1. Characterization of Serum Carboxylesterase Knockout Mice as a
Model for Organophosphorous Nerve Agent Research
Catherine A Hofstetter1, C Linn Cadieux1, Zachary Canter1 and Douglas M Cerasoli1.
1Physiology and Immunology, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD, United States.
INTRODUCTION
CONCLUSIONS
Table 3. Complete Blood Count analyses of KO and WT mouse blood.
CBC analysis was performed on a Sysmex XT 2000i. Top panel: absolute
cell counts (left) and percentages as a subset of total WBC (right) are
shown. Hemo.=Hemoglobin; Corp.=Corpuscular; MCHC=Mean corp.
hematocrit. Results (bottom row of each panel) are shown as ratios of
average KO values to average WT values. N=9 per sex/genotype cohort.
Table 4. Determination of the LD50 values of a panel of OPs in male and
female KO mice vs. control mice. All values were determined using the
up-down (Dixon-Massey, 1983) assay.
The protein serum carboxylesterase (sCaE) expressed by the Es-1 gene
found in mice and rats, but not in primates, confers increased protection
against G-type but not V-type OPs. We are breeding mice lacking the gene Es-1
(sCaE knockout [KO] mice) and wild-type (WT) counterparts, on the C57BL/6
background. sCaE plays no known physiological role in mice; we performed a
number of analyses which suggest that with the exception of a lack of sCaE
activity in their blood, KO mice exhibit similar physiological profiles to WT
mice. We have determined LD50 values of a panel of OP nerve agents in both
KO and WT mice using the Dixon and Massey method. LD50 values of the G-
series agents in the KO mice are 10-40% of those in WT mice, while LD50 values
for VX do not differ substantially between KO and WT mice. When administed
to KO mice, bioscavenger enzymes such as butyrylcholinesterase (BuChE)
provide protection against 2 LD50s of a variety of agents. Together, these data
support the hypothesis that sCaE KO mice are a more relevant small animal
model for predicting human responses to these OPs than guinea pigs, rats, or
WT mice.
OP nerve agent toxicity results from the inactivation of the enzyme
acetylcholinesterase (AChE) and subsequent accumulation of the
neurotransmitter acetylcholine, leading to convulsions and dysfunctions of the
respiratory and cardiac systems [1-2]. However, other enzymes, including
several carboxylesterases (CaEs), also react with OPs with relatively high
affinity.
The presence of a CaE isoform in the serum of mice, rats, and guinea pigs
[3] contributes to their enhanced resistance to some OPs relative to that of
primates. Because of the high concentration of serum CaE in mice and rats
relative to primates, these animal models are not ideal for predicting OP
susceptibility and medical countermeasure efficacy in humans.
Previous studies suggest that sCaE provides the majority of the enhanced
OP resistance of mice relative to primates [4-8]. We have established at the
USAMRICD a breeding colony of Es-1 KO mice generated on a C57BL/6
background [9]. These animals have been shown to have modestly increased
susceptibility to some OP pesticides and OP nerve agent compounds [10].
sCaE KO mice are physiologically and reproductively similar to WT mice; the
only measurable difference is the presence or absence of CaE in the plasma.
KO mice exhibit LD50 values for the G agents that are 20-40% those of WT
mice; sCaE provides significant protection against the G agents.
Eliminating expression of serum CaE in mice results in a susceptibility to G
agent toxicity that more closely mimics the human response, and injection
of BuChE protects against OP challenge.
sCaE KO mice appear to be a more relevant small animal model than
currently utilized ones for testing candidate bioscavenger enzymes as well as
other medical chemical countermeasures.
sCaE KO mice are currently being used to evaluate novel bioscavenger
enzyme safety, stability and efficacy.
ABSTRACT
RESULTS
DISCLAIMERS. The views expressed in this poster are those of the author(s) and do not reflect the official
policy of the Department of Army, Department of Defense, or the U.S. Government. The experimental
protocol was approved by the Animal Care and Use Committee at the United States Army Medical
Research Institute of Chemical Defense and all procedures were conducted in accordance with the
principles stated in the Guide for the Care and Use of Laboratory Animals (National Research Council,
2011), and the Animal Welfare Act of 1966 (P.L. 89-544), as amended. This research was supported by the
Defense Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division.
ORISE participants C.A.H. and C.L.C. were supported by an appointment to the Internship/Research
Participation Program for the US Army Medical Research Institute of Chemical Defense, administered by
the Oak Ridge Institute for Science and Education through an agreement between the US Department of
Energy and the USAMRICD. This research was supported by the Defense Threat Reduction Agency – Joint
Science and Technology Office, Medical S&T Division.
