Impact of low-level gestational exposure to organophosphate pesticides on neurobehavior in early infancy: a prospective study.

BACKGROUND: National data suggest widespread gestational exposure to organophosphate pesticides (OPs) based on the detection of OP metabolites in the urine of pregnant women. Associations with early infant neurobehavior are largely understudied, with only two studies reporting abnormal reflexes in newborns in association with gestational exposure to OPs. Our objective was to utilize biological markers of OP metabolites in pregnant women and a comprehensive assessment of infant neurobehavior to determine the association of gestational exposure to OPs with neurobehavioral outcomes during early infancy. METHODS: Among a cohort of 350 mother/infant pairs, we measured six common dialkylphosphate metabolites of OP pesticides in maternal urine, at two times during pregnancy (16 w & 26 w gestation), then calculated aggregate concentrations of diethylphosphate, dimethylphosphate, and total dialkyphosphate metabolites. We measured infant neurobehavior at about five weeks of age using the NICU Network Neurobehavioral Scale (NNNS), a comprehensive assessment of neurobehavior in young infants. Analyses of associations between gestational exposure to OPs and neurobehavior at five weeks included multiple linear and logistic regression. RESULTS: After adjustment for covariates, higher creatinine-corrected urinary concentrations of diethylphosphate metabolites were associated with improved attention and reduced lethargy and hypotonia in young infants. Higher creatinine-corrected urinary concentrations of total dialkylphosphate metabolites were associated with fewer signs of autonomic stress. Women who were white, married, had advanced education, and reported more frequent consumption of fresh fruits and vegetables had higher concentrations of OP metabolites during pregnancy. CONCLUSIONS: In this sample of pregnant women whose urinary concentrations of dialkylphosphate metabolites are representative of national exposure levels, we found no detrimental effects of gestational exposure to OPs on neurobehavioral outcomes among young infants. These results are important as they suggest there may be minimal to no detectable adverse impact of low level prenatal OP exposure on the neurobehavior of young infants.

Idiotypes expressed early in experimental Schistosoma mansoni infections predict clinical outcomes of chronic disease.

In murine Schistosoma mansoni infections, schistosome-specific cross-reactive idiotypes (CRI) are present in the sera of mice with moderate splenomegaly syndrome (MSS) at 20 wk after infection. In contrast, sera from animals that have the more severe hypersplenomegaly syndrome (HSS) at 20 wk of infection do not express these CRI in their sera. To examine when these regulatory CRI first appear in mice that eventually develop MSS, sera from infected animals were monitored for CRI from 1.5 to 20 wk of infection. In mice that eventually developed MSS, CRI were detected by 5 to 6 wk after infection, plateaued by 8 to 10 wk, and persisted through 20 wk of infection. Animals that developed HSS pathology or that died before 20 wk of infection never expressed CRI. Moreover, CRI levels present in the sera of mice at 6 wk of infection were inversely correlated with splenomegaly and hepatic fibrosis, but not with parasitologic measures, at 20 wk after infection. These results suggest that critical events occur very early in some schistosome infections that induce the production of regulatory idiotypes and that the presence or absence of these idiotypes predicts, and possibly determines, subsequent morbidity.

Quantification of Paraquat, MPTP, and MPP+ in brain tissue using microwave-assisted solvent extraction (MASE) and high-performance liquid chromatography-mass spectrometry.

Animal models, consistent with the hypothesis of direct interaction of paraquat (PQ) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) with specific areas of the central nervous system have been developed to study Parkinson's disease (PD) in mice. These models have necessitated the creation of an analytical method for unambiguous identification and quantitation of PQ and structurally similar MPTP and 1-methyl-4-phenylpyridinium ion (MPP+) in brain tissue. A method for determination of these compounds was developed using microwave-assisted solvent extraction (MASE) and liquid chromatography-mass spectrometry. Extraction solvent and microwave conditions such as power and time were optimized to produce recoveries of 90% for PQ 78% for MPTP and 97% for its metabolite MPP+. The chromatographic separation was performed on a C8, column and detection was carried out using an ion trap as an analyzer with electrospray ionization. Mass spectrometer parameters such as heated capillary temperature, spray voltage, capillary voltage and others were also optimized for each analyte. Analysis was done in selective ion-monitoring (SIM) mode using m/z 186 for PQ, m/z 174 for MPTP, and m/z 170 for MPP+. The method detection limit for paraquat in matrix was 100 pg, 40 pg for MPTP, and 20 pg MPP+.

