Robert J. Mioduszewski

Inhalation toxicity of Cyclosarin (GF) vapor in rats as a function of exposure concentration and duration: potency comparison to sarin (GB).

The inhalation toxicity of cyclohexyl methylphosphonofluoridate (GF) was examined in male and female Sprague-Dawley rats exposed by whole body in a dynamic 750-L chamber. The objectives of this study were to (1) generate GF vapor in a dynamic inhalation chamber system, starting in the lethal to near-lethal concentration range, (2) examine dose-response effects of inhaled GF vapor and analyze the relationship between concentration (C) and exposure duration (T) in determining probability of lethality, and (3) establish a lethal potency ratio between GF and the more volatile agent Sarin (GB). Using a syringe pump, GF vapor concentrations were generated for exposure times of 10, 60, and 240 min. Dose-response curves with associated slopes were determined for each exposure duration by the Bliss probit method. GF vapor exposures were associated with sublethal clinical signs such as tremors, convulsions, salivation, and miosis. Concentration-exposure time values for lethality in 50% of the exposed population (LCT(50)) were calculated for 24-h and 14-day postexposure periods for 10-, 60-, and 240-min exposures. In general, LCT(50) values were lower in female rats than males and increased with exposure duration; that is, CT was not constant over time. The GF LCT(50) values for female rats were 253 mg min/m(3) at 10 min, 334 mg min/m(3) at 60 min, and 533 mg min/m(3) at 240 min, while the values for males were 371, 396, and 585 mg min/m(3), respectively. The GB LCT(50) values for female rats were 235 mg min/m(3) at 10 min, 355 mg min/m(3) at 60 min, and 840 mg min/m(3) at 240 min, while the values for males were 316, 433, and 1296 mg min/m(3), respectively. At longer exposure durations, the LCT(50) for GF was less than that found for GB but at shorter exposure durations, the LCT(50) for GF was more than that found for GB. Empirical models, consisting of the toxic load model plus higher order terms, were developed and successfully fit to the data.

Pretreatment with human serum butyrylcholinesterase alone prevents cardiac abnormalities, seizures, and death in Göttingen minipigs exposed to sarin vapor

Human serum butyrylcholinesterase (Hu BChE) is a stoichiometric bioscavenger that is being developed as a prophylactic countermeasure against organophosphorus nerve agents. This study was designed to evaluate the efficacy of Hu BChE against whole-body inhalation exposure to a lethal dose of sarin (GB) vapor. Male Göttingen minipigs were subjected to: air exposure, GB vapor exposure, or pretreatment with Hu BChE followed by GB vapor exposure. Hu BChE was administered by i.m. injection 24 h prior to exposure to 4.1 mg/m(3) of GB vapor for 60 min. Electrocardiograms (ECG), electroencephalograms (EEG), and pupil size were recorded throughout exposure. Blood drawn before and throughout exposure was analyzed for blood gases, electrolytes, metabolites, acetylcholinesterase and BChE activities, and amount of GB present. Untreated animals exposed to GB vapor exhibited cardiac abnormalities and generalized seizures, ultimately succumbing to respiratory failure. Pretreatment with 3.0 or 6.5 mg/kg of Hu BChE delayed blood gas and acid-base disturbances and the onset of cardiac and neural toxic signs, but failed to increase survivability. Pretreatment with 7.5 mg/kg of Hu BChE, however, completely prevented toxic signs, with blood chemistry and ECG and EEG parameters indistinguishable from control during and after GB exposure. GB bound in plasma was 200-fold higher than plasma from pigs that did not receive Hu BChE, suggesting that Hu BChE scavenged GB in blood and prevented it from reaching other tissues. Thus, prophylaxis with Hu BChE alone not only increased survivability, but also prevented cardiac abnormalities and neural toxicity in minipigs exposed to a lethal dose of GB vapor.

Efficacy of human serum butyrylcholinesterase against sarin vapor.

Human serum butyrylcholinesterase (Hu BChE) is currently under advanced development as a pretreatment drug for organophosphate (OP) poisoning in humans. It was shown to protect mice, rats, guinea pigs, and monkeys against multiple LD(50) challenges of OP nerve agents by i.v. or s.c. bolus injections. Since inhalation is the most likely route of exposure to OP nerve agents on the battlefield or in public places, the aim of this study was to evaluate the efficacy of Hu BChE against whole-body inhalation exposure to sarin (GB) vapor. Male Göttingen minipigs were subjected to one of the following treatments: (1) air exposure; (2) GB vapor exposure; (3) pretreatment with 3 mg/kg of Hu BChE followed by GB vapor exposure; (4) pretreatment with 6.5 mg/kg of Hu BChE followed by GB vapor exposure; (5) pretreatment with 7.5 mg/kg of Hu BChE followed by GB vapor exposure. Hu BChE was administered by i.m. injection, 24h prior to whole-body exposure to GB vapor at a concentration of 4.1 mg/m(3) for 60 min, a dose lethal to 99% of untreated exposed pigs (LCt99). EEG, ECG, and pupil size were monitored throughout exposure, and blood drawn from a surgically implanted jugular catheter before and throughout the exposure period, was analyzed for acetylcholinesterase (AChE) and BChE activities, and the amount of GB present in plasma. All animals exposed to GB vapor alone or pretreated with 3 or 6.5 mg/kg of Hu BChE, died following exposure to GB vapor. All five animals pretreated with 7.5 mg/kg of Hu BChE survived the GB exposure. The amount of GB bound in plasma was 200-fold higher compared to that from plasma of pigs that did not receive Hu BChE, suggesting that Hu BChE was effective in scavenging GB in blood. Additionally, pretreatment with 7.5 mg/kg of Hu BChE prevented cardiac abnormalities and seizure activity observed in untreated animals and those treated with lower doses of Hu BChE.

