, Wen-Le; Xiang, Zhong-Huai; Zhang, Ze

2009-11-24

Carboxylesterase is a multifunctional superfamily and ubiquitous in all living organisms, including animals, plants, insects, and microbes. It plays important roles in xenobiotic detoxification, and pheromone degradation, neurogenesis and regulating development. Previous studies mainly used Dipteran Drosophila and mosquitoes as model organisms to investigate the roles of the insect COEs in insecticide resistance. However, genome-wide characterization of COEs in phytophagous insects and comparative analysis remain to be performed. Based on the newly assembled genome sequence, 76 putative COEs were identified in Bombyx mori. Relative to other Dipteran and Hymenopteran insects, alpha-esterases were significantly expanded in the silkworm. Genomics analysis suggested that BmCOEs showed chromosome preferable distribution and 55% of which were tandem arranged. Sixty-one BmCOEs were transcribed based on cDNA/ESTs and microarray data. Generally, most of the COEs showed tissue specific expressions and expression level between male and female did not display obvious differences. Three main patterns could be classified, i.e. midgut-, head and integument-, and silk gland-specific expressions. Midgut is the first barrier of xenobiotics peroral toxicity, in which COEs may be involved in eliminating secondary metabolites of mulberry leaves and contaminants of insecticides in diet. For head and integument-class, most of the members were homologous to odorant-degrading enzyme (ODE) and antennal esterase. RT-PCR verified that the ODE-like esterases were also highly expressed in larvae antenna and maxilla, and thus they may play important roles in degradation of plant volatiles or other xenobiotics. B. mori has the largest number of insect COE genes characterized to date. Comparative genomic analysis suggested that the gene expansion mainly occurred in silkworm alpha-esterases. Expression evidence indicated that the expanded genes were specifically expressed in midgut, integument and

Annotation and expression of carboxylesterases in the silkworm, Bombyx mori

PubMed Central

2009-01-01

Background Carboxylesterase is a multifunctional superfamily and ubiquitous in all living organisms, including animals, plants, insects, and microbes. It plays important roles in xenobiotic detoxification, and pheromone degradation, neurogenesis and regulating development. Previous studies mainly used Dipteran Drosophila and mosquitoes as model organisms to investigate the roles of the insect COEs in insecticide resistance. However, genome-wide characterization of COEs in phytophagous insects and comparative analysis remain to be performed. Results Based on the newly assembled genome sequence, 76 putative COEs were identified in Bombyx mori. Relative to other Dipteran and Hymenopteran insects, alpha-esterases were significantly expanded in the silkworm. Genomics analysis suggested that BmCOEs showed chromosome preferable distribution and 55% of which were tandem arranged. Sixty-one BmCOEs were transcribed based on cDNA/ESTs and microarray data. Generally, most of the COEs showed tissue specific expressions and expression level between male and female did not display obvious differences. Three main patterns could be classified, i.e. midgut-, head and integument-, and silk gland-specific expressions. Midgut is the first barrier of xenobiotics peroral toxicity, in which COEs may be involved in eliminating secondary metabolites of mulberry leaves and contaminants of insecticides in diet. For head and integument-class, most of the members were homologous to odorant-degrading enzyme (ODE) and antennal esterase. RT-PCR verified that the ODE-like esterases were also highly expressed in larvae antenna and maxilla, and thus they may play important roles in degradation of plant volatiles or other xenobiotics. Conclusion B. mori has the largest number of insect COE genes characterized to date. Comparative genomic analysis suggested that the gene expansion mainly occurred in silkworm alpha-esterases. Expression evidence indicated that the expanded genes were specifically

Detection of carboxylesterase and esterase activity in culturable gut bacterial flora isolated from diamondback moth, Plutella xylostella (Linnaeus), from India and its possible role in indoxacarb degradation.

PubMed

Ramya, Shanivarsanthe Leelesh; Venkatesan, Thiruvengadam; Srinivasa Murthy, Kottilingam; Jalali, Sushil Kumar; Verghese, Abraham

2016-01-01

Diamondback moth (DBM), Plutella xylostella (Linnaeus), is a notorious pest of brassica crops worldwide and is resistant to all groups of insecticides. The insect system harbors diverse groups of microbiota, which in turn helps in enzymatic degradation of xenobiotic-like insecticides. The present study aimed to determine the diversity of gut microflora in DBM, quantify esterase activity and elucidate their possible role in degradation of indoxacarb. We screened 11 geographic populations of DBM in India and analyzed them for bacterial diversity. The culturable gut bacterial flora underwent molecular characterization with 16S rRNA. We obtained 25 bacterial isolates from larvae (n=13) and adults (n=12) of DBM. In larval gut isolates, gammaproteobacteria was the most abundant (76%), followed by bacilli (15.4%). Molecular characterization placed adult gut bacterial strains into three major classes based on abundance: gammaproteobacteria (66%), bacilli (16.7%) and flavobacteria (16.7%). Esterase activity from 19 gut bacterial isolates ranged from 0.072 to 2.32μmol/min/mg protein. Esterase bands were observed in 15 bacterial strains and the banding pattern differed in Bacillus cereus - KC985225 and Pantoea agglomerans - KC985229. The bands were characterized as carboxylesterase with profenofos used as an inhibitor. Minimal media study showed that B. cereus degraded indoxacarb up to 20%, so it could use indoxacarb for metabolism and growth. Furthermore, esterase activity was greater with minimal media than control media: 1.87 versus 0.26μmol/min/mg protein. Apart from the insect esterases, bacterial carboxylesterase may aid in the degradation of insecticides in DBM. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Inhibition of carboxylesterase 1 is associated with cholesteryl ester retention in human THP-1 monocyte/macrophages

PubMed Central

Crow, J. Allen; Middleton, Brandy L.; Borazjani, Abdolsamad; Hatfield, M. Jason; Potter, Philip M.; Ross, Matthew K.

2008-01-01

Cholesteryl esters are hydrolyzed by cholesteryl ester hydrolase (CEH) yielding free cholesterol for export from macrophages. Hence, CEH has an important regulatory role in macrophage reverse cholesterol transport (RCT). CEH and human carboxylesterase 1 (CES1) appear to be the same enzyme. CES1 is inhibited by oxons, the bioactive metabolites of organophosphate (OP) pesticides. Here, we show that CES1 protein is robustly expressed in human THP-1 monocytes/macrophages and its biochemical activity inhibited following treatment of cell lysates and intact cells with chlorpyrifos oxon, paraoxon, or methyl paraoxon (with nanomolar IC50 values) or after immunodepletion of CES1 protein. CES1 protein expression in cells is unaffected by 24-h paraoxon treatment, suggesting the reduced hydrolytic activity is due to covalent inhibition of CES1 by oxons and not down-regulation of expression. Most significantly, treatment of cholesterol-loaded macrophages with either paraoxon (a non-specific CES inhibitor) or benzil (a specific CES inhibitor) caused enhanced retention of intracellular cholesteryl esters and a “foamy” phenotype, consistent with reduced cholesteryl ester mobilization. Thus, exposure to OP pesticides, which results in the inhibition of CES1, may also inhibit macrophage RCT, an important process in the regression of atherosclerosis. PMID:18762277

Carboxylesterase gene amplifications associated with insecticide resistance in Aedes albopictus: Geographical distribution and evolutionary origin

PubMed Central

Grigoraki, Linda; Pipini, Dimitra; Labbé, Pierrick; Chaskopoulou, Alexandra; Weill, Mylene; Vontas, John

2017-01-01

Background Aedes albopictus is one of the most invasive human disease vectors. Its control has been largely based on insecticides, such as the larvicide temephos. Temephos resistance has been associated with the up-regulation, through gene amplification, of two carboxylesterase (CCE) genes closely linked on the genome, capable of sequestering and metabolizing temephos oxon, the activated form of temephos. Principal findings Here, we investigated the occurrence, geographical distribution and origin of the CCE amplicon in Ae. albopictus populations from several geographical regions worldwide. The haplotypic diversity at the CCEae3a locus revealed high polymorphism, while phylogenetic analysis showed an absence of correlation between haplotype similarity and geographic origin. Two types of esterase amplifications were found, in two locations only (Athens and Florida): one, previously described, results in the amplification of both CCEae3a and CCEae6a; the second is being described for the first time and results in the amplification of CCEae3a only. The two amplification events are independent, as confirmed by sequence analysis. All individuals from Athens and Florida carrying the CCEae3a-CCEae6a co-amplicon share a common haplotype, indicating a single amplification event, which spread between the two countries. Significance The importance of passive transportation of disease vectors, including individuals carrying resistance mechanisms, is discussed in the light of efficient and sustainable vector control strategies. PMID:28394886

The environment shapes microbial enzymes: five cold-active and salt-resistant carboxylesterases from marine metagenomes.

PubMed

Tchigvintsev, Anatoli; Tran, Hai; Popovic, Ana; Kovacic, Filip; Brown, Greg; Flick, Robert; Hajighasemi, Mahbod; Egorova, Olga; Somody, Joseph C; Tchigvintsev, Dmitri; Khusnutdinova, Anna; Chernikova, Tatyana N; Golyshina, Olga V; Yakimov, Michail M; Savchenko, Alexei; Golyshin, Peter N; Jaeger, Karl-Erich; Yakunin, Alexander F

2015-03-01

Most of the Earth's biosphere is cold and is populated by cold-adapted microorganisms. To explore the natural enzyme diversity of these environments and identify new carboxylesterases, we have screened three marine metagenome gene libraries for esterase activity. The screens identified 23 unique active clones, from which five highly active esterases were selected for biochemical characterization. The purified metagenomic esterases exhibited high activity against α-naphthyl and p-nitrophenyl esters with different chain lengths. All five esterases retained high activity at 5 °C indicating that they are cold-adapted enzymes. The activity of MGS0010 increased more than two times in the presence of up to 3.5 M NaCl or KCl, whereas the other four metagenomic esterases were inhibited to various degrees by these salts. The purified enzymes showed different sensitivities to inhibition by solvents and detergents, and the activities of MGS0010, MGS0105 and MGS0109 were stimulated three to five times by the addition of glycerol. Screening of purified esterases against 89 monoester substrates revealed broad substrate profiles with a preference for different esters. The metagenomic esterases also hydrolyzed several polyester substrates including polylactic acid suggesting that they can be used for polyester depolymerization. Thus, esterases from marine metagenomes are cold-adapted enzymes exhibiting broad biochemical diversity reflecting the environmental conditions where they evolved.

Carboxylesterase Precursor (EST-1) Mediated the Fungicide Jinggangmycin-Suppressed Reproduction of Sogatella furcifera (Hemiptera: Delphacidae).

PubMed

Ge, Lin-Quan; Huang, Bo; Jiang, Yi-Ping; Gu, Hao-Tian; Xia, Ting; Yang, Guo-Qing; Liu, Fang; Wu, Jin-Cai

2017-10-01

The jinggangmycin (JGM) is a widely used fungicide for controlling the rice sheath blight, Rhizoctonia solani, in China. Previous experiments under lab conditions showed that JGM foliar spray suppressed Sogatella furcifera (Horvath) reproduction. However, the molecular mechanisms of JGM-driven changes in S. furcifera reproduction are unclear. Therefore, we selected carboxylesterase precursor (EST-1) as a target gene for silencing by RNAi based on gene expression profiles. The present results demonstrated that JGM and control + dsSfEST-1 treatments significantly reduced the number of eggs laid (down by 58% and 54%, respectively), oviposition period (down by 57% and 38%, respectively), and longevity (down by 32% and 38%, respectively) in adult females compared with untreated controls, while no pronounced differences in the preoviposition period were observed. Meanwhile, the dietary control + dsSfEST-1 treatment also severely impeded protein synthesis, specifically soluble ovarian protein content (down by 20% and 24%, respectively) and soluble sugar content (down by 42% and 35%, respectively), which led to stunted growth and reduced body weight in adult females. We thereby speculate that downregulated SfEST-1 expression may be one molecular mechanism underlying JGM-driven reproduction in S. furcifera. © The Author 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Carboxylesterase 1 Is Regulated by Hepatocyte Nuclear Factor 4α and Protects Against Alcohol- and MCD diet-induced Liver Injury.

PubMed

Xu, Jiesi; Xu, Yang; Li, Yuanyuan; Jadhav, Kavita; You, Min; Yin, Liya; Zhang, Yanqiao

2016-04-14

The liver is a major organ that controls hepatic and systemic homeostasis. Dysregulation of liver metabolism may cause liver injury. Previous studies have demonstrated that carboxylesterase 1 (CES1) regulates hepatic triglyceride metabolism and protects against liver steatosis. In the present study, we investigated whether CES1 played a role in the development of alcoholic liver disease (ALD) and methionine and choline-deficient (MCD) diet-induced liver injury. Both hepatocyte nuclear factor 4α (HNF4α) and CES1 were markedly reduced in patients with alcoholic steatohepatitis. Alcohol repressed both HNF4α and CES1 expression in primary hepatocytes. HNF4α regulated CES1 expression by directly binding to the proximal promoter of CES1. Global inactivation of CES1 aggravated alcohol- or MCD diet-induced liver inflammation and liver injury, likely as a result of increased production of acetaldehyde and reactive oxygen species and mitochondrial dysfunctions. Knockdown of hepatic CES1 exacerbated ethanol-induced steatohepatitis. These data indicate that CES1 plays a crucial role in protection against alcohol- or MCD diet-induced liver injury.

Inhibition of recombinant human carboxylesterase 1 and 2 and monoacylglycerol lipase by chlorpyrifos oxon, paraoxon and methyl paraoxon

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crow, J. Allen; Bittles, Victoria; Herring, Katye L.

2012-01-01

Oxons are the bioactivated metabolites of organophosphorus insecticides formed via cytochrome P450 monooxygenase-catalyzed desulfuration of the parent compound. Oxons react covalently with the active site serine residue of serine hydrolases, thereby inactivating the enzyme. A number of serine hydrolases other than acetylcholinesterase, the canonical target of oxons, have been reported to react with and be inhibited by oxons. These off-target serine hydrolases include carboxylesterase 1 (CES1), CES2, and monoacylglycerol lipase. Carboxylesterases (CES, EC 3.1.1.1) metabolize a number of xenobiotic and endobiotic compounds containing ester, amide, and thioester bonds and are important in the metabolism of many pharmaceuticals. Monoglyceride lipase (MGL,more » EC 3.1.1.23) hydrolyzes monoglycerides including the endocannabinoid, 2-arachidonoylglycerol (2-AG). The physiological consequences and toxicity related to the inhibition of off-target serine hydrolases by oxons due to chronic, low level environmental exposures are poorly understood. Here, we determined the potency of inhibition (IC{sub 50} values; 15 min preincubation, enzyme and inhibitor) of recombinant CES1, CES2, and MGL by chlorpyrifos oxon, paraoxon and methyl paraoxon. The order of potency for these three oxons with CES1, CES2, and MGL was chlorpyrifos oxon > paraoxon > methyl paraoxon, although the difference in potency for chlorpyrifos oxon with CES1 and CES2 did not reach statistical significance. We also determined the bimolecular rate constants (k{sub inact}/K{sub I}) for the covalent reaction of chlorpyrifos oxon, paraoxon and methyl paraoxon with CES1 and CES2. Consistent with the results for the IC{sub 50} values, the order of reactivity for each of the three oxons with CES1 and CES2 was chlorpyrifos oxon > paraoxon > methyl paraoxon. The bimolecular rate constant for the reaction of chlorpyrifos oxon with MGL was also determined and was less than the values determined for chlorpyrifos oxon

Effect of diet on carboxylesterase activity of tadpoles (Rhinella arenarum) exposed to chlorpyrifos.

PubMed

Attademo, A M; Sanchez-Hernandez, J C; Lajmanovich, R C; Peltzer, P M; Junges, C

2017-01-01

An outdoor microcosm was performed with tadpoles (Rhinella arenarum) exposed to 125μgL -1 chlorpyrifos and fed two types of food, i.e., lettuce (Lactuca sativa) and a formulated commercial pellet. Acetylcholinesterase (AChE) and carboxylesterase (CbE) activities were measured in liver and intestine after 10 days of pesticide exposure. Non-exposed tadpoles fed lettuce had an intestinal AChE activity almost two-fold higher than that of pellet-fed tadpoles. No significant differences were observed, however, in liver AChE activity between diets. Likewise, intestinal CbE activity - measured using two substrates, i.e. 1-naphthyl acetate (1-NA) and 4-nitrophenyl valerate (4-NPV) - was higher in tadpoles fed lettuce than in those fed pellets. However, the diet-dependent response of liver CbE activity was opposite to that in the intestine. Chlorpyrifos caused a significant inhibition of both esterase activities, which was tissue- and diet-specific. The highest inhibition degree was found in the intestinal AChE and CbE activities of lettuce-fed tadpoles (42-78% of controls) compared with pellet-fed tadpoles (<60%). Although chlorpyrifos significantly inhibited liver CbE activity of the group fed lettuce, this effect was not observed in the group fed pellets. In general, intestinal CbE activity was more sensitive to chlorpyrifos inhibition than AChE activity. This finding, together with the high levels of basal CbE activity found in the intestine, may be understood as a detoxification system able to reduce intestinal OP uptake. Moreover, the results of this study suggest that diet is a determinant factor in toxicity testing with tadpoles to assess OP toxicity, because it modulates levels of this potential detoxifying enzyme activity. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of chlorpyrifos on soil carboxylesterase activity at an aggregate-size scale.

PubMed

Sanchez-Hernandez, Juan C; Sandoval, Marco

2017-08-01

The impact of pesticides on extracellular enzyme activity has been mostly studied on the bulk soil scale, and our understanding of the impact on an aggregate-size scale remains limited. Because microbial processes, and their extracellular enzyme production, are dependent on the size of soil aggregates, we hypothesized that the effect of pesticides on enzyme activities is aggregate-size specific. We performed three experiments using an Andisol to test the interaction between carboxylesterase (CbE) activity and the organophosphorus (OP) chlorpyrifos. First, we compared esterase activity among aggregates of different size spiked with chlorpyrifos (10mgkg -1 wet soil). Next, we examined the inhibition of CbE activity by chlorpyrifos and its metabolite chlorpyrifos-oxon in vitro to explore the aggregate size-dependent affinity of the pesticides for the active site of the enzyme. Lastly, we assessed the capability of CbEs to alleviate chlorpyrifos toxicity upon soil microorganisms. Our principal findings were: 1) CbE activity was significantly inhibited (30-67% of controls) in the microaggregates (<0.25mm size) and smallest macroaggregates (<1.0 - 0.25mm), but did not change in the largest macroaggregates (>1.0mm) compared with the corresponding controls (i.e., pesticide-free aggregates), 2) chlorpyrifos-oxon was a more potent CbE inhibitor than chlorpyrifos; however, no significant differences in the CbE inhibition were found between micro- and macroaggregates, and 3) dose-response relationships between CbE activity and chlorpyrifos concentrations revealed the capability of the enzyme to bind chlorpyrifos-oxon, which was dependent on the time of exposure. This chemical interaction resulted in a safeguarding mechanism against chlorpyrifos-oxon toxicity on soil microbial activity, as evidenced by the unchanged activity of dehydrogenase and related extracellular enzymes in the pesticide-treated aggregates. Taken together, these results suggest that environmental risk

Global and local molecular dynamics of a bacterial carboxylesterase provide insight into its catalytic mechanism

PubMed Central

Yu, Xiaozhen; Sigler, Sara C.; Hossain, Delwar; Wierdl, Monika; Gwaltney, Steven R.; Potter, Philip M.; Wadkins, Randy M.

2013-01-01

Carboxylesterases (CEs) are ubiquitous enzymes responsible for the detoxification of xenobiotics. In humans, substrates for these enzymes are far-ranging, and include the street drug heroin and the anticancer agent irinotecan. Hence, their ability to bind and metabolize substrates is of broad interest to biomedical science. In this study, we focused our attention on dynamic motions of a CE from B. subtilis (pnbCE), with emphasis on the question of what individual domains of the enzyme might contribute to its catalytic activity. We used a 10 ns all-atom molecular dynamics simulation, normal mode calculations, and enzyme kinetics to understand catalytic consequences of structural changes within this enzyme. Our results shed light on how molecular motions are coupled with catalysis. During molecular dynamics, we observed a distinct C-C bond rotation between two conformations of Glu310. Such a bond rotation would alternately facilitate and impede protonation of the active site His399 and act as a mechanism by which the enzyme alternates between its active and inactive conformation. Our normal mode results demonstrate that the distinct low-frequency motions of two loops in pnbCE, coil_5 and coil_21, are important in substrate conversion and seal the active site. Mutant CEs lacking these external loops show significantly reduced rates of substrate conversion, suggesting this sealing motion prevents escape of substrate. Overall, the results of our studies give new insight into the structure-function relationship of CEs and have implications for the entire family of α/β fold family of hydrolases, of which this CE is a member. PMID:22127613

A Fungus-Inducible Pepper Carboxylesterase Exhibits Antifungal Activity by Decomposing the Outer Layer of Fungal Cell Walls.

PubMed

Seo, Hyo-Hyoun; Park, Ae Ran; Lee, Hyun-Hwa; Park, Sangkyu; Han, Yun-Jeong; Hoang, Quyen T N; Choi, Gyung Ja; Kim, Jin-Cheol; Kim, Young Soon; Kim, Jeong-Il

2018-05-01

Colletotrichum species are major fungal pathogens that cause devastating anthracnose diseases in many economically important crops. In this study, we observed the hydrolyzing activity of a fungus-inducible pepper carboxylesterase (PepEST) on cell walls of C. gloeosporioides, causing growth retardation of the fungus by blocking appressorium formation. To determine the cellular basis for the growth inhibition, we observed the localization of PepEST on the fungus and found the attachment of the protein on surfaces of conidia and germination tubes. Moreover, we examined the decomposition of cell-wall materials from the fungal surface after reaction with PepEST, which led to the identification of 1,2-dithiane-4,5-diol (DTD) by gas chromatography mass spectrometry analysis. Exogenous DTD treatment did not elicit expression of defense-related genes in the host plant but did trigger the necrosis of C. gloeosporioides. Furthermore, the DTD compound displayed protective effects on pepper fruits and plants against C. gloeosporioides and C. coccodes, respectively. In addition, DTD was also effective in preventing other diseases, such as rice blast, tomato late blight, and wheat leaf rust. Therefore, our results provide evidence that PepEST is involved in hydrolysis of the outmost layer of the fungal cell walls and that DTD has antifungal activity, suggesting an alternative strategy to control agronomically important phytopathogens.

Crystal Structures of Human Carboxylesterase 1 in Covalent Complexes with the Chemical Warfare Agents Soman and Tabun†,‡

PubMed Central

Fleming, Christopher D.; Edwards, Carol C.; Kirby, Stephen D.; Maxwell, Donald M.; Potter, Philip M.; Cerasoli, Douglas M.; Redinbo, Matthew R.

2008-01-01

The organophosphorus nerve agents sarin, soman, tabun, and VX exert their toxic effects by inhibiting the action of human acetylcholinesterase, a member of the serine hydrolase superfamily of enzymes. The current treatments for nerve agent exposure must be administered quickly to be effective and they often do not eliminate long-term toxic side effects associated with organophosphate poisoning. Thus, there is significant need for effective prophylactic methods to protect at-risk personnel from nerve agent exposure, and protein-based approaches have emerged as promising candidates. We present the 2.7 Å resolution crystal structures of the serine hydrolase human carboxylesterase 1 (hCE1), a broad-spectrum drug metabolism enzyme, in covalent acyl-enzyme intermediate complexes with the chemical weapons soman and tabun. The structures reveal that hCE1 binds stereoselectively to these nerve agents; for example, hCE1 appears to react preferentially with the 104-fold more lethal PS stereoisomer of soman relative to the PR form. In addition, structural features of the hCE1 active site indicate that the enzyme may be resistant to dead-end organophosphate aging reactions that permanently inactivate other serine hydrolases. Taken together, these data provide important structural details toward the goal of engineering hCE1 into an organophosphate hydrolase and protein-based therapeutic for nerve agent exposure. PMID:17407327

Characterization and functional analysis of a carboxylesterase gene associated with chlorpyrifos resistance in Nilaparvata lugens (Stål).

PubMed

Lu, Kai; Wang, Ying; Chen, Xia; Zhang, Zhichao; Li, Yue; Li, Wenru; Zhou, Qiang

2017-12-01

The widespread and extensive application of insecticides have promoted the development of resistance in the brown planthopper Nilaparvata lugens (Stål), one of the most important rice pests in Asia. To better understand the underlying molecular mechanisms of metabolic resistance to insecticides, a chlorpyrifos-resistant (CR) strain of N. lugens was selected and its possible resistance mechanism was investigated. Synergistic tests using carboxylesterases (CarEs) inhibitor triphenyl phosphate (TPP) decreased the resistance of N. lugens to chlorpyrifos, and CarE activities could be induced by low concentrations of chlorpyrifos. Subsequently, a gene putatively encoding CarE, namely NlCarE, predominant in the midgut and ovary was isolated and characterized. The expression levels of NlCarE were detected and compared between the CR and a susceptible (SS) strain of N. lugens. Consistent with the increased CarE activity, this gene was overexpressed in the CR strain compared to the SS strain. The transcript levels of NlCarE were up-regulated by chlorpyrifos exposure, showing dose- and time-dependent expression patterns. Furthermore, RNA interference (RNAi)-mediated knockdown of NlCarE followed by insecticide application significantly increased the susceptibility of N. lugens to chlorpyrifos. These results demonstrate that NlCarE plays an important role in chlorpyrifos detoxification and its overexpression may be involved in chlorpyrifos resistance in N. lugens. Copyright © 2017 Elsevier Inc. All rights reserved.

Metabolism of deltamethrin and cis- and trans-permethrin by human expressed cytochrome P450 and carboxylesterase enzymes.

PubMed

Hedges, Laura; Brown, Susan; MacLeod, A Kenneth; Vardy, Audrey; Doyle, Edward; Song, Gina; Moreau, Marjory; Yoon, Miyoung; Osimitz, Thomas G; Lake, Brian G

2018-06-04

The metabolism of the pyrethroids deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes. DLM, CPM and TPM were metabolised by human CYP2B6 and CYP2C19, with the highest apparent intrinsic clearance (CL int ) values for pyrethroid metabolism being observed with CYP2C19. Other CYP enzymes contributing to the metabolism of one or more of the three pyrethroids were CYP1A2, CYP2C8, CYP2C9*1, CYP2D6*1, CYP3A4 and CYP3A5. None of the pyrethroids were metabolised by CYP2A6, CYP2E1, CYP3A7 or CYP4A11. DLM, CPM and TPM were metabolised by both human CES1 and CES2 enzymes. Apparent CL int values for pyrethroid metabolism by CYP and CES enzymes were scaled to per gram of adult human liver using abundance values for microsomal CYP enzymes and for CES enzymes in liver microsomes and cytosol. TPM had the highest and CPM the lowest apparent CL int values for total metabolism (CYP and CES enzymes) per gram of adult human liver. Due to their higher abundance, all three pyrethroids were extensively metabolised by CES enzymes in adult human liver, with CYP enzymes only accounting for 2%, 10% and 1% of total metabolism for DLM, CPM and TPM, respectively.

Presence and inter-individual variability of carboxylesterases (CES1 and CES2) in human lung.

PubMed

Gabriele, Morena; Puccini, Paola; Lucchi, Marco; Vizziello, Anna; Gervasi, Pier Giovanni; Longo, Vincenzo

2018-04-01

Lungs are pharmacologically active organs and the pulmonary drug metabolism is of interest for inhaled drugs design. Carboxylesterases (CESs) are enzymes catalyzing the hydrolysis of many structurally different ester, amide and carbamate chemicals, including prodrugs. For the first time, the presence, kinetics, inhibition and inter-individual variations of the major liver CES isozymes (CES1 and CES2) were investigated in cytosol and microsomes of human lungs from 20 individuals using 4-nitrophenyl acetate (pNPA), 4-methylumbelliferyl acetate (4-MUA), and fluorescein diacetate (FD) as substrates the rates of hydrolysis (V max ) for pNPA and 4-MUA, unlike FD, were double in microsomes than in cytosol. In these cellular fractions, the V max of pNPA, as CES1 marker, were much greater (30-50-fold) than those of FD, as a specific CES2 marker. Conversely, the K m values were comparable suggesting the involvement of the same enzymes. Inhibition studies revealed that the FD hydrolysis was inhibited by bis-p-nitrophenylphosphate, phenylmethanesulfonyl fluoride, and loperamide (specific for CES2), whereas the pNPA and 4-MUA hydrolysis inhibition was limited. Inhibitors selective for other esterases missed having any effect on above-mentioned activities. In cytosol and microsomes of 20 lung samples, inter-individual variations were found for the hydrolysis of pNPA (2.5-5-fold), FD or 4-MUA (8-15-fold). Similar variations were also observed in CES1 and CES2 gene expression, although determined in a small number (n = 9) of lung samples. The identification of CES1 and CES2 and their variability in human lungs are important for drug metabolism and design of prodrugs which need to be activated in this organ. Copyright © 2018 Elsevier Inc. All rights reserved.

Fluorescein as a Visible-Light-Induced Oxidase Mimic for Signal-Amplified Colorimetric Assay of Carboxylesterase by an Enzymatic Cascade Reaction.

PubMed

Liu, Li; Sun, Chaoqun; Yang, Juan; Shi, Ying; Long, Yijuan; Zheng, Huzhi

2018-04-20

We have found that fluorescein possesses high visible-light-induced oxidase mimetic activity and could transform colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxidized TMB (oxTMB) without unstable and destructive H 2 O 2 under visible-light illumination. Instead, fluorescein uses oxygen as a mild and green electron acceptor, and its activity can be easily controlled by the switching "on/off" of visible light. In addition, the visible-light-induced catalytic mechanism was elucidated in detail and, as the main reactive species h + and O 2 .- accounted for TMB oxidation. Based on the fact that fluorescein diacetate (FDA) possessed no activity and generated active fluorescein in situ in the presence of carboxylesterase (CaE), a signal-amplified sensing platform through a cascade reaction for CaE detection was constructed. Our proposed sensing system displayed excellent analytical performance for the detection of CaE in a wide linear range from 0.040 to 20 U L -1 with a low detection limit of 0.013 U L -1 . This work not only changes the conventional concept that fluorescein is generally considered to be photocatalytically inert, but also provides a novel sensing strategy by tailoring the enzyme mimetic activity of fluorescein derivatives with analyte. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Human Carboxylesterase 1 Stereoselectively Binds the Nerve Agent Cyclosarin and Spontaneously Hydrolyzes the Nerve Agent Sarin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hemmert, Andrew C.; Otto, Tamara C.; Wierdl, Monika

Organophosphorus (OP) nerve agents are potent toxins that inhibit cholinesterases and produce a rapid and lethal cholinergic crisis. Development of protein-based therapeutics is being pursued with the goal of preventing nerve agent toxicity and protecting against the long-term side effects of these agents. The drug-metabolizing enzyme human carboxylesterase 1 (hCE1) is a candidate protein-based therapeutic because of its similarity in structure and function to the cholinesterase targets of nerve agent poisoning. However, the ability of wild-type hCE1 to process the G-type nerve agents sarin and cyclosarin has not been determined. We report the crystal structure of hCE1 in complex withmore » the nerve agent cyclosarin. We further use stereoselective nerve agent analogs to establish that hCE1 exhibits a 1700- and 2900-fold preference for the P{sub R} enantiomers of analogs of soman and cyclosarin, respectively, and a 5-fold preference for the P{sub S} isomer of a sarin analog. Finally, we show that for enzyme inhibited by racemic mixtures of bona fide nerve agents, hCE1 spontaneously reactivates in the presence of sarin but not soman or cyclosarin. The addition of the neutral oxime 2,3-butanedione monoxime increases the rate of reactivation of hCE1 from sarin inhibition by more than 60-fold but has no effect on reactivation with the other agents examined. Taken together, these data demonstrate that hCE1 is only reactivated after inhibition with the more toxic P{sub S} isomer of sarin. These results provide important insights toward the long-term goal of designing novel forms of hCE1 to act as protein-based therapeutics for nerve agent detoxification.« less

Biochemical characterisation of MdCXE1, a carboxylesterase from apple that is expressed during fruit ripening.

PubMed

Souleyre, Edwige J F; Marshall, Sean D G; Oakeshott, John G; Russell, Robyn J; Plummer, Kim M; Newcomb, Richard D

2011-05-01

Esters are an important component of apple (Malus×domestica) flavour. Their biosynthesis increases in response to the ripening hormone ethylene, but their metabolism by carboxylesterases (CXEs) is poorly understood. We have identified 16 members of the CXE multigene family from the commercial apple cultivar, 'Royal Gala', that contain all the conserved features associated with CXE members of the α/β hydrolase fold superfamily. The expression of two genes, MdCXE1 and MdCXE16 was characterised in an apple fruit development series and in a transgenic line of 'Royal Gala' (AO3) that is unable to synthesise ethylene in fruit. In wild-type MdCXE1 is expressed at low levels during early stages of fruit development, rising to a peak of expression in apple fruit at harvest maturity. It is not significantly up-regulated by ethylene in the skin of AO3 fruit. MdCXE16 is expressed constitutively in wild-type throughout fruit development, and is up-regulated by ethylene in skin of AO3 fruit. Semi-purified recombinant MdCXE1 was able to hydrolyse a range of 4-methyl umbelliferyl ester substrates that included those containing acyl moieties that are found in esters produced by apple fruit. Kinetic characterisation of MdCXE1 revealed that the enzyme could be inhibited by organophosphates and that its ability to hydrolyse esters showed increasing affinity (K(m)) but decreasing turnover (k(cat)) as substrate acyl carbon length increases from C2 to C16. Our results suggest that MdCXE1 may have an impact on apple flavour through its ability to hydrolyse relevant flavour esters in ripe apple fruit. Copyright © 2011 Elsevier Ltd. All rights reserved.

Identification and Expression Patterns of Putative Diversified Carboxylesterases in the Tea Geometrid Ectropis obliqua Prout

PubMed Central

Sun, Liang; Wang, Qian; Wang, Qi; Zhang, Yuxing; Tang, Meijun; Guo, Huawei; Fu, Jianyu; Xiao, Qiang; Zhang, Yanan; Zhang, Yongjun

2017-01-01

Carboxylesterases (CXEs) belong to a family of metabolic enzymes. Some CXEs act as odorant-degrading enzymes (ODEs), which are reportedly highly expressed in insect olfactory organs and participate in the rapid deactivation of ester pheromone components and plant volatiles. The tea geometrid Ectropis obliqua Prout produces sex pheromones consisting of non-ester functional compounds but relies heavily on acetic ester plant volatiles to search for host plants and locate oviposition sites. However, studies characterizing putative candidate ODEs in this important tea plant pest are still relatively scarce. In the present study, we identified 35 candidate EoblCXE genes from E. obliqua chemosensory organs based on previously obtained transcriptomic data. The deduced amino acid sequences possessed the typical characteristics of the insect CXE family, including oxyanion hole residues, the Ser-Glu-His catalytic triad, and the Ser active included in the conserved pentapeptide characteristic of esterases, Gly-X-Ser-X-Gly. Phylogenetic analyses revealed that the EoblCXEs were diverse, belonging to several different insect esterase clades. Tissue- and sex-related expression patterns were studied via reverse-transcription and quantitative real-time polymerase chain reaction analyses (RT- and qRT-PCR). The results showed that 35 EoblCXE genes presented a diversified expression profile; among these, 12 EoblCXEs appeared to be antenna-biased, two EoblCXEs were non-chemosensory organ-biased, 12 EoblCXEs were ubiquitous, and nine EoblCXEs showed heterogeneous expression levels among different tissues. Intriguingly, two EoblCXE genes, EoblCXE7 and EoblCXE13, were not only strongly localized to antennal sensilla tuned to odorants, such as the sensilla trichodea (Str I and II) and sensilla basiconica (Sba), but were also expressed in the putative gustatory sensilla styloconica (Sst), indicating that these two CXEs might play multiple physiological roles in the E. obliqua chemosensory

An antennal carboxylesterase from Drosophila melanogaster, esterase 6, is a candidate odorant-degrading enzyme toward food odorants.

PubMed

Chertemps, Thomas; Younus, Faisal; Steiner, Claudia; Durand, Nicolas; Coppin, Chris W; Pandey, Gunjan; Oakeshott, John G; Maïbèche, Martine

2015-01-01

Reception of odorant molecules within insect olfactory organs involves several sequential steps, including their transport through the sensillar lymph, interaction with the respective sensory receptors, and subsequent inactivation. Odorant-degrading enzymes (ODEs) putatively play a role in signal dynamics by rapid degradation of odorants in the vicinity of the receptors, but this hypothesis is mainly supported by in vitro results. We have recently shown that an extracellular carboxylesterase, esterase-6 (EST-6), is involved in the physiological and behavioral dynamics of the response of Drosophila melanogaster to its volatile pheromone ester, cis-vaccenyl acetate. However, as the expression pattern of the Est-6 gene in the antennae is not restricted to the pheromone responding sensilla, we tested here if EST-6 could play a broader function in the antennae. We found that recombinant EST-6 is able to efficiently hydrolyse several volatile esters that would be emitted by its natural food in vitro. Electrophysiological comparisons of mutant Est-6 null flies and a control strain (on the same genetic background) showed that the dynamics of the antennal response to these compounds is influenced by EST-6, with the antennae of the null mutants showing prolonged activity in response to them. Antennal responses to the strongest odorant, pentyl acetate, were then studied in more detail, showing that the repolarization dynamics were modified even at low doses but without modification of the detection threshold. Behavioral choice experiments with pentyl acetate also showed differences between genotypes; attraction to this compound was observed at a lower dose among the null than control flies. As EST-6 is able to degrade various bioactive odorants emitted by food and plays a role in the response to these compounds, we hypothesize a role as an ODE for this enzyme toward food volatiles.

Interaction of Ferulic Acid with Glutathione S-Transferase and Carboxylesterase Genes in the Brown Planthopper, Nilaparvata lugens.

PubMed

Yang, Jun; Sun, Xiao-Qin; Yan, Shu-Ying; Pan, Wen-Jun; Zhang, Mao-Xin; Cai, Qing-Nian

2017-07-01

Plant phenolics are crucial defense phytochemicals against herbivores and glutathione S-transferase (GST) and carboxylesterase (CarE) in herbivorous insects are well-known detoxification enzymes for such xenobiotics. To understand relationship between a plant phenolic and herbivore GST or CarE genes, we evaluated the relationship between a rice phenolic ferulic acid and resistance to brown planthopper (BPH, Nilaparvata lugens), and investigated the interaction of ferulic acid with GST or CarE genes in BPH. The results indicate that ferulic acid content in tested rice varieties was highly associated with resistance to BPH. Bioassays using artificial diets show that the phenolic acid toxicity to BPH was dose dependent and the LC 25 and LC 50 were 5.81 and 23.30 μg/ml at 72 hr, respectively. Activities of the enzymes BPH GST and CarE were increased at concentrations below the LC 50 of ferulic acid. Moreover, low ferulic acid concentrations (< LC 25 ) upregulated the transcriptional levels of NlGSTD1 and NlGSTE1 of the GST family and NlCE of the CarE family. By using dsRNA-induced gene silencing (DIGS) of GST or CarE, it was shown that suppressed expression levels of NlGSTD1, NlGSTE1 and NlCE were 14.6%-21.2%, 27.8%-34.2%, and 10.5%-19.8%, respectively. Combination of NlGSTD1, NlGSTE1 or NlCE knockdown with ferulic acid increased nymph mortality by 92.9%, 119.9%, or 124.6%, respectively. These results suggest that depletion of detoxification genes in herbivorous insects by plant-mediated RNAi technology might be a new potential resource for improving rice resistance to BPH.

Molecular and Functional Characterization of cDNAs Putatively Encoding Carboxylesterases from the Migratory Locust, Locusta migratoria

PubMed Central

Zhang, Jianqin; Li, Daqi; Ge, Pingting; Guo, Yaping; Zhu, Kun Yan; Ma, Enbo; Zhang, Jianzhen

2014-01-01

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. PMID:24722667

Activation of a camptothecin prodrug by specific carboxylesterases as predicted by quantitative structure-activity relationship and molecular docking studies.

PubMed

Yoon, Kyoung Jin P; Krull, Erik J; Morton, Christopher L; Bornmann, William G; Lee, Richard E; Potter, Philip M; Danks, Mary K

2003-11-01

7-Ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (irinotecan, CPT-11) is a camptothecin prodrug that is metabolized by carboxylesterases (CE) to the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38), a topoisomerase I inhibitor. CPT-11 has shown encouraging antitumor activity against a broad spectrum of tumor types in early clinical trials, but hematopoietic and gastrointestinal toxicity limit its administration. To increase the therapeutic index of CPT-11 and to develop other prodrug analogues for enzyme/prodrug gene therapy applications, our laboratories propose to develop camptothecin prodrugs that will be activated by specific CEs. Specific analogues might then be predicted to be activated, for example, predominantly by human liver CE(hCE1), by human intestinal CE (hiCE), or in gene therapy approaches using a rabbit liver CE (rCE). This study describes a molecular modeling approach to relate the structure of rCE-activated camptothecin prodrugs with their biological activation. Comparative molecular field analysis, comparative molecular similarity index analysis, and docking studies were used to predict the biological activity of a 4-benzylpiperazine derivative of CPT-11 [7-ethyl-10-[4-(1-benzyl)-1-piperazino]carbonyloxycamptothecin (BP-CPT)] in U373MG glioma cell lines transfected with plasmids encoding rCE or hiCE. BP-CPT has been reported to be activated more efficiently than CPT-11 by a rat serum esterase activity; however, three-dimensional quantitative structure-activity relationship studies predicted that rCE would activate BP-CPT less efficiently than CPT-11. This was confirmed by both growth inhibition experiments and kinetic studies. The method is being used to design camptothecin prodrugs predicted to be activated by specific CEs.

Hydrolysis of a series of parabens by skin microsomes and cytosol from human and minipigs and in whole skin in short-term culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jewell, Christopher; Prusakiewicz, Jeffery J.; Ackermann, Chrisita

2007-12-01

Parabens are esters of 4-hydroxybenzoic acid and used as anti-microbial agents in a wide variety of toiletries, cosmetics and pharmaceuticals. It is of interest to understand the dermal absorption and hydrolysis of parabens, and to evaluate their disposition after dermal exposure and their potential to illicit localised toxicity. The use of minipig as a surrogate model for human dermal metabolism and toxicity studies, justifies the comparison of paraben metabolism in human and minipig skin. Parabens are hydrolysed by carboxylesterases to 4-hydroxybenzoic acid. The effects of the carboxylesterase inhibitors paraoxon and bis-nitrophenylphosphate provided evidence of the involvement of dermal carboxylesterases inmore » paraben hydrolysis. Loperamide, a specific inhibitor of human carboxylesterase-2 inhibited butyl- and benzylparaben hydrolysis in human skin but not methylparaben or ethylparaben. These results show that butyl- and benzylparaben are more selective substrates for human carboxylesterase-2 in skin than the other parabens examined. Parabens applied to the surface of human or minipig skin were absorbed to a similar amount and metabolised to 4-hydroxybenzoic acid during dermal absorption. These results demonstrate that the minipig is a suitable model for man for assessing dermal absorption and hydrolysis of parabens, although the carboxylesterase profile in skin differs between human and minipig.« less

EstA from Arthrobacter nitroguajacolicus Rü61a, a thermo- and solvent-tolerant carboxylesterase related to class C beta-lactamases.

PubMed

Schütte, Marcus; Fetzner, Susanne

2007-03-01

The estA gene encoding a novel cytoplasmic carboxylesterase from Arthrobacter nitroguajacolicus Rü61a was expressed in Escherichia coli. Sequence analysis and secondary structure predictions suggested that EstA belongs to the family VIII esterases, which are related to class C beta-lactamases. The S-x-x-K motif that in beta-lactamases contains the catalytic nucleophile, and a putative active-site tyrosine residue are conserved in EstA. The native molecular mass of hexahistidine-tagged (His6) EstA, purified by metal chelate affinity chromatography, was estimated to be 95 kDa by gel filtration, whereas the His6EstA peptide has a calculated molecular mass of 42.1 kDa. The enzyme catalyzes the hydrolysis of short-chain phenylacyl esters and triglycerides, and shows weak activity toward 2-hydroxy- and 2-nitroacetanilide. Its catalytic activity was inhibited by the serine-specific effector phenylmethylsulfonyl fluoride, and by Cd2+ and Hg2+ ions. Maximum activity of His6EstA was observed at a pH of 9.5 and a temperature of 50 degrees C to 60 degrees C. The enzyme was fairly thermostable. After 19 days at 50 degrees C and after 24 hours at 60 degrees C, its residual relative esterase activity toward phenylacetate was still 53% and 30%, respectively. Exposure of His6EstA to buffer-solvent mixtures showed that the enzyme was inactivated by several high log P (hydrophobic) solvents, whereas it showed remarkable stability and activity in up to 30% (by volume) of polar (low log P) organic solvents such as dimethylsulfoxide, methanol, acetonitrile, acetone, and propanol.

Structure of an Insecticide Sequestering Carboxylesterase from the Disease Vector Culex quinquefasciatus: What Makes an Enzyme a Good Insecticide Sponge?

PubMed

Hopkins, Davis H; Fraser, Nicholas J; Mabbitt, Peter D; Carr, Paul D; Oakeshott, John G; Jackson, Colin J

2017-10-17

Carboxylesterase (CBE)-mediated metabolic resistance to organophosphate and carbamate insecticides is a major problem for the control of insect disease vectors, such as the mosquito. The most common mechanism involves overexpression of CBEs that bind to the insecticide with high affinity, thereby sequestering them before they can interact with their target. However, the absence of any structure for an insecticide-sequestering CBE limits our understanding of the molecular basis for this process. We present the first structure of a CBE involved in sequestration, Cqestβ2 1 , from the mosquito disease vector Culex quinquefasciatus. Lysine methylation was used to obtain the crystal structure of Cqestβ2 1 , which adopts a canonical α/β-hydrolase fold that has high similarity to the target of organophosphate and carbamate insecticides, acetylcholinesterase. Sequence similarity networks of the insect carboxyl/cholinesterase family demonstrate that CBEs associated with metabolic insecticide resistance across many species share a level of similarity that distinguishes them from a variety of other classes. This is further emphasized by the structural similarities and differences in the binding pocket and active site residues of Cqestβ2 1 and other insect carboxyl/cholinesterases. Stopped-flow and steady-state inhibition studies support a major role for Cqestβ2 1 in organophosphate resistance and a minor role in carbamate resistance. Comparison with another isoform associated with insecticide resistance, Cqestβ1, showed both enzymes have similar affinity to insecticides, despite 16 amino acid differences between the two proteins. This provides a molecular understanding of pesticide sequestration by insect CBEs and could facilitate the design of CBE-specific inhibitors to circumvent this resistance mechanism in the future.

Organ-specific carboxylesterase profiling identifies the small intestine and kidney as major contributors of activation of the anticancer prodrug CPT-11

PubMed Central

Hatfield, M. Jason; Tsurkan, Lyudmila; Garrett, Michael; Shaver, Timothy M.; Hyatt, Janice L.; Edwards, Carol C.; Hicks, Latorya D.; Potter, Philip M.

2010-01-01

The activation of the anticancer prodrug CPT-11, to its active metabolite SN-38, is primarily mediated by carboxylesterases (CE). In humans, three CEs have been identified, of which human liver CE (hCE1; CES1) and human intestinal CE (hiCE; CES2) demonstrate significant ability to hydrolyze the drug. However, while the kinetic parameters of CPT-11 hydrolysis have been measured, the actual contribution of each enzyme to activate the drug in biological samples has not been addressed. Hence, we have used a combination of specific CE inhibition and conventional chromatographic techniques to determine the amounts, and hydrolytic activity, of CEs present within human liver, kidney, intestinal and lung specimens. These studies confirm that hiCE demonstrates the most efficient kinetic parameters for CPT-11 activation, however, due to the high levels of hCE1 that are expressed in liver, the latter enzyme can contribute up to 50% of the total of drug hydrolysis in this tissue. Conversely, in human duodenum, jejunum, ileum and kidney, where hCE1 expression is very low, greater than 99% of the conversion of CPT-11 to SN-38 was mediated by hiCE. Furthermore, analysis of lung microsomal extracts indicated that CPT-11 activation was more proficient in samples obtained from smokers. Overall, our studies demonstrate that hCE1 plays a significant role in CPT-11 hydrolysis even though it is up to 100-fold less efficient at drug activation than hiCE, and that drug activation in the intestine and kidney are likely major contributors to SN-38 production in vivo. PMID:20833148

Inhibition of carboxylesterases by benzil (diphenylethane-1,2-dione) and heterocyclic analogues is dependent upon the aromaticity of the ring and the flexibility of the dione moiety.

PubMed

Hyatt, Janice L; Stacy, Vanessa; Wadkins, Randy M; Yoon, Kyoung Jin P; Wierdl, Monika; Edwards, Carol C; Zeller, Matthias; Hunter, Allen D; Danks, Mary K; Crundwell, Guy; Potter, Philip M

2005-08-25

Benzil has been identified as a potent selective inhibitor of carboxylesterases (CEs). Essential components of the molecule required for inhibitory activity include the dione moiety and the benzene rings, and substitution within the rings affords increased selectivity toward CEs from different species. Replacement of the benzene rings with heterocyclic substituents increased the K(i) values for the compounds toward three mammalian CEs when using o-nitrophenyl acetate as a substrate. Logarithmic plots of the K(i) values versus the empirical resonance energy, the heat of union of formation energy, or the aromatic stabilization energy determined from molecular orbital calculations for the ring structures yielded linear relationships that allowed prediction of the efficacy of the diones toward CE inhibition. Using these data, we predicted that 2,2'-naphthil would be an excellent inhibitor of mammalian CEs. This was demonstrated to be correct with a K(i) value of 1 nM being observed for a rabbit liver CE. In addition, molecular simulations of the movement of the ring structures around the dione dihedral indicated that the ability of the compounds to inhibit CEs was due, in part, to rotational constraints enforced by the dione moiety. Overall, these studies identify subdomains within the aromatic ethane-1,2-diones, that are responsible for CE inhibition.

Substrate specificity of xenobiotic metabolizing esterases in the liver of two catfish species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jaiswal, R.G.; Huang, T.L.; Obih, P.O.

1994-12-31

The preliminary studies were conducted on the characterization of substrate specificity in the liver microsomes and cytosol of two catfish species, Ictalurus punctatus and Ictalurus natalie. A series of five esters of p-nitrophenol were used as calorimetric substrates to assay the carboxylesterases. The substrate specificity of liver microsomal and cytosolic carboxylesterases were remarkably different from each other. The valerate ester of p-nitrophenol was most rapidly hydrolyzed by the microsomal carboxylesterases, whereas the prioponate ester was the best substrate for cytosolic carboxylesterases. The Ictalurus natalie catfish species were obtained from the Devil Swamp site of the Mississippi River Basin which ismore » known to be heavily contaminated with toxic and hazardous industrial wastes. These results will be discussed in relation to the responses of xenobiotic metabolizing esterases to environmental pollutants and their possible use as biomarkers.« less

E4bp4 regulates carboxylesterase 2 enzymes through repression of the nuclear receptor Rev-erbα in mice.

PubMed

Zhao, Mengjing; Zhang, Tianpeng; Yu, Fangjun; Guo, Lianxia; Wu, Baojian

2018-06-01

Carboxylesterases (CES) are a family of phase I enzymes that play an important role in xenobiotic clearance and lipid metabolism. Here, we investigate a potential role of E4 promoter-binding protein 4 (E4bp4) in regulation of Ces and CPT-11 (irinotecan, a first-line drug for treating colorectal cancer) pharmacokinetics in mice. Mouse hepatoma Hepa-1c1c7 cells were transfected with Rev-erbα expression plasmid or siRNA targeting E4bp4. The relative mRNA and protein levels of Ces enzymes in the cells or the livers of wild-type and E4bp4-deficient (E4bp4 -/- ) mice were determined by qPCR and Western blotting, respectively. Transcriptional regulation of Ces by E4bp4/Rev-erbα were investigated using luciferase reporter, mobility shift, and co-immunoprecipitation (Co-IP) assays. Pharmacokinetic studies were performed with wild-type and E4bp4 -/- mice after intraperitoneal injection of CPT-11. E4bp4 ablation down-regulated an array of hepatic Ces genes in mice. E4bp4 -/- mice also showed reduced Ces-mediated metabolism and elevated systemic exposure of CPT-11, a well-known Ces substrate. Consistently, E4bp4 knockdown reduced the expression of Ces genes (Ces2b, Ces2e and Ces2f) in Hepa-1c1c7 cells. Furthermore, Rev-erbα repressed the transcription of Ces2b, whereas E4bp4 antagonized this repressive action. Co-IP experiment confirmed a direct interaction between E4bp4 and Rev-erbα. Through a combination of promoter analysis and mobility shift assays, we demonstrated that Rev-erbα trans-repressed Ces (Ces2b) through its specific binding to the -767 to-754 bp promoter region. In conclusion, E4bp4 regulates Ces enzymes through inhibition of the transrepression activity of Rev-erbα, thereby impacting the metabolism and pharmacokinetics of Ces substrates. Copyright © 2018 Elsevier Inc. All rights reserved.

PYRETHROID INSECTICIDES: ISOFORM-DEPENDENT HYDROLYSIS, INDUCTION OF CYTOCHROME P450 3A4 AND EVIDENCE ON THE INVOLVEMENT OF THE PREGNANE X RECEPTOR

PubMed Central

Yang, Dongfang; Wang, Xiliang; Chen, Yi-tzai; Deng, Ruitang; Yan, Bingfang

2009-01-01

Pyrethroids account for more than one-third of the insecticides currently marketed in the world. In mammals, these insecticides undergo extensive metabolism by carboxylesterases and cytochrome P450s (CYPs). In addition, some pyrethroids are found to induce the expression of CYPs. The aim of this study was to determine whether pyrethroids induce carboxylesterases and CYP3A4, and whether the induction is correlated inversely with their hydrolysis. Human liver microsomes were pooled and tested for the hydrolysis of 11 pyrethroids. All pyrethroids were hydrolyzed by the pooled microsomes, but the hydrolytic rates varied by as many as 14 fold. Some pyrethroids such as bioresmethrin were preferably hydrolyzed by carboxylesterase HCE1, whereas others such as bifenthrin preferably by HCE2. In primary human hepatocytes, all pyrethroids except tetramethrin significantly induced CYP3A4. In contrast, insignificant changes were detected on the expression of carboxylesterases. The induction of CYP3A4 was confirmed in multiple cell lines including HepG2, Hop92 and LS180. Overall, the magnitude of the induction was correlated inversely with the rates of hydrolysis, but positively with the activation of the pregnane X receptor (PXR). Transfection of a carboxylesterase markedly decreased the activation of PXR, and the decrease was in agreement with carboxylesterase-based preference for hydrolysis. In addition, human PXR variants as well as rat PXR differed from human PXR (wild-type) in responding to certain pyrethroids (e.g., lambda-cyhalothrin), suggesting that induction of PXR target genes by these pyrethroids varies depending on polymorphic variants and the PXR species identity. PMID:19249324

Pyrethroid insecticides: isoform-dependent hydrolysis, induction of cytochrome P450 3A4 and evidence on the involvement of the pregnane X receptor.

PubMed

Yang, Dongfang; Wang, Xiliang; Chen, Yi-Tzai; Deng, Ruitang; Yan, Bingfang

2009-05-15

Pyrethroids account for more than one-third of the insecticides currently marketed in the world. In mammals, these insecticides undergo extensive metabolism by carboxylesterases and cytochrome P450s (CYPs). In addition, some pyrethroids are found to induce the expression of CYPs. The aim of this study was to determine whether pyrethroids induce carboxylesterases and CYP3A4, and whether the induction is correlated inversely with their hydrolysis. Human liver microsomes were pooled and tested for the hydrolysis of 11 pyrethroids. All pyrethroids were hydrolyzed by the pooled microsomes, but the hydrolytic rates varied by as many as 14 fold. Some pyrethroids such as bioresmethrin were preferably hydrolyzed by carboxylesterase HCE1, whereas others such as bifenthrin preferably by HCE2. In primary human hepatocytes, all pyrethroids except tetramethrin significantly induced CYP3A4. In contrast, insignificant changes were detected on the expression of carboxylesterases. The induction of CYP3A4 was confirmed in multiple cell lines including HepG2, Hop92 and LS180. Overall, the magnitude of the induction was correlated inversely with the rates of hydrolysis, but positively with the activation of the pregnane X receptor (PXR). Transfection of a carboxylesterase markedly decreased the activation of PXR, and the decrease was in agreement with carboxylesterase-based preference for hydrolysis. In addition, human PXR variants as well as rat PXR differed from human PXR (wild-type) in responding to certain pyrethroids (e.g., lambda-cyhalothrin), suggesting that induction of PXR target genes by these pyrethroids varies depending on polymorphic variants and the PXR species identity.

Functional and immunohistochemical characterization of CCEae3a, a carboxylesterase associated with temephos resistance in the major arbovirus vectors Aedes aegypti and Ae. albopictus.

PubMed

Grigoraki, Linda; Balabanidou, Vassileia; Meristoudis, Christos; Myridakis, Antonis; Ranson, Hilary; Swevers, Luc; Vontas, John

2016-07-01

Temephos is a major organophosphate (OP) larvicide that has been used extensively for the control of Aedes albopictus and Aedes aegypti, the major vectors for viral diseases, such as dengue fever, zika and chikungunya. Resistance to temephos has been recently detected and associated with the upregulation of carboxylesterases (CCEs) through gene amplification, in both species. Here, we expressed the CCEae3a genes which showed the most striking up-regulation in resistant Aedes strains, using the baculovirus system. All CCEae3a variants encoded functional enzymes, with high activity and preference for p-nitrophenyl butyrate, a substrate that was shown capable to differentiate temephos resistant from susceptible Aedes larvae. Enzyme kinetic studies showed that CCEae3as from both Ae. aegypti and Ae. albopictus (CCEae3a_aeg and CCEae3a_alb, respectively) strongly interact with temephos oxon and slowly released the OP molecule, indicating a sequestration resistance mechanism. No difference was detected between resistant and susceptible CCEae3a_aeg variants (CCEae3a_aegR and CCEae3a_aegS, respectively), indicating that previously reported polymorphism is unlikely to play a role in temephos resistance. HPLC/MS showed that CCEae3as were able to metabolize temephos oxon to the temephos monoester [(4-hydroxyphenyl) sulfanyl] phenyl O,O-dimethylphosphorothioate. Western blot and immunolocalization studies, based on a specific antibody raised against the CCEae3a_alb showed that the enzyme is expressed at higher levels in resistant insects, primarily in malpighian tubules (MT) and nerve tissues. Copyright © 2016 Elsevier Ltd. All rights reserved.

Pyrethroid insecticides: Isoform-dependent hydrolysis, induction of cytochrome P450 3A4 and evidence on the involvement of the pregnane X receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang Dongfang; Wang Xiliang; Chen Yitzai

2009-05-15

Pyrethroids account for more than one-third of the insecticides currently marketed in the world. In mammals, these insecticides undergo extensive metabolism by carboxylesterases and cytochrome P450s (CYPs). In addition, some pyrethroids are found to induce the expression of CYPs. The aim of this study was to determine whether pyrethroids induce carboxylesterases and CYP3A4, and whether the induction is correlated inversely with their hydrolysis. Human liver microsomes were pooled and tested for the hydrolysis of 11 pyrethroids. All pyrethroids were hydrolyzed by the pooled microsomes, but the hydrolytic rates varied by as many as 14 fold. Some pyrethroids such as bioresmethrinmore » were preferably hydrolyzed by carboxylesterase HCE1, whereas others such as bifenthrin preferably by HCE2. In primary human hepatocytes, all pyrethroids except tetramethrin significantly induced CYP3A4. In contrast, insignificant changes were detected on the expression of carboxylesterases. The induction of CYP3A4 was confirmed in multiple cell lines including HepG2, Hop92 and LS180. Overall, the magnitude of the induction was correlated inversely with the rates of hydrolysis, but positively with the activation of the pregnane X receptor (PXR). Transfection of a carboxylesterase markedly decreased the activation of PXR, and the decrease was in agreement with carboxylesterase-based preference for hydrolysis. In addition, human PXR variants as well as rat PXR differed from human PXR (wild-type) in responding to certain pyrethroids (e.g., lambda-cyhalothrin), suggesting that induction of PXR target genes by these pyrethroids varies depending on polymorphic variants and the PXR species identity.« less

Prilocaine- and lidocaine-induced methemoglobinemia is caused by human carboxylesterase-, CYP2E1-, and CYP3A4-mediated metabolic activation.

