Assessing the therapeutic efficacy of oxime therapies against percutaneous organophosphorus pesticide and nerve agent challenges in the Hartley guinea pig

Abstract

Given the rapid onset of symptoms from intoxication by organophosphate (OP) compounds, a quick-acting, efficacious therapeutic regimen is needed. A primary component of anti-OP therapy is an oxime reactivator to rescue OP-inhibited acetylcholinesterases. Male guinea pigs, clipped of hair, received neat applications of either VR, VX, parathion, or phorate oxon (PHO) at the 85^th percentile lethal dose, and, beginning with presentation of toxicosis, received the human equivalent dose therapy by intramuscular injection with two additional follow-on treatments at 3-hr intervals. Each therapy consisted of atropine free base at 0.4 mg/kg followed by one of eight candidate oximes. Lethality rates were obtained at 24 hr after VR, VX and PHO challenges, and at 48 hr after challenge with parathion. Lethality rates among symptomatic, oxime-treated groups were compared with that of positive control (OP-challenged and atropine-only treated) guinea pigs composited across the test days. Significant (p ≤ 0.05) protective therapy was afforded by 1,1-methylene bis(4(hydroxyimino- methyl)pyridinium) dimethanesulfonate (MMB4 DMS) against challenges of VR (p ≤ 0.001) and VX (p ≤ 0.05). Lethal effects of VX were also significantly (p ≤ 0.05) mitigated by treatments with oxo-[[1-[[4-(oxoazaniumylmethylidene)pyridin-1-yl] methoxymethyl]pyridin-4-ylidene]methyl]azanium dichloride (obidoxime Cl₂) and 1-(((4-(aminocarbonyl) pyridinio)methoxy)methyl)-2,4-bis((hydroxyimino)methyl)pyridinium dimethanesulfonate (HLö-7 DMS). Against parathion, significant protective therapy was afforded by obidoxime dichloride (p ≤ 0.001) and 1,1′-propane-1,3-diylbis{4-[(E)-(hydroxyimino)methyl]pyridinium} dibromide (TMB-4, p ≤ 0.01). None of the oximes evaluated was therapeutically effective against PHO. Across the spectrum of OP chemicals tested, the oximes that offered the highest level of therapy were MMB4 DMS and obidoxime dichloride.

INTRODUCTION

Exposure to organophosphorus (OP) chemicals has always been a potential poisoning threat to military personnel since the Second World War. However, this threat is now a growing concern for the civilian population today as well. Intoxication by OP chemicals can occur in instances of intentional and accidental release. OP chemicals are known inhibitors of acetylcholinesterase (AChE), resulting in increased acetylcholine levels in brain tissue and peripheral nerves that can lead to increased secretions, fasciculations, seizures, convulsions, respiratory distress, and death. Generally, OP chemical warfare nerve agents (CWNAs), such as sarin, soman, and VX, are much more toxic than bioactivated OP pesticides, such as paraoxon, chlorpyrifos-oxon and phorate oxon (PHO), even though the mechanism of toxicosis via cholinolergic hyperstimulation of parasympathetic nerves is the same. OPs also inhibit serum butyrylcholinesterase (BChE), an enzyme similar to AChE, that possesses detoxification, scavenging, and other properties (Lockridge, 2015; Chen et al., 2015).

Given the rapid onset of CWNA- and OP pesticide-induced toxicity, a quick-acting, efficacious therapeutic regimen is needed to counter these toxic effects. The primary components of the currently fielded therapies are (a) a competitive muscarinic receptor antagonist, atropine, to mitigate the excitotoxic effect at post-synaptic targets (Shutt and Bowes, 1979) and (b) an AChE oxime reactivator, pralidoxime chloride (2-PAM Cl), to augment OP dissociation from and reactivation of inhibited AChE. The combination of these two therapeutic strategies aims to reduce the hyperstimulation of parasympathetic nerves that results from the over-accumulation of acetylcholine (Harris and Stitcher, 1983; Eyer, 2003). For use in emergency and pre-hospital settings, the U.S. government currently fields these two drugs within a single two-chambered, self-propelled syringe through a common needle intramuscular (IM) injection delivery system (the Antidote Treatment - Nerve Agent Autoinjector (ATNAA) - http://www.meridianmeds.com/products-overview-antidote and the DuoDote® Autoinjector - http://www.meridianmeds. com/products-overview-duodote).

Although 2-PAM Cl is efficacious to some extent, efforts to replace it with an AChE oxime reactivator that would offer enhanced antidotal efficacy have nevertheless been ongoing for decades. Comparative oxime efficacy studies that have been performed in the past, both in vitro (Hallek and Szinicz, 1988; Worek et al., 2002; Worek et al., 2007; Luo et al., 2007; Radić et al., 2013) and in vivo (Clement, 1981; Bosković et al., 1984; Lundy et al., 1992; Dawson, 1994; Maxwell et al., 2008; Shih et al., 2009) were often conducted only on those compounds within the same chemical class, employed unrealistic administration routes, timing and doses (for both the challenge OP chemicals and/or the oxime candidate), or animal models not directly relevant to humans, specifically rodents with circulating plasma carboxylesterase (CaE). Consequently, it is difficult, if not impossible, to identify an “optimal” lead candidate oxime therapy that can potentially be considered for advanced drug development. Therefore, a rigorous direct efficacy comparison among leading oxime therapies independent of shared structural moiety, in a single animal model physiologically relevant to humans, and against a spectrum of both OP pesticides and CWNAs has been needed. Although work to address that data gap was recently published, those studies utilized subcutaneous (SC) OP challenges (Wilhelm et al., 2014). While SC administration of OP chemicals historically has been an accepted delivery method in this research field as this route ensures an exact exposure level, it is, nonetheless, an unrealistic model for emulating a “real world” route of exposure. As such, the current work seeks to extend those previously published findings by employing topical/percutaneous (PC) OP challenges to better simulate that “real world” scenario. In both the previous SC and current PC studies, the guinea pig was selected based on affordability, historical use, and most importantly, direct relevance to humans, e.g., both species have low levels of plasma CaE relative to other animal models (Bahar et al., 2012), and similarity of AChE protein sequence (Cadieux et al., 2010).

