Automated measurement of acetylcholinesterase activity in rat peripheral tissues

The accepted mechanism of toxicity of many organophosphorous and carbamate insecticides is inhibition of acetylcholinesterase activity. In mammals, part of the toxicity assessment usually includes monitoring blood and/or brain acetylcholinesterase inhibition. Other tissues, however, contain cholinesterase activity (i.e. acetyl- and butyryl-cholinesterase), and the inhibition of that activity may be informative for a full appraisal of the toxicity profile. The present group of studies first optimized the variables for extraction and solubilization of cholinesterase activity from various rat tissues and then refined an existing automated method, in order to differentially assess acetyl and butyrylcholinesterase activity in those tissues. All these studies were conducted using tissues from untreated, Long-Evans, adult rats. The first studies determined the effect of Triton X-100 or salt (NaCl) on the extraction and solubilization of cholinesterase activity from retina, brain, striated muscle, diaphragm, and heart: phosphate buffer plus detergent (1% Triton X-100) yielded the highest activity for most tissues. For striated muscle, however, slightly more activity was extracted if the phosphate buffer contained both 1% Triton X-100 and 0.5 M NaCl. It was also noted that the degree of homogenization of some tissues (e.g. striated muscle) must be increased for maximal solubilization of all cholinesterase activity. Subsequent studies developed a method for assessing the level of acetylcholinesterase, butyrylcholinesterase and total cholinesterase activity in these tissues using an automated analyzer. In conclusion, automated assay of acetylcholinesterase activity in cholinergically innervated tissues in the rat (other than brain) is achievable and relatively convenient.     

Neurotoxic esterase: characterization of the solubilized enzyme and the conditions for its solubilization from chicken brain microsomal membranes with ionic, zwitterionic, or nonionic detergents

Neurotoxic esterase (NTE) is a membrane-bound protein found in highest concentration in brain and lymphocytes. The enzyme has no known physiological function, but its organophosphorylation and aging in neural tissue are thought to trigger the pathogenesis of organophosphorus-induced delayed neuropathy (OPIDN). Solubilization of NTE from microsomal membranes from hen or chick brain was studied with ten detergents encompassing ionic, zwitterionic, or nonionic types. Corrected yields of NTE solubilized over a range of [detergent]/[protein] ratios were determined by dividing the activity not sedimenting in detergent at 100,000 g for 60 min at 4 degrees by the activity in the original microsomal fraction with no detergent present. Highest corrected yields were obtained with sodium cholate (44%), Triton X-100 (48%), and nonyl-GPS (57%). Partial loss of NTE activity occurred in the presence of detergent which could be prevented by the inclusion of asolectin in the solubilization preparation. NTE could not be solubilized by omitting detergent or by substituting 2 M NaCl for detergent. Mipafox pI50 values obtained from complete titration curves carried out on NTE solubilized in Triton X-100, sodium cholate, or sodium cholate/asolectin were indistinguishable from the value for native enzyme from brain homogenate. These results indicate that NTE exhibits the properties of an integral membrane protein with lipid dependence. The enzyme can be solubilized in good yield with a variety of detergents with retention of its characteristic differential inhibition by paraoxon and mipafox, a necessary prelude to bulk purification of the enzymatically active protein.     

Characterization of a phosphatidic acid phosphatase from rat brain cell membranes

