Hydrolysis Of Α-naphthyl Acetate Research Articles

Combined detoxification mechanisms and target mutation fail to confer a high level of resistance to organophosphates in Cydia pomonella (L.) (Lepidoptera: Tortricidae)

Despite the frequent and widespread applications of organophosphates against Cydia pomonella this species has developed low levels of resistance to this chemical group. Investigations concerning the mechanisms involved in resistance are scarce, and usually consider only one of the potential mechanisms. With the aim of a better understanding the resistance mechanisms and their possible interaction, four of these mechanisms were investigated simultaneously in one sensitive (Sv) and two resistant strains (Raz and Rdfb) of this insect. Resistant strains displayed an increased mixed function oxidase activity, whereas carboxylesterase activity varied upon the substrate used. The three strains had similar β-naphtyl acetate activity, and the hydrolysis of α-naphthyl acetate and p-nitrophenyl valerate was higher in the Sv strain. The p-nitrophenyl acetate activity was highest in the resistant strains and was strongly inhibited by azinphos and DEF. The Raz strain has a modified acetylcholinesterase (AChE), which resulted in a 0.7-, 3.2- and 21.2-fold decrease in the susceptibility to chlorpyriphos-ethyl-oxon, azinphos-methyl-oxon, and paraoxon-methyl, respectively. These combined resistance mechanisms only conferred to Raz a 0.6-, 7.9- and 3.1-fold resistance to the related insecticides. Organophosphates resistance in C. pomonella results from a combination of mechanisms including modified affinities to carboxylesterase substrates, and increased metabolisation of the insecticide. The apparent antagonism between increased functionalisation and reduced sensitivity of the AChE target is discussed.

HPMC effect on catalyzed hydrolysis of α-naphthyl acetate in cationic micellar systems

Micelle formation is an important property of surfactants. Micellar solutions of cetyltrimethylamonium bromide (CTAB) have been found to accelerate the hydrolysis of α-naphthyl acetate (α-NA) by o-iodosobenzoic acid (IBA), a strong nucleophile. Critical micelle concentration (CMC) of CTAB was determined in the absence and presence of various concentrations and grades of hydroxypropylmethyl cellulose (HPMC) using surface tension measurement. Reaction under pseudo-first-order condition was utilized for rate constant determination. The aggregation of CTAB in the polymer solution started at higher concentration than in the absence of the polymer. The observed rate constants for the degradation of α-NA in the presence of various concentrations of CTAB with and without HPMC were calculated. The presence of HPMC retarded the reaction rate. However, there was no obvious difference in the observed rate constant among the different grades of HPMC (Methocel E50 ®, Methocel E4M ®, Methocel E10M ®) while the higher the polymer concentration the lower the rate constant.

Effects of Cypermethrin Selection on Expression of Insecticide Resistance Mechanisms in the German Cockroach (Blattaria: Blattellidae)

A cross-resistant strain of German cockroach, Blattella germanica (L.), (Marietta) expressing multiple resistance mechanisms was subjected to selection pressure with cypermethrin. Resistance to cypermethrin increased incrementally from 3.6-fold in the parental strain to 35-fold after 4 rounds of selection. No significant changes were observed in cytochrome P450 content, aldrin epoxidase activity, α-naphthyl acetate hydrolysis (cytosolic fraction), or glutathione S-transferase (CDNB conjugation) activities. Although no significant differences were observed in cypermethrin metabolism, a trend toward greater detoxification among each successive generation was observed. Furthermore, methoxyresorufin O-demethylase activity (often associated with pyrethroid resistance) increased incrementally with each successive selection iteration. The average knockdown time increased incrementally from 37 min (parental strain) to 177 min, and there was a corresponding increase in the kdr allele frequency from 19% to 99% after 4 rounds of selection. The data indicated that kdr-type nerve insensitivity and enhanced metabolism by cytochromes P450 and hydrolases were the principle mechanisms of resistance after selection with cypermethrin.