The objective of this study was to determine, in atropinized guinea pigs, the two generally most efficacious candidates within a prescribed field of eight oximes when delivered by IM injection against topical challenges at the dose associated with the 85^th percentile of lethality (LD₈₅) of several OPs with lethality assessed at either 24 hr or 48 hr. Therapy was administered at the onset of clinical evidence of toxicosis, and repeated at 3-hr intervals for a total of three therapy sessions. The eight oximes were selected as a result of down-selecting from a compiled list of oximes (1) fielded in autoinjectors for military or civilian use (2) for which there was published evidence of broad-spectrum efficacy, and (3) that were recommended by experts in researching medical countermeasures against chemical warfare agents.

MATERIALS AND METHODS

The OP challenge materials and oxime therapies are listed in Table 1. Challenge materials included VR and VX, procured from the Edgewood Chemical and Biological Center, Aberdeen Proving Ground, MD, USA), parathion (ChemService, West Chester, PA, USA), and PHO (synthesized by Battelle). Oximes tested included 2-PAM Cl, HI-6 DMS, HLö-7 DMS, MINA, MMB4 DMS, obidoxime Cl₂, RS194B, and TMB-4 (Fig. 1).

Table 1.

Identification of challenge materials and therapies.

Challenge material or therapy	CAS RN†	Synonym(s)
VR	159939-87-4	S-[2-(diethylamino)ethyl] O-isobutyl methylphosphonothioate
VX	50782-69-9	S-[2-(diisopropylamino)ethyl] O-ethyl methylphosphonothioate
parathion	56-38-2	O,O-diethyl O-(4-nitrophenyl) phosphorothioate
phorate oxon (PHO)	2600-69-3	O,O-diethyl S-[(ethylsulfanyl)methyl] phosphorothioate
atropine free base	51-55-8	(3-endo)-8-Methyl-8-azabicyclo[3.2.1]oct-3-yl tropate
2-PAM Cl	51-15-0	2-hydroxyiminomethyl-1-methylpyridinium chloride in aqueous benzyl alcohol (20 mg/mL), pH 2.6
MMB-4 DMS	-	1,1-methylene bis[4(hydroxyimino- methyl)pyridinium) dimethanesulfonate
Obidoxime Cl2	114-90-9	Toxogonin®, or oxo-[[1-[[4-(oxoazaniumylmethylidene)pyridin-1-yl] methoxymethyl]pyridin-4-ylidene]methyl]azanium dichloride
HI-6 DMS	34433-31-3	asoxime, or 1-(((4-(aminocarbonyl) pyridinio)methoxy) methyl)-2- ((hydroxyimino)methyl) pyridinium dimethanesulfonate
HLö-7 DMS	120103-35-7	1-(((4-(aminocarbonyl) pyridinio)methoxy)methyl)-2,4- bis((hydroxyimino) methyl)pyridinium dimethanesulfonate
MINA	306-44-5	monoisonitrosoacetone
TMB-4	56-97-3	trimedoxime bromide, or 1,1′-propane-1,3-diylbis{4-[(E)-(hydroxyimino) methyl]pyridinium} dibromide
RS194B	-	N-(2-(azepan-1-yl)ethyl)-2-(hydroxyimino)acetamide

^†Chemical Abstracts Service registration number.

- none given.

Fig. 1.

Structures of eight oximes tested and atropine free base.

Atropine free base at 1.64 mg/mL in a citrate buffered saline solution (batch RP-526-1) and 2-PAM Cl (batch number RP-526-2) both from King Pharmaceuticals, St Louis, MO, USA, and MMB4 DMS (lot number 1004) were government furnished (Medical Countermeasure Systems Joint Project Management Office, Frederick, MD, USA). Obidoxime dichloride was purchased from Sigma Aldrich (Lot Number 1446981V, St Louis, MO, USA). HI-6 DMS, HLö-7 DMS, MINA, and TMB-4 were custom synthesized by Southwest Research Institute (San Antonio, TX, USA). RS194B was custom synthesized at the Scripps Research Institute (La Jolla, CA, USA). The described batches were used throughout the study for all agents. All oximes, except RS194B, were formulated for IM injection in normal (0.9%, w/v) saline. RS194B was dissolved in a mixture of concentrated (37%, w/w) hydrochloric acid diluted 1:1 (v/v) with distilled water, and the final oxime solution was adjusted to pH 7 using 6.25% (w/v) aqueous sodium hydroxide. This brought the sodium chloride concentration in the RS194B solution to 1.6%, w/v.

A total of 352 specific pathogen-free male Dunkin-Hartley guinea pigs (mean body weight = 447 g) were purchased from three Charles River facilities (Raleigh, NC, USA; Stone Ridge, NY, USA; Saint Constant, QC, Canada). Guinea pigs were identified with ear tags and pair housed before challenge in polycarbonate cages labeled with cage cards. During a 7-day quarantine, the guinea pigs were weighed and randomized by body weight into test days, each comprised of multiple treatment groups of three (controls) or eight (oxime) guinea pigs.

On the day prior to challenge, designated guinea pigs were anesthetized with a combination of 70 mg/kg ketamine and 10 mg/kg xylazine, and clipped of hair on both thighs and on the dorsum between the scapulae. From the same animals, ~0.7-mL baseline blood samples were collected via the lateral saphenous vein, placed in K₃ EDTA tubes, chilled, processed (as described below), and stored at ≤ −70°C prior to analysis.

One (for VR, VX and PHO) or two (for parathion) rubber “O” rings, 12-mm ID/20-mm OD, were affixed around each dorsal test site using cyanoacrylate adhesive. Percutaneous applications were semi-occluded by affixing a 20-mm disk of Durapore^® (Millipore, Billerica, MA, USA, Catalogue Number HVLP2932A) 0.45-μm filter paper cover onto each “O” ring using cyanoacrylate adhesive. The cover did not impede evaporation of challenge material but prevented disturbance of the test site by the guinea pig. After receiving the challenge dose, guinea pigs were singly housed in disposable plastic shoebox cages in the dosing hood with food and water made available.

