Concentration-dependent kinetics of acetylcholinesterase inhibition by the organophosphate paraoxon.

For decades the interaction of the anticholinesterase organophosphorus compounds with acetylcholinesterase has been characterized as a straightforward phosphylation of the active site serine (Ser-203) which can be described kinetically by the inhibitory rate constant k(i). However, more recently certain kinetic complexities in the inhibition of acetylcholinesterase by organophosphates such as paraoxon (O,O-diethyl O-(p-nitrophenyl) phosphate) and chlorpyrifos oxon (O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphate) have raised questions regarding the adequacy of the kinetic scheme on which k(i) is based. The present article documents conditions in which the inhibitory capacity of paraoxon towards human recombinant acetylcholinesterase appears to change as a function of oxon concentration (as evidenced by a changing k(i)), with the inhibitory capacity of individual oxon molecules increasing at lower oxon concentrations. Optimization of a computer model based on an Ordered Uni Bi kinetic mechanism for phosphylation of acetylcholinesterse determined k(1) to be 0.5 nM(-1)h(-1), and k(-1) to be 169.5 h(-1). These values were used in a comparison of the Ordered Uni Bi model versus a k(i) model in order to assess the capacity of k(i) to describe accurately the inhibition of acetylcholinesterase by paraoxon. Interestingly, the k(i) model was accurate only at equilibrium (or near equilibrium), and when the inhibitor concentration was well below its K(d) (pseudo first order conditions). Comparisons of the Ordered Uni Bi and k(i) models demonstrate the changing k(i) as a function of inhibitor concentrations is not an artifact resulting from inappropriate inhibitor concentrations.     

Strychnine-insensitive binding of [3H]glycine to synaptic membranes in rat brain, treated with Triton X-100

Binding of radiolabelled glycine, a putative inhibitory neurotransmitter in mammalian lower central structures, was examined by using the synaptic membranes of the brain of rat, treated with Triton X-100. This treatment with Triton markedly potentiated the binding of [3H]glycine detected at 2 degrees C and 30 degrees C. However, this binding was not affected by three different convulsants, strychnine, picrotoxin and bicuculline. The binding was saturable at 2 degrees C, with increasing concentrations of [3H]glycine up to 1 microM. Scatchard analysis revealed that the binding sites consisted of a single component with a Kd of 202 nM and a Bmax of 1.74 pmol/mg protein. The binding was inhibited, not only by various amino acids structurally related to glycine, including D- and L-serine and D-, L- and beta-alanine, but was also eliminated by some peptides containing glycine, such as gamma-D- and gamma-L-glutamylglycine, glycine methylester and N-methyl-glycine. In addition, the strychnine-insensitive binding of [3H]glycine was significantly abolished by numerous quinoxaline antagonists for excitatory amino acid receptors in the brain. These results suggest that synaptic membranes of brain, treated with Triton X-100, are useful to detect the strychnine-insensitive binding of [3H]glycine and superior to untreated membranes in terms of the freedom from the confounding effects of some endogenous amino acids.     

Some effects of non-ionic detergent triton X-100 on rat liver monoamine oxidase

The non-ionic detergent Triton X-100, an agent used to solubilize mitochondrial membrane monoamine oxidase (EC 1.4.3.4, MAO), has been shown to inhibit markedly MAO activity. The inhibition was non-competitive in nature. Triton X-100 changed the susceptibility of MAO toward clorgyline, a specific type A MAO inhibitor, and deprenyl, a type B inhibitor. Its effect on the temperature dependence of the initial velocity revealed that the transition temperatures for p-tyramine and serotonin (22°) and β-phenylethylamine (16° and 27°) were not changed. The stability of the MAO decreased considerably, however, in the presence of Triton X-100, and its inactivation was particularly pronounced somewhat higher temperatures.     