Esterase variation in Tribolium confusum (Coleoptera: Tenebrionidae): genetic analysis of interstrain crosses in relation to malathion resistance

Abstract General esterase activity variation among individuals and strains of Tribolium confusum (du Val) is mostly due to a single electrophoretic locus. A strain (CFxlH) selected for very strongly staining isozyme (EST2), was reciprocally crossed with the black mutant laboratory strain (CFbb) which has very weak EST2. EST2 is not linked to the black locus and is inherited as a monogenic, autosomal trait. Similar results were obtained when CFxlH was reciprocally crossed with the sister strain (CFxlL) selected for low activity. Slight deviations from Mendelian expectation in the latter crosses suggest that maternally inherited effects modify EST2 activity in these strains. CFbb and CFxlL both have low EST2 activity, and when intercrossed, all offspring had the EST2L pattern, suggesting that the EST2 loci in the two strains are the same or very tightly linked. Malathion resistance in one strain (MN61) seems to be inherited in a monofactorial fashion, with dominance of the resistant allele. MN61 is fixed for the high-activity EST2 phenotype. Addition of triphenyl phosphate (TPP) synergised the effects of the pesticide, indicating that malathion resistance is due to carboxylesterase activity. CFxlH and CFxlL had similar malathion resistance despite the dramatic difference in EST2 activity: resistance is not due to EST2, the major contributor to the hydrolysis of α-naphthyl acetate (and probably other naphthyl esters).

Malathion-specific resistance in a strain of the rust red grain beetle Cryptolestes ferrugineus (Coleoptera: Cucujidae)

AbstractA strain of Cryptolestes ferrugineus (Stephens) bred for malathion-specific resistance was found to be 650 fold resistant at LD50 when compared with a susceptible strain bred from the same stock. Resistance was more than 98% synergized by triphenyl phosphate and S,S,S-tributyl phosphorotrithioate, but unaffected by piperonyl butoxide. AChE inhibition by malaoxon varied slightly between the strains. Non-specific esterase activity as measured by the hydrolysis of α-naphthyl acetate was slightly reduced in the resistant strain whereas there were no inter-strain differences in the hydrolysis of β-naphthyl acetate. Products of in vitro metabolism of malathion were identified by thin-layer chromatography and gas chromatography-mass spectrometry as α- and β-malathion mono-acids. It was therefore concluded that resistance was due to the hydrolytic breakdown of malathion by a malathion-specific carboxylesterase. The rate of in vitro malathion hydrolysis was found to be 31 times greater in the resistant strain. In vitro inhibition studies indicated that resistance is attributable to a carboxylesterase unique to the resistant strain. The implications of these results are discussed in relation to work recently carried out on malathion-specific resistance in dipterous species.

Regulation of mouse liver microsomal esterases by clofibrate and sexual hormones.

Carboxylesterase activity was measured using six different substrates in microsomal preparations from female and ovariectomized female mice in order to evaluate the effects of female sex hormones on esterase expression. With three of the substrates (alpha-naphthyl acetate and esters 2 and 3), esterase activity was the same in both groups; however, with the others (rho-nitrophenyl acetate and esters 1 and 4), there was a small increase in activity in ovariectomized females, compared with intact females. Castration of males followed by treatment with testosterone caused only transient increases in activity for four of the substrates (alpha-naphthyl acetate and esters 1, 2, and 3) and no change in activity for the other two (rho-nitrophenyl acetate and ester 4). Treatment of male and female mice with the peroxisome proliferator clofibrate, with or without testosterone, resulted in increased hydrolysis of alpha-naphthyl acetate and rho-nitrophenyl acetate, but little change for the other substrates. Clofibrate also induced alpha-naphthyl acetate and rho-nitrophenyl acetate hydrolysis in castrated males, but clofibrate and testosterone administrated together resulted in significant increases of activity with all substrates, which were greater than the additive effects of the two compounds administered separately. These results indicate that clofibrate causes significant alterations in the regulation of esterase activity, whereas sex hormones only cause small changes. However, it would seem that testosterone can synergize the effect of clofibrate in castrated males, resulting in higher levels of activity than with clofibrate alone. Finally, an overall increase in esterase activity might be due to a large increase in the activity of a few esterases or to a small increase in many esterases. Enzyme staining of native polyacrylamide gels reveals that the latter is true, with the majority of esterases present in mouse liver microsomes being induced to a small degree by clofibrate.

Catalysis of α-naphthyl acetate hydrolysis by o-iodosobenzoic acid in micellar solutions of cetyltrimethylammonium bromide

Hydrolysis of α-naphthyl acetate proceeds at a very slow rate in solutions near neutral pH. This reaction was found to be accelerated when carried out in surfactant solutions of cetyltrimethylammonium bromide which also contained o-iodosobenzoic acid, a strong nucleophile. The reaction followed pseudo-first-order kinetics and the rate constant versus surfactant concentration profile exhibited the maximum typical of bimolecular reactions conducted in micellar solutions. The calculated entropy of activation for the reaction supported a bimolecular mechanism of hydrolysis. The binding constant of α-naphthyl acetate for the micelle and the rate constant in the micellar pseudophase were determined from kinetic data using the pseudophase model. The influences of added salt concentration, solution pH and nucleophile concentration were evaluated.

