[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.

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.

[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.

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.

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.

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.

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.

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.

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.