A novel tertiary pyridostigmine derivative [3-(N,N-dimethylcarbamyloxy)-1-methyl-Δ3-tetrahydropyridine]: Anticholinesterase properties and efficacy against soman

In an effort to develop an effective centrally acting pretreatment compound against organophosphorus poisons, the tertiary pyridostigmine (Pyr) derivative 3-(N,N-dimethylcarbamyloxy)-1-methyl-Δ³-tetrahydropyridine (THP) was synthesized and studied for its anticholinesterase properties, as well as its efficacy against soman intoxication in guinea pigs. Injection of THP (262 μg/kg, im) into adult male guinea pigs caused inhibition of blood (30%) and brain (25%) acetylcholinesterase (AChE), showing that THP penetrates the blood-brain barrier. Pyr (131 μg/kg, im) caused AChE inhibition in the blood (59%), but not in the brain. The inhibitory potencies of THP and Pyr were compared by determining their IC50 values for in vitro inhibition of both AChE (brain, erythrocyte) and pseudo-cholinesterase (plasma) in three mammalian species (guinea pig, rat, rabbit). THP, although effective in inhibiting both types of cholinesterase, was in general less potent than Pyr. Pretreatment of guinea pigs with THP 9262 μg/kg, im) plus Pyr (131 μg/kg, im), 30 min prior to subcutaneous soman challenge, with no antimuscarinic or oxime treatment, protected 60% of the animals against 2 × LD50 of soman. Neither THP nor Pyr alone was effective. The protective pretreatment regimen did not prevent convulsions, but shortened the recovery time in surviving animals (median recovery time 1.6 hr, compared to 24 hr in control and other groups of animals pretreated with THP or Pyr alone). A combination of THP and Pyr thus appears to provide a means of evaluating the relative importance of selective peripheral plus central vs peripheral AChE protection against soman.     

Acetylcholinesterase inhibition by (+)physostigmine and efficacy against lethality induced by soman

The optical isomer (+)Physostigmine [(+)Phy] is a very weak anticholinesterase. In a recent report, pretreatment with (+)Phy, at a dose which failed to inhibit acetylcholinesterase (AChE), and atropine provided efficacy against a lethal dose of sarin (SYNAPSE:2, 139, 1988). It was of interest to see whether (+)Phy could protect against soman at a dose which caused only marginal inhibition of the whole blood (WB) AChE in guinea pigs (GPs). (-)Phy (0.15 mg/kg, im) and (+)Phy (10.0 mg/kg, im) produced nearly 70% inhibition of WB AChE at 30 min whereas (+)Phy (0.15 mg/kg, im) caused only marginal inhibition. Groups of guinea pigs (20/group) were dosed, im, with (-)Phy (0.15 mg/kg), (+)Phy (0.15 mg/kg), (+)Phy (10.0 mg/kg) or vehicle (0.5 ml/kg) respectively in one thigh while the mild anticholinergic trihexyphenidyl (THP), 2.0 mg/kg, was injected into the other thigh of 10 animals from each of the respective groups. Thirty min after pretreatment, all animals were challenged with soman (60 micrograms/kg, sc; 2 LD50s); this dose of soman is lethal in unprotected animals. (-)Phy or (+)Phy (10 mg/kg) alone protected nearly 50% from soman lethality, and in combination with THP, all animals survived. In contrast, (+)Phy (0.15 mg/kg; alone or together with THP) was completely ineffective against a 2 LD50 challenge of soman. These data support the hypothesis that protection against soman-induced lethality is related to the degree of carbamylation of the AChE just prior to challenge.     

Acetylcholinesterase Inhibition by (+)Physostigmine and Efficacy against Lethality Induced by Soman

ABSTRACT

The optical isomer (+)Physostigmine [(+) Phy] is a very weak anticholinesterase. In a recent report, pretreatment with (+)Phy, at a dose which failed to inhibit acetylcholinesterase (AChE), and atropine provided efficacy against a lethal dose of sarin (SYNAPSE:2,139,1988). It was of interest to see whether (+)Phy could protect against soman at a dose which caused only marginal inhibition of the whole blood (WB) AChE in guinea pigs (GPs). (-)Phy (0.15 rog/kg, im) and (+)Phy (10.0 mg/kg, im) produced nearly 70% inhibition of WB AChE at 30 min whereas (+)Phy (0.15 rog/kg, im) caused only marginal inhibition. Groups of guinea pigs (20/group) were dosed, im, with (-)Phy (0.15 mg/kg), (+)Phy (0.15 rog/kg), (+)Phy (10.0 mg/kg) or vehicle (0.5 ml/kg) respectively in one thigh while the mild anticholinergic trihexyphenidyl (THP), 2.0 mg/kg, was injected into the other thigh of 10 animals from each of the respective groups. Thirty min after pretreatment, all animals were challenged with soman (60 ug/kg, sc; 2 LD₅₀S); this dose of soman is lethal in unprotected animals. (-)Phy or (+)Phy (10 rog/kg) alone protected nearly 50% from soman lethality, and in combination with THP, all animals survived. In contrast, (+)Phy (0.15 mq/kq; alone or together with THP) was completely ineffective against a 2 LD₅₀ challenge of soman. These data support the hypothesis that protection against soman-induced lethality is related to the degree of carbamylation of the AChE just prior to challenge.     