PubMed

Higuchi, Ryota; Fukami, Tatsuki; Nakajima, Miki; Yokoi, Tsuyoshi

2013-06-01

Prilocaine and lidocaine are classified as amide-type local anesthetics for which serious adverse effects include methemoglobinemia. Although the hydrolyzed metabolites of prilocaine (o-toluidine) and lidocaine (2,6-xylidine) have been suspected to induce methemoglobinemia, the metabolic enzymes that are involved remain uncharacterized. In the present study, we aimed to identify the human enzymes that are responsible for prilocaine- and lidocaine-induced methemoglobinemia. Our experiments revealed that prilocaine was hydrolyzed by recombinant human carboxylesterase (CES) 1A and CES2, whereas lidocaine was hydrolyzed by only human CES1A. When the parent compounds (prilocaine and lidocaine) were incubated with human liver microsomes (HLM), methemoglobin (Met-Hb) formation was lower than when the hydrolyzed metabolites were incubated with HLM. In addition, Met-Hb formation when prilocaine and o-toluidine were incubated with HLM was higher than that when lidocaine and 2,6-xylidine were incubated with HLM. Incubation with diisopropyl fluorophosphate and bis-(4-nitrophenyl) phosphate, which are general inhibitors of CES, significantly decreased Met-Hb formation when prilocaine and lidocaine were incubated with HLM. An anti-CYP3A4 antibody further decreased the residual formation of Met-Hb. Met-Hb formation after the incubation of o-toluidine and 2,6-xylidine with HLM was only markedly decreased by incubation with an anti-CYP2E1 antibody. o-Toluidine and 2,6-xylidine were further metabolized by CYP2E1 to 4- and 6-hydroxy-o-toluidine and 4-hydroxy-2,6-xylidine, respectively, and these metabolites were shown to more efficiently induce Met-Hb formation than the parent compounds. Collectively, we found that the metabolites produced by human CES-, CYP2E1-, and CYP3A4-mediated metabolism were involved in prilocaine- and lidocaine-induced methemoglobinemia.

Resistance in cholinesterase activity after an acute and subchronic exposure to azinphos-methyl in the freshwater gastropod Biomphalaria straminea.

PubMed

Bianco, Karina; Otero, Sofía; Oliver, Agustina Balazote; Nahabedian, Daniel; Kristoff, Gisela

2014-11-01

Organophosphorous and carbamates insecticides are ones of the most popular classes of pesticides used in agriculture. Its success relies on their high acute toxicity and rapid environmental degradation. These insecticides inhibit cholinesterase and cause severe effects on aquatic non-target species, particularly in invertebrates. Since the properties of cholinesterases may differ between species, it is necessary to characterize them before their use as biomarkers. Also organophosphorous and carbamates inhibit carboxylesterases and the use of both enzymes for biomonitoring is suggested. Azinphos-methyl is an organophosphorous insecticide used in several parts of the word. In Argentina, it is the most applied insecticide in fruit production in the north Patagonian region. It was detected with the highest frequency in superficial and groundwater of the region. This work aims to evaluate the sensitivity of B. straminea cholinesterases and carboxylesterases to the OP azinphos-methyl including estimations of 48 h NOEC and IC50 of the pesticide and subchronic effects at environmentally relevant concentrations. These will allow us to evaluate the possibility of using cholinesterase and carboxylesterase of B. straminea as sensitive biomarkers. Previously a partial characterization of these enzymes will be performed. As in most invertebrates, acetylthiocholine was the preferred hydrolyzed substrate of B. straminea ChE, followed by propionylthiocholine and being butyrylthiocholine hydrolysis very low. Cholinesterase activity of B. straminea was significantly inhibited by the selective cholinesterases inhibitor (eserine) and by the selective inhibitor of mammalian acethylcholinesterase (BW284c51). In contrast, iso-OMPA, a specific inhibitor of butyrylcholinesterase, did not inhibit cholinesterase activity. These results suggest that cholinesterase activity in total soft tissue of B. straminea corresponds to acethylcholinesterase. Carboxylesterases activity was one order of

Juvenile Hormone Induction of Esterases: A Mechanism for the Regulation of Juvenile Hormone Titer

PubMed Central

Whitmore, Donald; Whitmore, Elaine; Gilbert, Lawrence I.

1972-01-01

Within a few hours after injection of juvenile hormone into Hyalophora gloveri pupae, several fast-migrating carboxylesterases (EC 3.1.1.1) that are sensitive to diisopropylfluorophosphate appear in the hemolymph. Treatment of the pupae with puromycin or actinomycin D prevents the appearance of these hemolymph enzymes, suggesting de novo synthesis of the carboxylesterases. Of the several other compounds investigated, only a potent mimic of the juvenile hormone is able to induce these enzymes. When the induced enzymes are incubated in vitro with 14C-labeled juvenile hormone, the hormone is rapidly and efficiently degraded. It is suggested that these induced carboxylesterases play an important role in the regulation of juvenile hormone titer. Images PMID:4504374

Individual variability in esterase activity and CYP1A levels in Chinook salmon (Oncorhynchus tshawytscha) exposed to esfenvalerate and chlorpyrifos

PubMed Central

Wheelock, Craig E.; Eder, Kai J.; Werner, Inge; Huang, Huazhang; Jones, Paul D.; Brammell, Benjamin F.; Elskus, Adria A.; Hammock, Bruce D.

2006-01-01

Acetylcholinesterase (AChE) activity has traditionally been monitored as a biomarker of organophosphate (OP) and/or carbamate exposure. However, AChE activity may not be the most sensitive endpoint for these agrochemicals, because OPs can cause adverse physiological effects at concentrations that do not affect AChE activity. Carboxylesterases are a related family of enzymes that have higher affinity than AChE for some OPs and carbamates and may be more sensitive indicators of environmental exposure to these pesticides. In this study, carboxylesterase and AChE activity, cytochrome P4501A (CYP1A) protein levels, and mortality were measured in individual juvenile Chinook salmon (Oncorhynchus tshawytscha) following exposure to an OP (chlorpyrifos) and a pyrethroid (esfenvalerate). As expected, high doses of chlorpyrifos and esfenvalerate were acutely toxic, with nominal concentrations (100 and 1 μg/l, respectively) causing 100% mortality within 96 h. Exposure to chlorpyrifos at a high dose (7.3 μg/l), but not a low dose (1.2 μg/l), significantly inhibited AChE activity in both brain and muscle tissue (85% and 92% inhibition, respectively), while esfenvalerate exposure had no effect. In contrast, liver carboxylesterase activity was significantly inhibited at both the low and high chlorpyrifos dose exposure (56% and 79% inhibition, respectively), while esfenvalerate exposure still had little effect. The inhibition of carboxylesterase activity at levels of chlorpyrifos that did not affect AChE activity suggests that some salmon carboxylesterase isozymes may be more sensitive than AChE to inhibition by OPs. CYP1A protein levels were ∼30% suppressed by chlorpyrifos exposure at the high dose, but esfenvalerate had no effect. Three teleost species, Chinook salmon, medaka (Oryzias latipes) and Sacramento splittail (Pogonichthys macrolepidotus), were examined for their ability to hydrolyze a series of pyrethroid surrogate substrates and in all cases hydrolysis activity was

Individual variability in esterase activity and CYP1A levels in Chinook salmon (Oncorhynchus tshawytscha) exposed to esfenvalerate and chlorpyrifos

USGS Publications Warehouse

Wheelock, C.E.; Eder, K.J.; Werner, I.; Huang, H.; Jones, P.D.; Brammell, B.F.; Elskus, A.A.; Hammock, B.D.

2005-01-01

Acetylcholinesterase (AChE) activity has traditionally been monitored as a biomarker of organophosphate (OP) and/or carbamate exposure. However, AChE activity may not be the most sensitive endpoint for these agrochemicals, because OPs can cause adverse physiological effects at concentrations that do not affect AChE activity. Carboxylesterases are a related family of enzymes that have higher affinity than AChE for some OPs and carbamates and may be more sensitive indicators of environmental exposure to these pesticides. In this study, carboxylesterase and AChE activity, cytochrome P4501A (CYP1A) protein levels, and mortality were measured in individual juvenile Chinook salmon (Oncorhynchus tshawytscha) following exposure to an OP (chlorpyrifos) and a pyrethroid (esfenvalerate). As expected, high doses of chlorpyrifos and esfenvalerate were acutely toxic, with nominal concentrations (100 and 1 ??g/l, respectively) causing 100% mortality within 96 h. Exposure to chlorpyrifos at a high dose (7.3 ??g/l), but not a low dose (1.2 ??g/l), significantly inhibited AChE activity in both brain and muscle tissue (85% and 92% inhibition, respectively), while esfenvalerate exposure had no effect. In contrast, liver carboxylesterase activity was significantly inhibited at both the low and high chlorpyrifos dose exposure (56% and 79% inhibition, respectively), while esfenvalerate exposure still had little effect. The inhibition of carboxylesterase activity at levels of chlorpyrifos that did not affect AChE activity suggests that some salmon carboxylesterase isozymes may be more sensitive than AChE to inhibition by OPs. CYP1A protein levels were ???30% suppressed by chlorpyrifos exposure at the high dose, but esfenvalerate had no effect. Three teleost species, Chinook salmon, medaka (Oryzias latipes) and Sacramento splittail (Pogonichthys macrolepidotus), were examined for their ability to hydrolyze a series of pyrethroid surrogate substrates and in all cases hydrolysis activity was

Response of digestive enzymes and esterases of ecotoxicological concern in earthworms exposed to chlorpyrifos-treated soils.

PubMed

Sanchez-Hernandez, Juan C; Ríos, Juan Manuel; Attademo, Andrés M

2018-03-01

Assessment of organophosphorus (OP) pesticide exposure in non-target organisms rarely involves non-neural molecular targets. Here we performed a 30-d microcosm experiment with Lumbricus terrestris to determine whether the activity of digestive enzymes (phosphatase, β-glucosidase, carboxylesterase and lipase) was sensitive to chlorpyrifos (5 mg kg -1 wet soil). Likewise, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in the wall muscle and gastrointestinal tissues as indicators of OP exposure. Chlorpyrifos inhibited the acid phosphatase (34% of controls), carboxylesterase (25.6%) and lipase activities (31%) in the gastrointestinal content. However, in the gastrointestinal tissue, only the carboxylesterase and lipase activities were significantly depressed (42-67% carboxylesterase inhibition in the foregut and crop/gizzard, and 15% lipase inhibition in the foregut). Chlorpyrifos inhibited the activity of both cholinesterases in the gastrointestinal tissues, whereas the AChE activity was affected in the wall muscle. These results suggested chlorpyrifos was widely distributed throughout the earthworm body after 30 d of incubation. Interestingly, we found muscle carboxylesterase activity strongly inhibited (92% of control) compared with that detected in the gastrointestinal tissues of the same OP-exposed individuals. This finding was explained by the occurrence of pesticide-resistant esterases in the gastrointestinal tissues, which were evidenced by zymography. Our results suggest that digestive processes of L. terrestris may be altered by chlorpyrifos, as a consequence of the inhibitory action of the insecticide on some digestive enzymes.

Multifactorial resistance to aminopeptidase inhibitor prodrug CHR2863 in myeloid leukemia cells: down-regulation of carboxylesterase 1, drug sequestration in lipid droplets and pro-survival activation ERK/Akt/mTOR

PubMed Central

Verbrugge, Sue Ellen; Al, Marjon; Assaraf, Yehuda G.; Kammerer, Sarah; Chandrupatla, Durga M.S.H.; Honeywell, Richard; Musters, Rene P.J.; Giovannetti, Elisa; O'Toole, Tom; Scheffer, George L.; Krige, David; de Gruijl, Tanja D.; Niessen, Hans W.M.; Lems, Willem F.; Kramer, Pieternella A.; Scheper, Rik J.; Cloos, Jacqueline; Ossenkoppele, Gert J.; Peters, Godefridus J.; Jansen, Gerrit

2016-01-01

Aminopeptidase inhibitors are receiving attention as combination chemotherapeutic agents for the treatment of refractory acute myeloid leukemia. However, the factors determining therapeutic efficacy remain elusive. Here we identified the molecular basis of acquired resistance to CHR2863, an orally available hydrophobic aminopeptidase inhibitor prodrug with an esterase-sensitive motif, in myeloid leukemia cells. CHR2863 enters cells by diffusion and is retained therein upon esterase activity-mediated conversion to its hydrophilic active metabolite drug CHR6768, thereby exerting amino acid depletion. Carboxylesterases (CES) serve as candidate prodrug activating enzymes given CES1 expression in acute myeloid leukemia specimens. We established two novel myeloid leukemia sublines U937/CHR2863(200) and U937/CHR2863(5uM), with low (14-fold) and high level (270-fold) CHR2863 resistance. The latter drug resistant cells displayed: (i) complete loss of CES1-mediated drug activation associated with down-regulation of CES1 mRNA and protein, (ii) marked retention/sequestration of the prodrug, (iii) a substantial increase in intracellular lipid droplets, and (iv) a dominant activation of the pro-survival Akt/mTOR pathway. Remarkably, the latter feature coincided with a gain of sensitivity to the mTOR inhibitor rapamycin. These finding delineate the molecular basis of CHR2863 resistance and offer a novel modality to overcome this drug resistance in myeloid leukemia cells. PMID:26496029

Isoflavones enhance pharmacokinetic exposure of active lovastatin acid via the upregulation of carboxylesterase in high-fat diet mice after oral administration of Xuezhikang capsules.

PubMed

Feng, Dong; Ge, Chun; Tan, Zhao-Yi; Sun, Jian-Guo; Xie, Yuan; Yao, Lan; Yan, Cai-Xia; Aa, Ji-Ye; Wang, Guang-Ji

2018-06-19

Xuezhikang capsule (XZK) is a traditional Chinese medicine that contains lovastatin (Lv) for hyperlipidemia treatment, although it has fewer side effects than Lv. However, the pharmacokinetic mechanisms contributing to its distinct efficacy and low side effects are unclear. Mice were fed a high-fat diet (HFD) for 6 weeks to induce hyperlipidemia. We first conducted the pharmacokinetic studies in HFD mice following oral administration of Lv (10 mg/kg, i.g.) and found that HFD remarkably decreased the active form of Lv (the lovastatin acid, LvA) exposure in the circulation system, especially in the targeting organ liver, with a declined conversion from Lv to LvA, whereas the Lv (responsible for myotoxicity) exposure in muscle markedly increased. Then we compared the pharmacokinetic profiles of Lv in HFD mice after the oral administration of XZK (1200 mg/kg, i.g.) or an equivalent dose of Lv (10 mg/kg, i.g.). A higher exposure of LvA and lower exposure of Lv were observed after XZK administration, suggesting a pharmacokinetic interaction of some ingredients in XZK. Further studies revealed that HFD promoted the inflammation and inhibited carboxylesterase (CES) activities in the intestine and the liver, thus contributing to the lower transformation of Lv into LvA. In contrast, XZK inhibited the inflammation and upregulated CES in the intestine and the liver. Finally, we evaluated the effects of monacolins and phytosterols, the fractional extracts of isoflavones, on inflammatory LS174T or HepG2 cells, which showed that isoflavones inhibited inflammation, upregulated CES, and markedly enhanced the conversion of Lv into LvA. For the first time, we provide evidence that isoflavones and Lv in XZK act in concert to enhance the efficacy and reduce the side effects of Lv.

Homology modeling and metabolism prediction of human carboxylesterase-2 using docking analyses by GriDock: a parallelized tool based on AutoDock 4.0

NASA Astrophysics Data System (ADS)

Vistoli, Giulio; Pedretti, Alessandro; Mazzolari, Angelica; Testa, Bernard

2010-09-01

Metabolic problems lead to numerous failures during clinical trials, and much effort is now devoted to developing in silico models predicting metabolic stability and metabolites. Such models are well known for cytochromes P450 and some transferases, whereas less has been done to predict the activity of human hydrolases. The present study was undertaken to develop a computational approach able to predict the hydrolysis of novel esters by human carboxylesterase hCES2. The study involved first a homology modeling of the hCES2 protein based on the model of hCES1 since the two proteins share a high degree of homology (≅73%). A set of 40 known substrates of hCES2 was taken from the literature; the ligands were docked in both their neutral and ionized forms using GriDock, a parallel tool based on the AutoDock4.0 engine which can perform efficient and easy virtual screening analyses of large molecular databases exploiting multi-core architectures. Useful statistical models (e.g., r 2 = 0.91 for substrates in their unprotonated state) were calculated by correlating experimental pKm values with distance between the carbon atom of the substrate's ester group and the hydroxy function of Ser228. Additional parameters in the equations accounted for hydrophobic and electrostatic interactions between substrates and contributing residues. The negatively charged residues in the hCES2 cavity explained the preference of the enzyme for neutral substrates and, more generally, suggested that ligands which interact too strongly by ionic bonds (e.g., ACE inhibitors) cannot be good CES2 substrates because they are trapped in the cavity in unproductive modes and behave as inhibitors. The effects of protonation on substrate recognition and the contrasting behavior of substrates and products were finally investigated by MD simulations of some CES2 complexes.

ONTOGENIC EXPRESSION OF HUMAN CARBOXYLESTERASE-2 AND CYTOCHROME P450 3A4 IN LIVER AND DUODENUM: POSTNATAL SURGE AND ORGAN-DEPENDENT REGULATION1

PubMed Central

Chen, Yi-Tzai; Trzoss, Lynnie; Yang, Dongfang; Yan, Bingfang

2015-01-01

Human carboxylesterase-2 (CES2) and cytochrome P450 3A4 (CYP3A4) are two major drug metabolizing enzymes that play critical roles in hydrolytic and oxidative biotransformation, respectively. They share substrates but may have opposite effect on therapeutic potential such as the metabolism of the anticancer prodrug irinotecan. Both CES2 and CYP3A4 are expressed in the liver and the gastrointestinal tract. This study was conducted to determine whether CES2 and CYP3A4 are expressed under developmental regulation and whether the regulation occurs differentially between the liver and duodenum. A large number of tissues (112) were collected with majority of them from donors at 1-198 days of age. In addition, multi-sampling (liver, duodenum and jejunum) was performed in some donors. The expression was determined at mRNA and protein levels. In the liver, CES2 and CYP3A4 mRNA exhibited a postnatal surge (1 versus 2 months of age) by 2.7 and 29 fold, respectively. CYP3A4 but not CES2 mRNA in certain pediatric groups reached or even exceeded the adult level. The duodenal samples, on the other hand, showed a gene-specific expression pattern at mRNA level. CES2 mRNA increased with age but the opposite was true with CYP3A4 mRNA. The levels of CES2 and CYP3A4 protein, on the other hand, increased with age in both liver and duodenum. The multi-sampling study demonstrated significant correlation of CES2 expression between the duodenum and jejunum. However, neither duodenal nor jejunal expression correlated with hepatic expression of CES2. These findings establish that developmental regulation occurs in a gene and organ-dependent manner. PMID:25724353

Pharmacology of irinotecan.

PubMed

Kuhn, J G

1998-08-01

Irinotecan (CPT-11 [Camptosar]), a semisynthetic derivative of the plant alkaloid camptothecin, is bioactivated by carboxylesterases (EC3.1.1-) to the topoisomerase I inhibitor SN-38, a minor metabolite. Bioactivation of intravenously administered irinotecan by carboxylesterases occurs predominantly in the liver. Two human carboxylesterase isoforms responsible for SN-38 formation have been characterized. At relevant hepatic irinotecan concentrations up to 12 micrograms/mL, a low-Km isoform is responsible for irinotecan bioactivation. High concentrations of drugs commonly coadministered with irinotecan do not inhibit carboxylesterase activity. Intestinal carboxylesterases can also generate SN-38, followed by subsequent oral absorption. A second major polar metabolite of irinotecan, aminopentanecarboxylic acid (APC), is the product of CYP3A4-mediated oxidation of the terminal piperidine ring. APC is 100-fold less active than SN-38 as a topoisomerase I inhibitor and is a relatively weak inhibitor of acetylcholinesterase. SN-38 is eliminated mainly through conjugation by hepatic uridine glucuronosyltransferase (UGT*1.1), the same isoezyme responsible for glucuronidation of bilirubin. Grade 4 irinotecan-related toxicity (ie, neutropenia, diarrhea) has recently been reported in two patients with deficient UGT*1.1 activity. SN-38 glucuronide (SN-38G), which has only 1/100th the antitumor activity of SN-38, is actively secreted into the bile by a canalicular multispecific organic anion transporter. Deconjugation of SN-38G to SN-38 by beta-glucuronidase produced by the intestinal flora may contribute to enterohepatic recirculation of SN-38 and delayed intestinal toxicity.

Characterization of deltamethrin metabolism by rat plasma and liver microsomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anand, Sathanandam S.; Bruckner, James V.; Haines, Wendy T.

2006-04-15

Deltamethrin, a widely used type II pyrethroid insecticide, is a relatively potent neurotoxicant. While the toxicity has been extensively examined, toxicokinetic studies of deltamethrin and most other pyrethroids are very limited. The aims of this study were to identify, characterize, and assess the relative contributions of esterases and cytochrome P450s (CYP450s) responsible for deltamethrin metabolism by measuring deltamethrin disappearance following incubation of various concentrations (2 to 400 {mu}M) in plasma (esterases) and liver microsomes (esterases and CYP450s) prepared from adult male rats. While the carboxylesterase metabolism in plasma and liver was characterized using an inhibitor, tetra isopropyl pyrophosphoramide (isoOMPA), CYP450more » metabolism was characterized using the cofactor, NADPH. Michaelis-Menten rate constants were calculated using linear and nonlinear regression as applicable. The metabolic efficiency of these pathways was estimated by calculating intrinsic clearance (Vmax/Km). In plasma, isoOMPA completely inhibited deltamethrin biotransformation at concentrations (2 and 20 {mu}M of deltamethrin) that are 2- to 10-fold higher than previously reported peak blood levels in deltamethrin-poisoned rats. For carboxylesterase-mediated deltamethrin metabolism in plasma, Vmax = 325.3 {+-} 53.4 nmol/h/ml and Km = 165.4 {+-} 41.9 {mu}M. Calcium chelation by EGTA did not inhibit deltamethrin metabolism in plasma or liver microsomes, indicating that A-esterases do not metabolize deltamethrin. In liver microsomes, esterase-mediated deltamethrin metabolism was completely inhibited by isoOMPA, confirming the role of carboxylesterases. The rate constants for liver carboxylesterases were Vmax = 1981.8 {+-} 132.3 nmol/h/g liver and Km = 172.5 {+-} 22.5 {mu}M. Liver microsomal CYP450-mediated biotransformation of deltamethrin was a higher capacity (Vmax = 2611.3 {+-} 134.1 nmol/h/g liver) and higher affinity (Km = 74.9 {+-} 5.9 {mu}M) process than

Experimental Measurements for the Effect of Dilution Procedure in Blood Esterases as Animals Biomarker for Exposure to OP Compounds

PubMed Central

Abass, Kasim Sakran

2014-01-01

Organophosphate compounds can bind to carboxylesterase, which may lower the concentration of organophosphate pesticides at the target site enzyme, cholinesterase. It is unclear from the literature whether it is the carboxylesterase affinity for the organophosphate and/or the number of carboxylesterase molecules that is the dominant factor in determining the protective potential of carboxylesterase. The fundamental dilutions and kinetic effects of esterase enzyme are still poorly understood. This study aims to confirm and extend our current knowledge about the effects of dilutions on esterases activities in the blood for birds with respect to protecting the enzyme from organophosphate inhibition. There was significantly higher esterases activities in dilution 1 : 10 in the all blood samples from quail, duck, and chick compared to other dilutions (1 : 5, 1 : 15, 1 : 20, and 1 : 25) in all cases. Furthermore, our results also pointed to the importance of estimating different dilutions effects prior to using in birds as biomarker tools of environmental exposure. Concentration-inhibition curves were determined for the inhibitor in the presence of dilutions 1 : 5, 1 : 10, plus 1 : 15 (to stimulate carboxylesterase). Point estimates (concentrations calculated to produce 20, 50, and 80% inhibition) were compared across conditions and served as a measure of esterase-mediated detoxification. Results with well-known inhibitors (malathion) were in agreement with the literature, serving to support the use of this assay. Among the thiol-esters dilution 1 : 5 was observed to have the highest specificity constant (k cat/K m), and the K m and k cat values were 176 μM and 16,765 s−1, respectively, for S-phenyl thioacetate ester, while detected in dilution 1 : 15 was the lowest specificity constant (k cat/K m), and the K m and k cat values were 943 μM and 1154 s−1, respectively, for acetylthiocholine iodide ester. PMID:24864243

Molecular Basis of Valine-Citrulline-PABC Linker Instability in Site-Specific ADCs and Its Mitigation by Linker Design.

PubMed

Dorywalska, Magdalena; Dushin, Russell; Moine, Ludivine; Farias, Santiago E; Zhou, Dahui; Navaratnam, Thayalan; Lui, Victor; Hasa-Moreno, Adela; Casas, Meritxell Galindo; Tran, Thomas-Toan; Delaria, Kathy; Liu, Shu-Hui; Foletti, Davide; O'Donnell, Christopher J; Pons, Jaume; Shelton, David L; Rajpal, Arvind; Strop, Pavel

2016-05-01

The degree of stability of antibody-drug linkers in systemic circulation, and the rate of their intracellular processing within target cancer cells are among the key factors determining the efficacy of antibody-drug conjugates (ADC) in vivo Previous studies demonstrated the susceptibility of cleavable linkers, as well as auristatin-based payloads, to enzymatic cleavage in rodent plasma. Here, we identify Carboxylesterase 1C as the enzyme responsible for the extracellular hydrolysis of valine-citrulline-p-aminocarbamate (VC-PABC)-based linkers in mouse plasma. We further show that the activity of Carboxylesterase 1C towards VC-PABC-based linkers, and consequently the stability of ADCs in mouse plasma, can be effectively modulated by small chemical modifications to the linker. While the introduced modifications can protect the VC-PABC-based linkers from extracellular cleavage, they do not significantly alter the intracellular linker processing by the lysosomal protease Cathepsin B. The distinct substrate preference of the serum Carboxylesterase 1C offers the opportunity to modulate the extracellular stability of cleavable ADCs without diminishing the intracellular payload release required for ADC efficacy. Mol Cancer Ther; 15(5); 958-70. ©2016 AACR. ©2016 American Association for Cancer Research.

Influence of polychlorinated aromatic compounds on the biotransformation and toxicity of organophosphorus pesticides (OP) to the Daphnia magna

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tonkopii, V.; Zagrebin, A.; Sherstneva, L.

1995-12-31

The effect of different polychlorinated aromatics (DDT, Aroclor 1254, certain polychlorinated biphenyls and dibenzofurans) on the toxicity of OP (DDVP paraoxon, malaoxon) to Daphnia magna was studied. Pretreatment of daphnids with chlorinated compounds during 72 hours in nontoxic concentrations (1/5--1/20 CL{sub 50}) has been shown to reduce the toxicity of OP for hydrobionts. For study of influence of chlorinated compounds on biotransformation of OP the activity of enzymes which are hydrolyzing the OP was investigated in Daphnia`s homogenates or microsomes. The activity of carboxylesterase (tributyrinase, aliesterase) and arylesterase (phosphorylphosphatase) with usage as substrates accordingly {alpha}-naphthylacetate and paraoxon was measured. Besidesmore » that the activity of cholinesterase with application of propionylthiocholine as substrate was determined. After polychlorinated aromatic compounds treatment of daphnids activities of both aryl-and carboxylesterase increased markedly. It decreased the inhibition of Daphnia`s cholinesterase caused by incubation with OP in concentrations 0.5--1.0 CL{sub 50}. Thus the induction by chlorinate aromatics of OP metabolizing enzymes seems to play the important role in reduction of OP toxicity to Daphnia magna. Perhaps the aryl- and carboxylesterase of Daphnia can be used as biomarkers of pollution by polychlorinated aromatics in water.« less

Interactive toxicity of chlorpyrifos and parathion in neonatal rats: Role of esterases in exposure sequence-dependent toxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kacham, R.; Karanth, S.; Baireddy, P.

2006-01-15

We previously reported that sequence of exposure to chlorpyrifos and parathion in adult rats can markedly influence toxic outcome. In the present study, we evaluated the interactive toxicity of chlorpyrifos (8 mg/kg, po) and parathion (0.5 mg/kg, po) in neonatal (7 days old) rats. Rats were exposed to the insecticides either concurrently or sequentially (separated by 4 h) and sacrificed at 4, 8, and 24 h after the first exposure for biochemical measurements (cholinesterase activity in brain, plasma, and diaphragm and carboxylesterase activity in plasma and liver). The concurrently-exposed group showed more cumulative lethality (15/24) than either of the sequentialmore » dosing groups. With sequential dosing, rats treated initially with chlorpyrifos prior to parathion (C/P) exhibited higher lethality (7/23) compared to those treated with parathion before chlorpyrifos (P/C; 1/24). At 8 h after initial dosing, brain cholinesterase inhibition was significantly greater in the C/P group (59%) compared to the P/C group (28%). Diaphragm and plasma cholinesterase activity also followed a relatively similar pattern of inhibition. Carboxylesterase inhibition in plasma and liver was relatively similar among the treatment groups across time-points. Similar sequence-dependent differences in brain cholinesterase inhibition were also noted with lower binary exposures to chlorpyrifos (2 mg/kg) and parathion (0.35 mg/kg). In vitro and ex vivo studies compared relative oxon detoxification of carboxylesterases (calcium-insensitive) and A-esterases (calcium-sensitive) in liver homogenates from untreated and insecticide pretreated rats. Using tissues from untreated rats, carboxylesterases detoxified both chlorpyrifos oxon and paraoxon, while A-esterases only detoxified chlorpyrifos oxon. With parathion pretreatment, A-esterases still detoxified chlorpyrifos oxon while liver from chlorpyrifos pretreated rats had little apparent effect on paraoxon. We conclude that while neonatal rats

Esterase detoxification of acetylcholinesterase inhibitors by human or rat liver in vitro

EPA Science Inventory

Organophosphate (OP) and N-methylcarbamate pesticides inhibit acetylcholinesterase (AChE), but differences in metabolism and detoxification can influence potency of these pesticides across and within species. Carboxylesterase (CaE) and A-esterase (paraoxonase, PON) are considered...

Esterase detoxification of acetylcholinesterase inhibitors using human liver samples in vitro

EPA Science Inventory

Organophosphate (OP) and N-methylcarbamate pesticides inhibit acetylcholinesterase (AChE), but differences in metabolism and detoxification can influence potency of these pesticides across and within species. Carboxylesterase (CaE) and A-esterase (paraoxonase, PON1) are consider...

Correlation between Pesticide Resistance and Enzyme Activity in the Diamondback Moth, Plutella xylostella

PubMed Central

Gong, Ya-Jun; Wang, Ze-Hua; Shi, Bao-Cai; Kang, Zong-Jiang; Zhu, Liang; Jin, Gui-Hua; Weig, Shu-Jun

2013-01-01

The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is one of the most important pests that has developed high pesticide resistance. The resistances of five Chinese populations of this moth, four resistant strains (from Beijing, Henan, Fujian, and Guangdong) and one susceptible strain, to five pesticides were determined, and the activities of carboxylesterase, glutathione S-transferase, and acetylcholine esterase were tested in all five populations. The correlations between pesticide resistance and enzyme activity were analyzed. The results showed that the resistance status to the five pesticides was different among the five populations. The resistance ratios of the Beijing and Henan populations to spinosad were 5.84 and 8.22, respectively, and those to beta-cypermethrin were 4.91 and 4.98, respectively. These ratios were higher than those for the Fujian and Guangdong populations. The Fujian population was more sensitive to abamectin and chlorpyrifos than the susceptible population (the resistance ratios were 0.14 and 0.91, respectively); in fact, the median lethal concentration for P. xylostella was significantly higher for chlorpyrifos than that for any of the other four pesticides. The carboxylesterase activity in P. xylostella showed positive correlations with the resistance to spinosad, beta-cypermethrin, chlorpyrifos, and abamectin, but no correlation was observed between the carboxylesterase activity and resistance to emamectin benzoate, between glutathione S-transferase activity and resistance to any of the five pesticides tested, or between acetylcholine esterase activity and any of the pesticides except for emamectin benzoate. PMID:24766444

Determination of Human Hepatic CYP2C8 and CYP1A2 Age-Dependent Expression to Support Human Health Risk Assessment for Early Ages

EPA Science Inventory

Predicting age-specific metabolism is important for evaluating age-related drug and chemical sensitivity. Multiple cytochrome P450s and carboxylesterase enzymes are responsible for human pyrethroid metabolism. Complete ontogeny data for each enzyme are needed to support in vitro ...

Metabolism of aspirin and procaine in mice pretreated with O-4-nitrophenyl methyl(phenyl)phosphinate or O-4-nitrophenyl diphenylphosphinate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Joly, J.M.; Brown, T.M.

Concentrations of (carboxyl-/sup 14/C)procaine in blood of mice were increased threefold for 27 min by exposure to O-4-nitrophenyl diphenylphosphinate 2 hr prior to (carboxyl-/sup 14/C)procaine injection ip, while there was no effect of O-4-nitrophenyl methyl(phenyl)phosphinate pretreatment. There was no effect of either organophosphinate on the primary hydrolysis of (acetyl-l-/sup 14/C)aspirin when assessed by the expiration of (/sup 14/C)carbon dioxide; however, O-4-nitrophenyl diphenylphosphinate pretreatment produced transient increases in blood concentrations of both (carboxyl-/sup 14/C)aspirin and (carboxyl-/sup 14/C)salicylic acid following administration of (carboxyl-/sup 14/C)aspirin. Liver carboxylesterase activity in O-4-nitrophenyl diphenylphosphinate pretreated mice was 11% of control activity. These results indicate the potentialmore » for drug interaction with O-4-nitrophenyl diphenylphosphinate but not with O-4-nitrophenyl methyl(phenyl)phosphinate. It appears that liver carboxylesterase activity has a minor role in hydrolysis of aspirin in vivo, but may be more important in procaine metabolism.« less

Insecticide resistance of Anopheles sinensis and An. vagus in Hainan Island, a malaria-endemic area of China.

PubMed

Qin, Qian; Li, Yiji; Zhong, Daibin; Zhou, Ning; Chang, Xuelian; Li, Chunyuan; Cui, Liwang; Yan, Guiyun; Chen, Xiao-Guang

2014-03-03

Malaria is one of the most important public health problems in Southeast Asia, including Hainan Island, China. Vector control is the main malaria control measure, and insecticide resistance is a major concern for the effectiveness of chemical insecticide control programs. The objective of this study is to determine the resistance status of the main malaria vector species to pyrethroids and other insecticides recommended by the World Health Organization (WHO) for indoor residual sprays. The larvae and pupae of Anopheles mosquitoes were sampled from multiple sites in Hainan Island, and five sites yielded sufficient mosquitoes for insecticide susceptibility bioassays. Bioassays of female adult mosquitoes three days after emergence were conducted in the two most abundant species, Anopheles sinensis and An. vagus, using three insecticides (0.05% deltamethrin, 4% DDT, and 5% malathion) and following the WHO standard tube assay procedure. P450 monooxygenase, glutathione S-transferase and carboxylesterase activities were measured. Mutations at the knockdown resistance (kdr) gene and the ace-1 gene were detected by DNA sequencing and PCR-RFLP analysis, respectively. An. sinensis and An. vagus were the predominant Anopheles mosquito species. An. sinensis was found to be resistant to DDT and deltamethrin. An. vagus was susceptible to deltamethrin but resistant to DDT and malathion. Low kdr mutation (L1014F) frequency (<10%) was detected in An. sinensis, but no kdr mutation was detected in An. vagus populations. Modest to high (45%-75%) ace-1 mutation frequency was found in An. sinensis populations, but no ace-1 mutation was detected in An. vagus populations. Significantly higher P450 monooxygenase and carboxylesterase activities were detected in deltamethrin-resistant An. sinensis, and significantly higher P450 monooxygenase, glutathione S-transferase and carboxylesterase activities were found in malathion-resistant An. vagus mosquitoes. Multiple insecticide resistance was

Identification and Expression Profile of Multiple Genes in Response to Magnesium Exposure in Culex quinquefasciatus Larvae

DTIC Science & Technology

2010-11-01

Vol. 47, no. 6 Potter and Wadkins 2006; Cui et al. 2007a, 2007b; Cummins et al. 2007; Holmes et al. 2009). One study showed that the activities of...carboxylesterases in re- sponse to three insecticides (i.e.,malathion, propoxur, and permethrin) differed by mosquito species: high activities of...al. 2004). Chitotrio- sidase secreted by activated human macrophages is involved in the defense against chitin-containing pathogens such as fungi

Oxime-Induced Reactivation of Carboxylesterase Inhibited by Organophosphorus Compounds

DTIC Science & Technology

1993-05-13

detoxication enzyme for OP compounds (Maxwell, 1992a), when in the presence of an uncharged oxime, becomes even more effective because it is easily...Wolring, 1984). Therefore, oxime-induced reactivation of OP-inhibited CaE for protection by enhancement of OP detoxication occurs at approximately the

Genetic variation associated with increased insecticide resistance in the malaria mosquito, Anopheles coluzzii.

PubMed

Main, Bradley J; Everitt, Amanda; Cornel, Anthony J; Hormozdiari, Fereydoun; Lanzaro, Gregory C

2018-04-04

Malaria mortality rates in sub-Saharan Africa have declined significantly in recent years as a result of increased insecticide-treated bed net (ITN) usage. A major challenge to further progress is the emergence and spread of insecticide resistance alleles in the Anopheles mosquito vectors, like An. coluzzii. A non-synonymous mutation in the para voltage-gated sodium channel gene reduces pyrethroid-binding affinity, resulting in knockdown resistance (kdr). Metabolic mechanisms of insecticide resistance involving detoxification genes like cytochrome P450 genes, carboxylesterases, and glutathione S-transferases are also important. As some gene activity is tissue-specific and/or environmentally induced, gene regulatory variation may be overlooked when comparing expression from whole mosquito bodies under standard rearing conditions. We detected complex insecticide resistance in a 2014 An. coluzzii colony from southern Mali using bottle bioassays. Additional bioassays involving recombinant genotypes from a cross with a relatively susceptible 1995 An. coluzzii colony from Mali confirmed the importance of kdr and associated increased permethrin resistance to the CYP9K1 locus on the X chromosome. Significant differential expression of CYP9K1 was not observed among these colonies in Malpighian tubules. However, the P450 gene CYP6Z1 was overexpressed in resistant individuals following sublethal permethrin exposure and the carboxylesterase gene COEAE5G was constitutively overexpressed. The significant P450-related insecticide resistance observed in the 2014 An. coluzzii colony indicates that ITNs treated with the P450 inhibitor piperonyl butoxide (PBO) would be more effective in this region. The known insecticide resistance gene CYP6Z1 was differentially expressed exclusively in the context of sublethal permethrin exposure, highlighting the importance of tissue-specificity and environmental conditions in gene expression studies. The increased activity of the carboxylesterase

In Vitro Metabolism of the Brominated Flame Retardants 2-Ethylhexyl-2,3,4,5-Tetrabromobenzoate (TBB) and Bis(2-Ethylhexyl) 2,3,4,5-Tetrabromophthalate (TBPH) in Human and Rat Tissues

PubMed Central

Roberts, Simon C.; Macaulay, Laura J.; Stapleton, Heather M.

2012-01-01

Due to the phaseout of polybrominated diphenyl ether (PBDE) flame retardants, new chemicals, such as 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (TBB) and bis(2-ethylhexyl) 2,3,4,5-tetrabromophthalate (TBPH), have been used as replacements in some commercial flame retardant mixtures. Both chemicals have been detected in indoor dust at concentrations approaching the concentrations of PBDEs; however, little is known about their fate, metabolism, or toxicity. The goal of this study was to investigate the potential metabolism of these two brominated flame retardants in human and rat tissues by conducting in vitro experiments with liver and intestinal subcellular fractions. In all the experiments, TBB was consistently metabolized to 2,3,4,5-tetrabromobenzoic acid (TBBA) via cleavage of the 2-ethylhexyl chain without requiring any added cofactors. TBBA was also formed in purified porcine carboxylesterase, but at a much faster rate of 6.29 ± 0.58 nmol min-1 mg protein-1. The estimated Km and Vmax values for TBB metabolism in human microsomes were 11.1 ± 3.9 μM and 0.644 ± 0.144 nmol min-1 mg protein-1, respectively. A similar Km of 9.3 ± 2.2 μM was calculated for porcine carboxylesterase, indicating similar enzyme specificity. While the rapid formation of TBBA may reduce the bioaccumulation potential of TBB in mammals and may be useful as a biomarker of TBB exposure, the toxicity of this brominated benzoic acid is unknown and may be a concern based on its structural similarity to other toxic pollutants. In contrast to TBB, no metabolites of TBPH were detected in human or rat subcellular fractions. However, a metabolic product of TBPH, mono(2-ethylhexyl) tetrabromophthalate (TBMEHP), was formed in purified porcine carboxylesterase at an approximate rate of 1.08 pmol min-1 mg protein-1. No Phase II metabolites of TBBA or TBMEHP were observed. More research is needed to understand the in vivo toxicokinetics and health effects of these compounds given their current

The Activity of Cholinesterases in Diapausing and Flying Red Mason Bees Osmia bicornis (Megachilidae).

PubMed

Dmochowska-Slezak, Kamila; Zaobidna, Ewa; Domeracka, Joanna; Swiatkowska, Marta; Rusznica, Małgorzata; Zółtowska, Krystyna

2015-01-01

The red mason bee (Osmia bicornis) is a highly effective pollinator that is exposed to various xenobiotics. The organism's potential resistance to the toxic effects of xenobiotics can be determined based on cholinesterase activity. The activity of cholinesterases (ChEs) towards acetylcholine (ACh) and butyrylcholine (BCh) was determined in extracts of diapausing (between October and late March) and flying bees (May). In both males and females, enzyme activity was higher towards ACh than towards BCh. The ratio of ACh/BCh activity was determined in the range of 1.43 to 4.15 in diapausing females and 3.00 to 7.18 in diapausing males. No significant changes in ChE activity towards ACh were observed in females before December and in males before February. Enzyme activity towards ACh increased dynamically in the second half of March. Enzyme activity towards BCh remained stable in both sexes until mid-March, after which it increased significantly. Excluding mid-March, enzyme BCh activity was significantly higher in females than in males. The activity of carboxylesterase towards 4-p-nitrophenyl butyrate was determined in females to assess the involvement of non-specific esterases in the hydrolysis of choline esters. Carboxylesterase activity was low in comparison with cholinesterase activity, and it remained practically unchanged throughout diapause, suggesting that choline esters in female O. bicornis extracts were hydrolyzed mainly by acetylcholinesterases.

Inhibition of Xenobiotic-Degrading Hydrolases by Organophosphinates

DTIC Science & Technology

1986-07-01

TMB-4 doubled the rates of recovery. Concentrations of Lcarboxyl- CIprocaine in blood of mice were increased three-fold for 27 m I" exposure to 0-4...enzyme was found to have recovered 45.7% of its activity 24 h 35 after exposure to 4.87 x 10- 4 M EPP (Table 10). Neither rabbit liver carboxylesterase...case of a competitive mechanism of inhibition. It is possible that IPP and DPP were competitive inhibitors acting by occupation of the active site of

Fluorescein diacetate (FDA) and its analogue as substrates for Pi-class glutathione S-transferase (GSTP1) and their biological application.

PubMed

Fujikawa, Yuuta; Nampo, Taiki; Mori, Masaya; Kikkawa, Manami; Inoue, Hideshi

2018-03-01

Pi class glutathione S-transferase (GSTP1) is highly expressed in various cancerous cells and pre-neoplastic legions, where it is involved in apoptotic resistance or metabolism of several anti-tumour chemotherapeutics. Therefore, GSTP1 is a marker of malignant and pre-malignant cells and is a promising target for visualization and drug development. Here we demonstrate that fluorescein diacetate (FDA), a fluorescent probe used for vital staining, is a fluorescently activated by esterolytic activity of human GSTP1 (hGSTP1) selectively among various cytosolic GSTs. Fluorescence activation of FDA susceptible to GST inhibitors was observed in MCF7 cells exogenously overexpressing hGSTP1, but not in cells overexpressing hGSTA1 or hGSTM1. Inhibitor-sensitive fluorescence activation was also observed in several cancer cell lines endogenously expressing GSTP1, suggesting that GSTP1 is involved in FDA esterolysis in these cells. Among the FDA derivatives examined, FOMe-Ac, the acetyl ester of fluorescein O-methyl ether, was found to be a potential reporter for GSH-dependent GSTP1 activity as well as for carboxylesterase activity. Since GSTP1 is highly expressed in various types of cancer cells compared to their normal counterparts, improving the fluorogenic substrates to be more selective to the esterolysis activity of GSTP1 rather than carboxylesterases should lead to development of tools for detecting GSTP1-overexpressing cancer cells and investigating the biological functions of GSTP1. Copyright © 2017 Elsevier B.V. All rights reserved.

Trends in detoxification enzymes and heavy metal accumulation in ground beetles (Coleoptera: Carabidae) inhabiting a gradient of pollution.

PubMed

Stone, David; Jepson, Paul; Laskowski, Ryszard

2002-05-01

Non-specfic carboxylesterase and glutathione S-transferase activity was measured in the ground beetle, Pterosthicus oblongopunctatus (Coleoptera: Carabidae), from five sites along a gradient of heavy metal pollution. A previous study determined that beetles from the two most polluted sites (site codes OLK2 and OLK3) were more susceptible to additional stressors compared with beetles from the reference site (Stone et al., Environ. Pollut. 113, 239-244 2001), suggesting the possibility of physiological impairment. Metal body burdens in ground beetles from five sites along the gradient ranged from 79 to 201 microg/g Zn, 0.174 to 8.66 microg/g Pb and 1.14 to 10.8 microg/g Cd, whereas Cu seemed to be efficiently regulated regardless of metal levels in the soil. Beetle mid- and hindguts were homogenized and the soluble fraction containing glutathione S-transferase (GST) and carboxylesterase (CaE) was assayed using kinetic analyses. Significantly higher levels of GST were found only in female beetles from the most polluted sites (OLK2 and OLK3; P=0.049, P<0.001, respectively) compared with the reference site (OLK7). In addition, OLK3 females had significantly higher levels of CaE compared with the reference beetles (P=0.01). Male beetles did not differ in enzyme activity along the metal gradient. Overall, obvious trends in detoxification enzymes were not detected in ground beetles in association with metal body burdens.

Kinetics of Carboxylesterase: An Experiment for Biochemistry and Physical Chemistry Laboratory.

ERIC Educational Resources Information Center

Nichols, C. S.; Cromartie, T. H.

1979-01-01

Describes a convenient, inexpensive experiment in enzyme kinetics developed for the undergraduate biochemistry laboratory at the University of Virginia. Required are a single beam visible spectrophotometer with output to a recorder, a constant temperature, a commercially available enzyme, substrates, and buffers. (BT)

[The mechanism of the transport of organophosphorus compounds across the histo-hematic barriers].

PubMed

Miroshkina, V N; Kosmachev, A B; Salova, L S

1999-01-01

It was demonstrated in experiments on mice [correction of rats] that the transport of organophosphorus compounds (OPC) through membranes of the histohematic barriers (HHB) of the organism occurs by means of diffusion. The rate of this process depends on the interaction of OPC with the specific sites of binding with the tissues, among which the enzyme carboxylesterase plays an important part. It is suggested that both the rate and direction of OPC diffusion are determined by the relationship between the values of affinity of the ligands for the sites of their specific binding found on both sides of the HHB.

Status of Resistance of Bemisia tabaci (Hemiptera: Aleyrodidae) to Neonicotinoids in Iran and Detoxification by Cytochrome P450-Dependent Monooxygenases.

PubMed

Basij, M; Talebi, K; Ghadamyari, M; Hosseininaveh, V; Salami, S A

2017-02-01

Nine Bemisia tabaci (Gennadius) populations were collected from different regions of Iran. In all nine populations, only one biotype (B biotype) was detected. Susceptibilities of these populations to imidacloprid and acetamiprid were assayed. The lethal concentration 50 values (LC 50 ) for different populations showed a significant discrepancy in the susceptibility of B. tabaci to imidacloprid (3.76 to 772.06 mg l -1 ) and acetamiprid (4.96 to 865 mg l -1 ). The resistance ratio of the populations ranged from 9.72 to 205.20 for imidacloprid and 6.38 to 174.57 for acetamiprid. The synergistic effects of piperonylbutoxide (PBO) and S,S,S-tributylphosphorotrithioate (DEF) were evaluated for the susceptible (RF) and resistant (JR) populations for the determination of the involvement of cytochrome P450-dependent monooxygenase and carboxylesterase, respectively, in their resistance mechanisms. The results showed that PBO overcame the resistance of the JR population to both imidacloprid and acetamiprid, with synergistic ratios of 72.7 and 106.9, respectively. Carboxylesterase, glutathione S-transferase and cytochrome P450-dependent monooxygenase were studied biochemically, for the purpose of measuring the activity of the metabolizing enzymes in order to determine which enzymes are directly involved in neonicotinoid resistance. There was an increase in the activity of cytochrome P450-dependent monooxygenase up to 17-fold in the resistant JR population (RR = 205.20). The most plausible activity of cytochrome P450-dependent monooxygenase correlated with the resistances of imidacloprid and acetamiprid, and this suggests that cytochrome P450-dependent monooxygenase is the only enzyme system responsible for neonicotinoid resistance in the nine populations of B. tabaci.

Screening for and isolation and identification of malathion-degrading bacteria: cloning and sequencing a gene that potentially encodes the malathion-degrading enzyme, carboxylestrase in soil bacteria.

PubMed

Goda, Sayed K; Elsayed, Iman E; Khodair, Taha A; El-Sayed, Walaa; Mohamed, Mervat E

2010-11-01

Five malathion-degrading bacterial strains were enriched and isolated from soil samples collected from different agricultural sites in Cairo, Egypt. Malathion was used as a sole source of carbon (50 mg/l) to enumerate malathion degraders, which were designated as IS1, IS2, IS3, IS4, and IS5. They were identified, based on their morphological and biochemical characteristics, as Pseudomonas sp., Pseudomonas putida, Micrococcus lylae, Pseudomonas aureofaciens, and Acetobacter liquefaciens, respectively. IS1 and IS2, which showed the highest degrading activity, were selected for further identification by partial sequence analysis of their 16S rRNA genes. The 16S rRNA gene of IS1 shared 99% similarity with that of Alphaprotoebacterium BAL284, while IS2 scored 100% similarity with that of Pseudomonas putida 32zhy. Malathion residues almost completely disappeared within 6 days of incubation in IS2 liquid cultures. LC/ESI-MS analysis confirmed the degradation of malathion to malathion monocarboxylic and dicarboxylic acids, which formed as a result of carboxylesterase activity. A carboxylesterase gene (CE) was amplified from the IS2 genome by using specifically designed PCR primers. The sequence analysis showed a significant similarity to a known CE gene in different Pseudomonas sp. We report here the isolation of a new malathion-degrading bacteria from soils in Egypt that may be very well adapted to the climatic and environmental conditions of the country. We also report the partial cloning of a new CE gene. Due to their high biodegradation activity, the bacteria isolated from this work merit further study as potential biological agents for the remediation of soil, water, or crops contaminated with the pesticide malathion.

Evaluation of liver and brain esterases in the spotted gar fish (Lepisosteus oculatus) as biomarkers of effect in the lower Mississippi River basin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, T.L.; Obih, P.O.; Jaiswal, R.

1997-05-01

The responses of various xenobiotic metabolizing enzymes in fish models are rapidly evolving as important biomarkers for monitoring unacceptable levels of environmental contaminants. Ethoxyresorufin O-deethylase, a specific cytochrome P450-dependent monooxygenase, is often used as an indicator of polycyclic aromatic hydrocarbon pollution. Another class of enzymes which are potential biomarkers are the B-type esterases. These enzymes are sensitive to inhibition by organophosphates, and include the cholinesterases (ChE) and carboxylesterases. ChEs are further subdivided into acetylcholinesterase and butyryl cholinesterase. Among fish, AChE is predominantly localized in the brain and muscle, whereas, BuChE activity is found mainly in liver and plasma. The precisemore » physiological role of BuChE is unknown, although it has been regarded as a marker enzyme for glial or supportive cells or other non-neuronal elements. Inhibition of ChE activity has often been associated with exposure to organophosphate and carbamate insecticides and other neurotoxic xenobiotics. Chemicals other than carbarnates and organophosphates that are environmental contaminants can also affect the activity of ChEs. Carboxylesterases represent a heterogenous group of isozymes that can catalyze the hydrolysis of a wide range of xenobiotic esters, amides and thioesters. For most CaE, their natural substrates are unknown, therefore, their physiological functions remain to be elucidated. These enzymes (CaE) occur widely in most tissues and are generally found in high levels in the liver. The purpose of this research was to evaluate the liver and brain esterases in the spotted gar fish as biomarkers of effect to multiple contaminants in the lower Mississippi River basin. 15 refs., 3 figs., 2 tabs.« less

Effects of acute exposure to chlorpyrifos on cholinergic and non-cholinergic targets in normal and high-fat fed male C57BL/6J mice.

PubMed

Kondakala, Sandeep; Lee, Jung Hwa; Ross, Matthew K; Howell, George E

2017-12-15

The prevalence of obesity is increasing at an alarming rate in the United States with 36.5% of adults being classified as obese. Compared to normal individuals, obese individuals have noted pathophysiological alterations which may alter the toxicokinetics of xenobiotics and therefore alter their toxicities. However, the effects of obesity on the toxicity of many widely utilized pesticides has not been established. Therefore, the present study was designed to determine if the obese phenotype altered the toxicity of the most widely used organophosphate (OP) insecticide, chlorpyrifos (CPS). Male C57BL/6J mice were fed normal or high-fat diet for 4weeks and administered a single dose of vehicle or CPS (2.0mg/kg; oral gavage) to assess cholinergic (acetylcholinesterase activities) and non-cholinergic (carboxylesterase and endocannabinoid hydrolysis) endpoints. Exposure to CPS significantly decreased red blood cell acetylcholinesterase (AChE) activity, but not brain AChE activity, in both diet groups. Further, CPS exposure decreased hepatic carboxylesterase activity and hepatic hydrolysis of a major endocannabinoid, anandamide, in a diet-dependent manner with high-fat diet fed animals being more sensitive to CPS-mediated inhibition. These in vivo studies were corroborated by in vitro studies using rat primary hepatocytes, which demonstrated that fatty acid amide hydrolase and CES activities were more sensitive to CPS-mediated inhibition than 2-arachidonoylglycerol hydrolase activity. These data demonstrate hepatic CES and FAAH activities in high-fat diet fed mice were more potently inhibited than those in normal diet fed mice following CPS exposure, which suggests that the obese phenotype may exacerbate some of the non-cholinergic effects of CPS exposure. Copyright © 2017 Elsevier Inc. All rights reserved.

Mechanism of Resistance Acquisition and Potential Associated Fitness Costs in Amyelois transitella (Lepidoptera: Pyralidae) Exposed to Pyrethroid Insecticides.

PubMed

Demkovich, Mark; Siegel, Joel P; Higbee, Bradley S; Berenbaum, May R

2015-06-01

The polyphagous navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae), is the most destructive pest of nut crops, including almonds and pistachios, in California orchards. Management of this insect has typically been a combination of cultural controls and insecticide use, with the latter increasing substantially along with the value of these commodities. Possibly associated with increased insecticide use, resistance has been observed recently in navel orangeworm populations in Kern County, California. In studies characterizing a putatively pyrethroid-resistant strain (R347) of navel orangeworm, susceptibility to bifenthrin and β-cyfluthrin was compared with that of an established colony of susceptible navel orangeworm. Administration of piperonyl butoxide and S,S,S-tributyl phosphorotrithioate in first-instar feeding bioassays with the pyrethroids bifenthrin and β-cyfluthrin produced synergistic effects and demonstrated that cytochrome P450 monooxygenases and carboxylesterases contribute to resistance in this population. Resistance is therefore primarily metabolic and likely the result of overexpression of specific cytochrome P450 monooxygenases and carboxylesterase genes. Resistance was assessed by median lethal concentration (LC50) assays and maintained across nine generations in the laboratory. Life history trait comparisons between the resistant strain and susceptible strain revealed significantly lower pupal weights in resistant individuals reared on the same wheat bran-based artificial diet across six generations. Time to second instar was greater in the resistant strain than the susceptible strain, although overall development time was not significantly different between strains. Resistance was heritable and may have an associated fitness cost, which could influence the dispersal and expansion of resistant populations in nut-growing areas in California. © The Authors 2015. Published by Oxford University Press on behalf of Entomological

Possible mechanism for inhibition of morphine formation from 6-acetylmorphine after intake of street heroin.

PubMed

Andersson, Maria; Björkhem-Bergman, Linda; Beck, Olof

2015-07-01

Heroin is de-acetylated in the body to morphine in two steps. The intermediate 6-acetylmorphine (6-AM) is formed rapidly and is considered important for the pharmacological effect of heroin. In urine drug testing, an atypical pattern of morphine and 6-AM is known to occur in low frequency. The aim of this study was to investigate this atypical pattern in more detail and to identify responsible substances for a possible inhibition of the conversion from 6-AM to morphine. Urine samples were selected from a routine flow of samples sent for drug testing. Out of 695 samples containing morphine and 6-acetylmorphine, 11.5% had the atypical pattern of a 6-AM to morphine ratio above 0.26 as derived from a bimodal frequency distribution. An in vitro study of the conversion of 6-acetylmorphine to morphine in human liver homogenates demonstrated that a number of known carboxylesterase inhibitors were able to inhibit the reaction mimicking the situation in vivo. Compound 3 (3,6-Dimethoxy-4-acetoxy-5-[2-(N-methylacetamido)ethyl]phenanthrene) a substance formed from thebaine during the production of heroin was found to be a strong inhibitor. Liquid chromatography-mass spectrometry was used to identify possible inhibitors present in vivo. This part of the investigation demonstrated that several components may contribute to the effect. It is concluded that inhibition of liver carboxylesterase activity is a possible mechanism causing the atypical pattern and that one candidate compound is the result of the heroin production process. An inhibition of 6-AM metabolism is likely to increase the pharmacological effect of heroin and may be related to a higher risk of lethal toxicity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A stereospecific carboxyl esterase from Bacillus coagulans hosting nonlipase activity within a lipase-like fold.

PubMed

De Vitis, Valerio; Nakhnoukh, Cristina; Pinto, Andrea; Contente, Martina L; Barbiroli, Alberto; Milani, Mario; Bolognesi, Martino; Molinari, Francesco; Gourlay, Louise J; Romano, Diego

2018-03-01

Microbial carboxylesterases are important biocatalysts that selectively hydrolyze an extensive range of esters. Here, we report the biochemical and structural characterization of an atypical carboxylesterase from Bacillus coagulans (BCE), endowed with high enantioselectivity toward different 1,2-O-isopropylideneglycerol (IPG or solketal) esters. BCE efficiently catalyzes the production of enantiopure (S)-IPG, a chiral building block for the synthesis of β-blockers, glycerophospholipids, and prostaglandins; efficient hydrolysis was observed up to 65 °C. To gain insight into the mechanistic bases of such enantioselectivity, we solved the crystal structures of BCE in apo- and glycerol-bound forms at resolutions of 1.9 and 1.8 Å, respectively. In silico docking studies on the BCE structure confirmed that IPG esters with small acyl chains (≤ C6) were easily accommodated in the active site pocket, indicating that small conformational changes are necessary to accept longer substrates. Furthermore, docking studies suggested that enantioselectivity may be due to an improved stabilization of the tetrahedral reaction intermediate for the S-enantiomer. Contrary to the above functional data implying nonlipolytic functions, BCE displays a lipase-like 3D structure that hosts a "lid" domain capping the main entrance to the active site. In lipases the lid mediates catalysis through interfacial activation, a process that we did not observe for BCE. Overall, we present the functional-structural properties of an atypical carboxyl esterase that has nonlipase-like functions, yet possesses a lipase-like 3D fold. Our data provide original enzymatic information in view of BCE applications as an inexpensive, efficient biocatalyst for the production of enantiopure (S)-IPG. Coordinates and structure factors have been deposited in the Protein Data Bank (www.rcsb.org) under accession numbers 5O7G (apo-BCE) and 5OLU (glycerol-bound BCE). © 2017 Federation of European Biochemical

Design of a 15N Molecular Unit to Achieve Long Retention of Hyperpolarized Spin State

NASA Astrophysics Data System (ADS)

Nonaka, Hiroshi; Hirano, Masashi; Imakura, Yuki; Takakusagi, Yoichi; Ichikawa, Kazuhiro; Sando, Shinsuke

2017-01-01

Nuclear hyperpolarization is a phenomenon that can be used to improve the sensitivity of magnetic resonance molecular sensors. However, such sensors typically suffer from short hyperpolarization lifetime. Herein we report that [15N, D14]trimethylphenylammonium (TMPA) has a remarkably long spin-lattice relaxation time (1128 s, 14.1 T, 30 °C, D2O) on its 15N nuclei and achieves a long retention of the hyperpolarized state. [15N, D14]TMPA-based hyperpolarized sensor for carboxylesterase allowed the highly sensitive analysis of enzymatic reaction by 15N NMR for over 40 min in phophate-buffered saline (H2O, pH 7.4, 37 °C).

Cell-Bound Lipase and Esterase of Brevibacterium linens

PubMed Central

Sørhaug, Terje; Ordal, Z. John

1974-01-01

The activities of glycerol ester hydrolase, lipase (EC 3.1.1.3) and carboxylesterase, and esterase (EC 3.1.1.1) were determined for whole cell preparations of Brevibacterium linens by using the pH-stat assay. The culture growth liquors were inactive against the three substrates, tributyrin emulsion, triacetin, and methyl butyrate. Cells washed in water had less activity than cells washed in 5% NaCl; the ratio of activities was close to 1:2 for all strains using tributyrin emulsion as the substrate. For the esterase substrates, this relationship varied widely and was strain dependent. The ability to hydrolyze the two esterase substrates varied independently of the level of lipase activity. PMID:4824883

Selective inhibition of plant serine hydrolases by agrochemicals revealed by competitive ABPP.

