OpdA, a bacterial organophosphorus hydrolase, prevents lethality in rats after poisoning with highly toxic organophosphorus pesticides

Article abstract of DOI:10.1016/j.tox.2008.02.005

Organophosphorus (OP) pesticides poison more than 3,000,000 people every year in the developing world, mostly through intentional self-poisoning. Advances in medical therapy for OP poisoning have lagged, and current treatment is not highly effective with mortality of up to 40% in even the most advanced Western medical facilities. Administration of a broadly active bacterial OP hydrolase to patients in order to hydrolyze OPs in circulation might allow current therapies to be more effective. The objective of this work was to evaluate the efficacy of a new recombinant bacterial OP hydrolase (OpdA), cloned from Agrobacterium radiobacter, in rat models of two chemically distinct but highly toxic and rapidly acting OP pesticides: dichlorvos and parathion. Without OpdA treatment, median time to death in rats poisoned with 3× LD₅₀ of dichlorvos or parathion was 6 min and 25.5 min, respectively. Administration of a single dose of OpdA immediately after dichlorvos resulted in 100% survival at 24 h, with no additional antidotal therapy. After parathion poisoning, OpdA alone caused only a delay to death. However, an additional two doses of OpdA resulted in 62.5% survival at 24 h after parathion poisoning. In combination with pralidoxime therapy, a single dose of OpdA increased survival to 75% after parathion poisoning. Our results demonstrate that OpdA is able to improve survival after poisoning by two chemically distinct and highly toxic OP pesticides.

Full text of DOI:10.1016/j.tox.2008.02.005

Toxicology 247 (2008) 88–92
Contents lists available at ScienceDirect
Toxicology
journal homepage: www.elsevier.com/locate/toxicol
OpdA, a bacterial organophosphorus hydrolase, prevents lethality in rats after
poisoning with highly toxic organophosphorus pesticides
Steven B. Birda,∗, Tara D. Sutherlandb, Chip Greshamc, John Oakeshottb, Colin Scottb, Michael Eddlestond
a Department of Emergency Medicine, Division of Toxicology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA
b CSIRO Entomology, Canberra, ACT 2601, Australia
c Department of Medical Toxicology, Banner Good Samaritan Medical Center, Phoenix, AZ, USA
d Clinical Pharmacology Unit, University of Edinburgh and Scottish Poisons Information Bureau, Royal Infirmary of Edinburgh, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Organophosphorus (OP) pesticides poison more than 3,000,000 people every year in the developing world,
mostly through intentional self-poisoning. Advances in medical therapy for OP poisoning have lagged,
and current treatment is not highly effective with mortality of up to 40% in even the most advanced
Western medical facilities. Administration of a broadly active bacterial OP hydrolase to patients in order
to hydrolyze OPs in circulation might allow current therapies to be more effective. The objective of this
work was to evaluate the efficacy of a new recombinant bacterial OP hydrolase (OpdA), cloned from
Agrobacterium radiobacter, in rat models of two chemically distinct but highly toxic and rapidly acting OP
pesticides: dichlorvos and parathion. Without OpdA treatment, median time to death in rats poisoned
with 3× LD50 of dichlorvos or parathion was 6 min and 25.5 min, respectively. Administration of a single
dose of OpdA immediately after dichlorvos resulted in 100% survival at 24 h, with no additional antidotal
therapy. After parathion poisoning, OpdA alone caused only a delay to death. However, an additional
two doses of OpdA resulted in 62.5% survival at 24 h after parathion poisoning. In combination with
pralidoxime therapy, a single dose of OpdA increased survival to 75% after parathion poisoning. Our results
demonstrate that OpdA is able to improve survival after poisoning by two chemically distinct and highly
toxic OP pesticides.