Table 2. Blood chemistry panel analyses of KO and WT mice: Blood
samples were collected from KO and WT mice of each sex and a panel
of factors was analyzed on a Vitros 350. Results (bottom row of each
panel) are shown as ratios of average KO values to average WT values.
N=10 per sex/genotype cohort.
Dramatically different levels of sCaE activity were found in the plasma of KO vs.
WT mice, with WT mice having an average of over 10-fold higher levels. No clinical
physiological differences between KO and WT mice were found. For blood chemistry
panel analyses, ratios of KO:WT values ranged from 0.7 to 1.2, with the exception of
glucose. CBC analysis did not indicate any clinically relevant differences in RBC or
WBC count or immune function, and genechip analysis showed no differences in
gene expression patterns between mouse strains, although a sex-based difference
in kidney function was observed. No differences in the reproductive physiology of
these strains, including gestation length, size of litters, and number of pups that
survive to weaning, was observed (data not shown).
Sensitivity of KO mice to the G-type agents ranges from 2.5- to 5-fold greater
than that of WT mice, confirming that sCaE plays an important role in the reduced
sensitivity of WT mice to OP nerve agents. As predicted, V agent sensitivity does not
appear to be heavily impacted by removal of sCaE from the blood.
BuChE stability in the circulatory system appears to peak around 5 hours after
administration. As a proof-of-concept experiment, mice were administered 60
mg/kg of BuChE (the molar equivalent of 4 LD50s of GB in WT mice; a therapeutically
relevant dose) or 120 mg/kg and 5 hours later, at the apparent enzyme activity
peak, mice were challenged with 2 LD50s of one of a panel of OPs. 42/44 mice were
protected from OP agent lethality, demonstrating the utility of Es-1 KO mice in
assessment of the protective efficacy of enzyme-based bioscavengers.
DISCUSSION
Table 1. Phenotypic analysis of mice to determine genotypic status
Average CaE activity values with standard deviations are shown for
male and female KO, WT, and heterozygote mice, with averaged male
and female values for each genotype shown in the far right column.
Figures are derived from analysis of over 1400 mice.
Female Male Combined
H +/- 84.91 ± 38.44 78.91 ± 34.94 81.93 ± 36.69
WT +/+ 155.57 ± 68.39 121.90 ± 46.07 140.68 ± 61.18
KO -/- 13.76 ± 14.21 16.31 ± 14.95 14.97 ± 14.60
Table 5. Es-1 KO mice were injected with 60 mg/kg of purified human
butyrylcholinesterase (BuChE) and challenged 5 hours later with 2
LD50s of the indicated OPs. Numbers indicate fraction of surviving
animals at 24 hours.
Figure 3. Es-1 KO mice (n=4) were administered 60 mg/kg of purified
human butyrylcholinesterase (BuChE) via i.p. injection. Mice were
monitored to evaluate enzyme safety, and enzyme stability was
assessed via analysis of blood samples drawn at various post-
administration time points.
GA GB GD GF VX
M WT 220.1 111.5 121.0 206.5 18.7
F WT 442.1 187.4 136.8 360 24.1
M KO 128 25.0 23.4 76.7 20.8
F KO 145.6 40.5 25.4 84 17.3
KO:WT 0.4 0.2 0.2 0.3 0.9
Figure 1. Affymetrix genechip analysis: mRNA was isolated from tissue
samples (kidney, lung, liver, heart, and brain) from male/female WT/KO
mice, and hybridized to a genechip with probes representing every gene in
the mouse genome. Hybridized RNA can be visualized via fluorescence, and
those values can be represented on a PCA, shown here. Each colored set of
dots represents a user-defined set of samples, and a computer-generated
algorithm creates a spheroid that graphically represents the n-dimensional
set of sample mRNA expression.
Male Female Male Female
60 mg/kg BuChE 120 mg/kg BuChE
GA 2/2 2/2 2/2 2/2
GB 2/2 2/2 1/2 2/2
GD 2/2 2/2 2/2 2/2
GF 2/2 2/2 2/2 2/2
VX 3/4 4/4 2/2 2/2
Liver/Muscle function markers
Albumen
Total
Protein
Alkaline
Phos
Alanine
Aminotrans
Aspartate
Aminotrans
Gamma
Glutamyl Trans
Total
Bilirubin
g/L g/dL U/L U/L U/L U/L mg/dL
0.9 1.0 1.1 1.1 0.7 0.4 1.2
Kidney/Metabolic markers Electrolytes
Blood
Urea N
Creatine
Kinase
Creatinine E CO2 Glucose Calcium Chloride Potassium Sodium
mg/dL U/L mg/dL mmol/L mg/dL mg/dL mmol/L mmol/L mmol/L
1.0 1.0 1.2 1.1 0.9 1.0 1.0 1.1 1.1
White Blood Cells
Total WBC Neutrophils
Lympho
cytes
Monocytes Eosinophils Basophils
(*103/ul) # % # % # % # % # %
1.4 1.1 0.5 1.8 1.1 2.1 1.1 2.9 1.4 3.3 1.4
Red Blood Cells
Total RBC Hemo. C Hematocrit
Mean Corp.
