Body fluids from the rat exposed to chlorpyrifos induce cytotoxicity against the corresponding tissue-derived cells in vitro.

BACKGROUND: This study aims to establish an in vitro monitoring approach to evaluate the pesticide exposures. We studied the in vitro cytotoxicity of three different body fluids of rats to the respective corresponding tissue-derived cells. METHODS: Wistar rats were orally administrated daily with three different doses of chlorpyrifos (1.30, 3.26, and 8.15 mg/kg body weight/day, which is equal to the doses of 1/125, 1/50, and 1/20 LD50, respectively) for consecutive 90 days. Blood samples as well as 24-hour urine and fecal samples were collected and processed. Then, urine, serum, and feces samples were used to treat the correspondent cell lines, i.e., T24 bladder cancer cells, Jurkat lymphocytes, and HT-29 colon cancer cells respectively, which derived from the correspondent tissues that could interact with the respective corresponding body fluids in organism. Cell viability was determined by using MTT or trypan blue staining. RESULTS: The results showed that urine, serum, and feces extract of the rats exposed to chlorpyrifos displayed concentration- and time-dependent cytotoxicity to the cell lines. Furthermore, we found that the cytotoxicity of body fluids from the exposed animals was mainly due to the presence of 3, 4, 5-trichloropyrindinol, the major toxic metabolite of chlorpyrifos. CONCLUSIONS: These findings indicated that urine, serum, and feces extraction, especially urine, combining with the corresponding tissue-derived cell lines as the in vitro cell models could be used to evaluate the animal exposure to pesticides even at the low dose with no apparent toxicological signs in the animals. Thus, this in vitro approach could be served as complementary methodology to the existing toolbox of biological monitoring of long-term and low-dose exposure to environmental pesticide residues in practice.

[In vitro study of histocompatibility and clearance of hemoperfusion adsorbents].

Objective: To evaluate the histocompatibility and clearance of chlorpyrifos and its metabolite of activated charcoal and adsorption resin by in vitro study. Methods: Venous blood from volunteers were incubation with activated charcoal or adsorbent resins, cytometry parameters and plasma components were detected for evaluation the histocompatibility of adsorbents. Venous blood from volunteers mixed with chlorpyrifos and its metabolite were incubation with activated charcoal or adsorbent resins, plasma concentration of chlorpyrifos and its metabolite were detected for evaluation the efficacy of adsorbents. Results: Incubation tests show that the absorbents reduce the blood platelet (F=3.671, P<0.05) , serum glucose (F=10.564, P<0.05) , albumin (F=5.239, P<0.05) , uric acid (F=7.175, P<0.05) , creatinine (F=23.673, P<0.05) , T3 (F=11.161, P<0.05) and free T3 (F=10.256, P<0.05) . However, other cytometry parameters and plasma components were not influenced. Both activated charcoal and adsorbent resins could reduce the plasma concentration of chlorpyrifos (F=798.110, P<0.01) and its metabolite (F=1495.212, P<0.05) . Conclusion: In vitro test show that both activated charcoal and adsorbent resins could clear chlorpyrifos and its metabolite, however, could not influence main cytometry parameters and plasma components, the histocompatibility of adsorbents are satisfactory.

Prenatal organophosphate insecticide exposure and infant sensory function.

BACKGROUND: Occupational studies suggest that exposure to organophosphate insecticides (OPs) can lead to vision or hearing loss. Yet the effects of early-life exposure on visual and auditory function are unknown. Here we examined associations between prenatal OP exposure and grating visual acuity (VA) and auditory brainstem response (ABR) during infancy. METHODS: 30 OPs were measured in umbilical cord blood using gas chromatography tandem mass spectrometry in a cohort of Chinese infants. Grating visual acuity (VA) (n = 179-200) and auditory brainstem response (ABR) (n = 139-183) were assessed at 6 weeks, 9 months, and 18 months. Outcomes included VA score, ABR wave V latency and central conduction time, and head circumference (HC). Associations between sensory outcomes during infancy and cord OPs were examined using linear mixed models. RESULTS: Prenatal chlorpyrifos exposure was associated with lower 9-month grating VA scores; scores were 0.64 (95% CI: -1.22, -0.06) points lower for exposed versus unexposed infants (p = 0.03). The OPs examined were not associated with infant ABR latencies, but chlorpyrifos and phorate were both significantly inversely associated with HC at 9 months; HCs were 0.41 (95% CI: 0.75, 0.6) cm and 0.44 (95% CI: 0.88, 0.1) cm smaller for chlorpyrifos (p = 0.02) and phorate (p = 0.04), respectively. CONCLUSIONS: We found deficits in grating VA and HC in 9-month-old infants with prenatal exposure to chlorpyrifos. The clinical significance of these small but statistically significant deficits is unclear. However, the disruption of visual or auditory pathway maturation in infancy could potentially negatively affect downstream cognitive development.

