Longitudinal assessment of occupational exposures to the organophosphorous insecticides chlorpyrifos and profenofos in Egyptian cotton field workers.

Chlorpyrifos (CPF) and profenofos (PFF) are organophosphorus (OP) insecticides that are applied seasonally in Egypt to cotton fields. Urinary trichloro-2-pyridinol (TCPy), a specific CPF metabolite, and 4-bromo-2-chlorophenol (BCP), a specific PFF metabolite, are biomarkers of exposure, while inhibition of blood butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) activities are effect biomarkers that may be associated with neurotoxicity. Urinary TCPy and BCP and blood BChE and AChE activities were measured in 37 adult Egyptian Ministry of Agriculture workers during and after 9-17 consecutive days of CPF application followed by an application of PFF (9-11 days), and a second CPF application (5 days) in 2008. During the OP applications, urinary TCPy and BCP levels were significantly higher than baseline levels, remained elevated following the application periods, and were associated with an exposure related inhibition of blood BChE and AChE. Analysis of blood AChE levels before and after the PFF application period suggests that individual workers with peak BCP levels greater than 1000 µg/g creatinine exhibited further inhibition of blood AChE with PFF application, demonstrating that PFF exposure had a negative impact on AChE activity in this highly exposed worker population. While large interindividual differences in exposure were observed throughout this longitudinal study (peak urinary BCP and peak TCPy levels for individuals ranging from 13.4 to 8052 and 16.4 to 30,107 µg/g creatinine, respectively), these urinary biomarkers were highly correlated within workers (r=0.75, p<0.001). This suggests that the relative exposures to CPF and PFF were highly correlated for a given worker. The variable exposures between job classification and work site suggest that job title and work location should not be used as the sole basis for categorizing OP exposures when assessing neurobehavioral and other health outcomes in Egyptian cotton field workers. Together, these findings will be important in educating the Egyptian insecticide application workers in order to encourage the development and implementation of work practices and personal protective equipment to reduce their exposure to CPF and PFF.

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.

Mouse divalent metal transporter 1 is a copper transporter in HEK293 cells.

Divalent Metal Transporter 1 (DMT1) is an apical Fe transporter in the duodenum and is involved in endosomal Fe export. Four protein isoforms have been described for DMT1, two from mRNA with an iron responsive element (IRE) and two from mRNA without it. The sets of two begin in exon 1A or 2. We have characterized copper transport using mouse 2/-IRE DMT1 during regulated ectopic expression. HEK293 cells carrying a TetR:Hyg element were stably transfected with pDEST31 containing a 2/-IRE construct. (64)Cu(1+) incorporation in doxycycline treated cells exhibited 18.6 and 30.0-fold increases in Cu content, respectively when were exposed to 10 and 100 µM of extracellular Cu. Cu content was ~4-fold above that of parent cells or cells carrying just the vector. (64)Cu uptake in transfected cells pre-incubated with 5 µM of Cu-His revealed a Vmax and Km of 11.98 ± 0.52 pmol mg protein(-1) min(-1) and 2.03 ± 0.03 µM, respectively. Doxycycline-stimulated Cu uptake was linear with time. The rates of apical Cu uptake decreased and transepithelial transport increased when intracellular Cu increased. The optimal pH for Cu transport was 6.5; uptake of Cu was temperature dependent. Silver does not inhibit Cu uptake in cells carrying the vector. In conclusion, Cu uptake in HEK293 cells that over-expressed the 2/-IRE isoform of DMT1 transporter supports our earlier contention that DMT1 transports Cu as Cu(1+).

Characterization of α-cypermethrin exposure in Egyptian agricultural workers.

Pyrethroids are neurotoxic insecticides that exert their effects by prolonging the open time of sodium channels, which increases the duration of neuronal excitation. α-cypermethrin (αCM) is derived from the 8-stereoisomers that together make up the pyrethroid cypermethrin, which is one of the most common pyrethroids being used in agriculture throughout the world. The objective of this study was to characterize the occupational exposure to αCM in a cohort of Egyptian agriculture workers (n=37) before, during and after 6-10 consecutive days of application of αCM to cotton fields. Daily spot urine specimens were collected and analyzed by GC-MS NCI for the αCM metabolites 3-phenoxybenzoic acid (3-PBA) and cis-3-(2',2'-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (cis-DCCA). Prior to αCM application, median urinary levels of 3-PBA (4.59nmol/g creatinine) were greater than cis-DCCA (0.33nmole/g creatinine) demonstrating low background exposures to pyrethroids. During the application period for αCM, median urinary levels of both biomarkers increased (13.44nmol 3-PBA/g creatinine and 7.76nmol cis-DCCA/g creatinine) and ranged from 2.3-93.96nmol 3-PBA/g creatinine and 0.09-90.94nmol cis-DCCA/g creatinine, demonstrating that workers had a wide range of exposures to αCM. The data also demonstrate that pesticide applicators had greater exposures to αCM than workers who play a supporting role in the seasonal application of pesticides on the cotton crop. Urinary cis-DCCA and 3-PBA concentrations were elevated at 7-11 days after the cessation of αCM application, compared to baseline levels. This study is the first to use these biomarkers to quantify occupational exposures specifically to αCM. This urinary biomarker data will be useful for estimating daily internal dose, comparing exposures across job categories within the Egyptian pesticide application teams, and for modeling human exposures to αCM.

Metabolism of profenofos to 4-bromo-2-chlorophenol, a specific and sensitive exposure biomarker.

