Longitudinal assessment of chlorpyrifos exposure and self-reported neurological symptoms in adolescent pesticide applicators.

OBJECTIVES: Occupational exposure of organophosphorus pesticides, such as chlorpyrifos (CPF), in adolescents is of particular concern because of the potential vulnerability of the developing neurological system. The objectives of this study were to examine how neurological symptoms reported over the application season vary across time, whether these effects are reversible postapplication and if there are associations between CPF biomarkers and neurological symptoms in an adolescent study population. SETTING: The longitudinal study was conducted in two agricultural districts of Menoufia Governorate, Egypt between April 2010 and January 2011. PARTICIPANTS: Male adolescent participants, including CPF applicators (n=57) and non-applicators (n=38), were recruited. PRIMARY AND SECONDARY OUTCOME MEASURES: Self-reported data for 25 neurological symptoms were collected at 32 time points over the 8-month period before, during and after the application season. Additionally, urine and blood samples were collected to measure urine trichloro-2-pyridinol (TCPy), a CPF-specific biomarker and blood cholinesterase activity. RESULTS: Applicators and non-applicators report the highest numbers of symptoms during the application season, followed by a reduction in symptoms after the application ended. Applicators reported a greater percentage of neurological symptoms, relative to baseline, than non-applicators after accounting for potential covariates. Among the applicators, cumulative TCPy was positively and significantly associated with the average percentage of symptoms (B=4.56, 95% CI 3.29 to 5.84; p<0.001). Significant associations (p=0.03-0.07) between the change in butyrylcholinesterase activity from the preapplication to the postapplication season and several domains of neurological symptoms were also found, even after adjusting for potential covariates. CONCLUSIONS: These observations demonstrate changes in the reporting of symptoms across the application season, showing an increase in symptom reporting during application and recovery following the end of pesticide application. These findings reinforce the growing concern regarding the neurotoxic health effects of CPF in adolescent applicators in developing countries and the need for developing and implementing intervention programmes.

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.

PON1 status does not influence cholinesterase activity in Egyptian agricultural workers exposed to chlorpyrifos.

Animal studies have shown that paraoxonase 1 (PON1) genotype can influence susceptibility to the organophosphorus pesticide chlorpyrifos (CPF). However, Monte Carlo analysis suggests that PON1 genotype may not affect CPF-related toxicity at low exposure conditions in humans. The current study sought to determine the influence of PON1 genotype on the activity of blood cholinesterase as well as the effect of CPF exposure on serum PON1 in workers occupationally exposed to CPF. Saliva, blood and urine were collected from agricultural workers (n=120) from Egypt's Menoufia Governorate to determine PON1 genotype, blood cholinesterase activity, serum PON1 activity towards chlorpyrifos-oxon (CPOase) and paraoxon (POase), and urinary levels of the CPF metabolite 3,5,6-trichloro-2-pyridinol (TCPy). The PON1 55 (P≤0.05) but not the PON1 192 genotype had a significant effect on CPOase activity. However, both the PON1 55 (P≤0.05) and PON1 192 (P≤0.001) genotypes had a significant effect on POase activity. Workers had significantly inhibited AChE and BuChE after CPF application; however, neither CPOase activity nor POase activity was associated with ChE depression when adjusted for CPF exposure (as determined by urinary TCPy levels) and stratified by PON1 genotype. CPOase and POase activity were also generally unaffected by CPF exposure although there were alterations in activity within specific genotype groups. Together, these results suggest that workers retained the capacity to detoxify chlorpyrifos-oxon under the exposure conditions experienced by this study population regardless of PON1 genotype and activity and that effects of CPF exposure on PON1 activity are minimal.

Bioactivation of chlorpyrifos by CYP2B6 variants.

Chlorpyrifos (CPF), an organophosphorus (OP) pesticide, is bioactivated by cytochrome P450s (CYPs) to the active metabolite chlorpyrifos oxon (CPF-O). Given that human CYP2B6 has the highest intrinsic clearance (CL(int)) for CPF bioactivation, CYP2B6 polymorphisms may impact human susceptibility to CPF at real world environmental and occupational CPF exposure levels. CYP2B6.4,.5,.7, and .18 were over-expressed in mammalian COS-1 cells to assess the impact of CYP2B6 variants on the K(m) and V(max) for bioactivation of CPF. Cell lysates were incubated with CPF (0-100 µM) and the production of CPF-O was measured via HPLC analysis. CYP2B6 content was determined by western blot. CYP2B6.18 had neither detectable protein nor activity levels. The V(max) value for each remaining variant was significantly higher than wild-type (CYP2B6.1, V(max) 4.13 × 10(4) pmol/min/nmol CYP2B6), with CYP2B6.4,.5, and .7 having V(max) values of 4.52 × 10(5), 1.82 × 10(5), and 9.60 × 10(4) pmol/min/nmol CYP2B6, respectively. The K(m) values for these variants ranged from 0.39 to 1.09 µM and were not significantly different from wild-type. All active variants examined had significantly higher CL(int) than CYP2B6.1. Variants of CYP2B6 have altered capacity to bioactivate CPF and may affect individual susceptibility by altering the V(max) for CPF-O formation.