In order to simulate a real world mass casualty and triage care treatment scenario, therapy was administered only at the onset of clinical evidence of toxicosis, and repeated at 3-hr intervals for a total of three therapy sessions, each consisting of atropine free base administered at 0.4 mg/kg by IM injection in the right thigh and an oxime/saline solution at 146 μmol/kg (35 μmol/kg for TMB-4 due to toxicity at the higher level; see below) in the left thigh. The atropine dose was the recommended human equivalent dose (HED) for a 70-kg person receiving three DuoDote^® autoinjectors each containing 2.1 mg of atropine free base. The HED was calculated by multiplying the human dose, (3×2.1 mg)/70 kg, by 4.6, the ratio of guinea pig body surface area/kg body mass to that for human beings (CDER, U.S. F.D.A., 2002). An atropinization level of 0.4 mg/kg in the guinea pigs was equivalent to what a human victim of OP poisoning would be expected to receive in a pre-hospital setting without supportive care, i.e., no more than three administrations of the ATNAA or DuoDote® i.m. autoinjectors. Sustained atropinization is expected only upon arrival in the hospital. Atropine in the DuoDote^® autoinjector is sufficient for reducing painful ciliary muscle spasm, mitigating muscarinic hyperstimulation of secretory organs, and correcting bradycardia from cholinergic poisonings.

For most oximes, the target therapy dose given at each therapy session was equimolar to 2-PAM Cl in three autoinjectors given to a 70-kg human, equivalent to 25.71 mg/kg or 149 μmol/kg. An average of 146 μmol/kg was actually administered. The only exception to this was in the case of TMB-4 due to its toxicity at 146 μmol/kg, which was lethal within 15 min to all guinea pigs treated. The Atromat Automatic Injector 101-2080 (Shalon-Chemical Industries, Tel Aviv, Israel) contains 80 mg of TMB-4 (Bentur et al., 2006). Three autoinjectors administered to a 70-kg person, and adjusted by the surface area factor 4.6 for converting to a guinea pig human-equivalent dose was the revised target dose for TMB-4:

$$\frac{3\text{autoinjectors}\times \frac{80\text{mg}}{\text{autoinjector}}}{70\text{kg}}\times 4.6=15.8\text{mg}/\text{kg}=35\mathrm{\mu }\text{mol}/\text{kg}$$

The PC challenges, previously determined in similarly atropinized guinea pigs, were 24-hr LD₈₅s = 0.285 mg/kg VR, 0.161 mg/kg VX, and 121 mg/kg PHO, and for parathion the 48-hr LD₈₅ = 1,265 mg/kg. All guinea pigs were challenged topically between the scapulae at the PC LD₈₅ respective for the OP, and triplicate therapy was initiated in those that became symptomatic, consisting of atropine and either saline (controls) or an oxime in saline (test animals). The oxime solutions were formulated (Table 2) to maintain equivalent IM injection volumes across treatments, approximately 0.26 mL/kg.

Table 2.

Oxime therapy.

Oxime	Oxime therapy, each of three administrations		Concentration in saline (mg/mL)
	(μmol/kg)	(mg/kg)
2-PAM Cl	146	25.7	100
MMB-4 DMS	146	65.3	254
Obidoxime Cl2	146	52.3	203
HI-6 DMS	146	69.7	271
HLö-7 DMS	146	75.9	295
MINA	146	12.7	49
TMB-4	35†	15.8	61
RS194B	146	31.1	121

^†TMB-4 was given at a reduced therapy dose due to toxicity at 146 μmol/kg.

Observations of survivors were recorded at 0.25, 0.5, 1, 2, 4, 8, and 24 hr post-challenge. Parathion-challenged guinea pigs also were observed at 30 and 48 hr after challenge. If a guinea pig was found dead, the time and observation were recorded. After the final observation, each surviving animal was euthanized by catheter injection of euthanasia solution. A 1-mL volume of blood was collected immediately prior to euthanasia and designated the “terminal” sample. Whole blood samples were processed and analyzed as previously described (McGarry et al., 2013). Briefly, whole blood samples were treated with HemogloBind^™ (Biotech Support Group LLC, Monmouth Junction, NJ, USA) to remove hemoglobin, which interferes with the ChE activity assay due to spectral overlap. To prepare the HemogloBind^™ treated blood for ChE activity analysis, the samples were diluted twofold in phosphate buffered saline (PBS, pH 7.4, Sigma Aldrich, item P3813). Subsequently, samples were diluted with PBS an additional two-fold into the test plate by adding 100 μL of sample to a total volume of 200 μL in each well of a 96-well plate. The brains from animals surviving to 24 hr (48 hr for parathion) were collected, bisected sagittally, and rinsed to remove residual blood. One brain half was placed into a cassette, snap-frozen in liquid nitrogen and stored at ≤ −70°C until processed. The other half was placed into 10% neutral buffered formalin, paraffin-embedded, sectioned, and stained for both fluoro-jade and routine (H&E) histopathology. Since lethality was the primary endpoint of this study, many animals did not survive to yield samples of blood and brain. Brain samples were later thawed and assayed for acetylthiocholine (ATC) and butyrylthiocholine (BTC) hydrolysis activities. Brain samples were weighed, and an appropriate volume of tissue homogenization buffer (PBS plus 1% Triton X-100, Sigma Aldrich, item T8787) plus 1% protease inhibitor (Sigma Aldrich, item P8340 or Pierce, Dallas, TX, USA, item 88266) was added to dilute each sample to 150 mg/mL. The samples were then homogenized using a PowerGen High Throughput Homogenizer for approximately 2 min. The homogenized samples were placed on a rocker at 2–8°C and allowed to rock for ≥ 1 hr. Samples were then centrifuged at 10,000 x g at 4°C for 2 min. The supernatant was removed and retained at ≤ −70°C for cholinesterase activity analysis. Prior to cholinesterase activity analysis, the homogenate was thawed and diluted five-fold in PBS. Blood and brain cholinesterase activity was assessed using a modified spectrophotometric assay (Ellman et al.,1961). AChE and BChE activity levels were calculated using 1×10⁻³ M ATC or 3×10⁻³ M BTC as the substrate, respectively. Ellman’s Reagent [DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid), Sigma Aldrich, item D8130] at 5×10⁻⁴ M was used as the indicator. The extinction coefficient used in the calculations was 13,600 (M⁻¹ cm⁻¹).

One-sided Fisher’s exact tests were performed between lethality rates for aggregate saline control animals and oxime test animals for each OP to determine the significance of any protective effect attributable to the oxime alone. In the analysis of the cholinesterase data, the model pooled the data across all of the oximes within a challenge material in order to estimate a standard error adjusted for the number of observations.

RESULTS

Clinical signs and lethality rates

Lethality data are summarized in Table 3. Other clinical signs are not shown in tabulated form. Fig. 2 presents the mean time to onset of clinical signs.

Table 3.

Summary of oxime efficacies against topical challenges of selected organophosphates on non-sedated, atropinized guinea pigs: lethality rates with Fisher’s exact test probabilities.