PubMed

Kaschani, Farnusch; Nickel, Sabrina; Pandey, Bikram; Cravatt, Benjamin F; Kaiser, Markus; van der Hoorn, Renier A L

2012-01-15

Organophosphate and -phosphonates and their thio derivatives are often used in agroindustry as herbicides and insecticides, but their potential off-targets in the plant are poorly investigated. Here, we use competitive activity-based protein profiling (ABPP) of serine hydrolases (SHs) to detect targets of these agrochemicals and other compounds in Arabidopsis thaliana. Using broad-range and specific probes, and by overexpression of various SHs in planta, we are able to confirm eight SH-compound interactions, including selective inhibition of carboxylesterase CXE12, prolyloligopeptidase, methylesterase MES2 and tripeptidyl peptidase TPP2. These observations can be used for the design of novel probes and selective inhibitors and may help to assess physiological effects of agrochemicals on crop plants. Copyright © 2011 Elsevier Ltd. All rights reserved.

Selective Inhibition of Plant Serine Hydrolases by Agrochemicals Revealed by Competitive ABPP

PubMed Central

Kaschani, Farnusch; Nickel, Sabrina; Pandey, Bikram; Cravatt, Benjamin F.; Kaiser, Markus; van der Hoorn, Renier A. L.

2013-01-01

Organophosphate and –phosphonates and their thiol derivatives are often used in agroindustry as herbicides and insecticides, but their potential off-targets in the plant and their consumers are poorly investigated. Here, we use competitive Activity-based Protein Profiling (ABPP) of serine hydrolases (SHs) to detect targets of these agrochemicals and other compounds in Arabidopsis thaliana. Using broad-range and specific probes, and by overexpression of various SHs in planta, we are able to confirm eight SH-compound interactions, including selective inhibition of carboxylesterase CXE12, prolyloligopeptidase, methylesterase MES2 and tripeptidyl peptidase TPP2. These observations can be used for the design of novel probes and selective inhibitors and may help to assess physiological effects of agrochemicals on crop plants. PMID:21764588

Pyrethroid Resistance in Malaysian Populations of Dengue Vector Aedes aegypti Is Mediated by CYP9 Family of Cytochrome P450 Genes

PubMed Central

Ishak, Intan H.; Kamgang, Basile; Ibrahim, Sulaiman S.; Riveron, Jacob M.; Irving, Helen

2017-01-01

Background Dengue control and prevention rely heavily on insecticide-based interventions. However, insecticide resistance in the dengue vector Aedes aegypti, threatens the continued effectiveness of these tools. The molecular basis of the resistance remains uncharacterised in many endemic countries including Malaysia, preventing the design of evidence-based resistance management. Here, we investigated the underlying molecular basis of multiple insecticide resistance in Ae. aegypti populations across Malaysia detecting the major genes driving the metabolic resistance. Methodology/Principal Findings Genome-wide microarray-based transcription analysis was carried out to detect the genes associated with metabolic resistance in these populations. Comparisons of the susceptible New Orleans strain to three non-exposed multiple insecticide resistant field strains; Penang, Kuala Lumpur and Kota Bharu detected 2605, 1480 and 425 differentially expressed transcripts respectively (fold-change>2 and p-value ≤ 0.05). 204 genes were commonly over-expressed with monooxygenase P450 genes (CYP9J27, CYP6CB1, CYP9J26 and CYP9M4) consistently the most up-regulated detoxification genes in all populations, indicating that they possibly play an important role in the resistance. In addition, glutathione S-transferases, carboxylesterases and other gene families commonly associated with insecticide resistance were also over-expressed. Gene Ontology (GO) enrichment analysis indicated an over-representation of GO terms linked to resistance such as monooxygenases, carboxylesterases, glutathione S-transferases and heme-binding. Polymorphism analysis of CYP9J27 sequences revealed a high level of polymorphism (except in Joho Bharu), suggesting a limited directional selection on this gene. In silico analysis of CYP9J27 activity through modelling and docking simulations suggested that this gene is involved in the multiple resistance in Malaysian populations as it is predicted to metabolise

Pyrethroid Resistance in Malaysian Populations of Dengue Vector Aedes aegypti Is Mediated by CYP9 Family of Cytochrome P450 Genes.

PubMed

Ishak, Intan H; Kamgang, Basile; Ibrahim, Sulaiman S; Riveron, Jacob M; Irving, Helen; Wondji, Charles S

2017-01-01

Dengue control and prevention rely heavily on insecticide-based interventions. However, insecticide resistance in the dengue vector Aedes aegypti, threatens the continued effectiveness of these tools. The molecular basis of the resistance remains uncharacterised in many endemic countries including Malaysia, preventing the design of evidence-based resistance management. Here, we investigated the underlying molecular basis of multiple insecticide resistance in Ae. aegypti populations across Malaysia detecting the major genes driving the metabolic resistance. Genome-wide microarray-based transcription analysis was carried out to detect the genes associated with metabolic resistance in these populations. Comparisons of the susceptible New Orleans strain to three non-exposed multiple insecticide resistant field strains; Penang, Kuala Lumpur and Kota Bharu detected 2605, 1480 and 425 differentially expressed transcripts respectively (fold-change>2 and p-value ≤ 0.05). 204 genes were commonly over-expressed with monooxygenase P450 genes (CYP9J27, CYP6CB1, CYP9J26 and CYP9M4) consistently the most up-regulated detoxification genes in all populations, indicating that they possibly play an important role in the resistance. In addition, glutathione S-transferases, carboxylesterases and other gene families commonly associated with insecticide resistance were also over-expressed. Gene Ontology (GO) enrichment analysis indicated an over-representation of GO terms linked to resistance such as monooxygenases, carboxylesterases, glutathione S-transferases and heme-binding. Polymorphism analysis of CYP9J27 sequences revealed a high level of polymorphism (except in Joho Bharu), suggesting a limited directional selection on this gene. In silico analysis of CYP9J27 activity through modelling and docking simulations suggested that this gene is involved in the multiple resistance in Malaysian populations as it is predicted to metabolise pyrethroids, DDT and bendiocarb. The predominant

Development of organophosphate hydrolase activity in a bacterial homolog of human cholinesterase

NASA Astrophysics Data System (ADS)

Legler, Patricia; Boisvert, Susanne; Compton, Jaimee; Millard, Charles

2014-07-01

We applied a combination of rational design and directed evolution (DE) to Bacillus subtilis p-nitrobenzyl esterase (pNBE) with the goal of enhancing organophosphorus acid anhydride hydrolase (OPAAH) activity. DE started with a designed variant, pNBE A107H, carrying a histidine homologous with human butyrylcholinesterase G117H to find complementary mutations that further enhance its OPAAH activity. Five sites were selected (G105, G106, A107, A190, and A400) within a 6.7 Å radius of the nucleophilic serine O?. All 95 variants were screened for esterase activity with a set of five substrates: pNP-acetate, pNP-butyrate, acetylthiocholine, butyrylthiocholine, or benzoylthiocholine. A microscale assay for OPAAH activity was developed for screening DE libraries. Reductions in esterase activity were generally concomitant with enhancements in OPAAH activity. One variant, A107K, showed an unexpected 7-fold increase in its kcat/Km for benzoylthiocholine, demonstrating that it is also possible to enhance the cholinesterase activity of pNBE. Moreover, DE resulted in at least three variants with modestly enhanced OPAAH activity compared to wild type pNBE. A107H/A190C showed a 50-fold increase in paraoxonase activity and underwent a slow time- and temperature-dependent change affecting the hydrolysis of OPAA and ester substrates. Structural analysis suggests that pNBE may represent a precursor leading to human cholinesterase and carboxylesterase 1 through extension of two vestigial specificity loops; a preliminary attempt to transfer the Ω-loop of BChE into pNBE is described. pNBE was tested as a surrogate scaffold for mammalian esterases. Unlike butyrylcholinesterase and pNBE, introducing a G143H mutation (equivalent to G117H) did not confer detectable OP hydrolase activity on human carboxylesterase 1. We discuss the importance of the oxyanion-hole residues for enhancing the OPAAH activity of selected serine hydrolases.

Insecticide susceptibility status and major detoxifying enzymes' activity in Aedes albopictus (Skuse), vector of dengue and chikungunya in Northern part of West Bengal, India.

PubMed

Bharati, Minu; Saha, Dhiraj

2017-06-01

Mosquitoes belonging to Aedes genus, Aedes aegypti and Aedes albopictus transmit many globally important arboviruses including Dengue (DENV) and Chikungunya (CHIKV). Vector control with the use of insecticide remains the suitable method of choice to stop the transmission of these diseases. However, vector control throughout the world is failing to achieve its target results because of the worldwide development of insecticide resistance in mosquitoes. To assess the insecticide susceptibility status of Aedes albopictus from northern part of West Bengal, the susceptibility of eight different Aedes albopictus populations were tested against a commonly used larvicide (temephos) and some adulticides (malathion, deltamethrin and lambda cyhalothrin) along with the major insecticide detoxifying enzymes' activity in them. Through this study, it was revealed that most of the populations were found susceptible to temephos except Nagrakata (NGK) and Siliguri (SLG), which showed both a higher resistance ratio (RR 99 ) and a lower susceptibility, thereby reflecting the development of resistance against temephos in them. However, all tested adulticides caused 100% mortality in all the population implying their potency in control of this mosquito in this region of India. Through the study of carboxylesterase activity, it was revealed that the NGK population showed a 9.6 fold higher level of activity than susceptible population. The same population also showed a lower level of susceptibility and a higher resistance ratio (RR 99 ), indicating a clear correlation between susceptibility to temephos and carboxylesterase enzymes' activity in this population. This preliminary data reflects that the NGK population is showing a trend towards resistance development and with time, there is possibility that this resistance phenomenon will spread to other populations. With the recurrence of dengue and chikungunya, this data on insecticide susceptibility status of Aedes albopictus could help the

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

PubMed Central

Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

2013-01-01

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0. PMID:23685703

Direct imaging of ER calcium with targeted-esterase induced dye loading (TED).

PubMed

Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

2013-05-07

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca(2+) indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca(2+) indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca(2+) indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca(2+) indicator and a hydrophilic fluorescent dye/Ca(2+) complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0.

Analogous pleiotropic effects of insecticide resistance genotypes in peach-potato aphids and houseflies.

PubMed

Foster, S P; Young, S; Williamson, M S; Duce, I; Denholm, I; Devine, G J

2003-08-01

We show that single-point mutations conferring target-site resistance (kdr) to pyrethroids and DDT in aphids and houseflies, and gene amplification conferring metabolic resistance (carboxylesterase) to organophosphates and carbamates in aphids, can have deleterious pleiotropic effects on fitness. Behavioural studies on peach-potato aphids showed that a reduced response to alarm pheromone was associated with both gene amplification and the kdr target-site mutation. In this species, gene amplification was also associated with a decreased propensity to move from senescing leaves to fresh leaves at low temperature. Housefly genotypes possessing the identical kdr mutation were also shown to exhibit behavioural differences in comparison with susceptible insects. In this species, resistant individuals showed no positional preference along a temperature gradient while susceptible genotypes exhibited a strong preference for warmer temperatures.

Placental oxidative status in rural residents environmentally exposed to organophosphates.

PubMed

Chiapella, Graciela; Genti-Raimondi, Susana; Magnarelli, Gladis

2014-07-01

The impact of environmental organophosphate pesticide exposure on the placenta oxidative status was assessed. Placental samples were collected from women residing in an agricultural area during pesticide pulverization period, non-pulverization period and from control group. Carboxylesterase activity was significantly decreased in pulverization period group. Enzymatic and non-enzymatic defense system, the oxidative stress biomarkers and the nuclear factor erythroid 2-related factor levels showed no differences among groups. However, in the pulverization period group, an inverse association between catalase activity and placental index, a useful metric for estimating placental inefficiency, was found. This result suggests that catalase may serve as a potential placental biomarker of susceptibility to pesticides. Further studies designed from a gene-environment perspective are needed. Copyright © 2014 Elsevier B.V. All rights reserved.

Synthetic polyester-hydrolyzing enzymes from thermophilic actinomycetes.

PubMed

Wei, Ren; Oeser, Thorsten; Zimmermann, Wolfgang

2014-01-01

Thermophilic actinomycetes produce enzymes capable of hydrolyzing synthetic polyesters such as polyethylene terephthalate (PET). In addition to carboxylesterases, which have hydrolytic activity predominantly against PET oligomers, esterases related to cutinases also hydrolyze synthetic polymers. The production of these enzymes by actinomycetes as well as their recombinant expression in heterologous hosts is described and their catalytic activity against polyester substrates is compared. Assays to analyze the enzymatic hydrolysis of synthetic polyesters are evaluated, and a kinetic model describing the enzymatic heterogeneous hydrolysis process is discussed. Structure-function and structure-stability relationships of actinomycete polyester hydrolases are compared based on molecular dynamics simulations and recently solved protein structures. In addition, recent progress in enhancing their activity and thermal stability by random or site-directed mutagenesis is presented. © 2014 Elsevier Inc. All rights reserved.

Multiple insecticide resistance in Aedes aegypti (Diptera: Culicidae) populations compromises the effectiveness of dengue vector control in French Guiana.

PubMed

Dusfour, Isabelle; Thalmensy, Véronique; Gaborit, Pascal; Issaly, Jean; Carinci, Romuald; Girod, Romain

2011-05-01

In French Guiana, pyrethroids and organophosphates have been used for many years against Aedes aegypti. We aimed to establish both the resistance level of Ae. aegypti and the ultra low volume spray efficacy to provide mosquito control services with practical information to implement vector control and resistance management. Resistance to deltamethrin and fenitrothion was observed. In addition, the profound loss of efficacy of AquaK'othrine® and the moderate loss of efficacy of Paluthion® 500 were recorded. Fenitrothion remained the most effective candidate for spatial application in French Guiana until its removal in December 2010. Further investigation of the mechanism of resistance to deltamethrin demonstrated the involvement of mixed-function oxidases and, to a lesser extent, of carboxylesterases. However, these observations alone cannot explain the level of insecticide resistance we observed during tube and cage tests.

Identification of a new steroid degrading bacterial strain H5 from the Baltic Sea and isolation of two estradiol inducible genes.

PubMed

Sang, Yingying; Xiong, Guangming; Maser, Edmund

2012-03-01

The presence of steroid hormones in the aquatic environment is potentially threatening the population dynamics of all kinds of sea animals and public health. Environmental estrogens in water have been reported to be associated with abnormal sexual development and abnormal feminizing responses in some animals. New approaches for the bioremediation of steroid hormones from the environment are therefore urgently sought. We have previously isolated a steroid degrading bacterial strain (H5) from the Baltic Sea, at Kiel, Germany. In the present investigation, 16S rRNA analysis showed that marine strain H5 belongs to the genus Vibrio, family Vibrionaceae and class Gamma-Proteobacteria. To enable identification of steroid inducible genes from bacterial strain H5, a library was constructed of H5 chromosomal DNA fragments cloned into a fluorescent reporter (pKEGFP-2). A reporter plasmid pK3α-4.6-EGFP3 containing the estrogen-inducible gene 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) from Comamonas testosteroni (C. testosteroni) was created as a positive control. Steroid induction could be detected by a microplate fluorescence reader, when the plasmids were transformed into Escherichia coli (E. coli) HB101 cells. With our meta-genomic pKEGFP-2 approach, we identified two estradiol-inducible genes from marine strain H5, which are obviously involved in steroid degradation. Sequencing of the pKEGFP-2 inserts and data base research at NCBI revealed that one gene corresponds to 3-ketosteroid-delta-1-dehydrogenase from several Mycobacterium strains, while the other showed high similarity to carboxylesterase in Sebadella termitidis and Brachyspira murdochii. Both 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase are one of the first enzymes in steroid degradation. In addition, we identified a strain H5 specific DNA sequence of 480bp which allows sensitive PCR detection and quantification of strain H5 bacteria in "unknown" seawater samples. Currently, the

In vitro metabolism and drug-drug interaction potential of UTL-5g, a novel chemo- and radioprotective agent.

PubMed

Wu, Jianmei; Shaw, Jiajiu; Dubaisi, Sarah; Valeriote, Frederick; Li, Jing

2014-12-01

N-(2,4-dichlorophenyl)-5-methyl-1,2-oxazole-3-carboxamide (UTL-5g), a potential chemo- and radioprotective agent, acts as a prodrug requiring bioactivation to the active metabolite 5-methylisoxazole-3-carboxylic acid (ISOX). UTL-5g hydrolysis to ISOX and 2,4-dichloroaniline (DCA) has been identified in porcine and rabbit liver esterases. The purpose of this study was to provide insights on the metabolism and drug interaction potential of UTL-5g in humans. The kinetics of UTL-5g hydrolysis was determined in human liver microsomes (HLM) and recombinant human carboxylesterases (hCE1b and hCE2). The potential of UTL-5g and its metabolites for competitive inhibition and time-dependent inhibition of microsomal cytochrome P450 (P450) was examined in HLM. UTL-5g hydrolysis to ISOX and DCA in HLM were NADPH-independent, with a maximum rate of reaction (Vmax) of 11.1 nmol/min per mg and substrate affinity (Km) of 41.6 µM. Both hCE1b and hCE2 effectively catalyzed UTL-5g hydrolysis, but hCE2 exhibited ∼30-fold higher catalytic efficiency (Vmax/Km) than hCE1b. UTL-5g and DCA competitively inhibited microsomal CYP1A2, CYP2B6, and CYP2C19 (IC50 values <50 µM), and exhibited time-dependent inhibition of microsomal CYP1A2 with the inactivation efficiency (kinact/KI) of 0.68 and 0.51 minute(-1)·mM(-1), respectively. ISOX did not inhibit or inactivate any tested microsomal P450. In conclusion, hCE1b and hCE2 play a key role in the bioactivation of UTL-5g. Factors influencing carboxylesterase activities may have a significant impact on the pharmacological and therapeutic effects of UTL-5g. UTL-5g has the potential to inhibit P450-mediated metabolism through competitive inhibition or time-dependent inhibition. Caution is particularly needed for potential drug interactions involving competitive inhibition or time-dependent inhibition of CYP1A2 in the future clinical development of UTL-5g. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

In Vitro Metabolism and Drug-Drug Interaction Potential of UTL-5g, a Novel Chemo- and Radioprotective Agent

PubMed Central

Wu, Jianmei; Shaw, Jiajiu; Dubaisi, Sarah; Valeriote, Frederick

2014-01-01

N-(2,4-dichlorophenyl)-5-methyl-1,2-oxazole-3-carboxamide (UTL-5g), a potential chemo- and radioprotective agent, acts as a prodrug requiring bioactivation to the active metabolite 5-methylisoxazole-3-carboxylic acid (ISOX). UTL-5g hydrolysis to ISOX and 2,4-dichloroaniline (DCA) has been identified in porcine and rabbit liver esterases. The purpose of this study was to provide insights on the metabolism and drug interaction potential of UTL-5g in humans. The kinetics of UTL-5g hydrolysis was determined in human liver microsomes (HLM) and recombinant human carboxylesterases (hCE1b and hCE2). The potential of UTL-5g and its metabolites for competitive inhibition and time-dependent inhibition of microsomal cytochrome P450 (P450) was examined in HLM. UTL-5g hydrolysis to ISOX and DCA in HLM were NADPH-independent, with a maximum rate of reaction (Vmax) of 11.1 nmol/min per mg and substrate affinity (Km) of 41.6 µM. Both hCE1b and hCE2 effectively catalyzed UTL-5g hydrolysis, but hCE2 exhibited ∼30-fold higher catalytic efficiency (Vmax/Km) than hCE1b. UTL-5g and DCA competitively inhibited microsomal CYP1A2, CYP2B6, and CYP2C19 (IC50 values <50 µM), and exhibited time-dependent inhibition of microsomal CYP1A2 with the inactivation efficiency (kinact/KI) of 0.68 and 0.51 minute−1·mM−1, respectively. ISOX did not inhibit or inactivate any tested microsomal P450. In conclusion, hCE1b and hCE2 play a key role in the bioactivation of UTL-5g. Factors influencing carboxylesterase activities may have a significant impact on the pharmacological and therapeutic effects of UTL-5g. UTL-5g has the potential to inhibit P450-mediated metabolism through competitive inhibition or time-dependent inhibition. Caution is particularly needed for potential drug interactions involving competitive inhibition or time-dependent inhibition of CYP1A2 in the future clinical development of UTL-5g. PMID:25249693

Comparison of esterase gene amplification, gene expression and esterase activity in insecticide susceptible and resistant strains of the brown planthopper, Nilaparvata lugens (Stål).

PubMed

Vontas, J G; Small, G J; Hemingway, J

2000-12-01

Organophosphorus and carbamate insecticide resistance in Nilaparvata lugens is based on amplification of a carboxylesterase gene, Nl-EST1. An identical gene occurs in susceptible insects. Quantitative real-time PCR was used to demonstrate that Nl-EST1 is amplified 3-7-fold in the genome of resistant compared to susceptible planthoppers. Expression levels were similar to amplification levels, with 1-15-fold more Nl-EST1 mRNA in individual insects and 5-11-fold more Nl-EST1 mRNA in mass whole body homogenates of resistant females compared to susceptibles. These values corresponded to an 8-10-fold increase in esterase activity in the head and thorax of individual resistant insects. Although amplification, expression and activity levels of Nl-EST1 in resistant N. lugens were similar, the correlation between esterase activity and Nl-EST1 mRNA levels in resistant individuals was not linear.

Triazophos up-regulated gene expression in the female brown planthopper, Nilaparvata lugens.

PubMed

Bao, Yan-Yuan; Li, Bao-Ling; Liu, Zhao-Bu; Xue, Jian; Zhu, Zeng-Rong; Cheng, Jia-An; Zhang, Chuan-Xi

2010-09-01

The widespread use of insecticides has caused the resurgence of the brown planthopper, Nilaparvata lugens, in Asia. In this study, we investigated an organo-phosphorous insecticide, triazophos, and its ability to induce gene expression variation in female N. lugens nymphs just before emergence. By using the suppression subtractive hybridization method, a triazophos-induced cDNA library was constructed. In total, 402 differentially expressed cDNA clones were obtained. Real-time qPCR analysis confirmed that triazophos up-regulated the expression of six candidate genes at the transcript level in nymphs on day 3 of the 5th instar. These genes encode N. lugens vitellogenin, bystin, multidrug resistance protein (MRP), purine nucleoside phosphorylase (PNP), pyrroline-5-carboxylate reductase (P5CR) and carboxylesterase. Our results imply that the up-regulation of these genes may be involved in the induction of N. lugens female reproduction or resistance to insecticides.

Biochemical and Structural Insights into Enzymatic Depolymerization of Polylactic Acid and Other Polyesters by Microbial Carboxylesterases.

PubMed

Hajighasemi, Mahbod; Nocek, Boguslaw P; Tchigvintsev, Anatoli; Brown, Greg; Flick, Robert; Xu, Xiaohui; Cui, Hong; Hai, Tran; Joachimiak, Andrzej; Golyshin, Peter N; Savchenko, Alexei; Edwards, Elizabeth A; Yakunin, Alexander F

2016-06-13

Polylactic acid (PLA) is a biodegradable polyester derived from renewable resources, which is a leading candidate for the replacement of traditional petroleum-based polymers. Since the global production of PLA is quickly growing, there is an urgent need for the development of efficient recycling technologies, which will produce lactic acid instead of CO2 as the final product. After screening 90 purified microbial α/β-hydrolases, we identified hydrolytic activity against emulsified PLA in two uncharacterized proteins, ABO2449 from Alcanivorax borkumensis and RPA1511 from Rhodopseudomonas palustris. Both enzymes were also active against emulsified polycaprolactone and other polyesters as well as against soluble α-naphthyl and p-nitrophenyl monoesters. In addition, both ABO2449 and RPA1511 catalyzed complete or extensive hydrolysis of solid PLA with the production of lactic acid monomers, dimers, and larger oligomers as products. The crystal structure of RPA1511 was determined at 2.2 Å resolution and revealed a classical α/β-hydrolase fold with a wide-open active site containing a molecule of polyethylene glycol bound near the catalytic triad Ser114-His270-Asp242. Site-directed mutagenesis of both proteins demonstrated that the catalytic triad residues are important for the hydrolysis of both monoester and polyester substrates. We also identified several residues in RPA1511 (Gln172, Leu212, Met215, Trp218, and Leu220) and ABO2449 (Phe38 and Leu152), which were not essential for activity against soluble monoesters but were found to be critical for the hydrolysis of PLA. Our results indicate that microbial carboxyl esterases can efficiently hydrolyze various polyesters making them attractive biocatalysts for plastics depolymerization and recycling.

Controlling Plasma Stability of Hydroxamic Acids: A MedChem Toolbox.

PubMed

Hermant, Paul; Bosc, Damien; Piveteau, Catherine; Gealageas, Ronan; Lam, BaoVy; Ronco, Cyril; Roignant, Matthieu; Tolojanahary, Hasina; Jean, Ludovic; Renard, Pierre-Yves; Lemdani, Mohamed; Bourotte, Marilyne; Herledan, Adrien; Bedart, Corentin; Biela, Alexandre; Leroux, Florence; Deprez, Benoit; Deprez-Poulain, Rebecca

2017-11-09

Hydroxamic acids are outstanding zinc chelating groups that can be used to design potent and selective metalloenzyme inhibitors in various therapeutic areas. Some hydroxamic acids display a high plasma clearance resulting in poor in vivo activity, though they may be very potent compounds in vitro. We designed a 57-member library of hydroxamic acids to explore the structure-plasma stability relationships in these series and to identify which enzyme(s) and which pharmacophores are critical for plasma stability. Arylesterases and carboxylesterases were identified as the main metabolic enzymes for hydroxamic acids. Finally, we suggest structural features to be introduced or removed to improve stability. This work thus provides the first medicinal chemistry toolbox (experimental procedures and structural guidance) to assess and control the plasma stability of hydroxamic acids and realize their full potential as in vivo pharmacological probes and therapeutic agents. This study is particularly relevant to preclinical development as it allows obtaining compounds equally stable in human and rodent models.

Acaricidal activity of Ocimum basilicum and Spilanthes acmella against the ectoparasitic tick, Rhipicephalus (Boophilus) microplus (Arachinida: Ixodidae).

PubMed

Veeramani, V; Sakthivelkumar, S; Tamilarasan, K; Aisha, S O; Janarthanan, S

2014-09-01

The ectoparasitic tick, Rhipicephalus (Boophilus) microplus collected at various cattle farms in and around Chennai was subjected to treatment of different crude solvent extracts of leaves of Ocimum basilicum and Spilanthes acmella for acaricidal activity. Among various solvent extracts of leaves of O. basilicum and S. acmella used, chloroform extract of O. basilicum at concentrations between 6% and 10% exhibited 70% and 100% mortality of ticks when compared to control. The LC50 and LC90 values of the chloroform extract of leaves of O. basilicum treatment on the ticks after 24 h were observed as 5.46% and 7.69%. Quantitative and qualitative analysis of α- and β- carboxylesterase enzymes in the whole gut homogenate of cattle tick, R. microplus treated with chloroform extract of leaves of O. basilicum revealed higher level of activities for the enzymes. This indicated that there was an induced response in the tick, R. microplus against the toxic effects of the extract of O. basilicum.

Notum deacylates Wnts to suppress signalling activity

PubMed Central

Howell, Steve; Chang, Tao-Hsin; Liu, Yan; Feizi, Ten; Bineva, Ganka; O’Reilly, Nicola; Snijders, Ambrosius P.; Jones, E. Yvonne; Vincent, Jean-Paul

2015-01-01

Signalling by Wnts is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnts from the cell surface. However, this view fails to explain specificity since glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which likely help Notum colocalise with Wnts. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnts and thus constitutes the first known extracellular protein deacylase. PMID:25731175

Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase.

PubMed

Van Etten, R L; Waymack, P P

1991-08-01

The broad substrate specificity of a homogeneous isoenzyme of wheat germ acid phosphatase (WGAP) was extensively investigated by chromatographic, electrophoretic, NMR, and kinetic procedures. WGAP exhibited no divalent metal ion requirement and was unaffected upon incubation with EDTA or o-phenanthroline. A comparison of two catalytically homogeneous isoenzymes revealed little difference in substrate specificity. The specificity of WGAP was established by determining the Michaelis constants for a wide variety of substrates. p-Nitrophenyl phosphate, pyrophosphate, tripolyphosphate, and ATP were preferred substrates while lesser activities were seen toward sugar phosphates, trimetaphosphate, phosphoproteins, and (much less) phosphodiesters. An extensive table of Km and Vmax values is given. The pathway for the hydrolysis of trimetaphosphate was examined by colorimetric and 31P NMR methods and it was found that linear tripolyphosphate is not a free intermediate in the enzymatic reaction. In contrast to literature reports, homogeneous wheat germ acid phosphatase exhibits no measurable carboxylesterase activity, nor does it hydrolyze phenyl phosphonothioate esters or phytic acid at significant rates.

Protein-expression profiles in mouse blood-plasma following acute whole-body exposure to (137)Cs gamma rays.

PubMed

Rithidech, Kanokporn Noy; Honikel, Louise; Rieger, Robert; Xie, Weiping; Fischer, Thomas; Simon, Sanford R

2009-05-01

To compare the pattern of protein-expression profiles in blood-plasma after exposure of CBA/CaJ mice to 0 or 3 Gy of (137)Cs gamma rays. Two-dimensional electrophoresis gel coupled with mass spectrometry was used to analyze blood-samples collected at days 2 and 7 post-irradiation. At each sacrifice-time, alterations in expression-level of protein spots between control- and exposed-groups were analyzed statistically by the PDQuest software using Student's t-test (at the significance level of p < 0.05). Mass spectrometry was used to identify the identity of protein-spots with significantly altered expression-level. At day 2, 18 proteins were significantly up-regulated in exposed-mice. These included: alpha-2-Heremans-Schmid (HS)-glycoprotein, apolipoprotein (Apo)-AII-precursor, Apo-E, beta-2-glycoprotein-I, clusterin, fibrinogen-alpha-chain, fibrinogen-gamma-polypeptide, fetuin-B, haptoglobin, high-molecular-weight (HMW)-kininogen (Kng), low-MW-Kng, Kng1-precursor, liver-carboxylesterase-I-precursor, major-urinary-protein-6-precursor, mannose-binding-protein-C-precursor, mannose-binding-lectin-C, and prothrombin-precursor. Gelsolin was detected in control-mice only. At day 7, high expression-levels of 14 proteins were detected in control-mice (i.e., alpha-1-antitrypsin-precursor, carboxylesterase-N, cholesterol-7-alpha-hydroxylase, contraspin, coagulation-factor-II, coagulation-factor-XIII, gelsolin, immunoglobulin-G-heavy-chain, neurexin, prothrombin-precursor, protein-phosphatase, putative-calcium-influx-channel, vitamin-D-binding-protein, and 1110018G07Rik); while 15 proteins were highly expressed in exposed-mice. These included: alpha-1-acid-glycoprotein, alpha-2-HS-glycoprotein, alpha-1-protease-inhibitor-2, ApoA-IV, ApoC-I, ApoH, beta-1-globin, clusterin, complement-component-3, fibrinogen-beta-chain, HMW-Kng, major-histocompatibility-complex-class-Ia-H2-K, serine-(cysteine)-proteinase-inhibitor, retinoblastoma-associated-protein-140, and vascular

Design of Enzymatically Cleavable Prodrugs of a Potent Platinum-Containing Anticancer Agent

PubMed Central

Ding, Song; Pickard, Amanda J.; Kucera, Gregory L.

2014-01-01

Using a versatile synthetic approach, a new class of potential ester prodrugs of highly potent, but systemically too toxic, platinum–acridine anticancer agents was generated. The new hybrids contain a hydroxyl group, which has been masked with a cleavable lipophilic acyl moiety. Both butanoic (butyric) and bulkier 2-propanepentanoic (valproic) esters were introduced. The goals of this design were to improve the drug-like properties (e.g., logD) and to reduce the systemic toxicity of the pharmacophore. Two distinct pathways by which the target compounds undergo effective ester hydrolysis, the proposed activating step, have been confirmed: platinum-assisted, self-immolative ester cleavage in a low-chloride environment (LC-ESMS, NMR spectroscopy) and enzymatic cleavage by human carboxylesterase-2 (hCES-2) (LC-ESMS). The valproic acid ester derivatives are the first example of a metal-containing agent cleavable by the pro-drug-converting enzyme. They show excellent chemical stability and reduced systemic toxicity. Preliminary results from screening in lung adenocarcinoma cell lines (A549, NCI-H1435) suggest that the mechanism of the valproic esters may involve intracellular deesterification. PMID:25303639

Metabolomics identifies an inflammatory cascade involved in dioxin- and diet-induced steatohepatitis

PubMed Central

Matsubara, Tsutomu; Tanaka, Naoki; Krausz, Kristopher W.; Manna, Soumen K.; Kang, Dong Wook; Anderson, Erik R.; Luecke, Hans; Patterson, Andrew D.; Shah, Yatrik M.; Gonzalez, Frank J.

2012-01-01

Summary 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is among the most potent environmentally toxic compounds. Serum metabolomics identified azelaic acid-mono esters as significantly increased metabolites after TCDD treatment, due to down-regulation of hepatic carboxylesterase 3 (CES3, also known as triglyceride hydrolase) expression in an arylhydrocarbon receptor (AhR)-dependent manner in mice. The decreased CES3 expression was accomplished by TCDD-stimulated TGFβ-SMAD3 and IL6-STAT3 signaling, but not by direct AhR signaling. Methionine- and choline-deficient (MCD) diet-treated mice also showed enhanced serum azelaic acid-mono ester levels following attenuation of hepatic CES3 expression, while db/db mice did not, thus suggesting an association with steatohepatitis. Forced expression of CES3 reversed serum azelaic acid-mono ester/azelaic acid ratios and hepatic TGFβ mRNA levels in TCDD- and MCD diet-treated mice and ameliorated steatohepatitis induced by MCD diet. These results support the view that azelaic acid-mono esters are possible indicators of TCDD exposure and steatohepatitis, and suggest a link between CES3, TGFβ, and steatohepatitis. PMID:23140643

Influence of chronic oral intake of cannabis extract on oxidative and hydrolytic metabolism of xenobiotics in rat.

PubMed

Khanna, P; Gupta, M B; Gupta, G P; Sanwal, G G; Ali, B

1991-01-01

Dietary intake of petroleum ether extract of cannabis leaves by rats in doses of 158, 250 and 500 mg/kg in the first, second and third week, respectively, caused selective induction of hepatic microsomal carboxylesterases/amidases without affecting the renal hydrolytic activity. Acetanilide N-deacetylase, p-nitrophenylacetate (NPA) esterase and acetylsalicylic acid (ASA) esterase I and II (active at pH 5.5 and 7.4) were stimulated 125, 64, 82 and 60%, respectively, whereas the activities of procaine esterase and acetylaminofluorene (AAF) N-deacetylase remained unaltered. The hydrolysis of acetylcholine was also unchanged. Upon withdrawal of treatment microsomal hydrolytic activity receded to basal levels within 7 days. Curiously though, the two-fold induction of thiacetazone N-deacetylase (118%), a cytosolic hydrolase, remained largely undiminished (62%). An appraisal of the hepatic cytochrome P450 mediated oxidative metabolism revealed approximately three-fold induction of aromatic hydrocarbon hydroxylase (AHH) metabolizing benzo(a)pyrene whereas the N-demethylation of aminopyrene was unaffected. These activities were restored to normal when resin administration was discontinued.

A major urinary protein of the domestic cat regulates the production of felinine, a putative pheromone precursor.

PubMed

Miyazaki, Masao; Yamashita, Tetsuro; Suzuki, Yusuke; Saito, Yoshihiro; Soeta, Satoshi; Taira, Hideharu; Suzuki, Akemi

2006-10-01

Domestic cats spray urine with species-specific odor for territorial marking. Felinine (2-amino-7-hydroxy-5,5-dimethyl-4-thiaheptanoic acid), a putative pheromone precursor, is excreted in cat urine. Here, we report that cauxin, a carboxylesterase excreted as a major urinary component, regulates felinine production. In vitro enzyme assays indicated that cauxin hydrolyzed the felinine precursor 3-methylbutanol-cysteinylglycine to felinine and glycine. Cauxin and felinine were excreted age dependently after 3 months of age. The age-dependent increases in cauxin and felinine excretion were significantly correlated. In mature cats, cauxin and felinine levels were sex-dependently correlated and were higher in males than in females. In headspace gas of cat urine, 3-mercapto-3-methyl-1-butanol, 3-mercapto-3-methylbutyl formate, 3-methyl-3-methylthio-1-butanol, and 3-methyl-3-(2-methyldisulfanyl)-1-butanol were identified as candidates for felinine derivatives. These findings demonstrate that cauxin-dependent felinine production is a cat-specific metabolic pathway, and they provide information for the biosynthetic mechanisms of species-specific molecules in mammals.

A Characterization of Carboxylesterases in Rat and Guinea Pig - Their Heterogeneity and Role in Detoxication of Organophosphorus Compounds

DTIC Science & Technology

1993-09-01

those of plasma, are resistant to mol) from Amersham International (Amersham, inhibition by these very active anticholinesterases . U.K.); paraoxon...Subcutaneous and intraperitonal administration of paraoxon and DFP rapidly inhibited the CarbE activity in guinea pig plasma, much higher doses were...necessary to obtain a marked inhibition in lung and liver, and about 25% of (arbE activity in lung was resistant to inhibition. Gel filtration of lung homo

Liver-specific expression of carboxylesterase 1g/esterase-x reduces hepatic steatosis, counteracts dyslipidemia and improves insulin signaling.

PubMed

Bahitham, Wesam; Watts, Russell; Nelson, Randal; Lian, Jihong; Lehner, Richard

2016-05-01

Ces1g/Es-x deficiency in mice results in weight gain, insulin resistance, fatty liver and hyperlipidemia through upregulation of de novo lipogenesis and oversecretion of triacylglycerol (TG)-rich lipoproteins. Here, we show that restoration of Ces1g/Es-x expression only in the liver significantly reduced hepatic TG concentration accompanied by decreased size of lipid droplets, reduced secretion of very low-density lipoproteins and improved insulin-mediated signal transduction in the liver. Collectively, these results demonstrate that hepatic Ces1g/Es-x plays a critical role in limiting hepatic steatosis, very low-density lipoprotein assembly and in augmenting insulin sensitivity. Copyright © 2016 Elsevier B.V. All rights reserved.

Draft genome of the most devastating insect pest of coffee worldwide: the coffee berry borer, Hypothenemus hampei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vega, Fernando E.; Brown, Stuart M.; Chen, Hao

The coffee berry borer, Hypothenemus hampei, is the most economically important insect pest of coffee worldwide. We present an analysis of the draft genome of the coffee berry borer, the third genome for a Coleopteran species. The genome size is ca. 163 Mb with 19,222 predicted protein-coding genes. Analysis was focused on genes involved in primary digestion as well as gene families involved in detoxification of plant defense molecules and insecticides, such as carboxylesterases, cytochrome P450, gluthathione S-transferases, ATP-binding cassette transporters, and a gene that confers resistance to the insecticide dieldrin. A broad range of enzymes capable of degrading complexmore » polysaccharides were identified. We also evaluated the pathogen defense system and found homologs to antimicrobial genes reported in the Drosophila genome. Ten cases of horizontal gene transfer were identified with evidence for expression, integration into the H. hampei genome, and phylogenetic evidence that the sequences are more closely related to bacterial rather than eukaryotic genes. We find the draft genome analysis broadly expands our knowledge on the biology of a devastating tropical insect pest and suggests new pest management strategies.« less

Conjugates of γ-Carbolines and Phenothiazine as new selective inhibitors of butyrylcholinesterase and blockers of NMDA receptors for Alzheimer Disease.

PubMed

Makhaeva, Galina F; Lushchekina, Sofya V; Boltneva, Natalia P; Sokolov, Vladimir B; Grigoriev, Vladimir V; Serebryakova, Olga G; Vikhareva, Ekaterina A; Aksinenko, Alexey Yu; Barreto, George E; Aliev, Gjumrakch; Bachurin, Sergey O

2015-08-18

Alzheimer disease is a multifactorial pathology and the development of new multitarget neuroprotective drugs is promising and attractive. We synthesized a group of original compounds, which combine in one molecule γ-carboline fragment of dimebon and phenothiazine core of methylene blue (MB) linked by 1-oxo- and 2-hydroxypropylene spacers. Inhibitory activity of the conjugates toward acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and structurally close to them carboxylesterase (CaE), as well their binding to NMDA-receptors were evaluated in vitro and in silico. These newly synthesized compounds showed significantly higher inhibitory activity toward BChE with IC50 values in submicromolar and micromolar range and exhibited selective inhibitory action against BChE over AChE and CaE. Kinetic studies for the 9 most active compounds indicated that majority of them were mixed-type BChE inhibitors. The main specific protein-ligand interaction is π-π stacking of phenothiazine ring with indole group of Trp82. These compounds emerge as promising safe multitarget ligands for the further development of a therapeutic approach against aging-related neurodegenerative disorders such as Alzheimer and/or other pathological conditions.

Draft genome of the most devastating insect pest of coffee worldwide: the coffee berry borer, Hypothenemus hampei

PubMed Central

Vega, Fernando E.; Brown, Stuart M.; Chen, Hao; Shen, Eric; Nair, Mridul B.; Ceja-Navarro, Javier A.; Brodie, Eoin L.; Infante, Francisco; Dowd, Patrick F.; Pain, Arnab

2015-01-01

The coffee berry borer, Hypothenemus hampei, is the most economically important insect pest of coffee worldwide. We present an analysis of the draft genome of the coffee berry borer, the third genome for a Coleopteran species. The genome size is ca. 163 Mb with 19,222 predicted protein-coding genes. Analysis was focused on genes involved in primary digestion as well as gene families involved in detoxification of plant defense molecules and insecticides, such as carboxylesterases, cytochrome P450, gluthathione S-transferases, ATP-binding cassette transporters, and a gene that confers resistance to the insecticide dieldrin. A broad range of enzymes capable of degrading complex polysaccharides were identified. We also evaluated the pathogen defense system and found homologs to antimicrobial genes reported in the Drosophila genome. Ten cases of horizontal gene transfer were identified with evidence for expression, integration into the H. hampei genome, and phylogenetic evidence that the sequences are more closely related to bacterial rather than eukaryotic genes. The draft genome analysis broadly expands our knowledge on the biology of a devastating tropical insect pest and suggests new pest management strategies. PMID:26228545

The role of genetic background in susceptibility to chemical warfare nerve agents across rodent and non-human primate models.

PubMed

Matson, Liana M; McCarren, Hilary S; Cadieux, C Linn; Cerasoli, Douglas M; McDonough, John H

2018-01-15

Genetics likely play a role in various responses to nerve agent exposure, as genetic background plays an important role in behavioral, neurological, and physiological responses to environmental stimuli. Mouse strains or selected lines can be used to identify susceptibility based on background genetic features to nerve agent exposure. Additional genetic techniques can then be used to identify mechanisms underlying resistance and sensitivity, with the ultimate goal of developing more effective and targeted therapies. Here, we discuss the available literature on strain and selected line differences in cholinesterase activity levels and response to nerve agent-induced toxicity and seizures. We also discuss the available cholinesterase and toxicity literature across different non-human primate species. The available data suggest that robust genetic differences exist in cholinesterase activity, nerve agent-induced toxicity, and chemical-induced seizures. Available cholinesterase data suggest that acetylcholinesterase activity differs across strains, but are limited by the paucity of carboxylesterase data in strains and selected lines. Toxicity and seizures, two outcomes of nerve agent exposure, have not been fully evaluated for genetic differences, and thus further studies are required to understand baseline strain and selected line differences. Published by Elsevier B.V.

Determination of malathion and diazinon resistance by sequencing the Md alpha E7 gene from Guatemala, Colombia, Manhattan, and Thailand housefly (Musca domestica L.) strains.

PubMed

Taşkin, Vatan; Kence, Meral; Göçmen, Belgin

2004-04-01

Organophosphate (OP) insecticides (parathion/diazinon) resistance in housefly (Musca domestica L.) is associated with the change in carboxylesterase activity. The product of alpha E7 gene, which is a member of alpha-esterase gene cluster, is probably playing a role in detoxification of the xenobiotic esters. In parathion/diazinon resistant M. domestica species Gly137 to Asp substitution was found in the active center of the product of alpha E7 gene. In malathion (an OP) resistant M. domestica strains Trp251 to Ser substitution was identified in the active center of the Md alpha E7. In our research, to understand the allelic diversity of the Md alpha E7, the gene was partially sequenced from four different housefly strains from different localities (Guatemala, Manhattan (USA), Colombia (USA) and Thailand). It was found out that; in Thailand strain one allele has Cys residue at the position of 251, the other allele contains a Trp for the same site. In Colombia strain, one allele has Asp137, the other allele contains a Gly residue at this point. The Manhattan and Guatemala strains have Asp137 and Trp251 residues on their both alleles at these two different positions.

Draft genome of the most devastating insect pest of coffee worldwide: the coffee berry borer, Hypothenemus hampei

DOE PAGES

Vega, Fernando E.; Brown, Stuart M.; Chen, Hao; ...

2015-07-31

The coffee berry borer, Hypothenemus hampei, is the most economically important insect pest of coffee worldwide. We present an analysis of the draft genome of the coffee berry borer, the third genome for a Coleopteran species. The genome size is ca. 163 Mb with 19,222 predicted protein-coding genes. Analysis was focused on genes involved in primary digestion as well as gene families involved in detoxification of plant defense molecules and insecticides, such as carboxylesterases, cytochrome P450, gluthathione S-transferases, ATP-binding cassette transporters, and a gene that confers resistance to the insecticide dieldrin. A broad range of enzymes capable of degrading complexmore » polysaccharides were identified. We also evaluated the pathogen defense system and found homologs to antimicrobial genes reported in the Drosophila genome. Ten cases of horizontal gene transfer were identified with evidence for expression, integration into the H. hampei genome, and phylogenetic evidence that the sequences are more closely related to bacterial rather than eukaryotic genes. We find the draft genome analysis broadly expands our knowledge on the biology of a devastating tropical insect pest and suggests new pest management strategies.« less

Separating esterase targets of organophosphorus compounds in the brain by preparative chromatography.

PubMed

Mangas, I; Vilanova, E; Benabent, M; Estévez, J

2014-02-10

Low level exposure to organophosphorus esters (OPs) may cause long-term neurological effects and affect specific cognition domains in experimental animals and humans. Action on known targets cannot explain most of these effects by. Soluble carboxylesterases (EC 3.1.1.1) of chicken brain have been kinetically discriminated using paraoxon, mipafox and phenylmethyl sulfonylfluoride as inhibitors and phenyl valerate as a substrate. Three different enzymatic components were discriminated and called Eα, Eβ and Eγ. In this work, a fractionation procedure with various steps was developed using protein native separation methods by preparative HPLC. Gel permeation chromatography followed by ion exchange chromatography allowed enriched fractions with different kinetic behaviors. The soluble chicken brain fraction was fractionated, while total esterase activity, proteins and enzymatic components Eα, Eβ and Eγ were monitored in each subfraction. After the analysis, 13 fractions were pooled and conserved. Preincubation of the soluble chicken brain fraction of with the organophosphorus mipafox gave rise to a major change in the ion exchange chromatography profile, but not in the molecular exchanged chromatography profile, which suggest that mipafox permanently modifies the ionic properties of numerous proteins. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Metabolism of deltamethrin and cis- and trans-permethrin by rat and human liver microsomes, liver cytosol and plasma preparations.

PubMed

Hedges, Laura; Brown, Susan; Vardy, Audrey; Doyle, Edward; Yoon, Miyoung; Osimitz, Thomas G; Lake, Brian G

2018-04-19

The metabolism of deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in liver microsomes, liver cytosol and plasma from male Sprague-Dawley rats aged 15, 21 and 90 days and from adult humans. DLM and CPM were metabolised by rat hepatic microsomal cytochrome P450 (CYP) enzymes and to a lesser extent by microsomal and cytosolic carboxylesterase (CES) enzymes, whereas TPM was metabolised to a greater extent by CES enzymes. In human liver, DLM and TPM were mainly metabolised by CES enzymes, whereas CPM was metabolised by CYP and CES enzymes. The metabolism of pyrethroids by cytosolic CES enzymes contributes to the overall hepatic clearance of these compounds. DLM, CPM and TPM were metabolised by rat, but not human, plasma CES enzymes. This study demonstrates that the ability of male rats to metabolise DLM, CPM and TPM by hepatic CYP and CES enzymes and plasma CES enzymes increases with age. In all instances, apparent intrinsic clearance values were lower in 15 than in 90 day old rats. As pyrethroid-induced neurotoxicity is due to the parent compound, these results suggest that DLM, CPM and TPM may be more neurotoxic to juvenile than to adult rats.

De Novo Transcriptome Sequencing of Olea europaea L. to Identify Genes Involved in the Development of the Pollen Tube.

PubMed

Iaria, Domenico; Chiappetta, Adriana; Muzzalupo, Innocenzo

2016-01-01

In olive (Olea europaea L.), the processes controlling self-incompatibility are still unclear and the molecular basis underlying this process are still not fully characterized. In order to determine compatibility relationships, using next-generation sequencing techniques and a de novo transcriptome assembly strategy, we show that pollen tubes from different olive plants, grown in vitro in a medium containing its own pistil and in combination pollen/pistil from self-sterile and self-fertile cultivars, have a distinct gene expression profile and many of the differentially expressed sequences between the samples fall within gene families involved in the development of the pollen tube, such as lipase, carboxylesterase, pectinesterase, pectin methylesterase, and callose synthase. Moreover, different genes involved in signal transduction, transcription, and growth are overrepresented. The analysis also allowed us to identify members in actin and actin depolymerization factor and fibrin gene family and member of the Ca(2+) binding gene family related to the development and polarization of pollen apical tip. The whole transcriptomic analysis, through the identification of the differentially expressed transcripts set and an extended functional annotation analysis, will lead to a better understanding of the mechanisms of pollen germination and pollen tube growth in the olive.

Recovery study of cholinesterases and neurotoxic signs in the non-target freshwater invertebrate Chilina gibbosa after an acute exposure to an environmental concentration of azinphos-methyl.

PubMed

Cossi, Paula Fanny; Beverly, Boburg; Carlos, Luquet; Kristoff, Gisela

2015-10-01

Azinphos-methyl belongs to the class of organophosphate insecticides which are recognized for their anticholinesterase action. It is one of the most frequently used insecticides in the Upper Valley of Río Negro and Río Neuquén in Argentina, where agriculture represents the second most important economic activity. It has been detected in water from this North Patagonian region throughout the year and the maximum concentration found was 22.48 μg L(-1) during the application period. Chilina gibbosa is a freshwater gastropod widely distributed in South America, particularly in Patagonia, Argentina and in Southern Chile. Toxicological studies performed with C. gibbosa in our laboratory have reported neurotoxicity signs and cholinesterase inhibition after exposure to azinphos-methyl for 48 h. Recovery studies together with characterization of the enzyme and sensitivity of the enzyme to pesticides can improve the toxicological evaluation. However, little is known about recovery patterns in organisms exposed to organophosphates. The aim of the present work was to evaluate the recovery capacity (during 21 days in pesticide-free water) of cholinesterase activity and neurotoxicity in C. gibbosa after 48 h of exposure to azinphos-methyl. Also, lethality and carboxylesterase activity were registered during the recovery period. Regarding enzyme activities, after a 48-h exposure to 20 μg L(-1) of azinphos-methyl, cholinesterases showed an inhibition of 85% with respect to control, while carboxylesterases were not affected. After 21 days in pesticide-free water, cholinesterases continued to be inhibited (70%). Severe neurotoxicity signs were observed after exposure: 82% of the snails presented lack of adherence to vessels, 11% showed weak adherence, and 96% exhibited an abnormal protrusion of the head-foot region from shell. After 21 days in pesticide-free water, only 15% of the snails presented severe signs of neurotoxicity. However, during the recovery period significant

BmNPV resistance of silkworm larvae resulting from the ingestion of TiO₂ nanoparticles.

PubMed

Li, Bing; Xie, Yi; Cheng, Zhe; Cheng, Jie; Hu, Rengping; Gui, Suxin; Sang, Xuezi; Sun, Qingqing; Zhao, Xiaoyang; Sheng, Lei; Shen, Weide; Hong, Fashui

2012-12-01

Bombyx mori nucleopolyhedrovirus (BmNPV) causes infection in the silkworm that is often lethal. The infection is hard to prevent, partly because of the nature of the virus particles and partly because of the different strains of B. mori. Titanium dioxide nanoparticles (TiO₂ NPs) have been demonstrated to have antimicrobial properties. The present study investigated whether TiO₂ NPs added to an artificial diet can increase the resistance of B. mori larvae to BmNPV and examined the molecular mechanism behind any resistance shown. The results indicated that ingested TiO₂ NPs decreased reactive oxygen species and NO accumulation in B. mori larvae under BmNPV infection, which in turn led to a decrease in their growth inhibition and mortality. In addition, the TiO₂ NPs significantly promoted the expression of resistance-related genes, including those encoding superoxide dismutase, catalase, glutathione peroxidase, acetylcholine esterase, carboxylesterase, heat shock protein 21, glutathione S transferase o1, P53, and transferring and of genes encoding cytochrome p302 and nitric oxide synthase. These findings are a useful addition to the understanding of the mechanism of BmNPV resistance of B. mori larvae in response to TiO₂ NPs addition. Such information also provides a theoretical basis for the use of TiO₂ NPs in sericulture.

Hydrolytic Fate of 3/15-Acetyldeoxynivalenol in Humans: Specific Deacetylation by the Small Intestine and Liver Revealed Using in Vitro and ex Vivo Approaches

PubMed Central

Ajandouz, El Hassan; Berdah, Stéphane; Moutardier, Vincent; Bege, Thierry; Birnbaum, David Jérémie; Perrier, Josette; Di Pasquale, Eric; Maresca, Marc

2016-01-01

In addition to deoxynivalenol (DON), acetylated derivatives, i.e., 3-acetyl and 15-acetyldexynivalenol (or 3/15ADON), are present in cereals leading to exposure to these mycotoxins. Animal and human studies suggest that 3/15ADON are converted into DON after their ingestion through hydrolysis of the acetyl moiety, the site(s) of such deacetylation being still uncharacterized. We used in vitro and ex vivo approaches to study the deacetylation of 3/15ADON by enzymes and cells/tissues present on their way from the food matrix to the blood in humans. We found that luminal deacetylation by digestive enzymes and bacteria is limited. Using human cells, tissues and S9 fractions, we were able to demonstrate that small intestine and liver possess strong deacetylation capacity compared to colon and kidneys. Interestingly, in most cases, deacetylation was more efficient for 3ADON than 15ADON. Although we initially thought that carboxylesterases (CES) could be responsible for the deacetylation of 3/15ADON, the use of pure human CES1/2 and of CES inhibitor demonstrated that CES are not involved. Taken together, our original model system allowed us to identify the small intestine and the liver as the main site of deacetylation of ingested 3/15ADON in humans. PMID:27483321

Antifeedant Activity of Ginkgo biloba Secondary Metabolites against Hyphantria cunea Larvae: Mechanisms and Applications

PubMed Central

Ren, Lili; Chen, Fang; Feng, Yuqian

2016-01-01

Ginkgo biloba is a typical relic plant that rarely suffers from pest hazards. This study analyzed the pattern of G. biloba pest hazards in Beijing; tested the antifeedant activity of G. biloba extracts, including ginkgo flavonoids, ginkgolide, and bilobalide, against Hyphantria cunea larvae; determined the activities of glutathione transferase (GSTs), acetylcholinesterase (AChE), carboxylesterase (CarE) and mixed-functional oxidase (MFO), in larvae after feeding on these G. biloba secondary metabolites; and screened for effective botanical antifeedants in the field. In this study, no indicators of insect infestation were found for any of the examined leaves of G. biloba; all tested secondary metabolites showed significant antifeedant activity and affected the activity of the four larval detoxifying enzymes. Ginkgolide had the highest antifeedant activity and the most significant effect on the detoxifying enzymes (P<0.05). Spraying leaves with G. biloba extracts or ginkgolide both significantly repelled H. cunea larvae in the field (P<0.05), although the former is more economical and practical. This study investigated the antifeedant activity of G. biloba secondary metabolites against H. cunea larvae, and the results provide new insights into the mechanism of G. biloba pest resistance. This study also developed new applications of G. biloba secondary metabolites for effective pest control. PMID:27214257

Antifeedant Activity of Ginkgo biloba Secondary Metabolites against Hyphantria cunea Larvae: Mechanisms and Applications.

PubMed

Pan, Long; Ren, Lili; Chen, Fang; Feng, Yuqian; Luo, Youqing

2016-01-01

Ginkgo biloba is a typical relic plant that rarely suffers from pest hazards. This study analyzed the pattern of G. biloba pest hazards in Beijing; tested the antifeedant activity of G. biloba extracts, including ginkgo flavonoids, ginkgolide, and bilobalide, against Hyphantria cunea larvae; determined the activities of glutathione transferase (GSTs), acetylcholinesterase (AChE), carboxylesterase (CarE) and mixed-functional oxidase (MFO), in larvae after feeding on these G. biloba secondary metabolites; and screened for effective botanical antifeedants in the field. In this study, no indicators of insect infestation were found for any of the examined leaves of G. biloba; all tested secondary metabolites showed significant antifeedant activity and affected the activity of the four larval detoxifying enzymes. Ginkgolide had the highest antifeedant activity and the most significant effect on the detoxifying enzymes (P<0.05). Spraying leaves with G. biloba extracts or ginkgolide both significantly repelled H. cunea larvae in the field (P<0.05), although the former is more economical and practical. This study investigated the antifeedant activity of G. biloba secondary metabolites against H. cunea larvae, and the results provide new insights into the mechanism of G. biloba pest resistance. This study also developed new applications of G. biloba secondary metabolites for effective pest control.

Transcriptome mining: Multigene panel to test delousing drug response in the sea louse Caligus rogercresseyi.

PubMed

Valenzuela-Muñoz, V; Gallardo-Escárate, C

2016-02-01

Controlling infestations of copepodid ectoparasites in the salmon industry is increasingly problematic given higher instances of drug resistance or loss of sensitivity. Despite the importance of this issue, the molecular mechanisms and genes implicated in resistance/susceptibility are only scarcely understood. The objective of the present study was to identify and evaluate the expression levels of candidate genes associated with delousing drug response in the sea louse Caligus rogercresseyi. From RNA-seq data obtained for adult male and female sea lice, 62.48 M reads were assembled in 70,349 high-quality contigs. BLASTX analysis against UniprotKB/Swiss-Prot and the ESTs available for crustaceans in the NCBI database identified 870 transcripts previously related to genes associated with delousing drug response. Furthermore, 14 candidate genes were validated through RT-qPCR and were evaluated with deltamethrin and azamethiphos bioassays. The results evidenced an overregulation of genes involved in ion transport in salmon lice treated with deltamethrin, while those treated with azamethiphos evidenced an overregulation of genes such as cytochrome P450, Carboxylesterase, and acetylcholine receptors. The present study provides a multigene panel to test delousing drug response to pyrethroids and organophosphates in a highly prevalent pathogen of the Chilean salmon industry. Copyright © 2015 Elsevier B.V. All rights reserved.

Deep Sequencing of Pyrethroid-Resistant Bed Bugs Reveals Multiple Mechanisms of Resistance within a Single Population

PubMed Central

Adelman, Zach N.; Kilcullen, Kathleen A.; Koganemaru, Reina; Anderson, Michelle A. E.; Anderson, Troy D.; Miller, Dini M.

2011-01-01

A frightening resurgence of bed bug infestations has occurred over the last 10 years in the U.S. and current chemical methods have been inadequate for controlling this pest due to widespread insecticide resistance. Little is known about the mechanisms of resistance present in U.S. bed bug populations, making it extremely difficult to develop intelligent strategies for their control. We have identified bed bugs collected in Richmond, VA which exhibit both kdr-type (L925I) and metabolic resistance to pyrethroid insecticides. Using LD50 bioassays, we determined that resistance ratios for Richmond strain bed bugs were ∼5200-fold to the insecticide deltamethrin. To identify metabolic genes potentially involved in the detoxification of pyrethroids, we performed deep-sequencing of the adult bed bug transcriptome, obtaining more than 2.5 million reads on the 454 titanium platform. Following assembly, analysis of newly identified gene transcripts in both Harlan (susceptible) and Richmond (resistant) bed bugs revealed several candidate cytochrome P450 and carboxylesterase genes which were significantly over-expressed in the resistant strain, consistent with the idea of increased metabolic resistance. These data will accelerate efforts to understand the biochemical basis for insecticide resistance in bed bugs, and provide molecular markers to assist in the surveillance of metabolic resistance. PMID:22039447

Use of enzyme inhibitors to evaluate the conversion pathways of ester and amide prodrugs: a case study example with the prodrug ceftobiprole medocaril.

