Efectos in vivo e in vitro del insecticida organofosforado diazinón en la actividad colinesterasa del sistema nervioso de conejos (Orictolagus cuniculus) / In vivo and in vitro effects of the organophosphorus insecticide diazinon on the cholinesterase activity of the rabbit (Orictolagus cuniculus) nervous system

En el presente estudio se investigó la sensibilidad de la actividad colinesterasa del sistema nervioso del conejo (Orictolagus cuniculus) al insecticida diazinón tanto in vivo como in vitro. En el ensayo in vivo, los animales fueron expuestos oralmente a dosis únicas de diazinón (25 o 125 mg/kg) y tras 10 días fueron sacrificados para obtener el cerebro, el cerebelo y la médula espinal. En ellos se determinó la actividad colinesterasa con el fin de conocer sus valores basales en conejo (grupo control) al igual que el potencial de inhibición in vivo originado por exposición a este pesticida (grupos expuestos). Los valores basales de actividad colinesterasa, usando la acetiltiocolina como sustrato, fueron 258.5 ± 38.0, 242.4 ± 32.0 y 235.1 ± 27.2 nmol/min.mg proteínas para el cerebro, el cerebelo y la médula espinal, respectivamente. En los grupos expuestos a diazinón no se observó inhibición de la actividad colinesterasa en comparación con la actividad del grupo control. En el ensayo in vitro se expusieron homogeneizados de los tres tejidos nerviosos de conejos control a distintas concentraciones de diazinón (0.025, 0.05, 0.1, 0.2 y 0.4 mg/l). La actividad colinesterasa permaneció constantemente deprimida en relación concentración y tiempo dependiente a lo largo del periodo experimental (30 minutos, 9, 24 y 48 horas) en todos los tejidos evaluados. La Concentración Inhibitoria Media (CI50) de la actividad AChE para el diazinón se estimó en 0.154, 0.138 y 0.159 mg/l para el cerebro, el cerebelo y la médula espinal, respectivamente. Se concluye la necesidad de establecimiento de una actividad basal o de referencia en cada laboratorio cuando se pretenda utilizar esta actividad con fines diagnósticos de exposición a plaguicidas anticolinesterásicos (AU)

In the current study, the in vivo and in vitro sensitivity of nervous system cholinesterase activity to pesticide diazinon in rabbits (Orictolagus cuniculus) was investigated. In the in vivo assay, animals were orally exposed to single doses of diazinon (25 or 125 mg/kg) and after 10 days were sacrificed to obtain the brain, cerebellum and spinal cord. The cholinesterase activity in these tissues was determined to know its basal values in rabbits (control group) as well as the potential in vivo inhibition caused by exposure to this pesticide (exposed groups). The basal values of cholinesterase activity were 258.5 ± 38.0, 242.4 ± 32.0 and 235.1 ± 27.2 nmol/min.mg protein to brain, cerebellum and spinal cord,respectively, using acetylthiocholine as substrate. No inhibition of cholinesterase activity in the exposed groups compared with the control group was observed at the end of the assay. In the in vitro assay, the supernatants of the homogenized of tissues were exposed to 0.025, 0.05, 0.1, 0.2 and 0.4 mg/l of diazinon. The cholinesterase activity remained steadily depressed in a concentration and time dependent relationship throughout the experimental period (30 minutes, 9, 24 and 48 hours) in all tissues assessed. The Mean Inhibitory Concentration (IC50) of cholinesterase activity to diazinon was estimated in 0.154, 0.138 and 0.159 mg/l to brain, cerebellum and spinal cord, respectively. To determine a basal activity in each laboratory is necessary to use this enzymatic activity for diagnostic purposes of exposure to anticholinesterase pesticides (AU)

Cardiotoxicity in rabbits after a low-level exposure to diazinon, propoxur, and chlorpyrifos.