Effects of Whole-Body VX Vapor Exposure on Lethality in Rats

Male and female rats were whole-body exposed to VX vapor in a 1000-L single-pass exposure chamber. Estimated exposure dosages producing lethal (LCT50) effects in 50% of exposed male and female rats were established for 10, 60, and 240 min exposure durations. A potency comparison with GB and GF shows that VX becomes increasingly more potent than these G agents with increasing exposure duration. VX is approximately 4–30 times more potent than GB and 5–15 times more potent than GF. Gender differences in the estimated median dosages were not significant at the 10, 60, and 240 min exposure durations. An empirical toxic load model was developed and the toxic load exponent for lethality (n) in the equation Cn× T = k was determined to be n = 0.92. The VX–G regeneration assay was successfully used as a biomarker for the presence of VX in the blood plasma and RBC fractions of the blood 24 h postexposure.

Gender difference in the miotic potency of soman vapor in rats

The present study was undertaken to investigate the miotic potency of soman vapor in the rat, as well as gender differences in the miotic response to soman vapor that have been reported previously for other nerve agents. The results of the present study demonstrate that the miotic potency of soman vapor is significantly less than that of other nerve agents, and that female rats are 2.5–3.0 times more sensitive to soman vapor than male rats. The results also demonstrate that ocular acetylcholinesterase and butyrylcholinesterase activities differ between males and females, although this difference is not likely large enough to account for the observed gender difference.

Biomarkers of low-level exposure to soman vapor: comparison of fluoride regeneration to acetylcholinesterase inhibition.

The nerve agent O-pinacolyl methylphosphonofluoridate, also known as soman or by its military designation GD, is a highly toxic organophosphorous compound that exerts its effects through inhibition of the enzyme acetylcholinesterase (AChE). In the present study, a fluoride ion based regeneration assay was developed to quantify the level of soman present in the blood of rats following a low-level whole-body inhalation exposure. It was hypothesized that the amount of regenerated nerve agent in the blood would be dose dependent in rats subjected to a whole-body inhalation exposure to a low-level dose of soman vapor, and that the fluoride ion-based regeneration method would be more sensitive for the detection of a low-level exposure to soman vapor than the measurement of whole blood AChE activity. Regenerated soman was dose-dependently detected in both the red blood cells (RBCs) and plasma of exposed rats at all concentrations tested (0.033–0.280 mg/m3 for a 240-min exposure). Significant inhibition of whole blood AChE activity did not occur below a concentration of 0.101 mg/m3, and was only depressed by approximately 10–25% at concentrations ranging from 0.101 mg/m3 to 0.280 mg/m3. This study is the first to utilize a fluoride ion-based regeneration assay to demonstrate the dose-dependent increases in soman in the blood following whole-body inhalation exposure to low levels of vapor. Additionally, the results of the present study demonstrate that the fluoride ion based regeneration assay was approximately threefold more sensitive than the measurement of AChE activity in the blood for the detection of exposure to soman, and also that miosis is a more sensitive marker of soman exposure than inhibition of AChE activity.

Prophylaxis with human serum butyrylcholinesterase protects Göttingen minipigs exposed to a lethal high-dose of sarin vapor

Serum-derived human butyrylcholinesterase (Hu BChE) is a stoichiometric bioscavenger that is being developed as a potential prophylactic nerve agent countermeasure. Previously, we reported the prophylactic efficacy of Hu BChE in Göttingen minipigs against a whole-body exposure to 4.1mg/m(3) of sarin (GB) vapor, which produced lethality over 60min. Since the toxicity of nerve agent is concentration-dependent, in the present study, we investigated the toxic effects of an almost 3-fold higher rate of GB vapor exposure and the ability of Hu BChE to protect minipigs against this exposure. Male minipigs were subjected to: (1) air exposure; (2) GB vapor exposure; or (3) pretreatment with 7.5mg/kg of Hu BChE by i.m. injection, 24h prior to whole-body exposure to 11.4mg/m(3) of GB vapor for 10min. Electrocardiogram, electroencephalogram, and pupil size were monitored throughout exposure. Blood drawn before and throughout exposure was analyzed for blood gases, electrolytes, metabolites, acetylcholinesterase and BChE activities, and amount of GB bound to red blood cells and plasma. A novel finding was that saline-treated animals exposed to GB vapor did not develop any seizures, but manifested a variety of cardiac and whole blood toxic signs and rapidly died due to respiratory failure. Strikingly, pre-treatment with 7.5mg/kg of Hu BChE not only prevented lethality, but also avoided all cardiac toxic signs manifested in the non-treated cohort. Thus, Hu BChE alone can serve as an effective prophylactic countermeasure versus a lethal high-dose exposure to GB vapor.