We have characterized a phosphatidic acid phosphatase (PAP, EC 3.1.3.4) that is associated with cell membranes from rat brain using [³²P] phosphatidic acid as substrate in a simple assay. The enzyme could be activated by Triton X-100, cholic acid and Chaps and inhibited by Lubrol PX and sodium dodecyl sulfate. The optimal pH was between 6.0 and 7.0. Mg²⁺ was not essential for enzyme activity. The enzyme activity was decreased by about 50% by Ca²⁺ at concentrations of 0.1 to 1 mmol/1. Zn²⁺ inhibited the enzyme by 50% at concentrations of about 10 mol/l in the absence of, and 100 nmol/1 in the presence (3 mmol/1) of, Triton X-100. NaF decreased the activity by about 50% at concentrations between 0.3 and 1 mmol/l when Triton X-100 was added, but did not inhibit the enzyme if the detergent was not present. N-Ethylmaleimide (NEM) did not affect the enzyme. In the absence of Triton X-100, propranolol and metoprolol enhanced the PAP activity. In the presence of 3 mmol/1 Triton X-100, the enzyme was inhibited by about 50% by propranolol at a concentration of 10 mmol/l, whereas metoprolol caused only a slight inhibition of PAP. The K _m for phosphatidic acid was 150 mol/1 and was changed to 20 mol/1 by 3 mmol/1 Triton X-100 without the V _max being changed. Enzyme activity could be solubilized by 1–5% (w/v) Triton X-100. Gel filtration chromatography showed a M _r of 320000. This membrane-associated PAP from neuronal tissue probably belongs among the NEM-insensitive forms of PAP enzymes which have been proposed to play a role in transmembrane signal transduction via phospholipase D. Correspondence to: Ariane Hoer at the above address     

Concentration-dependent binding of chlorpyrifos oxon to acetylcholinesterase.

The organophosphorus insecticides have been known for many years to cause cholinergic crisis in humans as a result of the inhibition of the critical enzyme acetylcholinesterase. The interactions of the activated, toxic insecticide metabolites (termed oxons) with acetylcholinesterase have been studied extensively for decades. However, more recent studies have suggested that the interactions of certain anticholinesterase organophosphates with acetylcholinesterase are more complex than previously thought since their inhibitory capacity has been noted to change as a function of inhibitor concentration. In the present report, chlorpyrifos oxon (O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphate) was incubated with human recombinant acetylcholinesterase in the presence of p-nitrophenyl acetate in order to better characterize kinetically the interactions of this oxon with enzyme. Determination of the dissociation constant, Kd, and the phophorylation rate constant, k2, for chlorpyrifos oxon with a range of oxon and p-nitrophenyl acetate concentrations revealed that Kd, but not k2, changed as a function of oxon concentration. Changes in p-nitrophenyl acetate concentrations did not alter these same kinetic parameters. The inhibitory capacity of chlorpyrifos oxon, as measured by ki (k2/Kd), was also affected as a result of the concentration-dependent alterations in binding affinity. These results suggest that the concentration-dependent interactions of chlorpyrifos oxon with acetylcholinesterase resulted from a different mechanism than the concentration-dependent interactions of acetylthiocholine. In the latter case, substrate bound to the peripheral anionic site of acetylcholinesterase has been shown to reduce enzyme activity by blocking the release of the product thiocholine from the active site gorge. With chlorpyrifos oxon, the rate of release of 3,5,6-trichloro-2-pyridinol is irrelevant since the active site is not available to interact with other oxon molecules after phosphorylation of Ser-203 has occurred.     

Microsomal UDP-glucuronyltransferase in rat liver: oxidative activation

Activation of microsomal UDP-glucuronyltransferase (UDPGT) activity by treatment of hepatic microsomes with either detergents or Fe(3+)/ascorbate pro-oxidant system has been reported; however, definite mechanisms underlying these effects have not been clarified. In this work, we characterize Fe(3+)/ascorbate-induced activation of UDPGT activity prior to solubilization with Triton X-100 and after the oxidation process provoked the solubilization of the enzyme. We observed a time-dependent increase in UDPGT activity up to 20 min. incubation of the microsomes with Fe(3+)/ascorbate (3-times); after 20 min. incubation, however, we observed a time-dependent decrease in this activity to basal levels after 4 hr incubation. Treatment of microsomes with 0.1% Triton X-100 (5 min.) lead to a similar increase in UDPGT activity; higher detergent concentrations produced a dose-dependent decrease in this activity to basal levels with 1% Triton X-100. Interestingly, UDPGT activity was susceptible to activation only when associated to microsomal membranes and the loss of activation correlated with the solubilization of this activity. UDPGT activation by either Fe(3+)/ascorbate or Triton X-100 was correlated with an increase in p-nitrophenol apparent K(m) and V(max) values. This activation was prevented or reversed by the reducing agents glutathione, cysteine or dithiothreitol when it was induced by the Fe(3+)/ascorbate. Furthermore, the latter provoked a significant decrease in microsomal thiol content, effect not observed after treatment with Triton X-100. Our results suggest that the main mechanism responsible for Fe(3+)/ascorbate-induced UDPGT activation is likely to be the promotion of protein sulfhydryl oxidation; this mechanism appears to be different from detergent-induced UDPGT activation.     