Elevated esterase activity in resistant tufted apple bud moth, Platynota idaeusalis (Walker) (Lepidoptera: Tortricidae)

1. Esterase activity in the tufted apple bud moth was measured spectrophotometrically by the hydrolysis of α-naphthyl acetate. 2. Resistant populations from both laboratory and field exhibited significantly greater esterase activity than did the respective susceptible populations. 3. The resistant laboratory population had significantly higher esterase activity than the resistant field population, suggesting that this field population contained a mixture of resistant and susceptible individuals.

Correlation of azinphosmethyl resistance with detoxication enzyme activity in the light brown apple moth Epiphyas postvittana (Lepidoptera: Tortricidae)

Bioassay using diet incorporation of azinphosmethyl showed six strains of light brown apple moth to have varying levels of resistance to azinphosmethyl (3- to 15-fold) compared with three susceptible strains. Two of these strains were products of a selection and backcrossing program providing resistant insects that had a common genome with that of one of the susceptible strains. Individual male moths from each strain were tested for (i) glutathione S-transferase and (ii) non-specific esterase activity. Larvae were assayed for (iii) mixed function oxidase activity. Each enzyme group was assayed with two substrates. Significant positive regressions between LC 50 and enzyme activity were observed for all three groups: (i) r 2 = 0.84 for 3,4-dichloronitrobenzene conjugation, (ii) 0.64 for α-naphthyl acetate hydrolysis, 0.54 for α-naphthyl butyrate hydrolysis, and (iii) 0.88 for p-nitroanisole oxidation and 0.86 for aniline oxidation. No correlation with the LC 50 was found for 1-chloro-2,4-dinitrobenzene conjugation ( r 2 = 0.001). Activities of all three groups were significantly higher in the two backcross resistant strains compared to the susceptible parent strain ( P < 0.05). The gene(s) for high enzyme activity had thus been successfully transferred with the factor for resistance. The results suggest a close relationship between detoxication enzyme activity and azinphosmethyl resistance.

Flow-injection determination of paraoxon by inhibition of immobilized acetylcholinesterase

Two flow-injection methods (continuous-flow and stopped-flow) are proposed for the determination of paraoxon, applying the dual-injection technique and spectrophotometric detection. They are based on the inhibition of the acetylcholinesterase-catalysed hydrolysis of α-naphthyl acetate and subsequent reaction of the α-naphthol produced with p-nitrobenzenediazonium fluoroborate. For the continuous-flow system the calibration graph was linear from 5 × 10 −7 to 1.5 × 10 −5 M, the relative standard deviation (r.s.d.) ( n=6) for an 8 × 10 −6 M standard was 1.4%, the limit of detection (3σ) was 4 × 10 −7 M and the sample throughput was ca. 60 h −1. For the stopped-flow system the linear range was from 1 × 10 −8 to 4 × 10 −7 M, the r.s.d. for a 2.5 × 10 −7 M standard was 0.9%, the limit of detection was 8 × 10 −9 M and the sample throughput was 30 h −1.

Developmental changes in trans-permethrin and α-naphthyl acetate ester hydrolysis during the last larval instar of Pseudoplusia includens

The hydrolysis of trans-permethrin and α-naphthyl acatate by midgut, fat body, and cuticle homogenates from Pseudoplusia includens (Walker) was monitored during the development of the last instar. The midgut homogenates appeared to have two pH optima (7.6 and 8.6) for the hydrolysis of trans-permethrin, the fat body homogenates had one optimum (7.4–7.8), and the cuticle homogenates had a major optimum at 6.6. Hydrolysis of both substrates peaked during the late feeding stages for midgut and cuticle homogenates, although relative changes were not the same. Hydrolysis of trans-permethrin peaked during the late feeding stage in fat body homogenates, while hydrolysis of α-naphthyl acetate continually increased through the prepupal stage. Thus, the hydrolysis of α-naphthyl acetate is not necessarily associated with the hydrolysis of trans-permethrin. The LD 50 values for trans-permethrin on the different stages appeared to reflect the influence of hydrolysis.

