Hydrolysis of malathion by rabbit liver oligomeric and monomeric carboxylesterases

The hydrolysis of malation by rabbit liver oligomeric and monomeric carboxylesterases (CE's) (EC 3.1.1.1) results in the formation of a mixture of α- and β-monoacids. A new chromatographic procedure was utilized to investigate the formation of α- and β-monoacids. The oligomeric carboxylesterase (oCE) produced an $\text{α}\text{β}$ ratio of monoacids of 4.55, and the monomeric carboxylesterase (mCE) produced an $\text{α}\text{β}$ ratio of monoacids of 2.33. The ratios of α- and β-monoacids were independent of the initial concentration of malathion and remained constant over the time course of the reaction. Kinetic studies demonstrated that the K_m values were the same for the corresponding reactions which produced either α-monoacid or β-monoacid with the same enzyme. Since both carboxylesterases are electrophoretically pure, the kinetic data strongly supports the theory that the reactions which produced α- and β-monoacids are catalyzed by the same active site. Comparison of the k_cat and K_m values governing the hydrolysis of malathion by the two esterases, together with their relative abundance in liver, indicated that the oCE would be responsible for about 80 to 98% of the hydrolytic detoxication of malathion by rabbit liver.     

Subcellular distribution of malathion,phenthoate, and diethylsuccinate carboxylesterases in rat lungs

The subcellular distribution of malathion, phenthoate, and diethylsuccinate carboxylesterases was determined in the lungs of male Sprague-Dawley rats and in rats administered lung toxic doses of bromobenzene and paraquat. In control with the former two substrates, the highest activity was encountered in the 6500g and 12,000g pellets. In addition, significant activity was found in the 100,000g supernatant. These fractions hydrolyzed diethylsuccinate very slowly, and the major diethylsuccinate carboxylesterase activity was recovered in the 100,000g pellet. The bromobenzene treatment had no effect on these carboxylesterases; however, the paraquat treatment significantly decreased the phenthoate and diethylsuccinate carboxylesterases in the 100,000g pellet without altering the activity in the other fractions. The present study suggested that the subcellular distribution of malathion and phenthoate carboxylesterases is different from that in liver. The present study revealed that, in the lungs, the highest total activity for malathion and phenthoate carboxylesterases was found in the 100,000g supernatant, in comparison with liver, where the 100,000g pellet contains a much higher activity of these enzymes. The decrease in specific activities of diethylsuccinate and phenthoate carboxylesterases following the treatment with a pneumotoxicant may serve as an indicator of lung damage.     

Selective inhibition of separate esterases in rat and mouse liver microsomes hydrolyzing malathion,trans-permethrin,and cis-permethrin

Separate esterase activities of rat and mouse liver microsomes hydrolyzing malathion, trans-permethrin, and cis-permethrin were differentiated on the basis of their sensitivities to inhibition by paraoxon and α-naphthyl N-propylcarbamate (NPC). In rat liver microsomes, the malathionhydrolyzing activity was more sensitive to both inhibitors and showed a different time course of NPC inhibition than the activities hydrolyzing the permethrin isomers. Paraoxon completely inhibited trans-permethrin hydrolysis, but only partially inhibited that of cis-permethrin. The paraoxonsensitive trans- and cis-permethrin-hydrolyzing activities were not differentially inhibited, but separate inhibition curves were obtained for the inhibition of trans- and cis-permethrin hydrolysis by NPC. The mouse liver esterase activity hydrolyzing trans-permethrin showed a similar paraoxon sensitivity to that of rat liver, but that the paraoxon-sensitive portion of the cis-permethrinhydrolyzing activity was 5.5-fold less sensitive to paraoxon than the corresponding rat liver activity and was clearly differentiated from the mouse liver trans-permethrin-hydrolyzing activity. The mouse liver malathion-hydrolyzing activity was 100-fold less sensitive to paraoxon and 14-fold less sensitive to NPC than the corresponding rat liver activity. Rat and mouse liver esterase activities hydrolyzed trans- and cis-permethrin at similar rates under standard assay conditions, but mouse liver esterases were 10-fold less active in hydrolyzing malathion. The higher specific activity of rat liver malathion-hydrolyzing esterases resulted from the greater apparent affinity and maximum velocity for malathion hydrolysis. These results demonstrate that the hydrolysis of malathion, trans-permethrin, and cis-permethrin by rat and mouse liver microsomal preparations involves several esterases with differing substrate specificities and inhibitor sensitivities.     

