Resolving pathways of interaction of mipafox and a sarin analog with human acetylcholinesterase by kinetics, mass spectrometry and molecular modeling approaches.

The hydroxyl oxygen of the catalytic triad serine in the active center of serine hydrolase acetylcholinesterase (AChE) attacks organophosphorus compounds (OPs) at the phosphorus atom to displace the primary leaving group and to form a covalent bond. Inhibited AChE can be reactivated by cleavage of the Ser-phosphorus bond either spontaneously or through a reaction with nucleophilic agents, such as oximes. At the same time, the inhibited AChE adduct can lose part of the molecule by progressive dealkylation over time in a process called aging. Reactivation of the aged enzyme has not yet been demonstrated. Here, our goal was to study oxime reactivation and aging reactions of human AChE inhibited by mipafox or a sarin analog (Flu-MPs, fluorescent methylphosphonate). Progressive reactivation was observed after Flu-MPs inhibition using oxime 2-PAM. However, no reactivation was observed after mipafox inhibition with 2-PAM or the more potent oximes used. A peptide fingerprinted mass spectrometry (MS) method, which clearly distinguished the peptide with the active serine (active center peptide, ACP) of the human AChE adducted with OPs, was developed by MALDI-TOF and MALDI-TOF/TOF. The ACP was detected with a diethyl-phosphorylated adduct after paraoxon inhibition, and with an isopropylmethyl-phosphonylated and a methyl-phosphonylated adduct after Flu-MPs inhibition and subsequent aging. Nevertheless, nonaged nonreactivated complexes were seen after mipafox inhibition and incubation with oximes, where MS data showed an ACP with an NN diisopropyl phosphoryl adduct. The kinetic experiments showed no reactivation of activity. The computational molecular model analysis of the mipafox-inhibited hAChE plots of energy versus distance between the atoms separated by dealkylation showed a high energy demand, thus little aging probability. However, with Flu-MPs and DFP, where aging was observed in our MS data and in previously published crystal structures, the energy demand calculated in modeling was lower and, consequently, aging appeared as a more likely reaction. We document here direct evidence for a phosphorylated hAChE refractory to oxime reactivation, although we observed no aging.

Sensitivity analysis on a physiologically-based pharmacokinetic and pharmacodynamic model for diisopropylfluorophosphate-induced toxicity in mice and rats.

A physiologically-based pharmacokinetic and pharmacodynamic (PBPK/PD) model was recently developed to study the effect of diisopropylfluorophosphate (DFP) on acetylcholinesterase (AChE) activity in mouse and rat. That model takes into account relatively complex interactions involving many parameters, some of which may be uncertain and/or highly variable, especially those characterizing AChE activity after DFP intoxication. The primary objective of this study was to identify parameters that contribute most to the variability of AChE dynamics for model optimization against data. For this purpose, the influence of the variability of the rate constants for synthesis (K(syn)) and degradation (K(deg)) of AChE, and regeneration (K(reg)) and aging (K(age)) of inhibited AChE on the variability of AChE activity in mice and rat venous blood and brain was first calculated by a global sensitivity analysis. Next, the mouse PBPK/PD model was calibrated by optimizing the values of K(syn), K(deg), K(reg) and K(age). Thereafter, scale-up of the DFP-induced AChE activity was performed from mouse to rat. Validation of the rat model was performed by comparing the time course of venous blood and brain AChE activities from a Monte Carlo analysis to those obtained in vivo. Sensitivity analysis on the verified models showed that K(reg) and K(syn) were the most influential factors of AChE activity at shorter and longer durations, respectively, after DFP challenge. Scale-up of the AChE dynamics from mouse to rat was also successful, as evidenced by significant overlapping between the predicted 95(th) percentile confidence intervals and the experimental data.

Acyl peptide hydrolase, a serine proteinase isolated from conditioned medium of neuroblastoma cells, degrades the amyloid-beta peptide.

