Pharmacology & Toxicology 1992,70, 65-66.
A Purified Recombinant Organophosphorus Acid Anhydrase Protects Mice Against Soman C. A. Broomfield
Biochemical Pharmacology Branch, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5425, U.S.A. Received April 23, 1991; Accepted August 5, 1991) Abstract: Since pharmacologic treatments of organophosphorus anticholinesterases (OPs) are nearing their practical limit other types of treatment are being sought. One approach is the prophylactic administration of scavengers that will detoxify OPs before they reach their critical target site. Using mice that were sensitized to OPs by depletion of their serum carboxylesterase with cresylbenzodioxaphosphorinoxide (CBDP), we have shown that animals pretreated intravenously with a purified organophosphorus acid anhydride hydrolase (parathionase) (0.10 mg per g body wt.) are not measureably affected by up to 34.4 pg soman per kg, a dose more than double that which is lethal to untreated animals. This result indicates that this approach is worthy of exploration and development for protecting military personnel and agricultural workers against OP intoxication.
There has been considerable progress in the development of pretreatments and therapy against nerve agents. Nevertheless, soman and other organophosphorus anticholinesterases (OPs) remain a chemical warfare threat. There is also a concern that treatments for agricultural workers who are accidentally exposed to OP insecticides are less than satisfactory. Furthermore, we are approaching the limiting efficacy for pharmacologic intervention with the drugs now in development (Dunn & Sidell 1989). Any significant increase of protection without debilitating side effects must result from new approaches. One such approach is the prophylactic administration of scavengers capable of detoxifying OPs in the blood stream before they reach their critical targets. The use of rapidly reacting enzymes, such as acetylcholinesterase, butyrylcholinesterase and carboxylesterase has been suggested (Maxwell et al. 1991), and the feasibility of this approach has been demonstrated (Fonnum et al. 1985; Lenz et al. 1991). While those scavengers offer some protection against the nerve agents, including soman, they have in common the disadvantage that they all have high molecular weights and react 1 : l with the OPs. As a result, about 500 mg of scavenger must be active in the blood stream for each mg of OP to be neutralized. In addition, multiple exposures would deplete the available scavenger and would render the organism susceptible to intoxication. It would be preferable to introduce a small amount of effective enzyme into the blood stream to destroy larger amounts of OP. Recently it has become feasible to produce large amounts of an organophosphorus acid anhydride hydrolase (OPAH), also called parathion hydrolase, by expressing a gene from Pseudomonas diminuta MG in Escherichia coli (Serdar & Gibson 1985; Serdar et al. 1989). This purified enzyme was shown previously to inactivate OPs, including soman (Dumas et al. 1990). Therefore, we tested the feasibility of protecting mice against multiple lethal doses of soman by administering a high concentration of this enzyme intravenously and then challenging the animals repeatedly with the OP. Mice
and certain other rodents are resistant to OP poisoning due to the high levels of carboxylesterase in their plasma (Boscovic 1979). To compensate for this factor all animals were treated with cresylbenzodioxaphosphorin oxide (CBDP), two hours before the first soman challenge (McKay et al. 1971). The LDso of OPs in mice so treated are approximately the same as those seen in higher species (McKay et al. 1971; Maxwell et al. 1988; Sterri & Fonnum 1989), and therefore the protection achieved in this model is believed to approximate that expected in man.
Materials and Methods Animal care was in accordance with the principles prescribed in the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources, National Academy of Sciences-National Research Council. All of the mice were given identifying markings and injected subcutaneously (one p1 per g body weight, 2 mg per kg) with the CBDP solution, 2 mg/ml in propane diol. They were then divided into two groups of five and one hour after the CBDP treatment one group was injected intravenously with a solution of OPAH, 10 mg per ml in 20 mM Tris HCI, pH 8.5, 10 plig body wt. so as to produce a soman-hydrolyzing activity in the blood of 90.3 pmol per min./ml one hour after injection. They were then all placed together in the same cage, and beginning one hour after treatment with OPAH, they were treated subcutaneously in random order with soman, 100 pI of a 0.5 pg per ml solution in normal saline, at intervals of not less than 15 or more than 20 min. During the soman treatment experimental mice were not distinguished from controls in order to eliminate possible bias. Injections were discontinued whenever an animal began to show severe toxic signs. Those animals surviving after 5 hrs were tested for incapacitation on an inverted screen apparatus (Coughenour et al. 1977).
