Continuous biodegradation of parathion by immobilized Sphingomonas sp. in magnetically fixed-bed bioreactors and evaluation of the enzyme stability of immobilized bacteria.

Magnetically-modified Sphingomonas sp. was prepared using covalent binding of magnetic nanoparticles on to the cell surface. The magnetic modified bacteria were immobilized in the fixed-bed bioreactors (FBR) by internal and external magnetic fields for the biodetoxification of a model organophosphate, parathion: 93 % of substrate (50 mg parathion/l) was hydrolyzed at 0.5 ml/min in internal magnetic field fixed-bed bioreactor. The deactivation rate constants (at 1 ml/min) were 0.97 × 10(-3), 1.24 × 10(-3) and 4.17 × 10(-3) h(-1) for immobilized bacteria in external and internal magnetic field fixed-bed bioreactor and FBR, respectively. The deactivation rate constant for immobilized magnetically modified bacteria in external magnetic field fixed-bed bioreactor (EMFFBR) was 77 % lower than that of immobilized cells by entrapping method on porous basalt beads in FBR at 1 ml/min. Immobilized magnetic modified bacteria exhibited maximum enzyme stability in EMFFBR.

Partitioning and diffusion of parathion in human dermis.

A previously developed method employing the use of a dialysis membrane in series with human dermis tissue mounted in side-by-side diffusion cells was utilized to observe the effects of the presence of soluble proteins in the donor compartment on the measured transport parameters of parathion. In the presence of the dialysis membrane the partition coefficient was significantly lower and the diffusion coefficient significantly higher than those determined in its absence; however, the difference was less than that previously determined for the more highly protein bound compound, diclofenac. The result suggests the dialysis membrane method is important for studying permeants that are more than about 87% bound to soluble proteins in the dermis. The results are discussed in the context of a predictive model for partitioning and transport of low molecular weight solutes in human dermis.

Deadly drop--cholinergic syndrome from wearing a laundered uniform.

Unintentional organophosphate compound poisoning, although known, contamination of organophosphate compound through laundered uniform and subsequent transcutaneous absorption, in 30 children is reported herewith for its rarity. Emergency physicians have to recognize such entities clinically, confirm by laboratory means wherever possible, and intervene with appropriate measures.

Cytochrome P450-specific human PBPK/PD models for the organophosphorus pesticides: chlorpyrifos and parathion.

Organophosphorus pesticides (OPs) remain a potential concern to human health because of their continuing use worldwide. Phosphororthioate OPs like chlorpyrifos and parathion are directly activated and detoxified by various cytochrome P450s (CYPs), with the primary CYPs involved being CYP2B6 and CYP2C19. The goal of the current study was to convert a previously reported human pharmacokinetic and pharmacodynamic (PBPK/PD) model for chlorpyrifos, that used chlorpyrifos metabolism parameters from rat liver, into a human CYP based/age-specific model using recombinant human CYP kinetic parameters (V(max), K(m)), hepatic CYP content and plasma binding measurements to estimate new values for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibition and to use the model as a template for the development of a comparable parathion PBPK/PD model. The human CYP based/age-specific PBPK/PD models were used to simulate single oral exposures of adults (19 year old) and infants (1 year) to chlorpyrifos (10,000, 1000 and 100 µg/kg) or parathion (100, 25 and 5 µg/kg). Model simulations showed that there is an age dependency in the amount of blood cholinesterase inhibition observed, however additional age-dependent data are needed to further optimize age-specific human PBPK/PD modeling for these OP compounds. PBPK/PD model simulations estimated that a 4-fold increase or decrease in relative CYP2B6 and CYP2C19 content would produce a 9-22% inhibition in blood AChE activity following exposure of an adult to chlorpyrifos (1000 µg/kg). Similar model simulation produced an 18-22% inhibition in blood AChE activity following exposure of an adult to parathion (25 µg/kg). Individuals with greater CYP2B6 content and lower CYP2C19 content were predicted to be most sensitive to both OPs. Changes in hepatic CYP2B6 and CYP2C19 content had more of an influence on cholinesterase inhibition for exposures to chlorpyrifos than parathion, which agrees with previously reported literature that these CYPs are more reaction biased for desulfurization (activation) and dearylation (detoxification) of chlorpyrifos compared to parathion. The data presented here illustrate how PBPK/PD models with human enzyme-specific parameters can assist ongoing risk assessment efforts and aid in the identification of sensitive individuals and populations.

