Comparative effects of organophosphorus insecticides on the activities of acetylcholinesterase,diacylglycerol kinase,and phosphatidylinositol phosphodiesterase in rat brain microsomes

The activities of acetylcholinesterase, diacylglycerol kinase, and phosphatidylinositol phosphodiesterase in rat brain microsomes were measured in the presence and absence of the organophosphorus insecticides, parathion and diazinon, and their respective oxon analogs, paraoxon and diazoxon. Marked inhibition of acetylcholinesterase (by 45–99%) was observed in the presence of paraoxon (10^?2–10^?6M) and diazoxon (10^?2–10^?4M). Reduction of acetylcholinesterase activity (by 22–33%) was achieved with the parent insecticides at high concentrations only (10^?2M). In most cases, diacylglycerol kinase was insensitive to the pesticides. Marked stimulation of phosphatidylinositol phosphodiesterase (by 10–57%) was observed in the presence of all pesticides (10^?2–10^?3M). The phosphodiesterase exhibited slightly greater sensitivity to the parent compounds compared to the oxon derivatives. Stimulation of the phosphodiesterase by the insecticides was not correlated with acetylcholinesterase inhibition. Accordingly, the increase in phosphodiesterase activity was judged not to be acetylcholine mediated, but rather represented a direct effect of the pesticides on the enzyme or its microenvironment. Based on the present in vitro observations, it is proposed that certain organophosphorus pesticides may interfere with the normal process of synaptic transmission through both the inhibition of acetylcholinesterase and the stimulation of phosphatidylinositol phosphodiesterase. In view of the high concentrations of pesticides required to elicit the latter effect, interpretation of its physiological significance must await results from further studies performed in vivo.     

In vitro and in vivo inhibition of chicken brain neurotoxic esterase by leptophos analogs

Inhibition of chicken brain neurotoxic esterase (NTE) by a series of O-halogenated-phenyl-O-alkyl phenylphosphonates was studied in vitro. The “apparent” activity was found to consist of “true” NTE (sensitive to mipafox) plus a minor mipafox-resistant component. The pI₅₀ of O-(2,6-dichlorophenyl) O-methyl phenylphosphonate for “true” NTE was 6.65, whereas it was about 3 for mipafox-resistant hydrolysis of phenyl valerate. This compound is suitable as an alternative to mipafox in the assay of “true” NTE, whereas the use of leptophos oxon gives a less accurate measure. The ethoxy analogs are about as potent in vitro as the corresponding methoxy compounds. Leptophosoxon and ethoxyleptophosoxon are more potent in vitro inhibitors than desbromoleptophosoxon. Within a like group of chlorinated phenylphosphonates, a reasonable correlation between in vitro neurotoxic esterase inhibition of the oxon and in vivo delayed neurotoxic potential by the corresponding phosphonothionate exists. In vivo inhibition of “apparent” NTE from chicken brain, studied 24 hr after an oral dose, is dose dependent for leptophos, ethoxyleptophos, and desbromoleptophos, the latter one being a very potent in vivo inhibitor. Ethoxyleptophos and leptophos have about equal in vivo esterase inhibitory properties. For desbromoleptophos and leptophos there is good agreement between the minimum dose causing delayed neurotoxicity and the dose leading to substantial inhibition of “apparent” NTE; ethoxyleptophos, on the other hand, inhibits the esterase at a dose much lower than the one which is neurotoxic. Several possible explanations for this discrepancy are considered.     

Inhibition of neurotoxic esterase and acetylcholinesterase by organophosphorus compounds in selected ectothermic vertebrates

The sensitivity of brain acetylcholinesterase and neurotoxic esterase to inhibition by several organophosphorus compounds was studied in selected ectothermic vertebrates. These enzymes are associated with organophosphorus compound acute and delayed toxicity, respectively. In addition, the susceptibility of several of these species to delayed neurotoxicity induced by organophosphorus compounds was studied. Larvae of the gray treefrog, Southern leopard frog, and narrow-mouthed toad were exposed dermally to tri-o-tolyl phosphate or phenyl saliginen cyclic phosphate (PSCP); no symptoms of delayed neurotoxicity were observed in any of these animals up to 2 weeks after metamorphosis. No symptoms of delayed neurotoxicity were seen in juvenile bullfrogs exposed to multiple ip doses of PSCP. The specific activity of neurotoxic esterase was highest in the larval bullfrog, with juvenile channel catfish and adult mosquitofish demonstrating intermediate levels. The larval Southern leopard frog, adult Northern leopard frog, juvenile green treefrog, and adult marine toad exhibited extremely low activities. The specific activity of acetylcholinesterase was highest in the juvenile channel catfish. Neurotoxic esterase in the larval bullfrog was more sensitive to organophosphate inhibition than that in either fish. PSCP was a more potent neurotoxic esterase inhibitor than leptophos-oxon. The juvenile channel catfish had the acetylcholinesterase most sensitive to organophosphate inhibition. Under the conditions tested, no evidence of in vivo sensitivity to the organophosphate-induced delayed neurotoxicity phenomenon was observed.     

