Some Comments on the Selection of Human Intraspecies Uncertainty Factors

The Reference Dose (RfD) is used in the risk assessment of non-carcinogenic chemicals. It is derived by dividing a point of departure by the product of the uncertainty (UFs) and modifying factors (MFs). Separate UFs are used for different variables, e.g., intraspecies variation and, in general, each UF is an order of magnitude (10-fold). On the other hand, the MF is usually based on some known variable such as differences in absorption of a chemical from food and water and its default value is one. The USEPA's Integrated Risk Information System (IRIS) has 14 chemicals that have RfDs based on human studies. We examined those IRIS files to determine the rationale for setting human intraspecies uncertainty factors (UF_H). The first consideration was that the chemical had an adequate peer-reviewed human database. Without such, it would not be possible to derive an RfD based on human data. Ten of the 14 chemicals had an UF_H of 1 or 3; four of these were essential trace elements (ETEs). The rationales for using less than a 10-fold UF_H for the ETEs included; 1) nutritional data, 2) large human exposure groups, 3) minimal effect levels and/or 4) several studies with similar effect levels. For the other compounds, reasons included; 1) large human exposure groups, 2) a critical effect that was not adverse (cosmetic), 3) the most sensitive population was exposed, 4) the compound was on the FDA's “generally regarded as safe” (GRAS) list, 5) database uncertainties and 6) less-than-lifetime exposure adjusted for 70 years exposure. It is important to understand the reasons for selecting a UF_H of 1, or 3 as they will apply to future chemicals considered by the USEPA and other agencies.     

Inter- and Intra-Species Extrapolation in Risk Assessment of Different Classes of Pesticides

Risk assessors routinely use the reference dose (RfD) approach for non-cancer risk assessment. In this approach, No-Observed-Adverse-Effect-Level (NOAEL) is divided by the product of uncertainty factors (UFs) and, occasionally, an additional modifying factor (MF), each usually employed by default as factors of 10. In the present investigation, kinetic and dynamic data have been used in order to reduce uncertainties when establishing exposure guidelines for examples of chemicals representing four classes of pesticides (warfarin, lindane, carbaryl and parathion). An intensive search of databases was conducted for these pesticides, and toxicokinetic and toxicodynamic parameters in inter- and intra-species were evaluated. The kinetic and dynamic subfactors were less than the proposed values of Renwick and the International Programme on Chemical Safety (IPCS). The composite factors for all the examined pesticides were less than 100. The present study indicated that in setting exposure levels it is important to incorporate kinetic and dynamic data, as they become available, rather than rely on default uncertainty factors, which are imprecise in many cases.     

Chiral Separation of G‐type Chemical Warfare Nerve Agents via Analytical Supercritical Fluid Chromatography

Chemical warfare nerve agents (CWNAs) are extremely toxic organophosphorus compounds that contain a chiral phosphorus center. Undirected synthesis of G‐type CWNAs produces stereoisomers of tabun, sarin, soman, and cyclosarin (GA, GB, GD, and GF, respectively). Analytical‐scale methods were developed using a supercritical fluid chromatography (SFC) system in tandem with a mass spectrometer for the separation, quantitation, and isolation of individual stereoisomers of GA, GB, GD, and GF. Screening various chiral stationary phases (CSPs) for the capacity to provide full baseline separation of the CWNAs revealed that a Regis WhelkO1 (SS) column was capable of separating the enantiomers of GA, GB, and GF, with elution of the P(+) enantiomer preceding elution of the corresponding P(–) enantiomer; two WhelkO1 (SS) columns had to be connected in series to achieve complete baseline resolution. The four diastereomers of GD were also resolved using two tandem WhelkO1 (SS) columns, with complete baseline separation of the two P(+) epimers. A single WhelkO1 (RR) column with inverse stereochemistry resulted in baseline separation of the GD P(–) epimers. The analytical methods described can be scaled to allow isolation of individual stereoisomers to assist in screening and development of countermeasures to organophosphorus nerve agents. Chirality 26:817–824, 2014. © 2014 The Authors. Chirality published by John Wiley Periodicals, Inc.     