Oxime	Oxime dose, each admin. (μmol/kg)	Lethality rate (n·dead/n·dosed and treated) with decimal equivalent, and probability
		VR		VX		Parathion		Phorate oxon
		Lethality rate		Lethality rate		Lethality rate		Lethality rate
none	-	20/23	0.87	11/21	0.52	20/24	0.83	8/11	0.73
MMB4 DMS	146	1/7	0.14***	0/6	0.00*	8/8	1.00	5/7	0.71
obidoxime Cl2	146	8/8	1.00	0/8	0.00*	1/8	0.13***	3/5	0.60
2-PAM Cl	146	5/7	0.71	1/6	0.17	8/8	1.00	5/6	0.83
HLö-7 DMS	146	8/8	1.00	0/6	0.00*	7/7	1.00	1/1	1.00
HI-6 DMS	35	8/8	1.00	2/8	0.25	6/8	0.75	5/6	0.83
TMB-4	146	8/8	1.00	3/7	0.43	2/8	0.25**	3/4	0.75
MINA	146	7/8	0.88	6/8	0.75	7/7	1.00	1/2	0.50
RS194B	146	8/8	1.00	6/8	0.75	8/8	1.00	5/5	1.00

^‡Each p value is the probability of the difference between the oxime lethality rate and the “none” controls lethality rate being as much as that shown due purely to random sampling; a one-sided decision level of 0.05 was used. Statistically significant oxime effects are in bold font:

^*p ≤ 0.05;

^**p ≤ 0.01;

^***p ≤ 0.001.

Fig. 2.

Time to onset of cholinergic clinical signs in guinea pigs exposed topically to LD₈₅ of four organophosphates.

VR has a relatively low vapor pressure, 0.000293 torr at 18.1°C, and the PC threat from VR is considered persistent. Of 88 guinea pigs challenged with a topical application of VR at the 24-hr LD₈₅, 85 exhibited at least one clinical sign and received the first of three treatments at a geometric mean of 2.4 hr (range: 0.9 to 12 hr) after challenge. Among the controls, biologically significant (20% or more in frequency) clinical signs included salivation, test site fasciculations, tremors, lacrimation, and death (20/23 = 87% lethality). Clinical signs among oxime-treated groups were generally the same but with some obvious differences. Only MMB4 DMS significantly reduced 24-hr lethality, to 1/7 (0.14, p ≤ 0.001). Prostration appeared to be exacerbated by HI-6 DMS (75% versus 17% in controls at 4 hr). Both 2-PAM Cl and MMB4 DMS were associated with ataxia at 4 hr, but this was likely due to the improvement from prostration in the same animals.

VX has an even lower low vapor pressure, 0.000063 torr at 25°C, than VR. Of 88 guinea pigs challenged by topical application of VX at the 24-hr LD₈₅, 78 exhibited at least one clinical sign and received the first of three treatments at a geometric mean of 3.4 hr (range: 0.2 to 11 hr) after challenge. Among the controls, significant clinical signs included ataxia/lethargy, tremors, lacrimation, and death (11/21 = 52% lethality). MMB4 DMS, obidoxime dichloride, and HLö-7 DMS significantly (p ≤ 0.05) reduced 24-hr lethality to 0/6, 0/8, and 0/6, respectively. TMB-4 seemed to exacerbate lethargy (43% versus 10% in controls at 8 hr).

Of the 88 guinea pigs challenged with topical applications of parathion at the 48-hr LD₈₅, 86 exhibited at least one clinical sign and received the first of three treatments at a geometric mean of 13 hr (range: 3.9 to 36 hr) after challenge. This delay reflects the requirement for hepatic bioactivation of parathion to the much more toxic form, paraoxon. Among the controls, significant signs included salivation, test site fasciculations, dyspnea, ataxia/lethargy, tremors, lacrimation, hunched posture, and death (20/24 = 83% lethality). TMB-4 and obidoxime Cl₂ significantly reduced 48-hr lethality, to 2/8 (0.25, p ≤ 0.01) and 1/8 (0.13, p ≤ 0.001), respectively. HLö-7 DMS reduced ataxia at 24 and 30 hr. Elevated levels of salivation, ataxia, and tremors associated with TMB-4 at the end of the 48-hr observation period indicated that the animals were alive, but in poor health, compared to the controls, which in general had expired.

Of the 88 guinea pigs challenged by topical application of PHO at the 24-hr LD₈₅, 47 exhibited at least one clinical sign and received the first of three treatments at a geometric mean of 5 hr (range: 0.4 to 24 hr) after challenge. Many of the animals were disqualified because they died without presentation of abnormal clinical signs, or there was insufficient time to administer therapy. Among the controls, significant signs included death (8/11= 73% lethality). None of the oximes tested significantly reduced 24-hr lethality. No overtly beneficial effects of the oximes tested against PHO were detected, perhaps in part due to small sample sizes of treated animals.

Notably, although in terms of lethality MMB4 DMS was found to be the best of the eight oximes tested against VR and VX, obidoxime Cl₂ and TMB-4 were the most efficacious against parathion. Against PHO, none of the tested oximes offered any detectable protection.

Cholinesterase activity

ATC and BTC hydrolysis results (herein synonymous with AChE and BChE activity) are presented in Table 4 and Figs. 3 to 6.

Table 4.

Summary of oxime efficacies against topical challenges of selected organophosphates on non-sedated, atropinized guinea pigs: blood and brain cholinesterase activities.