Differences in the Toxicity of Soman in Various Strains of Mice

Differences in Toxicity of Soman in Various Strains of Mice.Clement, J.G., Hand, B.T. and Shiloff, J.D. (1981). Fundam.Appl. Toxicol. 1:419–420. The acute toxicity of somanwas assessed in eight strains of mice (ALAS, CD® -1, C57BL,CF1®, CFW® C3H, DBA and BALB/c). In fasted animals theLD₅₀ values for soman varied from 98 µg/kg in C57BL miceto 151 µg/kg in BALB/c mice. In general in non-fastedmice the soman LD⁵⁰ was not significantly changed except inALAS strain where the soman LD⁵⁰ value increased significantly.The different sensitivities to soman poisoning among the variousstrains does not appear to be due totally to differences inlevel of brain acetylcholinesterase. Fasting had no significanteffect on the activity of brain acetylcholinesterase and somantoxicity in CD® -1 mice whereas, upon fasting ALAS strainmice for 18 hr, there was a 25% decrease in brain acetylcholinesterasewhich could explain their increased sensitivity to soman however,it is possible that other biochemical changes may also playa role.     

Reduction by Pyridostigmine Pretreatment of the Efficacy of Atropine and 2-PAM Treatment of Sarin and VX Poisoning in Rodents

Reduction by Pyridostiginine Pretreatment of the Efficacy ofAtropine and 2-PAM Treatment of Sarin and VX Poisoning in Rodents.KOPLOVITZ, I., HARRIS, L. W., ANDERSON, D. R., LENNOX, W. J.,AND STEWART, J. R. (1992). Fundam. Appl. Toxicol. 18, 102–106. This study concerned the effect of pyridostigmine pretreatmenton (a) the antidotal efficacy of atropine and 2-PAM in sarin,tabun, and VX poisoning in mice and guinea pigs and on (b) theoxime-induced reactivation of VX-inhibited whole blood acetyicholinesterase(AChE) of guinea pigs. One hour prior to organophosphate (OP)challenge with sarin, tabun, or VX, animals were given oraldoses of pyridostiginine to induce approximately 30 and 60%inhibition of whole blood AChE; controls received vehicle. Micewere challenged im and guinea pigs sc with the OP compounds.Treatment with atropine (11.2 mg/kg to mice; 32 mg/kg to guineapigs) plus 2-PAM (25 mg/kg) was given im at 10 sec postchallengein mice and 1 min postchallenge in guinea pigs. In the reactivationexperiments, pyridostigmine or saline was given im to guineapigs 30 min prior to VX (8.24 µg/kg, sc), atropine (16mg/kg) was given im at 1 mm, and 2-PAM (25 mg/kg) at 16 minpostchallenge. Pyridostigmine significantly enhanced the efficacyof atropine and 2-PAM against tabun in both species. In contrast,pyridostigmine reduced or did not increase the efficacy of atropineand 2-PAM against sarin or VX in both species. Recovery of VX-inhibitedAChE by 2-PAM was decreased significantly in pyridostigminepretreated animals. The results suggest that pyridostigminepretreatment may adversely effect the efficacy of atropine and2-PAM as antidotes for VX and sarin intoxication.     

Prophylaxis against Soman Inhalation Toxicity in Guinea Pigs by Pretreatment Alone with Human Serum Butyrylcholinesterase

Human butyrylcholinesterase (HuBChE) has previously been shownto protect mice, rats, and monkeys against multiple lethal toxicdoses of organophosphorus (OP) anticholinesterases that werechallenged by iv bolus injections. This study examines the conceptof using a cholinesterase scavenger as a prophylactic measureagainst inhalation toxicity, which is the more realistic simulationof exposure to volatile OPs. HuBChE-treated awake guinea pigswere exposed to controlled concentration of soman vapors rangingfrom 417 to 430 µg/liter, for 45 to 70 s. The correlationbetween the inhibition of circulating HuBChE and the dose ofsoman administered by sequential iv injections and by respiratoryexposure indicated that the fraction of the inhaled dose ofsoman that reached the blood was 0.29. HuBChE to soman molarratio of 0.11 was sufficient to prevent the manifestation oftoxic signs in guinea pigs following exposure to 2.17 theinhaled LD50 dose of soman (ILD50, 101 µg/kg). A slightincrease in HuBChE:soman ratio (0.15) produced sign-free animalsafter two sequential respiratory exposures with a cumulativedose of 4.5 ILD50. Protection was exceptionally high and farsuperior to the currently used traditional approach that consistedof pretreatment with pyridostigmine and postexposure combinedadministration of atropine, benactyzine, and an oxime reactivator.Quantitative analysis of the results suggests that in vivo sequestrationof soman, and presumably other OPs, by exogenously administeredHuBChE, is independent of the species used or the route of challengeentry. This assuring conclusion significantly expands the databaseof the bioscavenger strategy that now offers a dependable extrapolationfrom animals to human.     