Studies on organophosphorus impurities in technical malathion: Inhibition of carboxylesterases and the stability of isomalathion

Four organophosphorus esters found as impurities in technical malathion were synthesized, and their abilities to inhibit monomeric and oligomeric carboxylesterases from rabbit liver, as well as type I and type II esterases from porcine liver, were studied. The equilibrium dissociation constant (K_d), phosphorylation constant (k₂), and bimolecular rate constant (k_i) were determined in the presence of substrate. Inhibition, as judged by the k_i, was in the following order: isomalathion > O,S,S-trimethyl phosphorodithioate > O,O,S-trimethyl phosphorothioate > O,O,S-trimethyl phosphorodithioate. All of the k₂ values were relatively small, indicating that the k_d values contribute most to the overall inhibitory power. Rabbit liver carboxylesterases are more sensitive to inhibition by isomalathion than porcine liver esterases, reflecting a species difference. Isomalathion undergoes nonenzymatic degradation in the presence of fluoride ions, producing diethyl thiomalate. A kinetic investigation of the nonenzymatic degradation of isomalathion was conducted, and reaction rate constants were determined in phosphate buffer at various molarities, temperatures, and pH values.     

The effect of ronnel and its oxygen analog on succinate oxidation by rat brain,liver homogenates,and mitochondria

The effect of the thiophosphoroorganic insecticide ronnel and its oxygen analog on oxygen consumption by homogenates, and on succinate dehydrogenase and cytochrome oxidase activities in the mitochrondrial fractions of rat brain and liver, was investigated. A marked inhibition of blood plasma cholinesterase activity by the oxygen analog was demonstrated, but acetylcholinesterase activity was not affected. In contrast to its thiophosphoroorganic precursor, the oxygen analog caused a significant decrease in oxygen consumption by rat brain and liver homogenates and a decrease in cytochrome oxidase activity in both solubilized mitochondrial fractions. The succinate dehydrogenase activity was reduced only slightly by the oxygen analog in the brain fraction, but was marked in the liver fractions. The results suggest that the toxic activity of ronnel in vivo may be ascribed to its oxygen analog which appears as a metabolite of the former.     

Effect of short-time malathion administration on glucose homeostasis in Wistar rat

The main objective of this study is to investigate the 24 h kinetic effect of acute administration of malathion, an organophosphorus insecticide, on glucose homeostasis in adult rats. A single dose of malathion (400 mg/kg, 1/5 of LD₅₀) was administered orally to rats, blood glucose was measured, liver and pancreas were removed to determine the level of hepatic glycogen, the activity of pancreatic acetylcholinesterase and butyrylcholinesterase, as well as the level of TBARs. Blood glucose increased in rats treated with a single dose of malathion. This effect was observed in the first hours of treatment, reached a 2.2 fold peak after 2 h, and then decreased after 4 h, followed by a decrease in the level of hepatic glycogen. The storage of glycogen starts from 6 to 12 h of administration. A decrease in cholinesterase activities was noted. The level of TBARs increased considerably in liver and pancreas. Results of this study can be explain by a stimulation of glycogenolysis and gluconeogenesis by liver, with a temporarily loss of endocrine functions of pancreas leading to hyperglycemia.     