Lethal cardiac complications leading to death and various arrhythmias have been reported after organophosphate and/or carbamate poisonings. The present study focuses on the long-term effects of repeated low-level exposure to diazinon, propoxur, and chlorpyrifos (CPF) on cardiac function in rabbits. The yearly based experimental scheme of exposure consisted of two oral administration periods, lasting 3 months and 1 month each, interrupted by an 8-month washout period (total duration 12 months). At the end of the experimental scheme, the rabbits underwent an echocardiographic evaluation under sedation, after which they were killed and the tissue and serum samples were collected. A mild localized cardiotoxic effect was established by echocardiography for the three pesticides tested. Severe histological alterations were identified, especially in the diazinon-treated animals in agreement with increased persistence of this pesticide established in the cardiac tissue. In addition, all pesticides tested increased the oxidative stress and oxidative modifications in the genomic DNA content of the cardiac tissues, each one following a distinct mechanism.

Safety prediction of topically exposed biocides using permeability coefficients and the desquamation rate at the stratum corneum.

Advances in the synthesis and utilization of new chemical compounds have led to improvements in our daily lives. However, new chemicals may be both beneficial and toxic. Thus, exposure to these new compounds should be restricted in an attempt to limit their potential toxicities. We predicted the safety of three biocides (p-cresol, diazinon and resmethrin) by comparing their skin permeability coefficients and desquamation rate (the counter flux of permeability coefficient for chemical compounds induced by skin turnover) following skin exposure. In vitro skin permeation experiments revealed that the permeability coefficients of diazinon and resmethrin were smaller than the desquamation rate; therefore, these biocides could not permeate the skin, which resulted in very low skin concentrations of these compounds. On the other hand, the skin concentration of p-cresol was high because of its higher permeability coefficient than the desquamation rate. Furthermore, low in vitro cell viability was reported for skin exposed to p-cresol. These results in the present study indicate that the method described herein is useful for predicting the toxicities of chemicals following their topical exposure.

Evidence for diazinon-mediated inhibition of cis-permethrin metabolism and its effects on reproductive toxicity in adult male mice.

The potential toxicity resulting from combinatorial effects of organophosphorus and pyrethroid insecticides are not completely known. We evaluated male reproductive toxicity in mice co-exposed to diazinon and cis-permethrin. Nine-week-old male Sv/129 mice were exposed to diazinon (10 µmol/kg/day) or cis-permethrin (90 µmol/kg/day) alone or in combination (100 µmol/kg/day), or vehicle (corn oil), for 6 weeks. Diazinon and the diazinon-permethrin mixture inhibited plasma and liver carboxylesterase activities. In the mixture group, urinary excretion of cis-permethrin metabolite 3-phenoxybenzoic acid decreased along with increased plasma and testicular concentrations of cis-permethrin, while excretion of diazinon metabolites, diethylphosphate and diethylthiophosphate, did not change, versus mice exposed to each chemical alone, which suggested that inhibition of carboxylesterase decreased the metabolic capacity to cis-permethrin. Though the co-exposure decreased testosterone biosynthesis, increased degenerate germ cells in seminiferous tubule and sperm morphological abnormalities versus controls more clearly than exposure to cis-permethrin alone, the expected potentiation of toxicity was not evident.

Toxicokinetic and toxicodynamic model for diazinon toxicity--mechanistic explanation of differences in the sensitivity of Daphnia magna and Gammarus pulex.

A mechanistic toxicokinetic and toxicodynamic model for acute toxic effects (immobilization, mortality) of the organothiophosphate insecticide diazinon in Daphnia magna is presented. The model was parameterized using measured external and internal (whole-body) concentrations of diazinon, its toxic metabolite diazoxon, and the inactive metabolite 2-isopropyl-6-methyl-4-pyrimidinol, plus acetylcholinesterase (AChE) activity measured during exposure to diazinon in vivo. The toxicokinetic and toxicodynamic model provides a coherent picture from exposure to the resulting toxic effect on an organism level through internally formed metabolites and the effect on a molecular scale. A very fast reaction of diazoxon with AChE (pseudo first-order inhibition rate constant k(i) = 3.3 h(-1)) compared with a slow formation of diazoxon (activation rate constant k(act) = 0.014 h(-1)) was responsible for the high sensitivity of D. magna toward diazinon. Recovery of AChE activity from inhibition was slow and rate-determining (99% recovery within 16 d), compared with a fast elimination of diazinon (99% elimination within 17 h). The obtained model parameters were compared with toxicokinetic and toxicodynamic parameters of Gammarus pulex exposed to diazinon from previous work. This comparison revealed that G. pulex is less sensitive because of a six times faster detoxification of diazinon and diazoxon and an approximately 400 times lower rate for damage accrual. These differences overcompensate the two times faster activation of diazinon to diazoxon in G. pulex compared to D. magna. The present study substantiates theoretical considerations that mechanistically based effect models are helpful to explain sensitivity differences among different aquatic invertebrates.