Inhibition of Hen Brain Acetylcholinesterase and Neurotoxic Esterase by Chlorpyrifos in Vivo and Kinetics of Inhibition by Chlorpyrifos Oxon in Vitro: Application to Assessment of Neuropathic Risk

Chlorpyrifos (CPS; O,O-diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate;Dursban) is a widely used broad-spectrum organophosphorus (OP)insecticide. Because some OP compounds can cause a sensory-motordistal axonopathy called OP compound-induced delayed neurotoxicity(OPIDN), CPS has been evaluated for this paralytic effect. Earlystudies of the neurotoxicity of CPS in young and adult hensreported reversible leg weakness but failed to detect OPIDN.More recently, a human case of mild OPIDN was reported to resultfrom ingestion of a massive dose (about 300 mg/kg) in a suicideattempt. Subsequent experiments in adult hens (the currentlyaccepted animal model of choice for studies of OPIDN) showedthat doses of CPS in excess of the LD₅₀ in atropine-treatedanimals inhibited brain neurotoxic esterase (NTE) and producedmild to moderate ataxia. Considering the extensive use of CPSand its demonstrated potential for causing OPIDN at supralethaldoses, additional data are needed to enable quantitative estimatesto be made of the neuropathic risk of this compound. Previouswork has shown that the ability of OP insecticides to causeacute cholinergic toxicity versus OPIDN can be predicted fromtheir relative tendency to inhibit the intended target, acetylcholinesterase(AChE), versus the putative neuropathic target, NTE, in braintissue. The present study was designed to clarify the magnitudeof neuropathic risk associated with CPS exposures by measuringhen brain AChE and NTE inhibition following dosing in vivo anddetermining the bimolecular rate constant of inhibition (k₁)for each enzyme by the active metabolite, CPS oxon (CPO), invitro. CPS administered to atropine-treated adult hens at 0,75, 150, and 300 mg/kg po in corn oil produced mean values forbrain AChE inhibition 4 days after dosing of 0, 58, 75, and86%, respectively, and mean values for brain NTE inhibitionof 0, 21, 40, and 77%, respectively. Only the high dose (sixtimes the unprotected LD₅₀ in hens) produced NTE inhibitionabove the presumed threshold of 70%, and these animals werein extremis from cholinergic toxicity at the time of euthanizationdespite continual treatment with atropine. When 150 mg/kg CPSpo in corn oil was given to atropine-treated hens on Day 0,inhibition on Days 1, 2,4, 8, and 16 for brain AChE was 86,82, 72, 44, and 29%, respectively, and for brain NTE was 30,28, 38, 29, and 6%, respectively. No signs of OPIDN were observedin any of the animals during the 16-day study period. Kineticstudies of the inhibition of hen brain AChE and NTE by CPO invitro demonstrated that CPO exhibits high potency and extraordinaryselectivity for its intended target, AChE. The k₁, values were15.5 µM^–1 min^–1 for AChE and 0.145 µM^–1min^–1 for NTE. The calculated fixed-time (20-min) I₅₀values were 2.24 nM for AChE and 239 nM for NTE, yielding anI₅₀ ratio for NTE/AChE of 107. These results may be comparedwith data compiled for other OP compounds with respect to NTE/AChEI₅₀ ratios and the corresponding doses required to produce OPIDNrelative to the LD₅₀. In general, NTE/AChE I₅₀ ratios greaterthan 1 indicate that the dose required to produce OPIDN is greaterthan the LD₅₀. Taken together, the results of this study indicatethat acute exposures to CPS would not be expected to cause OPIDNexcept under extreme conditions such as attempted suicides involvingmedically assisted survival of doses considerably in excessof the LD₅₀.     

Sublethal Acute Toxicity of Carbosulfan [2,3-dihydro-2,2-dimethyl-7-benzofuranyl(di-n-butylaminosulfenyl)(methyl)carbamate] in the Rat after Intravenous and Oral Exposures