Received 30 January 2008
Received in revised form 13 February 2008
Accepted 14 February 2008
Available online 10 March 2008
Keywords:
Organophosphorus (OP)
Hydrolase
Acetylcholinesterase (AChE)
Pralidoxime (2-PAM)
© 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
OPs inhibit acetylcholinesterase (AChE, EC 3.1.1.7), resulting
in overstimulation at cholinergic synapses. Clinical management
Occupational exposure and intentional self-poisoning with
organophosphorus (OP) pesticides are major global health prob-
lems (Jeyaratnam, 1990; Van der Hoek et al., 1998). The World
Health Organization estimates that as many as 3,000,000 people
per year are poisoned by pesticides; many are due to OP pesti-
cides, resulting in around 200,000 deaths (Jeyaratnam). Although
the greatest burden is borne by the developing world (Buckley et al.,
2004; Eddleston and Phillips, 2004), it is also an important cause
of fatal self-poisoning in developed countries (Bruyndonckx et al.,
2002). Highly toxic and widely available OPs such as parathion
also pose a threat to municipal water supplies from intentional or
unintentional contamination.
of moderate and severe poisoning is difficult, requiring pro-
longed intensive care and use of large doses of atropine, oxime
cholinesterase reactivators, and benzodiazepines (Eddleston et al.,
2007). However, these therapies are insufficient, not always avail-
able in the developing world, and do not prevent the post-poisoning
neurocognitive dysfunction that is common with severe poisonings
(Dunn and Sidell, 1989). Overall mortality after OP poisoning in the
developing world is as high as 25%, and in the most sophisticated
Western hospitals mortality is as high as 40% (Eyer et al., 2003). This
overall difference in mortality between the developed and devel-
oping world is due to the vast numbers of poisoned patients in the
agricultural areas of the developing world, the majority of whom
are not critically ill. However, OP pesticide poisoning is uncommon
in the developed world, and patients who ingest OP pesticides for
self-harm typically have substantial ingestions and are more likely
to be critically ill.
∗
Corresponding author. Tel.: +1 508 421 1422; fax: +1 508 421 1490.
E-mail addresses: birds@ummhc.org (S.B. Bird), tara.sutherland@csiro.au
(T.D. Sutherland), chipgresham@yahoo.com (C. Gresham), john@oakeshott.csiro.au
(J. Oakeshott), colin.scott@csiro.au (C. Scott), eddlestonm@yahoo.com
(M. Eddleston).
OpdA is a bacterial enzyme capable of hydrolyzing a wide variety
of OP pesticides in vitro (Fig. 1) (Yang et al., 2003). The addition of an
0300-483X/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.tox.2008.02.005

S.B. Bird et al. / Toxicology 247 (2008) 88–92
89
Fig. 1. Hydrolysis of dichlorvos (A) and parathion (B) by OpdA.
OP-degrading enzyme should improve the clinical results obtained
with standard therapies by decreasing the concentration of OP pes-
ticides in circulation. Clinical use of an enzyme with a broad range
of substrates would be useful in the event of poisoning with many
OPs, even when the identity of the pesticide is unknown.
We sought to determine the in vivo efficacy of OpdA in rat models
of two chemically distinct and highly toxic OP pesticides: dichlor-
vos and parathion. Demonstration of OpdA’s effectiveness should
provide the impetus for further development of this enzyme for
eventual use in humans.
curve of 0–10 ␮M nitrophenol (the ethyl parathion hydrolysis product) measured at
412 nM (using a Molecular Devices SpectraMax 190). The kinetic parameters for the
hydrolysis of ethyl parathion by OpdA were estimated using hyperbolic regression
(using the Hyper32.exe enzyme kinetic analysis software; a freeware package). The
kcat of OpdA with parathion was calculated to be 1500 s−1, and the Km was estimated
at 1 ␮M.
Dichlorvos assays were conducted in duplicate in assay buffer with 0–1 mM
dichlorvos and 19 nM OpdA. The hydrolysis products were detected by mass spec-
trometry (MS) using an Agilent G1969 LC/MS TOF after filtration through a liquid
chromatography (LC) guard column at a flow rate of 1 ml min−1 and 80% acetonitrile,
0.002% formic acid. A 5 ␮l sample was analyzed by MS, with the fragmentor set at
120 V. The mass spectral fragmentation patterns of authentic dichlorvos standards
(Sigma–Aldrich, Castle Hill, New South Wales, Australia) were used to validate the
identity of the substrates. The assay reactions were assayed at 3-min intervals for
12 min at each substrate concentration. The kinetic parameters for this reaction were
estimated using hyperbolic regression (as above) giving a Km of 183 ␮M and kcat of
2. Methods
All animals were acquired and cared for in accordance with the guidelines pub-
lished in the Guide for the Care and Use of Laboratory Animals (National Institutes of
Health Publications No. 85-23, Revised 1985). The Institutional Animal Care and Use
Committee of the University of Massachusetts Medical School approved the study
protocol.
