U.S. Army Edgewood Chemical Biological Center, AMSSB-RRT-TT (E3150), 5183 Blackhawk Road, Aberdeen Proving Ground, Maryland 21010-5424
Received August 14, 2001; accepted November 1, 2001
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ABSTRACT |
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Key Words: Sarin; inhalation; exposure concentration; rat; lethality; miosis; mydriasis; cholinesterase; LC50; LCT50.
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INTRODUCTION |
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Alternatively, ten Berge et al. (1986) demonstrated that:
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The primary objective of the present study was to model the relationship between GB vapor exposure concentration (C), duration of exposure (T) and the probability of a toxic effect (lethality). Developing such models is hampered by the scarcity of published data involving acute chemical agent vapor exposures beyond several minutes (NRC, 1997; Yee, 1996
). Estimating responses to exposures beyond a few minutes currently requires extrapolation based upon the assumptions of the theoretical dosage models cited above. This study tested whether the relationship between exposure concentration time and lethal response in rats exposed to GB vapor for 5360 min, could be adequately described by the above models.
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MATERIALS AND METHODS |
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The majority of the impurities in the CASARM GB consisted of 0.2% o,o'-diisopropyl methyl phosphonate (DIMP), 0.2% methyl phosphonic difluoride (DF), 0.3% methyl fluoridic acid (Fluor Acid), and 0.3% excess HF/F ion. Impurity percentages were based on mole ratios from acid-base titration.
Vapor generation.
The vapor generation system (Muse et al., 2000) consisted of a gas-tight syringe (Hamilton, Reno, NV), variable-rate syringe drive (Model 22, Harvard Apparatus Inc., South Natick, MA), and a vaporization apparatus. This system, located at the chamber inlet, was contained within a stainless steel glove box (23 in. long x 14 in. wide x 18 in. high) maintained under negative pressure (0.25 in. H2O). A Plexiglas door at the front of the glove box facilitated syringe loading and syringe drive adjustments during setup operations. Prior to chamber operation, liquid GB (undiluted) was loaded into a gas-tight syringe (Hamilton, Reno, NV), then the syringe was mounted onto the syringe drive. Once activated, the syringe drive provided a constant dispersal rate of GB (µl/min) through a flexible plastic line (
8 in. long) into a spray atomization system (Spray Atomization Nozzle
J SS, Spraying Systems Co., Wheaton, IL). The atomizer was modified by retrofitting a syringe needle (SS 25 gauge 3 in.) into the top of the sprayer to provide a smaller orifice. As liquid GB entered through the top of the sprayer, compressed air (3040 psi) entered through the side. The compressed air broke the liquid GB into fine droplets (< 15 µm diameter.), which then entered the chamber inlet. Due to the high volatility of GB (2.2 x 104 mg/m3 at 25°C), these droplets quickly evaporated, with the resulting vapor drawn through the exposure chamber.
Sampling/Monitoring Exposure Chamber GB Vapor
Vapor sampling/analysis.
Three methods were used to sample/monitor and analyze GB vapor concentration in the exposure chamber: (1) Edgewood bubblers (containing hexane)/ gas chromatograph with flame photometric detection (GC-FPD); (2) solid sorbent tubes (Tenax-TA)/gas chromatograph with flame ionization detection (GC-FID); and (3) a phosphorus monitor (HYFED, Model PH262) that provided a continuous strip chart record of rise, equilibrium, and decay of the chamber vapor concentration during an exposure.
GB vapor was sampled as it was drawn through a 750-liter dynamic airflow, whole-body inhalation exposure chamber located within a 20-m3 containment chamber. The exposure chamber was constructed of stainless steel with Plexiglas windows on each of the six sides. The interior of the exposure chamber was maintained under negative pressure (0.25 in. H2O), which was monitored with a calibrated magnehelix (Dwyer, Michigan City, IN). Room air (500650 l/min) was drawn through the chamber under negative pressure. A thermoanemometer (Model 8565, Alnor, Skokie, IL) was used to monitor chamber airflow at the chamber outlet. All samples were drawn from the same area (middle) of the chamber. Bubbler and solid sorbent tube samples were drawn after the chamber attained equilibration (t99) while the HYFED monitored the entire run. Bubbler samples were drawn from the chamber every hour, with each sampling period lasting 112 min. Solid sorbent tube samples were drawn from the chamber approximately every 15 min, with each sampling lasting 23 min. All sample flow rates for the bubbler and solid sorbent tube systems were controlled with calibrated mass flow controllers (Matheson Gas Products, Montgomeryville, PA). Typical flow rates were 0.91.0 l/min for the bubblers and 100 standard cubic centimeters per minute for the sorbent tubes. Due to solvent (hexane) evaporation during sampling, an in-line charcoal filter was installed between the bubbler and mass flow controller. This was to prevent the cooling effect of the solvent from affecting the mass flow sensor. Flow rates from both systems were verified before and after sampling by temporarily connecting a calibrated flow meter (DryCal®, Bios International, Pompton Plains, NJ) in-line to the sample stream.