Sublethal Acute Toxicity of Carbosulfan [2,3-dihydro-2,2-dimethyl-7-benzofuranyl(di-n-butylaminosulfenyl)(methyl)carbamate]in the Rat after Intravenous and Oral Exposures. RENZI, B. E.,AND KRIEGER, R. I. (l986). Fundwn. Appl. Toxicol. 6, 7–15.Sublethal toxicity of carcarbosulfan, 2,3-dihydro-2,2-dimethyl-benzofuranyl(di-n-butylaminosulfenyl)(methyl)carbamate,was evaluated in female Sprague-Dawley rats. Erythrocyte acetylcholinesterase(AChE) activity was maximally inhibited 1 min after iv administration(38, 23, and 15% of pretreatment activity after 86, 250, and690 µg/kg, respectively) and recovered by 4 hr. MaximumAChE inhibition (63% of pretreatment activity) was measured45 min after oral dosing (690 µg/kg) and activity recoveredafter 5 hr. Signs included urination, defecation, facial musclefasciculations, salivation, and tremors. Carbosulfan was lesstoxic when given orally. Metabolic activation of carbosulfanto carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranol methylcarbamate)was in vestigated by measuring plasma concentrations 4, 30,and 240 min after iv (80–120 or 620–640 µg/kg)and oral (540–700 or 2030–2190 µ/kg) dosagesof [carbonyl-¹⁴C]carbosulfan. Peak plasma concentrations weremeasured at 4 and 30 min after iv and oral exposure, respectively.Carbosulfan was rapidly activated to carbofuran. Reduction inAChE activity was better correlated (r = 0.97) with plasma concentrationof [carbosulfan + carbofuran] and plasma carbofuran (r = 0.96)than with plasma carbosulfan (r = 0.73). Signs generally occurredwhen AChE activity was less than 65% of pretreatment levels,corresponding to 40 pmol/ml [carbosulfan + carbofuran] in plasma.Based on regression analysis and metabolic studies, both carbosulfanand carbofuran contributed to the observed AChE inhibition;however, carbofuran, a more potent in vitro inhibitor and theusual predominant inhibitor in plasma, was responsible for mostof the erythrocyte AChE inhibition.     

Hypophagia-Induced Weight Loss in Mice, Rats, and Guinea Pigs Treated with 2,3,7,8-Tetrachlorodibenzo-p-dioxin

Hypophagia-lnduced Weight Loss in Mice, Rats, and Guinea PigsTreated with 2,3,7,8-Tetrachlorodibenzo-p-dioxin. KELLING, C.K, CHRISTIAN, B. J., INHORN, S. L., AND PETERSON, R. E. (1985).Fundam. Appl. Toxicol. 5, 700–712. C57BL/6 mice treatedwith 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 360 µ/kg)displayed a significant reduction in feed intake and body weightuntil just before death, when they developed ascites and subcutaneousedema. This caused body weight of the mice that died to suddenlyincrease during the terminal stage of toxicity. TCDD-treatedmice that survived did not develop ascites or edema, and maintaineda body weight that was slightly less than that of pair-fed mice.Cumulative lethality in TCDD-treated mice (69%) was greaterthan that of pair-fed controls (14%). In guinea pigs treatedwith TCDD (2 µ/kg) both the time course and magnitudeof hypophagja were closely associated with weight loss. Pair-fedguinea pigs did not lose quite as much weight as TCDD-treatedanimals because their total body water content was higher. Waterintake in pair-fed guinea pigs was greater than that of TCDD-treatedanimals. The time course and magnitude of lethality tended tobe similar in TCDD-treated guinea pigs (81%) and pair-fed controls(64%). In Fischer F-344 rats treated with TCDD (100 µg/kg)body weight loss was associated with a reduction in both feedand water intake. The time course and magnitude of weight lossin TCDD-treated and pair-fed rats was essentially identical.Lethality was higher in TCDD-treated rats (95%) than pair-fedcontrol animals (48%). Taken together, these findings suggestthat hypophagia is responsible for the loss of adipose and leantissue in mice, guinea pigs, and rats treated with a LD70–95dose of TCDD. Under these dosage conditions, weight loss contributesmore to the lethality of guinea pigs than to that of FischerF-344 rats or C57BL/6 mice     

Effects of subacute pretreatment with carbamate together with acute adjunct pretreatment against nerve agent exposure

Visual observations were made to compare the pretreatment benefits of subacute (75 micrograms/hr, sc) and acute (146 micrograms/kg, im, at 30 min) deliveries of physostigmine salicylate (Phy) against 2 or 5 LD50s (60 or 150 micrograms/kg, sc) of soman in guinea pigs; scopolamine, 80 micrograms/kg, im, was given routinely at 30 min. In a second set of studies, pretreatment with subacute carbamate [sc, Phy 36 micrograms/hr or pyridostigmine (Pyr), 50 micrograms/hr] and acute adjunct (im, scopolamine, 0.48 mg/kg, or trihexyphenidyl, 2 mg/kg) at 30 min, was used against soman (5 LD50s, sc) and VX (18.4 micrograms/kg, sc; 2 LD50s); atropine (16 mg/kg, im) and 2-PAM (25 mg/kg, im) were given at 1 min post soman. In all studies, lethality, % convulsing, convulsive/subconvulsive score, and recovery time were noted. Subacute dosing for 7 days was done via 14-day osmotic minipumps (OMPs). Results of the first set of studies indicate that subacute and acute deliveries of Phy give essentially comparable protection against 2 or 5 LD50s of soman. The second set of studies show that against soman, the adjuncts scopolamine and trihexyphenidyl when compared, and the carbamates, Phy and Pyr when compared, gave similar protective benefits as indicated by all four monitored measures of toxicity. Phy with either adjunct provided excellent protection against VX induced mortality and convulsions. With both carbamates, trihexyphenidyl gave similar protective benefits against VX. Scopolamine, however, under the conditions used herein, failed to act beneficially with Pyr against VX.     