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.     

The biochemical basis of resistance to organophosphorus insecticides in the sheep blowfly, Lucilia cuprina

The metabolism in vivo and in vitro of [¹⁴C]parathion and [¹⁴C]paraoxon was studied in a susceptible (LS) and an organophosphorus-resistant (Q) strain of the sheep blowfly, Lucilia cuprina. Both strains detoxified the insecticides in vivo via a number of pathways, but the resistant strain produced more of the metabolites diethyl phosphate and diethyl phosphorothionate. No difference was found between strains in the rate of penetration of the compounds used. Also, in vitro studies showed no difference between strains in the sensitivity of head acetylcholinesterase to inhibition by paraoxon. Both the microsomal and the 100,000g supernatant fractions degraded paraoxon, but resistance in Q could be explained by the eightfold greater rate of diethyl phosphate production with or without added NADPH. Parathion was also degraded to diethyl phosphorothionate by an NADPH-requiring enzyme in microsomal preparations from both strains. However, Q produced significantly more diethyl phosphorothionate in vivo than LS. It was concluded that organophosphorus resistance in Q was due mainly to a microsomal phosphatase hydrolyzing phosphate but not phosphorothionate esters, probably enhanced by a microsomal oxidase detoxifying the latter.     

Insensitive acetylcholinesterase,high glutathione-S-transferase,and hydrolytic activity as resistance factors in a tetrachlorvinphos-resistant strain of house fly

The gene producing insensitive acetylcholinesterase in a tetrachlorvinphos-resistant strain of house fly was introduced into the genome of a susceptible strain. The resistance to a number of organophosphorus insecticides, which was high in the original strain (234- and more than 800-fold for paraoxon and tetrachlorvinphos, respectively), was much lower in the resulting strain, ranging from 53-fold for paraoxon to 3.9-fold for tetrachlorvinphos. Synergists further decreased these factors. The reduction in sensitivity of the acetylcholinesterase in vitro was about 20-fold for both inhibitors. The Michaelis parameters for hydrolysis of acetylcholine and acetylthiocholine of the mutant and normal acetylcholinesterase showed only minor differences. An increased rate of metabolism of the insecticides was found in the original strain. In vitro, glutathione-dependent detoxication was from 9- to 120-fold that of a susceptible strain, depending on the insecticide and the conditions used. The enzyme de-alkylated parathion and methylparaoxon, but there was no de-arylation of parathion, although it is important in other strains. In vitro, paraoxon and methylparaoxon were also hydrolysed, and this is not so in the susceptible strain. The high resistance present in the original strain seems to be due to joint action of at least three mechanisms.     

Acephate toxicity,metabolism, and anticholinesterase activity in Heliothis virescens (F.) and Anthonomus grandis grandis (Boheman)

The toxicity (72 hr) of acephate and methamidophos to fourth-instar larvae of the tobacco budworm, Heliothis virescens (F.), was nearly equivalent. In contrast, toxicity (72 hr) of methamidophos to adult boll weevils, Anthonomus grandis grandis (Boheman), was substantially greater than that of acephate. The internal accumulation of acephate was greater for A. grandis grandis than for H. virescens at 24 and 48 hr post-treatment, as was excretion. Acephate was metabolized to methamidophos both in vivo and in vitro by H. virescens but not by A. grandis grandis. In vitro acetylcholinesterase inhibition by methamidophos was greater than that of acephate, but less than that of paraoxon for H. virescens, A. grandis grandis, and the electric eel. Treatment of H. virescens larvae with acephate resulted in increased in vivo acetylcholinesterase inhibition between 24 and 72 hr post-treatment, which was associated with a large increase in mortality. H. virescens treated with methamidophos showed greater mortality and greater acetylcholinesterase inhibition at earlier time periods than those treated with acephate. However, by 72 hr post-treatment, in vivo acetylcholinesterase inhibition by LD₅₀ doses of acephate and methamidophos were approximately equivalent. These results indicate that, for H. virescens, toxicity of acephate is directly related to its metabolism to methamidophos and subsequent acetylcholinesterase inhibition. Likewise, the differential toxicity of acephate and methamidophos to A. grandis grandis adults appears to be due to their inability to metabolize acephate to methamidophos.     