Comparative inhibition of the juvenile hormone esterases from Trichoplusia ni, Tenebrio molitor, and Musca domestica

Twenty-seven compounds were screened as potential inhibitors of juvenile hormone esterases. Of these compounds O-ethyl- S-phenyl phosphoramidothiolate provided the best inhibition for the cabbage looper, Trichoplusia ni (Hubner), and the yellow mealworm, Tenebrio molitor L., while the juvenile hormone esterases of the house fly, Musca domestica L., were best inhibited by a juvenoid carbamate (1-( m-phenoxy- N-ethyl carbamate)-3,7-dimethyl-7-methoxy-2 E-octene). The inhibition patterns of T. ni and T. molitor are similar, while those of M. domestica are relatively different. Further studies on the juvenile hormone and α-napthyl acetate esterases of T. ni showed that they could be differentially inhibited. Diisopropyl phosphorofluoridate and an alkyl trifluoromethyl ketone selectively inhibit the hydrolysis of α-naphthyl acetate and juvenile hormone, respectively, while O-ethyl- S-phenyl phosporamidothiolate inhibits both enzymes. The juvenile hormone esterases of T. ni also appear to be unique enzymes that are selective for juvenile-hormone-like molecules. The in vivo inhibition of T. ni juvenile hormone esterases by O-ethyl- S-phenyl phosphoramidothiolate slows the in vivo hydrolysis of juvenile hormone and results in delayed pupation and malformed larvae that resemble larval-pupal intermediates. Thus, the esterases involved in juvenile hormone metabolism appear to be important in juvenile hormone regulation.

Effects of ovarian steroid hormones on esterases in the rat endometrium.

A technique in which uterine luminal epithelium is separated from the remainder of the endometrium by rapidly vibrating everted uterine cornua in a 3-5 mM solution of EDTA has been developed. Examination of the hormonal sensitivities and physiological roles of the tissue components of the endometrium is thus facilitated. Biochemical and histochemical studies of epithelial, stromal and endometrial esterases have shown that the rate of hydrolysis of alpha-naphthyl acetate is significantly higher in epithelial and endometrial tissue extracts during pro-oestrus than at any other stage of the oestrous cycle. In ovariectomized animals, oestradiol-17 beta caused a 60% increase in the rate of esterase activity over that of control animals, whereas medroxyprogesterone acetate had no effect. These findings suggest that the variations in the levels of neutral lipids in the uterine luminal epithelium of non-pregnant mature female rats result from the periodic stimulation of the epithelial esterases by the cyclically increased levels of plasma oestrogens.

Genetic polymorphism of plasma carboxylesterases in the rabbit: Correlation with pharmacologic and toxicologic effects

An enzyme that degrades atropine is found in varying frequency in the sera and tissues of rabbits. Its presence or absence can readily be detected by the rate of hydrolysis of α-naphthyl acetate (α-NA). The frequency distributions of the plasma enzyme(s) hydrolyzing α-NA, atropine, procaine and benzoylcholine were markedly similar, raising questions regarding the genetic inheritance and whether these esters were hydrolyzed by 1 enzyme, by a group of closely related esterases or by distinctly different proteins. Techniques including electrophoresis, titrimetric and spectrophotometric analysis of substrate specificity and inhibition sensitivity were employed to characterize the plasma enzyme in adult male and female albino New Zealand rabbits. Atropinesterase was identified as a nonspecific carboxyl-esterase, occurring with a 50% frequency in either sex. Electrophoretically, 2 distinct phenotypes could be detected though the rabbit population could be divided into 3 phenotypes on the basis of rates of α-NA hydrolysis. Inhibition studies suggested that the hydrolysis of the ester-type drugs was the function of 1 genetically determined enzyme existing in multiple forms. The importance of this enzyme in toxicologic studies was confirmed by a study of atropine-induced mydriasis and procaine-induced convulsions in animals known to possess or be deficient in this plasma carboxylesterase.

A study of the effect of ovex on parathion toxicity in rats.

Oral administration (100 mg/kg) of the miticide ovex (p-chlorophenyl p-chlorobenzene-sulfonate) caused a decrease in the hexobarbital sleeping time of rats. Administration of this same dosage of ovex resulted in a significant decrease in the toxicity of orally administered parathion (100 mg/kg) to rats.The changes in rats noted in conjunction with the increased resistance to organophosphate toxicity in vivo were an increased liver size, an increased rate of in vitro α-naphthyl acetate hydrolysis by the 9000 × g liver supernatant.The hexane-extractable organophosphate detected in the liver of the ovex-pretreated rats was significantly lower than the hexane extractable organophosphate found in the liver of the control rats.

