Mosquito NADPH-cytochrome P450 oxidoreductase: kinetics and role of phenylalanine amino acid substitutions at leu86 and leu219 in CYP6AA3-mediated deltamethrin metabolism.

The NADPH-cytochrome P450 oxidoreductase (CYPOR) enzyme is a membrane-bound protein and contains both FAD and FMN cofactors. The enzyme transfers two electrons, one at a time, from NADPH to cytochrome P450 enzymes to function in the enzymatic reactions. We previously expressed in Escherichia coli the membrane-bound CYPOR (flAnCYPOR) from Anopheles minimus mosquito. We demonstrated the ability of flAnCYPOR to support the An. minimus CYP6AA3 enzyme activity in deltamethrin degradation in vitro. The present study revealed that the flAnCYPOR purified enzyme, analyzed by a fluorometric method, readily lost its flavin cofactors. When supplemented with exogenous flavin cofactors, the activity of flAnCYPOR-mediated cytochrome c reduction was increased. Mutant enzymes containing phenylalanine substitutions at leucine residues 86 and 219 were constructed and found to increase retention of FMN cofactor in the flAnCYPOR enzymes. Kinetic study by measuring cytochrome c-reducing activity indicated that the wild-type and mutant flAnCYPORs followed a non-classical two-site Ping-Pong mechanism, similar to rat CYPOR. The single mutant (L86F or L219F) and double mutant (L86F/L219F) flAnCYPOR enzymes, upon reconstitution with the An. minimus cytochrome P450 CYP6AA3 and a NADPH-regenerating system, increased CYP6AA3-mediated deltamethrin degradation compared to the wild-type flAnCYPOR enzyme. The increased enzyme activity could illustrate a more efficient electron transfer of AnCYPOR to CYP6AA3 cytochrome P450 enzyme. Addition of extra flavin cofactors could increase CYP6AA3-mediated activity supported by wild-type and mutant flAnCYPOR enzymes. Thus, both leucine to phenylalanine substitutions are essential for flAnCYPOR enzyme in supporting CYP6AA3-mediated metabolism.

Evaluation of deltamethrin kinetics and dosimetry in the maturing rat using a PBPK model.

Immature rats are more susceptible than adults to the acute neurotoxicity of pyrethroid insecticides like deltamethrin (DLM). A companion kinetics study (Kim et al., in press) revealed that blood and brain levels of the neuroactive parent compound were inversely related to age in rats 10, 21, 40 and 90 days old. The objective of the current study was to modify a physiologically based pharmacokinetic (PBPK) model of DLM disposition in the adult male Sprague-Dawley rat (Mirfazaelian et al., 2006), so blood and target organ dosimetry could be accurately predicted during maturation. Age-specific organ weights and age-dependent changes in the oxidative and hydrolytic clearance of DLM were modeled with a generalized Michaelis-Menten model for growth and the summary equations incorporated into the PBPK model. The model's simulations compared favorably with empirical DLM time-courses in plasma, blood, brain and fat for the four age-groups evaluated (10, 21, 40 and 90 days old). PND 10 pups' area under the 24-h brain concentration time curve (AUC(0-24h)) was 3.8-fold higher than that of the PND 90 adults. Our maturing rat PBPK model allows for updating with age- and chemical-dependent parameters, so pyrethroid dosimetry can be forecast in young and aged individuals. Hence, this model provides a methodology for risk assessors to consider age-specific adjustments to oral Reference Doses on the basis of PK differences.

Residues of lambda-cyhalothrin in tea.

Field experiments were conducted at two places in Tamil Nadu (India) during dry season of 2006 to determine the residues of lambda-cyhalothrin in fresh green tea leaves and black tea. Residues were quantified at different harvest intervals of '0' (3h), 1st, 3rd, 5th, 7th, 10th and 14th day after insecticide application. Persistence, dissipation pattern, half-life value and safe harvest interval of the insecticide in tea were calculated. Residues of lambda-cyhalothrin dissipated exponentially after application at both the locations and reached below the European Union maximum residue limit (MRL) of 1mg/kg on the 5th day. Lambda-cyhalothrin showed that like other insecticides it followed the first order dissipation kinetics. Half-life values varied from 2.8 to 3.5 days for lambda-cyhalothrin and a safety harvest interval of 5 days is suggested for tea at the recommended dosage.

Reference values for metabolites of pyrethroid and organophosphorous insecticides in urine for human biomonitoring in environmental medicine.

Pesticides are widely used throughout the world in agriculture to protect crops, and in public health to control diseases transmitted by vectors or intermediate hosts. After the prohibition of organochlorines, such as DDT, today mainly pyrethroids and organophosphorous insecticides are used. With reliable and sensitive analytical methods for detecting metabolites of organophosphorous and pyrethroid insecticides in urinary specimens of the general population several studies have been published on internal exposure to these insecticides of the population in Germany. In total, data on levels of metabolites of organophosphorous acids in urine of about 1200 children and adults have been published, as well as data on levels of pyrethroid metabolites in urine of about 2100 children and adults. In Germany, reference values for environmental pollutants related to the population are established continuously by the Human Biomonitoring Commission of the German Federal Environmental Agency, preferably based on data gained by representative studies. Reference values are defined as the 95th percentile, rounded off within the 95% confidence interval of the population studied. Since there is a need for reference values to characterise the population's exposure to organophosphates and pyrethroids, and since there are different studies available from Germany that agree quite well with data from other industrialised countries, the Commission has derived reference values from the available data, though none of the studies had fulfilled criteria on representativity. Reference values for metabolites of organophosphorous acids are as follows: DMP 135 microg/l, DMTP 160 microg/l and DEP 16 microg/l and for metabolites of pyrethroids: cis-Cl2CA 1 microg/l, trans-Cl2CA 2 microg/l and 3-PBA 2 microg/l. As the volume-related concentrations of organophosphate and pyrethroid metabolites show no significant age-dependence, the reference values derived are not age-stratified. Though based merely on statistical and not on toxicological data, levels analysed above the reference levels, when reliably measured (verified several times), should prompt environmental health practitioners to search for sources, within the bounds of proportionality. In addition to accidental poisoning, possible sources include indoor contamination following improper pest control operations in homes as well as in pets and food products contaminated by these pesticides.

