Role of carboxylesterase in protection against soman toxicity

Rats were injected intraperitoneally with phenobarbital (PB) and 3-methylcholanthrene (MC) which are microsomal enzyme inducers, and methyl iodide (MeI), cobalt chloride (CoCl2) and tri-o-cresyl phosphate (TOCP) which are inhibitors of the enzymes glutathione transferase, cytochrome (cyt) P-450 and carboxylesterase, respectively, and then challenged with soman (i.p.) to know its LD50. Pretreatment with PB and MC increased and TOCP decreased, whereas MeI as well as CoCl2 did not alter the LD50 value of soman in rats. The 1/2 LD50 dose of soman did not affect the liver microsomal cyt P-450 level, but significantly lowered carboxylesterase (CaE) and cholinesterase (ChE) activities in liver microsomes and in blood plasma. Induction of plasma CaE was more important than microsomal CaE in PB-mediated protection against soman toxicity. Gel filtration of plasma into four protein fractions for their relative soman binding capacity showed that a high-molecular-weight protein fraction (180,000 daltons on SDS-PAGE) which had no CaE activity could bind soman 6 times more than the low-molecular-weight CaE-containing protein fraction (60,000 daltons on SDS-PAGE).     

Alteration of hepatic carboxylesterase activity by soman: inhibition in vitro and enhancement in vivo

1. Hydrolysis of the drug esters procaine, chloramphenicol succinate, and prednisolone succinate was studied. Addition of soman to guinea pig liver microsomes caused a dose-dependent inhibition of hydrolysis of all three substrates; at the highest soman concentration (1 microM), ester hydrolysis was totally abolished. 2. Ester hydrolysis was also measured in liver microsomes from guinea pigs pretreated with soman at a low dose (10% of LD50) or at a high dose (90% of LD50) either 1 h or 12 h before killing. Plasma-cholinesterase activity was decreased in all pretreated animals. Liver carboxylesterase activity, measured with the three drug substrates and by hydrolysis of 4-nitrophenyl acetate was increased by all pretreatments. 3. This enhancing effect varies with the substrate and increases with dose of soman. The 12 h pretreatment produced a greater increase in activity than did the 1 h pretreatment. 4. These studies indicate that soman is a potent inhibitor of carboxylesterase activity in vitro but increases the activity of the liver enzyme when administered in vivo.     

抑制大鼠血浆羧酸酯酶对伊立替康代谢的体外研究

目的研究抑制大鼠血浆羧酸酯酶对伊立替康代谢的体外方法。方法建立伊立替康含量测定方法,考察不同pH和高中低质量浓度十二烷基硫酸钠对羧酸酯酶的抑制效果。结果降低血浆pH可以部分抑制羧酸酯酶的活性;随着十二烷基硫酸钠质量浓度的增加,羧酸酯酶活性逐渐降低,当质量浓度达到0.4g.mL-1时完全抑制活性。结论十二烷基硫酸钠可以抑制羧酸酯酶活性,提高药物血浆稳定性,便于分析测定。     

Iso-OMPA-induced potentiation of soman toxicity in rat correlates with the inhibition of plasma carboxylesterases

Recently, the question was raised as to why iso-OMPA, generally known as a selective irreversible inhibitor of butyrylcholinesterase (BuChE), potentiates soman toxicity in rats but not in mice. Mice are known to have higher carboxylesterase (CarbE) and lower BuChE activity in plasma than rat. It could be hypothesized that it is the iso-OMPA inhibition of plasma CarbE, and not of BuChE, which is responsible for potentiation of soman toxicity in iso-OMPA-pretreated rats. In order to test this hypothesis two doses of iso-OMPA were administered to rats prior to soman. The two doses were selected in such a way that both were high enough to inhibit more than 90% of plasma BuChE activity; plasma CarbE activity, however, was only slightly inhibited by the lower and substantially by the higher dose of iso-OMPA. Our results demonstrate that iso-OMPA-induced potentiation of soman toxicity correlates with the inhibition of CarbE and not with the inhibition of BuChE activity in rat plasma. Relative resistance of mice to iso-OMPA-induced potentiation of soman toxicity could therefore be explained by a higher proportion of CarbE activity remaining uninhibited after iso-OMPA pretreatment. By having their active centers unoccupied, CarbE molecules can bind soman and reduce its concentration in neuronal tissue and motor end-plates.This is a summarized report. The full material is available from the authors on request     