Challenge material	Oxime	Blood cholinesterase activity: portion of baseline activity at terminal collection (%)			Brain cholinesterase activity (U/g)
		n	ATC† Hydrolysis	BTC‡ Hydrolysis	n	ATC† Hydrolysis	BTC‡ Hydrolysis
			Mean (Standard Error) significance			Mean (Standard Error) significance
VR	Vehicle	3	56.0 (5.0)	85.8 (6.0)	3	1.16 (0.03)	1.57 (0.06)
	2-PAM Cl	2	54.7 (6.2)	74.8 (7.4)	2	1.14 (0.04)	1.60 (0.07)
	MMB4 DMS	6	55.6 (3.6)	74.7 (4.3)	6	1.13 (0.02)	1.62 (0.04)
	MINA	1	38.6 (8.7)	75.3 (10.4)	1	1.05 (0.05)	1.39 (0.1)
VX	Vehicle	10	27.4 (5.0)	41.0 (5.9)	8	0.71 (0.05)	0.96 (0.06)
	2-PAM Cl	5	63.1 (7.1)**	74.5 (8.4)*	5	0.96 (0.06)*	1.23 (0.08)
	HI-6 DMS	6	49.3 (6.5)	60.9 (7.7)	6	0.97 (0.06)**	1.28 (0.07)*
	Obidoxime Cl2	8	67.4 (5.6)***	73.8 (6.6)**	8	1.02 (0.05)***	1.35 (0.06)***
	TMB-4	4	44.0 (7.9)	47.7 (9.4)	4	0.53 (0.07)	0.89 (0.09)
	MMB4 DMS	6	54.4 (6.5)*	71.3 (7.7)*	6	0.95 (0.06)*	1.16 (0.07)
	HLo7 DMS	6	45.6 (6.0)	56.6 (7.1)	6	0.44 (0.05)**	0.68 (0.07)*
	MINA	2	26.2 (11.2)	36.9 (13.3)	0	-	-
	RS194B	2	36.8 (11.2)	49.5 (13.3)	0	-	-
Parathion	Vehicle	3	3.5 (1.1)	1.9 (0.4)	4	0.51 (0.06)	0.50 (0.06)
	HI-6 DMS	2	3.9 (1.4)	0.6 (0.5)	2	0.12 (0.08)**	0.07 (0.09)**
	Obidoxime Cl2	7	6.8 (0.7)	0.6 (0.3)*	7	0.59 (0.04)	0.57 (0.05)
	TMB-4	6	7.5 (0.8)*	1.0 (0.3)	6	0.13 (0.05)***	0.07 (0.05)***
PHO	Vehicle	3	13.2 (3.9)	9.2 (3.8)	2	0.20 (0)	0.19 (0.06)
	2-PAM Cl	1	18.6 (6.7)	6.2 (6.6)	0	-	-
	HI-6 DMS	1	7.1 (6.7)	3.9 (6.6)	1	0.12 (0)	0.11 (0.08)
	Obidoxime Cl2	2	28.9 (4.7)	9.2 (4.7)	2	0.22 (0)	0.18 (0.06)
	TMB-4	1	1.9 (6.7)	1.1 (6.6)	1	0.18 (0)	0.10 (0.08)
	MMB4 DMS	2	24.3 (4.7)	13.3 (4.7)	0	-	-
	MINA	1	13.6 (6.7)	7.5 (6.6)	1	0.22 (0)	0.22 (0.08)

^†ATC = acetylthiocholine;

^‡BTC = butyrylthiocholine

^*p ≤ 0.05;

^**p ≤ 0.01;

^***p ≤ 0.001

Note: Bold, italicized figures indicate exacerbated enzyme inhibition relative to controls for that challenge material. The analysis of variance model made use of the variability across groups within each challenge material, thereby making possible a standard error value for groups of size n = 1.

Fig. 3.

Acetylthiocholine and butyrylthiocholine hydrolysis rates in blood and brain collected from survivors exposed topically to LD₈₅ of VR. Filled circles (labeled by oxime and sample size) represent treatment group means with error bars of one standard error when n > 1. The inverted triangle in the upper right corner of the blood plot represents baseline relative ChE levels of 100%. Since there was no baseline brain sample collected, there could be no such AChE:BChE activity reference for the brain plot. The mean vehicle control brain values are represented with an inverted triangle with error bars.

Fig. 6.

Acetylthiocholine and butyrylthiocholine hydrolysis rates in blood and brain collected from survivors exposed topically to LD₈₅ of phorate oxon. Filled circles (labeled by oxime and sample size) represent treatment group means with error bars of one standard error when n > 1. The inverted triangle in the upper right corner of the blood plot represents baseline relative ChE levels of 100%. Since there was no baseline brain sample collected, there could be no such AChE:BChE activity reference for the brain plot. The mean vehicle control brain values are represented with an inverted triangle with error bars.

This study was designed with lethality as the primary endpoint, and the collection of blood and brain to assess the cholinergic status of the survivors, if any, was ancillary to the main objectives. Thus, the numbers of animals that became symptomatic, received at least one oxime treatment, and survived to the end of the observation period for tissue collection were somewhat limited. Data from animals that remained asymptomatic (and thereby did not receive therapy) were excluded from Table 4 and the figures. Excluding the atropine/saline animals that had not become symptomatic from the summary statistics reduced the control sample sizes as well and skewed the control summary data toward the more sensitive individuals to a particular OP challenge. However, this preserved the integrity of the statistical contrasts, that is, that they were performed among only symptomatic, treated animals.

Figs. 3 through 6 are each composites of two plots, one for terminal blood and one for brain. Within each plot, BChE and AChE activities are represented on the x and y axes, respectively, and the filled circles (labeled by oxime and sample size) represent treatment group means with error bars of one standard error. Mean terminal blood relative (baseline-normalized) AChE activity was plotted as a function of mean terminal relative BChE activity. The inverted triangle without error bars in the upper right corner represents, by definition, baseline relative ChE levels of 100%, and a solid line of slope = 1 was drawn to it from the origin in order to give a visual reference for determining which enzyme was more active in each treatment group. The relative distance from the blood baseline inverted triangle to the vehicle control inverted triangle represents the degree of cholinergic crisis remaining at 24 hr (48 hr for parathion) from the topical LD₈₅ challenge. Since there was no baseline brain sample collected, there could be no such AChE:BChE activity reference for brain. The mean vehicle control brain values are represented with an inverted triangle with error bars.

Main oxime effects: the relative distance from the vehicle controls to each oxime point in an upper right direction represents the degree of reactivation attributed to that oxime. Bold labels indicated oximes that significantly (p < 0.05) reactivated AChE and/or BChE. Table 4 lists those main effects and gives significance levels to each oxime/control contrast by enzyme.

VR

Only three of the 24 control guinea pigs challenged at the topical LD₈₅ of VR became symptomatic, were treated, and survived to 24 hr. Mean terminal blood relative AChE (56%) and BChE (86%) indicated preferential inhibition of AChE by VR. In brain, mean AChE and BChE activity rates were 1.16 and 1.57 U/g, respectively. Among the surviving animals, none of the three oximes (2-PAM Cl, MMB4 DMS and MINA) offered significant reactivation of either enzyme in either blood or brain.