Effect of CYP2B6*6 and CYP2C19*2 genotype on chlorpyrifos metabolism.

Chlorpyrifos (CPF) is a widely used organophosphorus (OP) pesticide. CPF is bioactivated by cytochrome P450s (CYPs) to the potent cholinesterase inhibitor chlorpyrifos oxon (CPF-O) or detoxified to 3,5,6-trichloro-2-pyridinol (TCPy). Human CYP2B6 has the highest reported Vmax)/Km (intrinsic clearance--CL(int)) for bioactivation while CYP2C19 has the highest reported CL(int) for detoxification of CPF. In this study, 22 human liver microsomes (HLMs) genotyped for common variants of these enzymes (CYP2B6*6 and CYP2C19*2) were incubated with 10 µM and 0.5 µM CPF and assayed for metabolite production. While no differences in metabolite production were observed in homozygous CYP2C19*2 HLMs, homozygous CYP2B6*6 specimens produced significantly less CPF-O than wild-type specimens at 10 µM (mean 144 and 446 pmol/min/mg, respectively). This correlated with reduced expression of CYP2B6 protein (mean 4.86 and 30.1 pmol/mg, for CYP2B6*6 and *1, respectively). Additionally, CYP2B6*1 and CYP2B6*6 were over-expressed in mammalian COS-1 cells to assess for the first time the impact of the CYP2B6*6 variant on the kinetic parameters of CPF bioactivation. The Vmax for CYP2B6*6 (1.05×105 pmol/min/nmol CYP2B6) was significantly higher than that of CYP2B6*1 (4.13×104 pmol/min/nmol CYP2B6) but the K(m) values did not differ (1.97 µM for CYP2B6*6 and 1.84 µM for CYP2B6*1) resulting in CL(int) rates of 53.5 and 22.5 nL/min/nmol CYP2B6 for *6 and *1, respectively. These data suggest that CYP2B6*6 has increased specific activity but reduced capacity to bioactivate CPF in HLMs compared to wild-type due to reduced hepatic protein expression, indicating that individuals with this genotype may be less susceptible to CPF toxicity.

Biomarkers of chlorpyrifos exposure and effect in Egyptian cotton field workers.

BACKGROUND: Chlorpyrifos (CPF), a widely used organophosphorus pesticide (OP), is metabolized to CPF-oxon, a potent cholinesterase (ChE) inhibitor, and trichloro-2-pyridinol (TCPy). Urinary TCPy is often used as a biomarker for CPF exposure, whereas blood ChE activity is considered an indicator of CPF toxicity. However, whether these biomarkers are dose related has not been studied extensively in populations with repeated daily OP exposures. OBJECTIVE: We sought to determine the relationship between blood ChE and urinary TCPy during repeated occupational exposures to CPF. METHODS: Daily urine samples and weekly blood samples were collected from pesticide workers (n=38) in Menoufia Governorate, Egypt, before, during, and after 9-17 consecutive days of CPF application to cotton fields. We compared blood butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) activities with the respective urinary TCPy concentrations in each worker. RESULTS: Average TCPy levels during the middle of a 1- to 2-week CPF application period were significantly higher in pesticide applicators (6,437 µg/g creatinine) than in technicians (184 µg/g) and engineers (157 µg/g), both of whom are involved in supervising the application process. We observed a statistically significant inverse correlation between urinary TCPy and blood BuChE and AChE activities. The no-effect level (or inflection point) of the exposure-effect relationships has an average urinary TCPy level of 114 µg/g creatinine for BuChE and 3,161 µg/g creatinine for AChE. CONCLUSIONS: Our findings demonstrate a dose-effect relationship between urinary TCPy and both plasma BuChE and red blood cell AChE in humans exposed occupationally to CPF. These findings will contribute to future risk assessment efforts for CPF exposure.