Subchronic to chronic exposure extrapolation: Toxicologic evidence for a reduced uncertainty factor

Acceptable human exposure guidelines for noncarcinogens are usually based on laboratory test no‐observed‐adverse‐effect levels (NOAELs), which are downwardly adjusted by fixed 10‐fold uncertainty factors. One such factor adjusts subchronic data to estimate corresponding values for lifetime exposures. Matched ninety‐day and two‐year toxicity studies in rodents were evaluated to biologically assess the sufficiency or excess of the 10‐fold uncertainty factor for subchronic‐to‐chronic data extrapolation. Studies conducted by the oral and inhalation routes of exposure were evaluated. NOAELs and lowest‐observed‐adverse‐effect levels (LOAELs) in the subchronic (sc) and chronic (c) studies were compared in terms of NOAEL and LOAEL ratios. The mean and median NOAEL_SC/NOAEL_C ratios for 23 oral studies were 2.4 and 2.0, respectively. Data from 9 inhalation studies yielded mean and median ratios of 4.5 and 4.0, respectively. Only one oral or inhalation study had a NOAEL_SC/NOAEL_C ratio as high as 10, and 22 of the 23 oral studies had ratios of 5 or less. These findings indicate that uncertainty factors less than 10 are sufficient to downwardly adjust subchronic data for lifetime exposures.     

Studies on the use of uncertainty factors in deriving RfDs

Risk assessment of exposure to chemicals having a toxic end point routinely uses the reference dose (RfD) approach based on uncertainty factors of 10. RfD model can be used with widely different databases. However, the quality of individual risk assessment is unequal among chemicals, often resulting in either an over‐ or underestimation of adverse health risk. The purpose of this investigation was to evaluate whether the magnitude of the 10X uncertainty factors has scientific merit against data from recent human and animal experimental studies. Although we assessed the use of uncertainty factors for representative chemicals from various classes of compounds, such as volatile organics, alcohols, gasoline components, and pesticides, we are presenting our findings for 24 chemicals.

A compilation and comparison of ratios between LOAEL/NOAEL (Lowest Observed Adverse Effect Level/No Observed Adverse Effect Level), and subchronic/chronic values were made. Although a 10X uncertainty factor is most commonly used in the risk assessment processes, an examination of the datasets which have been used to calculate RfDs suggests different values which are scientifically justifiable.     

Developmental toxicity evaluation of sodium thioglycolate administered topically to Sprague-Dawley (CD) rats and New Zealand White rabbits

BACKGROUND: Sodium thioglycolate, which has widespread occupational and consumer exposure to women from cosmetics and hair‐care products, was evaluated for developmental toxicity by topical exposure during the embryonic and fetal periods of pregnancy METHODS: Timed‐mated Sprague–Dawley rats (25/group) and New Zealand White (NZW) rabbits (24/group) were exposed to sodium thioglycolate in vehicle (95% ethanol:distilled water, 1:1) by unoccluded topical application on gestational days (GD) 6–19 (rats) or 6–29 (rabbits) for 6 hr/day, at 0, 50, 100, or 200 mg/kg body weight/day (rats) and 0, 10, 15, 25, or 65 mg/kg/day (rabbits). At termination (GD 20 rats; GD 30 rabbits), fetuses were examined for external, visceral, and skeletal malformations and variations. RESULTS: In rats, maternal topical exposure to sodium thioglycolate, at 200 mg/kg/day (the highest dose tested) on GD 6–19, resulted in maternal toxicity, including reduced body weights and weight gain, increased relative water consumption and one death. Treatment‐related increases in feed consumption and changes at the application site occurred at all doses, in the absence of increased body weights or body weight change. Fetal body weights/litter were decreased at 200 mg/kg/day, with no other embryo/fetal toxicity and no treatment‐related teratogenicity in any group. In rabbits, maternal topical exposure to sodium thioglycolate on GD 6–29 resulted in maternal dose‐related toxicity at the dosing site in all groups; no maternal systemic toxicity, embryo/fetal toxicity, or treatment‐related teratogenicity were observed in any group. CONCLUSIONS: A no observed adverse effect level (NOAEL) was not identified for maternal toxicity in either species with the dosages tested. The developmental toxicity NOAEL was 100 mg/kg/day (rats) and ≥65 mg/kg/day (rabbits; the highest dose tested). The clinical relevance of theses study results is uncertain because no data were available for levels, frequency, or duration of exposures in female workers or end users. Birth Defects Research Part B 68:144–161, 2003. © 2003 Wiley‐Liss, Inc.     