Bubbler tube.
The concentration of GB in the chamber was determined by collecting chamber air samples into Edgewood bubblers containing hexane, connected in series (Muse et al., 2000). The second bubbler (downstream), in a series of two, contained one-half the solvent volume of the upstream bubbler to increase sensitivity for analyzing the lower GB concentration (58% of the upstream bubbler) present in the downstream bubbler. Samples were drawn through glass sample lines (0.25 in. o.d.) at the rate of 0.91.0 l/min. The collected solvent was diluted to a known volume and injected into a gas chromatograph with flame photometric detection, (GC-FPD) phosphorus mode. External standards (GB/hexane) were injected into the GC-FPD to generate a calibration curve. A linear regression fit (r2 = 0.999) of the standard data was used to compute GB concentration.
Solid sorbent tube.
The automated solid sorbent tube sampling system consisted of four parts: a heated sample transfer line; heated external switching valve; thermal desorption unit; and gas chromatograph. A stainless steel sample line (1/16 in. o.d. x 0.004 in. i.d. x 6 feet long) extended from the middle of the chamber to an external sample valve. The sample line was commercially treated with a silica coating (Silicasteel® Restek, Bellefonte, PA) and covered with a heated (60°C) sample transfer line (CMS, Birmingham, AL). The combination line coating and heating minimized GB absorption onto sample surfaces. From the transfer line, the sample entered a heated (125°C) 6-port gas-switching valve (UWP, Valco Instruments, Houston, TX). In the bypass mode, GB vapor from the chamber continuously purged through the sample line and out to a charcoal filter. In the sample mode, the gas sample valve redirected GB vapors from the sample line to a Tenax TA sorbent tube located in the thermal desorption unit (ACEM-900, Dynatherm Analytical Instruments, Kelton, PA). The solid sorbent material used to trap GB vapor consisted of Tenax-TA. Temperature and flow programming within the Dynatherm desorbed GB from the sorbent tube for vapor injection directly onto the gas chromatograph (GC) for quantitation. Either flame ionization detection (FID) or flame photometric detection (FPD) was used, depending upon the level of sensitivity required.
To calibrate the solid sorbent tube sampling system, external standards (GB/hexane) were injected directly into the heated sample line of the Dynatherm. In this way, injected GB standards were put through the same sampling and analysis stream as the chamber samples. A linear regression fit (r2 = 0.999) of the standard data was used to compute chamber sample GB concentration.
Phosphorus monitor (HYFED).
GB levels in the chamber were continuously monitored with a phosphorus analyzer (HYFED, Model PH262, Columbia Scientific, Austin, TX). The analyzer output was recorded on a strip chart recorder, which showed the rise, equilibrium, and decay of the chamber vapor concentration during a run. In addition, it gave a close approximation of the amount of GB (µg/l) in the chamber based on data (bubbler and solid sorbent tube quantification with HYFED response) from previous chamber runs.
Animal Exposures
In conducting this study, investigators adhered to the Guide for the Care and Use of Laboratory Animals, National Institutes of Health Publication No. 86-23, 1985, as promulgated by the Committee on Revision of the Guide for Laboratory Animal Facilities and Care of the Institute of Laboratory Animal Resources, Commission of Life Sciences, National Research Council, Washington, DC. These investigations were also performed in accordance with the requirements of AR 70-18, Laboratory Animals, Procurement, Transportation, Use, Care, and Public Affairs, and the U.S. Army Edgewood Chemical and Biological Center Institutional Animal Care and Use Committee (IACUC).
Animal model.
Young adult male and female Sprague-Dawley rats (810 weeks) were purchased from Charles River Laboratories, Inc., Wilmington, MA. Rats were identified by tail tattoo and housed individually in plastic shoebox cages in an American Association for Accreditation of Laboratory Animal Care (AAALAC) accredited facility. Ambient holding conditions were maintained at 21 ± 3°C, 4070% relative humidity, and a 12:12 h light-dark cycle. Rats were provided with certified laboratory rat chow and water ad libitum (automatic watering system using a reverse osmosis process), except during vapor exposure. Animals were quarantined for at least 5 days prior to exposure.
Whole-body inhalation exposures.
