Studies on the toxicology of organophosphorus compounds (OP), including pesticides and nerve agents, have been complicated by the absence of a suitable laboratory animal model. Inexpensive animal models that mimic humans are needed to test the effects of exposure to OP agents and to identify antidotes. Rodents are the traditional laboratory model animals. The principal problem with rodents is the presence of carboxylesterase in their blood. For example, mouse plasma contains four esterases: acetylcholinesterase (AChE), butyrylcholinesterase (BChE), carboxylesterase and paraoxonase. Human and monkey plasma contain BChE and paraoxonase, but no carboxylesterase (1)
and only minute amounts of AChE. AChE, BChE, and carboxylesterase are all inhibited by OP toxicants. The LD50
of soman in mice is approximately 20 fold higher than in Rhesus monkeys (2)
. This difference in toxicity is attributed to the presence of carboxylesterase (ES1) in mouse plasma. ES1 acts as an OP bioscavenger, effectively reducing the amount of OP reaching biologically relevant targets.
In the past, the contribution of plasma carboxylesterase activity to nerve agent toxicity has been minimized by pretreating animals with the carboxylesterase inhibitor cresylbenzodioxaphosphorin oxide(3)
(CBDP). This strategy potentiated the toxicity of OP nerve agents, so that the LD50
dose became similar in mouse, rat, guinea pig, and rabbit where these animals differ in the amounts of carboxylesterase in plasma (2–8)
. Use of CBDP may be problematic because this agent inhibits not only carboxylesterase but also a number of plasma and tissue enzymes to varying degrees (9)
, thereby confounding analysis of OP effects.
Our goal was to produce a mouse that lacks plasma carboxylesterase (ES1) but has normal levels of carboxylesterase in liver, intestine, lung and other organs. Plasma carboxylesterase deficient mice have been previously reported in a screen of progeny from triethylenemelamine-treated male mice (10, 11)
. The Jackson Laboratory bred ES1e
males to C57BL/6J females to produce heterozygous embryos for cryopreservation. Before we started the project to make the ES1−/− mouse, we purchased live mice (stock number 000785), strain B6; D2-aEs1e/J. The mice and their progeny had plasma alpha-naphthyl acetate activity levels that were indistinguishable from wild type. However, ES1 deficient mice produced from this stock by another laboratory had 26% of normal activity with o
-nitrophenyl acetate and 1% of normal activity with irinotecan (12)
ES1 carboxylesterase is a 65 kDa glycoprotein synthesized by mouse liver. It constitutes the majority of esterase activity in mouse plasma (13)
. Inhibition of ES1 by OP agents is without apparent associated toxicity (9)
. It was anticipated that this animal model would more accurately mirror human response to OP exposure, because humans have no carboxylesterase in plasma, but do have carboxylesterase in liver, intestine, lung, and other organs (14, 15)
. We made an ES1 knockout mouse that is devoid of plasma carboxylesterase activity and tested its response to soman coumarin, a fluorogenic soman model compound. Soman coumarin was used because it is less toxic than authentic nerve agent and is therefore permitted in non-military laboratories. Soman coumarin inhibits human and bovine acetylcholinesterase(16, 17)
. Authentic soman, but not soman coumarin is hydrolyzed by human plasma paraoxonase and squid DFPase(16)
. Soman coumarin has been used to screen bacterial colonies for soman hydrolase activity where it was found to mimic authentic soman(18, 19)
. This work reports the phenotypes of wild-type ES1+/+, heterozygous (ES1+/−) and homozygous (ES1−/−) mice. The role of plasma carboxylesterase in protection against OP toxicity was tested by comparing the response of ES1−/− and ES1+/+ mice to soman coumarin.