Concentrations of urinary pesticide metabolites in small-scale farmers in Chiang Mai Province, Thailand.

Our research goal was to assess exposure to currently used pesticides among small-scale male farmers residing in two topographically different areas in Chiang Mai Province, Thailand. Farmers (N=136) were recruited from Pong Yaeng subdistrict (N=67) and Inthakhin subdistrict (N=69). Each farmer provided a morning urine void for the analysis of 30 urinary metabolites of insecticides, herbicides, and fungicides. Farmers in Pong Yaeng had significantly higher urinary concentrations of metabolites of organophosphorus insecticides and ethylene bisdithiocarbamates, while farmers from Inthakhin had significantly higher concentrations of malathion, 2,4-D, alachlor, and parathion or EPN metabolites. Based upon the metabolites measured in the urine of the farmers, chlorpyrifos and pyrethroid insecticides seemed to be commonly used across both communities; no significant differences in metabolite concentrations of these insecticides were observed between the two farmer groups. The presence of methamidaphos in the urine of farmers suggests that, despite a ban on its use, methamidaphos continues to be used in the communities. A similar finding with metabolites of methyl parathion must be further investigated. Overall, our results suggest that while each community may use different pesticides, Thai farmers are exposed to a wide variety of pesticides with a broad range in exposure magnitude. Furthermore, age, field size, crop production type, and the use of protective equipment were found to be potential factors influencing the degree of exposure.

Distribution and determinants of urinary biomarkers of exposure to organophosphate insecticides in Puerto Rican pregnant women.

Globally, human exposures to organophosphate (OP) insecticides may pose a significant burden to the health of mothers and their developing fetuses. Unfortunately, relevant data is limited in certain areas of the world concerning sources of exposure to OP insecticides in pregnant populations. To begin to address this gap in information for Puerto Rico, we studied repeated measures of urinary concentrations of 10 OP insecticide metabolites among 54 pregnant women from the northern karst region of the island. We also collected demographic data and self-reported information on the consumption of fruits, vegetables, and legumes in the past 48 h before urine collection and home pest-related issues. We calculated the distributions of the urinary biomarkers and compared them to women of reproductive age from the general U.S. population. We also used statistical models accounting for correlated data to assess within-subject temporal variability of the urinary biomarkers and to identify predictors of exposure. We found that for all but two metabolites (para-nitrophenol [PNP], diethylthiophosphate [DETP]), 50th or 95th percentile urinary concentrations (the metric that was used for comparison was based on the biomarker's detection frequency) of the other eight metabolites (3,5,6-trichloro-2-pyridinol [TCPY], 2-isopropyl-4-methyl-6-hydroxy-pyrimidine, malathion dicarboxylic acid, diethylphosphate, diethyldithiophosphate, dimethylphosphate, dimethylthiophosphate [DMTP], dimethyldithiophosphate) were somewhat lower in our cohort compared with similarly aged women from the continental United States. TCPY, PNP, DETP, and DMTP, which were the only urinary metabolites detected in greater than 50% of the samples, had poor reproducibility (intraclass correlation coefficient range: 0.19-0.28) during pregnancy. Positive predictors of OP insecticide exposure included: age; marital or employment status; consumption of cherries, grape juice, peanuts, peanut butter, or raisins; and residential application of pesticides. Further research is needed to understand what aspects of the predictors identified influence OP insecticide exposure during pregnancy.

Quantification of cyanuric acid residue in human urine using high performance liquid chromatography-tandem mass spectrometry.

Concern has increased about the resulting health effects of exposure to melamine and its metabolic contaminant, cyanuric acid, after infants in China were fed baby formula milk products contaminated with these compounds. We have developed a selective and sensitive analytical method to quantify the amount of cyanuric acid in human urine. The sample preparation involved extracting free-form cyanuric acid in human urine using anion exchange solid phase extraction. Cyanuric acid was separated from its urinary matrix components on the polymeric strong anion exchange analytical column; the analysis was performed by high performance liquid chromatography-tandem mass spectrometry using negative mode electrospray ionization interface. Quantification was performed using isotope dilution calibration covering the concentration range of 1.00-200ng/mL. The limit of detection was 0.60ng/mL and the relative standard deviations were 2.8-10.5% across the calibration range. The relative recovery of cyanuric acid was 100-104%. Our method is suitable to detect urinary concentrations of cyanuric acid caused by either environmental exposures or emerging poisoning events.