Acute Toxic Effects of Inhaled Dichlorvos Vapor on Respiratory Mechanics and Blood Cholinesterase Activity in Guinea Pigs

Using a modified noninvasive volume-displacement plethysmography system, we investigated the effects of inhaled dichlorvos (2,2-dimethyl dichlorovinyl phosphate, or DDVP) vapor on the respiratory mechanics and blood cholinesterase activity of guinea pigs. Data revealed significant dose-dependent changes in several pulmonary parameters. Animals exposed to a DDVP concentration of 35 mg/m3 did not show any significant changes in frequency, tidal volume, or minute ventilation. However, animals exposed to 55 mg/m3 DDVP showed significantly decreased respiratory frequency and significantly increased tidal volume with no significant changes in minute ventilation. Similarly, animals exposed to 75 mg/m3 DDVP showed significantly decreased respiratory frequency along with significantly increased tidal volume. The decreased respiratory frequency was large enough in the high exposure group to offset the increased tidal volume. This effect resulted in significantly decreased minute ventilation by the end of exposure, which remained attenuated 10 min after exposure. An analysis of whole-blood cholinesterase activity revealed significantly decreased activity for both acetylcholinesterase (AChE) and butylcholinesterase (BChE). Peak inhibition occurred for both enzymes at the end of exposure for all three concentrations and rapidly recovered within several minutes of exposure. Analysis of blood samples using gas chromatography–mass spectroscopy (GC-MS) revealed that minute ventilation may only play a minimal role in the dosimetry of inhaled DDVP vapor.

Kinetics of sarin (GB) following a single sublethal inhalation exposure in the guinea pig.

To improve toxicity estimates from sublethal exposures to chemical warfare nerve agents (CWNA), it is necessary to generate mathematical models of the absorption, distribution, and elimination of nerve agents. However, current models are based on representative data sets generated with different routes of exposure and in different species and are designed to interpolate between limited laboratory data sets to predict a wide range of possible human exposure scenarios. This study was performed to integrate CWNA sublethal toxicity data in male Duncan Hartley guinea pigs. Specific goal was to compare uptake and clearance kinetics of different sublethal doses of sarin (either 0.1 × or 0.4 × LC50) in blood and tissues of guinea pigs exposed to agent by acute whole-body inhalation exposure after the 60-min LC50 was determined. Arterial catheterization allowed repeated blood sampling from the same animal at various time periods. Blood and tissue levels of acetylcholinesterase, butyrylcholinesterase, and regenerated sarin (rGB) were determined at various time points during and following sarin exposure. The following pharmacokinetic parameters were calculated from the graph of plasma or RBC rGB concentration versus time: time to reach the maximal concentration; maximal concentration; mean residence time; clearance; volume of distribution at steady state; terminal elimination-phase rate constant; and area under plasma concentration time curve extrapolated to infinity using the WinNonlin analysis program 5.0. Plasma and RBC t1/2 for rGB was also calculated. Data will be used to develop mathematical model of absorption and distribution of sublethal sarin doses into susceptible tissues.

Behavioral evaluation of rats following low-level inhalation exposure to sarin ☆

We evaluated the effects, in rats, of single and multiple low-level inhalation exposures to sarin. Rats were trained on a variable-interval, 56 s (VI56) schedule of food reinforcement and then exposed to sarin vapor (1.7-4.0 mg/m(3) x 60 min) or air control. The exposures did not produce clinical signs of toxicity other than miosis. Subsequently, performance on the VI56 and acquisition of a radial-arm maze spatial memory task was evaluated over approximately 11 weeks. Single exposures did not affect performance on the VI56 and had little effect on acquisition of the radial-arm maze task. Multiple exposures (4.0 mg/m(3) x 60 min/day x 3) disrupted performance on the VI56 schedule during the initial post-exposure sessions. The disruption, however, resolved after several days. Multiple exposures also produced a deficit on the radial-arm maze task in that sarin-exposed rats tended to take it longer to complete the maze and to make more errors. The deficit, however, resolved during the first three weeks of acquisition. These results demonstrate that in rats, inhalation exposure to sarin at levels below those causing overt signs of clinical toxicity can produce cognitive and performance deficits. Furthermore, the observed deficits do not appear to be persistent.