149 s−1
.
2.4. Poisoning models
Animals were briefly anesthetized with isoflurane while a 24-gauge intravenous
lateral tail vein catheter was placed. Immediately upon awakening, 3 times the
oral LD50 for parathion (LD50 = 6 mg/kg) (Sigma–Aldrich, St. Louis, MO, USA) or
dichlorvos (LD50 = 50 mg/kg) (Sigma–Aldrich) suspended in peanut oil was given
by gavage feeding tube in a volume of 1.5 ml/kg. Three times the LD50 of the pes-
ticides were used in order to mimic severe human poisoning and to assure that
all, or nearly all, control animals would die, thereby decreasing the number of
animals needed to demonstrate statistical significance. All injections were given
via a 24-gauge catheter (Surflo catheter, Terumo Corporation, Somerset, NJ, USA)
through a lateral tail vein in volume of 0.5 ml. Nitrile gloves and chemical safety
goggles were worn when working with the OP pesticides or handling animals after
poisoning. Consumable supplies were soaked in a dilute OpdA solution in order
to hydrolyze any residual pesticide before disposal in a biohazards container for
collection by the institutional Environmental Health and Safety department. Ani-
mal carcasses were burned in an incinerator equipped with an afterburner and
scrubber.
2.1. Subjects
Male Wistar rats weighing 250 50 g were obtained from Charles River Labora-
tories (Wilmington, MA, USA). Animals were housed in pairs, maintained on 12-h
light:12-h dark cycle and provided food and water ad libitum except for 2 h prior to
experimentation.
2.2. Expression of highly active OpdA
OpdA is a metaloenzyme that is catalytically active with a variety of metal ions
(Zn2+, Mn2+, Co2+ or Cd2+) with highest activity toward organophosphate insec-
ticides when Co2+ is present in the active site (Jackson et al., 2006; Yang et al.,
2003). Therefore, highly active OpdA was prepared by expressing the enzyme in
the presence of cobalt as follows. OpdA discovery and cloning are described else-
where (Horne et al., 2002). The OpdA gene in the plasmid pCy76-opdA (Yang
et al., 2003) was transformed into electrocompetent E. coli DH5␣ cells. A 20 ml
starter culture in Luria–Bertani medium supplemented with 50 ␮g/ml ampicillin
(LB Amp) was inoculated then incubated at 30 ◦C for 8 h. This inoculum was used
to inoculate 1 l of Terrific broth medium (24 g/l of yeast extract, 12 g/l of tryp-
tone, 100 mM K2HPO4/KH2PO4, pH 7.0, 0.4% glycerol, and 50 ␮g/ml ampicillin)
supplemented with 1 mM CoCl2. After 30 h at 37 ◦C, cells were harvested and
resuspended in 50 mM HEPES buffer (pH 8.0). Cells were lysed using a French
Press, and cell debris removed by centrifugation at 30,000 × g for 30 min. The sol-
uble fraction was passed through a DEAE Fractogel column that does not bind
OpdA. Following 12-h dialysis against 50 mM HEPES (pH 7.0) the protein solu-
tion was loaded on to a Sulphopropyl-Sepharose column. Protein was using a
linear gradient from 0 to 1 M NaCl, with OpdA eluting at around 150 mM NaCl.
SDS/PAGE analysis of the eluted OpdA indicated a purity of greater than 95%.