VX

Only ten of the 24 control guinea pigs challenged at the topical LD₈₅ of VX became symptomatic, were treated, and survived to 24 hr. Mean terminal blood relative ChE activity rates in those ten animals indicated significant inhibition of AChE (27%) and BChE (41%). In order of decreasing distance of the oxime means from the vehicle controls, obidoxime Cl₂, 2-PAM Cl, and MMB4 DMS offered significant reactivation of one or both enzymes. Those three oximes and HI-6 DMS offered significant reactivation of either enzyme in brain. However, HLö-7 DMS appeared to significantly exacerbate brain ChE inhibition.

Parathion

Only three of the 24 control guinea pigs challenged at the topical LD₈₅ of parathion became symptomatic, were treated, and survived to 48 hr. Mean terminal blood relative ChE activity rates in that group were low and indicated significant inhibition of AChE (3.5%) and BChE (1.9%). Only TMB-4 offered statistically significant reactivation of blood AChE, which seemed trivial but was associated with significant reduction in lethality. Obidoxime dichloride also appeared to slightly mitigate blood AChE but exacerbated blood BChE inhibition (p < 0.05), and these paradoxical effects also were associated with significant reduction in lethality. None of the oximes offered significant reactivation of either enzyme in brain. However, both HI-6 DMS and TMB-4 appeared to significantly (p ≤ 0.01) exacerbate both AChE and BChE inhibition in brain.

Phorate oxon

Only three of the 24 control guinea pigs challenged at the topical LD₈₅ of PHO became symptomatic, were treated, and survived to 24 hr. The terminal blood relative ChE activity rate for those animals was low and indicated inhibition of AChE (13%) and BChE (9%). There were no significant oxime effects on PHO blood AChE or BChE inhibition levels. Brain tissue collected from the treated survivors offered limited data for statistical analysis.

Histopathology

A general theme found among studies involving near-lethal challenges of OPs is that animals will develop seizures and, unless quickly treated with centrally-acting countermeasures, suffer subsequent brain damage and neurobehavioral deficits. Typically, brain histopathology in severely affected animals indicate lesions such as acute neuronal necrosis in cortical and subcortical limbic structures (McDonough and Romano, 2007). Neural degeneration from OP-induced seizures can be histologically detected in histological preparations stained with fluoro-jade (Schmued et al., 1997).

Only eight animals (six challenged with VX and two with parathion) exhibited brain lesions, all characterized as acute neuronal necrosis, minimal to marked in severity, and generally not associated with an oxime treatment (Table 5). Notably however, both parathion lesions were from the TMB-4-treated animals that had exhibited significant brain ChE depression beyond that induced by the bioactivated form of the OP. Representative colored micrographs of both H&E and fluoro-jade staining are presented in Figs. 7 through 14.

Table 5.

Incidence (severity) of acute neuronal necrosis in non-sedated, atropinized guinea pigs.

Oxime	Topical challenge
	VX	PHO	Parathion	VR
saline	1 (marked)	-	-	-
	1 (moderate)
2-PAM Cl	-	-	-	-
MMB4 DMS	1 (moderate)	-	-	-
Obidoxime Cl2	-	-	-	-
HI-6 DMS	1 (marked)	-	-	-
RS194B	1 (marked)	-	-	-
MINA	-	-	-	-
TMB-4	-	-	2 (minimal)	-
HLö-7 DMS	1 (moderate)	-	-	-

Fig. 7.

Phorate oxon/atropine/saline, H&E 40x – Normal neurons in the hippocampus. Dashed arrows: Normal neurons – Neuronal nuclei are large and well-defined; neuron cell bodies are large with basophilic cytoplasm and distinct cell borders.

Fig. 14.

VX/atropine/saline, Fluoro Jade 40x – Neuronal necrosis in the cerebral cortex. Solid arrows: Neuronal necrosis – Neuron cell bodies fluoresce brightly and are slightly shrunken. Nuclei are not distinct. Dashed arrows: Normal neurons – Neuron cell bodies are dull green, with distinct cell borders.

Figs. 7 and 8, although collected from a control guinea pig challenged with PHO, are micrographs of normal hippocampus tissue. Likewise for Figs. 9 and 10, the micrographs are of normal cerebral cortex from a control guinea pig challenged with parathion. That those brains, representing the severely affected PHO and parathion control groups, were histologically normal suggested that PHO and parathion target sites could be primarily non-CNS. Figs. 11 and 12 represent neuronal necrosis in the hippocampus associated with TMB-4 treatment of a parathion-challenged guinea pig. It is important to note that similar lesions were not apparent in the parathion controls, nor among any other VR-, VX-, or PHO-challenged and TMB-4 treated animals. Figs. 13 and 14 are micrographs of marked neuronal necrosis in cerebral cortex from a control guinea pig challenged with VX.

Fig. 8.

Phorate oxon/atropine/saline, Fluoro Jade 40x – Normal neurons in the hippocampus. Dashed arrows: Normal neurons – Neuron cell bodies are dull green, with distinct cell borders.

Fig. 9.

Parathion/atropine/saline, H&E 40x – Normal neurons in the cerebral cortex. Dashed arrows: Normal neurons – Neuronal nuclei are large and well-defined; neuron cell bodies are large with scant, basophilic cytoplasm and distinct cell borders.

Fig. 10.

Parathion/atropine/saline, Fluoro Jade 40x – Normal neurons in the cerebral cortex. Dashed Arrows: Normal neurons – Neuron cell bodies are dull green with distinct cell borders and nuclei are large and ovoid with a granular appearance.

Fig. 11.

Parathion/atropine/TMB-4, H&E 40x – Neuronal necrosis in the hippocampus. Solid arrows: Neuronal necrosis – Neuronal nuclei are pyknotic (shrunken and deeply basophilic); neuron cell bodies are dark red and are slightly shrunken. Dashed arrows: Normal neurons – Neuronal nuclei are large and well-defined; neuron cell bodies are large with distinct cell borders.

Fig. 12.

Parathion/atropine/TMB-4, Fluoro Jade 40x – Neuronal necrosis in the hippocampus. Solid arrows: Neuronal necrosis – Neuron cell bodies fluoresce brightly and are slightly shrunken. Dashed arrows: Normal neurons – Neuron cell bodies are dull green with distinct cell borders.

Fig. 13.

