Grantee Research Project Results
2004 Progress Report: Center for Children’s Environmental Health Research – Mechanisms of Pesticide Neuro- and Immunotoxicity
EPA Grant Number: R831710C003Subproject: this is subproject number 003 , established and managed by the Center Director under grant R831710
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Center for Integrative Research on Childhood Leukemia and the Environment - 2015
Center Director: Metayer, Catherine
Title: Center for Children’s Environmental Health Research – Mechanisms of Pesticide Neuro- and Immunotoxicity
Investigators: Holland, Nina T.
Current Investigators: Holland, Nina T. , Tager, Ira , Lipsett, Michael , Casida, John
Institution: University of California - Berkeley
EPA Project Officer: Callan, Richard
Project Period: May 1, 2004 through October 31, 2008 (Extended to October 31, 2010)
Project Period Covered by this Report: May 1, 2004 through October 31, 2005
RFA: Centers for Children's Environmental Health and Disease Prevention Research (2003) RFA Text | Recipients Lists
Research Category: Human Health , Children's Health
Objective:
The objectives of this research project are to:
- Examine the in vitro effects of the organophosphate (OP) pesticide chlorpyrifos, the carbamate maneb, and dust mite allergen, alone and in combination, on the production of Th1/Th2 cytokines. Blood will be collected from five atopic adult donors, treated with the compounds in vitro, and the Th1/Th2 cytokine dose responses will be determined by Taqmanâ real-time polymerase chain reaction, flow cytometric detection of intracellular cytokines, and enzyme-linked immunosorbent assay (ELISA).
- Determine whether pesticide and allergen exposures differentially affect in vitro cytokine expression in lymphocytes of children versus those of adults. The treatment protocol optimized in Objective 1 for human lymphocytes will be used to investigate differences between the responses in cells of children and adults to pesticide and allergen exposures in vitro, using blood samples from 10 atopic maternal-child pairs recruited from the Center for Health Assessment of Mothers and Children of Salinas (CHAMACOS) cohort.
- Establish a mechanism-based new enzymatic assay for neuropathy target esterase (NTE) in human adult lymphocytes and neuroblastoma cell line. We will evaluate fully the new mechanism-based assay (currently only examined in cells of two adult donors) as comparable to the conventional procedure in fresh, frozen, and cryopreserved human cells, and in neuroblastoma cell line. We also will evaluate a modification of the assay, which has increased sensitivity and requires fewer cells.
- Determine the ontogenetic changes in NTE activity in human fetus, child, and adult. Evidence from animals indicates that NTE activity decreases with age. This differs from trends observed with other enzymes. We will measure NTE activity in cells from stored CHAMACOS maternal, child, and cord blood.
- Determine the sensitivity of NTE in fetal and adult lymphocytes to known NTE inhibitors and to test the relative sensitivity of NTE and acetylcholinesterase (AChE) to these inhibitors. Evidence indicates that younger animals are resistant to pesticide-delayed neurotoxicity compared to adults, even though the NTE inhibition is similar. We will treat human lymphocytes from children and adults with chlorpyrifos-oxon (CPO), mipafox, and phenyl saligenin cyclic phosphate in vitro and determine the dose-response relationship for NTE and AChE inhibition.
- Determine the sensitivity of NTE in fetal versus adult lymphocytes to widely used pesticides in their bioactivated form. Many of the inhibitors of NTE that have been studied are chemicals that are not used widely and not relevant to human exposure. Nine out of 20 of the pesticides most widely used in the Salinas Valley are potential NTE inhibitors. We will employ the in vitro system used for Objective 5 to evaluate the effect of these pesticides on NTE activity in cells of children and adults from the CHAMACOS cohort.
Progress Summary:
Overview
The primary goal of this research project is to investigate the mechanisms of pesticide neuro- and immunotoxicity and to evaluate differential cellular responses of neonates, young children, and adults to pesticide exposure. To understand potential associations between pesticide exposure and adverse neurological and respiratory outcomes being studied in the Community-Based Participatory Research, we will identify and investigate the roles of specific molecules involved in pathways we hypothesize to be causally linked to the development of these outcomes. Specifically, we will focus on two recently identified and thus less studied pathways of pesticide neuropathy and immunotoxicity: delayed and subtle forms of neuropathy that may be associated with NTE and altered Th1/Th2 cytokine production by immune cells in vivo and in vitro.
Objective 1. We expanded methods of immunological assessment by including two Luminex-based platforms for cytokine analysis of 10 different cytokines. We evaluated the effects of the pesticide chlorpyrifos (CPF) and its metabolitesCPO and 3,5,6-trichloro-2-pyridinol (TCP) on cytokine secretion, a biological marker of immune function. To model the exposure patterns of agricultural workers and their families, we designed our experiments to specifically address the effects of combined pesticide and bacterial endotoxin exposures on cytokine expression. Human whole blood was treated with CPO/TCP (1-10,000 μg/mL) and LPS (1.5-2.5 μg/mL), either singly or in combination, for 2-72 hours. At 2, 24, 48, and 72 hours supernatants were collected and assayed for production of interferon-g (IFN-γ) and interleukin-4 (IL-4) cytokines by ELISA. No expression of IFN-γ was detected in cultures treated with CPO or TCP alone, whereas cultures treated with pesticides in combination with LPS expressed higher levels of IFN-γ with some combinations inducing greater IFN-γ expression than LPS alone (p<0.01). IL-4 expression was observed only at the highest single dose that neared cytotoxicity. We confirmed these results using Luminex multiplex cytokine analysis.