Evaluation of chlorpyrifos toxicity through a 28-day study: Cholinesterase activity, oxidative stress responses, parent compound/metabolite levels, and primary DNA damage in blood and brain tissue of adult male Wistar rats.

In this 28 day-study, we evaluated the effects of the insecticide chlorpyrifos orally administered to Wistar rats at doses 0.160, 0.015, and 0.010 mg/kg b. w./day. Following treatment, total cholinesterase activity and activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were measured. Oxidative stress responses were evaluated using a battery of endpoints to establish lipid peroxidation, changes in total antioxidant capacity, level of reactive oxygen species (ROS), glutathione (GSH) level and activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase. Using HPLC-UV DAD analysis, levels of the parent compound and its main metabolite 3,5,6-trichloro-2-pyridinol in plasma and brain tissue were measured. The genotoxic effect was estimated using alkaline comet assay in leukocytes and brain tissue. The exposure did not result in significant effects on total cholinesterase, AChE and BChE activity in plasma and brain tissue. Lipid peroxidation slightly increased both in plasma and brain tissue. Total antioxidant capacity, ROS and GSH levels were marginally influenced by the exposure. Treatment led to significant increases of GSH-Px activity in blood, SOD activity in erythrocytes and a slight increase of catalase activity in plasma. HPLC-UV DAD analysis revealed the presence of both the parent compound and its main metabolite in the plasma of all of the experimental animals and brain tissue of the animals treated at the two higher doses. All of the tested doses of chlorpyrifos were slightly genotoxic, both to leukocytes and brain tissue. Our results call for further research using other sensitive biomarkers of effect, along with different exposure scenarios.

Pesticides contaminated dust exposure, risk diagnosis and exposure markers in occupational and residential settings of Lahore, Pakistan.

There are few studies documenting the dust loaded with pesticides as a potential non-dietary exposure source for occupational worker and populations living near agricultural farms and pesticides formulation plants. In present study we have evaluated the pesticide concentration in dust from potential sites and relevant health risk from dust ingestion. Furthermore, the effect of currently used pesticides was investigated on blood and urine parameters of subjects: farmer, factory worker, urban resident and rural resident and controlled subjects with presumably different levels of exposure. The urinary metabolites (TCPY and IMPY) were quantified as biomarkers of exposure to chlorpyrifos and diazinon in relation with biomarkers of effect including BuChE, LH, FSH, testosterone and oxidative stress. Results showed that chlorpyrifos and diazinon were present in higher concentration in dust and posed a high health risk to exposed subjects. The mean SOD value was high among the farmer (3048U/g Hb) followed by factory worker (1677.6U/g Hb). The urinary biomarkers - TCPY and IMPY- were found higher in exposed subjects as compared to control. Furthermore, testosterone was found in higher concentration in factory worker than control (12.63ng/ml vs 4.61ng/ml respectively). A decreased BuChE activity was noticed in occupational group and significant differences were observed in control verses exposed subjects. The PCA analysis evidenced the impact of pesticides on exposure biomarkers and male reproductive hormones. The study suggests that dust contaminated with pesticides engenders significant health risk particularly related to the nervous and endocrine system, not only for occupational workers exposed to direct ingestion but also for nearby residential community. Succinctly putting: Pesticides loaded dust in the city of Lahore, being a high priority concern for the government of Pakistan, demands to be addressed.

[Gas chromatography for measurement of chlorpyrifos in serum].