Quantification of dialkylphosphate metabolites of organophosphorus insecticides in human urine using 96-well plate sample preparation and high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry.

Organophosphorus (OP) pesticides kill by disrupting a targeted pest's brain and nervous systems. But if humans and other animals are sufficiently exposed, OP pesticides can have the same effect on them. We developed a fast and accurate high-performance liquid chromatography-tandem mass spectrometry method for the quantitative measurement of the following six common dialkylphosphate (DAP) metabolites of organophosphorus insecticides: dimethylphosphate (DMP), dimethylthiophosphate (DMTP), dimethyldithiophosphate (DMDTP), diethylphosphate, (DEP), diethylthiophosphate (DETP), and diethyldithiophosphate (DEDTP). The general sample preparation included 96-well plate solid phase extraction using weak anion exchange cartridges. The analytical separation was performed by high-performance liquid chromatography with a HILIC column. Detection involved a triple quadrupole mass spectrometer with an ESI probe in negative ion mode using multiple reaction monitoring. Repeated analyses of urine samples spiked at 150, 90 and 32 ng/mL with the analytes gave relative standard deviations of less than 22%. The extraction efficiency ranged from 40% to 98%. The limits of detection were in the range of 0.04-1.5 ng/mL. The throughput is 1152 samples per week, effectively quadrupling our previous throughput. The method is safe, quick, and sensitive enough to be used in environmental and emergency biological monitoring of occupational and nonoccupational exposure to organophosphates.

Measurement of ethyl methanesulfonate in human plasma and breast milk samples using high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry.

Ethyl methanesulfonate (EMS) is a mesylate ester, which is known to be a potent mutagen, teratogen, and possibly carcinogen. Mesylate esters have been found in pharmaceuticals as contaminants formed during the manufacturing process and may potentially pose an exposure hazard to humans. We have developed and validated a method for detection of trace amounts (ng/ml levels) of EMS in human plasma and breast milk. The samples were extracted by matrix solid-phase dispersion with ethyl acetate using Hydromatrix and the ASE 200 Accelerated Solvent Extractor. The extracts were separated by high-performance liquid chromatography (HPLC) using a HILIC column. The detection was performed with a triple quadrupole mass spectrometer (TSQ Quantum Ultra, Thermo Electron Corporation) using atmospheric pressure chemical ionization in negative-ion mode and multiple reaction monitoring. The use of a surrogate internal standard in combination with HPLC-MS/MS provided a high degree of accuracy and precision. The extraction efficiency was greater than 70%. Repeated analyses of plasma and breast milk samples spiked with high (100 ng/ml), medium (50 ng/ml) and low (5 ng/ml) concentrations of the analytes gave relative standard deviations of less than 12%. The limits of detection were in the range of 0.5-0.9 ng/ml for both matrices.

Method for measurement of the quaternary amine compounds paraquat and diquat in human urine using high-performance liquid chromatography-tandem mass spectrometry.

We have developed a highly selective and sensitive analytical method to quantify paraquat and diquat by use of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The sample preparation includes solid phase extraction that uses weak cation exchange cartridges. These highly charged dual quaternary amines were not retained by standard reversed phase columns, but they could be adequately separated through HPLC with a HILIC column. The detection was carried out with a triple quadrupole mass spectrometer with an electrospray ionization probe in positive ion mode in multiple reaction monitoring. Repeated analysis in human urine samples spiked with low (5 ng/ml), medium (15 ng/ml), and high (30 ng/ml) concentrations of the analytes yielded relative standard deviations of less than 9%. The extraction efficiencies ranged from 77.7% to 94.2%. The limits of detection were in the range of 1 ng/ml.

An improved high-performance liquid chromatography-tandem mass spectrometric method to measure atrazine and its metabolites in human urine.