Characterization of deltamethrin metabolism by rat plasma and liver microsomes.

Deltamethrin, a widely used type II pyrethroid insecticide, is a relatively potent neurotoxicant. While the toxicity has been extensively examined, toxicokinetic studies of deltamethrin and most other pyrethroids are very limited. The aims of this study were to identify, characterize, and assess the relative contributions of esterases and cytochrome P450s (CYP450s) responsible for deltamethrin metabolism by measuring deltamethrin disappearance following incubation of various concentrations (2 to 400 microM) in plasma (esterases) and liver microsomes (esterases and CYP450s) prepared from adult male rats. While the carboxylesterase metabolism in plasma and liver was characterized using an inhibitor, tetra isopropyl pyrophosphoramide (isoOMPA), CYP450 metabolism was characterized using the cofactor, NADPH. Michaelis-Menten rate constants were calculated using linear and nonlinear regression as applicable. The metabolic efficiency of these pathways was estimated by calculating intrinsic clearance (Vmax/Km). In plasma, isoOMPA completely inhibited deltamethrin biotransformation at concentrations (2 and 20 microM of deltamethrin) that are 2- to 10-fold higher than previously reported peak blood levels in deltamethrin-poisoned rats. For carboxylesterase-mediated deltamethrin metabolism in plasma, Vmax=325.3+/-53.4 nmol/h/ml and Km=165.4+/-41.9 microM. Calcium chelation by EGTA did not inhibit deltamethrin metabolism in plasma or liver microsomes, indicating that A-esterases do not metabolize deltamethrin. In liver microsomes, esterase-mediated deltamethrin metabolism was completely inhibited by isoOMPA, confirming the role of carboxylesterases. The rate constants for liver carboxylesterases were Vmax=1981.8+/-132.3 nmol/h/g liver and Km=172.5+/-22.5 microM. Liver microsomal CYP450-mediated biotransformation of deltamethrin was a higher capacity (Vmax=2611.3+/-134.1 nmol/h/g liver) and higher affinity (Km=74.9+/-5.9 microM) process than carboxylesterase (plasma or liver) detoxification. Genetically engineered individual rat CYP450s (Supersomes) were used to identify specific CYP450 isozyme(s) involved in the deltamethrin metabolism. CYP1A2, CYP1A1, and CYP2C11 in decreasing order of importance quantitatively, metabolized deltamethrin. Intrinsic clearance by liver CYP450s (35.5) was more efficient than that by liver (12.0) or plasma carboxylesterases (2.4).

An in vitro reporter gene assay method incorporating metabolic activation with human and rat S9 or liver microsomes.

A metabolic activation system with an S9 fraction or liver microsomes was applied to a reporter gene assay in vitro for the screening of estrogenicity of chemicals. The endpoint (luciferase) was luciferase induction in cells transfected with a reporter plasmid containing an estrogen-responsive element linked to the luciferase gene. Compounds were applied to the reporter gene assay system after pretreatment or simultaneous treatment with an S9 fraction or liver microsomes. Both trans-stilbene and methoxychlor themselves showed no or little estrogenicity, but when they were treated with an S9 fraction or liver microsomes, they demonstrated strong effects, indicating their metabolites to be estrogenic. When four pyrethroid insecticides were subjected to this assay system, however, they showed no estrogenicity even with liver microsome or S9 mix treatment.

Age-dependent differences in the susceptibility of rats to deltamethrin.

Separate groups of weanling and adult rats were exposed to both behaviorally active and lethal doses of deltamethrin to examine age-dependent toxicity of a pyrethroid over a wide dose range. The acoustic startle response (ASR) was selected for comparison at low doses since it is a sensitive, quantifiable biological indicator of pyrethroid effects in rats. Acute mortality was included for comparison at the upper limit of the dose-response. Deltamethrin was administered by gavage as a single dose in corn oil for all tests. Effects on the ASR were comparable in 21- and 72-day-old rats, with a 4-mg/kg dose decreasing ASR amplitude by approximately 50% (ED50) at both ages. By comparison LD50 values in 11-, 21- and 72-day old male rats were 5.1, 11, and 81 mg/kg, respectively. Thus, 11- and 21-day-old male rats were 16 and 7 times, respectively, more sensitive than adults to acute lethality. The concentration of deltamethrin was measured in whole-brain tissue from weanling and adult males treated with ED50 and LD50 doses. The brain concentration of deltamethrin at the ED50 dose of 4 mg/kg was higher in weanling rats than adults. This suggests a possible functional difference, with weanling rats being less susceptible than adults to a low dose. By comparison, there was an equivalent concentration of deltamethrin in brain tissue following an LD50 dose of 12 mg/kg in weanling rats and 80 mg/kg in adults. These results support age-related differences in pharmacokinetics as the basis for the markedly greater sensitivity of young rats to a lethal dose of deltamethrin.