Subcellular localization of neuronal nitric oxide synthase in rat small intestine

The subcellular localization of neuronal nitric oxide synthase (NOS I, EC 1.14.13.39) was investigated in the longitudinal muscle/myenteric plexus (LM/MP) preparation of rat small intestine. The presence of NOS I, inducible nitric oxide synthase (NOS II), and endothelial nitric oxide synthase (NOS III) was assessed after homogenization and low-speed centrifugation in a postnuclear supernatant by immunological detection after PAGE and Western blotting. Only NOS I was clearly present, whereas NOS II and NOS III were below detection limits. After high-speed centrifugation of the postnuclear supernatant, soluble and particulate fractions were obtained, and the presence of NOS I in these fractions was investigated by measurement of NOS I immunoreactivity and enzyme activity. We found that 90 +/- 1% of NOS I immunoreactivity and 97 +/- 1% of NOS enzyme activity were confined to the soluble fraction of the tissue. Further immunological analysis demonstrated that washing the particulate fraction revealed detectable amounts of NOS I only after concentration of the washing supernatant. Most particulate NOS I remained in the pellet and therefore represents cell organelle-associated enzyme. No NOS I immunoreactivity could be detected as a soluble protein within organelles of the cell. Particulate NOS I could in part be solubilized by Triton X-100 treatment, and the detection of Triton X-100-soluble NOS I was dependent on the antibody used. In conclusion, our results indicate that NOS I in the LM/MP preparation of rat small intestine is mainly soluble and that the particulate NOS I is partly an intrinsic membrane protein and can partly be solubilized by detergent treatment.     

UDP-glucuronosyltransferase(s) activities towards natural substrates in rat liver microsomes. Kinetic properties and influence of triton X-100 activation

We studied the in vitro capability of hepatic microsomal UDP-glucuronosyltransferase (UDPGT) in male rats to conjugate 22 natural xenobiotics which are known to be excreted as glucuronides in vivo. We clearly demonstrated that the Vmax can range in a decreasing scale for the following families of aglycones: 7-hydroxylated coumarins greater than 2-naphthol and phenols greater than monoterpenoid alcohols greater than 4-hydroxylated coumarins. The Km app. cannot be arranged in the same scale. This suggests that the catalytic mechanism of UDPGT is dependent on the hydroxyl group reactivity rather than on the binding interaction at the active site expressed by the Km app. The effects of various concentrations of detergent (Triton X-100) were determined on specificity (apparent Km) and activity (Vmax). For the 22 aglycones we showed that activation caused a variation in the Vmax which was a function of the concentration in detergent. The maximum of this activation did not always correspond to the same detergent/protein weight ratio. The impact of activation on Km app. was less clear since the variations observed were slightly different.     

水胺硫磷在大鼠肝微粒体的生物转化和代谢动力学

目的 探讨水胺硫磷在大鼠肝微粒体的体外代谢活化产物及代谢动力学特征。方法 应用液相色谱-四级杆-飞行时间质谱(LC/QTOF MS)筛查并鉴定水胺硫磷在大鼠肝微粒体孵育液中的氧化产物。用乙酰胆碱酯酶抑制法考察水胺硫磷及其氧化产物水胺氧磷的抑酶活性。应用液相色谱-三重四级杆串联质谱(LC/Triple-Q MS/MS)定量检测肝微粒体中的水胺硫磷及其代谢产物水胺氧磷,研究水胺硫磷及其产物的消长动力学和氧化产物生成的酶动力学。结果 筛查并鉴定了水胺硫磷在大鼠肝微粒体的氧化脱硫产物水胺氧磷。水胺氧磷对乙酰胆碱酯酶的抑制活性远高于水胺硫磷,IC₅₀值比水胺硫磷低4个数量级,表明水胺硫磷的氧化脱硫反应是一个代谢活化过程。水胺硫磷在大鼠肝微粒体中的半衰期(t_1/2)为14.6 min,外推得到体内肝清除率Cl_H为43.8 ml·min^-1·kg^-1。水胺氧磷的生成符合双相酶动力学模型,K_m,app1为1.12 μmol·L^-1,产物生产最大速率(V_max1)为0.43 μmol·min^-1·g ^-1;蛋白K_m,app2为67.92 μmol·L^-1,V_max2为1.28 μmol·min^-1·g ^-1蛋白。结论 水胺硫磷在大鼠肝微粒体中能快速代谢消除,生成抑酶活性更高的产物水胺氧磷而产生毒性。     