PubMed

Eichenbaum, Gary; Skibbe, Jennifer; Parkinson, Andrew; Johnson, Mark D; Baumgardner, Dawn; Ogilvie, Brian; Usuki, Etsuko; Tonelli, Fred; Holsapple, Jeff; Schmitt-Hoffmann, Anne

2012-03-01

An approach was developed that uses enzyme inhibitors to support the assessment of the pathways that are responsible for the conversion of intravenously administered ester and amide prodrugs in different biological matrices. The methodology was applied to ceftobiprole medocaril (BAL5788), the prodrug of the cephalosporin antibiotic, ceftobiprole. The prodrug was incubated in plasma, postmitochondrial supernatant fractions from human liver (impaired and nonimpaired), kidney, and intestine as well as erythrocytes, in the presence and absence of different enzyme inhibitors (acetylcholinesterase, pseudocholinesterase, retinyl palmitoyl hydrolase, serine esterases, amidases, and cholinesterase). Hydrolysis was rapid, extensive, and not dependent on the presence of β-nicotinamide-adenine dinucleotide phosphate (reduced form) in all matrices tested, suggesting the involvement of carboxylesterases but not P450 enzymes. Hydrolysis in healthy human plasma was rapid and complete and only partially inhibited in the presence of paraoxonase inhibitors or in liver from hepatic impaired patients, suggesting involvement of nonparaoxonase pathways. The results demonstrate the utility of this approach in confirming the presence of multiple conversion pathways of intravenously administered prodrugs and in the case of BAL5788 demonstrated that this prodrug is unlikely to be affected by genetic polymorphisms, drug interactions, or other environmental factors that might inhibit or induce the enzymes involved in its conversion. Copyright © 2011 Wiley Periodicals, Inc.

Influence of rice black streaked dwarf virus on the ecological fitness of non-vector planthopper Nilaparvata lugens (Hemiptera: Delphacidae).

PubMed

Xu, Hong-Xing; He, Xiao-Chan; Zheng, Xu-Song; Yang, Ya-Jun; Lu, Zhong-Xian

2014-08-01

Rice black streak dwarf virus (RBSDV) is transmitted by the small brown planthopper (SBPH), Laodelphax striatellus (Fallen). Non-vector rice brown planthopper (BPH), Nilaparvata lugens (Stål), shares the same host rice plants with SBPH in paddy fields. The changes in nutritional composition of rice plants infected by RBSDV and the ecological fitness of BPH feeding on the infected plants were studied under both artificial climate chamber and field conditions. Contents of 16 detected amino acids and soluble sugar in RBSDV infected rice plants were higher than those in the healthy ones. On the diseased plants BPH had significantly higher nymphal survival rates, nymphal duration of the males, weight of the female adults, as well as egg hatchability compared to BPH being fed on healthy plants. However, there was no obvious difference in female nymph duration, longevity and fecundity. Defense enzymes (superoxidase dismutase, SOD and catalase, CAT) and detoxifying enzymes (carboxylesterase, CAE and glutathione S-transferase, GST) in BPH adults fed on diseased plants had markedly higher activities. The results indicate rice plants infected by RBSDV improved the ecological fitness of the brown planthopper, a serious pest but not a transmitter of the RBSDV virus. © 2013 Institute of Zoology, Chinese Academy of Sciences.

RNA interference: Applications and advances in insect toxicology and insect pest management.

PubMed

Kim, Young Ho; Soumaila Issa, Moustapha; Cooper, Anastasia M W; Zhu, Kun Yan

2015-05-01

Since its discovery, RNA interference (RNAi) has revolutionized functional genomic studies due to its sequence-specific nature of post-transcriptional gene silencing. In this paper, we provide a comprehensive review of the recent literature and summarize the current knowledge and advances in the applications of RNAi technologies in the field of insect toxicology and insect pest management. Many recent studies have focused on identification and validation of the genes encoding insecticide target proteins, such as acetylcholinesterases, ion channels, Bacillus thuringiensis receptors, and other receptors in the nervous system. RNAi technologies have also been widely applied to reveal the role of genes encoding cytochrome P450 monooxygenases, carboxylesterases, and glutathione S-transferases in insecticide detoxification and resistance. More recently, studies have focused on understanding the mechanism of insecticide-mediated up-regulation of detoxification genes in insects. As RNAi has already shown great potentials for insect pest management, many recent studies have also focused on host-induced gene silencing, in which several RNAi-based transgenic plants have been developed and tested as proof of concept for insect pest management. These studies indicate that RNAi is a valuable tool to address various fundamental questions in insect toxicology and may soon become an effective strategy for insect pest management. Copyright © 2015 Elsevier Inc. All rights reserved.

Impact of insecticide exposure on the predation activity of the European earwig Forficula auricularia.

PubMed

Malagnoux, Laure; Capowiez, Yvan; Rault, Magali

2015-09-01

The European earwig Forficula auricularia is an effective predator in apple orchards. It is therefore crucial to study whether insecticides affect this natural pest control agent. Predation activity, i.e., the number of aphids eaten in 24 h, was determined under laboratory conditions after exposure of fourth-instar nymphs and adult earwigs to widely used insecticides (acetamiprid, chlorpyrifos-ethyl, deltamethrin, and spinosad), which were applied at the normal application rates. Inhibition of acetylcholinesterase and carboxylesterase activities were also measured as indicators of pesticide exposure. Predation activity decreased significantly in nymphs exposed to spinosad (62%) and chlorpyrifos-ethyl (98%) compared with controls. A similar response was found for both esterase activities. Spinosad had a stronger effect on AChE (-33%) whereas chlorpyrifos-ethyl affected CbE activity preferentially (-59%). Spinosad (20% of controls), acetamiprid (28%), and chlorpyrifos-ethyl (66%) also significantly decreased the predation behavior of adult male but not female (5 to 40%) earwigs. Adult AChE and CbE activities were also significantly reduced (28 to 67% of controls) in pesticide-exposed earwigs. Our results suggest that earwigs should be included in the environmental risk assessment framework for authorization of newly marketed plant protection products. Their predation behavior appears to be a sensitive and complementary biomarker.

Cross-induction of detoxification genes by environmental xenobiotics and insecticides in the mosquito Aedes aegypti: impact on larval tolerance to chemical insecticides.

PubMed

Poupardin, Rodolphe; Reynaud, Stéphane; Strode, Clare; Ranson, Hilary; Vontas, John; David, Jean-Philippe

2008-05-01

The effect of exposure of Aedes aegypti larvae to sub-lethal doses of the pyrethroid insecticide permethrin, the organophosphate temephos, the herbicide atrazine, the polycyclic aromatic hydrocarbon fluoranthene and the heavy metal copper on their subsequent tolerance to insecticides, detoxification enzyme activities and expression of detoxification genes was investigated. Bioassays revealed a moderate increase in larval tolerance to permethrin following exposure to fluoranthene and copper while larval tolerance to temephos increased moderately after exposure to atrazine, copper and permethrin. Cytochrome P450 monooxygenases activities were induced in larvae exposed to permethrin, fluoranthene and copper while glutathione S-transferase activities were induced after exposure to fluoranthene and repressed after exposure to copper. Microarray screening of the expression patterns of all detoxification genes following exposure to each xenobiotic with the Aedes Detox Chip identified multiple genes induced by xenobiotics and insecticides. Further expression studies using real-time quantitative PCR confirmed the induction of multiple CYP genes and one carboxylesterase gene by insecticides and xenobiotics. Overall, this study reveals the potential of xenobiotics found in polluted mosquito breeding sites to affect their tolerance to insecticides, possibly through the cross-induction of particular detoxification genes. Molecular mechanisms involved and impact on mosquito control strategies are discussed.

Does Resistance to Buprofezin Improve Heat and Cold Tolerance of Laodelphax striatellus (Hemiptera: Delphacidae)?

PubMed

Li, Yongteng; Zhang, Yueliang; Liu, Xiangdong; Guo, Huifang

2017-08-01

There is ample evidence that insecticide resistance causes fitness costs and benefits in pests, while the impact of insecticide resistance on thermotolerance of pests is mostly unclear. The Laodelphax striatellus (Fallén), is an important rice insect pest, which has developed resistance to buprofezin in China. Here, we investigated differences in heat tolerance and cold tolerance among L. striatellus lines with variable buprofezin resistance. The lethal time for 50% of the individuals to die (LT50) at 40 °C increased with an increase in buprofezin resistance level, whereas both the survival rate under -22 °C and the supercooling point of planthoppers did not differ significantly between resistant and susceptible strains. The metabolic enzyme carboxylesterase was found to have an association with buprofezin resistance. Our research showed that buprofezin resistance was positively related with heat tolerance in L. striatellus, but it had no effect on cold tolerance. Insecticide resistance in L. striatellus may therefore have broader implications for the ecology of L. striatellus, and the management of buprofezin resistance in this pest may be challenging. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

In Vivo and In Vitro Metabolites from the Main Diester and Monoester Diterpenoid Alkaloids in a Traditional Chinese Herb, the Aconitum Species

PubMed Central

Zhang, Min; Peng, Chong-sheng; Li, Xiao-bo

2015-01-01

Diester diterpenoid alkaloids (DDAs), such as aconitine (AC), mesaconitine (MA), and hypaconitine (HA), are both pharmacologically active compounds and toxic ingredients in a traditional Chinese herb, the Aconitum species. Many DDA metabolism studies have been performed to explore mechanisms for reducing toxicity in these compounds and in Aconitum species extracts for safe clinical administration. In this review, we summarize recent progress on the metabolism of toxic AC, MA, and HA and corresponding monoester diterpenoid alkaloids (MDAs) in the gastrointestinal tract and liver in different animal species and humans in vivo and/or in vitro, where these alkaloids are primarily metabolized by cytochrome P450 enzymes, carboxylesterases, and intestinal bacteria, which produces phase I metabolites, ester hydrolysed products, and lipoalkaloids. Furthermore, we classify metabolites detected in the blood and urine, where the aforementioned metabolites are absorbed and excreted. Less toxic MDAs and nontoxic alcohol amines are the primary DDA metabolites detected in the blood. Most other DDAs metabolites produced in the intestine and liver detected in the urine have not been reported in the blood. We propose an explanation for this nonconformity. Finally, taking AC, for instance, we generalize a process of toxicity reduction in the body after oral AC administration for the first time. PMID:25705235

Natural genomic amplification of cholinesterase genes in animals.

PubMed

Chatonnet, Arnaud; Lenfant, Nicolas; Marchot, Pascale; Selkirk, Murray E

2017-08-01

Tight control of the concentration of acetylcholine at cholinergic synapses requires precise regulation of the number and state of the acetylcholine receptors, and of the synthesis and degradation of the neurotransmitter. In particular, the cholinesterase activity has to be controlled exquisitely. In the genome of the first experimental models used (man, mouse, zebrafish and drosophila), there are only one or two genes coding for cholinesterases, whereas there are more genes for their closest relatives the carboxylesterases. Natural amplification of cholinesterase genes was first found to occur in some cancer cells and in insect species subjected to evolutionary pressure by insecticides. Analysis of the complete genome sequences of numerous representatives of the various metazoan phyla show that moderate amplification of cholinesterase genes is not uncommon in molluscs, echinoderms, hemichordates, prochordates or lepidosauria. Amplification of acetylcholinesterase genes is also a feature of parasitic nematodes or ticks. In these parasites, over-production of cholinesterase-like proteins in secreted products and the saliva are presumed to have effector roles related to host infection. These amplification events raise questions about the role of the amplified gene products, and the adaptation processes necessary to preserve efficient cholinergic transmission. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms. © 2017 International Society for Neurochemistry.

Biowaiver Monographs for Immediate-Release Solid Oral Dosage Forms: Enalapril.

PubMed

Verbeeck, Roger K; Kanfer, Isadore; Löbenberg, Raimar; Abrahamsson, Bertil; Cristofoletti, Rodrigo; Groot, D W; Langguth, Peter; Polli, James E; Parr, Alan; Shah, Vinod P; Mehta, Mehul; Dressman, Jennifer B

2017-08-01

Literature data relevant to the decision to allow a waiver of in vivo bioequivalence testing for the marketing authorization of immediate-release, solid oral dosage forms containing enalapril maleate are reviewed. Enalapril, a prodrug, is hydrolyzed by carboxylesterases to the active angiotensin-converting enzyme inhibitor enalaprilat. Enalapril as the maleate salt is shown to be highly soluble, but only 60%-70% of an orally administered dose of enalapril is absorbed from the gastrointestinal tract into the enterocytes. Consequently, enalapril maleate is a Biopharmaceutics Classification System class III substance. Because in situ conversion of the maleate salt to the sodium salt is sometimes used in production of the finished drug product, not every enalapril maleate-labeled finished product actually contains the maleate salt. Enalapril is not considered to have a narrow therapeutic index. With this background, a biowaiver-based approval procedure for new generic products or after major revisions to existing products is deemed acceptable, provided the in vitro dissolution of both test and reference preparation is very rapid (at least 85% within 15 min at pH 1.2, 4.5, and 6.8). Additionally, the test and reference product must contain the identical active drug ingredient. Copyright © 2017 American Pharmacists Association®. All rights reserved.

Computational design of a Diels-Alderase from a thermophilic esterase: the importance of dynamics

NASA Astrophysics Data System (ADS)

Linder, Mats; Johansson, Adam Johannes; Olsson, Tjelvar S. G.; Liebeschuetz, John; Brinck, Tore

2012-09-01

A novel computational Diels-Alderase design, based on a relatively rare form of carboxylesterase from Geobacillus stearothermophilus, is presented and theoretically evaluated. The structure was found by mining the PDB for a suitable oxyanion hole-containing structure, followed by a combinatorial approach to find suitable substrates and rational mutations. Four lead designs were selected and thoroughly modeled to obtain realistic estimates of substrate binding and prearrangement. Molecular dynamics simulations and DFT calculations were used to optimize and estimate binding affinity and activation energies. A large quantum chemical model was used to capture the salient interactions in the crucial transition state (TS). Our quantitative estimation of kinetic parameters was validated against four experimentally characterized Diels-Alderases with good results. The final designs in this work are predicted to have rate enhancements of ≈103-106 and high predicted proficiencies. This work emphasizes the importance of considering protein dynamics in the design approach, and provides a quantitative estimate of the how the TS stabilization observed in most de novo and redesigned enzymes is decreased compared to a minimal, `ideal' model. The presented design is highly interesting for further optimization and applications since it is based on a thermophilic enzyme ( T opt = 70 °C).

Endophytic fungi producing of esterases: Evaluation in vitro of the enzymatic activity using pH indicator

PubMed Central

Lisboa, Helen Cristina Fávero; Biasetto, Carolina Rabal; de Medeiros, João Batista; Araújo, Ângela Regina; Silva, Dulce Helena Siqueira; Teles, Helder Lopes; Trevisan, Henrique Celso

2013-01-01

A sensitive and efficient colorimetric method was optimized for detection of esterase enzymes produced by endophytic fungi for development of High-Throughput Screening (HTS). The fungi were isolated and obtained previously from plant species of Cerrado and Atlantic Forest located in areas of environmental preservation in the State of Sao Paulo / Brazil, as part of the project “Chemical and biological prospecting endophytic fungi associated to plant species of Cerrado and Atlantic Forest”. The compounds ethyl butyrate, ethyl acetate and methyl propionate were used as standards esters which were hydrolyzed by extracellular enzyme from endophytic fungi (EC. 3.1.1.1 - carboxyl-esterases) for production of carboxylic acids. Thus, the reduction of the pH increases the protonated indicator concentration (bromothymol blue), changing the color of the reaction medium (from blue to yellow), that can be observed and measured by spectrophotometry at 616 nm. The methodology with acid-base indicator was performed on 13 microorganisms, aiming Periconia atropurpurea as a potential source of esterase for biotransformation of short chain esters. The results also evidenced that this methodology showed to be efficient, fast, cheap, having low consumption of reagents and easy development, and can be applied to screen carboxylic-ester hydrolases in a large number of microorganisms. PMID:24516461

Molecular dynamics of detoxification and toxin-tolerance genes in brown planthopper (Nilaparvata lugens Stål., Homoptera: Delphacidae) feeding on resistant rice plants.

PubMed

Yang, Zhifan; Zhang, Futie; He, Qing; He, Guangcun

2005-06-01

To investigate the molecular response of brown planthopper, Nilaparvata lugens (BPH) to BPH-resistant rice plants, we isolated cDNA fragments of the genes encoding for carboxylesterase (CAR), trypsin (TRY), cytochrome P450 monooxygenase (P450), NADH-quinone oxidoreductase (NQO), acetylcholinesterase (ACE), and Glutathione S-transferase (GST). Expression profiles of the genes were monitored on fourth instar nymphs feeding on rice varieties with different resistance levels. Northern blot hybridization showed that, compared with BPH reared on susceptible rice TN1, expression of the genes for P450 and CAR was apparently up-regulated and TRY mRNA decreased in BPH feeding on a highly resistant rice line B5 and a moderately resistant rice variety MH63, respectively. Two transcripts of GST increased in BPH feeding on B5; but in BPH feeding on MH63, this gene was inducible and its expression reached a maximum level at 24 h, and then decreased slightly. The expression of NQO gene was enhanced in BPH on B5 plants but showed a constant expression in BPH on MH63 plants. No difference in ACE gene expression among BPH on different rice plants was detected by the RT-PCR method. The results suggest these genes may play important roles in the defense response of BPH to resistant rice.

Candidate Chemosensory Genes in the Stemborer Sesamia nonagrioides

PubMed Central

Glaser, Nicolas; Gallot, Aurore; Legeai, Fabrice; Montagné, Nicolas; Poivet, Erwan; Harry, Myriam; Calatayud, Paul-André; Jacquin-Joly, Emmanuelle

2013-01-01

The stemborer Sesamia nonagrioides is an important pest of maize in the Mediterranean Basin. Like other moths, this noctuid uses its chemosensory system to efficiently interact with its environment. However, very little is known on the molecular mechanisms that underlie chemosensation in this species. Here, we used next-generation sequencing (454 and Illumina) on different tissues from adult and larvae, including chemosensory organs and female ovipositors, to describe the chemosensory transcriptome of S. nonagrioides and identify key molecular components of the pheromone production and detection systems. We identified a total of 68 candidate chemosensory genes in this species, including 31 candidate binding-proteins and 23 chemosensory receptors. In particular, we retrieved the three co-receptors Orco, IR25a and IR8a necessary for chemosensory receptor functioning. Focusing on the pheromonal communication system, we identified a new pheromone-binding protein in this species, four candidate pheromone receptors and 12 carboxylesterases as candidate acetate degrading enzymes. In addition, we identified enzymes putatively involved in S. nonagrioides pheromone biosynthesis, including a ∆11-desaturase and different acetyltransferases and reductases. RNAseq analyses and RT-PCR were combined to profile gene expression in different tissues. This study constitutes the first large scale description of chemosensory genes in S. nonagrioides. PMID:23781142

Cholinesterases and neurotoxicity as highly sensitive biomarkers for an organophosphate insecticide in a freshwater gastropod (Chilina gibbosa) with low sensitivity carboxylesterases.

PubMed

Bianco, Karina; Yusseppone, María Soledad; Otero, Sofía; Luquet, Carlos; Ríos de Molina, María Del Carmen; Kristoff, Gisela

2013-11-15

In the Upper Valley of Río Negro and Río Neuquén in Argentina, agriculture represents the second most important economic activity. Azinphos-methyl has been found in water from this region throughout the year at a maximum concentration of 22.48 μg L(-1) during the application period. Toxicological studies on local non-target species have been performed mostly on vertebrates, while mollusks, which could be more sensitive, have not been studied so far. This work aims to characterize cholinesterase (ChE) and carboxilesterase (CE) activities of Chilina gibbosa, a freshwater gastropod native to southern Argentina and Chile. These enzymes, together with neurotoxicity signals, are evaluated herein after as sensitive biomarkers of exposure to azinphos-methyl at environmentally relevant concentrations. Effects of azinphos-methyl on antioxidant defenses: glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and glutathione S-transferase (GST) are also studied in order to complete a set of biomarkers with different sensitivity and specificity, to propose C. gibbosa as a sentinel species. The highest specific activity was obtained with acetylthiocholine as substrate, followed by propionylthiocholine (83% in comparison to acetylthiocholine) and butyrylthiocholine (19%).The lowest Km and the highest efficiency for ChE were obtained with acetylthiocholine. Regarding CEs activities, a higher efficiency was obtained with p-nitrophenyl butyrate than with p-nitrophenyl acetate. Eserine produced significant inhibition of ChE activity (81% with 0.001 mM and 98% with 1mM) while iso-OMPA did not produce any significant effect on ChE. Our results show that C. gibbosa ChE is very sensitive to azinphos-methyl (CI50 0.02 μg L(-1)) while CEs are inhibited at higher concentrations (CI50 1,000 μg L(-1)). CEs have been reported to be more sensitive to OPs than ChEs in most of the aquatic invertebrates protecting the organisms from neurotoxic effects. In contrast, C. gibbosa, has ChE which are much more sensitive to azinphos-methyl than CEs and shows marked signs of neurotoxicity. Regarding antioxidant defenses, GSH levels were significantly increased by 0.02 and 20 μg L(-1) azinphos-methyl (80 and 103%, respectively), CAT activity was increased 85% only at 0.02 μg L(-1) and SOD and GST did not show any significant response. Since ChE activity, neurotoxicity signs, GSH and CAT are sensitive biomarkers of acute exposure to azinphos-methyl at environmental concentrations C. gibbosa could be included as sentinel species in monitoring programs of pesticide hazard in regions of Argentina and Chile. Copyright © 2013 Elsevier B.V. All rights reserved.

Differential physiological effects of neonicotinoid insecticides on honey bees: A comparison between Apis mellifera and Apis cerana.

PubMed

Li, Zhiguo; Li, Meng; He, Jingfang; Zhao, Xiaomeng; Chaimanee, Veeranan; Huang, Wei-Fone; Nie, Hongyi; Zhao, Yazhou; Su, Songkun

2017-08-01

Acute toxicities (LD50s) of imidacloprid and clothianidin to Apis mellifera and A. cerana were investigated. Changing patterns of immune-related gene expressions and the activities of four enzymes between the two bee species were compared and analyzed after exposure to sublethal doses of insecticides. Results indicated that A. cerana was more sensitive to imidacloprid and clothianidin than A. mellifera. The acute oral LD50 values of imidacloprid and clothianidin for A. mellifera were 8.6 and 2.0ng/bee, respectively, whereas the corresponding values for A. cerana were 2.7 and 0.5ng/bee. The two bee species possessed distinct abilities to mount innate immune response against neonicotinoids. After 48h of imidacloprid treatment, carboxylesterase (CCE), prophenol oxidase (PPO), and acetylcholinesterase (AChE) activities were significantly downregulated in A. mellifera but were upregulated in A. cerana. Glutathione-S-transferase (GST) activity was significantly elevated in A. mellifera at 48h after exposure to imidacloprid, but no significant change was observed in A. cerana. AChE was downregulated in both bee species at three different time points during clothianidin exposure, and GST activities were upregulated in both species exposed to clothianidin. Different patterns of immune-related gene expression and enzymatic activities implied distinct detoxification and immune responses of A. cerana and A. mellifera to imidacloprid and clothianidin. Copyright © 2017 Elsevier Inc. All rights reserved.

Alterations in the skin of Labeo rohita exposed to an azo dye, Eriochrome black T: a histopathological and enzyme biochemical investigation.

PubMed

Srivastava, Ayan; Verma, Neeraj; Mistri, Arup; Ranjan, Brijesh; Nigam, Ashwini Kumar; Kumari, Usha; Mittal, Swati; Mittal, Ajay Kumar

2017-03-01

Histopathological changes and alterations in the activity of certain metabolic and antioxidant enzymes were analyzed in the head skin of Labeo rohita, exposed to sublethal test concentrations of the azo dye, Eriochrome black T for 4 days, using 24 h renewal bioassay method. Hypertrophied epithelial cells, increased density of mucous goblet cells, and profuse mucous secretion at the surface were considered to protect the skin from toxic impact of the azo dye. Degenerative changes including vacuolization, shrinkage, decrease in dimension, and density of club cells with simultaneous release of their contents in the intercellular spaces were associated to plug them, preventing indiscriminate entry of foreign matter. On exposure of fish to the dye, significant decline in the activity of enzymes-alkaline phosphatase, acid phosphatase, carboxylesterase, succinate dehydrogenase, catalase, and peroxidase-was associated with the binding of dye to the enzymes. Gradual increase in the activity of lactate dehydrogenase was considered to reflect a shift from aerobic to anaerobic metabolism. On transfer of azo dye exposed fish to freshwater, skin gradually recovers and, by 8 days, density and area of mucous goblet cells, club cells, and activity of the enzymes appear similar to that of controls. Alteration in histopathology and enzyme activity could be considered beneficial tool in monitoring environmental toxicity, valuable in the sustenance of fish populations.

Acetylcholinesterase Inhibitors: Pharmacology and Toxicology

PubMed Central

Čolović, Mirjana B; Krstić, Danijela Z; Lazarević-Pašti, Tamara D; Bondžić, Aleksandra M; Vasić, Vesna M

2013-01-01

Acetylcholinesterase is involved in the termination of impulse transmission by rapid hydrolysis of the neurotransmitter acetylcholine in numerous cholinergic pathways in the central and peripheral nervous systems. The enzyme inactivation, induced by various inhibitors, leads to acetylcholine accumulation, hyperstimulation of nicotinic and muscarinic receptors, and disrupted neurotransmission. Hence, acetylcholinesterase inhibitors, interacting with the enzyme as their primary target, are applied as relevant drugs and toxins. This review presents an overview of toxicology and pharmacology of reversible and irreversible acetylcholinesterase inactivating compounds. In the case of reversible inhibitors being commonly applied in neurodegenerative disorders treatment, special attention is paid to currently approved drugs (donepezil, rivastigmine and galantamine) in the pharmacotherapy of Alzheimer’s disease, and toxic carbamates used as pesticides. Subsequently, mechanism of irreversible acetylcholinesterase inhibition induced by organophosphorus compounds (insecticides and nerve agents), and their specific and nonspecific toxic effects are described, as well as irreversible inhibitors having pharmacological implementation. In addition, the pharmacological treatment of intoxication caused by organophosphates is presented, with emphasis on oxime reactivators of the inhibited enzyme activity administering as causal drugs after the poisoning. Besides, organophosphorus and carbamate insecticides can be detoxified in mammals through enzymatic hydrolysis before they reach targets in the nervous system. Carboxylesterases most effectively decompose carbamates, whereas the most successful route of organophosphates detoxification is their degradation by corresponding phosphotriesterases. PMID:24179466

Role of white adipose lipolysis in the development of NASH induced by methionine-and choline-deficient diet

PubMed Central

Tanaka, Naoki; Takahashi, Shogo; Fang, Zhong-Ze; Matsubara, Tsutomu; Krausz, Kristopher W.; Qu, Aijuan; Gonzalez, Frank J.

2014-01-01

Methionine- and choline-deficient diet (MCD) is a model for nonalcoholic steatohepatitis (NASH) in rodents. However, the mechanism of NASH development by dietary methionine/choline deficiency remains undetermined. To elucidate the early metabolic changes associated with MCD-NASH, serum metabolomic analysis was performed using mice treated with MCD and control diet for three days and one week, revealing significant increases in oleic and linoleic acids after MCD treatment. These increases were correlated with reduced body weight and white adipose tissue (WAT) mass, increased phosphorylation of hormone-sensitive lipase, and up-regulation of genes encoding carboxylesterase 3 and β2-adrenergic receptor in WAT, indicating accelerated lipolysis in adipocytes. The changes in serum fatty acids and WAT by MCD treatment were reversed by methionine supplementation, and similar alterations were detected in mice fed a methionine-deficient diet (MD), thus demonstrating that dietary methionine deficiency enhances lipolysis in WAT. MD treatment decreased glucose and increased fibroblast growth factor 21 in serum, thus exhibiting a similar metabolic phenotype as the fasting response. Comparison between MCD and choline-deficient diet (CD) treatments suggested that the addition of MD-induced metabolic alterations, such as WAT lipolysis, to CD-induced hepatic steatosis promotes liver injury. Collectively, these results demonstrate an important role for dietary methionine deficiency and WAT lipolysis in the development of MCD-NASH. PMID:25178843

Engineering Pseudomonas putida KT2440 for simultaneous degradation of organophosphates and pyrethroids and its application in bioremediation of soil.

PubMed

Zuo, Zhenqiang; Gong, Ting; Che, You; Liu, Ruihua; Xu, Ping; Jiang, Hong; Qiao, Chuanling; Song, Cunjiang; Yang, Chao

2015-06-01

Agricultural soils are usually co-contaminated with organophosphate (OP) and pyrethroid pesticides. To develop a stable and marker-free Pseudomonas putida for co-expression of two pesticide-degrading enzymes, we constructed a suicide plasmid with expression cassettes containing a constitutive promoter J23119, an OP-degrading gene (mpd), a pyrethroid-hydrolyzing carboxylesterase gene (pytH) that utilizes the upp gene as a counter-selectable marker for upp-deficient P. putida. By introduction of suicide plasmid and two-step homologous recombination, both mpd and pytH genes were integrated into the chromosome of a robust soil bacterium P. putida KT2440 and no selection marker was left on chromosome. Functional expression of mpd and pytH in P. putida KT2440 was demonstrated by Western blot analysis and enzyme activity assays. Degradation experiments with liquid cultures showed that the mixed pesticides including methyl parathion, fenitrothion, chlorpyrifos, permethrin, fenpropathrin, and cypermethrin (0.2 mM each) were degraded completely within 48 h. The inoculation of engineered strain (10(6) cells/g) to soils treated with the above mixed pesticides resulted in a higher degradation rate than in noninoculated soils. All six pesticides could be degraded completely within 15 days in fumigated and nonfumigated soils with inoculation. Theses results highlight the potential of the engineered strain to be used for in situ bioremediation of soils co-contaminated with OP and pyrethroid pesticides.

Metabolite profiling of carbamazepine and ibuprofen in Solea senegalensis bile using high-resolution mass spectrometry.

PubMed

Aceña, Jaume; Pérez, Sandra; Eichhorn, Peter; Solé, Montserrat; Barceló, Damià

2017-09-01

The widespread occurrence of pharmaceuticals in the aquatic environment has raised concerns about potential adverse effects on exposed wildlife. Very little is currently known on exposure levels and clearance mechanisms of drugs in marine fish. Within this context, our research was focused on the identification of main metabolic reactions, generated metabolites, and caused effects after exposure of fish to carbamazepine (CBZ) and ibuprofen (IBU). To this end, juveniles of Solea senegalensis acclimated to two temperature regimes of 15 and 20 °C for 60 days received a single intraperitoneal dose of these drugs. A control group was administered the vehicle (sunflower oil). Bile samples were analyzed by ultra-high-performance liquid chromatography-high-resolution mass spectrometry on a Q Exactive (Orbitrap) system, allowing to propose plausible identities for 11 metabolites of CBZ and 13 metabolites of IBU in fish bile. In case of CBZ metabolites originated from aromatic and benzylic hydroxylation, epoxidation, and ensuing O-glucuronidation, O-methylation of a catechol-like metabolite was also postulated. Ibuprofen, in turn, formed multiple hydroxyl metabolites, O-glucuronides, and (hydroxyl)-acyl glucuronides, in addition to several taurine conjugates. Enzymatic responses after drug exposures revealed a water temperature-dependent induction of microsomal carboxylesterases. The metabolite profiling in fish bile provides an important tool for pharmaceutical exposure assessment. Graphical abstract Studies of metabolism of carbamazepine and ibuprofen in fish.

The Peripheral Olfactory Repertoire of the Lightbrown Apple Moth, Epiphyas postvittana

PubMed Central

Thrimawithana, Amali H.; Crowhurst, Ross N.; Newcomb, Richard D.

2015-01-01

The lightbrown apple moth, Epiphyas postvittana is an increasingly global pest of horticultural crops. Like other moths, E. postvittana relies on olfactory cues to locate mates and oviposition sites. To detect these cues, moths have evolved families of genes encoding elements of the peripheral olfactory reception system, including odor carriers, receptors and degrading enzymes. Here we undertake a transcriptomic approach to identify members of these families expressed in the adult antennae of E. postvittana, describing open reading frames encoding 34 odorant binding proteins, 13 chemosensory proteins, 70 odorant receptors, 19 ionotropic receptors, nine gustatory receptors, two sensory neuron membrane proteins, 27 carboxylesterases, 20 glutathione-S-transferases, 49 cytochrome p450s and 18 takeout proteins. For the odorant receptors, quantitative RT-PCR corroborated RNAseq count data on steady state transcript levels. Of the eight odorant receptors that group phylogenetically with pheromone receptors from other moths, two displayed significant male-biased expression patterns, one displayed significant female-biased expression pattern and five were expressed equally in the antennae of both sexes. In addition, we found two male-biased odorant receptors that did not group with previously described pheromone receptors. This suite of olfaction-related genes provides a substantial resource for the functional characterization of this signal transduction system and the development of odor-mediated control strategies for horticultural pests. PMID:26017144

Simplified assays of lipolysis enzymes for drug discovery and specificity assessment of known inhibitors.

PubMed

Iglesias, Jose; Lamontagne, Julien; Erb, Heidi; Gezzar, Sari; Zhao, Shangang; Joly, Erik; Truong, Vouy Linh; Skorey, Kathryn; Crane, Sheldon; Madiraju, S R Murthy; Prentki, Marc

2016-01-01

Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. The glycerolipid/ fatty acid cycle, encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening, and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes, including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn-1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, α/β-hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purified enzymes. We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) also inhibit other lipases. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared with the corresponding mouse enzymes was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL, and the HSL inhibitors WWL11 and Compound 13f were effective against mouse enzyme but much less potent against human enzyme. Many of these lipase inhibitors also inhibited human CES1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

Simplified assays of lipolysis enzymes for drug discovery and specificity assessment of known inhibitors

PubMed Central

Iglesias, Jose; Lamontagne, Julien; Erb, Heidi; Gezzar, Sari; Zhao, Shangang; Joly, Erik; Truong, Vouy Linh; Skorey, Kathryn; Crane, Sheldon; Madiraju, S. R. Murthy; Prentki, Marc

2016-01-01

Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. The glycerolipid/ fatty acid cycle, encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening, and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes, including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn-1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, α/β-hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purified enzymes. We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) also inhibit other lipases. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared with the corresponding mouse enzymes was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL, and the HSL inhibitors WWL11 and Compound 13f were effective against mouse enzyme but much less potent against human enzyme. Many of these lipase inhibitors also inhibited human CES1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors. PMID:26423520

Higher capecitabine AUC in elderly patients with advanced colorectal cancer (SWOGS0030).

PubMed

Louie, S G; Ely, B; Lenz, H-J; Albain, K S; Gotay, C; Coleman, D; Raghavan, D; Shields, A F; Gold, P J; Blanke, C D

2013-10-01

The aging process is accompanied by physiological changes including reduced glomerular filtration and hepatic function, as well as changes in gastric secretions. To investigate what effect would aging have on the disposition of capecitabine and its metabolites, the pharmacokinetics between patients ≥70 years and <60 years were compared in SWOG0030. Twenty-nine unresectable colorectal cancer patients were stratified to either ≥70 or <60 years of age, where the disposition of capecitabine and its metabolites were compared. Notable increase in capecitabine area under the curve (AUC) was accompanied by reduction in capecitabine clearance in ≥70 years patients (P<0.05). No difference in 5'-deoxy-5-fluorocytidine, 5'-deoxy-5-fluorouridine (DFUR), and 5-fluorouracil (5FU) AUCs between the two age groups, suggesting that carboxylesterase and cytidine deaminase (CDA) activity was similar between the two age groups. These results suggest that metabolic enzymes involved in converting capecitabine metabolites are not altered by age. An elevation in capecitabine Cmax and reduction in clearance was seen in females, where capecitabine AUC was 40.3% higher in women. Elevation of DFUR Cmax (45%) and AUC (46%) (P<0.05) was also noted, suggesting that CDA activity may be higher in females. Increases in capecitabine Cmax and AUC was observed in patients ≥70 years when compared with younger patients who were >60 years.

B-esterase activities and blood cell morphology in the frog Leptodactylus chaquensis (Amphibia: Leptodactylidae) on rice agroecosystems from Santa Fe Province (Argentina).

PubMed

Attademo, Andrés M; Cabagna-Zenklusen, Mariana; Lajmanovich, Rafael C; Peltzer, Paola M; Junges, Celina; Bassó, Agustín

2011-01-01

Activity of B-esterases (BChE: butyrylcholinesterase and CbE: carboxylesterase using two model substrates: α-naphthyl acetate and 4-nitrophenyl valerate) in a native frog, Leptodactylus chaquensis from rice fields (RF1: methamidophos and RF2: cypermethrin and endosulfan sprayed by aircraft) and non-contaminated area (pristine forest) was measured. The ability of pyridine-2-aldoxime methochloride (2-PAM) to reactivate BChE levels was also explored. In addition, changes in blood cell morphology and parasite infection were determined. Mean values of plasma BChE activities were lower in samples from the two rice fields than in those from the reference site. CbE (4-nitrophenyl valerate) levels varied in the three sites studied, being highest in RF1. Frog plasma from RF1 showed positive reactivation of BChE activity after incubation with 2-PAM. Blood parameters of frogs from RF2 revealed morphological alterations (anisochromasia and immature erythrocytes frequency). Moreover, a major infection of protozoan Trypanosoma sp. in individuals from the two rice fields was detected. We suggest that integrated use of several biomarkers (BChE and CBEs, chemical reactivation of plasma with 2-PAM, and blood cell parameters) may be a promising procedure for use in biomonitoring programmes to diagnose pesticide exposure of wild populations of this frog and other native anuran species in Argentina.

Molecular characterisation of two α-esterase genes involving chlorpyrifos detoxification in the diamondback moth, Plutella xylostella.

PubMed

Xie, Miao; Ren, Na-Na; You, Yan-Chun; Chen, Wei-Jun; Song, Qi-Sheng; You, Min-Sheng

2017-06-01

Carboxylesterases (CarEs) are involved in metabolic detoxification of dietary and environmental xenobiotics in insects. However, owing to the complexity of the protein family, the involvement of CarEs in insecticide metabolism in Plutella xylostella has not been fully elucidated. This study aimed to characterise two CarE genes and assess their potential roles in response to chlorpyrifos in P. xylostella. Synergistic tests showed that triphenyl phosphate decreased the resistance of the third-instar larvae to chlorpyrifos. The treatment of the third-instar larvae with chlorpyrifos at the LC 30 dose led to a significant increase in CarE activity. Two CarE cDNAs (Pxae18 and Pxae28) were subsequently sequenced and characterised. Both genes were expressed predominantly in the larval midgut. Most importantly, two CarE genes showed significantly higher expression in the chlorpyrifos-resistant strain than in the susceptible strain. RNAi knockdown of Pxae18 and Pxae28 significantly increased the mortality to chlorpyrifos from 40% in the control to 73.8 and 63.3% respectively. RNAi knockdown of Pxae18 and Pxae28 significantly inhibited detoxification ability and increased the mortality in P. xylostella. The results indicate that these two CarE genes play important roles in the detoxification of chlorpyrifos in P. xylostella. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Human and soil exposure during mechanical chlorpyrifos, myclobutanil and copper oxychloride application in a peach orchard in Argentina.

PubMed

Berenstein, Giselle; Nasello, Soledad; Beiguel, Érica; Flores, Pedro; Di Schiena, Johanna; Basack, Silvana; Hughes, Enrique A; Zalts, Anita; Montserrat, Javier M

2017-05-15

The objective of this study was to measure the impact of the mechanized chlorpyrifos, copper oxychloride and myclobutanil application in a small peach orchard, on humans (operators, bystanders and residents) and on the productive soil. The mean Potential Dermal Exposure (PDE) of the workers (tractor drivers) was 30.8mL·h -1 ±16.4mL·h -1 , with no specific pesticide distribution on the laborers body. Although the Margin of Safety (MOS) factor for the application stage were above 1 (safe condition) for myclobutanil and cooper oxycloride it was below 1 for chlorpyrifos. The mix and load stage remained as the riskier operation. Pesticide found on the orchard soil ranged from 5.5% to 14.8% of the total chlorpyrifos, copper oxychloride and myclobutanil applied. Pesticide drift was experimentally measured, finding values in the range of 2.4% to 11.2% of the total pesticide applied. Using experimental drift values, bystander (for one application), resident (for 20 applications) and earthworm (for one application) risk indicators (RIs) were calculated for the chlorpyrifos plus copper oxychloride and for myclobutanil treatments for different distances to the orchard border. Earthworm RI was correlated with experimental Eisenia andrei ecotoxicological assays (enzymatic activities: cholinesterases, carboxylesterases and glutathione S-transferases; behavioral: avoidance and bait-lamina tests) with good correlation. Copyright © 2017 Elsevier B.V. All rights reserved.

Ethylphenidate as a selective dopaminergic agonist and methylphenidate-ethanol transesterification biomarker

PubMed Central

Patrick, Kennerly S.; Corbin, Timothy R.; Murphy, Cristina E.

2014-01-01

We review the pharmaceutical science of ethylphenidate (EPH) in the contexts of drug discovery; drug interactions; biomarker for dl-methylphenidate (MPH)-ethanol exposure; potentiation of dl-MPH abuse liability; contemporary “designer drug”; pertinence to the newer transdermal and chiral switch MPH formulations; as well as problematic internal standard. d-EPH selectively targets the dopamine transporter while d-MPH exhibits equipotent actions at dopamine and norepinephrine transporters. This selectivity carries implications for the advancement of tailored attention-deficit/hyperactivity disorder (ADHD) pharmacotherapy in the era of genome-based diagnostics. Abuse of dl-MPH often involves ethanol co-abuse. Carboxylesterase 1 enantioselectively transesterifies l-MPH with ethanol to yield l-EPH accompanied by significantly increased early exposure to d-MPH and rapid potentiation of euphoria. The pharmacokinetic component of this drug interaction can largely be avoided using dexmethylphenidate (dexMPH). This notwithstanding, maximal potentiated euphoria occurs following dexMPH-ethanol. C57BL/6 mice model dl-MPH-ethanol interactions: An otherwise depressive dose of ethanol synergistically increases dl-MPH stimulation; A sub-stimulatory dose of dl-MPH potentiates a low, stimulatory dose of ethanol; Ethanol elevates blood, brain and urinary d-MPH concentrations while forming l-EPH. Integration of EPH preclinical neuropharmacology with clinical studies of MPH-ethanol interactions provides a translational approach toward advancement of ADHD personalized medicine and management of comorbid alcohol use disorder. PMID:25303048

Enhancement of brain-targeting delivery of danshensu in rat through conjugation with pyrazine moiety to form danshensu-pyrazine ester.

PubMed

Hui, Ailing; Yin, Huayang; Zhang, Zheng; Zhou, An; Chen, Jingchao; Yang, Li; Wu, Zeyu; Zhang, Wencheng

2018-06-01

Tetramethylpyrazine was introduced to the structure of danshensu (DSS) as P-glycoprotein (P-gp)-inhibiting carrier, designing some novel brain-targeting DSS-pyrazine derivatives via prodrug delivery strategy. Following the virtual screening, three DSS-pyrazine esters (DT1, DT2, DT3) were selected because of their better prediction parameters related to brain-targeting. Among them, DT3 was thought to be a promising candidate due to its appropriate bioreversible property in vitro release assay. Further investigation with regard to DT3's brain-targeting effects in vivo was also reported in this study. High-performance liquid chromatography-diode array detection (HPLC-DAD) method was established for the quantitative determination of DT3 and DSS in rat plasma, brain homogenate after intravenous injection. In vivo metabolism of DT3 indicated that it was first converted into DT1, DT2, then the generation of DSS, which could be the result of carboxylesterase activity in rat blood and brain tissue. Moreover, the brain pharmacokinetics of DT3 was significantly altered with 2.16 times increase in half-life compared with that of DSS, and its drug targeting index (DTI) was up to 16.95. Above these data demonstrated that DT3 had better tendency of brain-targeting delivery, which would be positive for the treatment of brain-related disorders.

Calibration and validation of a physiologically based model for soman intoxication in the rat, marmoset, guinea pig and pig.

PubMed

Chen, Kaizhen; Seng, Kok-Yong

2012-09-01

A physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model has been developed for low, medium and high levels of soman intoxication in the rat, marmoset, guinea pig and pig. The primary objective of this model was to describe the pharmacokinetics of soman after intravenous, intramuscular and subcutaneous administration in the rat, marmoset, guinea pig, and pig as well as its subsequent pharmacodynamic effects on blood acetylcholinesterase (AChE) levels, relating dosimetry to physiological response. The reactions modelled in each physiologically realistic compartment are: (1) partitioning of C(±)P(±) soman from the blood into the tissue; (2) inhibition of AChE and carboxylesterase (CaE) by soman; (3) elimination of soman by enzymatic hydrolysis; (4) de novo synthesis and degradation of AChE and CaE; and (5) aging of AChE-soman and CaE-soman complexes. The model was first calibrated for the rat, then extrapolated for validation in the marmoset, guinea pig and pig. Adequate fits to experimental data on the time course of soman pharmacokinetics and AChE inhibition were achieved in the mammalian models. In conclusion, the present model adequately predicts the dose-response relationship resulting from soman intoxication and can potentially be applied to predict soman pharmacokinetics and pharmacodynamics in other species, including human. Copyright © 2011 John Wiley & Sons, Ltd.

A point mutation (L1015F) of the voltage-sensitive sodium channel gene associated with lambda-cyhalothrin resistance in Apolygus lucorum (Meyer-Dür) population from the transgenic Bt cotton field of China.

PubMed

Zhen, Congai; Gao, Xiwu

2016-02-01

In China, the green mirid bug, Apolygus lucorum (Meyer-Dür), has caused severe economic damage to many kinds of crops, especially the cotton and jujubes. Pyrethroid insecticides have been widely used for controlling this pest in the transgenic Bt cotton field. Five populations of A. lucorum collected from cotton crops at different locations in China were evaluated for lambda-cyhalothrin resistance. The results showed that only the population collected from Shandong Province exhibited 30-fold of resistance to lambda-cyhalothrin. Neither PBO nor DEF had obvious synergism when compared the synergistic ratio between SS and RR strain which was originated from the Shandong population. Besides, there were no statistically significant differences (p>0.05) in the carboxylesterase, glutathione S-transferase, or 7-ethoxycoumarin O-deethylase activities between the Shandong population and the laboratory susceptible strain (SS). The full-length sodium channel gene named AlVSSC encoding 2028 amino acids was obtained by RT-PCR and rapid amplification of cDNA ends (RACE). One single point mutation L1015F in the AlVSSC was detected only in the Shandong population. Our results revealed that the L1015F mutation associated with pyrethroid resistance was identified in A. lucorum populations in China. These results will be useful for the rational chemical control of A. lucorum in the transgenic Bt cotton field. Copyright © 2015 Elsevier Inc. All rights reserved.

9-Substituted acridine derivatives as acetylcholinesterase and butyrylcholinesterase inhibitors possessing antioxidant activity for Alzheimer's disease treatment.

PubMed

Makhaeva, Galina F; Lushchekina, Sofya V; Boltneva, Natalia P; Serebryakova, Olga G; Rudakova, Elena V; Ustyugov, Alexey A; Bachurin, Sergey O; Shchepochkin, Alexander V; Chupakhin, Oleg N; Charushin, Valery N; Richardson, Rudy J

2017-11-01

We investigated the inhibitory activity of 4 groups of novel acridine derivatives against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and carboxylesterase (CaE) using the methods of enzyme kinetics and molecular docking. Antioxidant activity of the compounds was determined using the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS + ) radical decolorization assay as their ability to scavenge free radicals. Analysis of the esterase profiles and antiradical activities of the acridine derivatives showed that 9-aryl(heteroaryl)-N-methyl-9,10-dihydroacridines have a high radical-scavenging activity but low potency as AChE and BChE inhibitors, whereas 9-aryl(heteroaryl)-N-methyl-acridinium tetrafluoroborates effectively inhibit cholinesterases but do not exhibit antiradical activity. In contrast, a group of derivatives of 9-heterocyclic amino-N-methyl-9,10-dihydroacridine has been found that combine effective inhibition of AChE and BChE with rather high radical-scavenging activity. The results of molecular docking well explain the observed features in the efficacy, selectivity, and mechanism of cholinesterase inhibition by the acridine derivatives. Thus, in a series of acridine derivatives we have found compounds possessing dual properties of effective and selective cholinesterase inhibition together with free radical scavenging, which makes promising the use of the acridine scaffold to create multifunctional drugs for the therapy of neurodegenerative diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Rhipicephalus (Boophilus) microplus strain Deutsch, 5 BAC clone sequencing, including two encoding Cytochrome P450s and one encoding CzEst9 carboxylesterase

USDA-ARS?s Scientific Manuscript database

The cattle tick, Rhipicephalus (Boophilus) microplus, has a genome over 2.4 times the size of the human genome, and with over 70% of repetitive DNA, this genome would prove very costly to sequence at today's prices and difficult to assemble and analyze. BAC clones give insight into the genome struct...

Determination of esterase activity and characterization of cholinesterases in the reef fish Haemulon plumieri.

PubMed

Leticia, Alpuche-Gual; Gerardo, Gold-Bouchot

2008-11-01

White grunt (Haemulon plumieri) has been proposed by the Mesoamerican Barrier Reef System (MBRS) Synoptic Monitoring Program as a bioindicator species. It is in this sense that the present study has a main goal to evaluate this organism's suitability as an indicator species. Individuals were captured during three seasons at the port of Sisal, Yucatan, Mexico which is located in an area that is considered to be weakly impacted by human activities such as agriculture or industry. Both cholinesterase (ChE) and carboxylesterase (CbE) activities were measured in brain, muscle, liver and eye of sampled individuals. Results indicated that ChE and CbE activities were greatest in the brain (256.3 ± 43) and in the liver (191 ± 21), respectively. Furthermore, ChEs detected in brain, liver and muscle were characterized, and results suggested that the acetylcholinesterase (AChE) type was more abundant relative to pseudocholinesterase (BChE) which was rare. In addition, K(m) and V(max) and IC(50) values were calculated from the Michaelis-Menten equation. Finally, an additional experiment in vitro showed a significant decrease in both ChE and CbE activities when different tissues were exposed to model xenobiotics, such as benzo[a]pyrene and Chlorpyrifos. In conclusion, findings from this study confirm the potential suitability of H. plumieri as an organic pollution bioindicator species, and thus of practical use for environmental biomonitoring purposes.

Cellular Transport of Esculin and Its Acylated Derivatives in Caco-2 Cell Monolayers and Their Antioxidant Properties in Vitro.

PubMed

Zhang, Mengmeng; Xin, Xuan; Lai, Furao; Zhang, Xiaoyuan; Li, Xiaofeng; Wu, Hui

2017-08-30

Esculin has many pharmacological effects, but these are difficult to observe after oral administration owing to poor lipid solubility. In our previous study, five acylated derivatives with different acyl chain lengths (EA, EP, EO, EL, and EM) were synthesized to improve the lipophilicity of esculin. In this study, the bioavailability and antioxidant activity of the five derivatives were investigated. The logP of esculin, EA, EP, EO, EL, and EM were -1.1 ± 0.1, -0.3 ± 0.14, 0.1 ± 0.17, 1.6 ± 0.09, 2.4 ± 0.11, and 2.8 ± 0.18, and their P app were 0.71 ± 0.02, 1.24 ± 0.18, 1.74 ± 0.11, 11.6 ± 3.6, 4.11 ± 1.03, and 2.64 ± 0.97 × 10 -6 cm/s, respectively. Besides, the bioavailability of EO, EL, and EM were seriously affected by carboxylesterase. The results of ABTS, ORAC, and DPPH assays indicated that the antiradical ability of the five derivatives did not exceed that of esculin. However, EA, EP, and EO showed more effective inhibition of AAPH-induced oxidative hemolysis than esculin did (p < 0.05), and EL and EM were less effective than esculin (p < 0.05). The mechanism was related to the distribution and localization of the derivatives in "oil-water interface" between the cytomembrane and the aqueous phase.

Evaluation of enzymes inhibition activities of medicinal plant from Burkina Faso.

PubMed

Bangou, Mindiédiba Jean; Kiendrebeogo, Martin; Meda, Nâg-Tiero Roland; Coulibaly, Ahmed Yacouba; Compaoré, Moussa; Zeba, Boukaré; Millogo-Rasolodimby, Jeanne; Nacoulma, Odile Germaine

2011-01-15

The aim of the present study was to evaluate some enzymes inhibitory effects of 11 plant species belonging to 9 families from Burkina Faso. Methanolic extracts were used for their Glutathione-s-transferase (GST), Acetylcholinesterase (AChE), Carboxylesterase (CES) and Xanthine Oxidase (XO) inhibitory activities at final concentration of 100 microg mL(-1). The total phenolics, flavonoids and tannins were also determined spectrophotometrically using Folin-Ciocalteu, AlCl3 and ammonium citrate iron reagents, respectively. Among the 11 species tested, the best inhibitory percentages were found with Euphorbia hirta, Sclerocarya birrea and Scoparia dulcis (inhibition > 40%) followed by Annona senegalensis, Annona squamosa, Polygala arenaria and Ceratotheca sesamoides (inhibition > 25%). The best total phenolic and tannin contents were found with S. birrea with 56.10 mg GAE/100 mg extract and 47.75 mg TAE/100 mg extract, respectively. E hirta presented the higher total flavonoids (9.96 mg QE/100 mg extract). It's was found that Sclerocarya birrea has inhibited all enzymes at more than 30% and this activity is correlated to total tannins contents. Contrary to S. birrea, the enzymatic activities of E. hirta and S. dulcis are correlated to total flavonoids contents. Present findings suggest that the methanolic extracts of those plant species are potential inhibitors of GST, AChE, CES and XO and confirm their traditional uses in the treatment of mental disorders, gout, painful inflammations and cardiovascular diseases.

Carbamate and Pyrethroid Resistance in the Akron Strain of Anopheles gambiae

PubMed Central

Mutunga, James M.; Anderson, Troy D.; Craft, Derek T.; Gross, Aaron D.; Swale, Daniel R.; Tong, Fan; Wong, Dawn M.; Carlier, Paul R.; Bloomquist, Jeffrey R.

2015-01-01

Insecticide resistance in the malaria vector, Anopheles gambiae is a serious problem, epitomized by the multi-resistant Akron strain, originally isolated in the country of Benin. Here we report resistance in this strain to pyrethroids and DDT (13-fold to 35-fold compared to the susceptible G3 strain), but surprisingly little resistance to etofenprox, a compound sometimes described as a “pseudo-pyrethroid.” There was also strong resistance to topically-applied commercial carbamates (45-fold to 81-fold), except for the oximes aldicarb and methomyl. Biochemical assays showed enhanced cytochrome P450 monooxygenase and carboxylesterase activity, but not that of glutathione-S-transferase. A series of substituted α,α,α,-trifluoroacetophenone oxime methylcarbamates were evaluated for enzyme inhibition potency and toxicity against G3 and Akron mosquitoes. The compound bearing an unsubstituted phenyl ring showed the greatest toxicity to mosquitoes of both strains. Low cross resistance in Akron was retained by all analogs in the series. Kinetic analysis of acetylcholinesterase activity and its inhibition by insecticides in the G3 strain showed inactivation rate constants greater than that of propoxur, and against Akron enzyme inactivation rate constants similar to that of aldicarb. However, inactivation rate constants against recombinant human AChE were essentially identical to that of the G3 strain. Thus, the acetophenone oxime carbamates described here, though potent insecticides that control resistant Akron mosquitoes, require further structural modification to attain acceptable selectivity and human safety. PMID:26047119

A Linkage Map and QTL Analysis for Pyrethroid Resistance in the Bed Bug Cimex lectularius

PubMed Central

Fountain, Toby; Ravinet, Mark; Naylor, Richard; Reinhardt, Klaus; Butlin, Roger K.

2016-01-01

The rapid evolution of insecticide resistance remains one of the biggest challenges in the control of medically and economically important pests. Insects have evolved a diverse range of mechanisms to reduce the efficacy of the commonly used classes of insecticides, and finding the genetic basis of resistance is a major aid to management. In a previously unstudied population, we performed an F2 resistance mapping cross for the common bed bug, Cimex lectularius, for which insecticide resistance is increasingly widespread. Using 334 SNP markers obtained through RAD-sequencing, we constructed the first linkage map for the species, consisting of 14 putative linkage groups (LG), with a length of 407 cM and an average marker spacing of 1.3 cM. The linkage map was used to reassemble the recently published reference genome, facilitating refinement and validation of the current genome assembly. We detected a major QTL on LG12 associated with insecticide resistance, occurring in close proximity (1.2 Mb) to a carboxylesterase encoding candidate gene for pyrethroid resistance. This provides another example of this candidate gene playing a major role in determining survival in a bed bug population following pesticide resistance evolution. The recent availability of the bed bug genome, complete with a full list of potential candidate genes related to insecticide resistance, in addition to the linkage map generated here, provides an excellent resource for future research on the development and spread of insecticide resistance in this resurging pest species. PMID:27733453

The central role of mosquito cytochrome P450 CYP6Zs in insecticide detoxification revealed by functional expression and structural modelling.

PubMed

Chandor-Proust, Alexia; Bibby, Jaclyn; Régent-Kloeckner, Myriam; Roux, Jessica; Guittard-Crilat, Emilie; Poupardin, Rodolphe; Riaz, Muhammad Asam; Paine, Mark; Dauphin-Villemant, Chantal; Reynaud, Stéphane; David, Jean-Philippe

2013-10-01

The resistance of mosquitoes to chemical insecticides is threatening vector control programmes worldwide. Cytochrome P450 monooxygenases (CYPs) are known to play a major role in insecticide resistance, allowing resistant insects to metabolize insecticides at a higher rate. Among them, members of the mosquito CYP6Z subfamily, like Aedes aegypti CYP6Z8 and its Anopheles gambiae orthologue CYP6Z2, have been frequently associated with pyrethroid resistance. However, their role in the pyrethroid degradation pathway remains unclear. In the present study, we created a genetically modified yeast strain overexpressing Ae. aegypti cytochrome P450 reductase and CYP6Z8, thereby producing the first mosquito P450-CPR (NADPH-cytochrome P450-reductase) complex in a yeast recombinant system. The results of the present study show that: (i) CYP6Z8 metabolizes PBAlc (3-phenoxybenzoic alcohol) and PBAld (3-phenoxybenzaldehyde), common pyrethroid metabolites produced by carboxylesterases, producing PBA (3-phenoxybenzoic acid); (ii) CYP6Z8 transcription is induced by PBAlc, PBAld and PBA; (iii) An. gambiae CYP6Z2 metabolizes PBAlc and PBAld in the same way; (iv) PBA is the major metabolite produced in vivo and is excreted without further modification; and (v) in silico modelling of substrate-enzyme interactions supports a similar role of other mosquito CYP6Zs in pyrethroid degradation. By playing a pivotal role in the degradation of pyrethroid insecticides, mosquito CYP6Zs thus represent good targets for mosquito-resistance management strategies.

Mechanisms on boron-induced alleviation of aluminum-toxicity in Citrus grandis seedlings at a transcriptional level revealed by cDNA-AFLP analysis.

PubMed

Zhou, Xin-Xing; Yang, Lin-Tong; Qi, Yi-Ping; Guo, Peng; Chen, Li-Song

2015-01-01

The physiological and biochemical mechanisms on boron (B)-induced alleviation of aluminum (B)-toxicity in plants have been examined in some details, but our understanding of the molecular mechanisms underlying these processes is very limited. In this study, we first used the cDNA-AFLP to investigate the gene expression patterns in Citrus grandis roots responsive to B and Al interactions, and isolated 100 differentially expressed genes. Results showed that genes related to detoxification of reactive oxygen species (ROS) and aldehydes (i.e., glutathione S-transferase zeta class-like isoform X1, thioredoxin M-type 4, and 2-alkenal reductase (NADP+-dependent)-like), metabolism (i.e., carboxylesterases and lecithin-cholesterol acyltransferase-like 4-like, nicotianamine aminotransferase A-like isoform X3, thiosulfate sulfurtransferase 18-like isoform X1, and FNR, root isozyme 2), cell transport (i.e., non-specific lipid-transfer protein-like protein At2g13820-like and major facilitator superfamily protein), Ca signal and hormone (i.e., calcium-binding protein CML19-like and IAA-amino acid hydrolase ILR1-like 4-like), gene regulation (i.e., Gag-pol polyprotein) and cell wall modification (i.e., glycosyl hydrolase family 10 protein) might play a role in B-induced alleviation of Al-toxicity. Our results are useful not only for our understanding of molecular processes associated with B-induced alleviation of Al-toxicity, but also for obtaining key molecular genes to enhance Al-tolerance of plants in the future.