Effects of Chlorpyrifos on High-Affinity Choline Uptake and [3H]Hemicholinium-3 Binding in Rat Brain

High, subcutaneous doses of the organophosphorus insecticidechlorpyrifos (CPF) in adult male rats can be well-tolerateddespite extensive and persistent acetylcholinesterase (AChE)inhibition. We propose that changes in acetylcholine synthesiscould modulate the toxicity associated with extensive AChE inhibitionfollowing CPF exposure. High-affinity choline uptake (HACU,the rate-limiting step in acetylcholine synthesis) and bindingto [³H]-hemicholinium-3 (HC-3, a specific ligand for the cholinetransporter) were chosen as indicators of acetylcholine synthesis.Female, Sprague-Dawley rats (220–280 g) were treated witheither vehicle (peanut oil, 2 ml/kg, sc) or CPF (280 mg/kg,2 ml/kg, sc), examined daily for clinical signs of toxicity,and sacrificed 1, 2, or 7 days later for neurochemical measurements{AChE inhibition, muscarinic receptor binding using [³H]quinuclidinylbenzilate (QNB) and [³H]cis-methyldioxolane (CD) as ligands,HACU and [³H]HC-3 binding} in frontal cortex. Despite extensiveAChE inhibition (90–93%) at all time points, relativelyminor degrees of overt toxicity were noted in CPF-treated rats.Binding to the non-selective muscarinic antagonist [³H]QNB wasreduced (10–34%), whereas binding to the putative m2-selectiveagonist [³H]CD was increased (15–23%) at all three timepoints. HACU was reduced (20%) in crude synaptosomes preparedfrom CPF-treated rats 1 day following exposure but no significantchanges were noted at 2 or 7 days after treatment. CPF-oxon,the active oxidative metabolite of CPF, was a weak inhibitorof HACU in vitro (IC₅₀>200 µM). Binding to [³H]HC-3was reduced in a dose-related manner 1 day after CPF exposure.Kinetic analyses of [³H]HC-3 binding 1 day after CPF (280 mg/kg)indicated a significant reduction in density {B_max: control,187±18 fmol/mg protein; CPF, 104±12 fmol/mg protein)with no apparent change in binding affinity (K_d: control, 25±3nM; CPF, 19±3 nM). These results suggest that a reductionin HACU/acetylcholine synthesis may contribute, along with compensatorychanges in cholinergic receptors, to the diminished toxicityfollowing extensive AChE inhibition by CPF.     

Subchronic Oral Toxicity of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in the Guinea Pig: Comparisons with a PCB-Containing Transformer Fluid Pyrolysate

Subchronic Oral Toxicity of 2,3,7,8-Tetrachlorodibenzo-p-dioxinin the Guinea Pig: Comparisons with a PCB-Containing TransformerFluid Pyrolysate. DECAPRIO, A. P., MCMARTIN, D. N., O'KEEFE,P. W., REJ, R., SILKWORTH, J. B., AND KAMINSKY, L. S. (1986).Fundam. Appl. Taxicol. 6,454–463. In contrast to the well-characterizedacute toxicity of the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin(2,3,7,8-TCDD) in the guinea pig, the effects of prolonged poexposure in this species are unknown. The present report describesthe results of administration to guinea pigs of 2,3,7,8-TCDDin the feed at levels of 0,2, 10, 76, or 430 ppt for up to 90days. Additional aims were to examine recovery following prolonged2,3,7,8-TCDD exposure in the guinea pig and to generate datato facilitate comparison of the previously reported toxicityof a transformer fluid pyrolysate with that of pure 2,3,7,8-TCDD.Animals receiving 430 pot 2,3,7,8-TCDD exhibited body weightloss, thymic atrophy, liver enlargement, and 60% mortality byDay 46 (males) and by Day 60 (females), when surviving animalsin this group were sacrificed. Total 2,3,7,8-TCDD consumptionwas approximately 1.3 and 1.9 µg/kg, respectively. Animalsreceiving 76 ppt 2,3,7,8-TCDD for 90 days (total 0.44 µg/kg)exhibited a decreased rate of body weight gain and increasedrelative (to body) liver weights. Male animals also displayeda reduction in relative thymus weights and elevated serum triglycerides,while females exhibited hepatocellular cytoplasmic inclusionbodies and lowered serum alanine arninotransferase activities.Toxic effects were generally similar to those observed afteracute 2,3,7,8-TCDD administration. No dose-related alterationswere seen in animals receiving either 10 ppt (total 0.06 µg/kg)or 2 ppt (total 0.01 µg/kg) for 90 days, establishinga no-observed-effect level of approximately 0.65 ng 2,3,7,8-TCDD/kg/day.In the recovery study, groups of guinea pigs were administered430 ppt 2,3,7,8-TCDD for 11,21, or 35 days and then allowedto recover for an additional 79,69, or 55 days, respectively.Treatment-related mortality in each group was 0, 10, and 70%,respectively, by Day 90. An effective LD5O of 0.8 µg 2,3,7,8-TCDD/kgfor prolonged exposure was calculated on the basis of theseresults, a value lower than those previously reported from thislaboratory for acute exposure. The results also suggested apossible lowering of the body weight "set point" following 2,3,7,8-TCDD exposure. Comparison of the present findings with thosepreviously reported for a trans former fluid pyrolysate containinga mixture of polychlorinated aromatic species indicated botha greater variety of toxic effects and flatter dose-responserelationships for the pyrolysate in the guinea pig.     