Pharmacokinetics of OpdA, an organophosphorus hydrolase, in the African green monkey.

Organophosphorus (OP) pesticides are a broad class of acetylcholinesterase inhibitors that are responsible for tremendous morbidity and mortality worldwide, contributing to an estimated 300,000 deaths annually. Current pharmacotherapy for acute OP poisoning includes the use of atropine, an oxime, and benzodiazepines. However, even with such therapy, the mortality from these agents are as high as 40%. Enzymatic hydrolysis of OPs is an attractive new potential therapy for acute OP poisoning. A number of bacterial OP hydrolases have been isolated. A promising OP hydrolase is an enzyme isolated from Agrobacterium radiobacter, named OpdA. OpdA has been shown to decrease lethality in rodent models of parathion and dichlorvos poisoning. However, pharmacokinetic data have not been obtained. In this study, we examined the pharmacokinetics of OpdA in an African Green Monkey model. At a dose of 1.2mg/kg the half-life of OpdA was approximately 40 min, with a mean residence time of 57 min. As expected, the half-life did not change with the dose of OpdA given: at doses of 0.15 and 0.45 mg/kg, the half-life of OpdA was 43.1 and 38.9 min, respectively. In animals subjected to 5 daily doses of OpdA, the residual activity that was measured 24h after each OpdA dose increased 5-fold for the 0.45 mg/kg dose and 11-fold for the 1.2mg/kg dose. OpdA exhibits pharmacokinetics favorable for the further development as a therapy for acute OP poisoning, particularly for hydrophilic OP pesticides. Future work to increase the half-life of OpdA may be beneficial.

Toward a better understanding of pesticide dermal absorption: diffusion model analysis of parathion absorption in vitro and in vivo.

Human skin absorption of radiolabeled parathion was studied in vitro at specific doses (mass loadings) of 0.4, 4.0, 41, or 117 microg/cm(2), with and without occlusion. The compound was applied in small volumes of acetone solution to split-thickness skin. Permeation of radiolabel into the receptor solutions was monitored for 76 h, after which the tissue was dissected and analyzed for residual radioactivity. For the 3 lower doses, cumulative permeation after 76 h was approximately dose-proportional, ranging from 28.5-30.5% of applied dose (unoccluded) to 45.5-55.7% (occluded). Total absorption, calculated as receptor fluid plus dermis content, followed a similar pattern. Both permeation rate and total absorption continued to increase up to the highest dose tested, consistent with results from other laboratories. These results are compared with predictions from a previously developed skin diffusion model (Kasting et al., 2008a). The model predicted total absorption to within a factor of 1.4 at 0.4 microg/cm(2) and 1.6 at 4 microg/cm(2), but substantially underpredicted absorption at the 2 higher doses. The analysis showed that parathion partitioned more favorably into the stratum corneum than the diffusion model prediction. Nevertheless, comparison of the model predictions to a previously reported human study showed that the skin absorption model, when corrected for surface losses occurring in vivo, satisfactorily described in vivo dermal absorption of parathion applied at 4 microg/cm(2) to various body sites.

Percutaneous penetration and absorption of parathion using human and pig skin models in vitro and human skin grafted onto nude mouse skin model in vivo.