Species-related differences in the inhibition of brain acetylcholinesterase by paraoxon and malaoxon

Interspecies comparisons indicate that fish are relatively more resistant to acute intoxication with parathion and paraoxon than are rodents. In contrast, fish are more sensitive to malathion and malaoxon. The following investigation was designed to determine if species-related differences in the sensitivity of brain acetylcholinesterase (AChE) to inhibition by paraoxon and malaoxon could contribute to the interspecies differences in toxicity. Brain AChE activity was significantly greater in fathead minnows and rainbow trout than in rats and mice. The fathead minnow and rainbow trout IC50 values for paraoxon were 228- to 1879-fold greater than the corresponding values for rat and mouse. Similarly, the Ki (bimolecular inhibition constant) was 159- to 1663-fold greater in rodents than in fish, which reflected both a higher KA (association constant) and kp (phosphorylation constant) in rodents. The rodent IC50 values for malaoxon were 30-80% that of the fish IC50, and the Ki was 30-50% greater in rodents than in fish. These data suggest that the greater sensitivity of rodent brain AChE to inhibition by paraoxon may contribute to the greater toxicity of parathion and paraoxon in rodents than in fish. In contrast, the lack of correlation between the inhibition of brain AChE by malaoxon and species-related differences in acute I D50 suggests that other factors, such as the limited carboxylesterase activity in fish, may be responsible for this species selectivity.     

头孢唑酮在TritonX-100体系微乳液中缓释的研究

目的研究了β-内酰胺类抗生素药物头抱唑酮在表面活性剂微乳液中的释放行为。方法制备TritonX-100体系的胶束和微乳液．用紫外分光光度法测定头抱唑酮在微乳液的释放速率。结果 FritonX-100／n—C10H21OH／H2O体系微乳液对头抱唑酮具有较好的缓释作用。结论表面活性剂体系微乳液可作为一种新的药物控制释放系统。     

头孢唑酮在Triton X-100体系囊泡中的包封和缓释

目的研究了β-内酰胺类抗生素药物头孢唑酮在表面活性剂囊泡中的包封和释放行为。方法超声Triton X-100体系层状液晶制备了囊泡,用超离心分离法和紫外分光光度法测定头孢唑酮的包封率和释放速率。结果Triton X-100/n-C10H21OH/H2O体系囊泡对头孢唑酮具有较好的包封和缓释作用。结论表面活性剂体系囊泡可作为一种新的药物控制释放系统。     

头孢唑酮在Triton X-100体系中的释放机制初探

目的 探讨抗生素药物头孢唑酮在表面活性剂体系中的释放机制.方法 测定了头孢唑酮在表面活性剂体系中的释放曲线,用数学模型对药物的释放曲线进行拟合.结果 头孢唑酮在TritonX,100体系的释放为非菲克扩散控制过程.结论 为表面活性剂体系中药物控制释放的深入研究提供了科学依据.     

Studies of monoamine oxidases: Effect of Triton X-100 and bile salts on monoamine oxidase in brain mitochondria

Triton X-100 and the bile salts, cholate and deoxycholate, detergents often used in the solubilization of monoamine oxidase (MAO) from mitochondria, have been found to cause an inhibition of the enzyme activity. With beef brain mitochondria, it was found that there was a differential effect of Triton X-100 on the putative MAO types A and B, with MAO-A being more susceptible to inhibition by Triton X-100. This was indicated by the greater loss of serotonin-deaminating than of phenyl ethylamine-deaminating activity in the presence of Triton X-100. Although the bile salts also caused substantial inactivation at concentrations above 0.1%, no differentiation between MAO types could be made. Kinetic studies of the inhibition by Triton X-100 indicated two different mechanisms were occurring with the two MAO types. The inhibition was competitive for MAO-A, but uncompetitive for MAO-B. Removal of Triton X-100 by co-polymer beads restored some, but not all of the activity for both MAO-A and MAO-B types. This suggests that the activity loss may have been due in part to inactivation when the enzyme was separated from the mitochondrial membrane.     