All animals were exposed (whole-body) to GB vapor in a 750-liter dynamic airflow inhalation chamber located within a 20-m3 containment chamber. During inhalation chamber operations, the airflow through the chamber was kept constant. The concentration-time profile generated with this type of inhalation chamber is described in a review by MacFarland (1987). His definition of exposure duration was the one used in this study: the interval from the start of the flow of agent into the chamber to the time point when the agent supply is stopped. The time required for the vapor concentration to reach 99% (denoted as t99) of its equilibrium value for each of the exposure durations is listed in Table 1. The t99 is also the time required for the chamber to lose 99% of its equilibrium concentration after the agent supply is stopped. The concentrations recorded in Table 1
are the equilibrium concentrations.
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Exposure groups consisted of 10 rats of one sex or 20 rats (10 of each sex) placed in stainless steel compartmentalized cages (20 in. wide x 14 in. long x 4 in. high), with each rat in a separate compartment. Rats were exposed to one of five concentrations (254 mg/m3) of GB vapor for one of seven exposure times (5, 10, 30, 60, 90, 240, or 360 min). Lethality and sublethal clinical signs (e.g., miosis, tremors, salivation, labored breathing, and convulsions) were monitored from an observation point outside the chamber during exposure, afterward within the first hour postexposure, and once daily thereafter for up to 14 days. Physical parameters monitored during exposure included chamber airflow (monitored continuously), as well as chamber room temperature and relative humidity. Immediately after completion of the exposure period, the chamber was purged with air for a minimum time of t99 (t99 = time for chamber to attain or lose 99% of its equilibrium concentration).
Experimental Design
This study consisted of two parts. In Part I, concentration-response curves were determined for each of four GB vapor exposure durations (10, 30, 90, and 240 min), with separate shipments of rats used for each duration. The first shipment was used for 10-min duration exposures, the next shipment for 30-min duration, and the last two shipments for 90- and 240-min durations, in that order. Additional exposure durations (5, 60, and 360 min) were conducted in Part II. In Part II, five shipments of rats were used. Each shipment was divided into three equal-sized groups, one group for each exposure duration. Thus, the five shipments yielded five separate exposure concentrations per exposure duration. The three groups were tested in random order. Concentrations for the three exposure durations (i.e., the 5, 60, and 360 min) were varied independently of each other. Selection of exposure concentration depended on the fractional mortality at the concentrations used for the previous shipments.
Blood sample collection.
Two blood samples were collected (within 24 h prior to exposure and within 60 min after exposure) from each surviving animal for the purpose of measuring cholinesterase activity in both RBC and plasma components. Tail vein blood samples were collected into glass tubes containing EDTA. Assays of red blood cell acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BuChE) activity were performed by the U.S. Army Medical Research Institute of Chemical Defense (USAMRICD), APG, Maryland, using a modification of the Ellman reference method (Ellman et al., 1961).
Observation of clinical signs.
Observations for toxic clinical signs were carried out during, immediately after, and once daily after exposure. Clinical signs of clonic or tonic movements (e.g., convulsions, tremors, muscle fasciculation), piloerection, gait abnormalities, overt respiratory abnormalities, and general appearance were also noted. Pupil sizes were monitored at least 24 h prior to exposure, at 12 h following whole-body GB vapor exposure, and at 1, 2, 3, and 7 days postexposure. Pupil size (diameter) was assessed using a simple microscope (Bausch and Lomb, 20x) with a reticule eyepiece insert under a 200-foot-candle light source, as monitored by a light meter (Davis, Model 401025, Extech Instruments, Waltham, MA). This procedure consisted of counting the number of reticule lines covering the pupil diameter (20 lines/mm or 0.05 mm between lines).
Data analysis.
A probit analysis program (a component of MINITAB®, Version 13) was used to generate a separate dose-response curve (with slope, intercept, and 95% fiducial limits) for each duration of exposure tested and to determine if sex differences exist in the sensitivity to the toxic effects of GB vapor exposure. Binary normal regression (multifactor probit analysis) was used to model the relative effects of exposure concentration and duration on probability of lethality. Differences in preexposure versus postexposure cholinesterase levels were expressed as a percent change resulting from treatment; thus, each individual rats served as its own control. This graded response was plotted against CT using linear regression to determine if correlations exist as indicated by significant regression coefficients.