Concentration-dependent interactions of the organophosphates chlorpyrifos oxon and methyl paraoxon with human recombinant acetylcholinesterase

For many decades it has been thought that oxygen analogs (oxons) of organophosphorus insecticides phosphorylate the catalytic site of acetylcholinesterase by a mechanism that follows simple Michaelis-Menten kinetics. More recently, the interactions of at least some oxons have been shown to be far more complex and likely involve binding of oxons to a second site on acetylcholinesterase that modulates the inhibitory capacity of other oxon molecules at the catalytic site. The current study has investigated the interactions of chlorpyrifos oxon and methyl paraoxon with human recombinant acetylcholinesterase. Both chlorpyrifos oxon and methyl paraoxon were found to have k(i)'s that change as a function of oxon concentration. Furthermore, 10 nM chlorpyrifos oxon resulted in a transient increase in acetylthiocholine hydrolysis, followed by inhibition. Moreover, in the presence of 100 nM chlorpyrifos oxon, acetylthiocholine was found to influence both the K(d) (binding affinity) and k(2) (phosphorylation constant) of this oxon. Collectively, these results demonstrate that the interactions of chlorpyrifos oxon and methyl paraoxon with acetylcholinesterase cannot be described by simple Michaelis-Menten kinetics but instead support the hypothesis that these oxons bind to a secondary site on acetylcholinesterase, leading to activation/inhibition of the catalytic site, depending on the nature of the substrate and inhibitor. Additionally, these data raise questions regarding the adequacy of estimating risk of low levels of insecticide exposure from direct extrapolation of insecticide dose-response curves since the capacity of individual oxon molecules at low oxon levels could be greater than individual oxon molecules in vivo associated with the dose-response curve.     

Interaction of non-ionic surfactants with hepatic CYP in Prochilodus scrofa

Cytochromes P450 (CYP) constitute a superfamily of hemeproteins that play a vital role in the metabolism of a wide variety of endogenous and xenobiotic compounds. Xenobiotic metabolism and the role of CYP are of particular interest in studies regarding the prevention of the damage caused by chemical pollutants. We investigated, in this study, the interaction of Triton X-100 and Tween 80 with CYP and antioxidant defenses in Curimbatá, a Brazilian fish. Aiming to clarify the effects of non-ionic surfactants in the monooxigenase system of fish through in vitro study, the effects of Triton X-100 and Tween 80 were analyzed using monooxygenases and antioxidant system as experimental model. Total CYP and EROD were strongly inhibited by Triton X-100 and Tween 80 in a concentration-dependent way; the content of CYP was reduced until zero while EROD activity was completely inhibited in the presence of Triton X-100 and more than 40% inhibited in the presence of Tween 80. Each surfactant causes a different effect on each antioxidant enzyme. No effect was detected in SOD activity in the presence of even Triton X-100 or Tween 80. Triton X-100 increase catalase activity, while Tween 80 decreases this enzyme activity. The molecular structure of the surfactants causes the alteration of this system, since they are able to interact with the microsomal protein, especially with monooxigenase's components, altering their conformation and, consequently destroying their function. Our results suggest that surfactants can interact with components of the microsomal system leading to inhibition of CYP. Therefore, CYP activity, which has been used as a biomarker of xenobiotic exposure, should be used as a marker in association with other enzymes.     