Effects of Triton X-100 nanoaggregates on dimerization and antioxidant activity of morin

Dimerization and antioxidant activity of morin in the Triton X-100 micelles were studied by electronic absorption, ATR-FTIR spectra, cyclic voltammetric, DSC, freeze-fracture TEM, molecular modeling and ab initio quantum calculations. Morin can be solubilized in the Triton X-100 micelles and show selective dimerization in Triton X-100 micelles with different structures. In Triton X-100 spherical micelles, morin always exists in the form of dimer, and in Triton X-100 rodlike micelles, it is always in the form of monomer. The solubilization of morin dimer in Triton X-100 spherical micelles changes the micelle morphology from spherical to cubelike, and the size of the single micelle is also increased, while morin monomer links the Triton X-100 rodlike micelles and forms a kind of network micelle structure with the size of the "rod" unchanged. Solubilized and concentrated in Triton X-100 micelles, morin can protect human serum albumin from the damage induced by hydroxyl radicals effectively and even can form a kind of protein complex with human serum albumin showing more thermal stability.     

Lipid peroxidation, oxidative stress and acetylcholinesterase in rat brain exposed to organophosphate and pyrethroid insecticides

Oxidative stress by increased production of reactive oxygen species has been implicated in the toxicity of many pesticides. Therefore, the aim of the present study was to investigate the effect of a broad spectrum insecticide, composed of a mixture of organophosphate plus pyrethroids (fenitrothion 25%, lambda cyhalothrin 2.5% and piperonyl butoxide 6%), on antioxidant status and oxidative stress biomarkers in rat brain. Different insecticide concentrations (0, 0.1, 1, 10, 100 and 1000 mM) were incubated with brain homogenate at 37 °C for time intervals (0, 30, 60, 120, 180 and 240 min). Exposure to insecticide mixture resulted in a significant increase (p < 0.05) in thiobarbituric acid reactive substances (TBARS), which might be associated with decreased levels of reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and acetylcholinesterase activities and beside protein content in rat brain. However, a significant induction of lactate dehydrogenase (LDH) activities was observed. The response was concentration and time dependent. Results showed that the used insecticides had the propensity to cause significant oxidative damage in rat brain, which is associated with marked perturbations in antioxidant defense system in addition to antioxidant enzymes can be used as potential biomarkers of toxicity associated with pesticides exposure.     

Combined antioxidant effects of rutin and vitamin C in Triton X-100 micelles

UV-vis spectra, fluorescence emission spectra and cyclic voltammetric measurements were used to study the influence of Vitamin C on the antioxidant of rutin in Triton X-100 micelles. Rutin can be located in Triton X-100 micelles spontaneously through hydrophobic force, and the binding constant K between rutin and Triton X-100 increases with the rutin concentration. The embedment of two hydroxyl groups on rutin into the more hydrophobic micellar microenvironment makes the oxidation of rutin harder and the radical scavenging activity decrease. With low concentration of Vitamin C, the antioxidant capacity of rutin against hydroxyl radical is enhanced, while that capacity is partly inhibited when the concentration of Vitamin C become higher.     

Morphological and physiological changes in Tetrahymena pyriformis for the in vitro cytotoxicity assessment of Triton X-100.

Non-ionic surfactants such as Triton X-100 have been widely used in industrial processing and in cleaning products for almost 50 years, being effective and economic emulsifying, wetting agents, dispersants and solubilizers. Cleaning products containing these surfactants are disposed of mainly by discharge into wastewater, which receives biological treatment in wastewater treatment systems. However, surface-active agents interact with eukaryotic cell membranes leading to biological damage at high concentrations. Tetrahymena pyriformis was used here as model organism to assess the effects of Triton X-100 through a series of in vitro cytotoxicity tests. Growth rates and morphological changes were, by their simplicity and reproducibility, the simplest toxicological assays. Cytoskeleton analysis seemed to be related with phagocytosis rate. Viability was evaluated by two different tests. Calcein AM/EthD-1 was used to assess T. pyriformis membrane damage during the 48-h experiment. The colorimetric MTT assay proved to be highly sensitive even at very short periods of Triton X-100 exposure. Tests performed in this study included simple and fast bioassays that provide overall information on the morphological and physiological state of cells exposed to different non-lytic and lytic concentrations of Triton X-100.     

Effects on the reproductive organs of feeding the non-ionic surfactant Triton X-100 to mice

A series of feeding experiments has shown Triton X-100 to induce cystic degeneration in the mouse ovary. Parallel experiments in ovariectomized mice revealed no evidence that Triton X-100 possesses oestrogenic activity. Long term exposure of female mice to this surfactant did not impair fertility.     