Inhibitors of Renin as Potential Therapeutic Agents

Abstract

The reaction of usual (U) and atypical (A) cholinesterase phenotypes was studied with six organophosphorus compounds, two pyridinium oximes (HI-6 and PAM-2) and with 4–4-bipyridine (4,4-BP). No difference in the inhibition rate constants for the two phenotypes was found with the progressive inhibitors tabun, sarin, paraoxon and soman. The other two progressive inhibitors, VX and the positively charged phosphostigmine, inhibited the U phenotype more strongly than the A phenotype.

The positively charged reversible inhibitor HI-6 showed a higher affinity for the U than for the A phenotype, while PAM-2 and the non-charged 4,4′-BP did not show a significant difference in their affinity towards the two enzymes.

Both phenotypes phosphylated by VX or sarin were reactivatable by HI-6 and PAM-2, and the A phenotype was always reactivated more slowly than the U phenotype. The paraoxon-inhibited phenotypes were reactivated at equal rates with PAM-2 but were not reactivated with HI-6. The phosphylated phenotypes did not reactivate spontaneously during one hour.

The effect of reversible inhibitors upon the rate of phosphylation (protection) was tested with HI-6 (for inhibition by soman, tabun and paraoxon) and with 4,4′-BP (for inhibition by soman). By applying the concentrations of the protectors equal to their enzyme/inhibitor dissociation constants, a better protection of the U than of the A phenotype was achieved by HI-6, but equal protection was given by 4,4′-BP.     

Enzymes for the homeland defense: optimizing phosphotriesterase for the hydrolysis of organophosphate nerve agents

Phosphotriesterase (PTE) from soil bacteria is known for its ability to catalyze the detoxification of organophosphate pesticides and chemical warfare agents. Most of the organophosphate chemical warfare agents are a mixture of two stereoisomers at the phosphorus center, and the S(P)-enantiomers are significantly more toxic than the R(P)-enantiomers. In previous investigations, PTE variants were created through the manipulation of the substrate binding pockets and these mutants were shown to have greater catalytic activities for the detoxification of the more toxic S(P)-enantiomers of nerve agent analogues for GB, GD, GF, VX, and VR than the less toxic R(P)-enantiomers. In this investigation, alternate strategies were employed to discover additional PTE variants with significant improvements in catalytic activities relative to that of the wild-type enzyme. Screening and selection techniques were utilized to isolate PTE variants from randomized libraries and site specific modifications. The catalytic activities of these newly identified PTE variants toward the S(P)-enantiomers of chromophoric analogues of GB, GD, GF, VX, and VR have been improved up to 15000-fold relative to that of the wild-type enzyme. The X-ray crystal structures of the best PTE variants were determined. Characterization of these mutants with the authentic G-type nerve agents has confirmed the expected improvements in catalytic activity against the most toxic enantiomers of GB, GD, and GF. The values of k(cat)/K(m) for the H257Y/L303T (YT) mutant for the hydrolysis of GB, GD, and GF were determined to be 2 × 10(6), 5 × 10(5), and 8 × 10(5) M(-1) s(-1), respectively. The YT mutant is the most proficient enzyme reported thus far for the detoxification of G-type nerve agents. These results support a combinatorial strategy of rational design and directed evolution as a powerful tool for the discovery of more efficient enzymes for the detoxification of organophosphate nerve agents.     