槲皮素对棉铃虫体内一些解毒酶系和靶标酶的诱导作用

用培养基混药法研究了槲皮素对棉铃虫羧酸酯酶、谷胱甘肽转移酶的诱导作用和对乙酰胆碱酯酶敏感度的影响。槲皮素诱导种群对甲基对硫磷的敏感度明显降低 ,对灭多威和溴氰菊酯的敏感度没有明显变化。诱导种群羧酸酯酶的活性提高 2～ 3倍 ,谷胱甘肽转移酶活性提高近 15倍 ,而乙酰胆碱酯酶对对氧磷的敏感度却提高了 3.5倍。说明槲皮素诱导种群对甲基对硫磷的敏感度降低是由于解毒酶系和乙酰胆碱酯酶综合作用的结果。     

Studies on hydrolases in various house fly strains and their role in malathion resistance

Aliesterase, carboxylesterase, and phosphorotriester hydrolase activities in six house fly strains were studied in relation to malathion resistance. Selection of two susceptible strains with malathion for three generations resulted in an increase in both carboxylesterase activity and LD₅₀ of malathion, indicating that the increased detoxication by the enzyme was the major mechanism selected for malathion resistance. With the highly resistant strains, however, the carboxylesterase activity alone was not sufficient to explain the resistance level, and the involvement of additional mechanisms, including phosphorotriester hydrolase activity, was suggested. The E₁ strain, which had high phosphorotriester hydrolase activity but normal or low carboxylesterase activity, showed a moderate level, i.e., sevenfold resistance. Upon DEAE-cellulose chromatography, two or three esterase peaks were resolved from susceptible, moderately resistant, and highly resistant strains. The substrate specificity, the sensitivity to paraoxon inhibition, and the $\text{α}\text{β}$ ratio of malathion hydrolysis were studied for each esterase peak from the different strains. The results suggested the existence of multiple forms of esterases with overlapping substrate specificity in the house fly.     

Synergetic effect of organic solvents and paraoxon on the immobilized acetylcholinesterase

The toxicity of organophosphorus pesticides is attributed to their ability to inhibit acetylcholinesterase (AChE), which catalyses the hydrolysis of the neurotransmitting agent acetylcholine (ACh). A simple, reproducible and stable AChE-based amperometric bioelectrode was used, for measuring the enzyme activity before and after the incubation with paraoxon dissolved in organic solvents.The inhibition percentage induced by a paraoxon in organic solvent solutions increases in the following sequence: acetonitrile < water < hexane, suggesting that the paraoxon repartition between the organic solvent and the essential water for enzyme activity plays an important role in establishing the analytical and kinetic parameters of the bioelectrode. Considering the AChE denaturation, induced only by the organic solvent, as a pseudo-inhibition process, the kinetic treatment of the bioelectrode response revealed that it follows a mixed competitive/non-competitive mechanism, while the inhibition due to a non-aqueous solution of paraoxon corresponds to a non-competitive/uncompetitive mechanism. In the last case, the synergetic effect of denaturation and inhibition of enzyme was dominated by the inhibitor presence.     

Toxicity in the brain of organophosphate insecticides: Comparison of the toxicities of metabolites with parent compounds using an intracerebral injection method

The toxicity in the brain of several parathion, fenthion, and fensulfothion insecticides and their toxic metabolites was determined by a technique of directly injecting the compounds into the region of the third ventricle of conscious mice, an area rich in cholinesterase activity. The results were compared on a body weight basis to the toxicity of these compounds when given by ip and oral routes. The results show that there is a direct relationship between the relative inhibition of cholinesterase activity in the brain by the organophosphates (e.g., methyl paraoxon, Sumioxon, and some members of the fenthion series) and the toxicity of these compounds in the brain. Methyl paraoxon and Sumioxon were found to be very toxic in the brain, Sumioxon being three to four times less toxic than methyl paraoxon. This is of the same order of effect of these compounds in inhibiting cholinesterases. It is concluded that any selective effects of Sumithion compared with methyl parathion must be due to the greater rate of metabolism of Sumithion to less toxic metabolites as well as to the lower toxicity of the oxon metabolite and not due to the relative rates of penetration of the toxic oxygen metabolites as previously suggested [J. Miyamoto, Agr. Biol. Chem.28, 422 (1964)]. A gas-liquid chromatographic method was employed to assess the distribution in the brain following intracerebral injection of the parathion-type compounds. The results suggest that there may be intracerebral metabolism of thionophosphates in vivo.     