Review of pesticide urinary biomarker measurements from selected US EPA children's observational exposure studies.

Children are exposed to a wide variety of pesticides originating from both outdoor and indoor sources. Several studies were conducted or funded by the EPA over the past decade to investigate children's exposure to organophosphate and pyrethroid pesticides and the factors that impact their exposures. Urinary metabolite concentration measurements from these studies are consolidated here to identify trends, spatial and temporal patterns, and areas where further research is required. Namely, concentrations of the metabolites of chlorpyrifos (3,5,6-trichloro-2-pyridinol or TCPy), diazinon (2-isopropyl-6-methyl-4-pyrimidinol or IMP), and permethrin (3-phenoxybenzoic acid or 3-PBA) are presented. Information on the kinetic parameters describing absorption and elimination in humans is also presented to aid in interpretation. Metabolite concentrations varied more dramatically across studies for 3-PBA and IMP than for TCPy, with TCPy concentrations about an order of magnitude higher than the 3-PBA concentrations. Temporal variability was high for all metabolites with urinary 3-PBA concentrations slightly more consistent over time than the TCPy concentrations. Urinary biomarker levels provided only limited evidence of applications. The observed relationships between urinary metabolite levels and estimates of pesticide intake may be affected by differences in the contribution of each exposure route to total intake, which may vary with exposure intensity and across individuals.

Toxicokinetic model describing bioconcentration and biotransformation of diazinon in Daphnia magna.

A toxicokinetic model for Daphnia magna , which simulates the internal concentration of the insecticide diazinon, its detoxification product 2-isopropyl-6-methyl-4-pyrimidinol, and its active metabolite diazoxon, is presented. During in vivo exposure to diazinon with and without inhibition of cytochrome P450 by piperonyl butoxide, the parent compound as well as its metabolites were quantified with high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) in extracts of D. magna . Rate constants of all relevant toxicokinetic steps were obtained by modeling the time course of the internal concentrations with a multicomponent first-order kinetics model. When cytochrome P450 was inhibited, the kinetic bioconcentration factor (BCF) of diazinon increased from 17.8 to 51.0 mL·g(ww)(-1). This clearly indicates that diazinon is biotransformed to a high degree by cytochrome P450 in D. magna . The dominant elimination step of diazinon was shown to be its oxidative dearylation to pyrimidinol (62% of total elimination) with a corresponding rate constant of 0.16 h(-1). In contrast, oxidative activation to diazoxon with a rate constant of 0.02 h(-1) amounted to only 8% of the total elimination. During exposure to diazinon, the active metabolite diazoxon could be detected only in very low concentrations (approximately 0.5% of the parent compound), presumably due to a very fast reaction with the target site acetylcholinesterase. During the exposure experiments (no feeding of daphnids), an exponential decline of the lipid content in D. magna with a first-order rate constant of 0.013 h(-1) was observed. For short exposure times (≤ 24 h), this had only a minor influence on the determined TK parameters. Such a TK model containing detailed biotransformation processes is an important tool for estimation of the toxic potential of chemicals, particularly, when active metabolites are formed inside an organism.

Genome-wide gene expression analysis in response to organophosphorus pesticide chlorpyrifos and diazinon in C. elegans.