OBJECTIVE: To investigate the method for the measurement of chlorpyrifos in serum by gas chromatography, and to provide a basis for emergency treatment of poisoning in clinical practice. METHODS: Venous blood (3.0 ml) was collected from patients. After coagulation, the blood samples were centrifuged at 4 000 r/min for 5 minutes, and 0.5 ml of serum was placed in a glass test tube with a cork; 4.0 ml of ethyl acetate was then added and mixed rapidly, and this solution was subjected to extraction for 5 minutes and centrifuged at 4 000 r/min for 10 minutes. The ethyl acetate layer was placed in a conical tube and extracted twice with the same method. The extract was mixed and blow-dried with nitrogen, and the residue was dissolved with 50.0 µl ethanol. Gas chromatography was used for measurement, with a sample size of 1 µl and a retention time of 9.609 minutes. RESULTS: The linear range of this method was 0.2~20.0 µg/ml, and the regression equation was y=2 372.6x+357.2(r=0.999 6). The detection limit of chlorpyrifos in serum was 0.05 µg/ml, and the recovery rate was 84.6%~102.4%. The relative standard deviation was 3.6%~4.8%, and the intra-day and inter-day relative standard deviations were 3.62%~5.10% and 3.77%~4.98%, respectively. CONCLUSION: This detection method is accurate, simple, and convenient, and can be used for the clinical diagnosis of chlorpyrifos poisoning.

Co-treatment of chlorpyrifos and lead induce serum lipid disorders in rats: Alleviation by taurine.

The aim of this study was to investigate the effects of taurine (TA) on serum lipid profiles following chronic coadministration of chlorpyrifos (CP) and lead acetate (Pb) in male Wistar rats. Fifty rats randomly distributed into five groups served as subjects. Distilled water (DW) was given to DW group, while soya oil (SO; 1 mL kg(-1)) was given to SO group. The TA group was treated with TA (50 mg kg(-1)). The CP + Pb group was administered sequentially with CP (4.25 mg kg(-1); 1/20th median lethal dose (LD50)) and Pb at 233.25 mg kg(-1) (1/20th LD50), while the TA + CP + Pb group received TA (50 mg kg(-1)), CP (4.25 mg kg(-1)), and Pb (233.25 mg kg(-1)) sequentially. The treatments were administered once daily by oral gavage for 16 weeks. The rats were euthanised, and the blood samples were collected at the termination of the study. Sera obtained from the blood samples were analyzed for total cholesterol, high-density lipoprotein cholesterol, triglycerides, and malondialdehyde, and also the activities of serum antioxidant enzymes including superoxide dismutase, catalase and glutathione peroxidase were analyzed. The low-density lipoprotein cholesterol, very-low-density lipoprotein cholesterol, and atherogenic index were calculated. The results showed that CP and Pb induced alterations in the serum lipid profiles and evoked oxidative stress. TA alleviated the disruptions in the serum lipid profiles of the rats partially by mitigating oxidative stress. It was concluded that TA may be used for prophylaxis against serum lipid disorders in animals that were constantly co-exposed to CP and Pb in the environment.

Prenatal exposure to the organophosphate pesticide chlorpyrifos and childhood tremor.

BACKGROUND: The organophosphate insecticide chlorpyrifos (CPF), widely used for agricultural purposes, has been linked to neurodevelopmental deficits. Possible motor effects at low to moderate levels of exposure have not been evaluated. METHODS: Prenatal exposure to CPF was measured in umbilical cord blood in a sample of 263 inner-city minority children, who were followed prospectively. At approximately 11 years of age (mean age 10.9 ± 0.85 years, range=9.0-13.9), during a neuropsychological assessment, children were asked to draw Archimedes spirals. These were rated by a senior neurologist specializing in movement disorders who was blind to CPF exposure level. RESULTS: Compared to all other children, those with prenatal CPF exposure in the upper quartile range (n=43) were more likely to exhibit mild or mild to moderate tremor (≥ 1) in either arm (p=0.03), both arms (p=0.02), the dominant arm (p=0.01), and the non-dominant arm (p=0.055). Logistic regression analyses showed significant CPF effects on tremor in both arms, either arm, the dominant arm (p-values <0.05), and the non-dominant arm (p=0.06), after adjustment for sex, age at testing, ethnicity, and medication. CONCLUSION: Prenatal CPF exposure is associated with tremor in middle childhood, which may be a sign of the insecticide's effects on nervous system function.

Chlorpyrifos Determined in Human Blood by UPLC-MS/MS and Its Application in Poisoning Cases.

