Evaluation of miosis, behavior and cholinesterase inhibition from low-level, whole-body vapor exposure to soman in African green monkeys (Chlorocebus sabeus).

BACKGROUND: Relatively little is known about the effects of very low-level exposures to nerve agents where few signs or symptoms are present. METHODS: African green monkeys (Chlorocebus sabeus) (n = 8) were exposed for 10 min, whole-body, to a single concentration of soman (0.028-0.891 mg/m³). RESULTS: EC50 values for miosis were determined to be 0.055 mg/m³ and 0.132 mg/m³ when defined as a 50 percent reduction in pupil area and diameter, respectively. In general, performance on a serial probe recognition task remained unchanged at lower concentrations, but responding was suppressed at the largest concentration tested. Soman produced concentration-dependent inhibition of acetylcholinesterase activity and, to a lesser extent, butyrylcholinesterase activity. CONCLUSIONS: These results characterize threshold soman exposure concentrations that produce miosis in the absence of other overt signs of toxicity and extend previous studies indicating that miosis is a valuable early indicator for the detection of soman vapor exposure.

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.

Determination of miosis threshold from whole-body vapor exposure to sarin in African green monkeys.

We determined the threshold concentration of sarin vapor exposure producing miosis in African green monkeys (Chlorocebus aethiops). Monkeys (n=8) were exposed to a single concentration of sarin (0.069-0.701mg/m3) for 10min. Changes in pupil size were measured from photographs taken before and after the exposure. Sarin EC50 values for miosis were determined to be 0.166mg/m3 when miosis was defined as a 50% reduction in pupil area and 0.469mg/m3 when miosis was defined as a 50% reduction in pupil diameter. Monkeys were also evaluated for behavioral changes from sarin exposure using a serial probe recognition test and performance remained essentially unchanged for all monkeys. None of the concentrations of sarin produced specific clinical signs of toxicity other than miosis. Sarin was regenerated from blood sampled following exposure in a concentration-dependent fashion. Consistent with a predominant inhibition of acetylcholinesterase (AChE), more sarin was consistently found in RBC fractions than in plasma fractions. Further, elimination of regenerated sarin from RBC fractions was slower than from plasma fractions. Blood samples following exposure also showed concentration-dependent inhibition of AChE activity and, to a lesser extent, butyrylcholinesterase activity. At the largest exposure concentration, AChE inhibition was substantial, reducing activity to approximately 40% of baseline. The results characterize sarin exposure concentrations that produce miosis in a large primate species in the absence of other overt signs of toxicity. Further, these results extend previous studies indicating that miosis is a valid early indicator for the detection of sarin vapor exposure.

Behavioral and biochemical evaluation of sub-lethal inhalation exposure to VX in rats.

We evaluated the effects of low-level inhalation exposures (whole body, 60min duration) to the chemical warfare nerve agent VX (0.016, 0.15, 0.30 or 0.45mg/m(3)) in rats. The range of concentrations was approximately equivalent to 0.02-0.62 times 1.0 LC50. Biochemical effects were assessed by evaluating blood acetylcholinesterase (AChE) activity and by a regeneration assay that quantified the amount of VX (as the G analog) present in blood. Behavioral effects were assessed using a variable-interval 56-s schedule of reinforcement (VI56), in which rats were trained to press a lever to receive a food reward. VI56 training was established before exposure and evaluations continued after exposure. Additionally, after exposure, acquisition and maintenance of an eight-arm radial maze (RAM) task was evaluated in which rats learned to locate the four arms of the maze that presented a single food pellet reward. Behavioral assessments were conducted over approximately 3 months following exposure. Transient miosis was observed following exposure to all concentrations of VX and exposures to the 0.45mg/m(3) concentration also produced mild and temporary signs of toxicity (i.e., slight tremor and ataxia) in some subjects. All concentrations of VX also inhibited circulating AChE and the highest concentration inhibited AChE activity to less than 10% of pre-exposure values. Regenerated VX-G was found in red blood cell (RBC) and plasma blood fractions. In this respect, more VX-G was seen in plasma than RBC. Only small disruptions were observed on the VI56 or RAM following some VX exposures. In general, however, behavioral effects were minor and not clearly systematic. Taken together these results demonstrate that largely asymptomatic exposures to VX vapors in rats can produce substantial biochemical effects while having only minor performance effects on a previously learned behavioral task and on the acquisition of a new behavioral task.

Effects of low-level inhalation exposure to cyclosarin on learned behaviors in Sprague-Dawley rats.

Behavioral and biochemical effects of low-level whole-body inhalation exposure to the chemical warfare nerve agent cyclosarin (GF) were evaluated. Sprague-Dawley rats were first trained on a variable-interval, 56-s (VI56) schedule of food reinforcement. The VI56 schedule specifies that a single lever press, following an average interval of 56 s, produces food reinforcement (i.e., a single food pellet). Subjects were then exposed to GF vapor at concentrations of 1.6-5.2 mg/m3, or air control, for 60 min. Following exposures, performance on the VI56 and acquisition and maintenance of a radial-arm maze (RAM) spatial memory task were evaluated during 55 test sessions over approximately 11 wk. GF exposures produced miosis in all subjects and other mild clinical signs of toxicity at the highest concentration. Convulsions were not observed in any subjects. GF exposures produced concentration-dependent decreases in acetylcholinesterase and butyrylcholinesterase activity. Additionally, blood assays revealed concentration-dependent levels of regenerated GF, thus verifying systemic exposure. The largest concentration of GF disrupted performance on the VI56 task. The deficit, however, resolved by the third postexposure test session. All subjects acquired, and maintained, performance on the RAM task, and no significant differences were seen as a result of GF exposure. No delayed effects from exposures were observed. These results demonstrate that, in rats, inhalation exposure to GF at levels below those producing convulsions and other severe clinical signs of toxicity may produce performance deficits on learned behaviors, but the deficits appear to not be persistent.