Organophosphorus pesticides (OPs) were originally designed to affect the nervous system by inhibiting the enzyme acetylcholinesterase, an important regulator of the neurotransmitter acetylcholine. Over the past years evidence is mounting that these compounds affect many other processes. Little is known, however, about gene expression responses against OPs in the nematode Caenorhabditis elegans. This is surprising because C. elegans is extensively used as a model species in toxicity studies. To address this question we performed a microarray study in C. elegans which was exposed for 72 hrs to two widely used Ops, chlorpyrifos and diazinon, and a low dose mixture of these two compounds. Our analysis revealed transcriptional responses related to detoxification, stress, innate immunity, and transport and metabolism of lipids in all treatments. We found that for both compounds as well as in the mixture, these processes were regulated by different gene transcripts. Our results illustrate intense, and unexpected crosstalk between gene pathways in response to chlorpyrifos and diazinon in C. elegans.

Toxicokinetic and toxicodynamic modeling explains carry-over toxicity from exposure to diazinon by slow organism recovery.

Carry-over toxicity occurs when organisms exposed to an environmental toxicant survive but carry some damage resulting in reduced fitness. Upon subsequently encountering another exposure event stronger effects are possible if the organisms have not yet fully recovered. Carry-over toxicity was observed after exposure of the freshwater amphipod Gammarus pulex to repeated pulses of diazinon with varying intervals. Uptake, biotransformation and depuration kinetics were determined. Metabolites were identified and quantified (diazoxon, 2-isopropyl-6-methyl-4-pyrimidinol, one nonidentified metabolite). Parameters of a process-based toxicokinetic-toxicodynamic model were determined by least-squares fitting followed by Markov Chain Monte Carlo parameter estimation. Model parametrization was based on the time-course of measured internal concentrations of diazinon and its metabolite diazoxon in combination with the pulsed toxicity experiment. Prediction intervals, which take the covariation between parameters into account, were calculated for bioaccumulation factors, organism recovery time and simulations of internal concentrations as well as the time-course of survival under variable exposure. Organism recovery time was 28 days (95% prediction interval 25-31 days), indicating the possibility for carry-over toxicity from exposure events several weeks apart. The slow organism recovery and carry-over toxicity was caused by slow toxicodynamic recovery; toxicokinetic processes alone would have resulted in a recovery time of only 1-2 days.

Role of food and clay particles in toxicity of copper and diazinon using Daphnia magna.

Toxicity changes in copper and diazinon were investigated in the presence of food, clay, and their mixture by using Daphnia magna. In sorption equilibrium experiments, copper was significantly attracted (>34% sorbed) to food, clay, and food-clay mixture due to their negative zeta potential, while diazinon was less sorbed (<11%). In the exposure test with food and clay particles, it was revealed that copper was remarkably reduced in the presence of clay particles indicating the change in bioavailability of copper by sorption to clay. This was considered as the primary mechanism for toxicity reduction whereas diazinon toxicity was food dependent in the analysis of toxicity using toxicity change index (TCI). It was also shown that certain foods could not only act as a sorbent to copper and diazinon, but also as a material of energy source to alleviate the toxic damage. Meanwhile, clay can be considered as a prominent sorbent to copper but not to diazinon and can inhibit the sorption interaction between foodstuffs and toxicants through the aggregation and sedimentation processes. Furthermore, clay particles, as shown in TCI analysis, may be a potentially risky material as a physiological stressor or a toxicant carrier in contaminated environments.

Development of a physiologically based pharmacokinetic and pharmacodynamic model to determine dosimetry and cholinesterase inhibition for a binary mixture of chlorpyrifos and diazinon in the rat.

Physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) models have been developed for the organophosphorus (OP) insecticides chlorpyrifos (CPF) and diazinon (DZN). It is anticipated that these OPs could interact at a number of important metabolic steps including: CYP450 mediated activation/detoxification, B-esterases [carboxylesterase (CaE), butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE)] or PON-1 (A-esterase) oxon detoxification. We developed a binary PBPK/PD model for CPF, DZN and their metabolites based on previously published models for the individual insecticides. The metabolic interactions (CYP450) between CPF and DZN were evaluated in vitro and suggests that CPF is more substantially metabolized to its oxon metabolite than DZN, which is consistent with observed in vivo potency (CPF>DZN). Each insecticide inhibited the other's in vitro metabolism in a concentration-dependent manner. The PBPK model code used to describe the metabolism of CPF and DZN was modified to reflect the type of CYP450 inhibition kinetics (i.e. competitive vs. non-competitive), while B-esterase metabolism was described as dose-additive, and no PON-1 interactions were assumed between CPF- and DZN-oxon with the enzyme. The binary model was then evaluated against previously published rodent dosimetry and cholinesterase (ChE) inhibition data for the mixture. The PBPK/PD model simulations of the acute oral exposure to single-mixtures (15 mg/kg) vs. binary-mixtures (15+15 mg/kg) of CFP and DZN resulted in no differences in the predicted pharmacokinetics of either the parent OPs or their respective metabolites, while cholinesterase inhibition was reasonably described using the dose-additive model. A binary oral dose of CPF+DZN (60+60 mg/kg) did result in observable changes in the DZN pharmacokinetics where C(max) was more reasonably fit by modifying the absorption parameters. It is anticipated that at low environmentally relevant binary doses, most likely to be encountered in occupational or environmental related exposures, that the pharmacokinetics are expected to be linear, and ChE inhibition dose-additive.

Toxicity of diazinon and its metabolites increases in diabetic rats.

The effect of diazinon (DZN) on the activities of cholinesterase (ChE) in plasma and acetylcholinesterase (AChE) in erythrocyte and brain was investigated in normal and streptozotocin-induced diabetic rats. Hepatic drug-metabolizing enzyme activity was also estimated by measuring the systemic clearance of antipyrine, and the expression of hepatic cytochrome P450 (CYP) 3A2 and CYP1A2, which is closely related to the metabolism from DZN to DZN-oxon, a strong inhibitor of both ChE and AChE. No significant differences in the activities of ChE in plasma and AChE in erythrocyte were observed between normal and diabetic rats. Treatment with DZN significantly decreased these activities in diabetic rats more than in normal rats 6h after injection (6.5 mg/kg). Treatment with DZN significantly decreased the activity of AChE in brain of diabetic rats than normal rats 3h after injection (65 mg/kg), although no significant difference in the activity was found between normal and diabetic rats. The urinary recovery of diethylphosphate (DEP), a metabolite of DZN-oxon, was significantly increased in diabetic rats, but that of diethylthiophosphate (DETP), a metabolite of DZN, was unchanged. Significant increases in the systemic clearance of antipyrine and protein levels of hepatic CYP1A2, not CYP3A2, were observed in diabetic rats. These results suggest the possibility that a metabolite of DZN, DZN-oxon, causes higher toxicity in diabetic rats due to the enhancement of hepatic CYP1A2-mediated metabolism of DZN.

Use of liquid chromatography quadrupole time-of-flight mass spectrometry in the elucidation of transformation products and metabolites of pesticides. Diazinon as a case study.

Liquid chromatography (LC) coupled to hybrid quadrupole time-of-flight (QTOF) mass spectrometry (MS) is a useful analytical tool in the elucidation and confirmation of transformation products (TPs)/metabolites of pesticides with a wide range of polarity, in both environmental and biological samples. Firstly, the versatility of LC allows the determination of very distinct TPs/metabolites as chromatographic conditions can be easily changed and optimized depending on the analytical problem. Secondly, the mass accuracy provided by the TOF analyser allows the assignment of a highly probable empirical formula for each compound and the differentiation between nominal isobaric compounds. Finally, the possibility of performing MS/MS spectra with accurate mass measurements can been used for the final characterization of the TPs/metabolites detected and for the differentiation of isomeric compounds. In this study, the insecticide diazinon was used as model compound, and its photodegradation and metabolism have been investigated by LC-QTOF-MS. On one hand, environmental spiked water was irradiated with a mercury lamp for 9 days, sampling 3-mL aliquots approximately every 12 h. On the other hand, both in vitro and in vivo metabolism experiments were carried out with different substrate concentrations and incubation times. After centrifugation, and protein precipitation in the in vitro and in vivo studies, 50-microL aliquots of both environmental and biological samples were directly injected into the LC electrospray ionization QTOF system. The most important transformation processes were found to be hydrolysis of the ester moiety, hydroxylation in the aromatic ring or in one of the alkylic groups, oxidation of the sulfur atom on the P=S cleavage or a combination of these processes, with the highest number of compounds being found in the photodegradation study. Very polar compounds, such as diethyl phosphate and diethyl thiophosphate, were detected after direct injection of the aqueous sample, which was feasible owing to the characteristics of the LC. In MS mode, mass errors were below 3 mDa, leading to an empirical formula for each compound. MS/MS spectra with accurate mass were used for the final elucidation of the compounds detected.