OBJECTIVE: To determine the chlorpyrifos in human blood by liquid chromatography-tandem mass spectrometry and to validate its application in poisoning cases. METHODS: The samples were extracted by a simple one-step protein precipitation procedure. Chromatography was performed on a Capcell Pack C18 MGII column (250 mm x 2.0 mm, 5 µm) using an isocratic elution of solvent A (0.1% formic acid-water with 2 mmol/L ammonium acetate) and solvent B (methanol with 2 mmol/L ammonium acetate) at 5:95 V:V). RESULTS: The linear ranged from 5 to 500 ng/mL (r = 0.998 7). The limit of detection (LOD) and the lower limit of quantification (LLOQ) were 2 ng/mL and 4 ng/mL, respectively. For this method, the precision and accuracy of intra-day and inter-day were < 10% and 97.44%-101.10%, respectively. The results in stability test of long-term frozen were satisfied. The matrix effect, recovery and process efficiency were 64.97%-86.81%, 76.70%-85.52%, and 55.57%-66.58%, respectively. CONCLUSION: This method can provide a rapid approach to chlorpyrifos extraction and determination in toxicological analysis of forensic and clinical treatment.

Chlorpyrifos Determined in Human Blood by UPLC-MS/MS and Its Application in Poisoning Cases / 法医学杂志

OBJECTIVE@#To determine the chlorpyrifos in human blood by liquid chromatography-tandem mass spectrometry and to validate its application in poisoning cases.@*METHODS@#The samples were extracted by a simple one-step protein precipitation procedure. Chromatography was performed on a Capcell Pack C18 MGII column (250 mm x 2.0 mm, 5 μm) using an isocratic elution of solvent A (0.1% formic acid-water with 2 mmol/L ammonium acetate) and solvent B (methanol with 2 mmol/L ammonium acetate) at 5:95 V:V).@*RESULTS@#The linear ranged from 5 to 500 ng/mL (r = 0.998 7). The limit of detection (LOD) and the lower limit of quantification (LLOQ) were 2 ng/mL and 4 ng/mL, respectively. For this method, the precision and accuracy of intra-day and inter-day were < 10% and 97.44%-101.10%, respectively. The results in stability test of long-term frozen were satisfied. The matrix effect, recovery and process efficiency were 64.97%-86.81%, 76.70%-85.52%, and 55.57%-66.58%, respectively.@*CONCLUSION@#This method can provide a rapid approach to chlorpyrifos extraction and determination in toxicological analysis of forensic and clinical treatment.

Intracerebroventricularly and systemically delivered inhibitor of brain CYP2B (C8-Xanthate), even following chlorpyrifos exposure, reduces chlorpyrifos activation and toxicity in male rats.

Chlorpyrifos is a pesticide that is metabolically activated to chlorpyrifos oxon (acetylcholinesterase inhibitor) primarily by the cytochrome P450 2B (CYP2B) enzyme subfamily in the liver and brain. We have previously shown that intracerebroventricular pretreatment with a CYP2B inhibitor, C8-Xanthate, can block chlorpyrifos toxicity. Here, we assessed whether delayed introduction of C8-Xanthate would still reduce toxicity and whether peripheral administration of C8-Xanthate could also inhibit chlorpyrifos activation in the brain and block toxicity. Male rats (N = 4-5/group) were either pretreated with C8-Xanthate (40 µg intracerebroventricular or 5 mg/kg intraperitoneal), or vehicle (ACSF or saline, respectively), 24 h before chlorpyrifos treatment (125 mg/kg subcutaneous) and then treated daily with inhibitor or vehicle until 7 days post-chlorpyrifos treatment. Additional groups received vehicle pretreatment, switching to C8-Xanthate 1, 2, 3, or 4 days after chlorpyrifos and then continuing with daily C8-Xanthate treatment until 7 days post-chlorpyrifos treatment. Neurotoxicity was assessed at baseline (before chlorpyrifos) and then daily after chlorpyrifos, using behavioral assessments (e.g., gait score). Neurochemical assays (e.g., serum and brain chlorpyrifos) were performed at the end of study. Pretreatment with C8-Xanthate completely prevented chlorpyrifos toxicity, and delayed introduction of C8-Xanthate reduced toxicity, even when started up to 4 days after chlorpyrifos treatment. Discontinuation of C8-Xanthate treatment 7 days post-chlorpyrifos treatment did not result in the reappearance of toxicity, tested through 10 days after chlorpyrifos treatment. These findings suggest that CYP2B inhibitor treatment, even days after chlorpyrifos exposure, and using a peripheral delivery route, may be useful as a therapeutic approach to reduce chlorpyrifos toxicity.