Considerable evidence indicates that the amyloid-beta (Abeta) peptide, a proteolytic fragment of the amyloid precursor protein, is the pathogenic agent in Alzheimer's disease (AD). A number of proteases have been reported as capable of degrading Abeta, among them: neprilysin, insulin-degrading enzyme, endothelin-converting enzyme-1 and -2, angiotensin-converting enzyme and plasmin. These proteases, originating from a variety of cell types, degrade Abeta of various conformational states and in different cellular locations. We report here the isolation of a serine protease from serum-free conditioned medium of human neuroblastoma cells. Tandem mass spectrometry (MS/MS)-based sequencing of the isolated protein identified acyl peptide hydrolase (APH; EC3.4.19.1) as the active peptidase. APH is one of four members of the prolyl oligopeptidase family of serine proteases expressed in a variety of cells and tissues, including erythrocytes, liver and brain, but its precise biological activity is unknown. Here, we describe the identification of APH as an Abeta-degrading enzyme, and we show that the degradation of Abeta by APH isolated from transfected cells is inhibited by APH-specific inhibitors, as well as by synthetic Abeta peptide. In addition, we cloned APH from human brain and from neuroblastoma cells. Most importantly, our results indicate that APH expression in AD brain is lower than in age-matched controls.

Bovine chromaffin cell cultures as model to study organophosporus neurotoxicity.

Based on the high level of phenyl valerate esterase activities, and in particular of neuropathy target esterase (NTE) found in bovine adrenal medulla, chromaffin cells culture have been proposed as an alternative model for the study of organophosphorus neurotoxicity. Organophosphorus-induced polyneuropathy is a syndrome related to the inhibition and further modification by organophosphorus compounds of NTE (a protein that displays phenyl valerate esterase activity resistant to mipafox and sensitive to paraoxon). Total phenyl valerate esterase activities found in homogenate, particulate and soluble fractions of bovine adrenal medulla were 5200+/-35, 5000+/-280 and 1700+/-260 mU/g tissue, respectively. Cultured chromaffin cells displayed a total hydrolysing activity of 41+/-5 mU/10(6) cells. Homogenates of bovine adrenal medulla displayed only about 6% of activity sensitive to paraoxon. Most of the phenyl valerate esterase activity inhibited by mipafox (a neuropathy inducing compound) was found in particulate fraction. Cultured chromaffin cells displayed kinetics of inhibition by mipafox similar to the kinetics displayed by homogenates of bovine adrenal medulla. We conclude that NTE could be assayed in this system by only using one inhibitor (mipafox) instead of two (paraoxon and mipafox). Also, the proposal is supported of using chromaffin cells as in vitro model for the study of the role of NTE and related esterases in organophosphorus-induced polyneuropathy.

The inhibition of the high sensitive peripheral nerve soluble esterases by mipafox. A new mathematical processing for the kinetics of inhibition of esterases by organophosphorus compounds.

In the study of organophosphorus (OP) sensitive enzymes, careful discrimination of specific components within a complex multienzymatic mixture is needed. However, standard kinetic analysis gives inconsistent results (i.e., apparently different kinetic constants at different inhibitor concentration) with complex multienzymatic mixtures. A strategy is now presented to obtain consistent kinetic parameters. In the peripheral nerve, soluble carboxylesterases measured with the substrate phenylvalerate (PV) are found with extremely high sensitivity to some inhibitors. Tissue preparations were preincubated with mipafox at nanomolar concentrations (up to 100 nM) for different inhibition times (up to 180 min). Inhibition data were analyzed with model equations of one or two sensitive (exponential) components, with or without resistant components. The most complex model was %act=A1e-k1It+A2e-k2It+AR (step 1). From the curve with the highest mipafox concentration (100 nM), the amplitude for the resistant component was determined as AR=15.1% (step 2). The model equation with a fixed AR value was again applied (step 3) to deduce the second-order inhibition rate constants (k1=2.6 x 10(6) M-1 min-1 and k2=0.28 x 10(6) M-1 min-1), being conserved consistently throughout all mipafox concentrations. Finally, using fixed values of AR, k1, and k2, the amplitudes for the two exponential (sensitive) components (A1 and A2) were re-estimated (A1=50.2% and A2=34.2%). The operational process was internally validated by the close similarity with values obtained by directly fitting with a three-dimensional model equation (activity versus time and inhibitor concentration) to the same inhibition data. Carboxylesterase fractions separated by preparative chromatography showed kinetic properties consistent with the kinetically discriminated components. As practical conclusion, for routine analysis of esterases in toxicological studies, a simplified procedure using the inhibition with mipafox at 30 nM, 1 microM, and 1 mM for 30 min is suggested to discriminate the main esterase components in soluble fraction preparations.