Mechanisms on Boron-Induced Alleviation of Aluminum-Toxicity in Citrus grandis Seedlings at a Transcriptional Level Revealed by cDNA-AFLP Analysis

PubMed Central

Zhou, Xin-Xing; Yang, Lin-Tong; Qi, Yi-Ping; Guo, Peng; Chen, Li-Song

2015-01-01

The physiological and biochemical mechanisms on boron (B)-induced alleviation of aluminum (B)-toxicity in plants have been examined in some details, but our understanding of the molecular mechanisms underlying these processes is very limited. In this study, we first used the cDNA-AFLP to investigate the gene expression patterns in Citrus grandis roots responsive to B and Al interactions, and isolated 100 differentially expressed genes. Results showed that genes related to detoxification of reactive oxygen species (ROS) and aldehydes (i.e., glutathione S-transferase zeta class-like isoform X1, thioredoxin M-type 4, and 2-alkenal reductase (NADP+-dependent)-like), metabolism (i.e., carboxylesterases and lecithin-cholesterol acyltransferase-like 4-like, nicotianamine aminotransferase A-like isoform X3, thiosulfate sulfurtransferase 18-like isoform X1, and FNR, root isozyme 2), cell transport (i.e., non-specific lipid-transfer protein-like protein At2g13820-like and major facilitator superfamily protein), Ca signal and hormone (i.e., calcium-binding protein CML19-like and IAA-amino acid hydrolase ILR1-like 4-like), gene regulation (i.e., Gag-pol polyprotein) and cell wall modification (i.e., glycosyl hydrolase family 10 protein) might play a role in B-induced alleviation of Al-toxicity. Our results are useful not only for our understanding of molecular processes associated with B-induced alleviation of Al-toxicity, but also for obtaining key molecular genes to enhance Al-tolerance of plants in the future. PMID:25747450

Anti-dengue efficacy of bioactive andrographolide from Andrographis paniculata (Lamiales: Acanthaceae) against the primary dengue vector Aedes aegypti (Diptera: Culicidae).

PubMed

Edwin, Edward-Sam; Vasantha-Srinivasan, Prabhakaran; Senthil-Nathan, Sengottayan; Thanigaivel, Annamalai; Ponsankar, Athirstam; Pradeepa, Venkatraman; Selin-Rani, Selvaraj; Kalaivani, Kandaswamy; Hunter, Wayne B; Abdel-Megeed, Ahmed; Duraipandiyan, Veeramuthu; Al-Dhabi, Naif Abdullah

2016-11-01

The current study investigated the toxic effect of the leaf extract compound andrographolide from Andrographis paniculata (Burm.f) against the dengue vector Ae. aegypti. GC-MS analysis revealed that andrographolide was recognized as the major chemical constituent with the prominent peak area compared with other compounds. All isolated toxic compounds were purified and confirmed through RP-HPLC against chemical standards. The larvicidal assays established at 25ppm of bioactive compound against the treated instars of Ae. Aegypti showed prominent mortality compared to other treated concentrations. The percent mortality of larvae was directly proportional to concentration. The lethal concentration (LC50) was observed at 12ppm treatment concentration. The bioactive andrographolide considerably reduced the detoxifying enzyme regulations of α- and β- carboxylesterases. In contrast, the levels of GST and CYP450 significantly increase in a dose dependent manner. The andrographolide also showed strong oviposition deterrence effects at the sub-lethal dose of 12ppm. Similarly, the mean number of eggs were also significantly reduced in a dose dependent manner. At the concentration of 12ppm the effective percentage of repellency was greater than 90% with a protection time of 15-210min, compared with control. The histopathology study displayed that larvae treated with bioactive andrographolide had cytopathic effects in the midgut epithelium compared with the control. The present study established that bioactive andrographolide served as a potential useful for dengue vector management. Copyright © 2016 Elsevier B.V. All rights reserved.

Biotransformation and bioactivation reactions - 2015 literature highlights.

PubMed

Baillie, Thomas A; Dalvie, Deepak; Rietjens, Ivonne M C M; Cyrus Khojasteh, S

2016-05-01

Since 1972, Drug Metabolism Reviews has been recognized as one of the principal resources for researchers in pharmacological, pharmaceutical and toxicological fields to keep abreast of advances in drug metabolism science in academia and the pharmaceutical industry. With a distinguished list of authors and editors, the journal covers topics ranging from relatively mature fields, such as cytochrome P450 enzymes, to a variety of emerging fields. We hope to continue this tradition with the current compendium of mini-reviews that highlight novel biotransformation processes that were published during the past year. Each review begins with a summary of the article followed by our comments on novel aspects of the research and their biological implications. This collection of highlights is not intended to be exhaustive, but rather to be illustrative of recent research that provides new insights or approaches that advance the field of drug metabolism. Abbreviations NAPQI N-acetyl-p-benzoquinoneimine ALDH aldehyde dehydrogenase AO aldehyde oxidase AKR aldo-keto reductase CES carboxylesterase CSB cystathionine β-synthase CSE cystathionine γ-lyase P450 cytochrome P450 DHPO 2,3-dihydropyridin-4-one ESI electrospray FMO flavin monooxygenase GSH glutathione GSSG glutathione disulfide ICPMS inductively coupled plasma mass spectrometry i.p. intraperitoneal MDR multidrug-resistant NNAL 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone oaTOF orthogonal acceleration time-of-flight PBK physiologically based kinetic PCP pentachlorophenol SDR short-chain dehydrogenase/reductase SULT sulfotransferase TB tuberculosis.

Molecular and biochemical characterization of a sand fly population from Sri Lanka: evidence for insecticide resistance due to altered esterases and insensitive acetylcholinesterase.

PubMed

Surendran, S N; Karunaratne, S H P P; Adams, Z; Hemingway, J; Hawkes, N J

2005-08-01

With an increasing incidence of cutaneous leishmaniasis in Sri Lanka, particularly in northern provinces, insecticide-mediated vector control is under consideration. Optimizing such a strategy requires the characterization of sand fly populations in target areas with regard to species composition and extant resistance, among other parameters. Sand flies were collected by human bait and cattle-baited net traps on Delft Island, used as an illegal transit location by many refugees returning to the north of Sri Lanka from southern India where leishmaniasis is endemic. For species identification, genomic DNA was extracted and a fragment of the ribosomal 18S gene amplified. The sequence from all flies analysed matched that of Phlebotomus argentipes Annandale & Brunetti, the primary vector in India and the most likely vector in Sri Lanka. Independent morphological analysis also identified P. argentipes. To establish the current susceptibility status of vector species, data were obtained at the biochemical level, from which potential cross-resistance to alternative insecticides can be predicted. The Delft Island collection was assayed for the activities of four enzyme systems involved in insecticide resistance (acetylcholinesterase, non-specific carboxylesterases, glutathione-S-transferases and cytochrome p450 monooxygenases), establishing baselines against which subsequent collections can be evaluated. There was preliminary evidence for elevated esterases and altered acetylcholinesterase in this population, the first report of these resistance mechanisms in sand flies to our knowledge, which probably arose from the malathion-based spraying regimes of the Anti-Malarial Campaign.

Identification and characterization of an esterase involved in malathion resistance in the head louse Pediculus humanus capitis.

PubMed

Kwon, Deok Ho; Kim, Ju Hyeon; Kim, Young Ho; Yoon, Kyong Sup; Clark, J Marshall; Lee, Si Hyeock

2014-06-01

Enhanced malathion carboxylesterase (MCE) activity was previously reported to be involved in malathion resistance in the head louse Pediculus humanus capitis (Gao et al., 2006 [8]). To identify MCE, the transcriptional profiles of all five esterases that had been annotated to be catalytically active were determined and compared between the malathion-resistant (BR-HL) and malathion-susceptible (KR-HL) strains of head lice. An esterase gene, designated HLCbE3, exhibited approximately 5.4-fold higher transcription levels, whereas remaining four esterases did not exhibit a significant increase in their transcription in BR-HL, indicating that HLCbE3 may be the putative MCE. Comparison of the entire cDNA sequences of HLCbE3 revealed no sequence differences between the BR-HL and KR-HL strains and suggested that no single nucleotide polymorphism is associated with enhanced MCE activity. Two copies of the HLCbE3 gene were observed in BR-HL, implying that the over-transcription of HLCbE3 is due to the combination of a gene duplication and up-regulated transcription. Knockdown of HLCbE3 expression by RNA interference in the BR-HL strain led to increases in malathion susceptibility, confirming the identity of HLCbE3 as a MCE responsible for malathion resistance in the head louse. Phylogenetic analysis suggested that HLCbE3 is a typical dietary esterase and belongs to a clade containing various MCEs involved in malathion resistance. Copyright © 2014 Elsevier Inc. All rights reserved.

Effect of methylparaben in Artemia franciscana.

PubMed

Comeche, Amparo; Martín-Villamil, María; Picó, Yolanda; Varó, Inma

2017-09-01

In this study, the toxicity of methylparaben (MeP) an emerging contaminant, was analysed in the sexual species Artemia franciscana, due to its presence in coastal areas and marine saltworks in the Mediterranean region. The acute toxicity (24h-LC 50 ) of MeP in nauplii was tested and its chronic effect (9days) evaluated by measuring survival and growth under two sublethal concentrations (0.0085 and 0.017mg/L). Also, the effect on several key enzymes involved in: antioxidant defences (catalase (CAT) and gluthathion-S-transferase (GST)), neural activity (cholinesterase (ChE)) and xenobiotic biotransformation (carboxylesterase (CbE), was assessed after 48h under sublethal exposure. The results of acute exposure indicate that MeP is harmful to A. franciscana (24h-LC 50 =36.7mg/L). MeP causes a decrease in CAT activity after 48h exposure to both concentration tested, that points out at the oxidative stress effect of MeP in A. franciscana. However, no significant effect on ChE, CbE and GST activities was found. In addition, MeP does not affect survival and growth in chronic exposure at the sublethal concentrations tested. The results of this study indicate that MeP is not a threat for A. franciscana under the experimental conditions used. Additional studies should be done considering long-term exposure and reproduction studies to analyse the potential risk of MeP as emerging contaminant in marine and hypersaline environments. Published by Elsevier Inc.

The Toxicity and Detoxifying Mechanism of Cycloxaprid and Buprofezin in Controlling Sogatella furcifera (Homoptera: Delphacidae)

PubMed Central

Chang, Xiaoli; Yuan, Yongda; Zhang, Tianshu; Wang, Dongsheng; Du, Xingbin; Wu, Xiangwen; Chen, Haixia; Chen, Yaozhong; Jiao, Yuetong; Teng, Haiyuan

2015-01-01

The effects of cycloxaprid (a modified neonicotinoid insecticide) and buprofezin (a thiadiazine insecticide) on mortality of the white-backed planthopper (WBPH), Sogatella furcifera, were determined in laboratory assays. Cycloxaprid killed WBPH nymphs and adults but buprofezin killed only nymphs, and cycloxaprid acted faster than buprofezin. One day after infestation, mortality of third-instar nymphs was >65% with cycloxaprid at 125 mg liter−1 but was <38% with buprofezin at 148 mg liter−1. By the 4th day after infestation, however, control of nymphs by the two insecticides was similar, and cycloxaprid at 125 mg liter−1 caused ≥80% mortality of adults but buprofezin at 148 mg liter−1 (the highest rate tested) caused almost no adult mortality. LC50 values for cycloxaprid were lowest with nymphs, intermediate with adult males, and highest with adult females. Although buprofezin was slower acting than cycloxaprid, its LC50 for nymphs 5 d after infestation was 3.79-fold lower than that of cycloxaprid. Mean carboxylesterase (CarE) specific activity of nymphal WBPH treated with cycloxaprid and buprofezin was higher than that of control, but there was no significant difference between cycloxaprid and control (no insecticide), and it was significantly higher for buprofezin than those of cycloxaprid and control. For glutathione S-transferase and mixed function oxygenase, the specific activity of nymphal WBPH treated with buprofezin was significantly higher than those of cycloxaprid and control, too. PMID:26175461

Insecticide resistance status of Myzus persicae in Greece: long-term surveys and new diagnostics for resistance mechanisms.

PubMed

Voudouris, Costas Ch; Kati, Amalia N; Sadikoglou, Eldem; Williamson, Martin; Skouras, Panagiotis J; Dimotsiou, Ourania; Georgiou, Stella; Fenton, Brian; Skavdis, George; Margaritopoulos, John T

2016-04-01

Myzus persicae nicotianae is an important pest in Greece, controlled mainly by neonicotinoids. Monitoring of the aphid populations for resistance mechanisms is essential for effective control. Two new RFLP-based diagnostics for the detection of the M918T (super-kdr pyrethroid resistance) and nAChR R81T (neonicotinoid resistance) mutations were applied, along with other established assays, on 131 nicotianae multilocus genotypes (MLGs) collected from tobacco and peach in Greece in 2012-2013. Furthermore, we present resistance data from aphid clones (>500, mainly nicotianae) collected in 2006-2007. About half of the clones tested with a diagnostic dose of imidacloprid were tolerant. The R81T mutation was not found in the 131 MLGs and 152 clones examined. Over half (58.6%) of a subset of 29 clones showed a 9-36-fold overexpression of CYP6CY3. M918T was found at low to moderate frequencies. The kdr and MACE mechanisms and carboxylesterase-based resistance were found at high frequency in all years. The aphid retains costly resistance mechanisms even in the absence of pressure from certain insecticides, which could be attributed to factors related to climate and genetic properties of the populations. The indication of build-up of resistance/tolerance to neonicotinoids, related to CYP6CY3 overexpression, is a matter of concern. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

In vitro human metabolism of permethrin isomers alone or as a mixture and the formation of the major metabolites in cryopreserved primary hepatocytes.

PubMed

Willemin, M-E; Kadar, A; de Sousa, G; Leclerc, E; Rahmani, R; Brochot, C

2015-06-01

In vitro metabolism of permethrin, a pyrethroid insecticide, was assessed in primary human hepatocytes. In vitro kinetic experiments were performed to estimate the Michaelis-Menten parameters and the clearances or formation rates of the permethrin isomers (cis- and trans-) and three metabolites, cis- and trans-3-(2,2 dichlorovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid (cis- and trans-DCCA) and 3-phenoxybenzoic acid (3-PBA). Non-specific binding and the activity of the enzymes involved in permethrin's metabolism (cytochromes P450 and carboxylesterases) were quantified. Trans-permethrin was cleared more rapidly than cis-permethrin with a 2.6-factor (25.7±0.6 and 10.1±0.3 μL/min/10(6) cells respectively). A 3-factor was observed between the formation rates of DCCA and 3-PBA obtained from trans- and cis-permethrin. For both isomers, the rate of formation of DCCA was higher than the one of 3-PBA. The metabolism of the isomers in mixture was also quantified. The co-incubation of isomers at different ratios showed the low inhibitory potential of cis- and trans-permethrin on each other. The estimates of the clearances and the formation rates in the co-incubation condition did not differ from the estimates obtained with a separate incubation. These metabolic parameters may be integrated in physiologically based pharmacokinetic (PBPK) models to predict the fate of permethrin and metabolites in the human body. Copyright © 2015 Elsevier Ltd. All rights reserved.

[Resistance mechanisms and cross-resistance of phoxim-resistant Frankliniella occidentalis Pergande population].

PubMed

Wang, Sheng-Yin; Zhou, Xian-Hong; Zhang, An-Sheng; Li, Li-Li; Men, Xing-Yuan; Zhang, Si-Cong; Liu, Yong-Jie; Yu, Yi

2012-07-01

To understand the resistance risks of Frankliniella occidentalis Pergande against phoxim, this paper studied the resistance mechanisms of phoxim-resistant F. occidentalis population against phoxim and the cross-resistance of the population against other insecticides. The phoxim-resistant population had medium level cross-resistance to chlorpyrifos, lambda-cyhalothrin, and methomyl, low level cross-resistance to chlorfenapyr, imidacloprid, emamectin-benzoate, and spinosad, but no cross-resistance to acetamiprid and abamectin. The synergists piperonyl butoxide (PBO), s, s, s-tributyl phosphorotrithioate (DEF), and triphenyl phosphate (TPP) had significant synergism (P < 0.05) on the toxicity of phoxim to the resistant (XK), field (BJ), and susceptible (S) populations, while diethyl maleate (DEM) had no significant synergism to XK and S populations but had significant synergism to BJ population. As compared with S population, the XK and BJ populations had significantly increased activities of mixed-functional oxidases P450 (2.79-fold and 1.48-fold), b, (2.88-fold and 1.88-fold), O-demethylase (2.60-fold and 1.68-fold), and carboxylesterase (2.02-fold and 1.61-fold, respectively), and XK population had a significantly increased acetylcholine esterase activity (3.10-fold). Both XK and BJ population had an increased activity of glutathione S-transferases (1.11-fold and 1.20-fold, respectively), but the increment was not significant. The increased detoxification enzymes activities in F. occidentalis could play an important role in the resistance of the plant against phoxim.

Ethylphenidate as a selective dopaminergic agonist and methylphenidate-ethanol transesterification biomarker.

PubMed

Patrick, Kennerly S; Corbin, Timothy R; Murphy, Cristina E

2014-12-01

We review the pharmaceutical science of ethylphenidate (EPH) in the contexts of drug discovery, drug interactions, biomarker for dl-methylphenidate (MPH)-ethanol exposure, potentiation of dl-MPH abuse liability, contemporary "designer drug," pertinence to the newer transdermal and chiral switch MPH formulations, as well as problematic internal standard. d-EPH selectively targets the dopamine transporter, whereas d-MPH exhibits equipotent actions at dopamine and norepinephrine transporters. This selectivity carries implications for the advancement of tailored attention-deficit/hyperactivity disorder (ADHD) pharmacotherapy in the era of genome-based diagnostics. Abuse of dl-MPH often involves ethanol coabuse. Carboxylesterase 1 enantioselectively transesterifies l-MPH with ethanol to yield l-EPH accompanied by significantly increased early exposure to d-MPH and rapid potentiation of euphoria. The pharmacokinetic component of this drug interaction can largely be avoided using dexmethylphenidate (dexMPH). This notwithstanding, maximal potentiated euphoria occurs following dexMPH-ethanol. C57BL/6 mice model dl-MPH-ethanol interactions: an otherwise depressive dose of ethanol synergistically increases dl-MPH stimulation; a substimulatory dose of dl-MPH potentiates a low, stimulatory dose of ethanol; ethanol elevates blood, brain, and urinary d-MPH concentrations while forming l-EPH. Integration of EPH preclinical neuropharmacology with clinical studies of MPH-ethanol interactions provides a translational approach toward advancement of ADHD personalized medicine and management of comorbid alcohol use disorder. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Effect of alcohol on skin permeation and metabolism of an ester-type prodrug in Yucatan micropig skin.

PubMed

Fujii, Makiko; Ohara, Rieko; Matsumi, Azusa; Ohura, Kayoko; Koizumi, Naoya; Imai, Teruko; Watanabe, Yoshiteru

2017-11-15

We studied the effect that three alcohols, ethanol (EA), propanol (PA), and isopropanol (IPA), have on the skin permeation of p-hydroxy benzoic acid methyl ester (HBM), a model ester-type prodrug. HBM was applied to Yucatan micropig skin in a saturated phosphate buffered solution with or without 10% alcohol, and HBM and related materials in receptor fluid and skin were determined with HPLC. In the absence of alcohol, p-hydroxy benzoic acid (HBA), a metabolite of HBM, permeated the skin the most. The three alcohols enhanced the penetration of HBM at almost the same extent. The addition of 10% EA or PA to the HBM solution led to trans-esterification into the ethyl ester or propyl ester of HBA, and these esters permeated skin as well as HBA and HBM did. In contrast, the addition of 10% IPA promoted very little trans-esterification. Both hydrolysis and trans-esterification in the skin S9 fraction were inhibited by BNPP, an inhibitor of carboxylesterase (CES). Western blot and native PAGE showed the abundant expression of CES in micropig skin. Both hydrolysis and trans-esterification was simultaneously catalyzed by CES during skin permeation. Our data indicate that the alcohol used in dermal drug preparations should be selected not only for its ability to enhance the solubility and permeation of the drug, but also for the effect on metabolism of the drug in the skin. Copyright © 2017 Elsevier B.V. All rights reserved.

Biodegradation of pyraclostrobin by two microbial communities from Hawaiian soils and metabolic mechanism.

PubMed

Chen, Xiaoxin; He, Sheng; Liang, Zhibin; Li, Qing X; Yan, Hai; Hu, Jiye; Liu, Xiaolu

2018-04-27

Pyraclostrobin has been widely and long-termly applicated to agricultural fields. The removal of pyraclostrobin from ecological environment has received wide attention. In this study, using sequential enrichments with pyraclostrobin as a sole carbon source, two microbial communities (HI2 and HI6) capable of catabolizing pyraclostrobin were obtained from Hawaiian soils. The microfloras analysis indicated that only Proteobacteria and Bacteroides could survive in HI2-soil after acclimatization, whereas the number of Proteobacteria in HI6-soil accounted for more than 99%. The percentages of Pseudomonas in the HI2 and HI6 microfloras were 69.3% and 59.3%, respectively. More than 99% of pyraclostrobin (C 0  = 100 mg L -1 ) was degraded by the HI2 and HI6 microorganisms within five days. A unique metabolite was identified by high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS/MS). A metabolic pathway involving carbamate hydrolysis was proposed. The tertiary amine group of pyraclostrobin was hydrolyzed to primary amine group with the decarboxylation, which facilitated pyraclostrobin detoxification because carboxylester was an important functional group. The metabolic mechanism suggested that Pseudomonas expressing carboxylesterase might be able to degrade carbamate chemicals. Therefore, Pseudomonas might be an ideal candidate for expression and cloning of carbamate-degrading gene in genomics studies. The current study would have important implications in detoxification and bioremediation of carbamates through the CN bond cleavage of methyl carbamate. Copyright © 2018 Elsevier B.V. All rights reserved.

Alteration of the Expression of Pesticide-Metabolizing Enzymes in Pregnant Mice: Potential Role in the Increased Vulnerability of the Developing Brain

PubMed Central

Fortin, Marie C.; Aleksunes, Lauren M.

2013-01-01

Studies on therapeutic drug disposition in humans have shown significant alterations as the result of pregnancy. However, it is not known whether pesticide metabolic capacity changes throughout pregnancy, which could affect exposure of the developing brain. We sought to determine the effect of pregnancy on the expression of hepatic enzymes involved in the metabolism of pesticides. Livers were collected from virgin and pregnant C57BL/6 mice at gestational days (GD)7, GD11, GD14, GD17, and postpartum days (PD)1, PD15, and PD30. Relative mRNA expression of several enzymes involved in the metabolism of pesticides, including hepatic cytochromes (Cyp) P450s, carboxylesterases (Ces), and paraoxonase 1 (Pon1), were assessed in mice during gestation and the postpartum period. Compared with virgin mice, alterations in the expression occurred at multiple time points, with the largest changes observed on GD14. At this time point, the expression of most of the Cyps involved in pesticide metabolism in the liver (Cyp1a2, Cyp2d22, Cyp2c37, Cyp2c50, Cyp2c54, and Cyp3a11) were downregulated by 30% or more. Expression of various Ces isoforms and Pon1 were also decreased along with Pon1 activity. These data demonstrate significant alterations in the expression of key enzymes that detoxify pesticides during pregnancy, which could alter exposure of developing animals to these chemicals. PMID:23223497

Efficient one-cycle affinity selection of binding proteins or peptides specific for a small-molecule using a T7 phage display pool.

PubMed

Takakusagi, Yoichi; Kuramochi, Kouji; Takagi, Manami; Kusayanagi, Tomoe; Manita, Daisuke; Ozawa, Hiroko; Iwakiri, Kanako; Takakusagi, Kaori; Miyano, Yuka; Nakazaki, Atsuo; Kobayashi, Susumu; Sugawara, Fumio; Sakaguchi, Kengo

2008-11-15

Here, we report an efficient one-cycle affinity selection using a natural-protein or random-peptide T7 phage pool for identification of binding proteins or peptides specific for small-molecules. The screening procedure involved a cuvette type 27-MHz quartz-crystal microbalance (QCM) apparatus with introduction of self-assembled monolayer (SAM) for a specific small-molecule immobilization on the gold electrode surface of a sensor chip. Using this apparatus, we attempted an affinity selection of proteins or peptides against synthetic ligand for FK506-binding protein (SLF) or irinotecan (Iri, CPT-11). An affinity selection using SLF-SAM and a natural-protein T7 phage pool successfully detected FK506-binding protein 12 (FKBP12)-displaying T7 phage after an interaction time of only 10 min. Extensive exploration of time-consuming wash and/or elution conditions together with several rounds of selection was not required. Furthermore, in the selection using a 15-mer random-peptide T7 phage pool and subsequent analysis utilizing receptor ligand contact (RELIC) software, a subset of SLF-selected peptides clearly pinpointed several amino-acid residues within the binding site of FKBP12. Likewise, a subset of Iri-selected peptides pinpointed part of the positive amino-acid region of residues from the Iri-binding site of the well-known direct targets, acetylcholinesterase (AChE) and carboxylesterase (CE). Our findings demonstrate the effectiveness of this method and general applicability for a wide range of small-molecules.

Pollution biomarkers in the spiny lizard (Sceloporus spp.) from two suburban populations of Monterrey, Mexico.

PubMed

Aguilera, Carlos; del Pliego, Pamela González; Alfaro, Roberto Mendoza; Lazcano, David; Cruz, Julio

2012-11-01

Environmental pollution may severely impact reptile species in urbanized areas. The magnitude of the impact is analyzed in the present study using lizard tail tips for the quantitative evaluation of enzymatic biomarkers of pollution. Spiny lizards (Sceloporus serrifer and S. torquatus) were collected from two suburban localities in the Monterrey metropolitan area, Mexico: Chipinque Ecological Park, a natural protected area, and El Carmen Industrial Park (IP), a highly polluted site. Different enzymes were used as biomarkers including: acetylcholinesterase (AChE), butyrylcholinesterase (BChE), carboxylesterase (CaE), alkaline phosphatase (ALP), acid phosphatase (ACP), superoxide dismutase (SOD) and glutathione S-transferase (GST). The levels of AChE, BChE and ACP activity were not significantly different between localities. AChE and BChE, commonly used as biomarkers of neurotoxic polluting agents (e.g. organophosphate pesticides) do not appear to be affecting the populations from the study locations. In contrast, the levels of CaE, GST, ALP and SOD were significantly different between the localities. These biomarkers are regularly associated with oxidative stress and processes of detoxification, and generally indicate pollution caused by heavy metals or hydrocarbons, which are common in industrial sites. The data resulting from the analysis of these biomarkers indicate that these polluting agents are affecting the populations of Sceloporus in IP. The present work validates the possibility of conducting additional ecotoxicological studies using biomarkers in combination with a nondestructive sampling technique in species of spiny lizards that are abundant in many North America areas.

A Linkage Map and QTL Analysis for Pyrethroid Resistance in the Bed Bug Cimex lectularius.

PubMed

Fountain, Toby; Ravinet, Mark; Naylor, Richard; Reinhardt, Klaus; Butlin, Roger K

2016-12-07

The rapid evolution of insecticide resistance remains one of the biggest challenges in the control of medically and economically important pests. Insects have evolved a diverse range of mechanisms to reduce the efficacy of the commonly used classes of insecticides, and finding the genetic basis of resistance is a major aid to management. In a previously unstudied population, we performed an F 2 resistance mapping cross for the common bed bug, Cimex lectularius, for which insecticide resistance is increasingly widespread. Using 334 SNP markers obtained through RAD-sequencing, we constructed the first linkage map for the species, consisting of 14 putative linkage groups (LG), with a length of 407 cM and an average marker spacing of 1.3 cM. The linkage map was used to reassemble the recently published reference genome, facilitating refinement and validation of the current genome assembly. We detected a major QTL on LG12 associated with insecticide resistance, occurring in close proximity (1.2 Mb) to a carboxylesterase encoding candidate gene for pyrethroid resistance. This provides another example of this candidate gene playing a major role in determining survival in a bed bug population following pesticide resistance evolution. The recent availability of the bed bug genome, complete with a full list of potential candidate genes related to insecticide resistance, in addition to the linkage map generated here, provides an excellent resource for future research on the development and spread of insecticide resistance in this resurging pest species. Copyright © 2016 Fountain et al.

Insecticide resistance in the sand fly, Phlebotomus papatasi from Khartoum State, Sudan.

PubMed

Hassan, Mo'awia Mukhtar; Widaa, Sally Osman; Osman, Osman Mohieldin; Numiary, Mona Siddig Mohammed; Ibrahim, Mihad Abdelaal; Abushama, Hind Mohammed

2012-03-07

Phlebotomus papatasi the vector of cutaneous leishmaniasis (CL) is the most widely spread sand fly in Sudan. No data has previously been collected on insecticide susceptibility and/or resistance of this vector, and a first study to establish a baseline data is reported here. Sand flies were collected from Surogia village, (Khartoum State), Rahad Game Reserve (eastern Sudan) and White Nile area (Central Sudan) using light traps. Sand flies were reared in the Tropical Medicine Research Institute laboratory. The insecticide susceptibility status of first progeny (F1) of P. papatasi of each population was tested using WHO insecticide kits. Also, P. papatasi specimens from Surogia village and Rahad Game Reserve were assayed for activities of enzyme systems involved in insecticide resistance (acetylcholinesterase (AChE), non-specific carboxylesterases (EST), glutathione-S-transferases (GSTs) and cytochrome p450 monooxygenases (Cyt p450). Populations of P. papatasi from White Nile and Rahad Game Reserve were sensitive to dichlorodiphenyltrichloroethane (DDT), permethrin, malathion, and propoxur. However, the P. papatasi population from Surogia village was sensitive to DDT and permethrin but highly resistant to malathion and propoxur. Furthermore, P. papatasi of Surogia village had significantly higher insecticide detoxification enzyme activity than of those of Rahad Game Reserve. The sand fly population in Surogia displayed high AChE activity and only three specimens had elevated levels for EST and GST. The study provided evidence for malathion and propoxur resistance in the sand fly population of Surogia village, which probably resulted from anti-malarial control activities carried out in the area during the past 50 years.

The central role of mosquito cytochrome P450 CYP6Zs in insecticide detoxification revealed by functional expression and structural modelling

PubMed Central

Chandor-Proust, Alexia; Bibby, Jaclyn; Régent-Kloeckner, Myriam; Roux, Jessica; Guittard-Crilat, Emilie; Poupardin, Rodolphe; Riaz, Muhammad Asam; Paine, Mark; Dauphin-Villemant, Chantal; Reynaud, Stéphane; David, Jean-Philippe

2013-01-01

The resistance of mosquitoes to chemical insecticides is threatening vector control programmes worldwide. Cytochrome P450 monooxygenases (CYPs) are known to play a major role in insecticide resistance, allowing resistant insects to metabolize insecticides at a higher rate. Among them, members of the mosquito CYP6Z subfamily, like Aedes aegypti CYP6Z8 and its Anopheles gambiae orthologue CYP6Z2, have been frequently associated with pyrethroid resistance. However, their role in the pyrethroid degradation pathway remains unclear. In the present study, we created a genetically modified yeast strain overexpressing Ae. aegypti cytochrome P450 reductase and CYP6Z8, thereby producing the first mosquito P450–CPR (NADPH-cytochrome P450-reductase) complex in a yeast recombinant system. The results of the present study show that: (i) CYP6Z8 metabolizes PBAlc (3-phenoxybenzoic alcohol) and PBAld (3-phenoxybenzaldehyde), common pyrethroid metabolites produced by carboxylesterases, producing PBA (3-phenoxybenzoic acid); (ii) CYP6Z8 transcription is induced by PBAlc, PBAld and PBA; (iii) An. gambiae CYP6Z2 metabolizes PBAlc and PBAld in the same way; (iv) PBA is the major metabolite produced in vivo and is excreted without further modification; and (v) in silico modelling of substrate–enzyme interactions supports a similar role of other mosquito CYP6Zs in pyrethroid degradation. By playing a pivotal role in the degradation of pyrethroid insecticides, mosquito CYP6Zs thus represent good targets for mosquito-resistance management strategies. PMID:23844938

Time- and concentration-dependent increases in cell proliferation in rats and mice administered vinyl acetate in drinking water.

PubMed

Valentine, Rudolph; Bamberger, J R; Szostek, B; Frame, S R; Hansen, J F; Bogdanffy, M S

2002-06-01

Chronic administration of vinyl acetate (VA) in drinking water to rats and mice has produced upper digestive tract neoplasms. These tumors were believed to arise from the intracellular metabolism of VA by carboxylesterases to cytotoxic and genotoxic compounds. We hypothesized that prolonged VA exposure at high concentrations would induce cytotoxicity and a restorative cell proliferation (CP). These endpoints were measured in F-344 rats and BDF1 mice administered drinking water containing 0, 1000, 5000, 10,000, or 24,000 ppm VA for 92 days. On test days, Days 1, 8, 29, and 92, upper digestive tract histopathology and oral cavity CP (pulsed 5-bromodeoxyuridine [BrdU] to measure S-phase DNA synthesis) were evaluated. Analysis of test solutions showed that VA spontaneously hydrolyzed, slowly releasing acetic acid and thereby lowering pH. Statistically significant, concentration-related increases in CP occurred in basal cells of the mandibular oral cavity mucosa of mice at 10,000 and 24,000 ppm but only after 92 days. CP increases were approximately 2.4- and 3.4-fold above controls and were considered to be toxicologically significant. Some statistically significant increases in CP were also measured in the oral cavity mucosa of rats; however, these changes were considered to be of equivocal biological relevance. No histopathological evidence of mucosal injury was seen in either species. The absence of cytotoxicity in the upper digestive tract mucosa suggests that the increased CP at high administered VA concentrations may be due to a mitogenic response, ostensibly from the loss of cell growth controls in oral cavity mucosa.

Insecticide resistance in the dengue vector Aedes aegypti from Martinique: distribution, mechanisms and relations with environmental factors.

PubMed

Marcombe, Sébastien; Mathieu, Romain Blanc; Pocquet, Nicolas; Riaz, Muhammad-Asam; Poupardin, Rodolphe; Sélior, Serge; Darriet, Frédéric; Reynaud, Stéphane; Yébakima, André; Corbel, Vincent; David, Jean-Philippe; Chandre, Fabrice

2012-01-01

Dengue is an important mosquito borne viral disease in Martinique Island (French West Indies). The viruses responsible for dengue are transmitted by Aedes aegypti, an indoor day-biting mosquito. The most effective proven method for disease prevention has been by vector control by various chemical or biological means. Unfortunately insecticide resistance has already been observed on the Island and recently showed to significantly reduce the efficacy of vector control interventions. In this study, we investigated the distribution of resistance and the underlying mechanisms in nine Ae. aegypti populations. Statistical multifactorial approach was used to investigate the correlations between insecticide resistance levels, associated mechanisms and environmental factors characterizing the mosquito populations. Bioassays revealed high levels of resistance to temephos and deltamethrin and susceptibility to Bti in the 9 populations tested. Biochemical assays showed elevated detoxification enzyme activities of monooxygenases, carboxylesterases and glutathione S-tranferases in most of the populations. Molecular screening for common insecticide target-site mutations, revealed the presence of the "knock-down resistance" V1016I Kdr mutation at high frequency (>87%). Real time quantitative RT-PCR showed the potential involvement of several candidate detoxification genes in insecticide resistance. Principal Component Analysis (PCA) performed with variables characterizing Ae. aegypti from Martinique permitted to underline potential links existing between resistance distribution and other variables such as agriculture practices, vector control interventions and urbanization. Insecticide resistance is widespread but not homogeneously distributed across Martinique. The influence of environmental and operational factors on the evolution of the resistance and mechanisms are discussed.

Plasma esterases in the tegu lizard Tupinambis merianae (Reptilia, Teiidae): impact of developmental stage, sex, and organophosphorus in vitro exposure.

PubMed

Basso, Agustín; Attademo, Andrés M; Lajmanovich, Rafael C; Peltzer, Paola M; Junges, Celina; Cabagna, Mariana C; Fiorenza, Gabriela S; Sanchez-Hernandez, Juan Carlos

2012-01-01

In this study, we determined normal serum butyrylcholinesterase (BChE) and carboxylesterase (CbE) activities in Tupinambis merianae in order to obtain reference values for organophosphorus pesticide monitoring. Forty-two T. merianae individuals were grouped by sex and size to identify potential differences in their enzyme levels to allow for proper representation of normal values for females, males, juveniles, and hatchlings. Mean CbE was determined using two model substrates: alpha-naphtylacetate (α-NA) and p-nitrophenyl valerate (4-NPV). BChE and CbE sensitivity to malaoxon (Mx) was also evaluated as well as the possibility of BChE reactivation with pyridine-2-aldoxime methochloride (2-PAM). Mean adult females' BChE was significantly higher than adult males, juveniles, and hatchlings. No significant differences were found between groups regarding CbE. CbE (4-NPV) activity showed slightly negative correlation with lizard snout-vent length, while BChE and CbE (α-NA) showed no correlation with body size. Apparent IC(50) values for BChE and CbE (α-NA) suggested different sensitivities among groups. CbE (4-NPV) could not be inhibited. All Mx-inhibited groups treated with 2-PAM in a final concentration of 2.8 mM showed clear signs of reactivation. In conclusion, the results demonstrate that (1) plasma esterase activity did not vary with age and sex, except for BChE activity, and (2) because biological and environmental variables could be confounding factors in the response of plasma cholinesterases, complementary biomarkers like CbE inhibition and oxime-induced reactivation of esterases are strongly recommended.

Effects of chlorpyrifos on enzymatic systems of Cydia pomonella (Lepidoptera: Tortricidae) adults.

PubMed

Parra Morales, Laura Beatriz; Alzogaray, Raúl Adolfo; Cichón, Liliana; Garrido, Silvina; Soleño, Jimena; Montagna, Cristina Mónica

2017-06-01

The control program of codling moth (Cydia pomonella L.) in the Río Negro and Neuquén Valley is intended to neonate larvae. However, adults may be subjected to sublethal pesticide concentrations generating stress which might enhance both mutation rates and activity of the detoxification system. This study assessed the exposure effects of chlorpyrifos on target enzyme and, both detoxifying and antioxidant systems of surviving adults from both a laboratory susceptible strain (LSS) and a field population (FP). The results showed that the FP was as susceptible to chlorpyrifos as the LSS and, both exhibited a similar chlorpyrifos-inhibitory concentration 50 (IC 50 ) of acetylcholinesterase (AChE). The FP displayed higher carboxylesterase (CarE) and 7-ethoxycoumarine O-deethylase (ECOD) activities than LSS. Both LSS and FP showed an increase on CarE activity after the exposure to low-chlorpyrifos concentrations, followed by enzyme inhibition at higher concentrations. There were no significant differences neither in the activities of glutathione S-transferases (GST), catalase (CAT) and superoxide dismutase (SOD) nor in the reduced glutathione (GSH) content between LSS and FP. Moreover, these enzymes were unaffected by chlorpyrifos. In conclusion, control adults from the FP exhibited higher CarE and ECOD activities than control adults from the LSS. AChE and CarE activities were the most affected by chlorpyrifos. Control strategies used for C. pomonella, such as rotations of insecticides with different modes of action, will probably delay the evolution of insecticide resistance in FPs from the study area. © 2015 Institute of Zoology, Chinese Academy of Sciences.

Biotransformation enzymes in the rodent nasal mucosa: the value of a histochemical approach.

PubMed Central

Bogdanffy, M S

1990-01-01

An increasing number of chemicals have been identified as being toxic to the nasal mucosa of rats. While many chemicals exert their effects only after inhalation exposure, others are toxic following systemic administration, suggesting that factors other than direct deposition on the nasal mucosa may be important in mechanisms of nasal toxicity. The mucosal lining of the nasal cavity consists of a heterogeneous population of ciliated and nonciliated cells, secretory cells, sensory cells, and glandular and other cell types. For chemicals that are metabolized in the nasal mucosa, the balance between metabolic activation and detoxication within a cell type may be a key factor in determining whether that cell type will be a target for toxicity. Recent research in the area of xenobiotic metabolism in nasal mucosa has demonstrated the presence of many enzymes previously described in other tissues. In particular, carboxylesterase, aldehyde dehydrogenase, cytochromes P-450, epoxide hydrolase, and glutathione S-transferases have been localized by histochemical techniques. The distribution of these enzymes appears to be cell-type-specific and the presence of the enzyme may predispose particular cell types to enhanced susceptibility or resistance to chemical-induced injury. This paper reviews the distribution of these enzymes within the nasal mucosa in the context of their contribution to xenobiotic metabolism. The localization of the enzymes by histochemical techniques has provided important information on the potential mechanism of action of esters, aldehydes, and cytochrome P-450 substrates known to injure the nasal mucosa. Images PLATE 1. PLATE 2. PLATE 3. PMID:2200661

Transcriptomic Survey of the Midgut of Anthonomus grandis (Coleoptera: Curculionidae)

PubMed Central

Salvador, Ricardo; Príncipi, Darío; Berretta, Marcelo; Fernández, Paula; Paniego, Norma; Sciocco-Cap, Alicia; Hopp, Esteban

2014-01-01

Abstract Anthonomus grandis Boheman is a key pest in cotton crops in the New World. Its larval stage develops within the flower bud using it as food and as protection against its predators. This behavior limits the effectiveness of its control using conventional insecticide applications and biocontrol techniques. In spite of its importance, little is known about its genome sequence and, more important, its specific expression in key organs like the midgut. Total mRNA isolated from larval midguts was used for pyrosequencing. Sequence reads were assembled and annotated to generate a unigene data set. In total, 400,000 reads from A. grandis midgut with an average length of 237 bp were assembled and combined into 20,915 contigs. The assembled reads fell into 6,621 genes models. BlastX search using the NCBI-NR database showed that 3,006 unigenes had significant matches to known sequences. Gene Ontology (GO) mapping analysis evidenced that A. grandis is able to transcripts coding for proteins involved in catalytic processing of macromolecules that allows its adaptation to very different feeding source scenarios. Furthermore, transcripts encoding for proteins involved in detoxification mechanisms such as p450 genes, glutathione-S-transferase , and carboxylesterases are also expressed. This is the first report of a transcriptomic study in A. grandis and the largest set of sequence data reported for this species. These data are valuable resources to expand the knowledge of this insect group and could be used in the design of new control strategies based in molecular information. PMID:25473064

Insecticide Resistance in the Dengue Vector Aedes aegypti from Martinique: Distribution, Mechanisms and Relations with Environmental Factors

PubMed Central

Marcombe, Sébastien; Mathieu, Romain Blanc; Pocquet, Nicolas; Riaz, Muhammad-Asam; Poupardin, Rodolphe; Sélior, Serge; Darriet, Frédéric; Reynaud, Stéphane; Yébakima, André; Corbel, Vincent; David, Jean-Philippe; Chandre, Fabrice

2012-01-01

Dengue is an important mosquito borne viral disease in Martinique Island (French West Indies). The viruses responsible for dengue are transmitted by Aedes aegypti, an indoor day-biting mosquito. The most effective proven method for disease prevention has been by vector control by various chemical or biological means. Unfortunately insecticide resistance has already been observed on the Island and recently showed to significantly reduce the efficacy of vector control interventions. In this study, we investigated the distribution of resistance and the underlying mechanisms in nine Ae. aegypti populations. Statistical multifactorial approach was used to investigate the correlations between insecticide resistance levels, associated mechanisms and environmental factors characterizing the mosquito populations. Bioassays revealed high levels of resistance to temephos and deltamethrin and susceptibility to Bti in the 9 populations tested. Biochemical assays showed elevated detoxification enzyme activities of monooxygenases, carboxylesterases and glutathione S-tranferases in most of the populations. Molecular screening for common insecticide target-site mutations, revealed the presence of the “knock-down resistance” V1016I Kdr mutation at high frequency (>87%). Real time quantitative RT-PCR showed the potential involvement of several candidate detoxification genes in insecticide resistance. Principal Component Analysis (PCA) performed with variables characterizing Ae. aegypti from Martinique permitted to underline potential links existing between resistance distribution and other variables such as agriculture practices, vector control interventions and urbanization. Insecticide resistance is widespread but not homogeneously distributed across Martinique. The influence of environmental and operational factors on the evolution of the resistance and mechanisms are discussed. PMID:22363529

Physiological and biochemical effects of botanical extract from Piper nigrum Linn (Piperaceae) against the dengue vector Aedes aegypti Liston (Diptera: Culicidae).

PubMed

Lija-Escaline, Jalasteen; Senthil-Nathan, Sengottayan; Thanigaivel, Annamalai; Pradeepa, Venkatraman; Vasantha-Srinivasan, Prabhakaran; Ponsankar, Athirstam; Edwin, Edward Sam; Selin-Rani, Selvaraj; Abdel-Megeed, Ahmed

2015-11-01

The leaves of Piper nigrum L. (Piperaceae) were evaluated for chemical constituents and mosquito larvicidal activity against the larvae of Aedes aegypti. GC and GC-MS analyses revealed that the crude extracts contain 16 compounds. Thymol (20.77%) and ç-elemene (10.42%) were identified as the major constituents followed by cyclohexene, 4-ethenyl-4-methyl-3-(1-methylethenyl)-1-(1 methylethyl)-, (3R-trans) (7.58%), 4,6-octadienoic acid, 2-acetyl-2-methyl-, ethyl ester (6.98), 2(3H)-furanone, 3,4-bis(1,3-benzodioxol-5-ylmethyl) dihydro-, (3R-trans) (6.95%), 1-naphthalenol, 1,2,3,4,4a,7,8,8a-octahydro-1,6-dimethyl-4-(1-methylethyl)-, [1R-(1à,4á,4aá,8aá)]-(Cedreanol) (5.30%), trans-2-undecen-1-ol (4.48%), phytol (4.22%), 1,6-cyclodecadiene, 1-methyl-5-methylene-8-(1-methylethyl)-,[s-(E,E)] (3.78%) and 2,6-dimethyl-3,5,7-octatriene-2-ol, Z,Z (2.39%). Larval mortality was observed after 3 h of exposure period. The crude extract showed remarkable larvicidal activity against Ae. aegypti (LC50 = 34.97). The larvae of Ae. aegypti exposed to the P. nigrum, significantly reduced the activities of α- and β-carboxylesterases and superdioxide. Further, P. nigrum extract was severely affecting the mosquito gut cellular organelles. Based on the results, the chemical constituents of crude extracts of P. nigrum can be considered as a new source of larvicide for the control of Ae. aegypti.

The targeted histone deacetylase inhibitor tefinostat (CHR-2845) shows selective in vitro efficacy in monocytoid-lineage leukaemias

PubMed Central

Zabkiewicz, Joanna; Gilmour, Marie; Hills, Robert; Vyas, Pares; Bone, Elizabeth; Davidson, Alan; Burnett, Alan; Knapper, Steven

2016-01-01

Tefinostat (CHR-2845) is a novel monocyte/macrophage-targeted histone deacetylase (HDAC) inhibitor which is cleaved into its active acid by the intracellular esterase human carboxylesterase-1 (hCE-1). The in vitro efficacy of tefinostat was characterised in cell lines and in a cohort of 73 primary AML and CMML samples. Dose-dependent induction of apoptosis and significant growth inhibitory effects were seen in myelomonocytic (M4), monocytic/monoblastic (M5) and CMML samples in comparison to non-monocytoid AML sub-types (p = 0.007). Importantly, no growth inhibitory effects were seen in normal bone marrow CD34+ cells exposed to AML-toxic doses of tefinostat in clonogenic assays. Expression of hCE-1 was measured by intracellular flow cytometry and immunoblotting across the cohort, with highest levels seen in M5 AML patients. hCE-1 levels correlated with significantly increased tefinostat sensitivity (low EC50) as measured by growth inhibition assays (p = 0.001) and concomitant elevation of the mature monocytoid marker CD14+. Strong induction of intracellular histone protein acetylation was observed in tefinostat-responsive samples, as were high levels of the DNA damage sensor γ-H2A.X, highlighting potential biomarkers of patient responsiveness. Synergistic interaction between tefinostat and the current standard treatment cytarabine was demonstrated in dose response and clonogenic assays using simultaneous drug addition in primary samples (median Combination Index value = 0.51). These data provide a strong rationale for the further clinical evaluation of tefinostat in monocytoid-lineage haematological neoplasms including CMML and monocyte-lineage AMLs. PMID:26934551

The Toxicity and Detoxifying Mechanism of Cycloxaprid and Buprofezin in Controlling Sogatella furcifera (Homoptera: Delphacidae).

PubMed

Chang, Xiaoli; Yuan, Yongda; Zhang, Tianshu; Wang, Dongsheng; Du, Xingbin; Wu, Xiangwen; Chen, Haixia; Chen, Yaozhong; Jiao, Yuetong; Teng, Haiyuan

2015-01-01

The effects of cycloxaprid (a modified neonicotinoid insecticide) and buprofezin (a thiadiazine insecticide) on mortality of the white-backed planthopper (WBPH), Sogatella furcifera, were determined in laboratory assays. Cycloxaprid killed WBPH nymphs and adults but buprofezin killed only nymphs, and cycloxaprid acted faster than buprofezin. One day after infestation, mortality of third-instar nymphs was >65% with cycloxaprid at 125 mg liter(-1) but was <38% with buprofezin at 148 mg liter(-1). By the 4th day after infestation, however, control of nymphs by the two insecticides was similar, and cycloxaprid at 125 mg liter(-1) caused ≥ 80% mortality of adults but buprofezin at 148 mg liter(-1) (the highest rate tested) caused almost no adult mortality. LC50 values for cycloxaprid were lowest with nymphs, intermediate with adult males, and highest with adult females. Although buprofezin was slower acting than cycloxaprid, its LC50 for nymphs 5 d after infestation was 3.79-fold lower than that of cycloxaprid. Mean carboxylesterase (CarE) specific activity of nymphal WBPH treated with cycloxaprid and buprofezin was higher than that of control, but there was no significant difference between cycloxaprid and control (no insecticide), and it was significantly higher for buprofezin than those of cycloxaprid and control. For glutathione S-transferase and mixed function oxygenase, the specific activity of nymphal WBPH treated with buprofezin was significantly higher than those of cycloxaprid and control, too. © The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.

Anthracycline-Formaldehyde Conjugates and Their Targeted Prodrugs

NASA Astrophysics Data System (ADS)

Koch, Tad H.; Barthel, Benjamin L.; Kalet, Brian T.; Rudnicki, Daniel L.; Post, Glen C.; Burkhart, David J.

The sequence of research leading to a proposal for anthracycline cross-linking of DNA is presented. The clinical anthracycline antitumor drugs are anthraquinones, and as such are redox active. Their redox chemistry leads to induction of oxidative stress and drug metabolites. An intermediate in reductive glycosidic cleavage is a quinone methide, once proposed as an alkylating agent of DNA. Subsequent research now implicates formaldehyde as a mediator of anthracycline-DNA cross-linking. The cross-link at 5'-GC-3' sites consists of a covalent linkage from the amino group of the anthracycline to the 2-amino group of the G-base through a methylene from formaldehyde, hydrogen bonding from the 9-OH to the G-base on the opposing strand, and hydrophobic interactions through intercalation of the anthraquinone. The combination of these interactions has been described as a virtual cross-link of DNA. The origin of the formaldehyde in vivo remains a mystery. In vitro, doxorubicin reacts with formaldehyde to give firstly a monomeric oxazolidine, doxazolidine, and secondly a dimeric oxazolidine, doxoform. Doxorubicin reacts with formaldehyde in the presence of salicylamide to give the N-Mannich base conjugate, doxsaliform. Doxsaliform is several fold more active in tumor cell growth inhibition than doxorubicin, but doxazolidine and doxoform are orders of magnitude more active than doxorubicin. Exploratory research on the potential for doxsaliform and doxazolidine as targeted cytotoxins is presented. A promising lead design is pentyl PABC-Doxaz, targeted to a carboxylesterase enzyme overexpressed in liver cancer cells and/or colon cancer cells.

Characterization of tannase protein sequences of bacteria and fungi: an in silico study.

PubMed

Banerjee, Amrita; Jana, Arijit; Pati, Bikash R; Mondal, Keshab C; Das Mohapatra, Pradeep K

2012-04-01

The tannase protein sequences of 149 bacteria and 36 fungi were retrieved from NCBI database. Among them only 77 bacterial and 31 fungal tannase sequences were taken which have different amino acid compositions. These sequences were analysed for different physical and chemical properties, superfamily search, multiple sequence alignment, phylogenetic tree construction and motif finding to find out the functional motif and the evolutionary relationship among them. The superfamily search for these tannase exposed the occurrence of proline iminopeptidase-like, biotin biosynthesis protein BioH, O-acetyltransferase, carboxylesterase/thioesterase 1, carbon-carbon bond hydrolase, haloperoxidase, prolyl oligopeptidase, C-terminal domain and mycobacterial antigens families and alpha/beta hydrolase superfamily. Some bacterial and fungal sequence showed similarity with different families individually. The multiple sequence alignment of these tannase protein sequences showed conserved regions at different stretches with maximum homology from amino acid residues 389-469 and 482-523 which could be used for designing degenerate primers or probes specific for tannase producing bacterial and fungal species. Phylogenetic tree showed two different clusters; one has only bacteria and another have both fungi and bacteria showing some relationship between these different genera. Although in second cluster near about all fungal species were found together in a corner which indicates the sequence level similarity among fungal genera. The distributions of fourteen motifs analysis revealed Motif 1 with a signature amino acid sequence of 29 amino acids, i.e. GCSTGGREALKQAQRWPHDYDGIIANNPA, was uniformly observed in 83.3 % of studied tannase sequences representing its participation with the structure and enzymatic function.

Activity changes of antioxidant and detoxifying enzymes in Tenebrio molitor (Coleoptera: Tenebrionidae) larvae infected by the entomopathogenic nematode Heterorhabditis beicherriana (Rhabditida: Heterorhabditidae).

PubMed

Li, Xingyue; Liu, Qizhi; Lewis, Edwin E; Tarasco, Eustachio

2016-12-01

Entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis are lethal parasites of many insect species. To investigate defensive mechanisms towards EPNs in relation to antioxidative and detoxifying enzymes, we chose Tenebrio molitor (Coleoptera: Tenebrionidae) as experimental insect. We studied the activity changes of superoxide dismutases (SODs), peroxidases (PODs), and catalases (CATs), as well as tyrosinase (TYR), acetylcholinesterase (AChE), carboxylesterase (CarE), and glutathione S-transferase (GSTs) for 40 h in T. molitor larvae infected with Heterorhabditis beicherriana infective juveniles (IJs) at 5 rates (0, 20, 40, 80, and 160 IJs/larva). We found that when T. molitor larvae infected with H. beicherriana at higher rates (80 and 160 IJs/larva), SOD activity quickly increased to more than 70 % higher than that control levels. The activities of POD and CAT increased after 24 h. TYR activity increased slowly at lower rates of infection for 16 h, followed by a slight decrease, and then increasing from 32 to 40 h. The other detoxifying enzymes (GST, CarE, and AChE) were enhanced at lower infection rates, but were inhibited at higher rates. Our results suggested that host antioxidative response and detoxification reactions played a central role in the defensive reaction to EPNs, and that this stress which was reflected by the higher level enzymes activity contributed to the death of hosts. Further study should explore the exact function of these enzymes using different species of EPNs and investigate the links between enzyme activity and host susceptibility to EPNs.

Repeated low-dose exposures to sarin, soman, or VX affect acoustic startle in guinea pigs.

PubMed

Smith, C D; Lee, R B; Moran, A V; Sipos, M L

2016-01-01

Chemical warfare nerve agents (CWNAs) are known to cause behavioral abnormalities in cases of human exposures and in animal models. The behavioral consequences of single exposures to CWNAs that cause observable toxic signs are particularly well characterized in animals; however, less is known regarding repeated smaller exposures that may or may not cause observable toxic signs. In the current study, guinea pigs were exposed to fractions (0.1, 0.2, or 0.4) of a medial lethal dose (LD50) of sarin, soman, or VX for two weeks. On each exposure day, and for a post-exposure period, acoustic startle response (ASR) was measured in each animal. Although relatively few studies use guinea pigs to measure behavior, this species is ideal for CWNA-related experiments because their levels of carboxylesterases closely mimic those of humans, unlike rats or mice. Results showed that the 0.4 LD50 doses of soman and VX transiently increased peak startle amplitude by the second week of injections, with amplitude returning to baseline by the second week post-exposure. Sarin also increased peak startle amplitude independent of week. Latencies to peak startle and PPI were affected by agent exposure but not consistently among the three agents. Most of the changes in startle responses returned to baseline following the cessation of exposures. These data suggest that doses of CWNAs not known to produce observable toxic signs in guinea pigs can affect behavior in the ASR paradigm. Further, these deficits are transient and usually return to baseline shortly after the end of a two-week exposure period. Published by Elsevier Inc.

Comparative analysis of mosquito (Diptera: Culicidae: Aedes aegypti Liston) responses to the insecticide Temephos and plant derived essential oil derived from Piper betle L.

PubMed

Vasantha-Srinivasan, Prabhakaran; Senthil-Nathan, Sengottayan; Ponsankar, Athirstam; Thanigaivel, Annamalai; Edwin, Edward-Sam; Selin-Rani, Selvaraj; Chellappandian, Muthiah; Pradeepa, Venkatraman; Lija-Escaline, Jalasteen; Kalaivani, Kandaswamy; Hunter, Wayne B; Duraipandiyan, Veeramuthu; Al-Dhabi, Naif Abdullah

2017-05-01

Resistance to treatments with Temephos or plant derived oil, Pb-CVO, between a field collected Wild Strain (WS) and a susceptible Laboratory Strain (LS) of Ae. aegypti were measured. The Temephos (0.1mg/L) showed the greatest percentage of mosquito mortality compared to Pb-CVO (1.5mg/L) in LS Ae. aegypti. However, WS Ae. aegypti was not significantly affected by Temephos (0.1mg/L) treatment compare to the Pb-CVO (1.5mg/L). However, both strains (LS and WS) when treated with Pb-CVO (1.5mg/L) displayed steady larval mortality rate across all instars. The LC 50 of Temephos was 0.027mg in LS, but increased in WS to 0.081mg/L. The LC 50 of Pb-CVO treatment was observed at concentrations of 0.72 and 0.64mg/L for LS and WS strains respectively. The enzyme level of α- and β-carboxylesterase was reduced significantly in both mosquito strains treated with Pb-CVO. Whereas, there was a prominent deviation in the enzyme ratio observed between LS and WS treated with Temephos. The GST and CYP450 levels were upregulated in the LS, but decreased in WS, after treatment with Temephos. However, treatment with Pb-CVO caused both enzyme levels to increase significantly in both the strains. Visual observations of the midgut revealed cytotoxicity from sub-lethal concentrations of Temephos (0.04mg/L) and Pb-CVO (1.0mg/L) in both strains of Ae. aegypti compared to the control. The damage caused by Temephos was slightly less in WS compared to LS mosquito strains. Copyright © 2017 Elsevier Inc. All rights reserved.

Investigation of insecticide-resistance status of Cydia pomonella in Chinese populations.

PubMed

Yang, X-Q; Zhang, Y-L

2015-06-01

The codling moth Cydia pomonella (L.) is an economically important fruit pest and it has been directly targeted by insecticides worldwide. Serious resistance to insecticides has been reported in many countries. As one of the most serious invasive pest, the codling moth has populated several areas in China. However, resistance to insecticides has not been reported in China. We investigated the insecticide-resistance status of four field populations from Northwestern China by applying bioassays, enzyme activities, and mutation detections. Diagnostic concentrations of lambda-cyhalothrin, chlorpyrifos-ethyl, carbaryl, and imidacloprid were determined and used in bioassays. Field populations were less susceptible to chlorpyrifos-ethyl and carbaryl than laboratory strain. Insensitive populations displayed an elevated glutathione S-transferases (GSTs) activity. Reduced carboxylesterase (CarE) activity was observed in some insecticide insensitive populations and reduced acetylcholinesterase activity was observed only in the Wuw population. The cytochrome P450 polysubstrate monooxygenases activities in four field populations were not found to be different from susceptible strains. Neither the known-resistance mutation F399V in the acetylcholinesterase (AChE) gene, ace1, nor mutations in CarE gene CpCE-1 were found in adult individuals from our field populations. Native-PAGE revealed that various CarE isozymes and AChE insensitivity were occurring among Chinese populations. Our results indicate that codling moth populations from Northwestern China were insensitivity to chlorpyrifos-ethyl and carbaryl. Increased GST activity was responsible for insecticides insensitivity. Decreased CarE activity, as well as the presence of CarE and AChE polymorphisms might also be involved in insecticides insensitivity. New management strategies for managing this pest are discussed.

Bisphenol A-Associated Alterations in the Expression and Epigenetic Regulation of Genes Encoding Xenobiotic Metabolizing Enzymes in Human Fetal Liver

PubMed Central

Nahar, Muna S.; Kim, Jung H.; Sartor, Maureen A.; Dolinoy, Dana C.