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.     

Activation and degradation of the phosphorothionate insecticides parathion and EPN by rat brain

Cytochrome P-450-dependent monooxygenases are known to activate phosphorothionate insecticides to their oxon (phosphate) analogs by oxidative desulfuration. These activations produced potent anticholinesterases, decreasing the I50 values to rat brain acetylcholinesterase almost 1000-fold (from the 10(-5) M range to the 10(-8) M range). Since the usual cause of death in mammals from organophosphorus insecticide poisoning is respiratory failure resulting, in part, from a failure of the respiratory control center of the brain, we investigated the ability of rat brain to activate and subsequently degrade two phosphorothionate insecticides, parathion (diethyl 4-nitrophenyl phosphorothioate) and EPN (ethyl 4-nitrophenyl phenylphosphonothioate). Microsomes from specific regions (cerebral cortex, corpus striatum, cerebellum, and medulla/pons) of the brains of male and female rats and from liver were incubated with the phosphorothionate and an NADPH-generating system. Oxon production was quantified indirectly by the amount of inhibition resulting in an exogenous source of acetylcholinesterase added to the incubation mixture as an oxon trap. The microsomal activation specific activity was low for brain when compared to liver [0.23 to 0.44 and 5.1 to 12.0 nmol.min-1.(g tissue)-1 respectively]. The mitochondrial fraction of the brain possessed an activation activity for parathion similar to that of microsomes [about 0.35 nmol.min-1.(g tissue)-1 for each fraction], but mitochondrial activity was slightly greater than microsomal activity for EPN activation [0.53 to 0.58 and 0.23 to 0.47 nmole.min-1.(g tissue)-1]. Whole homogenates were tested for their ability to degrade paraoxon and EPN-oxon (ethyl 4-nitrophenyl phenylphosphonate), quantitated by 4-nitrophenol production. Specific activity for oxon degradation in liver was greater than that in brain [31 to 74 and 1.1 to 10.7 nmole.min-1.(g tissue)-1 respectively]. Overall, the brain and liver had about 1.5- to 12-fold higher specific activities for degradation than activation depending on the compound used. These findings demonstrate that the brain possesses both phosphorothionate activation and oxon degradation abilities, both of which may be significant during exposures to organophosphorus insecticides.     

An active and water-soluble truncation mutant of the human UDP-glucuronosyltransferase 1A9

The UDP-glucuronosyltransferases (UGTs) are integral membrane proteins, and previous attempts to generate a water-soluble UGT by removing the single trans-membrane helix yielded inactive and membrane-bound proteins. We have now replaced the 45 C-terminal amino acids of the human UGT1A9, including its trans-membrane helix, with a fusion peptide ending with six His residues. Detergent-free extraction of insect cells expressing this mutant, UGT1A9Sol, released scopoletin glucuronidation activity into the supernatant, and subsequent ultracentrifugation did not sediment that activity. UGT1A9Sol was purified by immobilized metal affinity chromatography (IMAC) in the absence of detergents throughout the entire process. The IMAC purification increased somewhat the apparent K(m) of UGT1A9 toward scopoletin and rendered the enzyme sensitive to freezing. The activity of UGT1A9Sol in the cell extract was partly inhibited by Triton X-100, irrespective of the presence or absence of phospholipids. UGT1A9Sol exhibited a relatively high rate of scopoletin glucuronidation, whereas its activity toward 1-naphthol, entacapone, umbelliferone, and 4-nitrophenol was much lower. The kinetics and substrate specificity of UGT1A9Sol resembled the detergent-suspended full-length UGT1A9 rather than the membrane-bound UGT1A9. The apparent K(m) value of UGT1A9Sol for scopoletin was similar to that of the full-length UGT1A9 in the presence of detergent, but much higher than the respective value in the membrane-bound enzyme. The results suggest that either the detergent binding to the trans-membrane helix within the full-length UGT1A9, or the removal of this helix by gene manipulation, affect the interaction of the enzyme with its aglycone substrate in a similar manner.     

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.     

Cytosolic and membrane-bound nitric oxide synthase.