Substrate-specificity and toxicological significance of pyrethroid-hydrolyzing esterases of mouse liver microsomes

An esterase or esterases in acetone powder preparations of mouse liver microsomes hydrolyze the cyclopropanecarboxylate ester linkage of pyrethroid insecticide chemicals derived from primary alcohols. The rate of cleavage of (+)-trans-chrysanthemates with various alcohol moieties decreases in the following order: 5-propargyl-2-furylmethyl; 5-benzyl-3-furylmethyl (bioresmethrin); 3-phenoxybenzyl; tetrahydrophthalimidomethyl esters. The hydrolysis rate of benzylfurylmethyl esters with various acid moieties decreases in the order: (+)- or (?)-trans-chrysanthemate; (+)-trans-ethanochrysanthemate; tetramethylcyclopropanecarboxylate; (+)- or (?)-cis-chrysanthemate or (+)-cis-ethanochrysanthemate. The trans-isomers of chrysanthemates and ethanochrysanthemates are hydrolyzed from 2.6- to more than 50-fold more rapidly than the corresponding cis-isomers. This enzyme system does not hydrolyze secondary alcohol esters, i.e., allethronyl (+)-trans- and (+)-cis-chrysanthemates.On intraperitoneal administration to mice, the (+)-trans-chrysanthemate and -ethanochrysanthemate of benzylfurylmethanol are of very low toxicity relative to the corresponding (+)-cis-isomers and the tetramethylcyclopropanecarboxylate. S,S,S-tributyl phosphorotrithioate (DEF) pretreatment increases the toxicity of these five compounds by 2.6- to more than 188-fold, with the exception of bioresmethrin whose toxicity is not altered. When the toxicity is increased, it is probably the result of esterase inhibition since DEF strongly inhibits the esterase activity of fresh liver microsomes while the mixed-function oxidase system remains active. The oxidase system metabolizes the chrysanthemates more rapidly than the ethanochrysanthemates of benzylfuryl-methanol. Depending upon the pyrethroid involved, the esterase or the mixed-function oxidase system, or both may be responsible for limiting the toxicity of these pyrethroids to mice.     

Effects of diazinon at different doses on rat liver and pancreas tissues

Diazinon is one of the most widely used organophosphates in agriculture. Toxic effects of diazinon are due to the inhibition of acetylcholinesterase, an enzyme needed for proper nervous system function. This study was designed to investigate the effects of diazinon at different doses on pancreas and liver tissues and in which dose level diazinon shows its effects. Sixty male Wistar albino rats were included in this study. Animals were initially divided into control and diazinon given groups. There were 10 animals in the control group and 50 animals in diazinon administered group. The latter was divided into five equal subgroups: 25, 50, 100, 200 and 300 mg/kg of diazinon administered groups. Control group was given only saline. All animals in 300 mg/kg diazinon group died. After 24 h, rats were sacrificed under ether anesthesia. Tissue and blood samples were taken for biochemical and histopathological analysis. Sample tissues were examined under light microscope. In biochemical analysis, AST, ALT, LDH, amylase and lipase enzyme activities were measured. One-way ANOVA test was used to compare the groups. In 200 mg/kg diazinon given group, it has been observed some histopathological changes in pancreas and liver tissues. Cholinesterase activities were significantly decreased and alkaline phosphatase levels were increased in all diazinon given groups, when compared with the controls. There was statistically significant difference between the control and diazinon given groups by means of serum amylase, lipase, ALT and AST activities (p < 0.05). LDH activities were significantly increased in 100 and 200 mg diazinon given groups, when compared with the controls (p < 0.05). Histopathological changes were observed only in 200 mg diazinon given group. This evidence suggest that diazinon effect is dose dependent and this is possibly 10-15% of the LD₅₀ dose (200 mg/kg), which cause acute pancreatitis and histopathological changes in liver.     