We report an improved solid-phase extraction-high-performance liquid chromatography-tandem mass spectrometry method with isotope dilution quantification to measure seven atrazine metabolites in urine. The metabolites measured were hydroxyatrazine (HA), diaminochloroatrazine (DACT), desisopropylatrazine (DIA), desethylatrazine (DEA), desethylatrazine mercapturate (DEAM), atrazine mercapturate (ATZM), and atrazine (ATZ). Using offline mixed-mode reversed-phase/cation-exchange solid-phase extraction dramatically increased recovery and sensitivity by reducing the influence of matrix components during separation and analysis. DACT extraction recovery improved to greater than 80% while the other analytes had similar extraction efficiencies as previously observed. Limits of detection were lower than our previous method (0.05-0.19 ng/mL) with relative standard deviations less than 10%. The total runtime was shorter (18 min) than the previous on-line method, thus it is suitable for large-scale sample analyses. We increased the throughput of our method twofold by using the newer extraction technique.

Urinary elimination kinetics of acephate and its metabolite, methamidophos, in urine after acute ingestion.

INTRODUCTION: Acephate (AP) is a widely available organophosphorus (OP) insecticide considered to have low mammalian toxicity. In plants and insects, AP is metabolized extensively to methamidophos (MP), a more potent OP insecticide. The limited mammalian metabolism of AP to MP has been studied in laboratory rat models and suggests that initial formation of MP from AP may inhibit further formation. No case reports of human ingestion with urine AP and MP levels have been previously published. CASE REPORT: A 4-year-old male being evaluated for altered mental status and head trauma was noted to have muscarinic and nicotinic cholinergic signs. Further history suggested possible ingestion of a commercial AP product at an unknown time. Ingestion of AP was confirmed by the presence of urinary AP and MP and severely depressed red blood cell (RBC) cholinesterase and pseudocholinesterase activity levels. The patient initially received atropine in two 0.02 mg/kg IV boluses, then was started on 0.05 mg/kg IV per hour and titrated accordingly to clinical signs of cholinergic toxicity. Pralidoxime was also given at 20 mg/kg IV bolus, followed by an infusion of 10 mg/kg per hour. The patient required mechanical ventilation for 18 days and atropine infusion for 20 days. After a complicated intensive care unit course, he recovered and was discharged after a total of 32 days of hospitalization. METHODS: Four urine samples collected at different times were analyzed for AP and MP by using high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry. Kinetic calculations were performed by using standard equations. RESULTS: Suspected ingestion was confirmed by the presence of AP and MP in urine. The amount of MP found in urine suggests some limited human metabolism to this more toxic compound. CONCLUSIONS: Urinary elimination kinetics of AP demonstrates low metabolic conversion of AP to MP in humans.

Method for determination of acephate, methamidophos, omethoate, dimethoate, ethylenethiourea and propylenethiourea in human urine using high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry.

Because of increasing concern about widespread use of insecticides and fungicides, we have developed a highly sensitive analytical method to quantify urine-specific urinary biomarkers of the organophosphorus pesticides acephate, methamidophos, omethoate, dimethoate, and two metabolites from the fungicides alkylenebis-(dithiocarbamate) family: ethylenethiourea and propylenethiourea. The general sample preparation included lyophilization of the urine samples followed by extraction with dichloromethane. The analytical separation was performed by high-performance liquid chromatography (HPLC), and detection by a triple quadrupole mass spectrometer with and atmospheric pressure chemical ionization source in positive ion mode using multiple reaction monitoring and tandem mass spectrometry (MS/MS) analysis. Two different Thermo-Finnigan (San Jose, CA, USA) triple quadrupole mass spectrometers, a TSQ 7,000 and a TSQ Quantum Ultra, were used in these analyses; results are presented comparing the method specifications of these two instruments. Isotopically labeled internal standards were used for three of the analytes. The use of labeled internal standards in combination with HPLC-MS/MS provided a high degree of selectivity and precision. Repeated analysis of urine samples spiked with high, medium and low concentration of the analytes gave relative standard deviations of less than 18%. For all compounds the extraction efficiency ranged between 52% and 63%, relative recoveries were about 100%, and the limits of detection were in the range of 0.001-0.282 ng/ml.