A human life-stage physiologically based pharmacokinetic and pharmacodynamic model for chlorpyrifos: development and validation.

Sensitivity to some chemicals in animals and humans are known to vary with age. Age-related changes in sensitivity to chlorpyrifos have been reported in animal models. A life-stage physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model was developed to predict disposition of chlorpyrifos and its metabolites, chlorpyrifos-oxon (the ultimate toxicant) and 3,5,6-trichloro-2-pyridinol (TCPy), as well as B-esterase inhibition by chlorpyrifos-oxon in humans. In this model, previously measured age-dependent metabolism of chlorpyrifos and chlorpyrifos-oxon were integrated into age-related descriptions of human anatomy and physiology. The life-stage PBPK/PD model was calibrated and tested against controlled adult human exposure studies. Simulations suggest age-dependent pharmacokinetics and response may exist. At oral doses ⩾0.6mg/kg of chlorpyrifos (100- to 1000-fold higher than environmental exposure levels), 6months old children are predicted to have higher levels of chlorpyrifos-oxon in blood and higher levels of red blood cell cholinesterase inhibition compared to adults from equivalent doses. At lower doses more relevant to environmental exposures, simulations predict that adults will have slightly higher levels of chlorpyrifos-oxon in blood and greater cholinesterase inhibition. This model provides a computational framework for age-comparative simulations that can be utilized to predict chlorpyrifos disposition and biological response over various postnatal life stages.

Protein tyrosine adduct in humans self-poisoned by chlorpyrifos.

Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos.

Longitudinal assessment of chlorpyrifos exposure and effect biomarkers in adolescent Egyptian agricultural workers.

Chlorpyrifos (CPF) is applied seasonally in Egypt by adolescent agricultural workers and the extent of occupational exposure and the potential for environmental CPF exposure in this population is poorly understood. Adolescent pesticide applicators (n=57; 12-21 years of age) and age-matched non-applicators (n=38) from the same villages were followed for 10 months in 2010, spanning pre-application through post-application. Eight urine and five blood samples were collected from participants within this time period. Blood acetylcholinesterase and butyrylcholinesterase (BChE; exposure/effect biomarker) and urine 3,5,6-trichloro-2-pyridinol (TCPy; exposure biomarker) were used to assess occupational CPF exposures in pesticide applicators and environmental exposures in non-applicators. Applicators demonstrated significantly higher TCPy concentration and BChE depression than non-applicators throughout CPF application. This difference persisted for 4-7 weeks after the cessation of agricultural spraying. However, both groups exhibited significantly elevated TCPy and depressed BChE, compared with their respective baseline. The peak TCPy levels during the spray season (95% confidence interval (CI)) for non-applicators and applicators reached 16.8 (9.87-28.5) and 137 (57.4-329) ug/g creatinine, respectively. BChE levels (95% CIs) during the spray were as follows: 1.47 (1.28-1.68) for non-applicators and 0.47 (0.24-0.94) U/ml for applicators. The longitudinal assessment of CPF biomarkers provided robust measures of exposure and effect throughout CPF application in adolescents and revealed significant exposures in both applicators and non-applicators. Biomarker data in the non-applicators, which mirrored that of the applicators, indicated that non-applicators received environmental CPF exposures. This suggests that similar exposures may occur in other residents of this region during periods of pesticide application.

Pharmacokinetics and pharmacodynamics of chlorpyrifos and 3,5,6-trichloro-2-pyridinol in rat saliva after chlorpyrifos administration.