Effects of fungal and plant esterases on the fungicidal activity of nitrophenyl esters

Aryl esterases, present within leaves and fungal spores and on their surfaces, hydrolysed 2-naphthyl, 4-nitrophenyl and 4-(1-substituted alkyl)-2, 6-dinitrophenyl esters at rates which appeared to be determined by the nature of the ester group and by the alkyl substituents in the molecule. No simple relationship was found between partitioning (as measured by Rm values from reverse-phase t.l.c.) and susceptibility to hydrolysis by esterases. The most readily hydrolysed alkyldinitrophenyl esters are not those derived from parent nitrophenols with the greatest fungitoxicity in vivo or in vitro and the highly active 4-(1-cyclobutylalkyl)-2, 6-dinitrophenyl crotonates are amongst the least easily hydrolysed. It is suggested that alkyldinitrophenyl esters may have their own mode of action as powdery mildew fungicides which complements, and may even supplant, that resulting from hydrolysis to the parent alkyldinitrophenol. The proposed mechanism is disruption of the fungal cell membrane (as a result of surfactant properties) leading to leakage of essential cell components.     

Interaction of phosphodiesterase 5 inhibitor with malathion on rat brain mitochondrial-bound hexokinase activity

The main objective of this study was to investigate the possible protective effect of pentoxifylline as a phosphodiesterase 5 inhibitor used as a cardiovascular medication on malathion-induced changes on rat mitochondrial-bound hexokinase activity.Animals in four various groups received moderate toxic dose of malathion (200 mg/kg/day), effective dose of pentoxifylline (50 mg/kg/day) alone and in combination, and the control group that received only vehicle. All administrations were done intraperitoneally for one week. At the end of the experiment, the brain was removed and the mitochondria were isolated. Hexokinase (HK) activity, cellular lipid peroxidation (LPO) and total antioxidant capacity (TAC) were analyzed in brain mitochondria.Malathion noticeably decreased TAC and increased HK activity and LPO in the mitochondria whereas pentoxifylline significantly restored malathion-induced changes in LPO, HK, and TAC.The results of the present study indicate that phosphodiesterase 5 inhibition remarkably protects brain mitochondria from malathion-induced changes on HK activity and oxidative stress.     

Interaction of phosphodiesterase 5 inhibitor with malathion on rat brain mitochondrial-bound hexokinase activity

The main objective of this study was to investigate the possible protective effect of pentoxifylline as a phosphodiesterase 5 inhibitor used as a cardiovascular medication on malathion-induced changes on rat mitochondrial-bound hexokinase activity.Animals in four various groups received moderate toxic dose of malathion (200 mg/kg/day), effective dose of pentoxifylline (50 mg/kg/day) alone and in combination, and the control group that received only vehicle. All administrations were done intraperitoneally for one week. At the end of the experiment, the brain was removed and the mitochondria were isolated. Hexokinase (HK) activity, cellular lipid peroxidation (LPO) and total antioxidant capacity (TAC) were analyzed in brain mitochondria.Malathion noticeably decreased TAC and increased HK activity and LPO in the mitochondria whereas pentoxifylline significantly restored malathion-induced changes in LPO, HK, and TAC.The results of the present study indicate that phosphodiesterase 5 inhibition remarkably protects brain mitochondria from malathion-induced changes on HK activity and oxidative stress.     

Multiple forms of esterases in mouse,rat, and rabbit liver,and their role in hydrolysis of organophosphorus and pyrethroid insecticides

Six to seven esterases from mouse, rat, and rabbit liver microsomes were resolved by chromatofocusing in the pH range 7–4. Each esterase peak showed a different substrate specificity pattern with the substrates evaluated. Malathion and paraoxon hydrolysis always corresponded with p-nitrophenyl acetate and methylthiobutyrate hydrolysis, whereas the pattern of fenvalerate hydrolysis was more complicated. Phosphorotriester hydrolase activity was isolated, and was found to be more specific toward paraoxon than toward the other insecticides. Time-course studies of paraoxon hydrolysis indicated that the hydrolysis of paraoxon by carboxylesterase was an inhibitory reaction. This reaction and phosphorotriester hydrolase activity can serve as a detoxication reaction toward organophosphate insecticides.     