Characterization of canine hepatic and renal esterases.

The esterases of canine liver and kidney were separated electrophoretically into nine bands with identical migration patterns in both tissues. An additional pair of rapidly migrating anodic bands were observed in hepatic extracts. Based on substrate specificity, the predominant tissue esterases were identified as nonspecific carboxylesterases (aliesterases). No cholinesterase activity was detected in the tissue extracts. Kinetic characteristics determined for the hepatic and renal esterases included (1) optimal pH; (2) Km values for esters of α-naphthyl and p-nitrophenol; (3) average rates of hydrolysis of α-naphthyl acetate and p-nitrophenyl acetate by the tissue extracts. Inhibition studies revealed the presence of two types of esterase activity in each tissue: one type being sensitive to organophosphorus esters, the second being resistant. A study of preferential substrate hydrolysis in the presence of known characteristic activators and inhibitors of esterases revealed approximately 5% and 20% arylesterase activity in liver and kidney, respectively. The presence of arylesterase activity in these tissues was confirmed by the hydrolysis of paraoxon (E600).

Characterization of the esterases of canine serum.

Various techniques including electrophoresis, gel filtration, and titrimetric and spectrophotometric analysis with specific and nonspecific substrates and inhibitors were used to characterize the serum esterases of male and female dogs. Electrophoresis in starch gel yielded 10 distinct bands of activity: four butyrylcholinesterase bands, an esterolytically active albumin, and five bands of aliesterase and/or arylesterase activity. Gel filtration on Sephadex G-200 yielded two distinct peaks of activity, one containing the cholinesterase bands, and the second peak containing both arylesterase and aliesterase activity in close association with the serum albumin. Kinetic characteristics determined for the canine serum esterases included (1) optimal pH; (2) Km values for esters of choline, α-naphthol, and p-nitrophenol; and (3) average rates of hydrolysis of α-naphthyl acetate, butyrylcholine iodide, p-nitrophenyl acetate, and E600 by the sera of males and females. The sensitivity of the serum esterases to inhibition by various cations, and specific and nonspecific inhibitors was investigated. The organophosphate, E600, may be a highly specific substrate for the detection and quantification of arylesterase in a mixture of nonspecific carboxylesterases.

Subcellular localization of rat brain esterases.

The localization and properties of soluble and bound esterases of subcellular fractions of rat brain have been investigated. Bound esterases were extracted with 1% Triton X-100 and separated by starch gel electrophoresis. By these means a molecular population of isoenzymes was demonstrable that was quantitatively different from the isoenzyme population of the watersoluble esterase activity. The highest specific activity for α-naphthyl acetate hydrolysis was contained in the microsomal fraction and could be extracted by Triton. In contradistinction to whole brain and to other subcellular fractions, microsomes contained a molecular population of esterase isozymes which was qualitatively distinct from that of water extracts in that the fast moving A-type esterases were absent. In addition, there was present a heavy concentration of slow movinig B-esterase. Acetylcholinesterase could also be extracted from this fraction by Triton, migrated with B-esterase and actively hydrolyzed αnaphthyl acetate. Combined electron microscopic and quantitative chemical analysis of the subcellular fractions suggested that some bound nonspecific esterase may be localized in sub-synaptic membranes. The pI50 values for E 600 of the soluble and insoluble, Triton X-100-extracted and Triton X-100-insoluble esterases are, respectively, 5.3, 7.5, 7.5 and 7.4. It is noted that these results may be determined in part by the participation of acetylcholinesterase in hydrolysis of the substrate. Mitochondria are virtually devoid of esteratic activity. C-type esterase (CMB-activated, E 600-resistant) occurred in both the bound and soluble state. A-, B- and C-type esterases exist in both bound and soluble forms and their chemical properties appear to be independent of their site of localization. Histochemical studies indicate that the B-type esterase is localized predominantly to cytoplasm of neurons and to neuropil. A-esterase is localized to droplets (presumed lysosomes) of neurons and other cell types, e.g., pericytes. The histochemical localization of C-esterase could not be determined.

A Kinetic Study of the Aminolysis and Hydrolysis of α-Naphthyl Acetate

A Kinetic Study of the Aminolysis and Hydrolysis of α-Naphthyl Acetate