Recombinant paraoxonase 1 protects against sarin and soman toxicity following microinstillation inhalation exposure in guinea pigs

To explore the efficacy of paraoxonase 1 (PON1) as a catalytic bioscavenger, we evaluated human recombinant PON1 (rePON1) expressed in Trichoplusia ni larvae against sarin and soman toxicity using microinstillation inhalation exposure in guinea pigs. Animals were pretreated intravenously with catalytically active rePON1, followed by exposure to 1.2 X LCt₅₀ sarin or soman. Administration of 5 units of rePON1 showed mild increase in the blood activity of the enzyme after 30 min, but protected the animals with a significant increase in survival rate along with minimal signs of nerve agent toxicity. Recombinant PON1 pretreated animals exposed to sarin or soman prevented the reduction of blood O₂ saturation and pulse rate observed after nerve agent exposure. In addition, rePON1 pretreated animals showed significantly higher blood PON1, acetylcholinesterase (AChE), and butyrylcholinesterase activity after nerve agent exposure compared to the respective controls without treatments. AChE activity in different brain regions of rePON1 pretreated animals exposed to sarin or soman were also significantly higher than respective controls. The remaining activity of blood PON1, cholinesterases and brain AChE in PON1 pretreated animals after nerve agent exposure correlated with the survival rate. In summary, these data suggest that human rePON1 protects against sarin and soman exposure in guinea pigs.     

The effect of pyridostigmine pretreatment on oxime efficacy against intoxication by soman or VX in rats.

This study was done to assess the effects of pyridostigmine (PYR) on a) the accumulation of labelled VX and soman within the brain, b) the therapeutic efficacy of atropine and oxime (2-PAM or HI-6) against intoxication by VX and soman and c) oxime-induced reactivation of inhibited acetylcholinesterase (AChE). In all experiments, rats were given PYR (131 micrograms/kg, im; I70 dose for whole blood AChE) or vehicle 30 min prior to nerve agent. In estimating 3H-agent the accumulation in the brain or estimating blood AChE activity, sufficient soman (47 micrograms/kg, iv) or VX (21.3 micrograms/kg, iv) was given to inhibit 50% of brain AChE activity. In assessing therapeutic efficacy and oxime-induced reactivation of blood AChE, rats were pretreated with PYR, challenged with agent and treated with atropine (16 mg/kg, im) and HI-6 or 2-PAM (100 umoles/kg, im) 30 sec post agent. Whole blood was collected by tail bleeding to monitor peripheral AChE activity at various time points before and after PYR and challenge. Pyridostigmine failed to alter covalent binding of labelled VX or soman in the brain. The 24-hr survival data showed that PYR reduced the therapeutic benefit of atropine and oxime against VX intoxication (but not soman). Protective ratios in VX-challenged rats given vehicle or PYR and treated with atropine + 2-PAM decreased slightly from 2.5 to 2.1 (p > .05), whereas with atropine + HI-6 they decreased significantly from 3.8 to 2.4. Also, AChE reactivation by HI-6 in VX-challenged rats was greater (p < .05) in vehicle- than in PYR-pretreated rats. HI-6 significantly reactivated AChE activity in both pretreatment groups (PYR or vehicle) given soman. The data suggest that PYR decreases the overall recovery of inhibited AChE in VX-challenged rats given HI-6; under the conditions used, this adverse effect decreases atropine+oxime efficacy against VX-induced lethality.     