This study determined and compared the percutaneous penetration and absorption of an organophosphorus (OP) pesticide, parathion (PA), using three experimental skin models: namely the human abdominal- and pig-ear skin in vitro models and the Human Skin grafted onto a nude mouse (HuSki) in vivo model. The percentage of topically applied dose absorbed and the doses present in the stratum corneum and skin were systematically determined at 24 h under similar experimental conditions. The three experimental skin models were first compared. Then, the advantages of the HuSki model for in vivo PA skin absorption studies were evaluated compared with the pig in vivo model previously used by others. Lastly, the relevance of each skin model to predict the permeability of human skin to PA in vivo was assessed by comparing our results with previously published in vivo human volunteer values. It was demonstrated that (a) pig-ear skin is relevant for predicting the in vitro human abdominal skin absorption taking into account a 2-3 times higher skin permeability to PA, (b) using ethanol as the vehicle, the absorption of PA was 4-5 times higher in the HuSki model than in the pig model but supports the usefulness of the HuSki model to easy mass balance studies, (c) both human in vitro and HuSki models closely predict the in vivo human volunteer absorption at 24 h when acetone is used as a vehicle but the HuSki model overcomes the known limitations of in vitro models for studying the fate of PA in the different skin layers after topical application.

In vitro dermal absorption of methyl salicylate, ethyl parathion, and malathion: first responder safety.

In vitro tests with fresh dermatomed (0.3 to 0.4 mm thick) female breast skin and one leg skin specimen were conducted in Bronaugh flow-through Teflon diffusion cells with three chemicals used to simulate chemical warfare agents: 14C-radiolabeled methyl salicylate (MES), ethyl parathion (PT), and malathion (MT), at three dose levels (2, 20, and 200 mM). Tests were conducted at a skin temperature of 29 degrees C using a brief 30-min exposure to the chemical and a 6.5-h receivor collection period. Rapid absorption of all three chemicals was observed, with MES absorbed about 10-fold faster than PT and MT. For MES, PT, and MT, respectively, there was 32%, 7%, and 12% absorption into the receivor solution (Hank's HEPES buffered saline with 4% bovine serum albumin [BSA], pH 7.4) at the low dose (2 mM), 17%, 2%, and 3% at the medium dose (20 mM), and 11%, 1%, and 1% at the high dose (200 mM) levels. Including the skin depot for MES, PT, and MT, respectively, there was 40%, 41%, and 21% (low dose), 26%, 16%, and 8% (medium dose), and 13%, 19%, and 10% (high does) absorption. Efficacy of skin soap washing conducted at the 30 min exposure time ranged from 31% to 86%, varying by chemical and dose level. Skin depot levels were highest for the relatively lipophilic PT. "Pseudo" skin permeability coefficient (K(p)) data declined with dose level, suggesting skin saturation had occurred. An in-depth comparison with literature data was conducted and risk assessment of first responder exposure was briefly considered.

Organophosphate poisonings with parathion and dimethoate.

OBJECTIVE: Organophosphate toxicity is the leading cause of morbidity and death in poisoning by insecticides. The clinical symptoms of pesticide toxicity range from the classic cholinergic syndrome to flaccid paralysis and intractable seizures. The mainstays of therapy are atropine, oximes, benzodiazepines and supportive care. The toxicokinetics vary not only with the extent of exposure, but also with the chemical structure of the agent. PATIENTS: We report two cases of poisoning with parathion-ethyl and dimethoate. The patients developed a cholinergic syndrome immediately, accompanied by bradycardia and hypotension. INTERVENTIONS: The patients were admitted to the intensive care unit (ICU) a few hours after ingestion. Atropine was administered according to the cholinergic symptoms. The patients recovered in the ICU after 10-12 days and were discharged after 3 and 4 weeks. MEASUREMENTS AND RESULTS: Organophosphate blood and urine levels were determined on admission and during hospitalisation. The pesticides were rapidly distributed and slow elimination rate of the poisons was documented. In the case of parathion-ethyl the distribution half-life estimated was t(1/2alpha) = 3.1h while the terminal half-life was t(1/2beta) = 17.9 h. Using a one-compartment model for dimethoate the elimination half-life was t(1/2beta) = 30.4 h in plasma and 23.8 h in urine. The serum pseudo-cholinesterase activity was below the limit of detection at admission and recovered during the following 3weeks.

A model for linking the effects of parathion in soil to its degradation and bioavailability kinetics.