Assessing the dose-dependency of allometric scaling performance using physiologically based pharmacokinetic modeling.

The performance of allometric scaling of dose as a power of body weight under a variety of extrapolation conditions with respect to species, route, exposure intensity, and mechanism/mode of action, remains untested in many cases. In this paper, animal-human internal dose ratio comparisons have been developed for 12 chemicals (benzene, carbon tetrachloride, chloroform, diisopropylfluorophosphate, ethanol, ethylene oxide, methylene chloride, methylmercury, styrene, tetrachloroethene, trichloroethene, and vinyl chloride). This group of predominantly volatile and lipophilic chemicals was selected on the basis that their kinetics have been well-studied and can be predicted in mice, rats, and humans using physiologically based pharmacokinetic (PBPK) models. PBPK model predictions were compared to the allometric scaling predictions for interspecies extrapolation. Recommendations for the application of the allometric scaling are made with reference to internal dose measure (mode of action) and concentration level. The results of this assessment generally support the use of scaling factors recommended in the published literature, which includes scaling factors of 1.0 for risk assessments in which toxicity is attributed to the parent chemical or stable metabolite, and -0.75 for dose-response assessments in which toxicity is attributed to the formation of a reactive metabolite from an inhaled compound. A scaling factor of 0.75 is recommended for dose-response assessments of orally administered compounds in which toxicity is attributed to the parent chemical or stable metabolite and 1.0 for risk assessments in which toxicity is attributed to the formation of a reactive metabolite from a compound administered by the oral route. A dose-dependency in the results suggests that the scaling factors appropriate at high exposures may differ from those at low exposures, primarily due to the impact of saturable metabolism.

Properties of phenyl valerate esterase activities from chicken serum are comparable with soluble esterases of peripheral nerves in relation with organophosphorus compounds inhibition.

Chicken serum, the usual in vivo animal for testing organophosphorus delayed neuropathy, has long been reported not to contain a homologous activity of the neuronal neuropathy target esterase (NTE) activity when it is assayed according to standard methods as the phenyl valerate esterase (PVase) activity, which is resistant to paraoxon and sensitive to mipafox. However, a PVase activity (1000-1500 nmol/min/ml) can be measured in serum that is extremely sensitive to both paraoxon, a non-neuropathic organophosphorus compound and mipafox, a model neuropathy inducer. The inhibition was time progressive in both cases, suggesting a covalent phosphorilating reaction. The fixed time inhibition curves suggest at least two sensitive components. The IC50 for 30 min, at 37 degrees C are 6 and 51 nM for paraoxon and 4 and 110 nM for mipafox, for every sensitive component. When paraoxon was removed from a serum sample pretreated with the inhibitor, the paraoxon sensitive PVase activity was recovered, in spite of showing a time progressive inhibition suggesting that hydrolytic dephosphorylating reaction recovered at a significant rate. The reactivation of the phosphorylated enzyme could explain that the time progressive inhibitions curves for long time with paraoxon tend to reach a plateau depending on the inhibition concentration. However, with mipafox, the curve approached the same maximal inhibitions at all concentrations as expected for a permanent covalent irreversible phosphorylation, which is coherent with the observations that the activity remained inhibited after removing the inhibitor. Data of serum esterases described in this paper showed similar properties to those previously reported for peripheral nerve soluble phenylvalerate esterase: (1) extremely high sensitivity to paraoxon and mipafox; (2) time progressive kinetic with two sensitive components; (3) recovery of activity after removal of paraoxon; and (4) permanent inhibition with mipafox. These properties of serum esterases are very similar to those of soluble fraction of peripheral nerves. So, serum PVases could be considered as appropriate biomarkers, as a mirror for the neural soluble paraoxon and mipafox sensitive soluble esterases that could be used for biomonitoring purpose.