Volume
Mean Corp.
Hemo.
MCHC
(*10e6/ul)
1.1 1.1 1.0 1.0 1.0 1.0
Modified LD50 values for OP agents
REFERENCES
1. Koelle, G.B., et al., Cholinesterases and Anticholinesterases, in Handbuch der Experimentallen
Pharmackologie. 1963, Springer-Verlag: Berlin, Germany.
2. Taylor, P., Anticholinesterase Agents, in The Pharmacological Basis of Therapeutics, ed. A.G. Gilman, Rall,
T.W., and Taylor, P. 1990, New York: Macmillan. 131-149.
3. Genetta, T.L., et al., cDNA cloning of esterase 1, the major esterase activity in mouse plasma. Biochem. &
Biophys. Res.Comm., 1988. 151: p. 1364-1370.
4. Maxwell, D.M., et al., The effect of carboxylesterase inhibition on interspecies differences in soman toxicity.
Toxicol. Lett., 1987. 39(1): p. 35-42.
5. Shih, T.M. and McDonough, J.H., Efficacy of biperiden and atropine as anticonvulsant treatment for
organophosphorus nerve agent intoxication. Arch. Toxicol., 2000. 74: p. 165-172.
6. Maynard, R.L. and Beswick, F.W., Organophosphorus compounds as chemical warfare agents, in Clinical and
Experimental Toxicology of Organophosphates and Carbamates, B. Ballantyne and T.C. Marrs, Editors. 1992,
Butterworth: Oxford, London. p. 373.
7. Doctor, B.P., et al., Enzymes as pretreatment drugs for organophosphate toxicity. Neurosci. Biobehav. Rev.,
1991. 15(1): p. 123-128.
8. Cadieux, C.L., et al., Comparing the Susceptibility of Genetically Modified Mice to Intoxication by
Organophosphorus Nerve Agents. 2008: Experimental Biology, San Diego, CA.
9. Duysen, E.G., et al., Production of ES1 plasma carboxylesterase knockout mice for toxicity studies. Chem.
Res. Toxicol., 2011. 24(11): p. 1891-1898.
10. Duysen, E.G., et al., Differential sensitivity of plasma carboxylesterase-null mice to parathion, chlorpyrifos
and chlorpyrifos oxon, but not to diazinon, dichlorvos, diisopropylfluorophosphate, cresyl saligenin
phosphate, cyclosarin thiocholine, tabun thiocholine, and carbofuran. Chem. Biol. Interact., 2012. 195(3): p.
189-198.
LD50 values for OP agents, ug/kg
0
10
20
30
40
50
60
0 50 100 150 200
AvgngBuChEactivity(ug/ml)
Hours after BuChE administration
BuChE activity in male and female KO
mice after I.P. administration
Females
Males
Variable Sex Genotype
Mean ± Std
Deviation
95% Confidence Interval
Lower Bound Upper Bound
Center Time
Male
KO 128.55 ± 14.2 99.8 157.3
WT 155 ± 14.2 126.2 183.8
Female
KO 63.2 ± 14.2 34.4 92.0
WT 93.7 ± 14.2 64.9 122.5
Outside Time
Male
KO 471.4 ± 14.2 442.7 500.2
WT 445 ± 14.2 416.2 473.8
Female
KO 536.8 ± 14.2 508.0 565.6
WT 506.3 ± 14.2 477.5 535.0
Center Distance
Male
KO 9.0 ± 1.2 6.6 11.4
WT 9.6 ± 1.2 7.2 12.0
Female
KO 7.1 ± 1.2 4.8 9.5
WT 7.9 ± 1.2 5.6 10.4
Outside Distance
Male
KO 20.4 ± 3.0 14.3 26.4
WT 20.9 ± 3.0 14.9 27.0
Female
KO 38.9 ± 3.0 32.9 44.9
WT 25.3 ± 3.0 19.3 31.3
Figure 2. Behavioral analysis: Several behavioral tests were performed on WT
and KO mice. Results from the Open Field Test are shown above. No
statistically significant differences were found between the two strains.