Esterase-mediated malathion resistance in the human head louse, Pediculus capitis (Anoplura: Pediculidae)

Resistance in a dual malathion- and permethrin-resistant head louse strain (BR-HL) was studied. BR-HL was 3.6- and 3.7-fold more resistant to malathion and permethrin, respectively, compared to insecticide-susceptible EC-HL. S,S,S-Tributylphosphorotrithioate synergized malathion toxicity by 2.1-fold but not permethrin toxicity in BR-HL. Piperonyl butoxide did not synergize malathion or permethrin toxicity. Malathion carboxylesterase (MCE) activity was 13.3-fold and general esterase activity was 3.9-fold higher in BR-HL versus EC-HL. There were no significant differences in phosphotriesterase, glutathione S-transferase, and acetylcholinesterase activities between strains. There was no differential sensitivity in acetylcholinesterase inhibition by malaoxon. Esterases from BR-HL had higher affinities and hydrolysis efficiencies versus EC-HL using various naphthyl-substituted esters. Protein content of BR-HL females and males was 1.6- and 1.3-fold higher, respectively, versus EC-HL adults. Electrophoresis revealed two esterases with increased intensity and a unique esterase associated with BR-HL. Thus, increased MCE activity and over-expressed esterases appear to be involved in malathion resistance in the head louse.     

Inhibition of two acetylcholinesterases by the 4-nitrophenyl esters of methyl-, ethyl-, and isopropyl(phenyl)phosphinic acid

The inhibition of eel acetylcholinesterase and bovine erythrocyte acetylcholinesterase by the 4-nitrophenyl esters of methyl-, ethyl-, and isopropyl(phenyl)phosphinic acid (MPP, EPP, and IPP, respectively) was investigated at pH 6.90 in 0.067 M phosphate buffer (25.0°C) using stopped-flow instrumentation and automated data processing. Our evaluation of the dissociation constant, K_d, the unimolecular bonding rate constant, k₂, and the bimolecular reaction constant, k_i, are the first reported values for these constants for a homologous series of this class of organophosphorus compounds. The largest k₁ value (29,428 M^?1 sec^?1) was observed for the reaction of eel acetylcholinesterase with 4-nitrophenyl methyl(phenyl)phosphinate. The smallest k_i value (9.6 M^?1 sec^?1) was observed for the reaction of bovine erythrocyte acetylcholinesterase with 4-nitrophenyl isopropyl(phenyl)phosphinate.     

Acetylcholinesterase of Aphis citricola: Properties and significance in determining toxicity of systemic organophosphorus and carbamate compounds

In apterous adults of the spirea aphid, Aphis citricola van der Goot, the optimum conditions for determining acetylcholinesterase (AChE) activity consist of reaction mixture of 0.1 M phosphate buffer (pH 7.5), 10^?3M acetylthiocholine (ASCh), and enzyme extract equivalent to 80 ± 3 μg protein incubated for 15 min at 30°C. The K_m value for ASCh (6.7 × 10^?5M) was much lower than that of butyrylthiocholine (BuSCh) (1.25 × 10^?2M). The enzyme activity was almost completely inhibited by 10^?6M paraoxon or 10^?5M eserine and was 84% inhibited by 10^?5M BW284C51 (a specific AChE inhibitor). DTNB was found to inhibit the enzyme activity and was therefore added at the end of the reaction. AChE activity of A. citricola was inhibited in vitro and in vivo by dimethoxon > dimethoate, and aldicarb sulfoxide > aldicarb > aldicarb sulfone. The in vivo effect correlates well with the toxicity level of the various toxicants. A neurotoxicity index which combines both mortality and in vivo inhibition of the aphid AChE activity is suggested as a measure for determining the toxicity of organophosphorus and carbamate compounds toward aphids.     