Effect of organophosphate pesticide diazinon on expression and activity of intestinal P-glycoprotein.

Organophosphate insecticide diazinon is widely used in agricultural practices, submitting farmers to repeated exposure. Because efflux pumps, as P-glycoprotein (P-gp), serve both as natural defense mechanisms and influence the bioavailability and disposition of drugs, we analyzed the ability of diazinon to act as efflux modulator. Oral administration of diazinon (2-20 mg/kg, 5 days, or 10 mg/kg, 2-12 days) increased intestinal mdr1a mRNA of rats, in both dose- and time-dependent manner, and increased the expression of intestinal P-gp. Using the intestinal cell-line Caco-2, we found that 100 microM diazinon significantly inhibited digoxin and vinblastine secretive flux through the cell monolayers, whereas digoxin and vinblastine absorptive flux increased. The 25 microM diazinon was transported preferentially in basolateral (BL) to apical (AP) direction, suggesting a net secretion. The efflux rate significantly decreased in the presence of metabolic inhibitors sodium azide and 2-deoxy-d-glucose, P-gp inhibitors cyclosporin A and valspodar, but not in the presence of MRPs inhibitor MK571. Repeated exposure of Caco-2 cells to diazinon increased P-glycoprotein expression and activity. These results suggested the involvement of P-gp in the transfer of diazinon, leading to potential consequences for xenobiotic interactions, and showed that repeated exposure to low doses of pesticide may lead to up-regulated P-gp functions in the intestine of mammals.

Pharmacokinetic and pharmacodynamic interaction for a binary mixture of chlorpyrifos and diazinon in the rat.

Chlorpyrifos (CPF) and diazinon (DZN) are two commonly used organophosphorus (OP) insecticides and a potential exists for concurrent exposures. The primary neurotoxic effects from OP pesticide exposures result from the inhibition of acetylcholinesterase (AChE). The pharmacokinetic and pharmacodynamic impact of acute binary exposures of rats to CPF and DZN was evaluated in this study. Rats were orally administered CPF, DZN, or a CPF/DZN mixture (0, 15, 30, or 60 mg/kg) and blood (plasma and RBC), and brain were collected at 0, 3, 6, 12, and 24 h postdosing, urine was also collected at 24 h. Chlorpyrifos, DZN, and their respective metabolites, 3,5,6-trichloro-2-pyridinol (TCP) and 2-isopropyl-4-methyl-6-hydroxypyrimidine (IMHP), were quantified in blood and/or urine and cholinesterase (ChE) inhibition was measured in brain, RBC, and plasma. Coexposure to CPF/DZN at the low dose of 15/15 mg/kg did not alter the pharmacokinetics of CPF, DZN, or their metabolites in blood. A high binary dose of 60/60 mg/kg increased the C(max) and AUC and decreased the clearance for both parent compounds, likely due to competition between CPF and DZN for CYP450 metabolism. At lower doses, most likely to be encountered in occupational or environmental exposures, the pharmacokinetics were linear. A dose-dependent inhibition of ChE was noted in tissues for both the single and coexposures, and the extent of inhibition was plasma > RBC > or = brain. The overall relative potency for ChE inhibition was CPF/DZN > CPF > DZN. A comparison of the ChE response at the low binary dose (15/15 mg/kg), where there were no apparent pharmacokinetic interactions, suggested that the overall ChE response was additive. These experiments represent important data concerning the potential pharmacokinetic and pharmacodynamic interactions for pesticide mixtures and will provide needed insight for assessing the potential cumulative risk associated with occupational or environmental exposures to these insecticides.