Interaction of Soman with {beta}-Cyclodextrin

Interaction of Soman with ß-CycIodextrin. DESIRE,B., AND SAINT-ANDRE, S. (1986). Fun-dam. Appl. Toxicol. 7, 646-657.Of the following neurotoxic agents, pinacolyl methylphospho-nofluoridate(soman), isopropyl methylphosphonofluoridate (sarin) and ethylN, N-dimethyl-phosphoramidocyanidate (tabun), only soman wasinactivated appreciably at pH 7.40 by ß-cyclodextrin.The interaction of soman, a mixture of four stereoisomers designatedas C(+)P(–), C(–)P(–), C(+)P(+), and C(–)P(+),with cyclodextrins was revealed by methods based on (i) theirreversible inhibition of acetylcholinesterase (AChE) thatis phosphonylated chiefly by P(–)-isomers of racemic somanand (ii) continuous titration of fluoride ions released by somanusing a fluoride-specific electrode. Soman and ß-cyclodextrinform a 1:1 complex. At pH 7.40 and 25°C the dissociationconstant Kd of this complex and the rate constant k2 of cleavageof soman by ß-cyclodextrin are (0.53 ± 0.05)mM and (5.9 ± 0.6) x 10² min¹, respectively. The rateconstant k₂^max for the cleavage of soman by monoionized ß-cyclodextrinhas a value of 2.8 x 103 min¹ and the second order rate constantk₂^max/K_d 5.3 x 106 M^–1 min^–1. Consequently, somanis hydrolyzed about 2500 times faster by the monoanion of ß-cyclodextrin,than by the hydroxide ion. The cleavage of P(–)-somanby ß-cyclodextrin as estimated by AChE inhibitionproceeds apparently at the same rate for the C(–)P(–)-and C(+)P(–)-isomers. However, the release of fluorideions indicated a stereospecific rate of reaction, the P(-Hsomersreacting faster than the P(+)-isomers. At pH 7.40, the inactivationrate of soman by ß-cyclodextrin was as fast in humanplasma in vitro as in Tris buffer. This interaction betweensoman and ß-cyclodextrin, and other data from theliterature, suggests that the introduction of catalytic or noncatalyticgroups on ß-cyclodextrin might possibly make it abetter catalyst for soman inactivation through improvement inthe catalytic or in the binding process.     

Variable Effects of Soman on Macromolecular Secretion by Ferret Trachea

The purpose of this study was to examine the effect of the anticholinesteraseagent, soman, on macromolecular secretion by ferret trachea,in vitro We mounted pieces of ferret trachea in Ussing-typechambers. Secreted sulfated macro-molecules were radiolabeledby adding 500 µCi of ³³SO₄ to the submucosal medium andincubating for 17 hr. Soman added to the submucosal side produceda concentration-dependent increase in radiolabeled macromolecularrelease with a maximal secretory response (mean ± SD)of 202 ± 125% (n = 8) relative to the basal secretionrate at a concentration of 10^–7 m. The addition of either10^–6 M pralidoxime (acetyicholinesterase reactivator)or 10^–7 M atropine blocked the response to 10^–7m soman. At soman concentrations greater than 10^–7 M,secretion rate decreased and was not significantly differentfrom basal secretion. Additional experiments utilizing acetylcholineand the acetyicholinesterase inhibitor, physostigmine, suggestthat inhibition of secretion by high concentrations of somanmay be due to a secondary antagonistic effect of soman on muscannicreceptors.     

Action of Organophosphates on GABAA Receptor and Voltage-Dependent Chloride Channels

The effects of several organophosphates were studied on thebinding of t-[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS)to rat brain GABA_A receptor and receptor function as assayedby GABA-induced ³⁶Cl^– influx into membrane vesicles andon the binding of [³⁵S]TBPS to a voltage-dependent Cl^–channel in Torpedo californica electric organ. The organophosphateanticholinesterases diisopropylphosphorofluoridate, soman, sarin,tabun, and VX had little or no effect on GABA-regulated chloridechannels. They also had no effect on [³⁵S]TBPS binding to thevoltage-dependent chloride channel, except for soman which inhibitedit with an IC50 of 24 µM. Triphenyl phosphate was theonly one of three organophosphate flame retardants tested thatinhibited both GABA-regulated chloride channel and binding of[³⁵S]TBPS to the voltage-dependent chloride channel with IC50sof 18 and 13 µM, respectively. The industrial organophosphatetri-o-cresyl phosphate and the anticholinesterase organophosphateinsecticides leptophos, leptophos oxon, and O-ethyl O-4-nitrophenylphenylphosphonothioate inhibited GABA-regulated chloride channelsand bound with high affinity to the voltage-dependent chloridechannels (lC50 = 0.3 to 8.7 µM). There was no apparentcorrelation between the affinities of the GABA_A receptor chloridechannel or the voltage-dependent chloride channel for the differentorganophosphates and their potencies in inhibiting acetylcholinesteraseor in inducing delayed neurotoxicity. Nevertheless, althoughthe voltage-dependent chloride channel and/or GABA_A receptorare not primary targets for organophosphate anticholinesterasesand flame retardants, it is suggested that the inhibition ofthese two proteins by certain organophosphates may contributeto their toxicities.     