Role of a microsomal carboxylesterase in reducing the action of malathion in eggs of Triatoma infestans

A microsomal malathion carboxylesterase present in Triatoma infestans eggs was active from the first day of embryonic development. This microsomal egg malathionase (MEM) showed a unique band in polyacrilamide gel electrophoresis (PAGE) when malathion was used as substrate. In vivo metabolism of [¹⁴C]malathion during all embryonic development showed a 10% degradation due to carboxylesterases. The in vitro evaluation of the same metabolic pathway catalyzed by the microsomal fraction of T. infestans eggs showed partial inhibition by paraoxon. α- and β-malathion monoacids were identified as the main metabolites of the in vivo and in vitro metabolic pathways. The carboxylesterase band that appeared in PAGE (MEM) from the first day of embryonic development could be the main cause of malathion tolerance in T. infestans eggs.     

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.     

The relationship of esterases to organophosphorus insecticide tolerance in mosquitofish

The levels of peripheral acetylcholinesterase and carboxylesterases and their organophosphate sensitivities were studied in two populations of mosquitofish (Gambusia affinis). One population, highly resistant to organochlorine insecticides, demonstrated a low tolerance to parathion and methyl parathion. The organochlorine resistant fish possessed higher levels of both peripheral acetylcholinesterase and carboxylesterases. The sensitivities of these esterases to organophosphate inhibition were the same in both populations. The esterases were more sensitive to paraoxon than to methyl paraoxon. Carboxylesterases were far more sensitive to organophosphate inhibition than was acetylcholinesterase. Carboxylesterases, by their higher affinity for the organophosphates, may serve to protect acetylcholinesterase from inhibition.     

Factors affecting the sequential acquisition by Danish houseflies (musca domestica L.) of resistance to organophosphorus insecticides

The prolonged use of dimethoate, introduced into Denmark to control houseflies (Musca domestica L.) that had become resistant to parathion and diazinon, resulted ultimately in dimethoate resistance. Selection with dimethoate led to the disappearance of the hydrolytic phosphatase, a major mechanism of resistance to parathion and diazinon, and its replacement by the acetylcholinesterase AChER with somewhat decreased sensitivity to inhibition by organophosphorus (OP) insecticides. The hydrolytic phosphatase probably disappeared because low substrate turn-over made it ineffective against dimethoxon (O, O-dimethyl S-methylcarbamoylmethyl phosphorothioate, also known as omethoate). which accumulates at higher concentrations than paraoxon (diethyl4-nitrophenyl phosphate) in the haemolymph. Dimethoate selected AChER preferentially because it improved the chances of houseflies surviving against the relatively poor AChE inhibitor dimethoxon, whereas its relatively small insensitivity to OP insecticides, unimportant against good inhibitors such as paraoxon, prevented its selection by parathion.     

The effect of pretreatment with S,S,S-tributyl phosphorotrithioate on deltamethrin resistance and carboxylesterase activity in Aphis gossypii (Glover) (Homoptera: Aphididae)

The synergism of S,S,S-tributyl phosphorotrithioate (DEF) and its effect on carboxylesterase activity were investigated in deltamethrin-selected resistant (DRR) and susceptible (DSS) strains of cotton aphids, Aphis gossypii (Glover). Compared to the DSS strain, the DRR strain showed 23,900-fold resistance to deltamethrin, and 7560- and 99-fold cross-resistance to bifenthrin and ethofenprox, respectively. The synergist, DEF, increased the toxicity of both deltamethrin and bifenthrin, but not of ethofenprox when DEF was pretreated of 15 h. DEF exhibited significant inhibition on the carboxylesterase activity in the DRR strain, but no significant effect on that of the DSS strain in vitro. After the cotton aphids exposing to DEF, the carboxylesterase activity decreased gradually until 15 h and then gradually recovered until 24 h in the DRR strain, which fluctuated according to the effect of DEF on the deltamethrin toxicity detected using DEF pretreatment in the DRR strain. Therefore, our studies suggested that the effect of DEF on carboxylesterase was associated with deltamethrin resistance in the DRR strain.     

Inhibition and reactivation of chicken kynurenine formamidase: In vitro studies with organophosphates, N-alkylcarbamates,and phenylmethanesulfonyl fluoride

In studies on the mechanism of avian teratogenesis induced by organophosphorus and methylcarbamate insecticides, chicken liver kynurenine formamidase (KFase) was used as a convenient model for the more difficultly accessible but teratogenically relevant KFase of the yolk sac membrane. The parameters of in vitro KFase inhibition kinetics do not correlate with the teratogenic potency of various organophosphates and N-alkylcarbamates. The phosphorylated, carbamoylated, and sulfonylated enzymes are relatively resistant to reactivation of enzyme activity either spontaneously or by aldoximes or hydroxylamine. This refractory property may contribute to the prolonged in ovo KFase inhibition necessary for the NAD-deficiency type of teratogenesis.