Role of carboxylesterase in soman, sarin and tabun poisoning in rats

TOCP (triorthocresyl phosphate) inhibits carboxylesterase (CarbE) activity in rat plasma and liver and significantly increases soman, sarin and tabun toxicity. Application of these agents after TOCP caused strong additional inhibition of CarbE and cholinesterases (ChE) in rat red blood cells, plasma, liver, brain, diaphragm and intercostal muscle. After phenobarbital pretreatment, which induced CarbE activity in plasma and liver by 80% and that of plasma ChE by 33%, acute toxicity of soman and tabun was greatly decreased, while that of sarin remained unaffected.     

iso-OMPA-induced potentiation of soman toxicity in rat

Male Sprague-Dawley rats, injected with the irreversible acetylcholinesterase (AChE) inhibitor soman (pinacolyl methylphosphonofluoridate) 25 g/kg sc, showed no signs of toxicity. Pretreatment with iso-OMPA (tetra-isopropylpyrophosphoramide) 1 mg/kg sc 1 h before the soman administration, caused severe signs of hypercholinergic activity, similar to those seen with an acute signs-producing nonlethal dosage (100 g soman/kg sc). Within 1 h iso-OMPA alone significantly reduced the activity of carboxylesterases (CarbE) in all tissues studied and butyrylcholinesterase (BuChE) activity was significantly reduced in plasma (22%) and liver (27%). Soman (25 g/kg) alone significantly reduced the plasma activity of CarbE (15%), BuChE (53%) and AChE (18%), but had no effect on these enzymes of liver. The combined treatment of iso-OMPA and soman, however, reduced CarbE activity in liver (0%) and produced significantly greater effects than iso-OMPA or soman alone on AChE and BuChE in all the brain areas and skeletal muscles tested. The number of necrotic lesions found in skeletal muscles was many times higher with the combined treatment than seen with soman (25 g/kg) alone, and was equal to those seen with an acute toxicity signs-producing dose of soman. It is concluded that the observed iso-OMPA-induced potentiation of soman toxicity is probably caused via reduced nonspecific binding sites (BuChE and CarbE) for soman leading to greater inhibition of AChE.     

Diphenyl ditelluride effect on embryo/fetal development in mice: interspecies differences

It is well established that diphenyl ditelluride, (PhTe)(2), is a potent teratogen in rats, however, little is known about its effects on embryo/fetal development in mice. The present study was undertaken to investigate whether any differences exist on embryo/fetal development of mice exposed to (PhTe)(2) during distinctive periods of gestation compared to rats. Dams were treated subcutaneously (s.c.) with 0.12 or 60.0 mg/kg (PhTe)(2) on gestational day (GD) 4, 8 or 14. Cesarean section was performed on GD18 and external and skeletal alterations were examined. The lower dose did not affect any parameter evaluated in mouse fetuses. The maternal body weight for 60 mg/kg (PhTe)(2) groups, at all periods studied, was not affected. Maternal liver and spleen weights were increased at GD8. At GD14, maternal relative weight of kidney was also increased. A significant reduction in the number of implantation sites at GD4 was found. At GD4 and GD14, there was a reduction in the fetal weight and biometry. A few signs of reduced ossification in sternebrae and limbs were observed at GD14 in (PhTe)(2) group. In conclusion, (PhTe)(2) was not toxic to dams and affected some fetal endpoints only at the dose about 500-fold higher than the dose that was teratogenic in rats, suggesting a different developmental toxicity induced by (PhTe)(2) among species. Thus, the mice were less susceptible to toxic effects induced by (PhTe)(2) than were rats.     

Esterase activities and soman toxicity in developing rat

The activity of carboxylesterase and cholinesterase in plasma, liver and lung of young rats at different ages (5-31 days old) have been measured. The cholinesterase activity in the tissues of both sexes were almost constant during the development, and were similar to adult male activity. The carboxylesterase activity towards methyl butyrate and 4-nitrophenyl butyrate in plasma of both sexes increased from negligible (5 days old) to adult male value (31 days old). The carboxylesterase activities in liver increased markedly during the period, whereas the lung activities increased only slightly. The toxicity of soman was 6-7 fold higher in 5 days old rats compared to 30 days old rats. The decrease in toxicity correlated well with the increase in plasma carboxylesterase.     