Toxicogenomic Studies of Human Neural Cells Following Exposure to Organophosphorus Chemical Warfare Nerve Agent VX

Organophosphorus (OP) compounds represent an important group of chemical warfare nerve agents that remains a significant and constant military and civilian threat. OP compounds are considered acting primarily via cholinergic pathways by binding irreversibly to acetylcholinesterase, an important regulator of the neurotransmitter acetylcholine. Many studies over the past years have suggested that other mechanisms of OP toxicity exist, which need to be unraveled by a comprehensive and systematic approach such as genome-wide gene expression analysis. Here we performed a microarray study in which cultured human neural cells were exposed to 0.1 or 10 μM of VX for 1 h. Global gene expression changes were analyzed 6, 24, and 72 h post exposure. Functional annotation and pathway analysis of the differentially expressed genes has revealed many genes, networks and canonical pathways that are related to nervous system development and function, or to neurodegenerative diseases such as Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease. In particular, the neuregulin pathway impacted by VX exposure has important implications in many nervous system diseases including schizophrenia. These results provide useful information valuable in developing suitable antidotes for more effective prevention and treatment of, as well as in developing biomarkers for, VX-induced chronic neurotoxicity.     

Developmental Toxicity of the HMG‐CoA Reductase Inhibitor (PPD10558) in Rats and Rabbits

PPD10558 is an orally active, lipid‐lowering 3–hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitor (statin) being developed as a treatment for hypercholesterolemia in patients who have not been able to tolerate statins because of statin‐associated myalgia. We have studied the potential developmental toxicity effects of PPD10558 in pregnant rats and rabbits given daily oral doses during the period of organogenesis. Rats were dosed with 0, 20, 80, or 320 mg/kg/day from Gestation Day (GD) 6 to 17 and rabbits received dose levels of 0, 12.5, 25, or 50 mg/kg/day from GD 6 to 18. Additional groups in both studies served as toxicokinetic animals and received the PPD10558 in the same manner as the main study groups at the same dose levels. Blood samples were collected from toxicokinetic animals at designated time points on GD 6 and 17 in rats and GD 6 and 18 in rabbits. Fetal exposure in rats was assessed on GD 20. Maternal and developmental parameters were evaluated in rats and rabbits on GD 20 and GD 29, respectively. No maternal and developmental toxicity was observed at any of the dose levels used in the rat study. Evidence of fetal exposure was determined in fetal plasma with mean fetal concentrations of PPD10558 and the metabolite (PPD11901) found to be between 1 and 6% of the mean maternal concentrations. In rabbits, marked maternal toxicity including mortality (eight deaths; 1 dose at 25 and 7 at 50 mg/kg/day), abortions (2 at 25 mg/kg/day and 6 at 50 mg/kg/day) and reduction in gestation body weight, gestation body weight changes and decreased food consumption were observed. In addition, fetal body weights of the combined sexes were significantly reduced at 50 mg/kg/day in comparison with the controls. Mean peak exposure (Cmax) and total exposure (AUC(0–24)) of PPD11901 in both rats and rabbits were higher than that of PPD10558 on GD 6 and GD 17 at each of the three dose levels.. Based on the results of these studies, the no observed adverse effect level (NOAEL) for maternal and developmental toxicity in rats was considered to be ≥320 mg/kg/day, the highest dose level used in the study. The NOAEL for maternal and developmental toxicity in rabbits was 12.5 mg/kg/day and 25 mg/kg/day, respectively.     

U.S. EPA Reference Dose/Reference Concentration Methodology: Update on a Review of the Process

The U.S. Environmental Protection Agency (USEPA) has been reviewing several approaches to testing and risk assessment related to implementation of the Food Quality Protection Act (FQPA) and the Amendments to the Safe Drinking Water Act (SDWA), both signed into law in 1996. Based on recommendations from a review of issues related to children's health protection under these laws, the USEPA established the RfD Technical Panel to evaluate in depth the current reference dose (RfD) and reference concentration (RfC) process in general, and in particular with respect to how well children and other potentially sensitive subpopulations are protected. The RfD Technical Panel also was asked to consider scientific issues that have become of greater concern in RfD and RfC derivation (e.g., neurotoxicity, immunotoxicity), and to raise issues that should be explored or developed further for application in the RfD/RfC process. This paper provides the current status of the activities of the RfD Technical Panel. The Technical Panel has recommended that acute, short- term, and intermediate reference values should be set for chemicals, where possible, and that these values should be incorporated into the USEPA's Integrated Risk Information System (IRIS) Database. A review of current testing procedures is underway, including the endpoints assessed, life stages covered by exposure and outcome evaluation, and information that can be derived from current protocols on various durations of exposure. Data gaps identified for risk assessment include the types of pharmacokinetic data that should be collected, especially for developmental toxicity studies, the impact of aging on toxic responses occurring after early exposure as well as concomitant with exposure in old age, and information available on latency to response. The implications of the RfD Technical Panel's recommendations for various uncertainty factors are also being explored.     