2014-01-01

Alterations in xenobiotic metabolizing enzyme (XME) expression across the life course, along with genetic, nutritional, and environmental regulation, can influence how organisms respond to toxic insults. In this study, we investigated the hypothesis that in utero exposure to the endocrine active compound, bisphenol A (BPA), influences expression and epigenetic regulation of phase I and II XME genes during development. Using healthy 1st to 2nd trimester human fetal liver specimens quantified for internal BPA levels, we examined XME gene expression using PCR Array (n =8) and RNA-sequencing (n =12) platforms. Of the greater than 160 XME genes assayed, 2 phase I and 12 phase II genes exhibited significantly reduced expression with higher BPA levels, including isoforms from the carboxylesterase, catechol O-methyltransferase, glutathione S-transferase, sulfotransferase, and UDP-glucuronosyltransferase families. When the promoters of these candidate genes were evaluated in silico, putative binding sites for the E-twenty-six (ETS) and activator protein1 (AP1) related transcription factor families were identified and unique to 97% of all candidate transcripts. Interestingly, many ETS binding sites contain cytosine-guanine dinucleotides (CpGs) within their consensus sequences. Thus, quantitative analysis of CpG methylation of three candidate genes was conducted across n =50 samples. Higher BPA levels were associated with increased site-specific methylation at COMT (P <0.005) and increased average methylation at SULT2A1 (P <0.020) promoters. While toxicological studies have traditionally focused on high-dose effects and hormonal receptor mediated regulation, our findings suggest the importance of low-dose effects and nonclassical mechanisms of endocrine disruption during development. PMID:24214726

Analysis of expressed sequence tags from Actinidia: applications of a cross species EST database for gene discovery in the areas of flavor, health, color and ripening

PubMed Central

Crowhurst, Ross N; Gleave, Andrew P; MacRae, Elspeth A; Ampomah-Dwamena, Charles; Atkinson, Ross G; Beuning, Lesley L; Bulley, Sean M; Chagne, David; Marsh, Ken B; Matich, Adam J; Montefiori, Mirco; Newcomb, Richard D; Schaffer, Robert J; Usadel, Björn; Allan, Andrew C; Boldingh, Helen L; Bowen, Judith H; Davy, Marcus W; Eckloff, Rheinhart; Ferguson, A Ross; Fraser, Lena G; Gera, Emma; Hellens, Roger P; Janssen, Bart J; Klages, Karin; Lo, Kim R; MacDiarmid, Robin M; Nain, Bhawana; McNeilage, Mark A; Rassam, Maysoon; Richardson, Annette C; Rikkerink, Erik HA; Ross, Gavin S; Schröder, Roswitha; Snowden, Kimberley C; Souleyre, Edwige JF; Templeton, Matt D; Walton, Eric F; Wang, Daisy; Wang, Mindy Y; Wang, Yanming Y; Wood, Marion; Wu, Rongmei; Yauk, Yar-Khing; Laing, William A

2008-01-01

Background Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs). Results The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified. Conclusion This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia. PMID:18655731

Variability of esterase patterns in adult flies of the saltans species group of Drosophila (subgenus Sophophora).

PubMed

Bernardo, Alessandra Augusta; Bicudo, Hermione Elly Melara de Campos

2009-09-01

Esterases are known for their involvement in several physiological processes and high degree of polymorphism, in many organisms. Such polymorphism has been used to characterize species and species groups and to study genetic changes occurred in their evolutionary history. In the present study, the esterase patterns of 19 strains from 10 species representative of the five subgroups of the saltans species group were analyzed using polyacrylamide gel electrophoresis and alpha- and beta- naphthyl acetates as substrates. Fifty-one esterase bands were detected and classified as 31 alpha-esterases, 18 beta-esterases and two alpha/beta-esterases. On the basis of the inhibition patterns using Malathion and eserine sulfate, 34 bands were classified as carboxylesterases, 14 as acethylesterases and three as cholinesterases. Ten gene loci were tentatively established on the basis of data on band position in the gel, substrate preference and inhibition pattern. Twenty bands were species-specific, the remaining being shared by species from the same or different subgroups. Bands detected exclusively in males and bands with a different frequency or degree of expression between sexes were also detected. In the gels prepared for analysis of gene expression in the body parts (head, thorax and abdomen), the degree of expression of the beta-esterases was higher in the thorax, while the alpha-esterases were expressed predominantly in the abdomen and thorax. A global view of the data available at present on the esterases of the species from the saltans group and their degree of polymorphism are presented, as well as the possibility of using some beta-esterases, because of their characteristics in the gels, as markers for species identification.

Insecticide resistance in the sand fly, Phlebotomus papatasi from Khartoum State, Sudan

PubMed Central

2012-01-01

Background Phlebotomus papatasi the vector of cutaneous leishmaniasis (CL) is the most widely spread sand fly in Sudan. No data has previously been collected on insecticide susceptibility and/or resistance of this vector, and a first study to establish a baseline data is reported here. Methods Sand flies were collected from Surogia village, (Khartoum State), Rahad Game Reserve (eastern Sudan) and White Nile area (Central Sudan) using light traps. Sand flies were reared in the Tropical Medicine Research Institute laboratory. The insecticide susceptibility status of first progeny (F1) of P. papatasi of each population was tested using WHO insecticide kits. Also, P. papatasi specimens from Surogia village and Rahad Game Reserve were assayed for activities of enzyme systems involved in insecticide resistance (acetylcholinesterase (AChE), non-specific carboxylesterases (EST), glutathione-S-transferases (GSTs) and cytochrome p450 monooxygenases (Cyt p450). Results Populations of P. papatasi from White Nile and Rahad Game Reserve were sensitive to dichlorodiphenyltrichloroethane (DDT), permethrin, malathion, and propoxur. However, the P. papatasi population from Surogia village was sensitive to DDT and permethrin but highly resistant to malathion and propoxur. Furthermore, P. papatasi of Surogia village had significantly higher insecticide detoxification enzyme activity than of those of Rahad Game Reserve. The sand fly population in Surogia displayed high AChE activity and only three specimens had elevated levels for EST and GST. Conclusions The study provided evidence for malathion and propoxur resistance in the sand fly population of Surogia village, which probably resulted from anti-malarial control activities carried out in the area during the past 50 years. PMID:22397726

Effect of Different Administration Paradigms on Cholinesterase Inhibition following Repeated Chlorpyrifos Exposure in Late Preweanling Rats

PubMed Central

Carr, Russell L.; Nail, Carole A.

2008-01-01

Chlorpyrifos (CPS) is widely used in agricultural settings and residue analysis has suggested that children in agricultural communities are at risk of exposure. This has resulted in a large amount of literature investigating the potential for CPS-induced developmental neurotoxic effects. Two developmental routes of administration of CPS are orally in corn oil at a rate of 0.5 ml/kg and subcutaneously in dimethyl sulfoxide (DMSO) at a rate of 1.0 ml/kg. For comparison between these methods, rat pups were exposed daily from days 10 to 16 to CPS (5 mg/kg) either orally dissolved in corn oil or subcutaneously dissolved in DMSO, both at rates of either 0.5 or 1.0 ml/kg. A representative vehicle/route group was present for each treatment. Both the low and high volume CPS in DMSO subcutaneous groups were lower than that of the low and high volume CPS in oil oral groups. At 4 h following the final administration, serum carboxylesterase was inhibited > 90% with all treatments. For cholinesterase activity in the cerebellum, medulla-pons, forebrain, and hindbrain, and serum, inhibition in the CPS-oil groups was similar and inhibition in the CPS-DMSO groups was similar. However, significantly greater inhibition was present in the high volume CPS-DMSO group as compared to the CPS-oil groups. Inhibition in the low volume CPS-DMSO group was generally between that in the CPS-oil groups and the high volume CPS-DMSO group. These data suggest that using DMSO as a vehicle for CPS may alter the level of brain ChE inhibition. PMID:18703558

Cell Surface Expression of Bacterial Esterase A by Saccharomyces cerevisiae and Its Enhancement by Constitutive Activation of the Cellular Unfolded Protein Response▿ †

PubMed Central

Breinig, Frank; Diehl, Björn; Rau, Sabrina; Zimmer, Christian; Schwab, Helmut; Schmitt, Manfred J.

2006-01-01

Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg−1 protein for Kre1/EstA/Cwp2p and 72 mU mg−1 protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg−1 protein for Kre1/EstA/Cwp2p and 1.27 U mg−1 protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway. PMID:16980424

Cell surface expression of bacterial esterase A by Saccharomyces cerevisiae and its enhancement by constitutive activation of the cellular unfolded protein response.

PubMed

Breinig, Frank; Diehl, Björn; Rau, Sabrina; Zimmer, Christian; Schwab, Helmut; Schmitt, Manfred J

2006-11-01

Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg(-1) protein for Kre1/EstA/Cwp2p and 72 mU mg(-1) protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg(-1) protein for Kre1/EstA/Cwp2p and 1.27 U mg(-1) protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway.

Transcriptomic survey of the midgut of Anthonomus grandis (Coleoptera: Curculionidae).

PubMed

Salvador, Ricardo; Príncipi, Darío; Berretta, Marcelo; Fernández, Paula; Paniego, Norma; Sciocco-Cap, Alicia; Hopp, Esteban

2014-01-01

Anthonomus grandis Boheman is a key pest in cotton crops in the New World. Its larval stage develops within the flower bud using it as food and as protection against its predators. This behavior limits the effectiveness of its control using conventional insecticide applications and biocontrol techniques. In spite of its importance, little is known about its genome sequence and, more important, its specific expression in key organs like the midgut. Total mRNA isolated from larval midguts was used for pyrosequencing. Sequence reads were assembled and annotated to generate a unigene data set. In total, 400,000 reads from A. grandis midgut with an average length of 237 bp were assembled and combined into 20,915 contigs. The assembled reads fell into 6,621 genes models. BlastX search using the NCBI-NR database showed that 3,006 unigenes had significant matches to known sequences. Gene Ontology (GO) mapping analysis evidenced that A. grandis is able to transcripts coding for proteins involved in catalytic processing of macromolecules that allows its adaptation to very different feeding source scenarios. Furthermore, transcripts encoding for proteins involved in detoxification mechanisms such as p450 genes, glutathione-S-transferase, and carboxylesterases are also expressed. This is the first report of a transcriptomic study in A. grandis and the largest set of sequence data reported for this species. These data are valuable resources to expand the knowledge of this insect group and could be used in the design of new control strategies based in molecular information. © The Author 2014. Published by Oxford University Press on behalf of the Entomological Society of America.

Development of an eco-friendly mosquitocidal agent from Alangium salvifolium against the dengue vector Aedes aegypti and its biosafety on the aquatic predator.

PubMed

Thanigaivel, Annamalai; Vasantha-Srinivasan, Prabhakaran; Edwin, Edward-Sam; Ponsankar, Athirstam; Selin-Rani, Selvaraj; Chellappandian, Muthiah; Kalaivani, Kandaswamy; Senthil-Nathan, Sengottayan; Benelli, Giovanni

2018-04-01

Plant extracts with their enriched chemical constituents have established potential alternative mosquito control agents. In this research, we developed an eco-friendly mosquitocidal agent from Alangium salvifolium leaves against the dengue and Zika virus vector Aedes aegypti and we investigated its biosafety on the mosquito aquatic predator Toxorhynchites splendens. Results showed that the methanolic extract of A. salvifolium leaves was composed by eight main compounds, with major peak area for hexadecenoic acid (21.74%). LC 50 and LC 90 values calculated on Ae. aegypti fourth instar larvae were 104.80 and 269.15 ppm respectively. The methanolic extract tested at 100 ppm decreased the α-β carboxylesterase and SOD ratio significantly and upregulated the GST and CYP450 level. The A. salvifolium methanolic extract displayed significant repellent and adulticidal activity at 100 and 400 ppm respectively. The treatment with 100 ppm of the methanolic extract led to 210 min of protection from Ae. aegypti bites. Four hundred parts per million of the extract showed 98% adult mortality within 30 min from the treatment. Lastly, biosafety assays on the mosquito aquatic predator Tx. splendens showed that the toxicity of the A. salvifolium extract was significantly lower if compared to the cypermethrin-based treatments. The methanolic extract of A. salvifolium showed a maximum of 47.3% mortality rate at the concentration of 1000 ppm, while 0.7 ppm of cypermethrin achieved 91.3% mortality rate on Tx. splendens. Overall, our study enhances basic knowledge on how to improve natural larvicidal agents against dengue and Zika virus mosquito vector with harmless responses on non-target aquatic predators.

Avoidance behaviour response and esterase inhibition in the earthworm, Lumbricus terrestris, after exposure to chlorpyrifos.

PubMed

Martínez Morcillo, S; Yela, J L; Capowiez, Y; Mazzia, C; Rault, M; Sanchez-Hernandez, Juan C

2013-05-01

The avoidance response of earthworms to polluted soils has been standardised using a simple and low-cost test, which facilitates soil toxicity screening. In this study, the avoidance response of Lumbricus terrestris was quantified in chlorpyrifos-spiked soils, depending on the pesticide concentration and exposure duration. The inhibition of acetylcholinesterase (AChE) and carboxylesterase (CbE) activities was also determined as indirect measures of pesticide bioavailability. The effects of different chlorpyrifos concentrations were examined in a standardised test (two-chamber system) with 0.6, 3 and 15 mg/kg chlorpyrifos. A modification of the test involved a pre-exposure step (24, 48 or 72 h) in soils spiked with 15 mg/kg. In both protocols, earthworms were unable to avoid the contaminated soils. However, the esterase activities showed that all earthworms were exposed to chlorpyrifos. Acetylcholinesterase activity did not change in earthworms in the standardised behavioural test (0.58 ± 0.20 U/mg protein, mean ± SD; n = 72), whereas the CbE activity was significantly inhibited (62-87 % inhibition) in earthworms exposed to 3 and 15 mg/kg. In the modified test, earthworms had greatly inhibited AChE activity (0.088 ± 0.034 U/mg protein, n = 72), which was supported by reactivation of the inhibited enzyme activity in the presence of pralidoxime (2-PAM). Similarly, the CbE activity was significantly inhibited in earthworms with all treatments. This study suggests that the avoidance behaviour test for organophosphorus-contaminated soils could be supported by specific biomarkers to facilitate a better understanding of pesticide exposure and toxicity during this test.

Transcriptional responses in the hepatopancreas of Eriocheir sinensis exposed to deltamethrin

PubMed Central

Zhu, Fengjiao; Zeng, Liugen; Wang, Yulan; Lei, Xiaoqing; Yao, Yi; Hou, Yujie; Xu, Liangqing; Xiong, Chunxian; Yang, Xianle; Hu, Kun

2017-01-01

Deltamethrin is an important pesticide widely used against ectoparasites. Deltamethrin contamination has resulted in a threat to the healthy breeding of the Chinese mitten crab, Eriocheir sinensis. In this study, we investigated transcriptional responses in the hepatopancreas of E. sinensis exposed to deltamethrin. We obtained 99,087,448, 89,086,478, and 100,117,958 raw sequence reads from control 1, control 2, and control 3 groups, and 92,094,972, 92,883,894, and 92,500,828 raw sequence reads from test 1, test 2, and test 3 groups, respectively. After filtering and quality checking of the raw sequence reads, our analysis yielded 79,228,354, 72,336,470, 81,859,826, 77,649,400, 77,194,276, and 75,697,016 clean reads with a mean length of 150 bp from the control and test groups. After deltamethrin treatment, a total of 160 and 167 genes were significantly upregulated and downregulated, respectively. Gene ontology terms “biological process,” “cellular component,” and “molecular function” were enriched with respect to cell killing, cellular process, other organism part, cell part, binding, and catalytic. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes showed that the metabolic pathways were significantly enriched. We found that the CYP450 enzyme system, carboxylesterase, glutathione-S-transferase, and material (including carbohydrate, lipid, protein, and other substances) metabolism played important roles in the metabolism of deltamethrin in the hepatopancreas of E. sinensis. This study revealed differentially expressed genes related to insecticide metabolism and detoxification in E. sinensis for the first time and will help in understanding the toxicity and molecular metabolic mechanisms of deltamethrin in E. sinensis. PMID:28910412

Evidence for trade-offs in detoxification and chemosensation gene signatures in Plutella xylostella.

PubMed

Bautista, Ma Anita M; Bhandary, Binny; Wijeratne, Asela J; Michel, Andrew P; Hoy, Casey W; Mittapalli, Omprakash

2015-03-01

Detoxification genes have been associated with insecticide adaptation in the diamondback moth, Plutella xylostella. The link between chemosensation genes and adaptation, however, remains unexplored. To gain a better understanding of the involvement of these genes in insecticide adaptation, the authors exposed lines of P. xylostella to either high uniform (HU) or low heterogeneous (LH) concentrations of permethrin, expecting primarily physiological or behavioral selection respectively. Initially, 454 pyrosequencing was applied, followed by an examination of expression profiles of candidate genes that responded to selection [cytochrome P450 (CYP), glutathione S-transferase (GST), carboxylesterase (CarE), chemosensory protein (CSP) and odorant-binding protein (OBP)] by quantitative PCR in the larvae. Toxicity and behavioral assays were also conducted to document the effects of the two forms of exposure. Pyrosequencing of the P. xylostella transcriptome from adult heads and third instars produced 198,753 reads with 52,752,486 bases. Quantitative PCR revealed overexpression of CYP4M14, CYP305B1 and CSP8 in HU larvae. OBP13, however, was highest in LH. Larvae from LH and HU lines had up to five- and 752-fold resistance levels respectively, which could be due to overexpression of P450s. However, the behavioral responses of all lines to a series of permethrin concentrations did not vary significantly in any of the generations examined, in spite of the observed upregulation of CSP8 and OBP13. Expression patterns from the target genes provide insights into behavioral and physiological responses to permethrin and suggest a new avenue of research on the role of chemosensation genes in insect adaptation to toxins. © 2014 Society of Chemical Industry.

Implications of Efficient Hepatic Delivery by Tenofovir Alafenamide (GS-7340) for Hepatitis B Virus Therapy

PubMed Central

Wang, Ting; Park, Yeojin; Hao, Jia; Lepist, Eve-Irene; Babusis, Darius; Ray, Adrian S.

2015-01-01

Tenofovir alafenamide (TAF) is a prodrug of tenofovir (TFV) currently in clinical evaluation for treatment for HIV and hepatitis B virus (HBV) infections. Since the target tissue for HBV is the liver, the hepatic delivery and metabolism of TAF in primary human hepatocytes in vitro and in dogs in vivo were evaluated here. Incubation of primary human hepatocytes with TAF resulted in high levels of the pharmacologically active metabolite tenofovir diphosphate (TFV-DP), which persisted in the cell with a half-life of >24 h. In addition to passive permeability, studies of transfected cell lines suggest that the hepatic uptake of TAF is also facilitated by the organic anion-transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3, respectively). In order to inhibit HBV reverse transcriptase, TAF must be converted to the pharmacologically active form, TFV-DP. While cathepsin A is known to be the major enzyme hydrolyzing TAF in cells targeted by HIV, including lymphocytes and macrophages, TAF was primarily hydrolyzed by carboxylesterase 1 (CES1) in primary human hepatocytes, with cathepsin A making a small contribution. Following oral administration of TAF to dogs for 7 days, TAF was rapidly absorbed. The appearance of the major metabolite TFV in plasma was accompanied by a rapid decline in circulating TAF. Consistent with the in vitro data, high and persistent levels of TFV-DP were observed in dog livers. Notably, higher liver TFV-DP levels were observed after administration of TAF than those given TDF. These results support the clinical testing of once-daily low-dose TAF for the treatment of HBV infection. PMID:25870059

Intracellular Activation of Tenofovir Alafenamide and the Effect of Viral and Host Protease Inhibitors

PubMed Central

Bam, Rujuta A.; Willkom, Madeleine; Frey, Christian R.; Tsai, Luong; Stray, Kirsten M.; Yant, Stephen R.; Cihlar, Tomas

2015-01-01

Tenofovir alafenamide fumarate (TAF) is an oral phosphonoamidate prodrug of the HIV reverse transcriptase nucleotide inhibitor tenofovir (TFV). Previous studies suggested a principal role for the lysosomal serine protease cathepsin A (CatA) in the intracellular activation of TAF. Here we further investigated the role of CatA and other human hydrolases in the metabolism of TAF. Overexpression of CatA or liver carboxylesterase 1 (Ces1) in HEK293T cells increased intracellular TAF hydrolysis 2- and 5-fold, respectively. Knockdown of CatA expression with RNA interference (RNAi) in HeLa cells reduced intracellular TAF metabolism 5-fold. Additionally, the anti-HIV activity and the rate of CatA hydrolysis showed good correlation within a large set of TFV phosphonoamidate prodrugs. The covalent hepatitis C virus (HCV) protease inhibitors (PIs) telaprevir and boceprevir potently inhibited CatA-mediated TAF activation (50% inhibitory concentration [IC50] = 0.27 and 0.16 μM, respectively) in vitro and also reduced its anti-HIV activity in primary human CD4+ T lymphocytes (21- and 3-fold, respectively) at pharmacologically relevant concentrations. In contrast, there was no inhibition of CatA or any significant effect on anti-HIV activity of TAF observed with cobicistat, noncovalent HIV and HCV PIs, or various prescribed inhibitors of host serine proteases. Collectively, these studies confirm that CatA plays a pivotal role in the intracellular metabolism of TAF, whereas the liver esterase Ces1 likely contributes to the hepatic activation of TAF. Moreover, this work demonstrates that a wide range of viral and host PIs, with the exception of telaprevir and boceprevir, do not interfere with the antiretroviral activity of TAF. PMID:26503655

Stability of cytotoxic luteinizing hormone-releasing hormone conjugate (AN-152) containing doxorubicin 14-O-hemiglutarate in mouse and human serum in vitro: implications for the design of preclinical studies.

PubMed

Nagy, A; Plonowski, A; Schally, A V

2000-01-18

Recently, we developed a series of cytotoxic peptide conjugates containing 14-O-glutaryl esters of doxorubicin (DOX) or 2-pyrrolino-DOX (AN-201). Serum carboxylesterase enzymes (CE) can partially hydrolyze these conjugates in the circulation, releasing the cytotoxic radical, before the targeting is complete. CE activity in serum of nude mice is about 10 times higher than in human serum. Thus, we found that the t(1/2) of AN-152, an analog of luteinizing hormone-releasing hormone (LH-RH) containing DOX, at 0.3 mg/ml is 19. 49 +/- 0.74 min in mouse serum and 126.06 +/- 3.03 min in human serum in vitro. The addition of a CE inhibitor, diisopropyl fluorophosphate (DFP), to mouse serum in vitro significantly (P < 0. 01) prolongs the t(1/2) of AN-152 to 69.63 +/- 4.44 min. When DFP is used in vivo, 400 nmol/kg cytotoxic somatostatin analog AN-238 containing AN-201 is well tolerated by mice, whereas all animals die after the same dose without DFP. In contrast, DFP has no effect on the tolerance of AN-201. A better tolerance to AN-238 after DFP treatment is due to the selective uptake of AN-238 by somatostatin receptor-positive tissues. Our results demonstrate that the suppression of the CE activity in nude mice greatly decreases the toxicity of cytotoxic hybrids containing 2-pyrrolino-DOX 14-O-hemiglutarate and brings this animal model closer to the conditions that exist in humans. The use of DFP together with these peptide conjugates in nude mice permits a better understanding of their mechanism of action and improves the clinical predictability of the oncological and toxicological results.

Effects of two strobilurins (azoxystrobin and picoxystrobin) on embryonic development and enzyme activities in juveniles and adult fish livers of zebrafish (Danio rerio).

PubMed

Jia, Wei; Mao, Liangang; Zhang, Lan; Zhang, Yanning; Jiang, Hongyun

2018-09-01

Azoxystrobin and picoxystrobin are two primary strobilurin fungicides used worldwide. This study was conducted to test their effects on embryonic development and the activity of several enzyme in the zebrafish (Danio rerio). After fish eggs were separately exposed to azoxystrobin and picoxystrobin from 24 to 144 h post fertilization (hpf), the mortality, hatching, and teratogenetic rates were measured. Additionally, effects of azoxystrobin and picoxystrobin on activities of three important antioxidant enzymes [catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD)] and two primary detoxification enzymes [carboxylesterase (CarE) and glutathione S-transferase (GST)] and malondialdehyde (MDA) content in zebrafish larvae (96 h) and livers of adult zebrafish of both sexes were also assessed for potential toxicity mechanisms. Based on the embryonic development test results, the mortality, hatching, and teratogenetic rates of eggs treated with azoxystrobin and picoxystrobin all showed significant dose- and time-dependent effects, and the 144-h LC 50 values of azoxystrobin and picoxystrobin were 1174.9 and 213.8 μg L -1 , respectively. In the larval zebrafish (96 h) test, activities of CAT, POD, CarE, and GST and MDA content in azoxystrobin and picoxystrobin-treated zebrafish larvae increased significantly with concentrations of the pesticides compared with those in the control. We further revealed that azoxystrobin and picoxystrobin exposure both caused significant oxidative stress in adult fish livers and the changes differed between the sexes. Our results indicated that picoxystrobin led to higher embryonic development toxicity and oxidative stress than azoxystrobin in zebrafish and the male zebrafish liver had stronger ability to detoxify than that of the females. Copyright © 2018 Elsevier Ltd. All rights reserved.

RNA-sequencing quantification of hepatic ontogeny of phase-I enzymes in mice.

PubMed

Peng, Lai; Cui, Julia Y; Yoo, Byunggil; Gunewardena, Sumedha S; Lu, Hong; Klaassen, Curtis D; Zhong, Xiao-Bo

2013-12-01

Phase-I drug metabolizing enzymes catalyze reactions of hydrolysis, reduction, and oxidation of drugs and play a critical role in drug metabolism. However, the functions of most phase-I enzymes are not mature at birth, which markedly affects drug metabolism in newborns. Therefore, characterization of the expression profiles of phase-I enzymes and the underlying regulatory mechanisms during liver maturation is needed for better estimation of using drugs in pediatric patients. The mouse is an animal model widely used for studying the mechanisms in the regulation of developmental expression of phase-I genes. Therefore, we applied RNA sequencing to provide a "true quantification" of the mRNA expression of phase-I genes in the mouse liver during development. Liver samples of male C57BL/6 mice at 12 different ages from prenatal to adulthood were used for defining the ontogenic mRNA profiles of phase-I families, including hydrolysis: carboxylesterase (Ces), paraoxonase (Pon), and epoxide hydrolase (Ephx); reduction: aldo-keto reductase (Akr), quinone oxidoreductase (Nqo), and dihydropyrimidine dehydrogenase (Dpyd); and oxidation: alcohol dehydrogenase (Adh), aldehyde dehydrogenase (Aldh), flavin monooxygenases (Fmo), molybdenum hydroxylase (Aox and Xdh), cytochrome P450 (P450), and cytochrome P450 oxidoreductase (Por). Two rapidly increasing stages of total phase-I gene expression after birth reflect functional transition of the liver during development. Diverse expression patterns were identified, and some large gene families contained the mRNA of genes that are enriched at different stages of development. Our study reveals the mRNA abundance of phase-I genes in the mouse liver during development and provides a valuable foundation for mechanistic studies in the future.

Lepidopteran insect susceptibility to silver nanoparticles and measurement of changes in their growth, development and physiology.

PubMed

Yasur, Jyothsna; Rani, Pathipati Usha

2015-04-01

Increased use of nanomaterials in various fields of science has lead for the need to study the impact of nanomaterial on the environment in general and on insect and plant life in particular. We studied the impact of silver nanoparticles (AgNPs) on growth and feeding responses of two lepidopteran pests of castor plant (Ricinus communis L.) namely Asian armyworm, Spodoptera litura F. and castor semilooper, Achaea janata L. Larvae were fed with PVP coated-AgNPs treated castor leaf at different concentrations and their activity was compared to that of silver nitrate (AgNO3) treated leaf diets. Larval and pupal body weights decreased along with the decrease in the concentrations of AgNPs and AgNO3 in both the test insects. Low amounts of silver were accumulated in the larval guts, but major portion of it was eliminated through the feces. Ultrastructural studies of insect gut cell using Transmission Electron Microscopy (TEM) showed accumulation of silver nanoparticles in cell organelles. Changes in the antioxidative and detoxifying enzymes of the treated larva were estimated. The effect of treatments showed differences in the activities of detoxifying enzymes, carboxylesterases (CarE), glucosidases (Glu) and glutathione S-transferases (GST) in the larval gut. Activities of superoxide dismutase, catalase, and peroxidase were also altered in the larval bodies due to the AgNPs treatments, suggesting that exposure of larvae to nanoparticles induces oxidative stress, which is countered by antioxidant enzymes. Induction of these enzymes may be an effective detoxification mechanism by which the herbivorous insect defends itself against nanoparticle treatment. Copyright © 2014 Elsevier Ltd. All rights reserved.

Transcriptional response after exposure to domoic acid-producing Pseudo-nitzschia in the digestive gland of the mussel Mytilus galloprovincialis.

PubMed

Pazos, Antonio J; Ventoso, Pablo; Martínez-Escauriaza, Roi; Pérez-Parallé, M Luz; Blanco, Juan; Triviño, Juan C; Sánchez, José L

2017-12-15

Bivalve molluscs are filter feeding species that can accumulate biotoxins in their body tissues during harmful algal blooms. Amnesic Shellfish Poisoning (ASP) is caused by species of the diatom genus Pseudo-nitzschia, which produces the toxin domoic acid. The Mytilus galloprovincialis digestive gland transcriptome was de novo assembled based on the sequencing of 12 cDNA libraries, six obtained from control mussels and six from mussels naturally exposed to domoic acid-producing diatom Pseudo-nitzschia australis. After de novo assembly 94,727 transcripts were obtained, with an average length of 1015 bp and a N50 length of 761 bp. The assembled transcripts were clustered (homology > 90%) into 69,294 unigenes. Differential gene expression analysis was performed (DESeq2 algorithm) in the digestive gland following exposure to the toxic algae. A total of 1158 differentially expressed unigenes (absolute fold change > 1.5 and p-value < 0.05) were detected: 686 up-regulated and 472 down-regulated. Several membrane transporters belonging to the family of the SLC (solute carriers) were over-expressed in exposed mussels. Functional enrichment was performed using Pfam annotations obtained from the genes differentially expressed, 37 Pfam families were found to be significantly (FDR adjusted p-value < 0.1) enriched. Some of these families (sulfotransferases, aldo/keto reductases, carboxylesterases, C1q domain and fibrinogen C-terminal globular domain) could be putatively involved in detoxification processes, in the response against of the oxidative stress and in immunological processes. Protein network analysis with STRING algorithm found alteration of the Notch signaling pathway under the action of domoic acid-producing Pseudo-nitzschia. In conclusion, this study provides a high quality reference transcriptome of M. galloprovincialis digestive gland and identifies potential genes involved in the response to domoic acid. Copyright © 2017 Elsevier Ltd. All rights reserved.

Thiamethoxam Resistance in the House Fly, Musca domestica L.: Current Status, Resistance Selection, Cross-Resistance Potential and Possible Biochemical Mechanisms.

PubMed

Khan, Hafiz Azhar Ali; Akram, Waseem; Iqbal, Javaid; Naeem-Ullah, Unsar

2015-01-01

The house fly, Musca domestica L., is an important ectoparasite with the ability to develop resistance to insecticides used for their control. Thiamethoxam, a neonicotinoid, is a relatively new insecticide and effectively used against house flies with a few reports of resistance around the globe. To understand the status of resistance to thiamethoxam, eight adult house fly strains were evaluated under laboratory conditions. In addition, to assess the risks of resistance development, cross-resistance potential and possible biochemical mechanisms, a field strain of house flies was selected with thiamethoxam in the laboratory. The results revealed that the field strains showed varying level of resistance to thiamethoxam with resistance ratios (RR) at LC50 ranged from 7.66-20.13 folds. Continuous selection of the field strain (Thia-SEL) for five generations increased the RR from initial 7.66 fold to 33.59 fold. However, resistance declined significantly when the Thia-SEL strain reared for the next five generations without exposure to thiamethoxam. Compared to the laboratory susceptible reference strain (Lab-susceptible), the Thia-SEL strain showed cross-resistance to imidacloprid. Synergism tests revealed that S,S,S-tributylphosphorotrithioate (DEF) and piperonyl butoxide (PBO) produced synergism of thiamethoxam effects in the Thia-SEL strain (2.94 and 5.00 fold, respectively). In addition, biochemical analyses revealed that the activities of carboxylesterase (CarE) and mixed function oxidase (MFO) in the Thia-SEL strain were significantly higher than the Lab-susceptible strain. It seems that metabolic detoxification by CarE and MFO was a major mechanism for thiamethoxam resistance in the Thia-SEL strain of house flies. The results could be helpful in the future to develop an improved control strategy against house flies.

Thiamethoxam Resistance in the House Fly, Musca domestica L.: Current Status, Resistance Selection, Cross-Resistance Potential and Possible Biochemical Mechanisms

PubMed Central

Khan, Hafiz Azhar Ali; Akram, Waseem; Iqbal, Javaid; Naeem-Ullah, Unsar

2015-01-01

The house fly, Musca domestica L., is an important ectoparasite with the ability to develop resistance to insecticides used for their control. Thiamethoxam, a neonicotinoid, is a relatively new insecticide and effectively used against house flies with a few reports of resistance around the globe. To understand the status of resistance to thiamethoxam, eight adult house fly strains were evaluated under laboratory conditions. In addition, to assess the risks of resistance development, cross-resistance potential and possible biochemical mechanisms, a field strain of house flies was selected with thiamethoxam in the laboratory. The results revealed that the field strains showed varying level of resistance to thiamethoxam with resistance ratios (RR) at LC50 ranged from 7.66-20.13 folds. Continuous selection of the field strain (Thia-SEL) for five generations increased the RR from initial 7.66 fold to 33.59 fold. However, resistance declined significantly when the Thia-SEL strain reared for the next five generations without exposure to thiamethoxam. Compared to the laboratory susceptible reference strain (Lab-susceptible), the Thia-SEL strain showed cross-resistance to imidacloprid. Synergism tests revealed that S,S,S-tributylphosphorotrithioate (DEF) and piperonyl butoxide (PBO) produced synergism of thiamethoxam effects in the Thia-SEL strain (2.94 and 5.00 fold, respectively). In addition, biochemical analyses revealed that the activities of carboxylesterase (CarE) and mixed function oxidase (MFO) in the Thia-SEL strain were significantly higher than the Lab-susceptible strain. It seems that metabolic detoxification by CarE and MFO was a major mechanism for thiamethoxam resistance in the Thia-SEL strain of house flies. The results could be helpful in the future to develop an improved control strategy against house flies. PMID:25938578

A simple method for accurate endotracheal placement of an intubation tube in Guinea pigs to assess lung injury following chemical exposure.

PubMed

Nambiar, M P; Gordon, R K; Moran, T S; Richards, S M; Sciuto, A M

2007-01-01

ABSTRACT Guinea pigs are considered as the animal model of choice for toxicology and medical countermeasure studies against chemical warfare agents (CWAs) and toxic organophosphate pesticides because of the low levels of carboxylesterase compared to rats and mice. However, it is difficult to intubate guinea pigs without damaging the larynx to perform CWA inhalation experiments. We describe an easy technique of intubation of guinea pigs for accurate endotracheal placement of the intubation tube. The technique involves a speculum made by cutting the medium-size ear speculum in the midline leaving behind the intact circular connector to the otoscope. Guinea pigs were anesthetized with Telazol/meditomidine, the tongue was pulled using blunt forceps, and an otoscope attached with the specially prepared speculum was inserted gently. Insertion of the speculum raises the epiglottis and restrains the movements of vocal cord, which allows smooth insertion of the metal stylet-reinforced intubation tube. Accurate endotracheal placement of the intubation tube was achieved by measuring the length from the tracheal bifurcation to vocal cord and vocal cord to the upper front teeth. The average length of the trachea in guinea pigs (275 +/- 25 g) was 5.5 +/- 0.2 cm and the distance from the vocal cord to the front teeth was typically 3 cm. Coinciding an intubation tube marked at 6 cm with the upper front teeth accurately places the intubation tube 2.5 cm above the tracheal bifurcation. This simple method of intubation does not disturb the natural flora of the mouth and causes minimum laryngeal damage. It is rapid and reliable, and will be very valuable in inhalation exposure to chemical/biological warfare agents or toxic chemicals to assess respiratory toxicity and develop medical countermeasures.

RNA-seq of Rice Yellow Stem Borer Scirpophaga incertulas Reveals Molecular Insights During Four Larval Developmental Stages

PubMed Central

Renuka, Pichili; Madhav, Maganti S.; Padmakumari, Ayyagari Phani; Barbadikar, Kalyani M.; Mangrauthia, Satendra K.; Vijaya Sudhakara Rao, Kola; Marla, Soma S.; Ravindra Babu, Vemuri

2017-01-01

The yellow stem borer (YSB), Scirpophaga incertulas, is a prominent pest in rice cultivation causing serious yield losses. The larval stage is an important stage in YSB, responsible for maximum infestation. However, limited knowledge exists on the biology and mechanisms underlying the growth and differentiation of YSB. To understand and identify the genes involved in YSB development and infestation, so as to design pest control strategies, we performed de novo transcriptome analysis at the first, third, fifth, and seventh larval developmental stages employing Illumina Hi-seq. High-quality reads (HQR) of ∼229 Mb were assembled into 24,775 transcripts with an average size of 1485 bp. Genes associated with various metabolic processes, i.e., detoxification mechanism [CYP450, GSTs, and carboxylesterases (CarEs)], RNA interference (RNAi) machinery (Dcr-1, Dcr-2, Ago-1, Ago-2, Sid-1, Sid-2, Sid-3, and Sid-1-related gene), chemoreception (CSPs, GRs, OBPs, and ORs), and regulators [transcription factors (TFs) and hormones] were differentially regulated during the developmental stages. Identification of stage-specific transcripts made it possible to determine the essential processes of larval development. Comparative transcriptome analysis revealed that YSB has not evolved much with respect to the detoxification mechanism, but showed the presence of distinct RNAi machinery. The presence of strong specific visual recognition coupled with chemosensory mechanisms supports the monophagous nature of YSB. Designed expressed sequenced tags-simple-sequence repeats (EST-SSRs) will facilitate accurate estimation of the genetic diversity of YSB. This is the first report on characterization of the YSB transcriptome and the identification of genes involved in key processes, which will help researchers and industry to devise novel pest control strategies. This study also opens up a new avenue to develop next-generation resistant rice using RNAi or genome editing approaches. PMID

Methylphenidate side effect profile is influenced by genetic variation in the attention-deficit/hyperactivity disorder-associated CES1 gene.

PubMed

Johnson, Katherine A; Barry, Edwina; Lambert, David; Fitzgerald, Michael; McNicholas, Fiona; Kirley, Aiveen; Gill, Michael; Bellgrove, Mark A; Hawi, Ziarih

2013-12-01

A naturalistic, prospective study of the influence of genetic variation on dose prescribed, clinical response, and side effects related to stimulant medication in 77 children with attention-deficit/hyperactivity disorder (ADHD) was undertaken. The influence of genetic variation of the CES1 gene coding for carboxylesterase 1A1 (CES1A1), the major enzyme responsible for the first-pass, stereoselective metabolism of methylphenidate, was investigated. Parent- and teacher-rated behavioral questionnaires were collected at baseline when the children were medication naïve, and again at 6 weeks while they were on medication. Medication dose, prescribed at the discretion of the treating clinician, and side effects, were recorded at week 6. Blood and saliva samples were collected for genotyping. Single nucleotide polymorphisms (SNPs) were selected in the coding, non-coding and the 3' flanking region of the CES1 gene. Genetic association between CES1 variants and ADHD was investigated in an expanded sample of 265 Irish ADHD families. Analyses were conducted using analysis of covariance (ANCOVA) and logistic regression models. None of the CES1 gene variants were associated with the dose of methylphenidate provided or the clinical response recorded at the 6 week time point. An association between two CES1 SNP markers and the occurrence of sadness as a side effect of short-acting methylphenidate was found. The two associated CES1 markers were in linkage disequilibrium and were significantly associated with ADHD in a larger sample of ADHD trios. The associated CES1 markers were also in linkage disequilibrium with two SNP markers of the noradrenaline transporter gene (SLC6A2). This study found an association between two CES1 SNP markers and the occurrence of sadness as a side effect of short-acting methylphenidate. These markers were in linkage disequilibrium together and with two SNP markers of the noradrenaline transporter gene.

Diversity of hydrolases from hydrothermal vent sediments of the Levante Bay, Vulcano Island (Aeolian archipelago) identified by activity-based metagenomics and biochemical characterization of new esterases and an arabinopyranosidase.

PubMed

Placido, Antonio; Hai, Tran; Ferrer, Manuel; Chernikova, Tatyana N; Distaso, Marco; Armstrong, Dale; Yakunin, Alexander F; Toshchakov, Stepan V; Yakimov, Michail M; Kublanov, Ilya V; Golyshina, Olga V; Pesole, Graziano; Ceci, Luigi R; Golyshin, Peter N

2015-12-01

A metagenomic fosmid expression library established from environmental DNA (eDNA) from the shallow hot vent sediment sample collected from the Levante Bay, Vulcano Island (Aeolian archipelago) was established in Escherichia coli. Using activity-based screening assays, we have assessed 9600 fosmid clones corresponding to approximately 350 Mbp of the cloned eDNA, for the lipases/esterases/lactamases, haloalkane and haloacid dehalogenases, and glycoside hydrolases. Thirty-four positive fosmid clones were selected from the total of 120 positive hits and sequenced to yield ca. 1360 kbp of high-quality assemblies. Fosmid inserts were attributed to the members of ten bacterial phyla, including Proteobacteria, Bacteroidetes, Acidobateria, Firmicutes, Verrucomicrobia, Chloroflexi, Spirochaetes, Thermotogae, Armatimonadetes, and Planctomycetes. Of ca. 200 proteins with high biotechnological potential identified therein, we have characterized in detail three distinct α/β-hydrolases (LIPESV12_9, LIPESV12_24, LIPESV12_26) and one new α-arabinopyranosidase (GLV12_5). All LIPESV12 enzymes revealed distinct substrate specificities tested against 43 structurally diverse esters and 4 p-nitrophenol carboxyl esters. Of 16 different glycosides tested, the GLV12_5 hydrolysed only p-nitrophenol-α-(L)-arabinopyranose with a high specific activity of about 2.7 kU/mg protein. Most of the α/β-hydrolases were thermophilic and revealed a high tolerance to, and high activities in the presence of, numerous heavy metal ions. Among them, the LIPESV12_24 was the best temperature-adapted, retaining its activity after 40 min of incubation at 90 °C. Furthermore, enzymes were active in organic solvents (e.g., >30% methanol). Both LIPESV12_24 and LIPESV12_26 had the GXSXG pentapeptides and the catalytic triads Ser-Asp-His typical to the representatives of carboxylesterases of EC 3.1.1.1.

Transcription profiling of a recently colonised pyrethroid resistant Anopheles gambiae strain from Ghana

PubMed Central

Müller, Pie; Donnelly, Martin J; Ranson, Hilary

2007-01-01

Background Mosquito resistance to the pyrethroid insecticides used to treat bednets threatens the sustainability of malaria control in sub-Saharan Africa. While the impact of target site insensitivity alleles is being widely discussed the implications of insecticide detoxification – though equally important – remains elusive. The successful development of new tools for malaria intervention and management requires a comprehensive understanding of insecticide resistance, including metabolic resistance mechanisms. Although three enzyme families (cytochrome P450s, glutathione S-transferases and carboxylesterases) have been widely associated with insecticide detoxification the role of individual enzymes is largely unknown. Results Here, constitutive expression patterns of genes putatively involved in conferring pyrethroid resistance was investigated in a recently colonised pyrethroid resistant Anopheles gambiae strain from Odumasy, Southern Ghana. RNA from the resistant strain and a standard laboratory susceptible strain, of both sexes was extracted, reverse transcribed and labelled with either Cy3- or Cy5-dye. Labelled cDNA was co-hybridised to the detox chip, a custom-made microarray containing over 230 A. gambiae gene fragments predominantly from enzyme families associated with insecticide resistance. After hybridisation, Cy3- and Cy5-signal intensities were measured and compared gene by gene. In both females and males of the resistant strain the cytochrome P450s CYP6Z2 and CYP6M2 are highly over-expressed along with a member of the superoxide dismutase (SOD) gene family. Conclusion These genes differ from those found up-regulated in East African strains of pyrethroid resistant A. gambiae and constitute a novel set of candidate genes implicated in insecticide detoxification. These data suggest that metabolic resistance may have multiple origins in A. gambiae, which has strong implications for the management of resistance. PMID:17261191

The activity of detoxifying enzymes in the infective juveniles of Heterorhabditis bacteriophora strains: Purification and characterization of two acetylcholinesterases.

PubMed

Mohamed, Magda A; Mahdy, El-Sayed M E; Ghazy, Abd-El-Hady M; Ibrahim, Nihal M; El-Mezayen, Hatem A; Ghanem, Manal M E

2016-02-01

The infectivity and detoxifying enzyme activities including glutathione-S-transferase (GST), acetylcholinesterase (AChE) and carboxylesterase (CaE) are investigated in the infective juveniles (IJs) of six different strains of Heterorhabditis bacteriophora as a biocontrol agent against insect pests. The specific activities ranged from 10.8-29.8 and 50-220units/mg protein for GST and AChE, respectively; and from 24.7-129 and 22.6-77.3units/mg protein for CaE as estimated by P-nitrophenyl and α-naphthyl acetates, respectively. H. bacteriophora EM2 strain has the highest infectivity and the highest enzymatic activities as well. AChE is the predominant detoxifying enzyme that might imply its major role in the detoxification of insecticide(s). The isoenzyme pattern demonstrated two major slow-moving isoforms in all EPN strains examined. Purification of two AChE isoforms, AChEAII and AChEBI, from H. bacteriophora EM2 strain is performed by ammonium sulfate precipitation, gel filtration on Sephacryl S-200 and chromatography on DEAE-Sepharose. AChEAII and AChEBII have specific activities of 1207 and 1560unit/mg protein, native molecular weights of 180 and 68kDa, and are found in dimeric and monomeric forms, respectively. Both isoforms showed optimum activity at pH8.5 and 35°C. AChEBI exhibited higher thermal stability and higher activation energy than AChEAII. The enzymatic activities of purified AChEs are completely inhibited by Hg(+2) and Ni(+2) and greatly enhanced by Mn(+2). The substrate specificity, the relative efficiency of substrates hydrolysis, substrate inhibition and inhibition by BW284C51, but not by iso-OMPA, clearly indicated that they are true AChEs; their properties are compared with those recorded for insects as target hosts for H. bacteriophora EM2. Copyright © 2015 Elsevier Inc. All rights reserved.

Physiological responses of emerald ash borer larvae to feeding on different ash species reveal putative resistance mechanisms and insect counter-adaptations.

PubMed

Rigsby, C M; Showalter, D N; Herms, D A; Koch, J L; Bonello, P; Cipollini, D

2015-07-01

Emerald ash borer, Agrilus planipennis Fairmaire, an Asian wood-boring beetle, has devastated ash (Fraxinus spp.) trees in North American forests and landscapes since its discovery there in 2002. In this study, we collected living larvae from EAB-resistant Manchurian ash (Fraxinus mandschurica), and susceptible white (Fraxinus americana) and green (Fraxinus pennsylvanica) ash hosts, and quantified the activity and production of selected detoxification, digestive, and antioxidant enzymes. We hypothesized that differences in larval physiology could be used to infer resistance mechanisms of ash. We found no differences in cytochrome P450, glutathione-S-transferase, carboxylesterase, sulfotransferase, and tryptic BApNAase activities between larvae feeding on different hosts. Despite this, Manchurian ash-fed larvae produced a single isozyme of low electrophoretic mobility that was not produced in white or green ash-fed larvae. Additionally, larvae feeding on white and green ash produced two serine protease isozymes of high electrophoretic mobility that were not observed in Manchurian ash-fed larvae. We also found lower activity of β-glucosidase and higher activities of monoamine oxidase, ortho-quinone reductase, catalase, superoxide dismutase, and glutathione reductase in Manchurian ash-fed larvae compared to larvae that had fed on susceptible ash. A single isozyme was detected for both catalase and superoxide dismutase in all larval groups. The activities of the quinone-protective and antioxidant enzymes are consistent with the resistance phenotype of the host species, with the highest activities measured in larvae feeding on resistant Manchurian ash. We conclude that larvae feeding on Manchurian ash could be under quinone and oxidative stress, suggesting these may be potential mechanisms of resistance of Manchurian ash to EAB larvae, and that quinone-protective and antioxidant enzymes are important counter-adaptations of larvae for dealing with these resistance

Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination

PubMed Central

Rejón, Juan D.; Zienkiewicz, Agnieszka; Rodríguez-García, María Isabel; Castro, Antonio J.

2012-01-01

Background and Aims A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized. Methods The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods. Key Results Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles. Conclusions In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of

Joint toxic effects of triazophos and imidacloprid on zebrafish (Danio rerio).

PubMed

Wu, Shenggan; Li, Xinfang; Liu, Xinju; Yang, Guiling; An, Xuehua; Wang, Qiang; Wang, Yanhua

2018-04-01

Pesticide contamination is more often found as a mixture of different pesticides in water bodies rather than individual compounds. However, regulatory risk evaluation is mostly based on the effects of individual pesticides. In the present study, we aimed to investigate the individual and joint toxicities of triazophos (TRI) and imidacloprid (IMI) to the zebrafish (Danio rerio) using acute indices and various sublethal endpoints. Results from 96-h semi-static test indicated that the LC 50 values of TRI to D. rerio at multiple life stages (embryonic, larval, juvenile and adult stages) ranged from 0.49 (0.36-0.71) to 4.99 (2.06-6.81) mg a.i. L -1 , which were higher than those of IMI ranging from 26.39 (19.04-38.01) to 128.9 (68.47-173.6) mg a.i. L -1 . Pesticide mixtures of TRI and IMI displayed synergistic response to zebrafish embryos. Activities of carboxylesterase (CarE) and catalase (CAT) were significantly changed in most of the individual and joint exposures of pesticides compared with the control group. The expressions of 26 genes related to oxidative stress, cellular apoptosis, immune system, hypothalamic-pituitary-thyroid and hypothalamic-pituitary-gonadal axis at the mRNA level revealed that zebrafish embryos were affected by the individual or joint pesticides, and greater changes in the expressions of six genes (Mn-sod, CXCL-CIC, Dio1, Dio2, tsh and vtg1) were observed when exposed to joint pesticides compared with their individual pesticides. Taken together, the synergistic effects indicated that it was highly important to incorporate joint toxicity studies, especially at low concentrations, when assessing the risk of pesticides. Copyright © 2018 Elsevier Ltd. All rights reserved.

A genome survey of Moniliophthora perniciosa gives new insights into Witches' Broom Disease of cacao

PubMed Central

Mondego, Jorge MC; Carazzolle, Marcelo F; Costa, Gustavo GL; Formighieri, Eduardo F; Parizzi, Lucas P; Rincones, Johana; Cotomacci, Carolina; Carraro, Dirce M; Cunha, Anderson F; Carrer, Helaine; Vidal, Ramon O; Estrela, Raíssa C; García, Odalys; Thomazella, Daniela PT; de Oliveira, Bruno V; Pires, Acássia BL; Rio, Maria Carolina S; Araújo, Marcos Renato R; de Moraes, Marcos H; Castro, Luis AB; Gramacho, Karina P; Gonçalves, Marilda S; Neto, José P Moura; Neto, Aristóteles Góes; Barbosa, Luciana V; Guiltinan, Mark J; Bailey, Bryan A; Meinhardt, Lyndel W; Cascardo, Julio CM; Pereira, Gonçalo AG

2008-01-01

Background The basidiomycete fungus Moniliophthora perniciosa is the causal agent of Witches' Broom Disease (WBD) in cacao (Theobroma cacao). It is a hemibiotrophic pathogen that colonizes the apoplast of cacao's meristematic tissues as a biotrophic pathogen, switching to a saprotrophic lifestyle during later stages of infection. M. perniciosa, together with the related species M. roreri, are pathogens of aerial parts of the plant, an uncommon characteristic in the order Agaricales. A genome survey (1.9× coverage) of M. perniciosa was analyzed to evaluate the overall gene content of this phytopathogen. Results Genes encoding proteins involved in retrotransposition, reactive oxygen species (ROS) resistance, drug efflux transport and cell wall degradation were identified. The great number of genes encoding cytochrome P450 monooxygenases (1.15% of gene models) indicates that M. perniciosa has a great potential for detoxification, production of toxins and hormones; which may confer a high adaptive ability to the fungus. We have also discovered new genes encoding putative secreted polypeptides rich in cysteine, as well as genes related to methylotrophy and plant hormone biosynthesis (gibberellin and auxin). Analysis of gene families indicated that M. perniciosa have similar amounts of carboxylesterases and repertoires of plant cell wall degrading enzymes as other hemibiotrophic fungi. In addition, an approach for normalization of gene family data using incomplete genome data was developed and applied in M. perniciosa genome survey. Conclusion This genome survey gives an overview of the M. perniciosa genome, and reveals that a significant portion is involved in stress adaptation and plant necrosis, two necessary characteristics for a hemibiotrophic fungus to fulfill its infection cycle. Our analysis provides new evidence revealing potential adaptive traits that may play major roles in the mechanisms of pathogenicity in the M. perniciosa/cacao pathosystem. PMID:19019209

Production of Null Mutants in the Major Intestinal Esterase Gene (Ges-1) of the Nematode Caenorhabditis Elegans

PubMed Central

McGhee, J. D.; Birchall, J. C.; Chung, M. A.; Cottrell, D. A.; Edgar, L. G.; Svendsen, P. C.; Ferrari, D. C.

1990-01-01

The ges-1 gene of the nematode Caenorhabditis elegans codes for a nonspecific carboxylesterase that is expressed only in the intestinal lineage. This esterase has turned out to be a convenient biochemical marker for lineage-specific differentiation. In the present paper, we describe the production of several C. elegans strains that lack detectable activity of the ges-1 esterase. To isolate these ges-1 null strains, we first produced a strain of hermaphrodites in which the wild-type copy of the ges-1 gene was stably balanced over a previously isolated isoelectric focusing allele, ges-1(ca6); this parental strain was then mutagenized with EMS and isoelectric focusing gels were used to identify progeny populations that lacked either ges-1(+) or ges-1(ca6) esterase activity. This method is a straightforward and general approach to obtaining null mutations in any gene that has a biochemical or immunological assay. The ges-1 gene is not essential to worm survival, development or reproduction. Furthermore, lack of the ges-1 product has no obvious effect on the ability of worms (containing either normal or greatly reduced levels of acetylcholinesterases) to survive exposure to esterase inhibitors. The ges-1 gene product provides roughly half of the total esterase activity measured in crude extracts of L1 larvae or mixed worm populations. However, histochemical staining of individual ges-1(0) embryos shows that the ges-1 esterase is the first and essentially the only esterase to be produced during embryonic development, from the midproliferation phase up to at least the twofold stage of morphogenesis. These ges-1(0) strains now allow us to investigate the developmental control of the ges-1 gene by DNA-mediated transformation, in which the ges-1 gene acts as its own reporter. PMID:2379823

N-Acetylanthranilate Amidase from Arthrobacter nitroguajacolicus Rü61a, an α/β-Hydrolase-Fold Protein Active towards Aryl-Acylamides and -Esters, and Properties of Its Cysteine-Deficient Variant▿ †

PubMed Central

Kolkenbrock, Stephan; Parschat, Katja; Beermann, Bernd; Hinz, Hans-Jürgen; Fetzner, Susanne

2006-01-01

N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldine degradation by Arthrobacter nitroguajacolicus Rü61a. Sequence analysis and secondary structure predictions indicated that Amq is related to carboxylesterases and belongs to the α/β-hydrolase-fold superfamily of enzymes; inactivation of (His6-tagged) Amq by phenylmethanesulfonyl fluoride and diethyl pyrocarbonate and replacement of conserved residues suggested a catalytic triad consisting of S155, E235, and H266. Amq is most active towards aryl-acetylamides and aryl-acetylesters. Remarkably, its preference for ring-substituted analogues was different for amides and esters. Among the esters tested, phenylacetate was hydrolyzed with highest catalytic efficiency (kcat/Km = 208 mM−1 s−1), while among the aryl-acetylamides, o-carboxy- or o-nitro-substituted analogues were preferred over p-substituted or unsubstituted compounds. Hydrolysis by His6Amq of primary amides, lactams, N-acetylated amino acids, azocoll, tributyrin, and the acylanilide and urethane pesticides propachlor, propham, carbaryl, and isocarb was not observed; propanil was hydrolyzed with 1% N-acetylanthranilate amidase activity. The catalytic properties of the cysteine-deficient variant His6AmqC22A/C63A markedly differed from those of His6Amq. The replacements effected some changes in Kms of the enzyme and increased kcats for most aryl-acetylesters and some aryl-acetylamides by factors of about three to eight while decreasing kcat for the formyl analogue N-formylanthranilate by several orders of magnitude. Circular dichroism studies indicated that the cysteine-to-alanine replacements resulted in significant change of the overall fold, especially an increase in α-helicity of the cysteine-deficient protein. The conformational changes may also affect the active site and may account for the observed changes in kinetic properties. PMID:17041061

Cytochrome P450 isoforms in the Metabolism of Decursin and Decursinol Angelate from Korean Angelica

PubMed Central

ZHANG, Jinhui; LI, Li; TANG, Suni; HALE, Thomas W.; XING, Chengguo; JIANG, Cheng; LÜ, Junxuan

2016-01-01

We have shown that the in vitro hepatic microsomal metabolism of pyranocoumarin compound decursinol angelate (DA) to decursinol (DOH) exclusively requires cytochrome P450 enzymes (CYP) whereas the conversion of its isomer decursin (D) to DOH can be mediated by CYP and esterase(s). To provide insight into specific isoforms involved, here we show with recombinant human CYP that 2C19 was the most active at metabolizing D and DA in vitro followed by 3A4. With carboxylesterases (CES), D was hydrolyzed by CES2 but not CES1, and DA was resistant to both CES1 and CES2. In human liver microsomal preparation, general CYP inhibitor 1-aminobenzotriazole (ABT) and respective competitive inhibitors for 2C19 and 3A4, (+)-N-3-benzylnirvanol and ketoconazole, substantially retarded the metabolism of DA and, to a lesser extent, of D. In healthy human subjects from a single-dose pharmacokinetic study, 2C19 extensive metabolizer genotype (2C19*17 allele) tended to have less plasma DA AUC0–48h and poor metabolizer genotype (2C19*2 allele) tended to have greater DA AUC0–48h. In mice given a single dose of D/DA, pretreatment with ABT boosted the plasma and prostate levels of D and DA by more than an order of magnitude. Taken together, our findings suggest that CYP isoforms 2C19 and 3A4 may play a crucial role in the first pass liver metabolism of DA and, to a lesser extent, that of D in humans. Pharmacogenetics with respect to CYP genotypes and interactions among CYP inhibitor drugs and D/DA should therefore be considered in designing future translation studies of DA and/or D. PMID:26394652

Cytochrome P450 Isoforms in the Metabolism of Decursin and Decursinol Angelate from Korean Angelica.

PubMed

Zhang, Jinhui; Li, Li; Tang, Suni; Hale, Thomas W; Xing, Chengguo; Jiang, Cheng; Lü, Junxuan

2015-01-01

We have shown that the in vitro hepatic microsomal metabolism of pyranocoumarin compound decursinol angelate (DA) to decursinol (DOH) exclusively requires cytochrome P450 (CYP) enzymes, whereas the conversion of its isomer decursin (D) to DOH can be mediated by CYP and esterase(s). To provide insight into specific isoforms involved, here we show with recombinant human CYP that 2C19 was the most active at metabolizing D and DA in vitro followed by 3A4. With carboxylesterases (CES), D was hydrolyzed by CES2 but not CES1, and DA was resistant to both CES1 and CES2. In human liver microsomal (HLM) preparation, the general CYP inhibitor 1-aminobenzotriazole (ABT) and respective competitive inhibitors for 2C19 and 3A4, (+)-N-3-benzylnirvanol (NBN) and ketoconazole substantially retarded the metabolism of DA and, to a lesser extent, of D. In healthy human subjects from a single-dose pharmacokinetic (PK) study, 2C19 extensive metabolizer genotype (2C19*17 allele) tended to have less plasma DA AUC0-48h and poor metabolizer genotype (2C19*2 allele) tended to have greater DA AUC0-48h. In mice given a single dose of D/DA, pretreatment with ABT boosted the plasma and prostate levels of D and DA by more than an order of magnitude. Taken together, our findings suggest that CYP isoforms 2C19 and 3A4 may play a crucial role in the first pass liver metabolism of DA and, to a lesser extent, that of D in humans. Pharmacogenetics with respect to CYP genotypes and interactions among CYP inhibitor drugs and D/DA should therefore be considered in designing future translation studies of DA and/or D.

Accelerated evolution of CES7, a gene encoding a novel major urinary protein in the cat family.