Interactions of acetylcholine mustard with acetylcholinesterase.

The hydrolysis of acetylcholine and acetylcholine mustard by acetylcholinesterase was compared over a substrate concentration range of 1-10 mM. Reactions were allowed to proceed for 2 min at 25 degrees. Results of these experiments reveal that the substrates have similar affinities for the enzyme, whereas the maximum velocity for the hydrolysis of acetylcholine mustard was significantly lower than for acetylcholine. These findings suggest that acetylcholine mustard has the ability to inactive acetycholinesterase.     

高效液相色谱法测定质粒pUDKH中曲拉通X-100的含量

目的检测质粒pUDKH最终产品中去污剂曲拉通X 10 0 (TritonX 10 0 )的残留含量。方法用高效液相色谱法测定TritonX 10 0残留含量 ,色谱柱为C18柱 ,流动相为乙腈 水 (70∶30 ) ,流速为 1.0ml/min ,进样量 10 μl,检测波长 2 2 3nm。结果平均回收率为 10 0 .19% ,日内精密度为 4 .36 % ,日间精密度为 4 .17% ,TritonX 10 0在 1.2 5～2 0 μg/ml浓度范围内呈线性关系。pUDKH产品中的TritonX 10 0含量在 2 .2 7～ 3.0 0 μg/ml之间。TritonX 10 0的最低检测限可达 1.0 μg/ml。 结论此法有良好的准确度与精密度 ,供试品不需预处理 ,不受其它成分的干扰。     

The action of Triton X-100 and sodium dodecyl sulphate on lipid layers. Effect on monolayers and liposomes

The action of two detergents, Triton X-100 and sodium dodecyl sulphate (SDS), on large, unilamellar liposomes was determined as liposome size variation, polydispersity and ability to release a soluble marker from liposomes. Triton X-100 produced stronger effects than SDS. Nevertheless, these differences in behaviour of such detergents could not be deduced from the interaction of the detergents with monolayers of the same composition as liposomes.     

The action of Triton X-100 and sodium dodecyl sulphate on lipid layers. Effect on monolayers and liposomes

Abstract

The action of two detergents, Triton X-l 00 and sodium dodecyl sulphate (SDS), on large, unilamellar liposomes was determined as liposome size variation, polydispersity and ability to release a soluble marker from liposomes. Triton X-100 produced stronger effects than SDS. Nevertheless, these differences in behaviour of such detergents could not be deduced from the interaction of the detergents with monolayers of the same composition as liposomes.     

Recovery of acetylcholinesterase in cultured chick embryo muscle treated with paraoxon

Brief treatments with paraoxon (O,O-diethyl-p-nitrophenyl phosphate) irreversibly inhibited the acetylcholinesterase (AChE, acetylcholine hydrolase, EC 3.1.1.7) activity of cultured chick embryo muscle. Enzyme activity recovered as long as protein synthesis occurred, and was most rapid during the first 4 hr after paraoxon treatment. The initial recovery rate was related to the extent of initial inhibition of AChE activity: the more activity inhibited the more rapid the recovery. Differences noted between paraoxon-treated and untreated cultures during recovery included a 192 per cent increase in net AChE activity and an increase of 200 per cent in cell protein levels. AChE activity first appeared around the nuclei after paraoxon treatment, spread through the rest of the cell, and was released into the medium. The results suggest the presence of feedback control of the rapid recovery of AChE activity after organophosphate poisoning.     

Analysis of the process of cell degradation induced by Triton X-100 in Ehrlich ascites tumor cells

Analysis of the liberation degrees of intracellular materials from Ehrlich ascites tumor cells by treatment of Triton X-100 was performed. The liberation process has two stages. In the first stage, cytoplasmic inorganic phosphorus were liberated at a concentration of about 0.01% of the detergent, and in the second stage macromolecules such as lysosomal enzymes were liberated at a concentration of about 0.03% of the detergent. Morphologically, the cells began to swell in the first stage but were not stained by Trypan blue, and the cells in the second stage became fully swollen and ruptured. The staining curve of the swollen and ruptured cells was closely in parallel with the liberation curves of the macromolecules.