2.5. Data analysis
Blinded outcomes of interest were survival to 4 and 24 h. Grouped survival data
were compared using a two-tailed Fisher’s exact test. For 80% power to detect a
50% reduction in mortality, assuming an alpha of 0.05, 8 animals per group were
required. For all analyses, a p value of less than 0.05 was considered significant. All
statistical analyses were performed with GraphPad Prism software version 4 for Mac
(GraphPad Software, Inc., San Diego, CA, USA).
3. Results
The protein was stored in 50 mM HEPES (pH 7.0)/150 mM NaCl at
required.
4
◦C until
3.1. Safety of repeated doses of OpdA in rats
Since OpdA had not previously been administered to animals,
we first examined whether the hydrolase would elicit severe aller-
gic reactions in the rat at OpdA doses likely to be needed in efficacy
studies. 0.5 mg of OpdA (a 10-fold excess of OpdA estimated for
effective hydrolysis of dichlorvos based on extrapolations from in
vitro kinetic data) was injected into a tail vein of four rats once a
2.3. Ethyl parathion and dichlorvos activity assays with purified OpdA
Ethyl parathion OpdA assays were conducted in duplicate in assay buffer
(50 mM HEPES, 10% methanol, 1 mM CoCl2, pH 7.0) with an ethyl parathion range of
0–100 ␮M and 2 nM of purified OpdA. The initial rates of the reactions were deter-
mined by measuring the change in absorbance at 412 nM over time, until 10% of the
substrate had been converted. The rate of conversion was calculated from a standard

90
S.B. Bird et al. / Toxicology 247 (2008) 88–92
Fig. 2. Acute mortality after poisoning with 3× LD50 of dichlorvos or parathion.
Survival after poisoning with dichlorvos (ꢀ) or parathion (ꢁ) alone. Rats were given
0.5 ml of saline placebo IV concomitantly with poisoning by 3× LD50 dichlorvos or
parathion via gavage.
week for 4 weeks. Rats were continuously observed for 4 h after
each injection, then regularly for 24 h. No evidence of an allergic
reaction was observed in any rat, and all exhibited normal behavior
and normal weight gain over the 4-week period. With no evidence
of an allergic reaction, we proceeded to test the efficacy of OpdA
against dichlorvos and parathion.
3.2. Dichlorvos and parathion oral poisoning models
To mimic human poisoning, we developed oral poisoning mod-
els for parathion and dichlorvos in the Wistar rat. To assure
severe poisoning and at least a lethality of 90% in controls, all
rats received 3× LD50 parathion (LD50 ∼ 6 mg/kg) or dichlorvos
(LD50 ∼ 50 mg/kg) via gavage feeding tube. The LD50s for dichlorvos
and parathion were determined from exhaustive literature reviews.
All rats that received dichlorvos developed signs of cholinergic
toxicity (muscle fasciculations and tremors; gait disturbances; sali-
vation; and urination) within 3 min and died by 12 min (median
time to death 6 min), while all rats that received parathion devel-
oped cholinergic signs within 20 min and died by 35 min (median
time to death 25.5 min) (Fig. 2). This rapid onset of poisoning is
consistent with clinical experience of human poisoning with these
pesticides (Eyer et al., 2003; Peng et al., 2004).
Fig. 3. OpdA improves survival after poisoning with 3× LD50 of (A) dichlorvos, or
(B) parathion. (A) 24-h survival after poisoning with dichlorvos. Rats were given
0.15 mg/kg of OpdA or the same volume of saline placebo concomitantly with poi-
soning by 3× LD50 dichlorvos via gavage. (ꢀ) dichlorvos plus saline placebo; (ꢁ)
dichlorvos plus single dose 0.15 mg/kg OpdA. Each group contained 8 rats. (B) 24-h
survival after poisoning with parathion. Rats were given OpdA 0.15 mg/kg or placebo,
with or without 2-PAM, after poisoning with 3× LD50 parathion via gavage. (ꢀ)
Parathion plus saline placebo; (×) parathion plus single dose 0.15 mg/kg OpdA; (ꢁ)
parathion plus 3 doses 0.15 mg/kg OpdA; (ꢂ) parathion plus 2-PAM bolus and infu-
sion of 30 mg/kg/h for 24 h; (ꢃ) parathion plus single dose 0.15 mg/kg OpdA plus
2-PAM bolus and infusion of 30 mg/kg/h for 24 h. Each group contained 8 rats with
the exception of the 24-h 2-PAM alone group, which contained 10 rats.