PubMed

Li, Gang; Janecka, Jan E; Murphy, William J

2011-02-01

Cauxin is a novel urinary protein recently identified in the domestic cat that regulates the excretion of felinine, a pheromone precursor involved in sociochemical communication and territorial marking of domestic and wild felids. Understanding the evolutionary history of cauxin may therefore illuminate molecular adaptations involved in the evolution of pheromone-based communication, recognition, and mate selection in wild animals. We sequenced the gene encoding cauxin, CES7, in 22 species representing all major felid lineages, and multiple outgroups and showed that it has undergone rapid evolutionary change preceding and during the diversification of the cat family. A comparison between feline cauxin and orthologous carboxylesterases from other mammalian lineages revealed evidence of strong positive Darwinian selection within and between several cat lineages, enriched at functionally important sites of the protein. The higher rate of radical amino acid replacements in small felids, coupled with the lack of felinine and extremely low levels of cauxin in the urine of the great cats (Panthera), correlates with functional divergence of this gene in Panthera, and its putative loss in the snow leopard. Expression studies found evidence for several alternatively spliced transcripts in testis and brain, suggesting additional roles in male reproductive fitness and behavior. Our work presents the first report of strong positive natural selection acting on a major urinary protein of nonrodent mammals, providing evidence for parallel selection pressure on the regulation of pheromones in different mammalian lineages, despite the use of different metabolic pathways. Our results imply that natural selection may drive rapid changes in the regulation of pheromones in urine among the different cat species, which in turn may influence social behavior, such as territorial marking and conspecific recognition, therefore serving as an important mechanism for the radiation of this group

Thiamethoxam resistance selected in the western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae): cross-resistance patterns, possible biochemical mechanisms and fitness costs analysis.

PubMed

Gao, Cong-Fen; Ma, Shao-Zhi; Shan, Cai-Hui; Wu, Shun-Fan

2014-09-01

The western flower thrips (WFT) Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), an important pest of various crops in the world, has invaded China since 2003. To understand the risks and to determine possible mechanisms of resistance to thiamethoxam in WFT, a resistant strain was selected under the laboratory conditions. Cross-resistance and the possible biochemical resistance mechanisms were investigated in this study. A 15.1-fold thiamethoxam-resistant WFT strain (TH-R) was established after selection for 55 generations. Compared with the susceptible strain (TH-S), the selected TH-R strain showed extremely high level cross-resistance to imidaclothiz (392.1-fold) and low level cross-resistance to dinotefuran (5.7-fold), acetamiprid (2.9-fold) and emamectin benzoate (2.1-fold), respectively. No cross-resistance to other fourteen insecticides was detected. Synergism tests showed that piperonyl butoxide (PBO) and triphenyl phosphate (TPP) produced a high synergism of thiamethoxam effects in the TH-R strain (2.6- and 2.6-fold respectively). However, diethyl maleate (DEM) did not act synergistically with thiamethoxam. Biochemical assays showed that mixed function oxidase (MFO) activities and carboxylesterase (CarE) activity of the TH-R strain were 2.8- and 1.5-fold higher than that of the TH-S strain, respectively. When compared with the TH-S strain, the TH-R strain had a relative fitness of 0.64. The results show that WFT develops resistance to thiamethoxam after continuous application and thiamethoxam resistance had considerable fitness costs in the WFT. It appears that enhanced metabolism mediated by cytochrome P450 monooxygenases and CarE was a major mechanism for thiamethoxam resistance in the WFT. The use of cross-resistance insecticides, including imidaclothiz and dinotefuran, should be avoided for sustainable resistance management. Copyright © 2014 Elsevier Inc. All rights reserved.

Stability of cytotoxic luteinizing hormone-releasing hormone conjugate (AN-152) containing doxorubicin 14-O-hemiglutarate in mouse and human serum in vitro: Implications for the design of preclinical studies

PubMed Central

Nagy, Attila; Plonowski, Artur; Schally, Andrew V.

2000-01-01

Recently, we developed a series of cytotoxic peptide conjugates containing 14-O-glutaryl esters of doxorubicin (DOX) or 2-pyrrolino-DOX (AN-201). Serum carboxylesterase enzymes (CE) can partially hydrolyze these conjugates in the circulation, releasing the cytotoxic radical, before the targeting is complete. CE activity in serum of nude mice is about 10 times higher than in human serum. Thus, we found that the t1/2 of AN-152, an analog of luteinizing hormone-releasing hormone (LH-RH) containing DOX, at 0.3 mg/ml is 19.49 ± 0.74 min in mouse serum and 126.06 ± 3.03 min in human serum in vitro. The addition of a CE inhibitor, diisopropyl fluorophosphate (DFP), to mouse serum in vitro significantly (P < 0.01) prolongs the t1/2 of AN-152 to 69.63 ± 4.44 min. When DFP is used in vivo, 400 nmol/kg cytotoxic somatostatin analog AN-238 containing AN-201 is well tolerated by mice, whereas all animals die after the same dose without DFP. In contrast, DFP has no effect on the tolerance of AN-201. A better tolerance to AN-238 after DFP treatment is due to the selective uptake of AN-238 by somatostatin receptor-positive tissues. Our results demonstrate that the suppression of the CE activity in nude mice greatly decreases the toxicity of cytotoxic hybrids containing 2-pyrrolino-DOX 14-O-hemiglutarate and brings this animal model closer to the conditions that exist in humans. The use of DFP together with these peptide conjugates in nude mice permits a better understanding of their mechanism of action and improves the clinical predictability of the oncological and toxicological results. PMID:10639165

Transport properties of valsartan, sacubitril and its active metabolite (LBQ657) as determinants of disposition.

PubMed

Hanna, Imad; Alexander, Natalya; Crouthamel, Matthew H; Davis, John; Natrillo, Adrienne; Tran, Phi; Vapurcuyan, Arpine; Zhu, Bing

2018-03-01

1. The potential for drug-drug interactions of LCZ696 (a novel, crystalline complex comprising sacubitril and valsartan) was investigated in vitro. 2. Sacubitril was shown to be a highly permeable P-glycoprotein (P-gp) substrate and was hydrolyzed to the active anionic metabolite LBQ657 by human carboxylesterase 1 (CES1b and 1c). The multidrug resistance-associated protein 2 (MRP2) was shown to be capable of LBQ657 and valsartan transport that contributes to the elimination of either compound. 3. LBQ657 and valsartan were transported by OAT1, OAT3, OATP1B1 and OATP1B3, whereas no OAT- or OATP-mediated sacubitril transport was observed. 4. The contribution of OATP1B3 to valsartan transport (73%) was appreciably higher than that by OATP1B1 (27%), Alternatively, OATP1B1 contribution to the hepatic uptake of LBQ657 (∼70%) was higher than that by OATP1B3 (∼30%). 5. None of the compounds inhibited OCT1/OCT2, MATE1/MATE2-K, P-gp, or BCRP. Sacubitril and LBQ657 inhibited OAT3 but not OAT1, and valsartan inhibited the activity of both OAT1 and OAT3. Sacubitril and valsartan inhibited OATP1B1 and OATP1B3, whereas LBQ657 weakly inhibited OATP1B1 but not OATP1B3. 6. Drug interactions due to the inhibition of transporters are unlikely due to the redundancy of the available transport pathways (LBQ657: OATP1B1/OAT1/3 and valsartan: OATP1B3/OAT1/3) and the low therapeutic concentration of the LCZ696 analytes.

Development of biomarkers of exposure to xenobiotics in the honey bee Apis mellifera: application to the systemic insecticide thiamethoxam.

PubMed

Badiou-Bénéteau, Alexandra; Carvalho, Stephan M; Brunet, Jean-Luc; Carvalho, Geraldo A; Buleté, Audrey; Giroud, Barbara; Belzunces, Luc P

2012-08-01

This study describes the development of acetylcholinesterase (AChE), carboxylesterases (CaE1, CaE2, CaE3), glutathion-S-transferase (GST), alkaline phosphatase (ALP) and catalase (CAT) as enzyme biomarkers of exposure to xenobiotics such as thiamethoxam in the honey bee Apis mellifera. Extraction efficiency, stability under freezing and biological variability were studied. The extraction procedure achieved good recovery rates in one extraction step and ranged from 65 percent (AChE) to 97.3 percent (GST). Most of the enzymes were stable at -20°C, except ALP that displayed a slight but progressive decrease in its activity. Modifications of enzyme activities were considered after exposure to thiamethoxam at the lethal dose 50 percent (LD(50), 51.16 ng bee(-1)) and two sublethal doses, LD(50)/10 (5.12 ng bee(-1)) and LD(50)/20 (2.56 ng bee(-1)). The biomarker responses revealed that, even at the lowest dose used, exposure to thiamethoxam elicited sublethal effects and modified the activity of CaEs, GST, CAT and ALP. Different patterns of biomarker responses were observed: no response for AChE, an increase for GST and CAT, and differential effects for CaEs isoforms with a decrease in CaE1 and CaE3 and an increase in CaE2. ALP and CaE3 displayed contrasting variations but only at 2.56 ng bee(-1). We consider that this profile of biomarker variation could represent a useful fingerprint to characterise exposure to thiamethoxam in the honey bee A. mellifera. This battery of honey bee biomarkers might be a promising option to biomonitor the health of aerial and terrestrial ecosystems and to generate valuable information on the modes of action of pesticides. Copyright © 2012 Elsevier Inc. All rights reserved.

3D-fibroblast tissues constructed by a cell-coat technology enhance tight-junction formation of human colon epithelial cells.

PubMed

Matsusaki, Michiya; Hikimoto, Daichi; Nishiguchi, Akihiro; Kadowaki, Koji; Ohura, Kayoko; Imai, Teruko; Akashi, Mitsuru

2015-02-13

Caco-2, human colon carcinoma cell line, has been widely used as a model system for intestinal epithelial permeability because Caco-2 cells express tight-junctions, microvilli, and a number of enzymes and transporters characteristic of enterocytes. However, the functional differentiation and polarization of Caco-2 cells to express sufficient tight-junctions (a barrier) usually takes over 21 days in culture. This may be due to the cell culture environment, for example inflammation induced by plastic petri dishes. Three-dimensional (3D) sufficient cell microenvironments similar to in vivo natural conditions (proteins and cells), will promote rapid differentiation and higher functional expression of tight junctions. Herein we report for the first time an enhancement in tight-junction formation by 3D-cultures of Caco-2 cells on monolayered (1L) and eight layered (8L) normal human dermal fibroblasts (NHDF). Trans epithelial electric resistance (TEER) of Caco-2 cells was enhanced in the 3D-cultures, especially 8L-NHDF tissues, depending on culture times and only 10 days was enough to reach the same TEER value of Caco-2 monolayers after a 21 day incubation. Relative mRNA expression of tight-junction proteins of Caco-2 cells on 3D-cultures showed higher values than those in monolayer structures. Transporter gene expression patterns of Caco-2 cells on 3D-constructs were almost the same as those of Caco-2 monolayers, suggesting that there was no effect of 3D-cultures on transporter protein expression. The expression correlation between carboxylesterase 1 and 2 in 3D-cultures represented similar trends with human small intestines. The results of this study clearly represent a valuable application of 3D-Caco-2 tissues for pharmaceutical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination.

PubMed

Rejón, Juan D; Zienkiewicz, Agnieszka; Rodríguez-García, María Isabel; Castro, Antonio J

2012-10-01

A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized. The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods. Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles. In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma.

Temephos Resistance in Aedes aegypti in Colombia Compromises Dengue Vector Control

PubMed Central

Grisales, Nelson; Poupardin, Rodolphe; Gomez, Santiago; Fonseca-Gonzalez, Idalyd; Ranson, Hilary; Lenhart, Audrey

2013-01-01

Background Control and prevention of dengue relies heavily on the application of insecticides to control dengue vector mosquitoes. In Colombia, application of the larvicide temephos to the aquatic breeding sites of Aedes aegypti is a key part of the dengue control strategy. Resistance to temephos was recently detected in the dengue-endemic city of Cucuta, leading to questions about its efficacy as a control tool. Here, we characterize the underlying mechanisms and estimate the operational impact of this resistance. Methodology/Principal Findings Larval bioassays of Ae. aegypti larvae from Cucuta determined the temephos LC50 to be 0.066 ppm (95% CI 0.06–0.074), approximately 15× higher than the value obtained from a susceptible laboratory colony. The efficacy of the field dose of temephos at killing this resistant Cucuta population was greatly reduced, with mortality rates <80% two weeks after application and <50% after 4 weeks. Neither biochemical assays nor partial sequencing of the ace-1 gene implicated target site resistance as the primary resistance mechanism. Synergism assays and microarray analysis suggested that metabolic mechanisms were most likely responsible for the temephos resistance. Interestingly, although the greatest synergism was observed with the carboxylesterase inhibitor, DEF, the primary candidate genes from the microarray analysis, and confirmed by quantitative PCR, were cytochrome P450 oxidases, notably CYP6N12, CYP6F3 and CYP6M11. Conclusions/Significance In Colombia, resistance to temephos in Ae. aegypti compromises the duration of its effect as a vector control tool. Several candidate genes potentially responsible for metabolic resistance to temephos were identified. Given the limited number of insecticides that are approved for vector control, future chemical-based control strategies should take into account the mechanisms underlying the resistance to discern which insecticides would likely lead to the greatest control efficacy while

Effects of Sublethal Exposure to a Glyphosate-Based Herbicide Formulation on Metabolic Activities of Different Xenobiotic-Metabolizing Enzymes in Rats.

PubMed

Larsen, Karen; Najle, Roberto; Lifschitz, Adrián; Maté, María L; Lanusse, Carlos; Virkel, Guillermo L

2014-07-01

The activities of different xenobiotic-metabolizing enzymes in liver subcellular fractions from Wistar rats exposed to a glyphosate (GLP)-based herbicide (Roundup full II) were evaluated in this work. Exposure to the herbicide triggered protective mechanisms against oxidative stress (increased glutathione peroxidase activity and total glutathione levels). Liver microsomes from both male and female rats exposed to the herbicide had lower (45%-54%, P < 0.01) hepatic cytochrome P450 (CYP) levels compared to their respective control animals. In female rats, the hepatic 7-ethoxycoumarin O-deethylase (a general CYP-dependent enzyme activity) was 57% higher (P < 0.05) in herbicide-exposed compared to control animals. Conversely, this enzyme activity was 58% lower (P < 0.05) in male rats receiving the herbicide. Lower (P < 0.05) 7-ethoxyresorufin O-deethlyase (EROD, CYP1A1/2 dependent) and oleandomycin triacetate (TAO) N-demethylase (CYP3A dependent) enzyme activities were observed in liver microsomes from exposed male rats. Conversely, in females receiving the herbicide, EROD increased (123%-168%, P < 0.05), whereas TAO N-demethylase did not change. A higher (158%-179%, P < 0.01) benzyloxyresorufin O-debenzylase (a CYP2B-dependent enzyme activity) activity was only observed in herbicide-exposed female rats. In herbicide-exposed rats, the hepatic S-oxidation of methimazole (flavin monooxygenase dependent) was 49% to 62% lower (P < 0.001), whereas the carbonyl reduction of menadione (a cytosolic carbonyl reductase-dependent activity) was higher (P < 0.05). Exposure to the herbicide had no effects on enzymatic activities dependent on carboxylesterases, glutathione transferases, and uridinediphospho-glucuronosyltransferases. This research demonstrated certain biochemical modifications after exposure to a GLP-based herbicide. Such modifications may affect the metabolic fate of different endobiotic and xenobiotic substances. The pharmacotoxicological significance of these

Putative pathway of sex pheromone biosynthesis and degradation by expression patterns of genes identified from female pheromone gland and adult antenna of Sesamia inferens (Walker).

PubMed

Zhang, Ya-Nan; Xia, Yi-Han; Zhu, Jia-Yao; Li, Sheng-Yun; Dong, Shuang-Lin

2014-05-01

The general pathway of biosynthesis and degradation for Type-I sex pheromones in moths is well established, but some genes involved in this pathway remain to be characterized. The purple stem borer, Sesamia inferens, employs a pheromone blend containing components with three different terminal functional groups (Z11-16:OAc, Z11-16:OH, and Z11-16:Ald) of Type-I sex pheromones. Thus, it provides a good model to study the diversity of genes involved in pheromone biosynthesis and degradation pathways. By analyzing previously obtained transcriptomic data of the sex pheromone glands and antennae, we identified 73 novel genes that are possibly related to pheromone biosynthesis (46 genes) or degradation (27 genes). Gene expression patterns and phylogenetic analysis revealed that one desaturase (SinfDes4), one fatty acid reductase (SinfFAR2), and one fatty acid xtransport protein (SinfFATP1) genes were predominantly expressed in pheromone glands, and clustered with genes involved in pheromone synthesis in other moth species. Ten genes including five carboxylesterases (SinfCXE10, 13, 14, 18, and 20), three aldehyde oxidases (SinfAOX1, 2 and 3), and two alcohol dehydrogenases (SinfAD1 and 3) were expressed specifically or predominantly in antennae, and could be candidate genes involved in pheromone degradation. SinfAD1 and 3 are the first reported alcohol dehydrogenase genes with antennae-biased expression. Based on these results we propose a pathway involving these potential enzyme-encoding gene candidates in sex pheromone biosynthesis and degradation in S. inferens. This study provides robust background information for further elucidation of the genetic basis of sex pheromone biosynthesis and degradation, and ultimately provides potential targets to disrupt sexual communication in S. inferens for control purposes.

Development of coated liposomes loaded with ghrelin for nose-to-brain delivery for the treatment of cachexia.

PubMed

Salade, Laurent; Wauthoz, Nathalie; Deleu, Magali; Vermeersch, Marjorie; De Vriese, Carine; Amighi, Karim; Goole, Jonathan

2017-01-01

The aim of the present study was to develop a ghrelin-containing formulation based on liposomes coated with chitosan intended for nose-brain delivery for the treatment of cachexia. Among the three types of liposomes developed, anionic liposomes provided the best results in terms of encapsulation efficiency (56%) and enzymatic protection against trypsin (20.6% vs 0% for ghrelin alone) and carboxylesterase (81.6% vs 17.2% for ghrelin alone). Ghrelin presented both electrostatic and hydrophobic interactions with the anionic lipid bilayer, as demonstrated by isothermal titration calorimetry. Then, anionic liposomes were coated with N -(2-hydroxy) propyl-3-trimethyl ammonium chitosan chloride. The coating involved a size increment from 146.9±2.7 to 194±6.1 nm, for uncoated and coated liposomes, respectively. The ζ-potential was similarly increased from -0.3±1.2 mV to 6±0.4 mV before and after coating, respectively. Chitosan provided mucoadhesion, with an increase in mucin adsorption of 22.9%. Enhancement of permeation through the Calu3 epithelial monolayer was also observed with 10.8% of ghrelin recovered in the basal compartment in comparison to 0% for ghrelin alone. Finally, aerosols generated from two nasal devices (VP3 and SP270) intended for aqueous dispersion were characterized with either coated or uncoated liposomes. Contrarily to the SP270 device, VP3 device showed minor changes between coated and uncoated liposome aerosols, as shown by their median volume diameters of 38.4±5.76 and 37.6±5.74 µm, respectively. Overall, the results obtained in this study show that the developed formulation delivered by the VP3 device can be considered as a potential candidate for nose-brain delivery of ghrelin.

Development of coated liposomes loaded with ghrelin for nose-to-brain delivery for the treatment of cachexia

PubMed Central

Salade, Laurent; Wauthoz, Nathalie; Deleu, Magali; Vermeersch, Marjorie; De Vriese, Carine; Amighi, Karim; Goole, Jonathan

2017-01-01

The aim of the present study was to develop a ghrelin-containing formulation based on liposomes coated with chitosan intended for nose–brain delivery for the treatment of cachexia. Among the three types of liposomes developed, anionic liposomes provided the best results in terms of encapsulation efficiency (56%) and enzymatic protection against trypsin (20.6% vs 0% for ghrelin alone) and carboxylesterase (81.6% vs 17.2% for ghrelin alone). Ghrelin presented both electrostatic and hydrophobic interactions with the anionic lipid bilayer, as demonstrated by isothermal titration calorimetry. Then, anionic liposomes were coated with N-(2-hydroxy) propyl-3-trimethyl ammonium chitosan chloride. The coating involved a size increment from 146.9±2.7 to 194±6.1 nm, for uncoated and coated liposomes, respectively. The ζ-potential was similarly increased from -0.3±1.2 mV to 6±0.4 mV before and after coating, respectively. Chitosan provided mucoadhesion, with an increase in mucin adsorption of 22.9%. Enhancement of permeation through the Calu3 epithelial monolayer was also observed with 10.8% of ghrelin recovered in the basal compartment in comparison to 0% for ghrelin alone. Finally, aerosols generated from two nasal devices (VP3 and SP270) intended for aqueous dispersion were characterized with either coated or uncoated liposomes. Contrarily to the SP270 device, VP3 device showed minor changes between coated and uncoated liposome aerosols, as shown by their median volume diameters of 38.4±5.76 and 37.6±5.74 µm, respectively. Overall, the results obtained in this study show that the developed formulation delivered by the VP3 device can be considered as a potential candidate for nose–brain delivery of ghrelin. PMID:29238190

Esterases activity in the axolotl Ambystoma mexicanum exposed to chlorpyrifos and its implication to motor activity.

PubMed

Robles-Mendoza, Cecilia; Zúñiga-Lagunes, Sebastian R; Ponce de León-Hill, Claudia A; Hernández-Soto, Jesús; Vanegas-Pérez, Cecilia

2011-10-01

The axolotl Ambystoma mexicanum is a neotenic salamander considered a good biological model due to its ability to regenerate limbs, tail, brain and heart cells. Nevertheless, severe reduction of A. mexicanum wild populations in the lacustrine area of Xochimilco, the natural habitat of the axolotl, could be related to several environmental pressures as the presence of organophosphate pesticides (OPPs), intensively applied in agricultural activities in Xochimilco. Thus the aim of this study was to evaluate the effect of environmentally realistic chlorpyrifos (CPF) concentrations, a OPP commonly used in this zone, on esterases activity (acetylcholinesterase and carboxylesterase) and bioconcentration of CPF and to relate them with the motor activity of A. mexicanum juveniles. Axolotls were exposed 48 h to 0.05 and 0.1mg CPF/L, and the responses were evaluated at the end of the CPF exposure. Results suggest that CPF is bioconcentrated into axolotls and that the CPF internal concentrations are related with the observed inhibition activity of AChE (>50%) and CbE (≈ 50%). CPF concentration responsible of the inhibition of the 50% of AChE activity (IC50) was estimated in 0.04 mg CPF/L; however IC50 for CbE activity was not possible to calculate since inhibition levels were lower than 50%, results that suggest a higher resistance of CbE enzymatic activity to CPF. However, motor activity was a more sensitive endpoint to CPF poisoning since time that axolotls spent active and walking, frequency and speed of swimming, frequency of prey attack were reduced >90% of control groups. The motor activity alterations in the axolotl could be related with the registered esterases inhibition. Thus important alterations on axolotls were identified even at short time and low concentrations of CPF exposure. Also, it was possible to link biochemical responses as esterases activity with higher levels of biological organization as behavior. This study provides tools for the regulation of the

RNA-Seq and molecular docking reveal multi-level pesticide resistance in the bed bug

PubMed Central

2012-01-01

Background Bed bugs (Cimex lectularius) are hematophagous nocturnal parasites of humans that have attained high impact status due to their worldwide resurgence. The sudden and rampant resurgence of C. lectularius has been attributed to numerous factors including frequent international travel, narrower pest management practices, and insecticide resistance. Results We performed a next-generation RNA sequencing (RNA-Seq) experiment to find differentially expressed genes between pesticide-resistant (PR) and pesticide-susceptible (PS) strains of C. lectularius. A reference transcriptome database of 51,492 expressed sequence tags (ESTs) was created by combining the databases derived from de novo assembled mRNA-Seq tags (30,404 ESTs) and our previous 454 pyrosequenced database (21,088 ESTs). The two-way GLMseq analysis revealed ~15,000 highly significant differentially expressed ESTs between the PR and PS strains. Among the top 5,000 differentially expressed ESTs, 109 putative defense genes (cuticular proteins, cytochrome P450s, antioxidant genes, ABC transporters, glutathione S-transferases, carboxylesterases and acetyl cholinesterase) involved in penetration resistance and metabolic resistance were identified. Tissue and development-specific expression of P450 CYP3 clan members showed high mRNA levels in the cuticle, Malpighian tubules, and midgut; and in early instar nymphs, respectively. Lastly, molecular modeling and docking of a candidate cytochrome P450 (CYP397A1V2) revealed the flexibility of the deduced protein to metabolize a broad range of insecticide substrates including DDT, deltamethrin, permethrin, and imidacloprid. Conclusions We developed significant molecular resources for C. lectularius putatively involved in metabolic resistance as well as those participating in other modes of insecticide resistance. RNA-Seq profiles of PR strains combined with tissue-specific profiles and molecular docking revealed multi-level insecticide resistance in C. lectularius

Insecticide resistance in disease vectors from Mayotte: an opportunity for integrated vector management

PubMed Central

2014-01-01

Background Mayotte, a small island in the Indian Ocean, has been affected for many years by vector-borne diseases. Malaria, Bancroftian filariasis, dengue, chikungunya and Rift Valley fever have circulated or still circulate on the island. They are all transmitted by Culicidae mosquitoes. To limit the impact of these diseases on human health, vector control has been implemented for more than 60 years on Mayotte. In this study, we assessed the resistance levels of four major vector species (Anopheles gambiae, Culex pipiens quinquefasciatus, Aedes aegypti and Aedes albopictus) to two types of insecticides: i) the locally currently-used insecticides (organophosphates, pyrethroids) and ii) alternative molecules that are promising for vector control and come from different insecticide families (bacterial toxins or insect growth regulators). When some resistance was found to one of these insecticides, we characterized the mechanisms involved. Methods Larval and adult bioassays were used to evaluate the level of resistance. When resistance was found, we tested for the presence of metabolic resistance through detoxifying enzyme activity assays, or for target-site mutations through molecular identification of known resistance alleles. Results Resistance to currently-used insecticides varied greatly between the four vector species. While no resistance to any insecticides was found in the two Aedes species, bioassays confirmed multiple resistance in Cx. p. quinquefasciatus (temephos: ~ 20 fold and deltamethrin: only 10% mortality after 24 hours). In An. gambiae, resistance was scarce: only a moderate resistance to temephos was found (~5 fold). This resistance appears to be due only to carboxyl-esterase overexpression and not to target modification. Finally, and comfortingly, none of the four species showed resistance to any of the new insecticides. Conclusions The low resistance observed in Mayotte’s main disease vectors is particularly interesting, because it leaves a

Dissecting the organ specificity of insecticide resistance candidate genes in Anopheles gambiae: known and novel candidate genes.

PubMed

Ingham, Victoria A; Jones, Christopher M; Pignatelli, Patricia; Balabanidou, Vasileia; Vontas, John; Wagstaff, Simon C; Moore, Jonathan D; Ranson, Hilary

2014-11-25

The elevated expression of enzymes with insecticide metabolism activity can lead to high levels of insecticide resistance in the malaria vector, Anopheles gambiae. In this study, adult female mosquitoes from an insecticide susceptible and resistant strain were dissected into four different body parts. RNA from each of these samples was used in microarray analysis to determine the enrichment patterns of the key detoxification gene families within the mosquito and to identify additional candidate insecticide resistance genes that may have been overlooked in previous experiments on whole organisms. A general enrichment in the transcription of genes from the four major detoxification gene families (carboxylesterases, glutathione transferases, UDP glucornyltransferases and cytochrome P450s) was observed in the midgut and malpighian tubules. Yet the subset of P450 genes that have previously been implicated in insecticide resistance in An gambiae, show a surprisingly varied profile of tissue enrichment, confirmed by qPCR and, for three candidates, by immunostaining. A stringent selection process was used to define a list of 105 genes that are significantly (p ≤0.001) over expressed in body parts from the resistant versus susceptible strain. Over half of these, including all the cytochrome P450s on this list, were identified in previous whole organism comparisons between the strains, but several new candidates were detected, notably from comparisons of the transcriptomes from dissected abdomen integuments. The use of RNA extracted from the whole organism to identify candidate insecticide resistance genes has a risk of missing candidates if key genes responsible for the phenotype have restricted expression within the body and/or are over expression only in certain tissues. However, as transcription of genes implicated in metabolic resistance to insecticides is not enriched in any one single organ, comparison of the transcriptome of individual dissected body parts cannot

Chemical characterization of territorial marking fluid of male Bengal tiger, Panthera tigris.

PubMed

Burger, B V; Viviers, M Z; Bekker, J P I; le Roux, M; Fish, N; Fourie, W B; Weibchen, G

2008-05-01

The territorial marking fluid of the male Bengal tiger, Panthera tigris, consists of a mixture of urine and a small quantity of lipid material that may act as a controlled-release carrier for the volatile constituents of the fluid. Using gas chromatography and gas chromatography-mass spectrometry, 98 volatile compounds and elemental sulfur were identified in the marking fluid. Another 16 volatiles were tentatively identified. The majority of these compounds were alkanols, alkanals, 2-alkanones, branched and unbranched alkanoic acids, dimethyl esters of dicarboxylic acids, gamma- and delta-lactones, and compounds containing nitrogen or sulfur. Several samples of the marking fluid contained pure (R)-3-methyl-2-octanone, (R)-3-methyl-2-nonanone, and (R)-3-methyl-2-decanone, but these ketones were partly or completely racemized in other samples. The gamma-lactone (S)-(+)-(Z)-6-dodecen-4-olide and the C(8) to C(16) saturated (R)-gamma-lactones and (S)-delta-lactones were present in high enantiomeric purities. The chiral carboxylic acids, 2-methylnonanoic acid, 2-methyldecanoic acid, 2-methylundecanoic acid, and 2-ethylhexanoic acid were racemates. Cadaverine, putrescine, and 2-acetylpyrroline, previously reported as constituents of tiger urine, were not detected. The dominant contribution of some ketones, fatty acids, and lactones to the composition of the headspace of the marking fluid suggests that these compounds may be important constituents of the pheromone. Although it constitutes only a small proportion, the lipid fraction of the fluid contained larger quantities of the volatile organic compounds than the aqueous fraction (urine). The lipid derives its role as controlled-release carrier of the chemical message left by the tiger, from its affinity for the volatiles of the marking fluid. Six proteins with masses ranging from 16 to 69 kDa, inter alia, the carboxylesterase-like urinary protein known as cauxin, previously identified in the urine of the domestic cat

Low Level Chlorpyrifos Exposure Increases Anandamide Accumulation in Juvenile Rat Brain in the Absence of Brain Cholinesterase Inhibition

PubMed Central

Carr, Russell L.; Graves, Casey A.; Mangum, Lee C.; Nail, Carole A.; Ross, Matthew K.

2014-01-01

The prevailing dogma is that chlorpyrifos (CPF) mediates its toxicity through inhibition of cholinesterase (ChE). However, in recent years, the toxicological effects of developmental CPF exposure have been attributed to an unknown non-cholinergic mechanism of action. We hypothesize that the endocannabinoid system may be an important target because of its vital role in nervous system development. We have previously reported that repeated exposure to CPF results in greater inhibition of fatty acid amide hydrolase (FAAH), the enzyme that metabolizes the endocannabinoid anandamide (AEA), than inhibition of either forebrain ChE or monoacylglycerol lipase (MAGL), the enzyme that metabolizes the endocannabinoid 2-arachidonylglycerol (2-AG). This exposure resulted in the accumulation of 2-AG and AEA in the forebrain of juvenile rats; however, even at the lowest dosage level used (1.0 mg/kg), forebrain ChE inhibition was still present. Thus, it is not clear if FAAH activity would be inhibited at dosage levels that do not inhibit ChE. To determine this, 10 day old rat pups were exposed daily for 7 days to either corn oil or 0.5 mg/kg CPF by oral gavage. At 4 and 12 h post-exposure on the last day of administration, the activities of serum ChE and carboxylesterase (CES) and forebrain ChE, MAGL, and FAAH were determined as well as the forebrain AEA and 2-AG levels. Significant inhibition of serum ChE and CES was present at both 4 and 12 h. There was no significant inhibition of the activities of forebrain ChE or MAGL and no significant change in the amount of 2-AG at either time point. On the other hand, while no statistically significant effects were observed at 4 h, FAAH activity was significantly inhibited at 12 h resulting in a significant accumulation of AEA. Although it is not clear if this level of accumulation impacts brain maturation, this study demonstrates that developmental CPF exposure at a level that does not inhibit brain ChE can alter components of

Combined Transcriptomic and Proteomic Analysis of the Posterior Salivary Gland from the Southern Blue-Ringed Octopus and the Southern Sand Octopus.

PubMed

Whitelaw, Brooke L; Strugnell, Jan M; Faou, Pierre; da Fonseca, Rute R; Hall, Nathan E; Norman, Mark; Finn, Julian; Cooke, Ira R

2016-09-02

This study provides comprehensive proteomic profiles from the venom producing posterior salivary glands of octopus (superorder Octopodiformes) species. A combined transcriptomic and proteomic approach was used to identify 1703 proteins from the posterior salivary gland of the southern blue-ringed octopus, Hapalochlaena maculosa and 1300 proteins from the posterior salivary gland of the southern sand octopus, Octopus kaurna. The two proteomes were broadly similar; clustering of proteins into orthogroups revealed 937 that were shared between species. Serine proteases were particularly diverse and abundant in both species. Other abundant proteins included a large number of secreted proteins, many of which had no known conserved domains, or homology to proteins with known function. On the basis of homology to known venom proteins, 23 putative toxins were identified in H. maculosa and 24 in O. kaurna. These toxins span nine protein families: CAP (cysteine rich secretory proteins, antigen 5, parthenogenesis related), chitinase, carboxylesterase, DNase, hyaluronidase, metalloprotease, phospholipase, serine protease and tachykinin. Serine proteases were responsible for 70.9% and 86.3% of putative toxin expression in H. maculosa and O. kaurna, respectively, as determined using intensity based absolute quantification (iBAQ) measurements. Phylogenetic analysis of the putative toxin serine proteases revealed a similar suite of diverse proteins present in both species. Posterior salivary gland composition of H. maculosa and O. kaurna differ in several key aspects. While O. kaurna expressed the proteinaceous neurotoxin, tachykinin, this was absent from H. maculosa, perhaps reflecting the acquisition of a potent nonproteinaceous neurotoxin, tetrodotoxin (TTX) produced by bacteria in the salivary glands of that species. The dispersal factor, hyaluronidase was particularly abundant in H. maculosa. Chitinase was abundant in both species and is believed to facilitate

CES2, ABCG2, TS and Topo-I primary and synchronous metastasis expression and clinical outcome in metastatic colorectal cancer patients treated with first-line FOLFIRI regimen.

PubMed

Silvestris, Nicola; Simone, Giovanni; Partipilo, Giulia; Scarpi, Emanuela; Lorusso, Vito; Brunetti, Anna Elisabetta; Maiello, Evaristo; Paradiso, Angelo; Mangia, Anita

2014-09-05

Enzymatic activation of irinotecan (CPT-11) is due to carboxylesterase (CES), and its pharmacological behavior is influenced by drug resistance-related proteins. We previously reported that the clinical response and prognosis of metastatic colorectal cancer (mCRC) patients did not differ in tumors with different thymidylate synthase (TS) or topoisomerase-I (Topo-I) expression. Using immunohistochemistry (IHC), we evaluated the biological role of CES2 and the expression of breast cancer resistance protein (BCRP/ABCG2) in 58 consecutive mCRC patients, who had undergone a first-line CPT-11/5-FU/leucovirin (FOLFIRI) regimen. The expression of these proteins was also examined in a group of synchronous lymph nodes and liver metastases. Furthermore, all samples were revaluated for TS and Topo-I expression. High expression of CES2, ABCG2, TS and Topo-I was observed in 55%, 56%, 38% and 49% of patients, respectively. There was a significant association between high TS and high ABCG2 expression (p = 0.049). Univariate analysis showed that only TS expression significantly impacted on time to progression (p = 0.005). Moreover, Cox' multivariate analysis revealed that TS expression was significantly associated with overall survival (p = 0.01). No significant correlation was found between investigated markers expression and clinical response. Topo-I expression resulted in being significantly higher in liver metastases with respect to the corresponding primary tumors (p < 0.0001), emphasizing the role of Topo-I expression in metastatic cancer biology. In primary tumor tissues, CES2 expression tended to be higher than that observed in liver metastasis tissues (p = 0.05). These preliminary data may suggest CES2 over-expression as a potential marker of malignant phenotype. In light of these findings, we suggest that Topo-I expression together with TS expression could be associated with metastatic progression of CRC. Further studies are warranted with the aim of evaluating the

Exploring the molecular basis of insecticide resistance in the dengue vector Aedes aegypti: a case study in Martinique Island (French West Indies).

PubMed

Marcombe, Sébastien; Poupardin, Rodolphe; Darriet, Frederic; Reynaud, Stéphane; Bonnet, Julien; Strode, Clare; Brengues, Cecile; Yébakima, André; Ranson, Hilary; Corbel, Vincent; David, Jean-Philippe

2009-10-26

The yellow fever mosquito Aedes aegypti is a major vector of dengue and hemorrhagic fevers, causing up to 100 million dengue infections every year. As there is still no medicine and efficient vaccine available, vector control largely based on insecticide treatments remains the only method to reduce dengue virus transmission. Unfortunately, vector control programs are facing operational challenges with mosquitoes becoming resistant to commonly used insecticides. Resistance of Ae. aegypti to chemical insecticides has been reported worldwide and the underlying molecular mechanisms, including the identification of enzymes involved in insecticide detoxification are not completely understood. The present paper investigates the molecular basis of insecticide resistance in a population of Ae. aegypti collected in Martinique (French West Indies). Bioassays with insecticides on adults and larvae revealed high levels of resistance to organophosphate and pyrethroid insecticides. Molecular screening for common insecticide target-site mutations showed a high frequency (71%) of the sodium channel 'knock down resistance' (kdr) mutation. Exposing mosquitoes to detoxification enzymes inhibitors prior to bioassays induced a significant increased susceptibility of mosquitoes to insecticides, revealing the presence of metabolic-based resistance mechanisms. This trend was biochemically confirmed by significant elevated activities of cytochrome P450 monooxygenases, glutathione S-transferases and carboxylesterases at both larval and adult stages. Utilization of the microarray Aedes Detox Chip containing probes for all members of detoxification and other insecticide resistance-related enzymes revealed the significant constitutive over-transcription of multiple detoxification genes at both larval and adult stages. The over-transcription of detoxification genes in the resistant strain was confirmed by using real-time quantitative RT-PCR. These results suggest that the high level of

Transcriptome Analysis in Venom Gland of the Predatory Giant Ant Dinoponera quadriceps: Insights into the Polypeptide Toxin Arsenal of Hymenopterans

PubMed Central

Chong, Cheong-Meng; Leung, Siu Wai; Prieto-da-Silva, Álvaro R. B.; Havt, Alexandre; Quinet, Yves P.; Martins, Alice M. C.; Lee, Simon M. Y.; Rádis-Baptista, Gandhi

2014-01-01

Background Dinoponera quadriceps is a predatory giant ant that inhabits the Neotropical region and subdues its prey (insects) with stings that deliver a toxic cocktail of molecules. Human accidents occasionally occur and cause local pain and systemic symptoms. A comprehensive study of the D. quadriceps venom gland transcriptome is required to advance our knowledge about the toxin repertoire of the giant ant venom and to understand the physiopathological basis of Hymenoptera envenomation. Results We conducted a transcriptome analysis of a cDNA library from the D. quadriceps venom gland with Sanger sequencing in combination with whole-transcriptome shotgun deep sequencing. From the cDNA library, a total of 420 independent clones were analyzed. Although the proportion of dinoponeratoxin isoform precursors was high, the first giant ant venom inhibitor cysteine-knot (ICK) toxin was found. The deep next generation sequencing yielded a total of 2,514,767 raw reads that were assembled into 18,546 contigs. A BLAST search of the assembled contigs against non-redundant and Swiss-Prot databases showed that 6,463 contigs corresponded to BLASTx hits and indicated an interesting diversity of transcripts related to venom gene expression. The majority of these venom-related sequences code for a major polypeptide core, which comprises venom allergens, lethal-like proteins and esterases, and a minor peptide framework composed of inter-specific structurally conserved cysteine-rich toxins. Both the cDNA library and deep sequencing yielded large proportions of contigs that showed no similarities with known sequences. Conclusions To our knowledge, this is the first report of the venom gland transcriptome of the New World giant ant D. quadriceps. The glandular venom system was dissected, and the toxin arsenal was revealed; this process brought to light novel sequences that included an ICK-folded toxins, allergen proteins, esterases (phospholipases and carboxylesterases), and lethal

Insecticidal Activity of Melaleuca alternifolia Essential Oil and RNA-Seq Analysis of Sitophilus zeamais Transcriptome in Response to Oil Fumigation.

PubMed

Liao, Min; Xiao, Jin-Jing; Zhou, Li-Jun; Liu, Yang; Wu, Xiang-Wei; Hua, Ri-Mao; Wang, Gui-Rong; Cao, Hai-Qun

2016-01-01

The cereal weevil, Sitophilus zeamais is one of the most destructive pests of stored cereals worldwide. Frequent use of fumigants for managing stored-product insects has led to the development of resistance in insects. Essential oils from aromatic plants including the tea oil plant, Melaleuca alternifolia may provide environmentally friendly alternatives to currently used pest control agents. However, little is known about molecular events involved in stored-product insects in response to plant essential oil fumigation. M. alternifolia essential oil was shown to possess the fumigant toxicity against S. zeamais. The constituent, terpinen-4-ol was the most effective compound for fumigant toxicity. M. alternifolia essential oil significantly inhibited the activity of three enzymes in S. zeamais, including two detoxifying enzymes, glutathione S-transferase (GST), and carboxylesterase (CarE), as well as a nerve conduction enzyme, acetylcholinesterase (AChE). Comparative transcriptome analysis of S. zeamais through RNA-Seq identified a total of 3,562 differentially expressed genes (DEGs), of which 2,836 and 726 were up-regulated and down-regulated in response to M. alternifolia essential oil fumigation, respectively. Based on gene ontology (GO) analysis, the majority of DEGs were involved in insecticide detoxification and mitochondrial function. Furthermore, an abundance of DEGs mapped into the metabolism pathway in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database were associated with respiration and metabolism of xenobiotics, including cytochrome P450s, CarEs, GSTs, and ATP-binding cassette transporters (ABC transporters). Some DEGs mapped into the proteasome and phagosome pathway were found to be significantly enriched. These results led us to propose a model of insecticide action that M. alternifolia essential oil likely directly affects the hydrogen carrier to block the electron flow and interfere energy synthesis in mitochondrial respiratory chain

Health Status of Sand Flathead (Platycephalus bassensis), Inhabiting an Industrialised and Urbanised Embayment, Port Phillip Bay, Victoria as Measured by Biomarkers of Exposure and Effects.

PubMed

Baker, Jarrad K; Long, Sara M; Hassell, Kathryn L; Pettigrove, Vincent J; Gagnon, Marthe M

2016-01-01

Port Phillip Bay, Australia, is a large semi-closed bay with over four million people living in its catchment basin. The Bay receives waters from the Yarra River which drains the city of Melbourne, as well as receiving the discharges of sewage treatment plants and petrochemical and agricultural chemicals. A 1999 study demonstrated that fish inhabiting Port Phillip Bay showed signs of effects related to pollutant exposure despite pollution management practices having been implemented for over a decade. To assess the current health status of the fish inhabiting the Bay, a follow up survey was conducted in 2015. A suite of biomarkers of exposure and effects were measured to determine the health status of Port Phillip Bay sand flathead (Platycephalus bassensis), namely ethoxyresorufin-O-deethylase (EROD) activity, polycyclic aromatic hydrocarbons (PAH) biliary metabolites, carboxylesterase activity (CbE) and DNA damage (8-oxo-dG). The reduction in EROD activity in the present study suggests a decline in the presence of EROD activity-inducing chemicals within the Bay since the 1990s. Fish collected in the most industrialised/urbanised sites did not display higher PAH metabolite levels than those in less developed areas of the Bay. Ratios of PAH biliary metabolite types were used to indicate PAH contaminant origin. Ratios indicated fish collected at Corio Bay and Hobsons Bay were subjected to increased low molecular weight hydrocarbons of petrogenic origin, likely attributed to the close proximity of these sites to oil refineries, compared to PAH biliary metabolites in fish from Geelong Arm and Mordialloc. Quantification of DNA damage indicated a localised effect of exposure to pollutants, with a 10-fold higher DNA damage level in fish sampled from the industrial site of Corio Bay relative to the less developed site of Sorrento. Overall, integration of biomarkers by multivariate analysis indicated that the health of fish collected in industrialised areas was compromised

New insights on molecular interactions of organophosphorus pesticides with esterases.

PubMed

Mangas, Iris; Estevez, Jorge; Vilanova, Eugenio; França, Tanos Celmar Costa

2017-02-01

Organophosphorus compounds (OPs) are a large and diverse class of chemicals mainly used as pesticides and chemical weapons. People may be exposed to OPs in several occasions, which can produce several distinct neurotoxic effects depending on the dose, frequency of exposure, type of OP, and the host factors that influence susceptibility and sensitivity. These neurotoxic effects are mainly due to the interaction with enzyme targets involved in toxicological or detoxication pathways. In this work, the toxicological relevance of known OPs targets is reviewed. The main enzyme targets of OPs have been identified among the serine hydrolase protein family, some of them decades ago (e.g. AChE, BuChE, NTE and carboxylesterases), others more recently (e.g. lysophospholipase, arylformidase and KIA1363) and others which are not molecularly identified yet (e.g. phenylvalerate esterases). Members of this family are characterized by displaying serine hydrolase activity, containing a conserved serine hydrolase motif and having an alpha-beta hydrolase fold. Improvement in Xray-crystallography and in silico methods have generated new data of the interactions between OPs and esterases and have established new methods to study new inhibitors and reactivators of cholinesterases. Mass spectrometry for AChE, BChE and APH have characterized the active site serine adducts with OPs being useful to detect biomarkers of OPs exposure and inhibitory and postinhibitory reactions of esterases and OPs. The purpose of this review is focus specifically on the interaction of OP with esterases, mainly with type B-esterases, which are able to hydrolyze carboxylesters but inhibited by OPs by covalent phosphorylation on the serine or tyrosine residue in the active sites. Other related esterases in some cases with no-irreversible effect are also discussed. The understanding of the multiple molecular interactions is the basis we are proposing for a multi-target approach for understanding the

A novel glucan-binding protein with lipase activity from the oral pathogen Streptococcus mutans.

PubMed

Shah, Deepan S H; Russell, Roy R B

2004-06-01

Streptococcus mutans produces extracellular glucosyltransferases (GTFs) that synthesize glucans from sucrose. These glucans are important in determining the permeability properties and adhesiveness of dental plaque. GTFs and the GbpA glucan-binding protein are characterized by a binding domain containing a series of 33-amino-acid repeats, called 'A' repeats. The S. mutans genome sequence was searched for ORFs containing 'A' repeats, and one novel gene, gbpD, which appears to be unique to the mutans group of streptococci, was identified. The GbpD sequence revealed the presence of three 'A' repeats, in the middle of the protein, and a novel glucan-binding assay showed that GbpD binds to dextran with a K(D) of 2-3 nM. Construction of truncated derivatives of GbpD confirmed that the 'A' repeat region was essential for binding. Furthermore, a gbpD knockout mutant was modified in the extent of aggregation induced by polymers derived from sucrose. The N-terminus of GbpD has a signal sequence, followed by a region with no homologues in the public databases, while the C-terminus has homology to the alpha/beta hydrolase family (including lipases and carboxylesterases). GbpD contains the two regions typical of these enzymes: a GxSxG active site 'lipase box' and an 'oxyanion hole'. GbpD released free fatty acids (FFAs) from a range of triglycerides in the presence of calcium, indicating a lipase activity. The glucan binding/lipase bifunctionality suggested the natural substrate for the enzyme may be a surface macromolecule consisting of carbohydrate linked to lipid. The gbpD mutant was less hydrophobic than wild-type and pure recombinant GbpD reduced the hydrophobicity of S. mutans and another plaque bacterium, Streptococcus sanguinis. GbpD bound to and released FFA from lipoteichoic acid (LTA) of S. sanguinis, but had no effect on LTA from S. mutans. These results raise the intriguing possibility that GbpD may be involved in direct interspecies competition within the plaque

Inheritance mode and mechanisms of resistance to imidacloprid in the house fly Musca domestica (Diptera:Muscidae) from China.

PubMed

Ma, Zhuo; Li, Jing; Zhang, Yi; Shan, Chao; Gao, Xiwu

2017-01-01

Imidacloprid is a neonicotinoid insecticide that is effective against house fly, Musca domestica L., which is a major pest with the ability to develop resistance to insecticides. In the present study, we investigated the inheritance mode, the cross-resistance pattern and the mechanisms of resistance to imidacloprid. A near-isogenic house fly line (N-IRS) with 78-fold resistance to imidacloprid was used to demonstrate the mode of inheritance. The overlapping confidence limits of LC50 values and the slopes of the log concentration-probit lines between the reciprocal F1 and F1' progenies suggest that imidacloprid resistance is inherited autosomally in the house fly. There was incomplete dominant inheritance in the F1 and F1' progenies, based on dominance values of 0.77 and 0.75, respectively. A monogenic inheritance model revealed that imidacloprid resistance is governed by more than one factor. Compared to the field strain (CFD), the N-IRS strain developed more cross-resistance to chlorfenapyr and no cross-resistance to chlorpyrifos and acetamiprid, but showed negative cross-resistance to beta-cypermethrin and azamethiphos. Three synergists, diethyl malate (DEM), s,s,s-tributylphosphorotrithioate (DEF), and piperonyl butoxide (PBO), showed significant synergism against to imidacloprid (4.55-, 4.46- and 3.34-fold respectively) in the N-IRS strain. However, both DEM and PBO had no synergism and DEF only exhibited slight synergism in the CSS strain. The activities of carboxylesterase (CarE), glutathione S-transferases (GSTs) and cytochrome P450 in the N-IRS strain were significantly higher than in the CSS strain. But similar synergistic potential of DEF to imidacloprid between the CSS and N-IRS strain suggested that GSTs and cytochrome P450 played much more important role than esterase for the N-IRS strain resistance to imidacloprid. These results should be helpful for developing an improved management strategy to delay the development of imidacloprid resistance in

Are deep-sea organisms dwelling within a submarine canyon more at risk from anthropogenic contamination than those from the adjacent open slope? A case study of Blanes canyon (NW Mediterranean)

NASA Astrophysics Data System (ADS)

Koenig, Samuel; Fernández, Pilar; Company, Joan B.; Huertas, David; Solé, Montserrat

2013-11-01

Due to their geomorphological structure and proximity to the coastline, submarine canyons may act as natural conduit routes for anthropogenic contaminants that are transported from surface waters to the deep-sea. Organisms dwelling in these canyon environments might thus be at risk of experiencing adverse health effects due to higher pollution exposure. To address this question, chemical and biochemical analyses were conducted on two of the most abundant deep-sea fish species in the study area, namely Alepocephalus rostratus and Lepidion lepidion, and the most abundant deep-sea commercial decapod crustacean Aristeus antennatus sampled inside Blanes canyon (BC) and on the adjacent open slope (OS). Persistent organic pollutants (POPs) levels, including polychlorinated biphenyl (PCB), dichlorodiphenyltrichloroethane (DDT) and derivatives, hexachlorocyclohexanes (HCHs) and hexachlorobenzene (HCB) were determined in muscle tissue of selected samples from 900 m and 1500 m depth. Potential effects resulting from contaminant exposure were determined using hepatic biomarkers such as ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-deethylase (PROD), catalase (CAT), carboxylesterase (CbE), glutathione-S-transferase (GST), total glutathione peroxidase (GPX), glutathione reductase (GR) and superoxide-dismutase (SOD) enzyme activities and lipid peroxidation levels (LP). L. lepidion and A. antennatus tissues exhibited higher POP levels inside BC compared to the OS at 900 m depth. These findings were consistent with biomarker data (i.e. enzymatic response to presence of contaminant agents). Elevated xenobiotic-metabolizing (EROD and PROD) and antioxidant enzymes (CAT and GPX) indicated higher contaminant exposure in both species caught within BC. No difference in POP accumulation between sites was observed in L. lepidion at 1500 m depth, nor in biomarker data, suggesting that the pollution gradient was less pronounced at greater depths. This trend was further corroborated

Insecticide resistance in disease vectors from Mayotte: an opportunity for integrated vector management.

PubMed

Pocquet, Nicolas; Darriet, Frédéric; Zumbo, Betty; Milesi, Pascal; Thiria, Julien; Bernard, Vincent; Toty, Céline; Labbé, Pierrick; Chandre, Fabrice

2014-07-01

Mayotte, a small island in the Indian Ocean, has been affected for many years by vector-borne diseases. Malaria, Bancroftian filariasis, dengue, chikungunya and Rift Valley fever have circulated or still circulate on the island. They are all transmitted by Culicidae mosquitoes. To limit the impact of these diseases on human health, vector control has been implemented for more than 60 years on Mayotte. In this study, we assessed the resistance levels of four major vector species (Anopheles gambiae, Culex pipiens quinquefasciatus, Aedes aegypti and Aedes albopictus) to two types of insecticides: i) the locally currently-used insecticides (organophosphates, pyrethroids) and ii) alternative molecules that are promising for vector control and come from different insecticide families (bacterial toxins or insect growth regulators). When some resistance was found to one of these insecticides, we characterized the mechanisms involved. Larval and adult bioassays were used to evaluate the level of resistance. When resistance was found, we tested for the presence of metabolic resistance through detoxifying enzyme activity assays, or for target-site mutations through molecular identification of known resistance alleles. Resistance to currently-used insecticides varied greatly between the four vector species. While no resistance to any insecticides was found in the two Aedes species, bioassays confirmed multiple resistance in Cx. p. quinquefasciatus (temephos: ~ 20 fold and deltamethrin: only 10% mortality after 24 hours). In An. gambiae, resistance was scarce: only a moderate resistance to temephos was found (~5 fold). This resistance appears to be due only to carboxyl-esterase overexpression and not to target modification. Finally, and comfortingly, none of the four species showed resistance to any of the new insecticides. The low resistance observed in Mayotte's main disease vectors is particularly interesting, because it leaves a range of tools useable by vector control

Comparative toxicity of methidathion and glyphosate on early life stages of three amphibian species: Pelophylax ridibundus, Pseudepidalea viridis, and Xenopus laevis.

PubMed

Güngördü, Abbas

2013-09-15

The assessments of pesticide toxicity on nontarget organisms have largely been focused on the determination of median lethal concentration (LC50) values using single/laboratory species. Although useful, these studies cannot describe the biochemical mechanisms of toxicity and also cannot explain the effects of pesticides on natural species. In this study, the toxic effects of glyphosate and methidathion were evaluated comparatively on early developmental stages of 3 anurans-2 natural (Pelophylax ridibundus, Pseudepidalea viridis) and 1 laboratory species (Xenopus laevis). The 96-h LC50 values for methidathion and glyphosate were determined as 25.7-19.6 mg active ingredient (AI)/L for P. viridis, 27.4-22.7 mg AI/L for P. ridibundus, and 15.3-5.05 mg AI/L for X. laevis tadpoles. Furthermore, as early signs of intoxication, glutathione S-transferase (GST), acetylcholinesterase (AChE), carboxylesterase (CaE), glutathione reductase, lactate dehydrogenase, and aspartate aminotrasferase were assayed in 4-day-old tadpoles after 96-h pesticide exposure. The GST induction after 3.2mg AI/L methidathion exposure was determined to be 173%, 83%, and 38% of control, and the AChE inhibition for the same dose was determined to be 86%, 96%, and 30% of control for P. ridibundus, P. viridis, and X. laevis, respectively. Unlike the application of methidathion, all enzyme activities showed statistically significant increases on glyphosate exposure compared to controls. However, these increases in enzyme activities were not shown to be parallel with the increase of concentration. The levels of increases of GST and AChE were determined to be 111% and 31% for P. ridibundus, 13% and 51% for P. viridis, and 15% and 36% for X. laevis after 3.2mg AI/L glyphosate exposure, respectively. The findings of the study suggest that the most sensitive species to pesticide exposure is X. laevis. The selected biomarker enzymes AChE, CaE, and GST are useful in understanding the toxic mechanisms of these

Enzyme characteristics of aminotransferase FumI of Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B₁.

PubMed

Hartinger, Doris; Schwartz, Heidi; Hametner, Christian; Schatzmayr, Gerd; Haltrich, Dietmar; Moll, Wulf-Dieter

2011-08-01

Fumonisins are carcinogenic mycotoxins that are frequently found as natural contaminants in maize from warm climate regions around the world. The aminotransferase FumI is encoded as part of a gene cluster of Sphingopyxis sp. MTA144, which enables this bacterial strain to degrade fumonisin B(1) and related fumonisins. FumI catalyzes the deamination of the first intermediate of the catabolic pathway, hydrolyzed fumonisin B(1). We used a preparation of purified, His-tagged FumI, produced recombinantly in Escherichia coli in soluble form, for enzyme characterization. The structure of the reaction product was studied by NMR and identified as 2-keto hydrolyzed fumonisin B(1). Pyruvate was found to be the preferred co-substrate and amino group receptor (K (M) = 490 μM at 10 μM hydrolyzed fumonisin B(1)) of FumI, but other α-keto acids were also accepted as co-substrates. Addition of the co-enzyme pyridoxal phosphate to the enzyme preparation enhanced activity, and saturation was already reached at the lowest tested concentration of 10 μM. The enzyme showed activity in the range of pH 6 to 10 with an optimum at pH 8.5, and in the range of 6°C to 50°C with an optimum at 35°C. The aminotransferase worked best at low salt concentration. FumI activity could be recovered after preincubation at pH 4.0 or higher, but not lower. The aminotransferase was denatured after preincubation at 60°C for 1 h, and the residual activity was also reduced after preincubation at lower temperatures. At optimum conditions, the kinetic parameters K (M) = 1.1 μM and k (cat) = 104/min were determined with 5 mM pyruvate as co-substrate. Based on the enzyme characteristics, a technological application of FumI, in combination with the fumonisin carboxylesterase FumD for hydrolysis of fumonisins, for deamination and detoxification of hydrolyzed fumonisins seems possible, if the enzyme properties are considered.

Health Status of Sand Flathead (Platycephalus bassensis), Inhabiting an Industrialised and Urbanised Embayment, Port Phillip Bay, Victoria as Measured by Biomarkers of Exposure and Effects

PubMed Central

Baker, Jarrad K.; Long, Sara M.; Hassell, Kathryn L.; Pettigrove, Vincent J.

2016-01-01

Port Phillip Bay, Australia, is a large semi-closed bay with over four million people living in its catchment basin. The Bay receives waters from the Yarra River which drains the city of Melbourne, as well as receiving the discharges of sewage treatment plants and petrochemical and agricultural chemicals. A 1999 study demonstrated that fish inhabiting Port Phillip Bay showed signs of effects related to pollutant exposure despite pollution management practices having been implemented for over a decade. To assess the current health status of the fish inhabiting the Bay, a follow up survey was conducted in 2015. A suite of biomarkers of exposure and effects were measured to determine the health status of Port Phillip Bay sand flathead (Platycephalus bassensis), namely ethoxyresorufin-O-deethylase (EROD) activity, polycyclic aromatic hydrocarbons (PAH) biliary metabolites, carboxylesterase activity (CbE) and DNA damage (8-oxo-dG). The reduction in EROD activity in the present study suggests a decline in the presence of EROD activity-inducing chemicals within the Bay since the 1990s. Fish collected in the most industrialised/urbanised sites did not display higher PAH metabolite levels than those in less developed areas of the Bay. Ratios of PAH biliary metabolite types were used to indicate PAH contaminant origin. Ratios indicated fish collected at Corio Bay and Hobsons Bay were subjected to increased low molecular weight hydrocarbons of petrogenic origin, likely attributed to the close proximity of these sites to oil refineries, compared to PAH biliary metabolites in fish from Geelong Arm and Mordialloc. Quantification of DNA damage indicated a localised effect of exposure to pollutants, with a 10-fold higher DNA damage level in fish sampled from the industrial site of Corio Bay relative to the less developed site of Sorrento. Overall, integration of biomarkers by multivariate analysis indicated that the health of fish collected in industrialised areas was compromised

Differential Influences of Ethanol on Early Exposure to Racemic Methylphenidate Compared with Dexmethylphenidate in Humans

PubMed Central

Straughn, Arthur B.; Reeves, Owen T.; Bernstein, Hilary; Bell, Guinevere H.; Anderson, Erica R.; Malcolm, Robert J.

2013-01-01

Enantioselective hydrolysis of oral racemic methylphenidate (dl-MPH) by carboxylesterase 1 (CES1) limits the absolute bioavailability of the pharmacologically active d-MPH isomer to approximately 30% and that of the inactive l-MPH to only 1–2%. Coadministration of dl-MPH with ethanol results in elevated d-MPH plasma concentrations accompanied by CES1-mediated enantioselective transesterification of l-MPH to l-ethylphenidate (EPH). The present study tested the hypothesis that administration of the pure isomer dexmethylphenidate (d-MPH) will overcome the influence of ethanol on d-MPH absorption by eliminating competitive CES1-mediated presystemic metabolism of l-MPH to l-EPH. Twenty-four healthy volunteers received dl-MPH (0.3 mg/kg) or d-MPH (0.15 mg/kg), with or without ethanol (0.6 g/kg). During the absorption phase of dl-MPH, concomitant ethanol significantly elevated d-MPH plasma concentrations (44–99%; P < 0.005). Furthermore, immediately following the ethanol drink the subjective effects of “high,” “good,” “like,” “stimulated,” and overall “effect” were significantly potentiated (P ≤ 0.01). Plasma l-EPH concentrations exceeded those of l-MPH. Ethanol combined with pure d-MPH did not elevate plasma d-MPH concentrations during the absorption phase, and the ethanol-induced potentiation of subjective effects was delayed relative to dl-MPH-ethanol. These findings are consistent with l-MPH competitively inhibiting presystemic CES1 metabolism of d-MPH. Ethanol increased the d-MPH area under the curve (AUC)0-inf by 21% following dl-MPH (P < 0.001) and 14% for d-MPH (P = 0.001). In men receiving d-MPH-ethanol, the d-MPH absorption partial AUC0.5–2 hours was 2.1 times greater and the time to maximum concentration (Tmax) occurred 1.1 hours earlier than in women, consistent with an increased rate of d-MPH absorption reducing hepatic extraction. More rapid absorption of d-MPH carries implications for increased abuse liability. PMID:23104969

Structural Characterization and Reversal of the Natural Organophosphate Resistance of a D-Type Esterase, Saccharomyces cerevisiae S-Formylglutathione Hydrolase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Legler,P.; Kumaran, D.; Swaminathan, S.