Nerve agent hydrolysis activity designed into a human drug metabolism enzyme

Organophosphorus (OP) nerve agents are potent suicide inhibitors of the essential neurotransmitter-regulating enzyme acetylcholinesterase. Due to their acute toxicity, there is significant interest in developing effective countermeasures to OP poisoning. Here we impart nerve agent hydrolysis activity into the human drug metabolism enzyme carboxylesterase 1. Using crystal structures of the target enzyme in complex with nerve agent as a guide, a pair of histidine and glutamic acid residues were designed proximal to the enzyme's native catalytic triad. The resultant variant protein demonstrated significantly increased rates of reactivation following exposure to sarin, soman, and cyclosarin. Importantly, the addition of these residues did not alter the high affinity binding of nerve agents to this protein. Thus, using two amino acid substitutions, a novel enzyme was created that efficiently converted a group of hemisubstrates, compounds that can start but not complete a reaction cycle, into bona fide substrates. Such approaches may lead to novel countermeasures for nerve agent poisoning.     

Isotope dilution LC/MS/MS for the detection of nerve agent exposure in urine

Organophosphorus nerve agents (OPNA), chemically related to and derived from organophosphate insecticides, constitute a clear and present threat to both military and civilian targets. Military regimes and terrorist organizations have demonstrated the will and ability to produce mass casualties by dispersing organophosphorus nerve agents, which, in turn could terrorize populations and overwhelm healthcare systems. A high throughput, robust and sensitive analytical protocol has been developed for the quantitation of the urinary metabolites of sarin (GB), soman (GD), VX, Russian VX (RVX) and cyclohexylsarin (GF) utilizing solid phase extraction (SPE) followed by High Performance Liquid Chromatography (HPLC)-isotope dilution tandem mass spectrometry (LC/MS/MS). The method has demonstrated linearity and reproducibility (1-200 ng/mL) for all analytes and has a Limit of Quantitation (LOQ)< or =0.5 ng/mL for all analytes (S/N> or =10/1). The method was validated by performing 20 individual analyses over 10 days by five scientists with all values falling within two standard deviations of the mean.     

Limitations and challenges in treatment of acute chemical warfare agent poisoning

Recent news from Syria on a possible use of chemical warfare agents made the headlines. Furthermore, the motivation of terrorists to cause maximal harm shifts these agents into the public focus. For incidents with mass casualties appropriate medical countermeasures must be available. At present, the most important threats arise from nerve agents and sulfur mustard. At first, self-protection and protection of medical units from contamination is of utmost importance. Volatile nerve agent exposure, e.g. sarin, results in fast development of cholinergic crisis. Immediate clinical diagnosis can be confirmed on-site by assessment of acetylcholinesterase activity. Treatment with autoinjectors that are filled with 2 mg atropine and an oxime (at present obidoxime, pralidoxime, TMB-4 or HI-6) are not effective against all nerve agents. A more aggressive atropinisation has to be considered and more effective oximes (if possible with a broad spectrum or a combination of different oximes) as well as alternative strategies to cope with high acetylcholine levels at synaptic sites should be developed. A further gap exists for the treatment of patients with sustained cholinergic crisis that has to be expected after exposure to persistent nerve agents, e.g. VX. The requirement for long-lasting artificial ventilation can be reduced with an oxime therapy that is optimized by using the cholinesterase status for guidance or by measures (e.g. scavengers) that are able to reduce the poison load substantially in the patients.     