VX/atropine/saline, H&E 40x – Neuronal necrosis in the cerebral cortex. Solid arrows: Neuronal necrosis – Neuronal nuclei are pyknotic (shrunken and deeply basophilic); neuron cell bodies are dark red and are slightly shrunken, pulling away from the surrounding neutrophil. Dashed arrows: Normal neurons – Neuronal nuclei are large and well-defined; neuron cell bodies are large with scant, basophilic cytoplasm and distinct cell borders.

DISCUSSION

Monomeric human AChE is a large (67,796 Da), typically membrane-bound hydrolase that terminates neuro-transmission at the synapses of cholinergic neurons by catalyzing the hydrolysis of the neurotransmitter, acetylcholine. Like other serine proteases and serine hydrolases, the reactive site in AChE is a catalytic triad, situated within AChE near the bottom of a 20-Å deep gorge. The rim of the gorge is rich in anionic residues that attract the quaternary amine of acetylcholine and channel the substrate along 14 aromatic amino acid residues that line the gorge toward an acyl binding pocket, oxyanion hole, and catalytic triad. The hydrolysis of acetylcholine to produce acetate and choline proceeds through a two-step acylation/deacylation process. The oxyanion hole forms two hydrogen bonds with the carbonyl oxygen of acetylcholine. This interaction stabilizes the substrate and ultimately the transition state, while serine and histidine serve to perform a nucleophilic attack and general acid-base catalysis, respectively. The acylated serine is subsequently deacylated through a hydrolysis reaction, and the catalytic site is regenerated (Zhang et al., 2002). This process can occur at one of the fastest turnover rates found in nature, approaching diffusion limited rates and hydrolyzing over 5,000 substrate units per second (Cooper et al., 2003).

If an OP molecule enters the gorge, the OP can bind in a manner similar to that described for the acetylcholine substrate discussed above. AChE that is inhibited by an OP cannot functionally hydrolyze acetylcholine. Unless AChE can be promptly reactivated, accumulation of acetylcholine can result in overstimulation of the cholinergic receptors, cholinergic crisis, and ultimately death. Reactivation of AChE through the use of oximes is only possible provided the OP has not “aged”. Aging is a dealkylation reaction of the OP-AChE adduct. Aging typically occurs when the O-C bond is hydrolyzed to yield a carbocation on the alkyl leaving group and a negatively charged phospho-oxygen (Li et al., 2007). This negatively charged oxygen prevents the nucleophilic attack and subsequent reactivation by oximes. Furthermore, because “aging is due to the dealkylation of the alkoxyl group of the residue bound to the enzyme, the rate of aging is proportional to the electron-donating capacity of the alkyl group” (Sun et al., 1979). The aging half-time can be as little as 1.3 min for soman-inhibited human AChE (Vale, 2007) and up to approximately 3 hr for sarin (Worek et al., 2004) and 48 hr for VX (Sidell and Groff, 1967).

Figure 1 shows that MMB4, HLö-7, HI-6, obidoxime, and TMB-4 are bis-quaternary oximes, whereas 2-PAM, RS194B, and MINA are mono-functional oximes. In terms of relative protective efficacy, the bis nature of the larger molecules gives them an advantage, as one end can interact with the rim of the gorge while the other end can extend into the gorge (Harel et al., 1993). Dual binding may help to shield AChE from OP attack, as well as out-compete and/or dislodge OP molecules interacting at sites within the gorge. Reactivation depends on (1) molecular spacing, as smaller alkyl substituents across similar OPs are generally associated with increased oxime protective efficacy, (2) relative absence of steric hindrance presented by an AChE-OP conjugate to an approaching oxime molecule, as well as (3) the nucleophilicity of the oxime molecule. The presence of an isonicotinamide group on an oxime (HLö-7, HI-6) has been associated with enhanced efficacy against nerve agents (Maxwell et al., 2008), but the mechanism for this is not known.

The therapeutic efficacy of MMB4 DMS against a topical LD₈₅ challenge of VR was evident in terms of 24-hr or 48-hr lethality, but not by ChE reactivation in terminal blood or brain. There were no brain lesions observed in VR-challenged animals. Obidoxime Cl₂ and MMB4 DMS were significantly effective against VX, evident in terms of both lethality and blood and brain ChE reactivation. HLö-7 DMS reduced lethality but exacerbated brain AChE and BChE inhibition, suggesting its mobility through the blood-brain barrier. 2-PAM Cl nearly eliminated lethality and reactivated VX-inhibited blood and brain ChE. HI-6 DMS effects on lethality and blood ChEs were marginal, yet demonstrated reactivation of brain ChEs. Neuronal necrosis was infrequently scattered in the VX controls and across half of the oxime groups without suggesting any cause/effect patterns. As expected, topical parathion challenges did not elicit immediate signs, and treatments were typically delayed by ~23 hr. Parathion doses needed to achieve the LD₈₅ were typically in excess of 0.5 mL for a 400-g guinea pig. Only TMB-4 and obidoxime Cl₂ offered significant improvement in survival against parathion, and TMB-4 also offered some blood AChE reactivation. Paradoxically however, TMB-4 and HI-6 DMS were strongly associated with decreased brain AChE and BChE activity relative to controls, and the only two histological instances of neuronal necrosis among parathion-challenged animals were observed in two of the three TMB-4-treated animals. Notably, the absence of similar lesions in the parathion controls and VX/TMB-4, VR/TMB-4, and PHO/TMB-4 animals suggested an isolated interaction of parathion and TMB-4. Further study of the possible untoward effect of neuronal necrosis with TMB-4 and exacerbation of cholinesterase inhibition by TMB-4 and HI-6 DMS in cases of parathion poisoning warrants further investigation, since TMB-4 is used in Israel and HI-6 dichloride is used in Canada and Sweden (Thiermann et al., 2013). The progression of clinical signs caused by topical challenges of PHO, once it began, was rapid and severe. Just over half (47/88 = 53%) of PHO-challenged animals could be treated under the prescribed regimen before they succumbed, and none of the oximes offered protection by any of the endpoints measured.

In general, the two oximes that offered the best protection were obidoxime Cl₂ and MMB4 DMS. Obidoxime Cl₂ also was efficacious against parathion, but none of the oximes tested was significantly effective against PHO in terms of promoting overall survival.

Although an aim of this study was to corroborate the findings of the previously published SC study (Wilhelm et al., 2014) and extend them to a scenario involving a realistic route of OP exposure (i.e., PC), the data collected here suggest such a direct correlation of an oxime’s efficacy is oxime-specific. Table 6 contrasts in terms of lethality rates how oxime efficacies varied depending on the challenge route of administration for the two OPs common to both this work and the Wilhelm et al. work, namely, VX and PHO (note that the G-agents, sarin, soman, tabun, and cyclosarin were not evaluated dermally in this study due to the volatile nature of those chemicals).