Sensors have been developed for noninvasive biomonitoring of the organophosphate pesticide chlorpyrifos (CPF), and previous studies have suggested consistent partitioning of 3,5,6-trichloro-2-pyridinol (TCPy), a metabolite of CPF, into saliva after exposure to TCPy. The objective of this study was to quantitatively evaluate in vivo pharmacokinetics and pharmacodynamics of CPF and TCPy in saliva after CPF administration. Rats were coadministered CPF (0.5-5mg/kg) and pilocarpine (~13 mg/kg) iv. Saliva and blood were collected, and levels of CPF, TCPy, and cholinesterase (ChE) activity were quantified. Experimental results suggest that CPF is rapidly metabolized after iv administration. Formation of TCPy from administered CPF at the low dose (0.5 mg/kg) was slower than from higher CPF doses, potentially due to differences in plasma protein binding to CPF. CPF was measured in saliva only at the first time point sampled (0-15 min), indicating low partitioning and rapid metabolism. After formation, TCPy pharmacokinetics were very similar in blood and saliva. Saliva/blood TCPy concentration ratios were not affected by TCPy concentration in blood, saliva flow rate, or salivary pH and were consistent with previous studies. ChE activity in plasma demonstrated a dose-dependent decrease, and ChE activity in saliva was extremely variable and demonstrated no dose relationship. A physiologically based pharmacokinetic and pharmacodynamic model for CPF was modified and predicted the data reasonably well. It is envisioned that a combination of biomonitoring compounds like TCPy in saliva coupled with computational modeling will form an approach to measure pesticide exposure to susceptible human populations such as agricultural workers.

Does the home environment and the sex of the child modify the adverse effects of prenatal exposure to chlorpyrifos on child working memory?

Prenatal exposure to chlorpyrifos (CPF), an organophosphorus insecticide, has long been associated with delayed neurocognitive development and most recently with decrements in working memory at age 7. In the current paper, we expanded the previous work on CPF to investigate how additional biological and social environmental factors might create or explain differential neurodevelopmental susceptibility, focusing on main and moderating effects of the quality of the home environment (HOME) and child sex. We evaluate how the quality of the home environment (specifically, parental nurturance and environmental stimulation) and child sex interact with the adverse effects of prenatal CPF exposure on working memory at child age 7years. We did not observe a remediating effect of a high quality home environment (either parental nurturance or environmental stimulation) on the adverse effects of prenatal CPF exposure on working memory. However, we detected a borderline significant interaction between prenatal exposure to CPF and child sex (B (95% CI) for interaction term=-1.714 (-3.753 to 0.326)) suggesting males experience a greater decrement in working memory than females following prenatal CPF exposure. In addition, we detected a borderline interaction between parental nurturance and child sex (B (95% CI) for interaction term=1.490 (-0.518 to 3.499)) suggesting that, in terms of working memory, males benefit more from a nurturing environment than females. To our knowledge, this is the first investigation into factors that may inform an intervention strategy to reduce or reverse the cognitive deficits resulting from prenatal CPF exposure.

Organophosphate pesticide levels in blood and urine of women and newborns living in an agricultural community.

Organophosphate pesticides are widely used and recent studies suggest associations of in utero exposures with adverse birth outcomes and neurodevelopment. Few studies have characterized organophosphate pesticides in human plasma or established how these levels correlate to urinary measurements. We measured organophosphate pesticide metabolites in maternal urine and chlorpyrifos and diazinon in maternal and cord plasma of subjects living in an agricultural area to compare levels in two different biological matrices. We also determined paraoxonase 1 (PON1) genotypes (PON1(192) and PON1(-108)) and PON1 substrate-specific activities in mothers and their newborns to examine whether PON1 may affect organophosphate pesticide measurements in blood and urine. Chlorpyrifos levels in plasma ranged from 0-1,726 ng/mL and non-zero levels were measured in 70.5% and 87.5% of maternal and cord samples, respectively. Diazinon levels were lower (0-0.5 ng/mL); non-zero levels were found in 33.3% of maternal plasma and 47.3% of cord plasma. Significant associations between organophosphate pesticide levels in blood and metabolite levels in urine were limited to models adjusting for PON1 levels. Increased maternal PON1 levels were associated with decreased odds of chlorpyrifos and diazinon detection (odds ratio(OR): 0.56 and 0.75, respectively). Blood organophosphate pesticide levels of study participants were similar in mothers and newborns and slightly higher than those reported in other populations. However, compared to their mothers, newborns have much lower quantities of the detoxifying PON1 enzyme suggesting that infants may be especially vulnerable to organophosphate pesticide exposures.

Simultaneous analysis of chlorpyrifos and cypermethrin in cord blood plasma by online solid-phase extraction coupled with liquid chromatography-heated electrospray ionization tandem mass spectrometry.