Results and Discussion Experimental animals that had been treated with OPAH received an average of 31.1 Fg/kg of soman over a period of 5 hr and none of them displayed any signs of intoxication. All control mice died between two and five hours after
C. A. BROOMFIELD
Table 1. Reactions of individual animals’ to soman challenge.
Animal no. 7 4 8 9 2
Wt. (g) 26.6 29.9 28.2 27.0 26.5
Role’ Con Con Con Con Con
Mean dose for control group 1
6 3 10
30.5 28.8 27.4 26.0 24.1
EXP EXP EXP EXP EXP
Mean dose for experimental group
Dose recd. (Pdkd 7.6 10.2 14.7 15.3 24.8
T/D’ (hr) 2.0 2.7 4.7 5.0 5.0
14.5 27.8 29.5 31.0 32.7 34.4
n/a n/a n/a n/a n/a
range (Dumas et al. 1990). Therefore we administered a fairly high concentration of enzyme in this experiment and delivered an incremental challenge so that the enzyme would have time to hydrolyze the OP. Work is currently in progress to discover or develop an enzyme with a higher affinity and a higher turnover number for soman so that a lower concentration of enzyme can be used and protection can be realized against a large single dose. Nevertheless, these results demonstrate that the concept of protecting animals against OP anticholinesterases by pretreatment with catalytic scavengers is potentially useful. Acknowledgement The purified enzyme used in this study was generously provided by Dr. C. M. Serdar, AMGEN, Thousand Oaks, CA, U.S.A.
1 As described in the “experimental” section, all mice were pre-
treated with CBDP (2 mg/kg subcutaneously); experimental animals also received an intravenous dose of 100 pg OPAH/g body wt. before challenge with soman doeses commenced. Exp: mice treated with OPAH; Con: mice treated only with CBDP. Elapsed time between beginning of soman injections and death of the animal.
injections of soman were begun with a mean lethal dose of 14.5 pg/kg. Thirty min. after the last injection the surviving animals were tested for incapacitation by an inverted screen test; all of them passed. They also passed the inverted screen test again the following morning with no evidence of toxic signs of any kind. While the number of animals used in this experiment was small it is clear that the enzyme afforded significant protection against soman intoxication. The administration of soman was not carried beyond 5 hr due to possible recovery of carboxylesterase activity, which might confuse the results; the half-time for recovery is at least 12 hr (Clement 1989). It is particularly significant that the enzyme-treated animals were apparently unaffected by doses of soman that were more than double those that killed the control animals. The median lethal dose (14.5 pg/kg) observed for these control animals was somewhat higher than that found (10 pg/ kg) for CBDP-treated mice challenged with higher single doses (McKay et al. 1971). Since the agent was administered in sublethal increments we believe that the endogenous OPAH present in the plasma of mice (Harris et al. 1984) was able to detoxify part of it before it reached the critical acetylcholinesterase sites. Soman is able to react with acetylcholinesterase within the course of a single circulation time (Maxwell et al. 1988). Therefore any effective scavenger must have a high affinity for the OP or be present at a very high concentration. Stoichiometric scavengers such as acetylcholinesterase, butyrylcholinesterase or carboxylesterase have the advantage of very high affinity enabling them to neutralize larger single doses of OP. Unfortunately, this is coupled with a low capacity, so that much more protein must be used. The K, is rather high for the enzyme used in this study with soman as the substrate, in the millimolar
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