Function of plasma and microsomal enzymes in soman toxicity

Such organophosphorous (OP) nerve agents as sarin (isopropyl methylphosphonofluoridate) and soman (pinacolyl methylphosphonofluoridate) are effective inhibitors of acetylcholinesterases (AChE), butyrylcholinesterases (BChE) and carboxylesterases (CaE). The acute toxicity of these compounds in mammals is known to be mediated through inhibition of AChEs, which leads to increased acetylcholine (ACh) levels. The aim of this study was to compare the significance of the plasma CaEs, microsomal CaEs and CYP450 enzymes in detoxification of soman with and without physostigmine treatment. The mice received physostigmine (0.1 mg/kg body wt) intravenously (i.v.) 10 min prior to the intraperitoneal (i.p.) injection of soman (0.400-0.650 mg/kg body wt in olive oil). To avoid possible signs of poisoning, the animals received atropine sulfate (37.5 mg/kg body wt in saline) subcutaneously (s.c.) immediately after the soman administration. In the present study, the inhibitory effect of soman was greater in plasma CaE than in hepatic microsomal CaE fraction. In addition, soman or the combination of soman-physostigmine had no remarkable effect on the microsomal CaE or P4502B activities. In spite of this, however, the microsomal CaEs might offer more protection against multiple LD50s of soman.     

The specificity of carboxylesterase protection against the toxicity of organophosphorus compounds.

The ability of endogenous carboxylesterase (CaE) to protect against the lethal effects of a variety of organophosphorus (OP) compounds was examined in rats. The in vivo protection provided by endogenous CaE was measured by the difference in the LD50 values of OP compounds in control rats and rats whose CaE activity had been inhibited by sc injection with 2 mg/kg of 2-(O-cresyl)-4H-1,3,2-benzodioxaphosphorin-2-oxide. Endogenous CaE provided significant protection against the in vivo toxicity of soman, sarin, tabun, and paraoxon, but not against dichlorvos, diisopropyl fluorophosphate, or ethoxymethyl-S-[2-(diisopropylamino)ethyl] thiophosphonate (VX). The relationship between the in vivo CaE protection against OP compounds and their relative reactivities with CaE and acetylcholinesterase (AChE) was evaluated by measuring the in vitro bimolecular rate constants (ki) for inhibition of plasma CaE and brain AChE. Except for VX, ki values for CaE inhibition varied less than 10-fold while ki values for AChE inhibition varied 10(5)-fold. The degree of in vivo inhibition of CaE by equitoxic doses of the OP compounds increased as the CaE/AChE ki ratio increased. However, the protective ratio of the LD50 values in control vs CaE-inhibited rats decreased as the CaE/AChE ki ratio increased. This inverse relationship between in vivo CaE protection and relative in vitro reactivity for CaE suggested that CaE detoxication is more important for highly toxic OP compounds (i.e., compounds with high AChE ki values and low LD50 values) than for less toxic compounds.     

Stereoselectivity of enzymes involved in toxicity and detoxification of soman

The fate of the four stereoisomers of soman [0-(1,2,2-trimethylpropyl)-methyl-fluoro phosphonate] has been studied a) in vivo in mouse blood and liver after IP injection of 0.75 × LD₅₀ R_c- and S_c-soman respectively, and b) in vitro upon incubation wih acetyl- und pseudocholinesterase, aliesterase and phosphorylphosphatase. The analytical method used is based on gas chromatography — mass spectrometry with deuterated internal standard.Most soman disappeared very rapidly from blood and liver. In liver, S_CR_P and R_CR_P, the two isomers that preferentially react with cholinesterase, could be detected. The level of S_CR_P, which was higher than that of R_CR_P, could be followed for 17–18 h. In blood only S_CR_P could be detected. The amounts found were fairly constant during the time period 2 min to 4h, and it could even be detected 17–18 h after soman administration.     