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RESULTS |
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Blood Cholinesterase Activity
Both red blood cell acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BuChE) activities were inhibited as a result of exposure to various combinations of GB vapor concentration and time (Fig. 2). Most responses appeared to fall between 5 and 30% of pretreatment baseline. These data are limited to samples collected from rats surviving up to 60 min after exposure to GB vapor. Each postexposure response is expressed as percent of baseline (i.e., each animal's postexposure AChE or BuChE activity is expressed as a percent of its pretreatment value collected at 24 h prior to exposure). Except for AChE activity in female rats, there was a statistically significant (p < 0.05) correlation between ChE activity and CT. However, the fits for male AChE (r2 = 0.04), female AChE (r2 = 0.00), male BuChE (r2 = 0.31), and female BuChE (r2 = 0.51) versus CT were very poor and could not be used for predictive purposes. Median pretreatment levels of BuChE activity were consistently higher (p < 0.001) in female (1750 U/ml) than in male rats (424 U/ml), as determined by the Mann-Whitney Rank Sum test. However, no differences were noted between pretreatment male and female AChE activity.
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DISCUSSION |
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To examine the curvature in the plot of log(LCT50) versus log(Time), only the data from Part II were used. Starting with the 12 terms constant, cLogC, cLogT, (cLogC)2, (cLogT)2, cLogC*cLogT, Sex (coded 1 for male and 1 for female), Sex*cLogC, Sex*cLogT, Sex*(cLogC)2, Sex*(cLogT)2, and Sex*cLogC*cLogT, where cLogC = centered log(Concentration) and cLogT = centered log(Time), the least significant term (largest p value) was deleted followed by reanalysis. This process was iterated until all terms were significant (p < 0.05). The centering (subtracting the mean) of log(C) and log(T) reduces multicollinearity in the model. The backwards elimination procedure resulted in the following model:
For probability of lethality, let Y = normit (normit = Probit -5), then
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The Sex*cLogC term in the interaction model, Equation 3, is of marginal significance (p = .033). When the model (3) is fit to the Part I data, the Sex*cLogC term is not significant (p = .184), whereas the other terms of Equation 3
are highly significant (p < .001). Part I data should be valid to test the Sex*cLogC term, because both sexes were exposed together in nearly all the chamber runs. Thus, the data are unclear about whether the Sex*cLogC term should be included in the model. Equations 4 and 5
are extensions of the toxic load model. They are referred to here as the interaction model because of the presence of the interaction term cLogC*cLogT.
The interaction term, log(T)*log(C), allows the distribution of individual tolerances to both concentration (at a fixed exposure duration) and exposure duration (at a fixed concentration) to be lognormal. The presence of the term [log(Time)] squared or the term [log(Concentration)] squared in the model would imply that the distribution of individual tolerances to exposure duration or exposure concentration, respectively, cannot be lognormal. The model (3) is an empirical model and is limited to the conditions of the experiment from which it was derived. These conditions include the strain of rat, the age, diet, and health status of the rats, the activity level of the rats during exposure, and concentration-time profiles to which the rats were exposed.
The curvature in the interaction model fit of Part II data is roughly equivalent to the curvature in the fit of Part I data. The experimental design for Part I could not definitively rule out the possible effects of season and shipment of rat. However, the similarity in curvature between the fits of Part I and Part II indicates that these effects are minor.
For comparison, a toxic load expression was also used to fit the data from both Part I and Part II:
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Male versus Female Sensitivity to GB Vapor-Induced Lethality
Female rats were more sensitive to the lethal effects of GB vapor than males in this study, based on the significance (p < 0.001) of the Sex term in Equation 3. Also, probit analysis with sex as a factor was performed for each duration (Mioduszewski et al., 2001
), and it was found that the concentration-response for each exposure time could be differentiated by sex (with the exception of a few instances [30 and 60 min]). In addition, a review of the clinical sign data suggests that clinical signs of toxicity appeared earliest in females, as a group, and progressed to more severe levels earlier than in male rats. These findings are consistent with those of Callaway and Blackburn (1954), who reported (for 1-min exposures) that female rats were almost twice as sensitive to the lethal effects of GB vapor than males. McPhail (1953) reported that the male mouse was more sensitive to GB vapor than the female, but the reverse was true for the hamster and the rat. In addition, female rats have been shown to be more sensitive than males to the lethal effects of Soman (Sket, 1993
). In female rats, the LD50 for Soman (GD) was only about half that of males. This pattern was also reported for lethal exposure of rats to some organophosphate (OP) insecticides (Sheets et al., 1997
).