Effects of β-pinene,a nonsubstituted monoterpene,on rat liver mitochondria

β-Pinene uncouples oxidative phosphorylation and inhibits respiration in isolated rat liver mitochondria. The uncoupling effects are observed at lower concentrations (100 to 200 μM) than the inhibition of respiration (400 μM). At low concentrations, the effects observed could be explained by an increase of the passive permeability of the mitochondrial membrane produced by the terpene. Higher concentrations seemed to inhibit respiration through an effect on the electron transport chain. At the highest concentrations tested (600 to 1200 μM), β-pinene seemed to produce a partial resealing of the mitochondrial membrane. All effects can be explained by the interaction of β-pinene with the mitochondrial membrane. Other hydrophobic molecules tested do not show the effects of β-pinene or limonene on mitochondria.     

Effect of a mixture of iprobenfos and malathion on the development of malathion resistance in the mosquito Culex pipiens pallens Coq

A malathion-resistant (RM) strain of Culex pipiens pallens Coq was obtained by successively selecting a field population with malathion in the laboratory. The synergistic effect of iprobenfos on malathion toxicity and alpha-naphthyl acetate (alpha-NA) esterase assay revealed that malathion resistance in the RM strain was associated with increased alpha-NA esterase activity and the synergism was mainly due to the inhibition by iprobenfos of this activity. There was no difference in alpha-NA esterase activity between the larvae and female adults in the susceptible (S) strain, but the activity in the adults was 13-fold higher than in the larvae of the RM strain. To understand the effect of the application of a mixture of iprobenfos and malathion on the evolution of malathion resistance, an artificial strain (Syn) was generated by mixing the RM and S strains with 0.1 frequency of the malathion-resistant individuals. The offspring of the Syn strain were divided into two sub-strains, Rm and Rm+ibp, which were successively treated with, respectively, malathion alone and malathion + iprobenfos (1:2) at LC70. In the mixture, the fungicide iprobenfos acted as a synergist of malathion. After treatment for 10 generations, the resistance level to malathion was 317.4-fold for the Rm sub-strain, whereas for the Rm+ibp sub-strain it was only 38.9-fold, compared with the Syn strain. Similar results were obtained by measurement of alpha-NA esterase activity from both larvae and female adults. The alpha-NA esterase activities in larvae and female adults at F10 generation were 2.6- and 10.9-fold from the Rm+ibp sub-strain and 5.7- and 98.5-fold from the Rm sub-strain, respectively, compared with the Syn strain. The above results suggested that iprobenfos, although it cannot completely stop or prevent the onset of malathion resistance, could dramatically delay its evolution.     

Determination of malathion on tomatoes and brinjals by multiband chromatoplates

A one step clean-up for malathion in extracts of tomatoes and brinjals using a multiband chromatoplate and its quantitative estimation by measuring spot areas are described.     

Detection of single-base substitution in an esterase gene and its linkage to malathion resistance in the parasitoid Anisopteromalus calandrae (Hymenoptera: Pteromalidae)

Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae) is an important parasitoid of stored-grain insect pests. Partial cDNA sequences of an esterase-like enzyme have been obtained from a malathion-resistant (R) strain and a susceptible (S) strain of this wasp. A single-base substitution in the R strain has been confirmed by using PCR amplification of specific allele (PASA) to amplify genomic DNA extracted from individual resistant and susceptible parents, F₁ hybrids from double reciprocal crosses, and progeny from backcrosses. The R allele appeared to be inherited in a strict Mendelian fashion in both diploid female and haploid male progeny. The esterase fragment co-segregated with resistance in these crosses and backcrosses. Female wasps in a mixed population of A calandrae that survived a malathion screen carried the R allele for the esterase-like enzyme, while those wasps that died did not have the R allele. The single base-pair mutation, guanine in the R strain and thymine in the S strain, presumably results in a tryptophan-to-glycine amino acid substitution in the encoded protein. We do not know how these amino acid substitutions may relate to functional differences in the enzyme. However, this esterase gene or another linked esterase gene may encode the resistance-associated malathion detoxifying activity in the R strain. © 1999 Society of Chemical Industry     