The effects of blood flow and detoxification on in vivo cholinesterase inhibition by soman in rats

The in vivo time course of cholinesterase inhibition was measured in brain, lung, spleen, hind limb skeletal muscle, diaphragm, intestine, kidney, heart, liver, and plasma of rats receiving 90 micrograms/kg soman, im. This dose of soman produced severe respiratory depression and transient hypertension, but no significant changes in the cardiac output or heart rate of anesthetized rats. The rate and maximal extent of in vivo cholinesterase inhibition by soman varied widely among the tissues. Although cardiac output was unchanged by soman administration, the blood flow in heart, brain, and lung (bronchial arterial flow and arteriovenous shunts) was increased, whereas blood flow in spleen, kidney, and skeletal muscle was decreased. The relative importance of tissue blood flow, tissue levels of cholinesterase and acetylcholinesterase, and tissue levels of soman-detoxifying enzymes (diisopropyl-fluorophosphatase and carboxylesterase) in determining the in vivo rate and maximal extent of cholinesterase inhibition was examined by multiple regression analysis. The best multiple regression model for the maximal extent of cholinesterase inhibition could explain only 63% of the observed variation. The best multiple regression model for the in vivo rate of cholinesterase inhibition contained three independent variables (blood flow, carboxylesterase, and cholinesterase) and could account for 94% of the observed variation. Of these three variables blood flow was the most important, accounting for 79% of the variation in the in vivo rate of cholinesterase inhibition. This suggests that it may be possible to use a flow-limited physiological pharmacokinetic model to describe the kinetics of in vivo cholinesterase inhibition by soman.     

Analysis of mammalian carboxylesterase inhibition by trifluoromethylketone-containing compounds

Carboxylesterases (CE) are ubiquitous enzymes that hydrolyze numerous ester-containing xenobiotics, including complex molecules, such as the anticancer drugs irinotecan (CPT-11) and capecitabine and the pyrethroid insecticides. Because of the role of CEs in the metabolism of many exogenous and endogenous ester-containing compounds, a number of studies have examined the inhibition of this class of enzymes. Trifluoromethylketone-containing (TFK) compounds have been identified as potent CE inhibitors. In this article, we present inhibition constants for 21 compounds, including a series of sulfanyl, sulfinyl, and sulfonyl TFKs with three mammalian CEs, as well as human acetyl- and butyrylcholinesterase. To examine the nature of the slow tight-binding inhibitor/enzyme interaction, assays were performed using either a 5-min or a 24-h preincubation period. Results showed that the length of the preincubation interval significantly affects the inhibition constants on a structurally dependent basis. The TFK-containing compounds were generally potent inhibitors of mammalian CEs, with Ki values as low as 0.3 nM observed. In most cases, thioether-containing compounds were more potent inhibitors then their sulfinyl or sulfonyl analogs. QSAR analyses demonstrated excellent observed versus predicted values correlations (r2 ranging from 0.908-0.948), with cross-correlation coefficients (q2) of approximately 0.9. In addition, pseudoreceptor models for the TKF analogs were very similar to structures and models previously obtained using benzil- or sulfonamide-based CE inhibitors. These studies indicate that more potent, selective CE inhibitors, containing long alkyl or aromatic groups attached to the thioether chemotype in TFKs, can be developed for use in in vivo enzyme inhibition.     

Effect of pretreatment with sodium phenobarbital on the toxicity of soman in mice