Parathion is an insecticide of a group of highly toxic organophosphorus compounds. To investigate the dissipation and toxicological impact of parathion [O,O-diethyl O-(4-nitrophenyl) phosphorothioate] and its highly toxic metabolite, paraoxon, soil laboratory experiments were conducted in columns during a 19-d experiment under variably saturated conditions. Water and pesticide transport, sorption, and biodegradation of parathion were measured in three soil pools (soluble phase, weakly and strongly sorbed phases) using C-labeled pesticide. The effects of parathion and its metabolite on the mobility of soil nematodes were observed and then modeled with an effective variable, which combined pesticide concentration and time of application. Results showed that parathion was highly sorbed and slowly degraded to a mixture of metabolites. The parent compound and its metabolites remained located in the top 0.06-m soil layer. A kinetic model describing the sorption, biodegradation, and allocation into different soil pools of parathion and its metabolites was coupled with heat and water transport equations to predict the fate of parathion in soil. Simulated results were in agreement with experimental data, showing that the products remained in the upper soil layers even in the case of long-term (11-mo) simulation. The strongly sorbed fraction may be regarded as a pesticide reservoir that regularly provides pesticide to the weakly sorbed phase, and then, liquid phase, respectively. From both modeling and observations, no major toxicological damage of parathion and paraoxon to soil nematodes was found, although some effects on nematodes were possible, but at the soil surface only (0.01- and 0.02-m depth).

A physiologically based pharmacokinetic model of organophosphate dermal absorption.

The rate and extent of dermal absorption are important in the analysis of risk from dermal exposure to toxic chemicals and for the development of topically applied drugs, barriers, insect repellents, and cosmetics. In vitro flow-through cells offer a convenient method for the study of dermal absorption that is relevant to the initial processes of dermal absorption. This study describes a physiologically based pharmacokinetic (PBPK) model developed to simulate the absorption of organophosphate pesticides, such as parathion, fenthion, and methyl parathion through porcine skin with flow-through cells. Parameters related to the structure of the stratum corneum and solvent evaporation rates were independently estimated. Three parameters were optimized based on experimental dermal absorption data, including solvent evaporation rate, diffusivity, and a mass transfer factor. Diffusion cell studies were conducted to validate the model under a variety of conditions, including different dose ranges (6.3-106.9 microg/cm2 for parathion; 0.8-23.6 microg/cm2 for fenthion; 1.6-39.3 microg/cm2 for methyl parathion), different solvents (ethanol, 2-propanol and acetone), different solvent volumes (5-120 microl for ethanol; 20-80 microl for 2-propanol and acetone), occlusion versus open to atmosphere dosing, and corneocyte removal by tape-stripping. The study demonstrated the utility of PBPK models for studying dermal absorption, which can be useful as explanatory and predictive tools that may be used for in silico hypotheses generation and limited hypotheses testing. The similarity between the overall shapes of the experimental and model-predicted flux/time curves and the successful simulation of altered system conditions for this series of small, lipophilic compounds indicated that the absorption processes that were described in the model successfully simulated important aspects of dermal absorption in flow-through cells. These data have direct relevance to topical organophosphate pesticide risk assessments.

Role of metabolism in parathion-induced hepatotoxicity and immunotoxicity.

The objective of this study was to investigate whether metabolic activation of parathion by cytochrome P-450s (CYPs) was responsible for pesticide-induced hepatotoxicity and immunotoxicity. Initially, to investigate parathion metabolism in vitro, the production of paraoxon and p-nitrophenol, major metabolites of parathion, was determined by high-performance liquid chromatography (HPLC). Subsequently, metabolic fate and CYP enzymes involved in the metabolism of parathion were partially monitored in rat liver microsomes in the presence of the NADPH-generating system. Among others, phenobarbital (PB)-induced microsomes produced the metabolites paraoxon and p-nitrophenol to the greatest extent, indicating the involvement of CYP 2B in parathion metabolism. When female BALB/c mice were treated orally with 1, 4, or 16 mg/kg of parathion in corn oil once, parathion suppressed the antibody response to sheep red blood cells. To further investigate a possible role of metabolic activation by CYP enzymes in parathion-induced toxicity, female BALB/c mice were pretreated intraperitoneally with 40 mg/kg PB for 3 d, followed by a single oral treatment with 16 mg/kg parathion. PB pretreatment produced a decrease in hepatic glutathione content and increases in hepatotoxic paramenters in parathion-treated mice with no changes in the antibody response. In addition, greater p-nitrophenol amounts were produced when mice were pretreated with PB, compared to treatment with parathion alone. These results indicate that parathion-induced hepatotoxicity might be differentiated from immunotoxicity in mice.