Percutaneous absorption of topical N,N-diethyl-m-toluamide (DEET): effects of exposure variables and coadministered toxicants.

Exposure to N,N-diethyl-m-toluamide (DEET) commonly occurs in the general population and has been implicated as a contributory factor to the Gulf War Illness. The focus of the present studies was to determine the effect of coexposure factors, potentially encountered in a military environment, that could modulate transdermal flux of topically applied DEET. Factors investigated were vehicle, dose, coexposure to permethrin, low-level sulfur mustard, occlusion, and simultaneous systemic exposure to pyridostigmine bromide and the nerve agent stimulant diisopropylfluorophosphate (DFP). Studies were conducted using the isolated perfused porcine skin flap (IPPSF), with a few mechanistically oriented studies conducted using in vitro porcine skin and silastic membrane diffusion cells. DEET was quantitated using high-performance liquid chromatography. The vehicle-control transdermal DEET flux in the IPPSF was approximately 2 micrograms/cm2/h for both 7.5 and 75% DEET concentrations, a value similar to that reported in humans. DEET absorption was enhanced by coinfusion of pyridostigmine bromide and DFP, by the presence of sulfur mustard, or by dosing under complete occlusion. The greatest increase in baseline flux was fivefold. In vitro diffusion cell studies indicated that silastic membranes had two orders of magnitude greater permeability than porcine skin, and showed vehicle effects on flux that were not detected in the IPPSF. These results suggest that coexposure to a number of chemicals that potentially could be encountered in a military environment may modulate the percutaneous absorption of topically applied DEET beyond that seen for normal vehicles at typically applied concentrations.

Nanocrystalline metal oxides as destructive adsorbents for organophosphorus compounds at ambient temperatures.

Nanocrystals of magnesium oxide react with organophosphorus compounds at room temperature by dissociative chemisorption, which we term "destructive adsorption". This process involves cleavage of P-O and P-F bonds (but not P-C bonds) and immobilization of the resultant molecular fragments. These ultrafine powders have unusual crystalline shapes and possess high surface concentrations of reactive edge/corner and defect sites, and thereby display higher surface reactivity, normalized for surface area, than typical polycrystalline material. This high surface reactivity coupled with high surface area allows their use for effective decontamination of chemical warfare agents and related toxic substances. Herein data is presented for paraoxon, diisopropylfluorophosphate (DFP), and (CH3CH2O)2P(O)CH2-SC6H5 (DEPTMP). Solid-state NMR and IR spectroscopy indicate that all OR and F groups dissociate; this leaves bound -PO4, -F, and -OR groups for paraoxon, DFP, and DEPTMP, respectively. For paraoxon, it was shown that one monolayer reacts. For DEPTMP, the OR groups dissociate, but not the P-CH2SC6H5 group. The nanocrystalline MgO reacts much faster and in higher capacity than typical activated carbon samples, which physisorb but do not destructively adsorb these phosphorous compounds.

Purification, cloning and characterization of a GPI inositol deacylase from Trypanosoma brucei.

Inositol acylation is an obligatory step in glycosylphosphatidylinositol (GPI) biosynthesis whereas mature GPI anchors often lack this modification. The GPI anchors of Trypanosoma brucei variant surface glycoproteins (VSGs) undergo rounds of inositol acylation and deacylation during GPI biosynthesis and the deacylation reactions are inhibited by diisopropylfluorophosphate (DFP). Inositol deacylase was affinity labelled with [3H]DFP and purified. Peptide sequencing was used to clone GPIdeAc, which encodes a protein with significant sequence and hydropathy similarity to mammalian acyloxyacyl hydrolase, an enzyme that removes fatty acids from bacterial lipopolysaccharide. Both contain a signal sequence followed by a saposin domain and a GDSL-lipase domain. GPIdeAc(-/-) trypanosomes were viable in vitro and in animals. Affinity-purified HA-tagged GPIdeAc was shown to have inositol deacylase activity. However, total inositol deacylase activity was only reduced in GPIdeAc(-/-) trypanosomes and the VSG GPI anchor was indistinguishable from wild type. These results suggest that there is redundancy in T.brucei inositol deacylase activity and that there is another enzyme whose sequence is not recognizably related to GPIdeAc.