2008-01-01

Saccharomyces cerevisiae expresses a 67.8 kDa homodimeric serine thioesterase, S-formylglutathione hydrolase (SFGH), that is 39.9% identical with human esterase D. Both enzymes possess significant carboxylesterase and S-formylglutathione thioesterase activity but are unusually resistant to organophosphate (OP) inhibitors. We determined the X-ray crystal structure of yeast (y) SFGH to 2.3 Angstroms resolution by multiwavelength anomalous dispersion and used the structure to guide site-specific mutagenesis experiments addressing substrate and inhibitor reactivity. Our results demonstrate a steric mechanism of OP resistance mediated by a single indole ring (W197) located in an enzyme 'acyl pocket'. The W197I substitution enhances ySFGH reactivity with paraoxon bymore » >1000-fold (kiW197I = 16 {+-} 2 mM-1 h-1), thereby overcoming natural OP resistance. W197I increases the rate of OP inhibition under pseudo-first-order conditions but does not accelerate OP hydrolysis. The structure of the paraoxon-inhibited W197I variant was determined by molecular replacement (2.2 Angstroms); it revealed a stabilized sulfenic acid at Cys60. Wild-type (WT) ySFGH is inhibited by thiol reactive compounds and is sensitive to oxidation; thus, the cysteine sulfenic acid may play a role in the regulation of a 'D-type' esterase. The structure of the W197I variant is the first reported cysteine sulfenic acid in a serine esterase. We constructed five Cys60/W197I variants and show that introducing a positive charge near the oxyanion hole, W197I/C60R or W197I/C60K, results in a further enhancement of the rates of phosphorylation with paraoxon (ki = 42 or 80 mM-1 h-1, respectively) but does not affect the dephosphorylation of the enzyme. We also characterized three histidine substitutions near the oxyanion hole, G57H, L58H, and M162H, which significantly decrease esterase activity.« less

In vivo inhibition of PC-3 human androgen-independent prostate cancer by a targeted cytotoxic bombesin analogue, AN-215.

PubMed

Plonowski, A; Nagy, A; Schally, A V; Sun, B; Groot, K; Halmos, G

2000-11-15

The effectiveness of chemotherapy targeted to bombesin (BN) receptors was evaluated in nude mice bearing PC-3 human androgen-independent prostate cancers. Cytotoxic BN analogue AN-215, consisting of 2-pyrrolinodoxorubicin (AN-201) linked to BN-like carrier peptide RC-3094, was injected i.v. at 150 nmol/kg on days 1, 11 and 21. After treatment with AN-215, tumor volume was 69% (p < 0.01) smaller than that in controls and tumor doubling time was extended from 8.5 +/- 0.7 days to 20.3 +/- 3.5 days (p < 0.05). Cytotoxic radical AN-201, carrier RC-3094 and their unconjugated mixture administered at the same dosage were ineffective. The mortality rate was 12.5% in the AN-201 group and 16.7% in the group treated with the mixture, but no deaths occurred in mice receiving AN-215. Because the ester bond linking AN-201 to the carrier molecule is hydrolyzed much faster in mouse serum than in human serum, in the second experiment we investigated the tolerance to AN-215 and its effect in nude mice bearing PC-3 tumors after pharmacological inhibition of serum carboxylesterases. Two applications of AN-201 at 200 nmol/kg were lethal, whereas no mortality was observed after 4 injections of AN-215 at the same dose. Administration of 200 nmol/kg AN-215 on days 1, 7, 17 and 26 again produced 69% tumor inhibition. BN receptors on membranes of PC-3 tumors were detected by (125)I-[Tyr(4)]BN binding, and expression of mRNA for BRS-3 and GRP-R subtypes was also found. AN-215 showed a high affinity to PC-3 tumors, displacing the radioligand at an IC(50) of 12.95 +/- 0.35 nM. Because BN receptors are present on primary and metastatic prostate cancer, targeted chemotherapy with AN-215 might benefit patients with advanced prostatic carcinoma who relapsed androgen ablation. Copyright 2000 Wiley-Liss, Inc.

Investigating the Molecular Mechanisms of Organophosphate and Pyrethroid Resistance in the Fall Armyworm Spodoptera frugiperda

PubMed Central

Carvalho, Renato A.; Omoto, Celso; Field, Linda M.; Williamson, Martin S.; Bass, Chris

2013-01-01

The fall armyworm Spodoptera frugiperda is an economically important pest of small grain crops that occurs in all maize growing regions of the Americas. The intensive use of chemical pesticides for its control has led to the selection of resistant populations, however, to date, the molecular mechanisms underlying resistance have not been characterised. In this study the mechanisms involved in the resistance of two S. frugiperda strains collected in Brazil to chlorpyrifos (OP strain) or lambda-cyhalothrin (PYR strain) were investigated using molecular and genomic approaches. To examine the possible role of target-site insensitivity the genes encoding the organophosphate (acetylcholinesterase, AChE) and pyrethroid (voltage-gated sodium channel, VGSC) target-site proteins were PCR amplified. Sequencing of the S. frugiperda ace-1 gene identified several nucleotide changes in the OP strain when compared to a susceptible reference strain (SUS). These result in three amino acid substitutions, A201S, G227A and F290V, that have all been shown previously to confer organophosphate resistance in several other insect species. Sequencing of the gene encoding the VGSC in the PYR strain, identified mutations that result in three amino acid substitutions, T929I, L932F and L1014F, all of which have been shown previously to confer knockdown/super knockdown-type resistance in several arthropod species. To investigate the possible role of metabolic detoxification in the resistant phenotype of the OP and PYR stains all EST sequences available for S. frugiperda were used to design a gene-expression microarray. This was then used to compare gene expression in the resistant strains with the susceptible reference strain. Members of several gene families, previously implicated in metabolic resistance in other insects were found to be overexpressed in the resistant strains including glutathione S-transferases, cytochrome P450s and carboxylesterases. Taken together these results provide

Inheritance mode and mechanisms of resistance to imidacloprid in the house fly Musca domestica (Diptera:Muscidae) from China

PubMed Central

Zhang, Yi; Shan, Chao

2017-01-01

Imidacloprid is a neonicotinoid insecticide that is effective against house fly, Musca domestica L., which is a major pest with the ability to develop resistance to insecticides. In the present study, we investigated the inheritance mode, the cross-resistance pattern and the mechanisms of resistance to imidacloprid. A near-isogenic house fly line (N-IRS) with 78-fold resistance to imidacloprid was used to demonstrate the mode of inheritance. The overlapping confidence limits of LC50 values and the slopes of the log concentration-probit lines between the reciprocal F1 and F1’ progenies suggest that imidacloprid resistance is inherited autosomally in the house fly. There was incomplete dominant inheritance in the F1 and F1’ progenies, based on dominance values of 0.77 and 0.75, respectively. A monogenic inheritance model revealed that imidacloprid resistance is governed by more than one factor. Compared to the field strain (CFD), the N-IRS strain developed more cross-resistance to chlorfenapyr and no cross-resistance to chlorpyrifos and acetamiprid, but showed negative cross-resistance to beta-cypermethrin and azamethiphos. Three synergists, diethyl malate (DEM), s,s,s-tributylphosphorotrithioate (DEF), and piperonyl butoxide (PBO), showed significant synergism against to imidacloprid (4.55-, 4.46- and 3.34-fold respectively) in the N-IRS strain. However, both DEM and PBO had no synergism and DEF only exhibited slight synergism in the CSS strain. The activities of carboxylesterase (CarE), glutathione S-transferases (GSTs) and cytochrome P450 in the N-IRS strain were significantly higher than in the CSS strain. But similar synergistic potential of DEF to imidacloprid between the CSS and N-IRS strain suggested that GSTs and cytochrome P450 played much more important role than esterase for the N-IRS strain resistance to imidacloprid. These results should be helpful for developing an improved management strategy to delay the development of imidacloprid resistance in

Hormone-dependence of sarin lethality in rats: Sex differences and stage of the estrous cycle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Carl D., E-mail: carl.d.smith179.mil@mail.mil; Wright, Linnzi K.M.; Garcia, Gregory E.

Chemical warfare nerve agents (CWNAs) are highly toxic compounds that cause a cascade of symptoms and death, if exposed casualties are left untreated. Numerous rodent models have investigated the toxicity and mechanisms of toxicity of CWNAs, but most are limited to male subjects. Given the profound physiological effects of circulating gonadal hormones in female rodents, it is possible that the daily cyclical fluctuations of these hormones affect females' sensitivity to the lethal effects of CWNAs, and previous reports that included female subjects did not control for the stage of the hormonal cycle. The aim of the current study was tomore » determine the 24-hour median lethal dose (LD{sub 50}) of the CWNA sarin in male, ovariectomized (OVEX) female, and female rats during different stages of the estrous cycle (diestrus, proestrus, and estrus). Additionally, baseline activity levels of plasma acetylcholinesterase, butyrylcholinesterase, and carboxylesterase were measured to determine differences among the groups. Results indicated that females in proestrus had a significantly higher LD{sub 50} of sarin compared to OVEX and estrous females. Although some sex differences were observed in the activity levels of plasma esterases, they were not consistent and likely not large enough to significantly affect the LD{sub 50}s. These results suggest that hormonal cyclicity can influence the outcome of CWNA-related studies using female rodents, and that this variability can be minimized by controlling for the stage of the cycle. Additional research is necessary to determine the precise mechanism of the observed differences because it is unlikely to be solely explained by plasma esterase activity. - Highlights: • The LD{sub 50} of sarin was determined in female rats throughout the stages of the estrous cycle. • Females in proestrus had a significantly higher LD{sub 50} compared to estrous or ovariectomized females. • No sex differences were observed between male and

Investigating the molecular mechanisms of organophosphate and pyrethroid resistance in the fall armyworm Spodoptera frugiperda.

PubMed

Carvalho, Renato A; Omoto, Celso; Field, Linda M; Williamson, Martin S; Bass, Chris

2013-01-01

The fall armyworm Spodoptera frugiperda is an economically important pest of small grain crops that occurs in all maize growing regions of the Americas. The intensive use of chemical pesticides for its control has led to the selection of resistant populations, however, to date, the molecular mechanisms underlying resistance have not been characterised. In this study the mechanisms involved in the resistance of two S. frugiperda strains collected in Brazil to chlorpyrifos (OP strain) or lambda-cyhalothrin (PYR strain) were investigated using molecular and genomic approaches. To examine the possible role of target-site insensitivity the genes encoding the organophosphate (acetylcholinesterase, AChE) and pyrethroid (voltage-gated sodium channel, VGSC) target-site proteins were PCR amplified. Sequencing of the S. frugiperda ace-1 gene identified several nucleotide changes in the OP strain when compared to a susceptible reference strain (SUS). These result in three amino acid substitutions, A201S, G227A and F290V, that have all been shown previously to confer organophosphate resistance in several other insect species. Sequencing of the gene encoding the VGSC in the PYR strain, identified mutations that result in three amino acid substitutions, T929I, L932F and L1014F, all of which have been shown previously to confer knockdown/super knockdown-type resistance in several arthropod species. To investigate the possible role of metabolic detoxification in the resistant phenotype of the OP and PYR stains all EST sequences available for S. frugiperda were used to design a gene-expression microarray. This was then used to compare gene expression in the resistant strains with the susceptible reference strain. Members of several gene families, previously implicated in metabolic resistance in other insects were found to be overexpressed in the resistant strains including glutathione S-transferases, cytochrome P450s and carboxylesterases. Taken together these results provide

Repeated Gestational Exposure of Mice to Chlorpyrifos Oxon Is Associated with Paraoxonase 1 (PON1) Modulated Effects in Maternal and Fetal Tissues

PubMed Central

Co, Aila L.; Hay, Ariel M.; MacDonald, James W.; Bammler, Theo K.; Farin, Federico M.; Costa, Lucio G.; Furlong, Clement E.

2014-01-01

Chlorpyrifos oxon (CPO), the toxic metabolite of the organophosphorus (OP) insecticide chlorpyrifos, causes developmental neurotoxicity in humans and rodents. CPO is hydrolyzed by paraoxonase-1 (PON1), with protection determined by PON1 levels and the human Q192R polymorphism. To examine how the Q192R polymorphism influences fetal toxicity associated with gestational CPO exposure, we measured enzyme inhibition and fetal-brain gene expression in wild-type (PON1+/+), PON1-knockout (PON1−/−), and tgHuPON1R192 and tgHuPON1Q192 transgenic mice. Pregnant mice exposed dermally to 0, 0.50, 0.75, or 0.85 mg/kg/d CPO from gestational day (GD) 6 through 17 were sacrificed on GD18. Biomarkers of CPO exposure inhibited in maternal tissues included brain acetylcholinesterase (AChE), red blood cell acylpeptide hydrolase (APH), and plasma butyrylcholinesterase (BChE) and carboxylesterase (CES). Fetal plasma BChE was inhibited in PON1−/− and tgHuPON1Q192, but not PON1+/+ or tgHuPON1R192 mice. Fetal brain AChE and plasma CES were inhibited in PON1−/− mice, but not in other genotypes. Weighted gene co-expression network analysis identified five gene modules based on clustering of the correlations among their fetal-brain expression values, allowing for correlation of module membership with the phenotypic data on enzyme inhibition. One module that correlated highly with maternal brain AChE activity had a large representation of homeobox genes. Gene set enrichment analysis revealed multiple gene sets affected by gestational CPO exposure in tgHuPON1Q192 but not tgHuPON1R192 mice, including gene sets involved in protein export, lipid metabolism, and neurotransmission. These data indicate that maternal PON1 status modulates the effects of repeated gestational CPO exposure on fetal-brain gene expression and on inhibition of both maternal and fetal biomarker enzymes. PMID:25070982

Increased expressions of genes and proteins involved in mitochondrial oxidation and antioxidant pathway in adipose tissue of pigs selected for a low residual feed intake.

PubMed

Louveau, I; Vincent, A; Tacher, S; Gilbert, H; Gondret, F

2016-12-01

Adipose tissue is a primary sensor for nutrient availability and regulates many functions including feed intake and energy homeostasis. This study was undertaken to determine the molecular responses of adipose tissue to differences in feed intake and feed efficiency. Subcutaneous adipose tissue was collected from two lines of pigs divergently selected for residual feed intake (RFI), a measure of feed efficiency defined as the difference between actual and expected feed intake, and from a subset of high-RFI pigs that were feed-restricted at the level of the voluntary feed intake of low-RFI pigs during the growing-finishing period. Transcriptomics analyses indicated that the number of genes that were differentially expressed ( < 0.01) between low- and high-RFI pigs ( = 8 per group at each stage) in adipose tissue was much lower when pigs were considered at 19 kg (postweaning) than at 115 kg BW (market weight). Extended investigations were performed at 115 kg BW to compare low-RFI ( = 8), high-RFI ( = 8), and feed-restricted high-RFI ( = 8) pigs. They included in silico pathway analyses of the differentially expressed (DE) genes ( < 0.01) and a complementary proteomic investigation to list adipose proteins with a differential abundance ( < 0.10). Only 23% of the DE genes were affected by both RFI and feed restriction. This indicates that the responses of adipose tissue to RFI difference shared only some common mechanisms with feed intake modulation, notably the regulation of cell cycle (including ) and transferase activity pathway. Two carboxylesterase genes (, ) involved in lipolysis, were among the most overexpressed genes in the low-RFI pigs; they were also affected by feed restriction within the high-RFI line. About 60% of the molecular changes between low- and high-RFI pigs were specific to genetic divergence in feed efficiency, independently of feed intake. Different genes and proteins known to be associated with mitochondrial oxidative metabolism were

Insecticidal Activity of Melaleuca alternifolia Essential Oil and RNA-Seq Analysis of Sitophilus zeamais Transcriptome in Response to Oil Fumigation

PubMed Central

Liao, Min; Xiao, Jin-Jing; Zhou, Li-Jun; Liu, Yang; Wu, Xiang-Wei; Hua, Ri-Mao; Wang, Gui-Rong; Cao, Hai-Qun

2016-01-01

Background The cereal weevil, Sitophilus zeamais is one of the most destructive pests of stored cereals worldwide. Frequent use of fumigants for managing stored-product insects has led to the development of resistance in insects. Essential oils from aromatic plants including the tea oil plant, Melaleuca alternifolia may provide environmentally friendly alternatives to currently used pest control agents. However, little is known about molecular events involved in stored-product insects in response to plant essential oil fumigation. Results M. alternifolia essential oil was shown to possess the fumigant toxicity against S. zeamais. The constituent, terpinen-4-ol was the most effective compound for fumigant toxicity. M. alternifolia essential oil significantly inhibited the activity of three enzymes in S. zeamais, including two detoxifying enzymes, glutathione S-transferase (GST), and carboxylesterase (CarE), as well as a nerve conduction enzyme, acetylcholinesterase (AChE). Comparative transcriptome analysis of S. zeamais through RNA-Seq identified a total of 3,562 differentially expressed genes (DEGs), of which 2,836 and 726 were up-regulated and down-regulated in response to M. alternifolia essential oil fumigation, respectively. Based on gene ontology (GO) analysis, the majority of DEGs were involved in insecticide detoxification and mitochondrial function. Furthermore, an abundance of DEGs mapped into the metabolism pathway in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database were associated with respiration and metabolism of xenobiotics, including cytochrome P450s, CarEs, GSTs, and ATP-binding cassette transporters (ABC transporters). Some DEGs mapped into the proteasome and phagosome pathway were found to be significantly enriched. These results led us to propose a model of insecticide action that M. alternifolia essential oil likely directly affects the hydrogen carrier to block the electron flow and interfere energy synthesis in

Toxicological effects of chemical constituents from Piper against the environmental burden Aedes aegypti Liston and their impact on non-target toxicity evaluation against biomonitoring aquatic insects.

PubMed

Vasantha-Srinivasan, Prabhakaran; Thanigaivel, Annamalai; Edwin, Edward-Sam; Ponsankar, Athirstam; Senthil-Nathan, Sengottayan; Selin-Rani, Selvaraj; Kalaivani, Kandaswamy; Hunter, Wayne B; Duraipandiyan, Veeramuthu; Al-Dhabi, Naif Abdullah

2018-04-01

Dengue is the most rapidly spreading mosquito-borne viral disease in the world. The mosquito, Aedes aegypti, also spreads Yellow fever, Chikungunya, and Zika virus. As the primary vector for dengue, Ae. aegypti now occurs in over 20 countries and is a serious concern with reports of increasing insecticide resistance. Developing new treatments to manage mosquitoes are needed. Formulation of crude volatile oil from Piper betle leaves (Pb-CVO) was evaluated as a potential treatment which showed larvicidal, ovipositional, and repellency effects. Gut-histology and enzyme profiles were analyzed post treatment under in-vitro conditions. The Pb-CVO from leaves of field collected plants was obtained by steam distillation and separated through rotary evaporation. The Pb-CVO were evaluated for chemical constituents through GC-MS analyses revealed 20 vital compounds. The peak area was establish to be superior in Eudesm-7(11)-en-4-ol (14.95%). Pb-CVO were determined and tested as four different concentrations (0.25, 0.5, 1.0, and 1.5 mg/L) of Pb-CVO towards Ae. aegypti. The larvicidal effects exhibited dose dependent mortality being greatest at 1.5 mg Pb-CVO/10 g leaves. The LC 50 occurred at 0.63 mg Pb-CVO/L. Larva of Ae. aegypti exposed to Pb-CVO showed significantly reduced digestive enzyme actions of α- and β-carboxylesterases. In contrast, GST and CYP450 enzyme levels increased significantly as concentration increased. Correspondingly, oviposition deterrence index and egg hatch of Ae. aegypti exposed to sub-lethal doses of Pb-CVO demonstrated a strong effect suitable for population suppression. Repellency at 0.6 mg Pb-CVO applied as oil had a protection time of 15-210 min. Mid-gut histological of Ae. aegypti larvae showed severe damage when treated with 0.6 mg of Pb-CVO treatment compared to the control. Non-toxic effects against aquatic beneficial insects, such as Anisops bouvieri and Toxorhynchites splendens, were observed at the highest concentrations, exposed

An enhanced in vivo stable isotope labeling by amino acids in cell culture (SILAC) model for quantification of drug metabolism enzymes.

PubMed

MacLeod, A Kenneth; Fallon, Padraic G; Sharp, Sheila; Henderson, Colin J; Wolf, C Roland; Huang, Jeffrey T-J

2015-03-01

Many of the enzymes involved in xenobiotic metabolism are maintained at a low basal level and are only synthesized in response to activation of upstream sensor/effector proteins. This induction can have implications in a variety of contexts, particularly during the study of the pharmacokinetics, pharmacodynamics, and drug-drug interaction profile of a candidate therapeutic compound. Previously, we combined in vivo SILAC material with a targeted high resolution single ion monitoring (tHR/SIM) LC-MS/MS approach for quantification of 197 peptide pairs, representing 51 drug metabolism enzymes (DME), in mouse liver. However, as important enzymes (for example, cytochromes P450 (Cyp) of the 1a and 2b subfamilies) are maintained at low or undetectable levels in the liver of unstimulated metabolically labeled mice, quantification of these proteins was unreliable. In the present study, we induced DME expression in labeled mice through synchronous ligand-mediated activation of multiple upstream nuclear receptors, thereby enhancing signals for proteins including Cyps 1a, 2a, 2b, 2c, and 3a. With this enhancement, 115 unique, lysine-containing, Cyp-derived peptides were detected in the liver of a single animal, as opposed to 56 in a pooled sample from three uninduced animals. A total of 386 peptide pairs were quantified by tHR/SIM, representing 68 Phase I, 30 Phase II, and eight control proteins. This method was employed to quantify changes in DME expression in the hepatic cytochrome P450 reductase null (HRN) mouse. We observed compensatory induction of several enzymes, including Cyps 2b10, 2c29, 2c37, 2c54, 2c55, 2e1, 3a11, and 3a13, carboxylesterase (Ces) 2a, and glutathione S-transferases (Gst) m2 and m3, along with down-regulation of hydroxysteroid dehydrogenases (Hsd) 11b1 and 17b6. Using DME-enhanced in vivo SILAC material with tHR/SIM, therefore, permits the robust analysis of multiple DME of importance to xenobiotic metabolism, with improved utility for the study of

Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna.

PubMed

Liu, Hong-cui; Yuan, Bing-qiang; Li, Shao-nan

2016-02-01

To yield cholinesterase (ChE) from prokaryotic expression, the ChE gene that belongs to Daphnia magna was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using forward primer 5'-CCCYGGNGCSAT GATGTG-3' and reverse primer 5'-GYAAGTTRGCCCAATATCT-3'. To express the gene, one sequence of the amplified DNA, which was able to encode a putative protein containing two conserved carboxylesterase domains, was connected to the prokaryotic expression vector PET-29a(+). The recombinant vector was transformed into Escherichia coil BL21 (DE3). Protein expression was induced by isopropy-D-thiogalactoside. The expressed ChE was used as an immunogen to immunize BALB/c mice. The obtained antibodies were tested for their specificity towards crude enzymes from species such as Alona milleri, Macrobrachium nipponense, Bombyx mori, Chironomus kiiensis, Apis mellifera, Eisenia foetida, Brachydanio rerio, and Xenopus laevis. Results indicated that the antibodies had specificity suitable for detecting ChE in Daphnia magna. A type of indirect and non-competitive enzyme-linked immunosorbent assay (IN-ELISA) was used to test the immunoreactive content of ChE (ChE-IR) in Daphina magna. The detection limit of the IN-ELISA was found to be 14.5 ng/ml at an antiserum dilution of 1:22 000. Results from tests on Daphnia magna exposed to sublethal concentrations of triazophos indicated a maximal induction of 57.2% in terms of ChE-IR on the second day after the animals were exposed to a concentration of 2.10 μg/L triazophos. Testing on animals acclimatized to a temperature of 16 °C indicated that ChE-IR was induced by 16.9% compared with the ChE-IR content detected at 21 °C, and the rate of induction was 25.6% at 10 °C. The IN-ELISA was also used to test the stability of ChE-IR in collected samples. Repeated freezing and thawing had no influence on the outcome of the test. All these results suggest that the polyclonal antibodies developed against the recombinant ChE are as

Exploring the molecular basis of insecticide resistance in the dengue vector Aedes aegypti: a case study in Martinique Island (French West Indies)

PubMed Central

Marcombe, Sébastien; Poupardin, Rodolphe; Darriet, Frederic; Reynaud, Stéphane; Bonnet, Julien; Strode, Clare; Brengues, Cecile; Yébakima, André; Ranson, Hilary; Corbel, Vincent; David, Jean-Philippe

2009-01-01

Background The yellow fever mosquito Aedes aegypti is a major vector of dengue and hemorrhagic fevers, causing up to 100 million dengue infections every year. As there is still no medicine and efficient vaccine available, vector control largely based on insecticide treatments remains the only method to reduce dengue virus transmission. Unfortunately, vector control programs are facing operational challenges with mosquitoes becoming resistant to commonly used insecticides. Resistance of Ae. aegypti to chemical insecticides has been reported worldwide and the underlying molecular mechanisms, including the identification of enzymes involved in insecticide detoxification are not completely understood. Results The present paper investigates the molecular basis of insecticide resistance in a population of Ae. aegypti collected in Martinique (French West Indies). Bioassays with insecticides on adults and larvae revealed high levels of resistance to organophosphate and pyrethroid insecticides. Molecular screening for common insecticide target-site mutations showed a high frequency (71%) of the sodium channel 'knock down resistance' (kdr) mutation. Exposing mosquitoes to detoxification enzymes inhibitors prior to bioassays induced a significant increased susceptibility of mosquitoes to insecticides, revealing the presence of metabolic-based resistance mechanisms. This trend was biochemically confirmed by significant elevated activities of cytochrome P450 monooxygenases, glutathione S-transferases and carboxylesterases at both larval and adult stages. Utilization of the microarray Aedes Detox Chip containing probes for all members of detoxification and other insecticide resistance-related enzymes revealed the significant constitutive over-transcription of multiple detoxification genes at both larval and adult stages. The over-transcription of detoxification genes in the resistant strain was confirmed by using real-time quantitative RT-PCR. Conclusion These results suggest

Biologically Based Methods for Control of Fumonisin-Producing Fusarium Species and Reduction of the Fumonisins.

PubMed

Alberts, Johanna F; van Zyl, Willem H; Gelderblom, Wentzel C A

2016-01-01

Infection by the fumonisin-producing Fusarium spp. and subsequent fumonisin contamination of maize adversely affect international trade and economy with deleterious effects on human and animal health. In developed countries high standards of the major food suppliers and retailers are upheld and regulatory controls deter the importation and local marketing of fumonisin-contaminated food products. In developing countries regulatory measures are either lacking or poorly enforced, due to food insecurity, resulting in an increased mycotoxin exposure. The lack and poor accessibility of effective and environmentally safe control methods have led to an increased interest in practical and biological alternatives to reduce fumonisin intake. These include the application of natural resources, including plants, microbial cultures, genetic material thereof, or clay minerals pre- and post-harvest. Pre-harvest approaches include breeding for resistant maize cultivars, introduction of biocontrol microorganisms, application of phenolic plant extracts, and expression of antifungal proteins and fumonisin degrading enzymes in transgenic maize cultivars. Post-harvest approaches include the removal of fumonisins by natural clay adsorbents and enzymatic degradation of fumonisins through decarboxylation and deamination by recombinant carboxylesterase and aminotransferase enzymes. Although, the knowledge base on biological control methods has expanded, only a limited number of authorized decontamination products and methods are commercially available. As many studies detailed the use of natural compounds in vitro, concepts in reducing fumonisin contamination should be developed further for application in planta and in the field pre-harvest, post-harvest, and during storage and food-processing. In developed countries an integrated approach, involving good agricultural management practices, hazard analysis and critical control point (HACCP) production, and storage management, together with

Biologically Based Methods for Control of Fumonisin-Producing Fusarium Species and Reduction of the Fumonisins

PubMed Central

Alberts, Johanna F.; van Zyl, Willem H.; Gelderblom, Wentzel C. A.

2016-01-01

Infection by the fumonisin-producing Fusarium spp. and subsequent fumonisin contamination of maize adversely affect international trade and economy with deleterious effects on human and animal health. In developed countries high standards of the major food suppliers and retailers are upheld and regulatory controls deter the importation and local marketing of fumonisin-contaminated food products. In developing countries regulatory measures are either lacking or poorly enforced, due to food insecurity, resulting in an increased mycotoxin exposure. The lack and poor accessibility of effective and environmentally safe control methods have led to an increased interest in practical and biological alternatives to reduce fumonisin intake. These include the application of natural resources, including plants, microbial cultures, genetic material thereof, or clay minerals pre- and post-harvest. Pre-harvest approaches include breeding for resistant maize cultivars, introduction of biocontrol microorganisms, application of phenolic plant extracts, and expression of antifungal proteins and fumonisin degrading enzymes in transgenic maize cultivars. Post-harvest approaches include the removal of fumonisins by natural clay adsorbents and enzymatic degradation of fumonisins through decarboxylation and deamination by recombinant carboxylesterase and aminotransferase enzymes. Although, the knowledge base on biological control methods has expanded, only a limited number of authorized decontamination products and methods are commercially available. As many studies detailed the use of natural compounds in vitro, concepts in reducing fumonisin contamination should be developed further for application in planta and in the field pre-harvest, post-harvest, and during storage and food-processing. In developed countries an integrated approach, involving good agricultural management practices, hazard analysis and critical control point (HACCP) production, and storage management, together with

Pharmacokinetic drug interactions with clopidogrel: updated review and risk management in combination therapy

PubMed Central

Wang, Zhi-Yu; Chen, Meng; Zhu, Ling-Ling; Yu, Lu-Shan; Zeng, Su; Xiang, Mei-Xiang; Zhou, Quan

2015-01-01

Background Coprescribing of clopidogrel and other drugs is common. Available reviews have addressed the drug–drug interactions (DDIs) when clopidogrel is as an object drug, or focused on combination use of clopidogrel and a special class of drugs. Clinicians may still be ignorant of those DDIs when clopidogrel is a precipitant drug, the factors determining the degree of DDIs, and corresponding risk management. Methods A literature search was performed using PubMed, MEDLINE, Web of Science, and the Cochrane Library to analyze the pharmacokinetic DDIs of clopidogrel and new P2Y12 receptor inhibitors. Results Clopidogrel affects the pharmacokinetics of cerivastatin, repaglinide, ferulic acid, sibutramine, efavirenz, and omeprazole. Low efficacy of clopidogrel is anticipated in the presence of omeprazole, esomeprazole, morphine, grapefruit juice, scutellarin, fluoxetine, azole antifungals, calcium channel blockers, sulfonylureas, and ritonavir. Augmented antiplatelet effects are anticipated when clopidogrel is coprescribed with aspirin, curcumin, cyclosporin, St John’s wort, rifampicin, and angiotensin-converting enzyme inhibitors. The factors determining the degree of DDIs with clopidogrel include genetic status (eg, cytochrome P540 [CYP]2B6*6, CYP2C19 polymorphism, CYP3A5*3, CYP3A4*1G, and CYP1A2-163C.A), species differences, and dose strength. The DDI risk does not exhibit a class effect, eg, the effects of clopidogrel on cerivastatin versus other statins, the effects of proton pump inhibitors on clopidogrel (omeprazole, esomeprazole versus pantoprazole, rabeprazole), the effects of rifampicin on clopidogrel versus ticagrelor and prasugrel, and the effects of calcium channel blockers on clopidogrel (amlodipine versus P-glycoprotein-inhibiting calcium channel blockers). The mechanism of the DDIs with clopidogrel involves modulating CYP enzymes (eg, CYP2B6, CYP2C8, CYP2C19, and CYP3A4), paraoxonase-1, hepatic carboxylesterase 1, P-glycoprotein, and organic anion

Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna *

PubMed Central

Liu, Hong-cui; Yuan, Bing-qiang; Li, Shao-nan

2016-01-01

To yield cholinesterase (ChE) from prokaryotic expression, the ChE gene that belongs to Daphnia magna was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using forward primer 5'-CCCYGGNGCSAT GATGTG-3' and reverse primer 5'-GYAAGTTRGCCCAATATCT-3'. To express the gene, one sequence of the amplified DNA, which was able to encode a putative protein containing two conserved carboxylesterase domains, was connected to the prokaryotic expression vector PET-29a(+). The recombinant vector was transformed into Escherichia coil BL21 (DE3). Protein expression was induced by isopropy-D-thiogalactoside. The expressed ChE was used as an immunogen to immunize BALB/c mice. The obtained antibodies were tested for their specificity towards crude enzymes from species such as Alona milleri, Macrobrachium nipponense, Bombyx mori, Chironomus kiiensis, Apis mellifera, Eisenia foetida, Brachydanio rerio, and Xenopus laevis. Results indicated that the antibodies had specificity suitable for detecting ChE in Daphnia magna. A type of indirect and non-competitive enzyme-linked immunosorbent assay (IN-ELISA) was used to test the immunoreactive content of ChE (ChE-IR) in Daphina magna. The detection limit of the IN-ELISA was found to be 14.5 ng/ml at an antiserum dilution of 1:22 000. Results from tests on Daphnia magna exposed to sublethal concentrations of triazophos indicated a maximal induction of 57.2% in terms of ChE-IR on the second day after the animals were exposed to a concentration of 2.10 μg/L triazophos. Testing on animals acclimatized to a temperature of 16 °C indicated that ChE-IR was induced by 16.9% compared with the ChE-IR content detected at 21 °C, and the rate of induction was 25.6% at 10 °C. The IN-ELISA was also used to test the stability of ChE-IR in collected samples. Repeated freezing and thawing had no influence on the outcome of the test. All these results suggest that the polyclonal antibodies developed against the recombinant ChE are as

Shengjiang Xiexin Decoction Alters Pharmacokinetics of Irinotecan by Regulating Metabolic Enzymes and Transporters: A Multi-Target Therapy for Alleviating the Gastrointestinal Toxicity.

PubMed

Guan, Huan-Yu; Li, Peng-Fei; Wang, Xiao-Ming; Yue, Jia-Jing; He, Yang; Luo, Xiao-Mei; Su, Mei-Feng; Liao, Shang-Gao; Shi, Yue

2017-01-01

Shengjiang Xiexin decoction (SXD), a classic traditional Chinese medical formula chronicled in Shang Han Lun , is used in modern clinical practice to decrease gastrointestinal toxicity induced by the chemotherapeutic drug irinotecan (CPT-11). In this study, the effect of SXD on the pharmacokinetics of CPT-11 and its active metabolites (SN-38 and SN-38G), and the underlying mechanisms were further examined. An ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the simultaneous quantification of CPT-11, SN-38, and SN-38G in the plasma, bile, liver, intestine, and intestinal contents of control and SXD-pre-treated rats after intravenous administration of CPT-11. SXD pretreatment increased the area under the curve (AUC) and the initial plasma concentration (C 0 ) of CPT-11 but decreased the plasma clearance (CL). The AUC and the maximum plasma concentration (C max ) of SN-38 decreased, whereas the C max of SN-38G increased. Compared with that of the control group, the biliary excretion of CPT-11, SN-38, and SN-38G was inhibited. The CPT-11, SN-38, and SN-38G concentrations in the liver, intestine, and intestinal contents were different between the two groups. Furthermore, the hepatic expression of multidrug resistance-associated protein-2 (Mrp-2), P-glycoprotein (P-gp), and carboxylesterase 2 (CES2) was significantly down-regulated by SXD, while the hepatic and jejunal uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) expression was elevated. The hydrolysis of CPT-11 to SN-38 by CES and the glucuronidation of SN-38 to SN-38G by UGT were affected by liver and jejunum S9 fractions from rats pre-treated with SXD. Therefore, this study demonstrated for the first time that SXD could alter the pharmacokinetics of CPT-11 and its metabolites to alleviate CPT-11-induced diarrhea. And the underlying mechanism of drug interaction between CPT-11 and SXD involves decreasing hepatic Mrp-2 and P

Association between copy number variation losses and alcohol dependence across African American and European American ethnic groups.

PubMed

Ulloa, Alvaro E; Chen, Jiayu; Vergara, Victor M; Calhoun, Vince; Liu, Jingyu

2014-05-01

Copy number variations (CNVs) are structural genetic mutations consisting of segmental gains or losses in DNA sequence. Although CNVs contribute substantially to genomic variation, few genetic and imaging studies report association of CNVs with alcohol dependence (AD). Our purpose is to find evidence of this association across ethnic populations and genders. This work is the first AD-CNV study across ethnic groups and the first to include the African American (AA) population. This study considers 2 CNV data sets, one for discovery (2,345 samples) and the other for validation (239 samples), both including subjects with AD and healthy controls of European and African ancestry. Our analysis assesses the association between AD and CNV losses across ethnic groups and gender by examining the effect of overall losses across the whole genome, collective losses within individual cytogenetic bands, and specific losses in CNV regions. Results from the discovery data set showed an association between CNV losses within 16q12.2 and AD diagnosis (p = 4.53 × 10(-3) ). An overlapping CNV region from the validation data set exhibited the same direction of effect with respect to AD (p = 0.051). This CNV region affects the genes CES1p1 and CES1, which are members of the carboxylesterase (CES) family. The enzyme encoded by CES1 is a major liver enzyme that typically catalyzes the decomposition of ester into alcohol and carboxylic acid and is involved in drug or xenobiotics, fatty acid, and cholesterol metabolisms. In addition, the most significantly associated CNV region was located at 9p21.2 (p = 1.9 × 10(-3) ) in our discovery data set. Although not observed in the validation data set, probably due to small sample size, this result might hold potential connection to AD given its connection with neuronal death. In contrast, we did not find any association between AD and the overall total losses or the collective losses within individual cytogenetic bands. Overall, our study provides

Levels of insecticide resistance to deltamethrin, malathion, and temephos, and associated mechanisms in Aedes aegypti mosquitoes from the Guadeloupe and Saint Martin islands (French West Indies).

PubMed

Goindin, Daniella; Delannay, Christelle; Gelasse, Andric; Ramdini, Cédric; Gaude, Thierry; Faucon, Frédéric; David, Jean-Philippe; Gustave, Joël; Vega-Rua, Anubis; Fouque, Florence

2017-02-10

In the Guadeloupe and Saint Martin islands, Aedes aegypti mosquitoes are the only recognized vectors of dengue, chikungunya, and Zika viruses. For around 40 years, malathion was used as a mosquito adulticide and temephos as a larvicide. Since the European Union banned the use of these two insecticide molecules in the first decade of the 21st century, deltamethrin and Bacillus thuringiensis var. israelensis are the remaining adulticide and larvicide, respectively, used in Guadeloupe. In order to improve the management of vector control activities in Guadeloupe and Saint Martin, we investigated Ae. aegypti resistance to and mechanisms associated with deltamethrin, malathion, and temephos. Ae. aegypti mosquitoes were collected from six different localities of Guadeloupe and Saint Martin. Larvae were used for malathion and temephos bioassays, and adult mosquitoes for deltamethrin bioassays, following World Health Organization recommendations. Knockdown resistance (Kdr) genotyping for V1016I and F1534C mutations, and expression levels of eight enzymes involved in detoxification mechanisms were examined in comparison with the susceptible reference Bora Bora strain. Resistance ratios (RR 50 ) calculated for Ae. aegypti larvae showed high resistance levels to temephos (from 8.9 to 33.1-fold) and low resistance levels to malathion (from 1.7 to 4.4-fold). Adult females displayed moderate resistance levels to deltamethrin regarding the time necessary to affect 50% of individuals, varying from 8.0 to 28.1-fold. Molecular investigations on adult mosquitoes showed high resistant allele frequencies for V1016I and F1534C (from 85 to 96% and from 90 to 98%, respectively), as well as an overexpression of the glutathione S-transferase gene, GSTe2, the carboxylesterase CCEae3a, and the cytochrome genes 014614, CYP6BB2, CYP6M11, and CYP9J23. Ae. aegypti populations from Guadeloupe and Saint Martin exhibit multiple resistance to organophosphates (temephos and malathion), and

Comparison of human and guinea pig acetylcholinesterase sequences and rates of oxime-assisted reactivation.

PubMed

Cadieux, C Linn; Broomfield, Clarence A; Kirkpatrick, Melanie G; Kazanski, Meghan E; Lenz, David E; Cerasoli, Douglas M

2010-09-06

Poisoning via organophosphorus (OP) nerve agents occurs when the OP binds and inhibits the enzyme acetylcholinesterase (AChE). This enzyme is responsible for the metabolism of the neurotransmitter acetylcholine (ACh) which transmits signals between nerves and several key somatic regions. When AChE is inhibited, the signal initiated by ACh is not properly terminated. Excessive levels of ACh result in a cholinergic crisis, and in severe cases can lead to death. Current treatments for OP poisoning involve the administration of atropine, which blocks ACh receptors, and oximes, which reactivate AChE after inhibition. Efforts to improve the safety, efficacy, and broad spectrum utility of these treatments are ongoing and usually require the use of appropriate animal model systems. For OP poisoning, the guinea pig (Cavia porcellus) is a commonly used animal model because guinea pigs more closely mirror primate susceptibility to OP poisoning than do other animals such as rats and mice. This is most likely because among rodents and other small mammals, guinea pigs have a very low relative concentration of serum carboxylesterase, an enzyme known to bind OPs in vitro and to act as an endogenous bioscavenger in vivo. Although guinea pigs historically have been used to test OP poisoning therapies, it has been found recently that guinea pig AChE is substantially more resistant to oxime-mediated reactivation than human AChE. To examine the molecular basis for this difference, we reverse transcribed mRNA encoding guinea pig AChE, amplified the resulting cDNA, and sequenced this product. The nucleotide and deduced amino acid sequences of guinea pig AChE were then compared to the human version. Several amino acid differences were noted, and the predicted locations of these differences were mapped onto a structural model of human AChE. To examine directly how these differences affect oxime-mediated reactivation of AChE after inhibition by OPs, human and guinea pig red blood cell

Metabolic inactivation of 2-oxiranylmethyl 2-ethyl-2,5-dimethylhexanoate (C10GE) in skin, lung and liver of human, rat and mouse.

PubMed

Boogaard, P J; van Elburg, P A; de Kloe, K P; Watson, W P; van Sittert, N J

1999-10-01

The inactivation of 2-oxiranylmethyl 2-ethyl-2,5-dimethylhexanoate (C10GE), one of the most abundant isomers of the epoxy-resin Carduras E-10 glycidyl ester, was studied in subcellular fractions of human, C3H mouse and F344 rat liver, lung and skin. C10GE is chemically very stable and resistant to aqueous hydrolysis, but it was rapidly metabolized in both cytosolic and microsomal fractions of all organs by epoxide hydrolase (EH)-catalysed hydrolysis of the epoxide moiety as well as carboxylesterase (CE)-catalysed hydrolysis of the ester bond. In cytosol the epoxide group was also efficiently conjugated with glutathione, catalysed by glutathione S-transferase (GST), but this conjugation was much less important than hydrolysis in human as well as rodent samples. Although CE-catalysed hydrolysis of C10GE would theoretically give rise to the formation of glycidol, a directly acting mutagen, it is highly unlikely that any significant level of glycidol would occur in vivo since reported rates of inactivation of glycidol exceed the total rate of hydrolysis of C10GE. The overall rates of inactivation in vitro decreased in the following order: mouse > rat > human. Scaling of the data in vitro to clearances in vivo suggests that the detoxifying capacity in the rodents is similar and about an order of magnitude greater than in human. Nevertheless, the rate of inactivation is 2-3 orders of magnitude greater than for simple epoxides such as butadiene monoxide and about one order of magnitude higher than for the diglycidyl ether of bisphenol A (BADGE). The transdermal penetration and metabolism of [14C]-C10GE was studied in fresh full-thickness mouse, and dermatomized human and rat skin. Of the total radioactivity applied on the skin, only 0.24+/-0.06 (SD), 1.8+/-0.2 and 6.8+/-0.6% penetrated through human, mouse and rat skin respectively. The corresponding apparent skin permeability constants were 0.81, 6.42 and 26.4 x 10(-6) cm/h. During transdermal penetration, [14C]-C10GE

Functional characterization of the Hyles euphorbiae hawkmoth transcriptome reveals strong expression of phorbol ester detoxification and seasonal cold hardiness genes.

PubMed

Barth, M Benjamin; Buchwalder, Katja; Kawahara, Akito Y; Zhou, Xin; Liu, Shanlin; Krezdorn, Nicolas; Rotter, Björn; Horres, Ralf; Hundsdoerfer, Anna K

2018-01-01

The European spurge hawkmoth, Hyles euphorbiae (Lepidoptera, Sphingidae), has been intensively studied as a model organism for insect chemical ecology, cold hardiness and evolution of species delineation. To understand species isolation mechanisms at a molecular level, this study aims at determining genetic factors underlying two adaptive ecological trait candidates, phorbol ester (TPA) detoxification and seasonal cold acclimation. A draft transcriptome of H. euphorbiae was generated using Illumina sequencing, providing the first genomic resource for the hawkmoth subfamily Macroglossinae. RNA expression levels in tissues of experimental TPA feeding larvae and cooled pupae was compared to levels in control larvae and pupae using 26 bp RNA sequence tag libraries (DeepSuperSAGE). Differential gene expression was assessed by homology searches of the tags in the transcriptome. In total, 389 and 605 differentially expressed transcripts for detoxification and cold hardiness, respectively, could be identified and annotated with proteins. The majority (22 of 28) of differentially expressed detox transcripts of the four 'drug metabolism' enzyme groups (cytochrome P450 (CYP), carboxylesterases (CES), glutathione S-transferases (GST) and lipases) are up-regulated. Triacylglycerol lipase was significantly over proportionally annotated among up-regulated detox transcripts. We record several up-regulated lipases, GSTe2, two CESs, CYP9A21, CYP6BD6 and CYP9A17 as candidate genes for further H. euphorbiae TPA detoxification analyses. Differential gene expression of the cold acclimation treatment is marked by metabolic depression with enriched Gene Ontology terms among down-regulated transcripts almost exclusively comprising metabolism, aerobic respiration and dissimilative functions. Down-regulated transcripts include energy expensive respiratory proteins like NADH dehydrogenase, cytochrome oxidase and ATP synthase. Gene expression patterns show shifts in carbohydrate

Enzymes mediating resistance to lambda-cyhalothrin in Eriopis connexa (Coleoptera: Coccinellidae).

PubMed

Rodrigues, Agna R S; Siqueira, Herbert A A; Torres, Jorge B

2014-03-01

demonstrated strong inhibition of type B carboxylesterase associated with insecticide resistance, the results reported here do not rule out metabolism by cytochrome P450-dependent microsomal monooxygenases as a factor conferring E. connexa resistance to lambda-cyhalothrin and confirmed that PBO may also act by inhibiting esterases of insects. Copyright © 2014 Elsevier Inc. All rights reserved.

Binary mixtures of azinphos-methyl oxon and chlorpyrifos oxon produce in vitro synergistic cholinesterase inhibition in Planorbarius corneus.

PubMed

Cacciatore, Luis Claudio; Kristoff, Gisela; Verrengia Guerrero, Noemí R; Cochón, Adriana C

2012-07-01

In this study, the cholinesterase (ChE) and carboxylesterase (CES) activities present in whole organism homogenates from Planorbarius corneus and their in vitro sensitivity to organophosphorous (OP) pesticides were studied. Firstly, a characterization of ChE and CES activities using different substrates and selective inhibitors was performed. Secondly, the effects of azinphos-methyl oxon (AZM-oxon) and chlorpyrifos oxon (CPF-oxon), the active oxygen analogs of the OP insecticides AZM and CPF, on ChE and CES activities were evaluated. Finally, it was analyzed whether binary mixtures of the pesticide oxons cause additive, antagonistic or synergistic ChE inhibition in P. corneus homogenates. The results showed that the extracts of P. corneus preferentially hydrolyzed acetylthiocholine (AcSCh) over propionylthiocholine (PrSCh) and butyrylthiocholine (BuSCh). Besides, AcSCh hydrolyzing activity was inhibited by low concentrations of BW284c51, a selective inhibitor of AChE activity, and also by high concentrations of substrate. These facts suggest the presence of a typical AChE activity in this species. However, the different dose-response curves observed with BW284c51 when using PrSCh or BuSCh instead of AcSCh suggest the presence of at least another ChE activity. This would probably correspond to an atypical BuChE. Regarding CES activity, the highest specific activity was obtained when using 2-naphthyl acetate (2-NA), followed by 1-naphthyl acetate (1-NA); p-nitrophenyl acetate (p-NPA), and p-nitrophenyl butyrate (p-NPB). The comparison of the IC(50) values revealed that, regardless of the substrate used, CES activity was approximately one order of magnitude more sensitive to AZM-oxon than ChE activity. Although ChE activity was very sensitive to CPF-oxon, CES activity measured with 1-NA, 2-NA, and p-NPA was poorly inhibited by this pesticide. In contrast, CES activity measured with p-NPB was equally sensitive to CPF-oxon than ChE activity. Several specific binary

Effect of surfactant in mitigating cadmium oxide nanoparticle toxicity: Implications for mitigating cadmium toxicity in environment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Balmuri, Sricharani Rao

Cadmium (Cd), classified as human carcinogen, is an extremely toxic heavy metal pollutant, and there is an increasing environmental concern for cadmium exposure through anthropogenic sources including cigarette smoke. Though Cd based nanoparticles such as cadmium oxide (CdO) are being widely used in a variety of clinical and industrial applications, the toxicity of CdO nanoparticles has not been well characterized. Herein we report the toxicity of CdO nanoparticles employing zebrafish as a model. Two different CdO nanoparticles were prepared, calcination of Cd(OH){sub 2} without any organic molecule (CdO-1) and calcination of Cd-citrate coordination polymer (CdO-2), to evaluate and compare themore » toxicity of these two different CdO nanoparticles. Results show that zebrafish exposed to CdO-2 nanoparticles expressed reduced toxicity as judged by lower oxidative stress levels, rescue of liver carboxylesterases and reduction in metallothionein activity compared to CdO-1 nanoparticles. Histopathological observations also support our contention that CdO-1 nanoparticles showed higher toxicity relative to CdO-2 nanoparticles. The organic unit of Cd-citrate coordination polymer might have converted into carbon during calcination that might have covered the surface of CdO nanoparticles. This carbon surface coverage can control the release of Cd{sup 2+} ions in CdO-2 compared to non-covered CdO-1 nanoparticles and hence mitigate the toxicity in the case of CdO-2. This was supported by atomic absorption spectrophotometer analyses of Cd{sup 2+} ions release from CdO-1 and CdO-2 nanoparticles. Thus the present study clearly demonstrates the toxicity of CdO nanoparticles in an aquatic animal and also indicates that the toxicity could be substantially reduced by carbon coverage. This could have important implications in terms of anthropogenic release and environmental pollution caused by Cd and human exposure to Cd{sup 2+} from sources such as cigarette smoke. - Highlights: �

Parallel metatranscriptome analyses of host and symbiont gene expression in the gut of the termite Reticulitermes flavipes

PubMed Central

Tartar, Aurélien; Wheeler, Marsha M; Zhou, Xuguo; Coy, Monique R; Boucias, Drion G; Scharf, Michael E

2009-01-01

Background Termite lignocellulose digestion is achieved through a collaboration of host plus prokaryotic and eukaryotic symbionts. In the present work, we took a combined host and symbiont metatranscriptomic approach for investigating the digestive contributions of host and symbiont in the lower termite Reticulitermes flavipes. Our approach consisted of parallel high-throughput sequencing from (i) a host gut cDNA library and (ii) a hindgut symbiont cDNA library. Subsequently, we undertook functional analyses of newly identified phenoloxidases with potential importance as pretreatment enzymes in industrial lignocellulose processing. Results Over 10,000 expressed sequence tags (ESTs) were sequenced from the 2 libraries that aligned into 6,555 putative transcripts, including 171 putative lignocellulase genes. Sequence analyses provided insights in two areas. First, a non-overlapping complement of host and symbiont (prokaryotic plus protist) glycohydrolase gene families known to participate in cellulose, hemicellulose, alpha carbohydrate, and chitin degradation were identified. Of these, cellulases are contributed by host plus symbiont genomes, whereas hemicellulases are contributed exclusively by symbiont genomes. Second, a diverse complement of previously unknown genes that encode proteins with homology to lignase, antioxidant, and detoxification enzymes were identified exclusively from the host library (laccase, catalase, peroxidase, superoxide dismutase, carboxylesterase, cytochrome P450). Subsequently, functional analyses of phenoloxidase activity provided results that were strongly consistent with patterns of laccase gene expression. In particular, phenoloxidase activity and laccase gene expression are mostly restricted to symbiont-free foregut plus salivary gland tissues, and phenoloxidase activity is inducible by lignin feeding. Conclusion To our knowledge, this is the first time that a dual host-symbiont transcriptome sequencing effort has been conducted in a

Rhodnius prolixus supergene families of enzymes potentially associated with insecticide resistance.

PubMed

Schama, Renata; Pedrini, Nicolás; Juárez, M Patricia; Nelson, David R; Torres, André Q; Valle, Denise; Mesquita, Rafael D

2016-02-01

Chagas disease or American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite, Trypanosoma cruzi. Once known as an endemic health problem of poor rural populations in Latin American countries, it has now spread worldwide. The parasite is transmitted by triatomine bugs, of which Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae) is one of the vectors and a model organism. This species occurs mainly in Central and South American countries where the disease is endemic. Disease prevention focuses on vector control programs that, in general, rely intensely on insecticide use. However, the massive use of chemical insecticides can lead to resistance. One of the major mechanisms is known as metabolic resistance that is associated with an increase in the expression or activity of detoxification genes. Three of the enzyme families that are involved in this process - carboxylesterases (CCE), glutathione s-transferases (GST) and cytochrome P450s (CYP) - are analyzed in the R. prolixus genome. A similar set of detoxification genes to those of the Hemipteran Acyrthosiphon pisum but smaller than in most dipteran species was found in R. prolixus genome. All major CCE classes (43 genes found) are present but the pheromone/hormone processing class had fewer genes than usual. One main expansion was detected on the detoxification/dietary class. The phosphotriesterase family, recently associated with insecticide resistance, was also represented with one gene. One microsomal GST gene was found and the cytosolic GST gene count (14 genes) is extremely low when compared to the other hemipteran species with sequenced genomes. However, this is similar to Apis mellifera, a species known for its deficit in detoxification genes. In R. prolixus 88 CYP genes were found, with representatives in the four clans (CYP2, CYP3, CYP4 and mitochondrial) usually found in insects. R. prolixus seems to have smaller species-specific expansions of CYP genes than

Plasma B-esterase activities in European raptors.

PubMed

Roy, Claudie; Grolleau, Gérard; Chamoulaud, Serge; Rivière, Jean-Louis

2005-01-01

B-esterases are serine hydrolases composed of cholinesterases, including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and carboxylesterase (CbE). These esterases, found in blood plasma, are inhibited by organophosphorus (OP) and carbamate (CB) insecticides and can be used as nondestructive biomarkers of exposure to anticholinesterase insecticides. Furthermore, B-esterases are involved in detoxification of these insecticides. In order to establish the level of these enzymes and to have reference values for their normal activities, total plasma cholinesterase (ChE), AChE and BChE activities, and plasma CbE activity were determined in 729 European raptors representing 20 species, four families, and two orders. The diurnal families of the Falconiforme order were represented by Accipitridae and Falconidae and the nocturnal families of the Strigiforme order by Tytonidae and Strigidae. Intraspecies differences in cholinesterase activities according to sex and/or age were investigated in buzzards (Buteo buteo), sparrowhawks (Accipiter nisus), kestrels (Falco tinnunculus), barn owls (Tyto alba), and tawny owls (Strix aluco). Sex-related differences affecting ChE and AChE activities were observed in young kestrels (2-3-mo-old) and age-related differences in kestrels (ChE and AChE), sparrowhawks (AChE), and tawny owls (ChE, AChE, and BChE). The interspecies analysis yielded a negative correlation between ChE activity and body mass taking into account the relative contribution of AChE and BChE to ChE activity, with the exception of the honey buzzard (Pernis apivorus). The lowest ChE activities were found in the two largest species, Bonelli's eagle (Hieraaetus fasciatus) and Egyptian vulture (Neophron percnopterus) belonging to the Accipitridae family. The highest ChE activities were found in the relatively small species belonging to the Tytonidae and Strigidae families and in honey buzzard of the Accipitridae family. Species of the Accipitridae, Tytonidae, and

Blood cholinesterases as human biomarkers of organophosphorus pesticide exposure.

PubMed

Nigg, H N; Knaak, J B

2000-01-01

phosphorothioates and phosphorodithioates (e.g., parathion and azinphosmethyl, respectively) to the more toxic oxons (P = O(S to O)). In some cases, P-450 isozymes catalyze the oxidative cleavage of P-O-aryl bonds (e.g., parathion, methyl parathion, fenitrothion, and diazinon) to form inactive water-soluble alkyl phosphates and aryl leaving groups that are readily conjugated with glucuronic or sulfuric acids and excreted. In addition to the P-450 isozymes, mammalian tissues contain ('A' and 'B') esterases capable of reacting with OPs to produce hydrolysis products or phosphorylated enzymes. 'A'-esterases hydrolyze OPs (i.e., oxons), while 'B'-esterases with serine at the active center are inhibited by OPs. OPs possessing carboxylesters, such as malathion and isofenphos, are hydrolyzed by the direct action of 'B'-esterases (i.e., carboxylesterase, CaE). Metabolic pathways shown for isofenphos, parathion, and malathion define the order in which these reactions occur, while Michaelis-Menten kinetics define reaction parameters (Vmax, K(m)) for the enzymes and substrates involved, and rates of inhibition of 'B'-esterases (kis, bimolecular rate constants) by OPs and their oxons. OPs exert their insecticidal action by their ability to inhibit AChE at the cholinergic synapse, resulting in the accumulation of acetylcholine. The extent to which AChE or other 'B'-esterases are inhibited in workers is dependent upon the rate the OP pesticide is activated (i.e., oxon formation), metabolized to nontoxic products by tissue enzymes, its affinity for AChE and other 'B'-esterases, and esterase concentrations in tissues. Rapid recovery of OP BChE inhibition may be related to reactivation of inhibited forms. AChE, BChE, and CaE appear to function in vivo as scavengers, protecting workers against the inhibition of AChE at synapses. Species sensitivity to OPs varies widely and results in part from binding affinities (Ka) and rates of phosphorylation (kp) rather than rates of activation and detoxif

Final amended report on the safety assessment of Methylparaben, Ethylparaben, Propylparaben, Isopropylparaben, Butylparaben, Isobutylparaben, and Benzylparaben as used in cosmetic products.

PubMed

2008-01-01

Parabens is the name given to a group of p-hydroxybenzoic acid (PHBA) esters used in over 22,000 cosmetics as preservatives at concentrations up to 0.8% (mixtures of parabens) or up to 0.4% (single paraben). The group includes Methylparaben, Ethylparaben, Propylparaben, Isopropylparaben, Butylparaben, Isobutylparaben, and Benzylparaben. Industry estimates of the daily use of cosmetic products that may contain parabens were 17.76 g for adults and 378 mg for infants. Parabens in cosmetic formulations applied to skin penetrate the stratum corneum in inverse relation to the ester chain length. Carboxylesterases hydrolyze parabens in the skin. Parabens do not accumulate in the body. Serum concentrations of parabens, even after intravenous administration, quickly decline and remain low. Acute toxicity studies in animals indicate that parabens are not significantly toxic by various routes of administration. Subchronic and chronic oral studies indicate that parabens are practically nontoxic. Numerous genotoxicity studies, including Ames testing, dominant lethal assay, host-mediated assay, and cytogenic assays, indicate that the Parabens are generally nonmutagenic, although Ethylparaben and Methylparaben did increase chromosomal aberrations in a Chinese Hamster ovary cell assay. Ethylparaben, Propylparaben, and Butylparaben in the diet produced cell proliferation in the forestomach of rats, with the activity directly related to chain length of the alkyl chain, but Isobutylparaben and Butylparaben were noncarcinogenic in a mouse chronic feeding study. Methylparaben was noncarcinogenic when injected subcutaneously in mice or rats, or when administered intravaginally in rats, and was not cocarcinogenic when injected subcutaneously in mice. Propylparaben was noncarcinogenic in a study of transplacental carcinogenesis. Methylparaben was nonteratogenic in rabbits, rats, mice, and hamsters, and Ethylparaben was nonteratogenic in rats. Parabens, even at levels that produce maternal