Physicochemical and biological data for the development of predictive organophosphorus pesticide QSARs and PBPK/PD models for human risk assessment.

A search of the scientific literature was carried out for physiochemical and biological data [i.e., IC50, LD50, Kp (cm/h) for percutaneous absorption, skin/water and tissue/blood partition coefficients, inhibition ki values, and metabolic parameters such as Vmax and Km] on 31 organophosphorus pesticides (OPs) to support the development of predictive quantitative structure-activity relationship (QSAR) and physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) models for human risk assessment. Except for work on parathion, chlorpyrifos, and isofenphos, very few modeling data were found on the 31 OPs of interest. The available percutaneous absorption, partition coefficients and metabolic parameters were insufficient in number to develop predictive QSAR models. Metabolic kinetic parameters (Vmax, Km) varied according to enzyme source and the manner in which the enzymes were characterized. The metabolic activity of microsomes should be based on the kinetic activity of purified or cDNA-expressed cytochrome P450s (CYPs) and the specific content of each active CYP in tissue microsomes. Similar requirements are needed to assess the activity of tissue A- and B-esterases metabolizing OPs. A limited amount of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterase (CaE) inhibition and recovery data were found in the literature on the 31 OPs. A program is needed to require the development of physicochemical and biological data to support risk assessment methodologies involving QSAR and PBPK/PD models.

Human parathion poisoning. A toxicokinetic analysis.

The mortality rate of suicidal parathion poisoning is particularly high, the onset of fulminant cholinergic signs, and the patients frequently present to the emergency physician with life-threatening symptoms. Despite this uniformity, subsequent clinical course differs significantly among patients, mostly not as a result of different delays in treatment or insufficiency of primary care. Probably, the differences depend on the amount of poison absorbed and/or the disposition of the active poison, paraoxon. We followed the toxicokinetics of parathion and tried to quantify the actual poison load. To this end, we monitored parathion-intoxicated patients (patients requiring artificial ventilation) for plasma levels of parathion and paraoxon along with the activity of erythrocyte acetylcholinesterase and its reactivatability. Plasma obidoxime concentrations were followed as well as the cumulative urinary para-nitrophenol conjugate excretion as a measure of total poison load. All patients received a standard obidoxime scheme of a 250 mg bolus dose intravenously, followed by continuous infusion with 750 mg per 24 hours as long as reactivation could be expected (usually 1 week). All other treatment was instituted as judged by the physician. It was recommended to use atropine at low doses to achieve dry mucous membranes, no bronchoconstriction and no bradycardia. Usually 1-2 mg/h were sufficient. Seven selected cases are presented exemplifying toxicokinetic peculiarities. All patients were severely intoxicated, while the amount of parathion absorbed varied widely (between 0.12 and 4.4 g; lethal dose 0.02-0.1 g) and was generally much lower than anticipated from the reports of relatives. It remains open whether the discrepancies between reports and findings were due to exaggeration or to effective decontamination (including spontaneous vomiting, gastric lavage and activated charcoal). Absorption of parathion from the gastrointestinal tract was sometimes retarded, up to 5 days, resulting in fluctuating plasma profiles. The volume of distribution at steady-state (Vdss) of parathion was around 20 L/kg. Post-mortem analysis in one patient revealed a 66-fold higher parathion concentration in fat tissue compared with plasma, 16 days after ingestion. Biotransformation of parathion varied widely and was severely retarded in one patient receiving fluconazole during worsening of renal function, while phenobarbital (phenobarbitone) sedation (two cases) had apparently no effect. The proportion of plasma parathion to paraoxon varied from 0.3-30, pointing also to varying paraoxon elimination, as illustrated by one case with particularly low paraoxonase-1 activity. Obidoxime was effective at paraoxon concentrations below 0.5 microM, provided aging was not too advanced. This concentration correlated poorly with the paration concentration or the poison load. The data are discussed in light of the pertinent literature.