Mutagenesis of organophosphorus hydrolase to enhance hydrolysis of the nerve agent VX.

Organophosphorus hydrolase (OPH) is capable of hydrolyzing a wide variety of organophosphorus pesticides and chemical warfare agents. However, the hydrolytic activity of OPH against the warfare agent VX is less than 0.1% relative to its activity against parathion and paraoxon. Based on the crystal structure of OPH and the similarities it shares with acetylcholinesterase, eight OPH mutants were constructed with the goal of increasing OPH activity toward VX. The activities of crude extracts from these mutants were measured using VX, demeton-S methyl, diisopropylfluoro-phosphate, ethyl parathion, paraoxon, and EPN as substrates. One mutant (L136Y) displayed a 33% increase in the relative VX hydrolysis rate compared to wild type enzyme. The other seven mutations resulted in 55-76% decreases in the relative rates of VX hydrolysis. There was no apparent relationship between the hydrolysis rates of VX and the rates of the other organophosphorus compounds tested.

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism.

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.

Homology modeling and identification of serine 160 as nucleophile of the active site in a thermostable carboxylesterase from the archaeon Archaeoglobus fulgidus.

The hyperthermophilic Archaeon Archaeoglobus fulgidus has a gene (AF1763) which encodes a thermostable carboxylesterase belonging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. Based on secondary structure predictions and a secondary structure-driven multiple sequence alignment with remote homologous proteins of known three-dimensional structure, we previously hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and esterases and identified Ser160, Asp 255 and His285 as the putative members of the catalytic triad. In this paper we report the building of a 3D model for this enzyme based on the structure of the homologous brefeldin A esterase from Bacillus subtilis whose structure has been recently elucidated. The model reveals the topological organization of the fold corroborating our predictions. As regarding the active-site residues, Ser160, Asp255 and His285 are located close each other at hydrogen bond distances. The catalytic role of Ser160 as the nucleophilic member of the triad is demonstrated by the [(3)H]diisopropylphosphofluoridate (DFP) active-site labeling and sequencing of a radioactive peptide containing the signature sequence GDSAGG.

Organophosphorus acid anhydrolase in slime mold, duckweed and mung bean: a continuing search for a physiological role and a natural substrate.

Recently, and for the first time, a diisopropylphosphorofluoridate (DFP)-hydrolyzing enzyme, i.e. an organophosphorus acid anhydrolase (OPAA), has been reported in a plant-source. Based on this and other suggestive evidence, the ability of three plant sources and a protist to hydrolyze DFP and 1,2,2-trimethylpropyl methylphosphonofluoridate (Soman) were tested, and the effects of Mn2+ and ethylenediamine tetraacetate (EDTA) on this activity. The plants are duckweed (Lemna minor), giant duckweed (Spirodela oligorhiza), and germinated mung bean (Vigna radiata); the protist is a slime mold (Dictyostelium discoidium). The tests are based on a crude classification of OPAAs as 'squid type' (DFP hydrolyzed more rapidly than Soman) and all of the others termed by us, with questionable justification, as 'Mazur type' (Soman hydrolyzed more rapidly than DFP). Of the two duckweeds, Spirodela oligorhiza hydrolyzes Soman but not DFP, and Lemna minor does not hydrolyze either substrate. In contrast to the report of Yu and Sakurai, mung bean does not hydrolyze DFP and hydrolyzes Soman with a 5-fold stimulation by Mn2+ and a marked inhibition by EDTA. The slime mold hydrolyzes Soman more rapidly than DFP (but does hydrolyze DFP) and the hydrolysis is Mn2+ stimulated. The failure of these plant sources to hydrolyze DFP is similar to the behavior of OPAA from Bacillus stearothermophilus.