OpdA demonstrates prolonged efficacy against dichlrovos and
parathion The prolonged in vivo enzymatic activity of an OP hydro-
lase is essential if it is to be used effectively against OPs, like
parathion, that partition out of the blood and into adipose tissue
and other physiologic compartments. To test the duration of OpdA’s
effect versus parathion and dichlorvos, 250 g male Wistar rats were
given 1.5 mg/kg i.v. OpdA, followed by 3× LD50 oral dichlorvos 30,
60, or 90 min later. All rats survived to 24 h and no animals exhibited
any signs of OP toxicity (p = 0.0002 vs. placebo). When these exper-
iments were repeated using 3× LD50 parathion, again all animals
survived to 24 h (p = 0.0002 vs. placebo). Lastly, a cohort of 4 rats
was given 1.5 mg/kg of OpdA 180 min before parathion poisoning.
Again, all rats survived to 24 h. Therefore, OpdA maintains clinically
relevant enzymatic activity in vivo for several hours.
3.3. Efficacy of OpdA versus dichlorvos and parathion
Because of the difference in speed of poisoning onset, we gave
a single dose of 0.15 mg/kg OpdA (or an equal volume of 0.9% nor-
mal saline) intravenously immediately after giving dichlorvos and
immediately or 10 min after giving parathion. We chose 0.15 mg/kg
of OpdA in order to give a margin of error based upon an esti-
mated minimum dose of 0.05 mg/kg OpdA derived from in vitro
data with dichlorvos, while remaining within the range of larger
OpdA doses given in the preliminary safety dosing studies. All
dichlorvos-poisoned rats that received OpdA survived to 4 h and
24 h (p = 0.0002 vs. placebo). Delaying OpdA administration to
3 min after dichlorvos administration resulted in complete loss of
OpdA efficacy (Fig. 3A).
A single dose of OpdA either immediately or 10 min after poi-
soning had no survival effect on parathion-poisoned rats, with all
dead at 4 h. However, repeat doses of 0.15 mg/kg OpdA at 45 min and
90 min after poisoning improved survival: all animals survived to
4 h after this repeated treatment with OpdA (p = 0.0002 vs. placebo).
Twenty-four hours after poisoning 5/8 animals were alive (OpdA vs.
placebo group, p = 0.026) (Fig. 3B).
3.4. OpdA improves efficacy of pralidoxime versus parathion
High OP concentrations are thought to limit the effectiveness
of oxime cholinesterase reactivators in OP poisoning (Eddleston
et al., 2005; Sogorb et al., 2004). To address this issue we treated
parathion-poisoned rats with either pralidoxime chloride (2-PAM)
alone, or 2-PAM with OpdA. Rats received a bolus dose of 30 mg/kg

S.B. Bird et al. / Toxicology 247 (2008) 88–92
91
Table 1
Physicochemical properties of the studied pesticides
OP pesticide
Chemistry
WHO toxicity class
Rat oral LD50 (mg/kg)a
Human mortality (%)
Fat solubility (Kow log p)b
Dichlorvos
Parathion
Aliphatic
Aromatic
Dimethyl
Diethyl
Oxon
Thion
Ib
Ia
25–100
2–13
34
40
Low (1.43)
High (3.83)
a
Values vary according to source: Crop Protection Handbook 2003, 2nd WHO Classification of Pesticide Toxicity.
National Toxicology Program, Forest Stewardship Council Pesticides Policy 2007.
b
Table 2
Enzymatic activity of OpdA vs. OPs representative of different chemical classes
Enzyme
Chemistry of OP
Ethyl-aromatic
thion (parathion)
Methyl-aromatic thion
(methyl parathion)
Methyl-aromatic oxon
(methyl paraoxon)
Methyl-aliphatic
(dichlorvos)
Km
kcat
Km
kcat
Km
kcat
Km
kcat
OpdA
110
1500
100
1200
230
2500
183
149
Km is expressed in ␮M and kcat in s−1
.