GB Vapor Effects on Blood AChE and BuChE
The most commonly accepted mechanism by which nerve agents are believed to induce acute toxicity is by inhibition of AChE activity in target tissues. AChE normally limits the action of endogenous acetylcholine (the chemical mediator of junctional transmission at cholinergic synapses). The resulting cholinergic hyperactivity is often expressed in a variety of toxic clinical signs. Although red blood cell (RBC) and plasma cholinesterase activities are routinely monitored as a sensitive index of exposure to anticholinesterase agents, they by no means imply anticholinesterase intoxication (Koelle, 1994). Sidell (1992) suggests that activity of the circulating ChE does not parallel the activity of ChE in tissue and that tissue function can be reasonably normal even with minimal blood ChE activity. If an OP compound is administered in low concentration levels over a long period, blood levels of ChE of an animal can drop to near zero, yet the animal survives. If blood levels of ChE are caused to rapidly drop to zero, the animal dies. Consequently, as confirmed by the results of the present study, poor correlation is expected between probability of toxic signs in the lethal range and exposure dosage.
It has been speculated that serum esterases (phosphatases, carboxylesterases, plasma BuChE, and red blood cell AChE) and plasma proteins may reduce OP availability by binding it or hydrolyzing the compound to a less toxic metabolite (Ecobichon and Comeau, 1973). The most important OP scavengers in rodents are carboxylesterases (CaEs) in plasma and organs, whereas endogenous plasma BuChE has only a minor role as a detoxification route for OPs (Clement, 1984
; Grubic et al., 1988
). Maxwell et al. (1987a) suggested this may be due to the greater frequency of CaE binding sites.
Sex differences in the activity of BuChE and CaE but not (AChE) have been reported both in rodents (Sterri and Fonnum, 1989; Sterri et al., 1985
) and in man (Jensen et al., 1995
). Sterri and Fonnum (1989) speculated that the 2-fold higher BuChE activity in female rat plasma is probably due to a doubling in the number of enzyme molecules, which also should double the number of binding sites for OPs. In the present study, female rats were more sensitive than males to GB vapor-induced toxic effects, even though the female rat preexposure mean BuChE activity was approximately four times higher than the corresponding BuChE activity in male rats. If BuChE was effectively minimizing the availability of GB, less toxicity would be expected in female rats. Although Sarin appears to be binding to BuChE in the present study (Fig. 2
), BuChE activity was not inhibited as effectively in female rats as in males, as suggested by higher incidences of toxic signs in females (Table 1
). Because CaE activity was not measured in this study, it would be difficult to speculate about the possible role of CaE in accounting for sex differences in sensitivity to GB toxicity. The greater importance of CaEs versus BuChEs as a detoxifying resource of OPs, has been emphasized by several investigators (Clement, 1984
; Maxwell et al., 1987a
,b
).
Pupil Response to GB Vapor Exposure
Insofar as the GB vapor concentrations used in this study were selected for estimating the LC50, it is not surprising that maximal pupil constriction was seen in all rats during the first 24 h after exposure. However, the marked and consistent reversal of this response (Fig. 3), progressing temporarily to mydriasis, was not expected, as such responses are rarely reported in the literature. In Figure 3
, there is a clear separation of pupil response (at 2 days postexposure) based upon exposure time group (5, 60, 240, and 360 min). Nevertheless, there are difficulties in interpreting the dose-response relationship for mydriasis due to possible confounding of sex and concentration, because the males were sometimes exposed to higher vapor concentrations. In addition, there were different proportions of survivors among various categories (male vs. female, and different concentration/exposure time groups [see Table 1
]). Lastly, the entire time profile of pupil diameter change was not recorded. Measurements were only made preexposure and postexposures at 1 h, 1 day, 2 days, and 7 days. Thus, it is unclear whether the maximum mydriasis was observed, and therefore it cannot be determined how it depends on exposure conditions.
GB vapor-induced changes in pupil diameter in the present study may likely be a local ocular effect of GB. It is possible that GB exposure altered the balance between sympathetic versus parasympathetic control over pupil size, which changed over time after exposure. It can be speculated that sometime after the start of exposure (within 24 h), the cholinergic component predominated in the absence of AChE activity, resulting in miosis. Within 23 days after exposure, cholinergic desensitization occurred, resulting in noradrenergic dominance and subsequent mydriasis. Beyond 3 days postexposure, homeostasis between opposing sympathetic and parasympathetic control of pupil size was restored.
Summary
This study evaluated the adequacy of traditional models in predicting the toxicity of Sarin vapor exposure in the rat, given various exposure conditions. Incidental data were also collected on changes in blood cholinesterase activity and pupil response due to Sarin vapor exposure. The lethality data generated from this study were used in formulating models for estimating the probability of Sarin vapor-induced lethality in a rodent model, given a combination of exposure concentration and duration. The findings of the present study are consistent with the need to expand beyond dependence on Haber's rule, as advocated by ten Berge et al. (1986), Yee (1996), and Miller et al. (2000).
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ACKNOWLEDGMENTS |
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NOTES |
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