Comparison of low-level sarin and cyclosarin vapor exposure on pupil size of the Gottingen minipig: effects of exposure concentration and duration.

The current studies estimated effective (miosis) concentrations of the nerve agents' sarin (GB) and cyclosarin (GF) as a function of exposure duration in the Gottingen minipig and determined dependency of the median effective dosage (ECT50) over time. Male and female Gottingen minipigs were exposed to various concentrations of vapor GB or GF for 10, 60, or 180 min. Infrared images of the pig's pupil before, during, and after nerve agent exposure were captured digitally and pupil area was quantified. An animal was classified "positive" for miosis if there was a 50% reduction in pupil area (as compared to baseline) at any time during or after the GB or GF exposure. Maximum likelihood estimation was used on the resulting quantal data to calculate ECT50 (miosis) values, with approximate 95% confidence intervals, for each of the six gender-exposure duration groups. As a group, male minipigs were significantly more sensitive to the pupil constricting effects of GF than were female minipigs. In male minipigs, GF is approximately equipotent to GB for 60-min exposures and more potent for 10- and 180-min exposures. In the female minipig GF is slightly more potent than GB for 10-min exposures but then progressively becomes less potent over the 60- and 180-min durations of exposure. The values of the toxic load exponents were essentially independent of the model fits used: 1.32 +/- 0.18 for GB exposures and 1.60 +/- 0.22 for GF exposures. Since neither of these intervals overlaps 1, Haber's rule is not an appropriate time-dependence model for these data sets.

Determination of threshold adverse effect doses of percutaneous VX exposure in African green monkeys.

Percutaneous exposure to the chemical warfare nerve agent VX was evaluated in African green monkeys (n=9). Doses of VX (7.5-100 µg/kg) were applied to the skin for 60 min and residual agent was quantified (before decontamination) to estimate the absorbed dose. Monkeys were evaluated for the presence or absence of clinical signs of toxicity and blood was sampled periodically (30 min--12 weeks) following exposure to measure the degree of circulating acetylcholinesterase (AChE) inhibition. Monkeys were also evaluated for behavioral changes from VX exposure using a serial probe recognition (SPR) task. The lowest observable adverse effect level (LOAEL) for the production of major clinical signs was determined to be 42.22 µg/kg (absorbed dose estimate=17.36 µg/kg) and the LOAEL for AChE inhibition was 13.33 µg/kg (absorbed dose estimate=6.53 µg/kg). Behavioral performance was unaffected at doses that, while producing substantial AChE inhibition, did not produce clinical signs. VX represents a substantial threat as a contact hazard and these results complement previous studies using the percutaneous route of exposure with VX and extend the findings to a non-human primate species.

Assessment of low level whole-body soman vapor exposure in rats.

We evaluated biochemical and behavioral effects of single, low-level exposures to the chemical warfare nerve agent soman (GD). Male Sprague-Dawley rats were trained on a variable-interval, 56-sec schedule of food reinforcement (VI56). The schedule specifies that a single lever press, following an average interval of 56 s, produces food reinforcement (i.e., a single food pellet). After training, rats received a single 60 min exposure to soman vapor at concentrations of 1.0-7.0 mg/m(3), or air control (n=8 for each treatment condition). Blood was sampled before and after the exposure. Following exposures, performance on the VI56 was evaluated for approximately 11 weeks. Additionally, the acquisition and maintenance of a radial-arm maze (RAM) spatial memory task were evaluated in the same subjects during the same 11-week period. Soman exposures produced miosis in all subjects but were otherwise essentially asymptomatic. That is, no convulsions or major signs of toxicity were observed in any subjects, a result consistent with a low-level concentration. Soman exposures produced significant and concentration-dependent decreases in circulating acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity. Soman exposures also produced concentration-dependent levels of regenerated soman in plasma and red blood cell fractions that served to verify the systemic exposure and estimate the total body burden. Soman exposure did not disrupt performance on the VI56 schedule as responding was maintained at pre-exposure levels throughout the 11-week period in all treatment groups. All subjects acquired, and maintained, performance on the RAM task and no significant differences were observed as a result of soman exposure. That is, soman-exposed rats learned the RAM task at the same general rate and to the same general level of accuracy as air-control rats. No delayed effects from exposures were observed. These results demonstrate that, in rats, single exposures to soman vapors at levels that produce substantial AChE and BChE inhibition, but below those producing convulsions and other severe clinical signs of toxicity, may not produce observable effects on the performance of a previously learned task or the acquisition of a new task.