Global changes in the expression patterns of RNA isolated from the hippocampus and cortex of VX exposed mice

One of the established activities of the nerve agent VX is inhibition of the enzyme acetylcholinesterase (AChE). This inhibition affects the cholinergic nervous system by decreasing the activity of the neurotransmitter-hydrolyzing enzyme cholinesterase (ChE). In an effort to gain a more comprehensive understanding of the molecular pathways affected by low-level exposure to VX, an expression profiling approach was used to identify genes with altered RNA expression patterns after exposure.Specifically, mice were exposed to 0.1, 0.2, 0.4, and 0.6 LD50 VX for a period of 2 weeks. At 2 h, 72 h, and 2 weeks after the final exposure, RNA was isolated from both the hippocampus and the cortex. Changes in gene expression levels were assessed by DNA microarray technology and grouped according to their expression patterns. Data presented here demonstrate that 2 weeks postexposure all up-regulated gene expression has returned to pre-exposure levels, including genes related to the central nervous system. Additionally, this investigation has revealed non-AChE pathway genes involved in other neuronal functions that display altered expression profiles after VX exposure.     

Effects of Repeated Low-Dose Exposure of the Nerve Agent VX on Monoamine Levels in Different Brain Structures in Mice

In a previous report, alterations of the serotonin metabolism were previously reported in mice intoxicated with repeated low doses of soman. In order to better understand the effects induced by repeated low-dose exposure to organophosphorus compounds on physiological and behavioural functions, the levels of endogenous monoamines (serotonin and dopamine) in different brain areas in mice intoxicated with sublethal dose of (O-ethyl-S-[2(di-isopropylamino) ethyl] methyl phosphonothioate) (VX) were analysed by HPLC method with electrochemical detection. Animals were injected once a day for three consecutive days with 0.10 LD₅₀ of VX (5 μg/kg, i.p). Neither severe signs of cholinergic toxicity nor pathological changes in brain tissue of exposed animals were observed. Cholinesterase (ChE) activity was only inhibited in plasma (a maximum of 30 % inhibition 24 h after the last injection of VX), but remained unchanged in the brain. Serotonin and dopamine (DA) metabolism appeared significantly modified. During the entire period of investigation, at least one of the three parameters investigated (i.e. DA and DOPAC levels and DOPAC/DA ratio) was modified. During the toxic challenge, an increase of the serotonin metabolism was noted in hippocampus (HPC), hypothalamus/thalamus, pons medulla and cerebellum (CER). This increase was maintained 4 weeks after exposure in HPC, pons medulla and CER whereas a decrease in cortex 3 weeks after the toxic challenge was observed. The lack of correlation between brain ChE activity and neurochemical outcomes points out to independent mechanisms. The involvement in possibly long-lasting behavioural disorders is discussed.     

Replacing the Default Values of 10 With Data-Derived Values: A Comparison of Two Different Data-Derived Uncertainty Factors for Boron

A “safe” or sub-threshold dose is often estimated for oral toxicity of substances in order to protect humans from adverse health effects. This dose is referred to by several terms: reference dose (RfD), tolerable daily intake (TDI), and acceptable daily intake (ADI). Similarly, tolerable concentration (TC), and reference concentration (RfC) are commonly used terms for a “safe” concentration for inhalation. The process of deriving these doses generally involves identifying a no observed, or lowest observed adverse effect level (NOAEL or LOAEL) in animals, or humans, and application of uncertainty factors to account for the extrapolation from laboratory animals to humans and/or from an average human to a sensitive human. Public health agencies have begun to consider using a data derived approach, which uses available toxicokinetic and toxicodynamic data in the determination of uncertainty factors, rather than relying on the standard default values. Recently two different tolerable daily intake risk values were derived by two different World Health Organization (WHO) work groups. The International Programme on Chemical Safety, and the Working Group on Chemical Substances in Drinking Water both used the approach developed by Renwick (1993); however, the two groups interpreted and used the available data differently. The result was a difference of over twofold in the total uncertainty factor used. This review compares and contrasts the two approaches used by these WHO work groups.     