2-PAM 10 min after parathion administration, followed by a con-
tinuous IV infusion of 30 mg/kg/h (standard 2-PAM doses used in
previous OP rat studies) for 24 h. 2-PAM treatment alone resulted
in 4 of 10 animals surviving to 4 h and 1 of 10 animals surviving
to 24 h (24-h 2-PAM infusion vs. placebo, p = 1.00). After a single
IV dose of 0.15 mg/kg OpdA administered concomitantly with the
2-PAM bolus and infusion, 8 of 8 animals survived to 4 h, and 6 of 8
animals survived to 24 h (p = 0.007 compared to single dose OpdA
alone; p = 0.013 compared to 2-PAM alone).
required after the first few hours or days. Therefore, theoretical con-
cerns regarding prolonged re-exposure to a bacterial OP hydrolase
such as OpdA are likely not applicable.
OpdA was surprisingly effective against dichlorvos and
parathion, two quite different OP insecticides. In vitro enzymatic
studies had previously suggested that OpdA would be poorly effec-
tive against dimethylated oxon OPs, such as dichlorvos (Horne
et al., 2002). The Kcat of OpdA towards dichlorvos is 149 26 s−1
at room temperature, compared to ∼1500 s−1 towards parathion
(Table 2). The similar clinical responses observed after dichlorvos
and parathion poisoning suggest that in vitro catalytic activities
above a certain threshold may not be important in determining
the clinical effectiveness of an OP hydrolase.
4. Discussion
Since their first description in 1946 (Mazur, 1946), several OP
degrading enzymes have been isolated. These enzymes include
phosphotriesterase (PTE, aka OPH) (Lewis et al., 1988), paraox-
onase (PON) (Ortigoza-Ferado et al., 1984), DFPase (Ahmad and
Forgash, 1976), sarinase (Adie, 1956), and OpdA. OpDA is a par-
ticularly good candidate enzyme for clinical applications, partly
because it is a very efficient hydrolase, performing at near diffusion-
limited rates towards its favored substrates in vitro. In addition, and
unlike some of the other OP-degrading enzymes, OpdA is active
against a wide range of phosphotriester OP pesticides, including
substrates with a phosphoryl sulfur (rather than an oxygen) and
substrates with both methoxy and ethoxy groups. One consequence
of the breadth of OpdA’s substrate range is that treatments based
on OpdA have a high probability of success against OP poison-
ing even when the exact identity of the OP pesticide is unknown.
Additionally, phosphoryl sulfur containing OP pesticides (such as
parathion) can, in principle, be hydrolyzed before they are acti-
vated by P450s, so long as they are available to the enzyme in
circulation.
As a test for broad-spectrum hydrolytic activity, we sought
to test the efficacy and safety of OpdA in two very different rat
models of severe oral OP poisoning. We chose parathion and
dichlorvos because their different chemical structures result in
variable requirements for bioactivation and differences in fat sol-
ubility (Table 1). However, both pesticides are highly toxic, fast
acting, widely used globally; parathion and dichlorvos are the two
most commonly used OPs in Chinese suicides, where an estimated
170,000 deaths occur annually from pesticide poisoning (Eddleston
and Phillips, 2004; Phillips et al., 2002), and therefore highly clini-
cally relevant.
Despite excellent efficacy against dichlorvos, a single dose of
OpdA was not effective against parathion whether OpdA was given
concomitantly with poisoning or 10 min later. Differences in pesti-
cide toxicokinetics andneed for bioactivation ofthetwo OPsexplain
the differences in speed of poisoning onset and the effectiveness
of OpdA: dichlorvos does not require bioactivation (because of its
oxon group) and is therefore immediately toxic. In addition, dichlor-
vos is rapidly present at high concentrations in the blood due to
its low distribution into fat, and is therefore available for hydrol-
ysis by OpdA. Parathion, in contrast, requires bioactivation from a
thion to oxon and distributes rapidly to fat. This latter characteristic
decreases the amount available in the circulation for OpdA hydrol-
ysis and provides a reservoir for subsequent leaching of parathion
back into circulation. Three doses of OpdA at 45-min intervals
were needed to counter parathion returning from the fat into the
blood.