Cytotoxic activated macrophages were sonicated and centrifuged. The activity of nitric oxide (NO) synthase was present in the supernatant and independent of Ca2+. The pellets were washed three times and treated with buffer containing 0.1% Triton X-100 or buffer alone, followed by centrifugation. The supernatant containing Triton X-100 showed NO synthase activity that was dependent on Ca2+, whereas the supernatant without the detergent had little activity. These data suggest that there are two forms of NO synthase: cytosolic and membrane-bound enzymes.     

The effect of detergents on the purified flavin-containing monooxygenase of mouse liver, kidney and lungs.

1. The effect of various commonly used membrane solubilizing detergents on the activity of the microsomal xenobiotic metabolizing enzyme, the flavin-containing monooxygenase (FMO) purified from mouse liver, kidney and lungs was determined. 2. Regardless of the type of detergent used, the effect on the enzyme activity was variable depending on the type of substrate used. 3. Emulgen 911 concentrations of up to 10% had very little effect on thiobenzamide-S-oxidation by liver, kidney or lung FMO. 4. While Emulgen 911 increased substrate dependent NADPH oxidation rate by thiourea and thioacetamide, it drastically reduced the activity toward the organophosphorous compounds, disulfoton, fenthion, fonofos and phorate at low concentrations. 5. Activities of fenthion, phorate and fonofos were decreased by 80, 65 and 55% by the inclusion of 0.25% Emulgen 911 in the assay mixture. 6. This decline in FMO activity for phorate was evident regardless of the type of detergent used. In contrast, thiourea dependent NADPH oxidation rate in the presence of various detergents was variable. 7. Thiourea oxidation rate was decreased by cholate and Zwittergent 3-12, whereas it was increased in the presence of Emulgen 911, Triton X-100 and Tween 20. 8. This study shows that before FMO activity is determined in the presence of detergents their effects should be carefully evaluated.     

Effect of triton X-100 on the conjugation of tetrahydrocortisone, in vitro

The detergent, Triton X-100, increased the conjugation of estrone, estradiol and tetrahydrocortisone (THE) by uridine diphosphate glucuronyl transferase (UDPGT) in rat liver microsomes; the maximal increase of the conjugation of these substrates was measured when the concentration of Triton in the incubation mixtures was 0.05 per cent. However, the magnitude of the increase and the effect seen with varying Triton concentration were substrate dependent, which is consistent with the hypothesis that multiple forms of UDPGT may be present in hepatic microsomes. The effects of various compounds which had previously been shown to either increase or decrease the conjugation of THE in non-activated enzyme preparations were re-examined in Triton-activated preparations. Compounds such as β-diethylaminoethyldiphenylpropylacetate (SKF-525A) and 7-hydroxychlorpromazine which inhibited conjugation in non-activated preparations also inhibited conjugation in Triton-activated preparations. Alternatively, the demethylated metabolites of chlorpromazine, which increased activity in non-treated preparations, decreased activity slightly in preparations maximally stimulated by Triton. Bisubstrate kinetic analysis of the THE conjugation of UDPGT also revealed differences between the properties of the non-treated and Triton-activated enzyme preparations. Triton activation caused an increase in the V_max of the reaction in the forward direction while having an insignificant effect on the dissociation constant for THE.     

In vitro sensitivity of cholinesterases and [3H]oxotremorine-M binding in heart and brain of adult and aging rats to organophosphorus anticholinesterases