Health effects of diazinon on a family.

There is increasing evidence of permanent sequalae from acute organophosphate poisoning. We report on accidental diazinon overexposure with acute organophosphate poisoning through cutaneous absorption and inhalation followed by persistent neurological effects. In addition, we observed skeletal and endocrine effects likely attributable to the diazinon poisoning. A family of seven was exposed to diazinon in June 1999 over a two-day period. The pesticide company mistakenly used diazinon to heavily spray the inside of the home instead of permethrin. The applicator applied the pesticide over the entire surface of the floor, carpeting, furniture, and clothing in closets to eradicate an infestation of fleas. Acute symptoms in the family members included headaches, nausea, skin irritation, runny nose, and vomiting. The family was first evaluated at 3 months and then 3 years after the acute poisoning. There were persisting neurological symptoms of memory loss, decreased concentration, irritability, and personality changes of varying degrees in all family members. Objective neurological findings of impaired balance, reaction time, color vision, slotted pegboards and trials making were present in the three older children who could be tested. Neuropsychological evaluation revealed evidence of organic brain dysfunction in all seven family members. Bone growth difficulties are present in four of five children. One child has delayed menarche.

Physiologically based pharmacokinetic/pharmacodynamic model for the organophosphorus pesticide diazinon.

Diazinon (DZN) is an organophosphorus pesticide with the possibility for widespread exposures. The toxicological effects of DZN are primarily mediated through the effects of its toxic metabolite, DZN-oxon on acetylcholinesterases, which results in accumulation of acetylcholine at neuronal junctions. A physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) model was developed to quantitatively assess the kinetics of DZN and its metabolites in blood and the inhibition of cholinesterases in plasma, RBC, brain, and diaphragm. Focused in vivo pharmacokinetic studies were conducted in male Sprague-Dawley rats and the data were used to refine the model. No overt toxicity was noted following doses up to 100mg/kg. However, cholinesterases in plasma, RBC, brain and diaphragm were substantially inhibited at doses of 50 mg/kg. In plasma, total cholinesterase was inhibited to less than 20% of control by 6 h post dosing with 100 mg/kg. Inhibition of brain acetylcholinesterase (AChE) following 100 mg/kg exposures was approximately 30% of control by 6 h. Diaphragm butyrylcholinesterase (BuChE) inhibition following 100 mg/kg dosing was to less than 20% of control by 6 h. The PBPK/PD model was used to describe the concentrations of DZN and its major, inactive metabolite, 2-isopropyl-4-methyl-6-hydroxypyrimidine (IMHP) in plasma and urinary elimination of IMHP. The fit of the model to plasma, RBC, brain, and diaphragm total cholinesterase and BuChE activity was also assessed and the model was further validated by fitting data from the open literature for intraperitoneal, intravenous, and oral exposures to DZN. The model was shown to quantitatively estimate target tissue dosimetry and cholinesterase inhibition following several routes of exposures. This model further confirms the usefulness of the model structure previously validated for chlorpyrifos and shows the potential utility of the model framework for other related organophosphate pesticides.

A comparative study of an accelerated life-test model and a toxicokinetics-based model for the analysis of Porcellio scaber survival data.

Statistical models have long been used for reliability analysis and risk assessment. In the present study, an accelerated life-test model was used to analyze a set of dose-time-response data obtained with the terrestrial isopod Porcellio scaber. Survival data were experimentally obtained by exposing P. scaber to diazinon (a nonpersistent insecticide) at six concentrations between 2 and 11.31 microg/g (toxicant/soil). Survival data are presented on a weekly basis. The accelerated life-test model assumed a log-normal distribution and constant variance across all diazinon concentrations. Model parameters were obtained by maximum likelihood estimation. The accelerated life-test model was compared to a toxicokinetics-based model reported in the literature. Survival predictions made by both models were compared with the observed data. Both the accelerated life-test model and the toxicokinetics-based model underestimated toxicity at a diazinon concentration of 8 microg/g. Overall, however, the accelerated life-test model outperformed the toxicokinetics-based model, with survival predictions closer to the observed data in most cases and a stronger correlation between predicted and observed survivals. However, as a statistical model, the accelerated life-test model did not reveal mechanistic information, and only statistical and distributional interpretations of its model parameters could be made.