[The mechanism of the transport of organophosphorus compounds across the histo-hematic barriers]. / K voprosu o mekhanizme transorta fosfororganicheskikh soedinenii cherez gisto-gematicheskie bar'ery.

It was demonstrated in experiments on mice [correction of rats] that the transport of organophosphorus compounds (OPC) through membranes of the histohematic barriers (HHB) of the organism occurs by means of diffusion. The rate of this process depends on the interaction of OPC with the specific sites of binding with the tissues, among which the enzyme carboxylesterase plays an important part. It is suggested that both the rate and direction of OPC diffusion are determined by the relationship between the values of affinity of the ligands for the sites of their specific binding found on both sides of the HHB.

P450 enzyme CYP2B catalyzes the detoxification of diisopropyl fluorophosphate.

Phenobarbital and some other enzyme-inducers are known to reduce organophosphate toxicity. One suggested mechanism is the induction of liver cytochrome P450 enzymes catalyzing monooxygenation reactions. The aim of the present study was to elucidate the cytochrome P450 subfamily, or P450 isoenzyme(s), participating in the detoxification of diisopropyl fluorophosphate (DFP) in the rat. DFP resulted in a type I spectrum in liver microsomes from phenobarbital- or RP 52028-treated rats (binding constants 0.32 and 0.17 microM, respectively) and in a purified P450 preparation enriched with CYP2B. The spectrum was reversible by metyrapone, an inhibitor of the CYP2B enzyme subfamily. The 7-pentoxyresorufin O-dealkylase activity was inhibited by DFP in liver microsomes from phenobarbital- or RP 52028-treated rats and in a reconstituted system containing the purified CYP2B preparation. In microsomes from phenobarbital-pretreated rats, the inhibition was of a mixed type, i.e., competitive-non-competitive (Km = 0.5 microM; Ki = 6 microM). The microsomal fractions of livers from phenobarbital- or RP 52028-treated rats detoxified DFP effectively in vitro, as measured by a decrease in the DFP inhibition of cholinesterase activity. This detoxification was antagonized by metyrapone and by an antibody raised against purified CYP2B preparation. Clotrimazole, an inhibitor of P450 enzymes, inhibited the detoxification of DFP in rat liver in vivo. A genetically-modified hamster cell line expressing CYP2B1 oxidized NADPH in the presence of DFP. No such oxidation was detected in the parent cell line. These studies suggest that CYP2B1 metabolizes DFP and may significantly contribute to the detoxification of this organophosphate in vivo.

Physiologically based pharmacokinetic model for the inhibition of acetylcholinesterase by organophosphate esters.

Organophosphate (OP) exposure can be lethal at high doses while lower doses may impair performance of critical tasks. The ability to predict such effects for realistic exposure scenarios would greatly improve OP risk assessment. To this end, a physiologically based model for diisopropylfluorophosphate (DFP) pharmacokinetics and acetylcholinesterase (AChE) inhibition was developed. DFP tissue/blood partition coefficients, rates of DFP hydrolysis by esterases, and DFP-esterase bimolecular inhibition rate constants were determined in rat tissue homogenates. Other model parameters were scaled for rats and mice using standard allometric relationships. These DFP-specific parameter values were used with the model to simulate pharmacokinetic data from mice and rats. Literature data were used for model validation. DFP concentrations in mouse plasma and brain, as well as AChE inhibition and AChE resynthesis data, were successfully simulated for a single iv injection. Effects of repeated, subcutaneous DFP dosing on AChE activity in rat plasma and brain were also well simulated except for an apparent decrease in basal AChE activity in the brain which persisted 35 days after the last dose. The psychologically based pharmacokinetic (PBPK) model parameter values specific for DFP in humans, for example, tissue/blood partition coefficients, enzymatic and nonenzymatic DFP hydrolysis rates, and bimolecular inhibition rate constants for target enzymes were scaled from rodent data or obtained from the literature. Good agreement was obtained between model predictions and human exposure data on the inhibition of red blood cell AChE and plasma butyrylcholinesterase after an intramuscular injection of 33 micrograms/kg DFP and at 24 hr after acute doses of DFP (10-54 micrograms/kg), as well as for repeated DFP exposures.(ABSTRACT TRUNCATED AT 250 WORDS)

Concentration-flux relations for a multicellular biological membrane with metabolism.