Table 6.

Contrast of topical versus subcutaneous challenges of VX and phorate oxon at respective 24-hr LD₈₅ for assessment of relative oxime efficacies in non-sedated, atropinized guinea pigs: lethality rates with Fisher’s exact test probabilities^‡ against no-oxime controls.

Oxime	Oxime dose each administration (μmol/kg)	Lethality rate (n·dead/n·dosed and treated) with decimal equivalent, and significance
		VX				Phorate oxon
		Topical		Subcutaneous		Topical		Subcutaneous
		Lethality rate		Lethality rate		Lethality rate		Lethality rate
none	-	11/21	0.52	33/64	0.52	8/11	0.73	62/64	0.97
MMB4 DMS	146	0/6	0.00*	0/8	0.00*	5/7	0.71	2/8	0.25*
obidoxime Cl2 Cl2	146	0/8	0.00*	1/8	0.13*	3/5	0.60	0/8	0.00*
2-PAM Cl	146	1/6	0.17	0/8	0.00*	5/6	0.83	1/8	0.13*
HLö-7 DMS	146	0/6	0.00*	0/8	0.00*	1/1	1.00	0/8	0.00*
HI-6 DMS	146	2/8	0.25	2/8	0.25	5/6	0.83	7/8	0.88
TMB-4	35	3/7	0.43	0/8	0.00*	3/4	0.75	6/8	0.75
MINA	146	6/8	0.75	2/8	0.25	1/2	0.50	8/8	1.00
RS194B	146	6/8	0.75	4/8	0.50	5/5	1.00	7/8	0.88

^‡Each p value is the probability of the difference between the oxime lethality rate and the “none” controls lethality rate being as much as that shown due purely to random sampling; a one-sided decision level of 0.05 was used. Statistically significant oxime effects are in bold font:

^*p ≤ 0.05

In addition to the challenge route of administration, there were several important procedural differences between the current topical exposures work and the referenced SC exposures work. In this topical study, atropine/oxime therapy was delayed until onset of clinical signs of cholinergic intoxication and repeated twice thereafter at 3-hr intervals, whereas therapy was given at 1 min after the SC challenges and not repeated. The designed paradigm for PC exposure, therefore, was comprised of three total administrations of the therapy. Again, the intent of this approach was to simulate the real world pre-hospital mass casualty and triage treatment care scenario. Against VX, the two test models agreed well for every oxime but TMB-4. Thus for VX, the route of challenge appeared to have little effect on oxime efficacy, and the SC results could have predicted the topical outcome fairly well. However, oxime efficacies seemed much more affected by the route of PHO challenge. Against a topical challenge of PHO, none of the oximes tested was significantly effective in reducing lethality rate, but four oximes were significantly effective against SC PHO challenges. Why were the VX results relatively route-independent, whereas the PHO results were strongly associated with how the challenge was administered? Physicochemical properties that are strong determinants for skin penetration include molecular weight (MW) and the base 10 logarithm of the octanol/water solubility ratio [log(P_OW)] (Guy et al., 1985; Nielsen et al., 2004; Czerwinski et al., 2006). Although values for these parameters are similar for VX [MW = 267 Da, log(P_OW) = 2.09] and PHO [MW = 244 Da, log(P_OW) = 2.07], PHO was slower than VX by approximately 100 min in causing a significant clinical sign, as shown in Fig. 2. The timing of OP intoxication also depends on the kinetics of AChE inhibition/reactivation and aging of the AChE/OP adduct. The human erythrocyte AChE/VX adduct is known to be readily reactivated by HLö-7, HI-6, obidoxime and pralidoxime (Worek et al., 2004), with second-order rate constant k_r2 for these ranging from 8 to 60 M⁻¹ min⁻¹, which is an intermediate range relative to AChE/methamidofos (readily reactivated, 40 to 160 mM⁻¹ min⁻¹) and AChE/tabun (difficult to reactivate, 0 to 0.5 mM⁻¹ min⁻¹). The aging half-life of guinea pigs erythrocyte AChE/VX is 380 hr (Luo et al., 2007). Thus for VX, whether an oxime was essentially co-administered with the SC challenge or given hours later, oxime efficacy was the same against biologically equivalent challenges.

Unfortunately, similar kinetics data are not currently available for PHO, but we hope to address this research gap in the near future. It may be that the AChE/PHO adduct had aged to the point that therapy administered at the onset of signs from a topical challenge was too late to be useful. Another possibility, if PHO were found to be a very slow inhibitor, is that the SC test model, in which therapy was given practically simultaneously with the challenge, presented more opportunity for the protective oximes obidoxime Cl₂ and HLö-7 DMS to have significant, direct interactions with either (a) target site receptors in a blocking fashion or (b) PHO before it could produce inhibition. The data pose interesting questions regarding the reaction kinetics, reversibility, and aging of AChE/PHO adducts.

In general, the two oximes that offered the best protection against the spectrum of OP chemicals evaluated here, VX, VR and parathion, were obidoxime Cl₂ and MMB4 DMS, but neither of those was significantly effective against PHO in terms of promoting overall survival. Further studies on the OP chemical PHO are necessary to understand the mechanism behind the lack of efficacy observed here.

Fig. 4.

Acetylthiocholine and butyrylthiocholine hydrolysis rates in blood and brain collected from survivors exposed topically to LD₈₅ of VX. Filled circles (labeled by oxime and sample size) represent treatment group means with error bars of one standard error when n > 1. The inverted triangle in the upper right corner of the blood plot represents baseline relative ChE levels of 100%. Since there was no baseline brain sample collected, there could be no such AChE:BChE activity reference for the brain plot. The mean vehicle control brain values are represented with an inverted triangle with error bars.

Fig. 5.

Acetylthiocholine and butyrylthiocholine hydrolysis rates in blood and brain collected from survivors exposed topically to LD₈₅ of parathion. Filled circles (labeled by oxime and sample size) represent treatment group means with error bars of one standard error when n > 1. The inverted triangle in the upper right corner of the blood plot represents baseline relative ChE levels of 100%. Since there was no baseline brain sample collected, there could be no such AChE:BChE activity reference for the brain plot. The mean vehicle control brain values are represented with an inverted triangle with error bars.