Oxidative metabolism of lindane and its isomers with microsomes from rat liver and house fly abdomen

Lindane and other hexachlorocyclohexane isomers produced 2,4,6-trichlorophenol as the major oxidative metabolite when incubated in the presence of NADPH under aerobic condition. A mechanism for the formation of 2,4,6-trichlorophenol is proposed, which includes direct oxygenation of the cyclohexane ring. The proposed mechanism is supported by data from studies of model chemical reactions of the pentachlorocyclohexanol isomers. Pathways leading to 1,2,4-trichlorobenzene, tetrachlorobenzene isomers, 2,4,5-trichlorophenol, and tetrachlorophenol isomers are discussed, and are considered to include the route through pentachlorocyclohexene and hexachlorocyclohexene. Reductive dechlorination of lindane under anaerobic condition was observed using microsomes and NADPH.     

Effects of the herbicides benzoylpropethyl and flampropisopropyl on rat liver mitochondria: An alteration in membrane fluidity?

The action of the herbicides benzoylpropethyl and flampropisopropyl, and the corresponding unesterified acids was studied in rat liver mitochondria. The herbicides were found to (a) inhibit the mitochondrial electron transfer in complex III or at the level of ubiquinone (I₅₀ of 4 nmol mg protein^?1 for flampropisopropyl and 18 for benzoylpropethyl with succinate as a substrate); (b) have an additional (however less sensitive) site of inhibition near succinate dehydrogenase; and (c) interfere with energy transfer. Sensitivity was increased 2- (benzoylpropethyl) and 3.5-fold (flampropisopropyl) as the rats age increased from 12–13 weeks to 23–26 weeks. The free acids were far less effective. Since the herbicides benzoylpropethyl and flampropisopropyl are readily hydrolyzed by animals and since the free acids are less effective than the herbicides, it explains why these potentially harmful compounds have a low acute toxicity. Swelling studies in isoosmotic salts suggested that the two herbicides decrease membrane fluidity, an action which was assumed to be responsible for the electron transfer inhibition and, via inhibition of phosphate transport, the interference with energy transfer.     

棉铃虫酯酶两个位点突变对三种杀虫剂代谢活性的影响

为研究棉铃虫酯酶对有机磷和拟除虫菊酯类杀虫剂的代谢活性,在前期对酯酶001F和001G酶活性研究的基础上,进一步采用荧光法和高效液相色谱法测定了酯酶及其突变体（A127位和F238位突变）对二甲基-4甲基伞形酮磷酸酯（dMUP）、二乙基-7羟基香豆素磷酸酯（dECP）及顺式氰戊菊酯（2．s,d5）的酶促代谢活性。酯酶001F和001G对有机磷的酶促反应常数（kcat值）在2．0×10^-3／min～5．2×10^-3／min之间,后者对顺式氰戊菊酯的代谢活性为10．19μmol·min-1·μmol-1;001F的突变提高了对有机磷的代谢活性,其中001FA127D和001F238L对dMUP的kcat值至少是突变前的2倍;001G的突变却降低了对dECP的代谢活性,但001GF238L对dMUP的活性有所提高。此外,突变体中001F舢。阳对顺式氰戊菊酯的活性与突变前接近,而001G心蹦。则降低到突变前的1／2,其它突变体未检测到活性。结果表明,酯酶001F和001G对杀虫剂有代谢活性,A127位和F238位的突变对酶活性有显著影响。