Pretreatment with sodium phenobarbital induces hepatic microsomal enzymes which are responsible for the metabolic breakdown of a large number of endogenous and exogenous chemical compounds. A previous study [K. P. DuBois and F. K. Kinoshita, Proc. Soc. exp. Biol. Med.129, 699 (1968)]reported that phenobarbital pretreatment reduced the toxicity of various organophosphorus anticholinesterases; however, the exact mechanism for the increased detoxification was not investigated. In this study, the effect of phenobarbital pretreatment on the toxicity of soman was investigated. Male mice were injected daily for 4 days with sodium phenobarbital (100 mg/kg, i.p.) and used in the various experiments 24 hr after the last injection. Phenobarbital pretreatment produced a significant increase in liver weight and decreased the sodium pentobarbital (75 mg/kg, i.p.) induced sleep-time to 41 min compared to 141 min in controls. The lethality of soman was reduced following phenobarbital pretreatment. In control mice, the soman 24hr LD₅₀ values (μg/kg) were 130, 393 and 42 following s.c., i.p. and i.v. administration, respectively, whereas in phenobarbital-pretreated mice the soman 24 hr LD₅₀ values (μg/kg) were 261, 746 and 63 following s.c., i.p. and i.v. administration respectively. Acetylcholinesterase activity was increased in the plasma (90%) but not in brain or diaphragm following phenobarbital pretreatment. Liver somanase activity was not affected. Liver aliesterase and serum aliesterase were both increased significantly following phenobarbital pretreatment. An increase in the amount of nonspecific binding sites for soman (esterases in liver and plasma) and not an increase in the metabolism of soman in vivo probably accounts for the protection afforded by phenobarbital pretreatment in mice.     

Neurobehavioral toxicity with low doses of sarin and soman

The acute effects of single subtoxic doses (1/48-1/9 of LD50) of two potent organophosphates (OPs), sarin (12.5 and 50 micrograms/kg i.p.) and soman (4 and 20 micrograms/kg i.p.), were studied on behavior, motor performance and nociception in male Wistar rats. On the elevated plus-maze with two open + two closed arms, higher doses of soman and sarin decreased the proportion of entries made onto open arms (p less than 0.05), while the total number of entries onto open + closed arms was unchanged. On the narrow elevated horizontal bridge, the latencies to reach the safe platform were prolonged with the higher dose of sarin (p less than 0.05) but not with that of soman. On the broad and rod bridges, the latencies of OP-treated rats did not differ significantly from those of controls. OPs did not significantly impair either learning frequency in one-trial passive avoidance test, rotarod performance or nociception in hot plate test. The results suggest that in acutely nontoxic doses sarin and soman affect the behavior of rats, and that the action profiles of the OPs differ from each other. Both soman and sarin change the behavior of rats in the plus-maze test but only sarin seems likely to impair motor coordination/balance.     

Prevention of soman toxicity after the continuous administration of physostigmine

Protective effects of continuous administration of physostigmine alone, or in addition to scopolamine, against soman-induced toxicity were studied in guinea pigs. The results clearly demonstrated that treatment with physostigmine continuously via implanted mini-osmotic pumps for 4 or 7 days prior to soman exposure significantly protected from soman-induced mortality. In vehicle-infused guinea pigs, tremors, convulsions and loss of righting reflex occurred prior to their deaths induced by soman. Although all of the guinea pigs which received physostigmine pretreatment for 4 days prior to soman administration also displayed soman-induced tremors and convulsions, the onsets of these symptoms were significantly delayed. When animals continuously treated with physostigmine received injections of scopolamine 10 min prior to soman injections, there was a decreased incidence of all three toxicity symptoms as well as an increase in the latency to onset of tremors. Scopolamine was also able to reverse toxicity symptoms when soman was administered earlier. In animals which had been continuously treated with physostigmine via mini-osmotic pumps, the protective action against soman-induced toxicity was still apparent. On the contrary, acute physostigmine administration failed to protect against soman lethality. The present results suggest that the prophylactic uses of physostigmine via mini-osmotic pumps might be more useful than the acute bolus administration of physostigmine.     

The hypertensive response to soman and its relation to brain acetylcholinesterase inhibition

Intravenous injection of soman in the rat produced a rapid and dose related increase in blood pressure. The dose response curve was very steep, threshold responses occurring after intravenous injection of 10 micrograms/kg, and maximum increases of about 50 mmHg occurring after 40 micrograms/kg. Heart rate also generally increased. An increase in blood pressure also followed injection of soman subcutaneously, intramuscularly, intraperitoneally and into the cerebral ventricles, although the onset was slower and higher doses were required. The magnitude of the pressor response was correlated with the degree of AChE activity in the cortex, hypothalamus and brain stem, but not in the striatum. The pressor response was aborted or prevented by atropine, but not by methylatropine. It also was prevented by phenoxybenzamine. Atropine increased survival following an LD50 dose of soman; phenoxybenzamine prevented the pressor response but did not alter the survival rate.