Profenofos is a direct acting phosphorothioate organophosphorus (OP) pesticide capable of inhibiting ß-esterases such as acetylcholinesterase, butyrylcholinesterase, and carboxylesterase. Profenofos is known to be detoxified to the biologically inactive metabolite, 4-bromo-2-chlorophenol (BCP); however, limited data are available regarding the use of urinary BCP as an exposure biomarker in humans. A pilot study conducted in Egyptian agriculture workers, demonstrated that urinary BCP levels prior to application (3.3-30.0 µg/g creatinine) were elevated to 34.5-3,566 µg/g creatinine during the time workers were applying profenofos to cotton fields. Subsequently, the in vitro enzymatic formation of BCP was examined using pooled human liver microsomes and recombinant human cytochrome P-450s (CYPs) incubated with profenofos. Of the nine human CYPs studied, only CYPs 3A4, 2B6, and 2C19 were able to metabolize profenofos to BCP. Kinetic studies indicated that CYP 2C19 has the lowest Km, 0.516 µM followed by 2B6 (Km=1.02 µM) and 3A4 (Km=18.9µM). The Vmax for BCP formation was 47.9, 25.1, and 19.2 nmol/min/nmol CYP for CYP2B6, 2C19, and 3A4, respectively. Intrinsic clearance (Vmax/Km) values of 48.8, 46.9, and 1.02 ml/min/nmol CYP 2C19, 2B6, and 3A4, respectively, indicate that CYP2C19 and CYP2B6 are primarily responsible for the detoxification of profenofos. These findings support the use of urinary BCP as a biomarker of exposure to profenofos in humans and suggest polymorphisms in CYP 2C19 and CYP 2B6 as potential biomarkers of susceptibility.

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.

Effect of glutamate and riluzole on manganese-induced apoptotic cell signaling in neuronally differentiated mouse P19 Cells.

Excess exposure to Mn causes a neurological disorder known as manganism which is similar to dystonic movements associated with Parkinson's disease. Manganism is largely restricted to occupations in which high atmospheric levels are prevalent which include Mn miners, welders and those employed in the ferroalloy processing or related industrial settings. T1 weighted MRI images reveal that Mn is deposited to the greatest extent in the globus pallidus, an area of the brain that is presumed to be responsible for the major CNS associated symptoms. Neurons within the globus pallidus receive glutamatergic input from the subthalamic nuclei which has been suggested to be involved in the toxic actions of Mn. The neurotoxic actions of Mn and glutamate are similar in that they both affect calcium accumulation in the mitochondria leading to apoptotic cell death. In this paper, we demonstrate that the combination of Mn and glutamate potentiates toxicity of neuronally differentiated P19 cells over that observed with either agent alone. Apoptotic signals ROS, caspase 3 and JNK were increased in an additive fashion when the two neurotoxins were combined. The anti-glutamatergic drug, riluzole, was shown to attenuate these apoptotic signals and prevent P19 cell death. Results of this study confirm, for the first time, that Mn toxicity is potentiated in the presence of glutamate and that riluzole is an effective antioxidant which protects against both Mn and glutamate toxicity.

DMT1: which metals does it transport?

DMT1-Divalent Metal (Ion) Transporter 1 or SLC11A2/DCT1/Nramp2 - transports Fe2+ into the duodenum and out of the endosome during the transferrin cycle. DMTI also is important in non-transferrin bound iron uptake. It plays similar roles in Mn2+ trafficking. Voltage clamping showed that six other metals evoked currents, but it is unclear if these metals are substrates for DMT1. This report summarizes progress on which metals DMT1 transports, focusing on results from the authors' labs. We recently cloned 1A/+IRE and 2/-IRE DMT1 isoforms to generate HEK293 cell lines that express them in a tetracycline-inducible fashion, then compared induced expression to uninduced expression and to endogenous DMT1 expression. Induced expression increases approximately 50x over endogenous expression and approximately 10x over uninduced levels. Fe2+, Mn2+, Ni2+ and Cu1+ or Cu2+ are transported. We also explored competition between metal ions using this system because incorporation essentially represents DMT1 transport and find this order for transport affinity: Mn>?Cd>?Fe>Pb-Co-Ni>Zn. The effects of decreased DMT1 also could be examined. The Belgrade rat has diminished DMT1 function and thus provides ways of testing. A series of DNA constructs that generate siRNAs specific for DMT1 or certain DMT1 isoforms yield another way to test DMT1-based transport.

DMT1: Which metals does it transport?

DMT1 _ Divalent Metal (Ion) Transporter 1 or SLC11A2/DCT1/Nramp2 _ transports Fe2+ into the duodenum and out of the endosome during the transferrin cycle. DMT1 also is important in non-transferrin bound iron uptake. It plays similar roles in Mn2+ trafficking. Voltage clamping showed that six other metals evoked currents, but it is unclear if these metals are substrates for DMT1. This report summarizes progress on which metals DMT1 transports, focusing on results from the authors' labs. We recently cloned 1A/+IRE and 2/-IRE DMT1 isoforms to generate HEK293 cell lines that express them in a tetracycline-inducible fashion, then compared induced expression to uninduced expression and to endogenous DMT1 expression. Induced expression increases about 50x over endogenous expression and about 10x over uninduced levels. Fe2+, Mn2+, Ni2+ and Cu1+ or Cu2+ are transported. We also explored competition between metal ions using this system because incorporation essentially represents DMT1 transport and find this order for transport affinity: Mn>?Cd>?Fe>Pb Co Ni>Zn. The effects of decreased DMT1 also could be examined. The Belgrade rat has diminished DMT1 function and thus provides ways of testing. A series of DNA constructs that generate siRNAs specific for DMT1 or certain DMT1 isoforms yield another way to test DMT1-based transport.