An approach for the quantitative consideration of genetic polymorphism data in chemical risk assessment: examples with warfarin and parathion.

In recent years, a great deal of research has been conducted to identify genetic polymorphisms. One focus has been to characterize variability in metabolic enzyme systems that could impact internal doses of pharmaceuticals or environmental pollutants. Methods are needed for using this metabolic information to estimate the resulting variability in tissue doses associated with chemical exposure. We demonstrate here the use of physiologically based pharmacokinetic (PBPK) modeling in combination with Monte Carlo analysis to incorporate information on polymorphisms into the analysis of toxicokinetic variability. Warfarin and parathion were used as case studies to demonstrate this approach. Our results suggest that polymorphisms in the PON1 gene, that give rise to allelic variants of paraoxonase, which is involved in the metabolism of paraoxon (a metabolite of parathion), make only a minor contribution to the overall variability in paraoxon tissue dose, while polymorphisms in the CYP2C9 gene, which gives rise to allelic variants of the major metabolic enzyme for warfarin, account for a significant portion of the overall variability in (S)-warfarin tissue dose. These analyses were used to estimate chemical-specific adjustment factors (CSAFs) for the human variability in toxicokinetics for both parathion and warfarin. Implications of alternatives in the calculation of CSAFs are explored. Key decision points for applying the PBPK-Monte Carlo approach to evaluate toxicokinetic variability for other chemicals are also discussed.

Continuous system modeling of equilibrium dialysis for determinations of tissue partitioning of parathion and paraoxon.

A tissue/blood partition coefficient, defined as the ratio of tissue chemical concentration to that of the venous outflow of the tissue when at equilibrium, is an important parameter required for physiological based pharmacokinetic models. While many techniques have been developed to quantify tissue/blood partition coefficients for various chemicals, there is no single best approach for their determination. In the current study, equilibrium dialysis of the organophosphorus insecticide parathion and its active metabolite paraoxon was undertaken to assess their partitioning into rat liver. A mass balance analysis of the contents of the dialysis cells suggested that significant levels of parathion and paraoxon were bound to the dialysis membranes. There was no evidence of metabolism of either parathion or paraoxon by the very dilute liver homogenate utilized in the dialysis. In order to investigate the potential impact of binding of a chemical to dialysis membrane during determination of partition coefficients, a computer model of a dialysis system was constructed. The model assumed that all processes occurring within the dialysis cell were first or second order in nature, and that binding to the dialysis membrane occurred symmetrically on both sides of the membrane. Variations in the total number of simulated binding sites on dialysis membrane revealed that increasing the degree of membrane binding resulted in decreased compound on the homogenate and buffer sides of the dialysis cells. However, the final tissue/buffer partition coefficient was unaffected by these alterations in membrane binding, although increased membrane binding prolonged the incubation time required to achieve equilibrium. These simulations suggest that loss of a compound to membrane binding does not preclude the use of equilibrium dialysis for determination of tissue/buffer, and therefore tissue/blood, partition coefficients, provided the dialysis system is allowed to proceed to equilibrium.

Genetic polymorphisms in assessing interindividual variability in delivered dose.

Increasing sophistication in methods used to account for human variability in susceptibility to toxicants has been one of the success stories in the continuing evolution of risk assessment science. Genetic polymorphisms have been suggested as an important contributor to overall human variability. Recently, data on polymorphisms in metabolic enzymes have been integrated with physiologically based pharmacokinetic (PBPK) modeling as an approach to determining the resulting overall variability. We present an analysis of the potential contribution of polymorphisms in enzymes modulating the disposition of four diverse compounds: methylene chloride, warfarin, parathion, and dichloroacetic acid. Through these case studies, we identify key uncertainties likely to be encountered in the use of polymorphism data and highlight potential simplifying assumptions that might be required to test the hypothesis that genetic factors are a substantive source of human variability in susceptibility to environmental toxicants. These uncertainties include (1) the relative contribution of multiple enzyme systems, (2) the extent of induction/inhibition through coexposure, (3) allelic frequencies of major ethnic groups, (4) the absence of chemical-specific data on the kinetic parameters for the different allelic forms of key enzymes, (5) large numbers of low-frequency alleles, and (6) uncertainty regarding differences between in vitro and in vivo kinetic data. Our effort sets the stage for the acquisition of critical data and further integration of polymorphism data with PBPK modeling as a means to quantitate population variability.