Advantages of the WRAIR whole blood cholinesterase assay: Comparative analysis to the micro-Ellman, Test-mate ChE™, and Michel (ΔpH) assays

Red blood cell AChE (RBC-AChE) and plasma BChE can be used as sensitive biomarkers to detect exposure to OP nerve agents, pesticides, and cholinergic drugs. In a comparative study, RBC-AChE and serum BChE activities in whole blood was obtained from forty seven healthy male and female human volunteers, and then exposed separately ex vivo to three OP nerve agents (soman (GD), sarin (GB) and VX) to generate a wide range of inhibition of AChE and BChE activity (up to 90% of control). These samples were measured using four different ChE assays: (i) colorimetric microEllman (using DTNB at 412 nm), (ii) Test-mate ChE field kit (also based on the Ellman assay), (iii) Michel (delta pH), and (iv) the Walter Reed Army Institute of Research Whole Blood (WRAIR WB) cholinesterase assay. The WRAIR assay is a modified Ellman method using DTP at 324 nm (which minimizes hemoglobin interference and improves sensitivity), and determines AChE and BChE in a small whole blood sample simultaneously.Scatter plots of RBC-AChE activities were determined using the WRAIR ChE assay versus the micro-Ellman, Test-mateTM and Michel after exposure to varying concentrations of soman, sarin and VX. Regression analyses yielded mostly linear relationships with high correlations (r2 = 0.83–0.93) for RBC-AChE values in the WRAIR assay compared to the alternate methods. For the plasma BChE measurements, individual human values were significantly more variable (as expected), resulting in lower correlations using WRAIR ChE versus the alternate assays (r2 values 0.5 – 0.6).To circumvent the limitations of simple correlation analysis, Bland and Altman analysis for comparing two independent measurement techniques was performed. For example, a Bland and Altman plot of the ratio of the WRAIR whole blood AChE and Michel AChE (plotted on the y-axis) vs. the average of the two methods (x-axis) shows that the majority of the individual AChE values are within ± 1.96 S.D. of the mean difference, indicating that the two methods may be used interchangeably with a high degree of confidence. The WRAIR ChE assay can be thus be used as a reliable inter-conversion assay when comparing results from laboratory-based (Michel) and field-based (Test-mateTM ChE kit), which use different methodology and report in different units of AChE activity.     

Advantages of the WRAIR whole blood cholinesterase assay: comparative analysis to the micro-Ellman, Test-mate ChE, and Michel (DeltapH) assays

Red blood cell AChE (RBC-AChE) and plasma BChE can be used as sensitive biomarkers to detect exposure to OP nerve agents, pesticides, and cholinergic drugs. In a comparative study, RBC-AChE and serum BChE activities in whole blood was obtained from forty seven healthy male and female human volunteers, and then exposed separately ex vivo to three OP nerve agents (soman (GD), sarin (GB) and VX) to generate a wide range of inhibition of AChE and BChE activity (up to 90% of control). These samples were measured using four different ChE assays: (i) colorimetric microEllman (using DTNB at 412 nm), (ii) Test-mate ChE field kit (also based on the Ellman assay), (iii) Michel (delta pH), and (iv) the Walter Reed Army Institute of Research Whole Blood (WRAIR WB) cholinesterase assay. The WRAIR assay is a modified Ellman method using DTP at 324 nm (which minimizes hemoglobin interference and improves sensitivity), and determines AChE and BChE in a small whole blood sample simultaneously. Scatter plots of RBC-AChE activities were determined using the WRAIR ChE assay versus the micro-Ellman, Test-mateTM and Michel after exposure to varying concentrations of soman, sarin and VX. Regression analyses yielded mostly linear relationships with high correlations (r2 = 0.83-0.93) for RBC-AChE values in the WRAIR assay compared to the alternate methods. For the plasma BChE measurements, individual human values were significantly more variable (as expected), resulting in lower correlations using WRAIR ChE versus the alternate assays (r2 values 0.5 - 0.6). To circumvent the limitations of simple correlation analysis, Bland and Altman analysis for comparing two independent measurement techniques was performed. For example, a Bland and Altman plot of the ratio of the WRAIR whole blood AChE and Michel AChE (plotted on the y-axis) vs. the average of the two methods (x-axis) shows that the majority of the individual AChE values are within +/- 1.96 S.D. of the mean difference, indicating that the two methods may be used interchangeably with a high degree of confidence. The WRAIR ChE assay can be thus be used as a reliable inter-conversion assay when comparing results from laboratory-based (Michel) and field-based (Test-mateTM ChE kit), which use different methodology and report in different units of AChE activity.