Organophosphorus (OP) insecticides elicit toxicity via acetylcholinesterase inhibition, allowing acetylcholine accumulation and excessive stimulation of cholinergic receptors. Some OP insecticides bind to additional macromolecules including butyrylcholinesterase and cholinergic receptors. While neurotoxicity from OP anticholinesterases has been extensively studied, effects on cardiac function have received less attention. We compared the in vitro sensitivity of acetylcholinesterase, butyrylcholinesterase and [³H]oxotremorine-M binding to muscarinic receptors in the cortex and heart of adult (3 months) and aging (18 months) rats to chlorpyrifos, methyl parathion and their active metabolites chlorpyrifos oxon and methyl paraoxon. Using selective inhibitors, the great majority of cholinesterase in brain was defined as acetylcholinesterase, while butyrylcholinesterase was the major cholinesterase in heart, regardless of age. In the heart, butyrylcholinesterase was markedly more sensitive than acetylcholinesterase to inhibition by chlorpyrifos oxon, and butyrylcholinesterase in tissues from aging rats was more sensitive than enzyme from adults, possibly due to differences in A-esterase mediated detoxification. Relatively similar differences were noted in brain. In contrast, acetylcholinesterase was more sensitive than butyrylcholinesterase to methyl paraoxon in both heart and brain, but no age-related differences were noted. Both oxons displaced [³H]oxotremorine-M binding in heart and brain of both age groups in a concentration-dependent manner. Chlorpyrifos had no effect but methyl parathion was a potent displacer of binding in heart and brain of both age groups. Such OP and age-related differences in interactions with cholinergic macromolecules may be important because of potential for environmental exposures to insecticides as well as the use of anticholinesterases in age-related neurological disorders.     

A comparative study on the relationship between various toxicological endpoints in Caenorhabditis elegans exposed to organophosphorus insecticides

The toxicity of 10 organophophorus (OP) insecticides-acephate, dimethoate, dichlorvos, dicrotophos, monocrotophos, methamidophos, phosphamidon, omethoate, phosdrin, and trichlorfon-was evaluated in Caenorhabditis elegans using lethality, movement, and acetylcholinesterase (AChE) activity as the endpoints after a 4-hr- exposure period. The OP insecticides tested showed LC50 values ranging from 0.039 mM (for dichlorovs) to 472.8 mM (for methamidophos). The order of toxicity for lethality and movement was not significantly different when tested using the rank order correlation coefficient. AChE activity was markedly affected by all the OP insecticide exposures that caused significant inhibition in movement, indicating that the mechanism of toxicity of OP insecticides in C. elegans is the same as in higher animals. All OP insecticides induced greater than 50% inhibition of AChE at the lowest tested OP insecticide concentration resulting in inhibition in movement. While a significant correlation was evident between LC50 values in C. elegans and the LD50 values in rats for the 10 OP insecticides studied, a correlation was not evident between EC50 values in C. elegans and LD50 values in rats. Overall, the two endpoints, LC50 and movement, were more reliable and easier to perform than measurement of AChE activity in C. elegans for determining the toxicity of OP insecticides. Further, ranking of these endpoints with respect to the OP insecticides studied indicates that these parameters in C. elegans are predictive of OP insecticides mammalian neurotoxicity.     

S-oxygenation of the thioether organophosphate insecticides phorate and disulfoton by human lung flavin-containing monooxygenase 2

Phorate and disulfoton are organophosphate insecticides containing three oxidizable sulfurs, including a thioether. Previous studies have shown that only the thioether is oxygenated by flavin-containing monooxygenase (FMO) and the sole product is the sulfoxide with no oxygenation to the sulfone. The major FMO in lung of most mammals, including non-human primates, is FMO2. The FMO2*2 allele, found in all Caucasians and Asians genotyped to date, codes for a truncated, non-functional, protein (FMO2.2A). Twenty-six percent of individuals of African descent and 5% of Hispanics have the FMO2*1 allele, coding for full-length, functional protein (FMO2.1). We have here demonstrated that the thioether-containing organophosphate insecticides, phorate and disulfoton, are substrates for expressed human FMO2.1 with Km of 57 and 32 microM, respectively. LC/MS confirmed the addition of oxygen and formation of a single polar metabolite for each chemical. MS/MS analysis confirmed the metabolites to be the respective sulfoxides. Co-incubations with glutathione did not reduce yield, suggesting they are not highly electrophilic. As the sulfoxide of phorate is a markedly less effective acetylcholinesterase inhibitor than the cytochrome P450 metabolites (oxon, oxon sulfoxide or oxon sulfone), humans possessing the FMO2*1 allele may be more resistant to organophosphate-mediated toxicity when pulmonary metabolism is an important route of exposure or disposition.