Protein binding of isofluorophate in vivo after coexposure to multiple chemicals.

Full toxicologic profiles of chemical mixtures, including dose-response extrapolations to realistic exposures, is a prohibitive analytical problem, even for a restricted class of chemicals. We present an approach to probing in vivo interactions of pesticide mixtures at relevant low doses using a monitor compound to report the response of biochemical pathways shared by mixture components. We use accelerator mass spectrometry (AMS) to quantify [14C]-diisopropylfluorophosphate as a tracer at attomole levels with 1-5% precision after coexposures to parathion (PTN), permethrin (PER), and pyridostigmine bromide separately and in conjunction. Pyridostigmine shows an overall protective effect against tracer binding in plasma, red blood cells, muscle, and brain that is not explained as competitive protein binding. PTN and PER induce a significant 25-30% increase in the amount of tracer reaching the brain with or without pyridostigmine. The sensitivity of AMS for isotope-labeled tracer compounds can be used to probe the physiologic responses of specific biochemical pathways to multiple compound exposures.

Glucose feeding exacerbates parathion-induced neurotoxicity.

Excessive dietary intake of sugars could alter various biotransformation processes and the pharmacological and toxicological properties of numerous xenobiotics. In the present study, the effects of glucose supplementation were examined on the neurotoxicity of the organophosphorus (OP) pesticide parathion (PS) and its active metabolite, paraoxon (PO), a potent inhibitor of acetylcholinesterase (AChE). Rats (n = 6-12/treatment group) were given free access to tap water or 15% glucose (w/v) in tap water beginning 7 d prior to OP toxicant exposure. Food, caloric intake, and body weight were measured daily. Animals were challenged with either PS (4.5, 9, or 18 mg/kg, sc) or PO (0.3 0.5, or 0.7 mg/kg, sc) and clinical signs of neurotoxicity (i.e., autonomic dysfunction, involuntary movements) were recorded daily for the following 13 d. Glucose feeding was associated with a dramatic drop (approximately 50%) in feed intake and an increase (approximately 20% in total caloric consumption over the 7 d prior to OP exposure. Functional toxicity associated with PS exposure was increased in glucose-fed (GF) rats, but the glucose diet had no apparent effect on clinical signs of toxicity following PO treatment. Glucose feeding increased the magnitude of AChE inhibition in the frontal cortex and plasma at lower dosages (i.e., 4.5 and 9 mg/kg) 3 d following PS treatment. Time-course studies (3, 7, and 11 d after PS exposure, 18 mg/kg, sc) indicated significantly greater brain and plasma AChE inhibition in glucose-fed animals at later time points. In contrast, glucose feeding had no effect on the degree of AChE inhibition following PO exposure. Neither liver microsomal oxidative desulfuration of PS, nor liver or plasma paraoxonase, nor liver or plasma carboxylesterase activities were measurably affected by glucose feeding. Downregulation of muscarinic receptors 7 d after PS exposure (18 mg/kg, sc) was more extensive in GF rats. It is postulated that excessiveglucose consumption decreases the intake of other dietary components, in particular amino acids, limiting the de novo synthesis of AChE and consequent recovery of synaptic transmission. Due to the shorter duration of inhibition following PO exposure, sponta neous reactivation of AChE may be more important than de novo protein synthesis in recovery of function, and thus with the effects of glucose feeding on its toxicity. Individuals that derive a large proportion of their calories from sugars may be at higher risk of acute toxicity from organophosphorus pesticides such as PS.

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.