Chlorpyrifos and cypermethrin are the most used insecticides in Taiwan. Exposure to both pesticides has been associated with reproductive and developmental health effects in humans and animals. This study describes an online solid-phase extraction coupled with liquid chromatography-heated electrospray ionization tandem mass spectrometry (online SPE-LC/HESI/MS/MS) method to analyze chlorpyrifos and cypermethrin in cord blood of pregnant women. Calibration curves showed good linearity (r² > 0.998) for both pesticides within the range of 0.1-100 ppb. Limits of detection (LODs) were 0.01 and 0.05 ppb and recoveries in cord blood were 97.2 ± 4.8% and 93.5 ± 9.5% for chlorpyrifos and cypermethrin respectively. After analysis of 396 samples, the mean concentrations of chlorpyrifos and cypermethrin were 0.38 and 1.08 ppb respectively. These results demonstrate that LC/HESI/MS/MS is effective for the simultaneous analysis of chlorpyrifos and cypermethrin in cord blood with excellent sensitivity and specificity and may also be effective for high throughput assay in future epidemiology studies.

In vitro age-dependent enzymatic metabolism of chlorpyrifos and chlorpyrifos-oxon in human hepatic microsomes and chlorpyrifos-oxon in plasma.

Age-dependent chlorpyrifos (CPF) metabolism was quantified by in vitro product formation in human hepatic microsomes (ages 13 days to 75 years) and plasma (ages 3 days to 43 years) with gas chromatography-mass spectrometry. Hepatic CPF cytochrome P450 desulfuration [CPF to chlorpyrifos-oxon (CPF-oxon)] and dearylation (CPF to 3,5,6-trichloro-2-pyridinol) V(max) values were 0.35 ± 0.21 and 0.73 ± 0.38 nmol · min(-1) · mg microsomal protein (-1) (mean ± S.D.), respectively. The mean (±S.D.) hepatic CPF-oxon hydrolysis (chlorpyrifos-oxonase [CPFOase]) V(max) was 78 ± 44 nmol · min(-1) · mg microsomal protein (-1). None of these hepatic measures demonstrated age-dependent relationships on a per microsomal protein basis using linear regression models. Ratios of CPF bioactivation to detoxification (CPF desulfuration to dearylation) V(max) values were consistent across ages. CPFOase in plasma demonstrated age-dependent increases on a volume of plasma basis, as did total plasma protein levels. Mean (±S.D.) CPF-oxon hydrolysis V(max) values for children <6 months of age and adults (≥16 years) were 1900 ± 660 and 6800 ± 1600 nmol · min(-1) · ml(-1), respectively, and at environmental exposure levels, this high- capacity enzyme is likely to be sufficient even in infants. Plasma samples were phenotyped for paraoxonase status, and frequencies were 0.5, 0.4, and 0.1 for QQ, QR, and RR phenotypes, respectively. These results will be integrated into a physiologically based pharmacokinetic and pharmacodynamic model for CPF and, once integrated, will be useful for assessing biological response to CPF exposures across life stages.

Dichlorvos, chlorpyrifos oxon and Aldicarb adducts of butyrylcholinesterase, detected by mass spectrometry in human plasma following deliberate overdose.

The goal of this study was to develop a method to detect pesticide adducts in tryptic digests of butyrylcholinesterase in human plasma from patients poisoned by pesticides. Adducts to butyrylcholinesterase in human serum may serve as biomarkers of pesticide exposure because organophosphorus and carbamate pesticides make a covalent bond with the active site serine of butyrylcholinesterase. Serum samples from five attempted suicides (with dichlorvos, Aldicarb, Baygon and an unknown pesticide) and from one patient who accidentally inhaled dichlorvos were analyzed. Butyrylcholinesterase was purified from 2 ml serum by ion exchange chromatography at pH 4, followed by procainamide affinity chromatography at pH 7. The purified butyrylcholinesterase was denatured, digested with trypsin and the modified peptide isolated by HPLC. The purified peptide was analyzed by multiple reaction monitoring in a QTRAP 4000 mass spectrometer. This method successfully identified the pesticide-adducted butyrylcholinesterase peptide in four patients whose butyrylcholinesterase was inhibited 60-84%, but not in two patients whose inhibition levels were 8 and 22%. It is expected that low inhibition levels will require analysis of larger serum plasma volumes. In conclusion, a mass spectrometry method for identification of exposure to live toxic pesticides has been developed, based on identification of pesticide adducts on the active site serine of human butyrylcholinesterase.