Induction of Ethylenediamine Hypersensitivity in the Guinea Pig and the Development of ELISA and Lymphocyte Blastogenesis Techniques for Its Characterization

Ethylenediamine (EDA) is reported to be a poorly characterizediatrogenic and occupational contact sensitizer. To better characterizeEDA hypersensitivity, a guinea pig model was employed in whichthe animals were exposed epicutaneously to simulate conditionsof human exposure, and selected immune parameters were measured.Induction of hypersensitivity was by the Buehler occluded patchmethod (6 hr application/day, once a week for 3 consecutiveweeks) to 10, 20, 30, or 407% EDA, using either an ethanol oracetone/corn oil vehicle. Fourteen days after the last induction,guinea pigs were challenged by patch application of 2% EDA (nonirritating).The incidence of responders for erythema in the 10% EDA (ethanol)treatment group was 83 and 50% at 24 and 48 hr, respectively.In the 10% EDA (acetone/corn oil) group the corresponding valueswere 50 and 17%. For 20, 30, and 40% EDA, in either vehicle,the incidence oferythema was 83 to 100%. Severity grades (scale= 0–3) for cutaneous reactions to increasing concentrationsof EDA in ethanol ranged from 0.8 to 2.5; those for EDA in acetone/cornoil ranged from 0.6 to 2.8. Using an enzyme-linked immunosorbentassay developed to detect the predominant serum antibodies toEDA, it was shown that guinea pigs treated by patch applicationdid not produce the main allergic antibody lgG specific forEDA. However, intradermal administration of an EDA–guineapig serum albumin conjugate (EDA–GSA) to guinea pigs presensitizedby patch application resulted in antibody production by 39 and86% of the animals, at the initial and second dosing, respectively.An in vitro blastogenesis assay, using peripheral blood lymphocytesfrom EDA-sensitized guinea pigs, was developed to identify specificchemical allergens implicated inin vivo sensitization. Maximumtritiated thymidine ([³H]TdR) incorporation by lymphocytes stimulatedin vitro with EDA–GSA was observed on Day 7. Optimal antigenconcentration for maximum lymphocyte proliferation ranged from5 to 50 µg/ml, the major variation being attributableto interanimal differences. These results indicate that epicutaneousapplication of EDA in the guinea pig induces a Type IV delayedhypersensitivity; immunological memory to the hapten is maintainedin cultured lymphocytes, suggesting the potential usefulnessof the lymphocyte transformation test for in vitro diagnosisof chemically induced hypersensitivity in humans.     

Treatment of organophosphate poisoning in pigs: antidote administration by a new binary autoinjector

 The therapeutic effectiveness of a new binary autoinjector containing 500 mg HI-6 and 2 mg atropine sulphate was tested in anesthetized pigs poisoned by a lethal dose of soman i.v. (9 μg/kg per 20 min). Pharmacokinetics and pharmacodynamics of HI-6 were studied concomitantly on administration of HI-6 alone, together with atropine sulphate, or together with atropine sulphate during soman intoxication. Cardiopulmonary parameters were monitored and serum concentrations of oxime and acetylcholinesterase (AChE) were measured in blood samples taken at intervals over a 6-h period postinjection. Five minutes after the start of soman infusion, mean AChE activity was decreased to 27±4.3% of baseline and signs of poisoning appeared. The antidotes, HI-6 and atropine sulphate, were then administered i.m. One minute after this injection there was a transient significant increase in AChE activity of 76±8.2% of baseline (p<0.01). It then again decreased and remained suppressed throughout the experiment. Mean respiratory rate was significantly decreased (p<0.01) to 20±3.2% of baseline after 20 min of soman infusion and remained low during the rest of the experiment. The poisoning signs were counteracted 15–20 min after antidote therapy and all pigs survived soman intoxication without ventilatory assistance. Administration of either atropine or atropine and soman had no significant effect on the pharmacokinetics of HI-6 in anesthetized pigs. Received: 20 February 1995/Accepted: 3 May 1995     

Effects of 4-pyridine aldoxime on nerve agent-inhibited acetylcholinesterase activity in guinea pigs

Methoxime (MMB-4) is a leading candidate oxime acetylcholinesterase (AChE) reactivator to replace pralidoxime (2-PAM) for therapeutic treatment of nerve agent intoxication. 4-Pyridine aldoxime (4-PA) is a synthetic starting material, a breakdown product, and a probable metabolite of MMB-4. There is a possibility that 4-PA may adversely interact with the nerve agent, thereby affecting nerve agent toxicity and biological AChE activity. This study evaluated the effects of 4-PA on sarin (GB)-, cyclosarin (GF)-, and VX-induced toxicity and AChE activity in blood, brain, and peripheral tissues of guinea pigs. Animals were pretreated with atropine methyl nitrate (1.0 mg/kg, im) 15 min prior to subcutaneous administration with 1.0× LD₅₀ of GB, GF, or VX and then treated 15 min after the administration of nerve agents with 4-PA (3.5, 7.0, or 14.0 mg/kg, im). The dose–response effects of 4-PA alone were also examined. Toxic signs and lethality were monitored, blood and tissues were collected, and AChE activities were determined at 60 min after nerve agent administration. Under the condition of this study, all animals exposed to nerve agents exhibited some degree of toxic signs such as salivation, lacrimation, rhinorrhea, and convulsions. 4-PA at the three doses tested neither induced toxic signs nor altered the toxicity of GB, GF, or VX at the 1.0× LD₅₀ exposure dose. Additionally, it did not modify the AChE activity in blood, brain, and peripheral tissues by itself or affect the AChE activity inhibited by a 1.0× LD₅₀ dose of these three nerve agents in guinea pigs.