2-PAM is considered by most researchers and clinicians to be an
integral part of therapy after OP poisoning (Eddleston et al., 2007).
However, a 2-PAM bolus followed by continuous 24-h infusion was
ineffective as a treatment for parathion poisoning. The addition of
a single bolus dose of OpdA markedly increased the effectiveness
of 2-PAM. This result suggests that early use of OpdA lowers the
amount of pesticide that reaches the fat, improving 2-PAM’s ability
to reactivate AChE. The combination of OpdA and 2-PAM may well
improve outcomes for other fat soluble OPs that have a long half-
life, such as fenthion (Eddleston et al., 2005), that are also associated
with severe poisonings in the developing world.
With dichlorvos poisoning, OpdA was required immediately to
have a beneficial effect, which will make it difficult for it be useful
clinically for very fast acting pesticides. However, it is possible that
OpdA may be effective at later time points in poisoning with lower
dose of OP pesticides. OpdA may also be effective in poisoning with
slowly activated and poorly fat-soluble OPs, such as dimethoate, for
which there is more time for OpdA administration.
We found no evidence of allergic reactions to this bacterial
hydrolase in rats. Clinical use of another microbial enzyme, strep-
tokinase, has shown that repeated administration is safe and
effective within 5 days or after 1 year (Fears et al., 1992). In the acute
OP pesticide-poisoning scenario, it is unlikely that OpdA would be

92
S.B. Bird et al. / Toxicology 247 (2008) 88–92
Eddleston, M., Phillips, M.R., 2004. Self poisoning with pesticides. BMJ 328, 42–44.
Given the profound human health burden caused by OP pesti-
Eyer, F., Meischner, V., Kiderlen, D., Thiermann, H., Worek, F., Haberkorn, M., Felgen-
hauer, N., Zilker, T., Eyer, P., 2003. Human parathion poisoning A toxicokinetic
analysis. Toxicol. Rev. 22, 143–163.
Fears, R., Ferres, H., Glasgow, E., Standring, R., Hogg, K.J., Gemmill, J.D., Burns,
J.M., Rae, A.P., Dunn, F.G., Hillis, W.S., 1992. Monitoring of streptokinase resis-
tance titre in acute myocardial infarction patients up to 30 months after giving
streptokinase or anistreplase and related studies to measure specific antistrep-
tokinase IgG. Br. Heart J. 68, 167–170.
Horne, I., Sutherland, T.D., Harcourt, R.L., Russell, R.J., Oakeshott, J.G., 2002. Iden-
tification of an opd (organophosphate degradation) gene in an Agrobacterium
isolate. Appl. Environ. Microbiol. 68, 3371–3376.
Jackson, C.J., Carr, P.D., Kim, H.K., Liu, J.W., Herrald, P., Mitic, N., Schenk, G., Smith,
C.A., Ollis, D.L., 2006. Anomalous scattering analysis of Agrobacterium radiobacter
phosphotriesterase: the prominent role of iron in the heterobinuclear active site.
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cide poisoning, and potential terrorist use of OP pesticides, these
studies should provide the impetus for accelerated research and
human trials of broad spectrum, low-cost therapeutic OP hydro-
lases such as OpdA.
Acknowledgments
Support for this research was provided by NIEHS grant K08
ES012897, the Emergency Medicine Foundation, and the Orphan
Medical/Jazz Pharmaceuticals research award from the American
College of Medical Toxicology. Its contents are solely the respon-
sibility of the authors and do not necessarily represent the official
views of the NIEHS.
Jeyaratnam, J., 1990. Acute pesticide poisoning: a major global health problem. World
Health Stat. Q. 43, 139–144.
Lewis, V.E., Donarski, W.J., Wild, J.R., Raushel, F.M., 1988. Mechanism and stere-
ochemical course at phosphorus of the reaction catalyzed by
phosphotriesterase. Biochemistry 27, 1591–1597.
a bacterial
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