Biotransformation of chlorpyrifos and diazinon by human liver microsomes and recombinant human cytochrome P450s (CYP).

The cytochrome P450 (CYP)-mediated biotransformation of the organophosphorothioate insecticides chlorpyrifos and diazinon was investigated. Rates of desulphuration to the active oxon metabolite (chlorpyrifos-oxon and diazinon-oxon) and dearylation to non-toxic hydrolysis products were determined in human liver microsome preparations from five individual donors and in recombinant CYP enzymes. Chlorpyrifos and diazinon underwent desulphuration in human liver microsome with mean Km = 30 and 45 microM and V(max) = 353 and 766 pmol min(-1) mg(-1), respectively. Dearylation of these compounds by human liver microsome proceeded with Km = 12 and 28 microM and V(max) = 653 and 1186 pmol min(-1) mg(-1), respectively. The apparent intrinsic clearance (V(max)/Km) of dearylation was 4.5- and 2.5-fold greater than desulphuration for chlorpyrifos and diazinon, respectively. Recombinant human CYP2B6 possessed the highest desulphuration activity for chlorpyrifos, whereas CYP2C19 had the highest dearylation activity. In contrast, both desulphuration and dearylation of diazinon were catalysed at similar rates, in the rank order CYP2C19 > CYP1A2 > CYP2B6 > CYP3A4. Both organophosphorothioates were more readily detoxified (dearylation) than bioactivated (desulphuration) in all human liver microsome preparations. However, the role of individual CYP enzymes in these two biotransformation pathways varied according to the structure of the organophosphorothioate, which was reflected in different activation/detoxification ratios for chlorpyrifos and diazinon. Variability in activity of individual CYP enzymes may influence interindividual sensitivity to the toxic effects of chlorpyrifos and diazinon.

Resistance to coumaphos and diazinon in Boophilus microplus (Acari: Ixodidae) and evidence for the involvement of an oxidative detoxification mechanism.

The levels of resistance to two organophosphate acaricides, coumaphos and diazinon, in several Mexican strains of Boophilus microplus (Canestrini) were evaluated using the FAO larval packet test. Regression analysis of LC50 data revealed a significant cross-resistance pattern between those two acaricides. Metabolic mechanisms of resistance were investigated with synergist bioassays. Piperonyl butoxide (PBO) reduced coumaphos toxicity in susceptible strains, but synergized coumaphos toxicity in resistant strains. There was a significant correlation between PBO synergism ratios and the coumaphos resistance ratios. The results suggest that an enhanced cytochrome P450 monooxygenase (cytP450)-mediated detoxification mechanism may exist in the resistant strains, in addition to the cytP450-mediated metabolic pathway that activates coumaphos. PBO failed to synergize diazinon toxicity in resistant strains, suggesting the cytP450 involved in detoxification were specific. Triphenylphosphate (TPP) synergized toxicity of both acaricides in both susceptible and resistant strains, and there was no correlation between TPP synergism ratios and the LC50 estimates for either acaricide. Esterases may not play a major role in resistance to coumaphos and diazinon in those strains. Bioassays with diethyl maleate (DEM) revealed a significant correlation between DEM synergism ratios and LC50 estimates for diazinon, suggesting a possible role for glutathione S-transferases in diazinon detoxification. Resistance to coumaphos in the Mexican strains of B. microplus was likely to be conferred by both a cytP450-mediated detoxification mechanism described here and the mechanism of insensitive acetylcholinesterases reported elsewhere. The results of this study also underscore the potential risk of coumaphos resistance in B. microplus from Mexico to the U.S. cattle fever tick eradication program.