Objective 2. Efforts to address this objective will begin when biological sample collection from 60-month-old children has been completed (expected in February 2005). All scopes of work have been prepared and refresher training of laboratory and field staff is in progress.
Objective 3. The NTE research has focused on developing a mechanism-based assay. The old procedure used a model substrate, phenyl valerate, when the actual endogenous substrate is now known to be lysolecithin. Developmental studies are underway to use lysolecithin directly in assaying NTE, now known as NTE-lysophospholipase.
Objectives 4-6. Preparation and validation of methods for these objectives are in progress.
Obstacles Encountered
The delay in funding and complicated logistics of the move of the Laboratory of Children’s Environmental Health to a new larger space at the Marchant building, allocated by the University of California at Berkeley, have been difficulties for this study.
Significance
To date, virtually no data have been published on cytokine induction or inhibition by pesticides in human cells in vitro. The novelty of these objectives includes their use of in vitro treatment to study mixtures of pesticides and allergens—exposures which are typical of children (such as in our cohort) who live in an agricultural community. Our study aims to conduct multiple assessments of cytokine expression in the cells of young children during critical periods of maturation of their immune systems. Immunological biomarkers in young children have not been well explored and thus, this study also will shed light on the ontogeny of the human immune system. Finally, an important advantage of our approach is that we will be able to use in vitro cell cultures derived from cryopreserved pediatric CHAMACOS samples as a model for interpretation of in vivo observations of immunological markers and respiratory effects in young children from the same cohort. Thus, the proposed research plan is innovative in that it will produce new information, augmenting a limited body of literature, and will be relevant to our interpretation of findings in the Center’s respiratory component.
Objective 5 focuses on the comparative assessment of the dynamics of NTE enzymatic activity from birth through early childhood and during adulthood. The primary reason for the lack of data on this subject is that repeated sampling of young infants and their mothers is necessary. Thus, the opportunity presented by the CHAMACOS cohort to conduct this comparative assessment is unique and will generate novel data. These data will be important in understanding mechanisms of neurotoxicity in the early stages of life. A new radioactivity-based method for analyses of NTE was established for mouse brains to be adopted in the next several months for frozen human lymphocytes.
Future Activities:
We plan to conduct the challenging final validation of all methods of sample collection and analyses for the new assays, including NTE from children and flow cytometry in preparation for the 5 year (60 months) collection starting in February 2005. The major focus will be on conducting in vitro experiments with OP (CPF and malathion) and their metabolites, alone and in combination with endotoxin and allergen in adult blood from laboratory volunteers.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other subproject views: | All 21 publications | 14 publications in selected types | All 13 journal articles |
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Other center views: | All 168 publications | 142 publications in selected types | All 134 journal articles |
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Casida JE, Quistad GB. Organophosphate toxicology: safety aspects of nonacetylcholinesterase secondary targets. Chemical Research in Toxicology 2004;17(8):983-998. |
R831710 (2004) R831710 (2005) R831710C003 (2004) |
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Duramad P, McMahon CW, Hubbard A, Eskenazi B, Holland NT. Flow cytometric detection of intracellular TH1/TH2 cytokines using whole blood: validation of immunologic biomarker for use in epidemiologic studies. Cancer Epidemiology, Biomarkers & Prevention 2004;13(9):1452-1458. |
R831710 (2004) R831710 (2005) R831710 (Final) R831710C003 (2004) |
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Quistad GB, Casida JE. Lysophospholipase inhibition by organophosphate toxicants. Toxicology and Applied Pharmacology 2004;196(3):319-326. |
R831710 (2004) R831710 (2005) R831710C003 (2004) |
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Supplemental Keywords:
organophosphate, carbamate, dithiocarbamate, mixtures, allergen, environmental management, health, pesticides, children’s health, health risk assessment, pesticide types, risk assessment, human health risk assessment, agricultural community, airway disease, allergen, assessment of exposure, childhood respiratory disease, children’s environmental health, community-based intervention, environmental health hazard, environmental risks, exposure assessment, health effects, immune system effects, insecticides, outreach and education, pesticide exposure,, RFA, Health, Scientific Discipline, ENVIRONMENTAL MANAGEMENT, PESTICIDES, Health Risk Assessment, Children's Health, Pesticide Types, Risk Assessment, health effects, pesticide exposure, community-based intervention, immune system effects, airway disease, environmental risks, respiratory problems, Human Health Risk Assessment, assessment of exposure, childhood respiratory disease, insecticides, children's environmental health, environmental health hazard, outreach and education, agricultural community, allergenRelevant Websites:
http://ehs.sph.berkeley.edu/chamacos Exit
Progress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R831710 Center for Integrative Research on Childhood Leukemia and the Environment - 2015 Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R831710C001 Center for Children’s Environmental Health Research – CHAMACOS Community Based Research Project
R831710C002 Center for Children’s Environmental Health Research – Pesticide Exposure Assessment Project
R831710C003 Center for Children’s Environmental Health Research – Mechanisms of Pesticide Neuro- and Immunotoxicity
R831710C004 Center for Children’s Environmental Health Research – Community Outreach and Translation Core
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.
Project Research Results
13 journal articles for this subproject
Main Center: R831710
168 publications for this center
134 journal articles for this center