A mathematical model is described for the simultaneous diffusion and metabolism of a chemical penetrating a multicellular biological membrane such as skin. Metabolism is assumed to follow saturable Michaelis-Menten kinetics, which leads to nonlinear relationships between the applied concentration and the metabolic and diffusive fluxes through the membrane. Approximate concentration-flux relations are derived under limiting conditions, and a computational method is described for the general case. The major barrier to dermal penetration of very lipophilic molecules is thought to be the viable tissues (viable epidermis and some of the dermis) underlying the stratum corneum, and some molecules are known to be metabolized by enzymes within these tissues. It is proposed to use the model to describe penetration and metabolism of such lipophilic molecules within the viable tissues of the skin.

Physiologically based pharmacokinetic and pharmacodynamic model for the inhibition of acetylcholinesterase by diisopropylfluorophosphate.

Organophosphate (OP) exposure can be lethal at high doses while lower doses may impair performance of critical tasks. The ability to predict such effects for realistic exposure scenarios would expedite OP risk assessment. To this end, a physiologically based model for diisopropylfluorophosphate (DFP) pharmacokinetics and acetylcholinesterase (AChE) inhibition was developed in mammals. DFP tissue:blood partition coefficients, rates of DFP hydrolysis by esterases, and DFP-esterase bimolecular inhibition rate constants were determined in rat tissue homogenates. Other model parameters were scaled for rats and mice using standard allometric relationships. These DFP-specific parameter values were used with the model to simulate expected in vivo pharmacokinetic data from mice and rats. Literature data were used for model validation. DFP concentrations in mouse plasma and brain were successfully simulated after a single iv injection (B.R. Martin, 1985, Toxicol. Appl. Pharmacol. 77, 275-284). AChE inhibition and AChE resynthesis data from this study were also simulated. Effects of repeated, subcutaneous DFP dosing on AChE activity in rat plasma and brain (H. Michalek, A. Meneguz, and G.M. Bisso, 1982, Arch. Toxicol., Suppl. 5, 116-119; M.E. Traina and L.A. Serpietri, 1984, Biochem. Pharmacol. 33, 645-653) were also simulated well, but the return of brain AChE activity to basal levels after cessation of repeated dosing was not as well described. The initial model structure returned brain AChE activity to the original level, while in the laboratory studies brain AChE never returned to basal levels, even at 35 days after the last dose. These data suggest modulation of AChE synthesis with prolonged DFP exposure. This study demonstrated the possibility of using a model based on mammalian physiology and biochemistry to simulate in vivo data on DFP pharmacokinetics and AChE inhibition. Scaling of the model between rats and mice was also successful. The approach holds promise for predictive simulation of organophosphate-mediated AChE inhibition in humans.

Distribution and fate of diethyl malonate and diisopropyl fluorophosphate on pig skin in vitro.

The in vitro distribution and fate of [14C]diethyl malonate and [14C]diisopropyl fluorophosphate were evaluated on normal and heat-treated pig skin. The extent of hydrolysis from the skin surface, skin, and receptor fluid was determined. A significant skin-mediated hydrolysis (15-35% of applied dose) was observed for diethyl malonate in normal skin, but not in heat-treated skin. These results indicated that a heat labile process (e.g., enzymatic hydrolysis) was in part responsible for the degradation of diethyl malonate after topical application to normal skin. Heat treatment tripled the skin penetration of diisopropyl fluorophosphate and reduced the amount of recovered hydrolysis product, diisopropyl phosphoric acid. Enzymatic and spontaneous hydrolysis, as well as impurity, accounted for the presence of degradation product.