Grantee Research Project Results
2002 Progress Report: Environmental Factors in the Etiology of Autism; Molecular and Cellular Mechanisms of Autism
EPA Grant Number: R829388C006Subproject: this is subproject number 006 , established and managed by the Center Director under grant R829388
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: UC Davis Center for Children's Environmental Health and Disease Prevention
Center Director: Van de Water, Judith
Title: Environmental Factors in the Etiology of Autism; Molecular and Cellular Mechanisms of Autism
Investigators: Pessah, Isaac N. , Gershwin, M. Eric , Goth, Samuel R. , Matsumura, Fumio , Van de Water, Judith
Current Investigators: Pessah, Isaac N. , Goth, Samuel R. , Van de Water, Judith
Institution: University of California - Davis
EPA Project Officer: Aja, Hayley
Project Period: September 30, 2001 through September 29, 2002
Project Period Covered by this Report: September 30, 2001 through September 29, 2002
RFA: Centers for Children's Environmental Health and Disease Prevention Research (2001) RFA Text | Recipients Lists
Research Category: Children's Health , Human Health
Objective:
How thimerosal and polybrominated diphenylethers, major environmental concerns to childhood neurodevelopment, influences peripheral immune cells in the B6 murine model has been a primary focus in year 1. Work has begun on assessing the influence of these xenobiotics on the growth and development of glial/hippocampal neurons in culture. The long-term goal is to define common mechanisms in immune and neural cells responsible for developmental toxicity of these environmental agents. Our specific aims are: 1)To define differences in the response of peripheral immune cells (PMBCs) isolated from autistic and non-autistic children in their sensitivity and/or pattern of cell activation and cytokine secretion when challenged in vitro with vaccine antigens, and xenobiotics of environmental relevance (thimerosal, PCBs, and BDEs), singly and in combination.2) Elucidate the mechanisms by which sub-toxic concentrations of (thimerosal, PCBs, and BDEs), singly and in combination, alter growth and activation properties of immune cells from rodents.3) Elucidate the mechanisms by which sub-toxic concentrations of (thimerosal, PCBs, and BDEs), singly and in combination, influence glia/neural cell interactions leading to altered patterns of dendritic spine growth, dendritic branching and synaptogenesis in hippocampal cells from rodents.
Progress Summary:
We are anticipating beginning work on PMBCs from the cohort in September. Although Dr. Van de Water has begun studies on cellular biomarkers of autism using tissue samples banked by the M.I.N.D Institute, we have decided to delay work on Aim 1 until samples are available from the cohort beginning in September as initially planned. However progress made on Aims II and III (described below) looking at the molecular and cellular mechanisms by which thimerosal and halogenated organics influence growth, maturation and function of immune and neuron/glia cultures from murine tissues. These have already produced important new information and are invaluable to execution and interpretation of Aim I.
We have focused our initial studies on the immunotoxicology of thimerosal, PCBs and BDEs toward dendritic cells. We felt this was warranted since dendritic cells (DC) reside in most peripheral tissues, particularly at sites of interface with the environment (e.g., skin and mucosae). Although DC make up 1-2% of the total immune cell number, they contribute a “frontline” towards engaging immune defenses by taking up antigens in peripheral tissues, processing them into proteolytic peptides, and loading these peptides onto major histocompatibility complex (MHC) class I and II molecules. DC then migrate to secondary lymphoid organs and become competent to present antigens to T lymphocytes, thus initiating antigen-specific immune responses, or immunological tolerance. Antigen presentation by dendritic cells is finely regulated: antigen uptake, intracellular transport and degradation, and the traffic of MHC molecules are different in dendritic cells as compared to other antigen-presenting cells. These specializations account for dendritic cells’ unique role in the initiation of primary immune responses and the induction of tolerance, and provide a strong rationale for examining the actions of our test xenobiotics on DC maturation and function.
Using MHCII and CD11c as selection markers, we have isolated DC from murine bone marrow and fetal liver in sufficient numbers to test specific hypotheses concerning whether thimerosal and other xenobiotics of concern to childhood autism influence DC maturation and their ability to activate T-cells. Within 20h in medium containing GM-CSF, approximately one third of DC possessed immunological (class II MHC) and morphological (dendrites) markers of maturation. Under basal conditions the majority of cells were small (as indicated by low forward scatter), exhibited high cell viability (indicated by excluding propidium iodide (PI) staining), and ~30% elaborated the DC maturation marker CD86. In response to LPS, a promoter of DC maturation, the mean cell size significantly increased and the fraction of DC exhibiting CD86 doubled (52%). Brominated diphenylethers (BDE) widely used as flame retardants in the United States are an emerging concern because their non-coplanar structures may exhibit a similar spectrum of developmental neurotoxicity already documented for ortho-substituted polychlorinated biphenyls (PCBs). Although 20h exposure to BDE4 did not increase mean cell size, nor enhance cytotoxicity compared to medium control, BDE4 consistently (n=5) promoted maturation of DCs -as measured by class II MHC and CD86 expression levels (20-50%) at concentrations as low as 500 nM (Fig 2, third column). Non-coplanar PCB95 (2,2’,3,5’,6 pentachlorobiphenyl) produced similar effects as BDE4 on DC cell maturation, indicating similar mechanisms.
Until it was discontinued in 2001, thimerosal has been widely used to preserve multiuse vaccine formulations. The widespread use of thimerosal in hundreds of other products including medications and cosmetics persists, although little is known about its immunotoxic and neurotoxic properties. We have explored the actions of this organic mercurial on DC maturation. Most significantly as little as 500nM thimerosal (20h) had dramatic influence on DC cultures (Fig 2, fourth column). 20h exposure to thimerosal completely abrogated DC maturation measured by CD86 and class II MHC expression (Fig 2, fourth column). Cell size of thimerosal treated DCs was small compared to medium controls, indicating a failure of smaller progenitor cells to mature into DCs (compare w/LPS treated cells). Many of the thimerosal treated cells were permeable to propidium iodide, indicating cell death. These are the first results defining the immunotoxicity of thimerosal on this important class of antigen presenting cell and provides impetus for more detailed studies of mechanism and relevance to autism.
Non-coplanar PCBs are widely known to disrupt Ca2+ signaling in muscle and neurons. We have previously published that the ryanodine receptor (RyR) is a selective target of non-coplanar PCBs in these tissue. Furthermore the mechanism appears to require association of RyR with a major immunophilin (FKBP12 or FKBP12.6). Because immature DC were recently shown to express functional RyR1, and BDE share several structural similarities to PCB, we examined the possible influence of BDE4 on RyR1. BDE4 like PCB95, exhibited high efficacy toward enhancing RyR1 activation, whereas BDE15 (4,4’-dibromodiphenylether) and PCB66 lacked activity. These results indicate that ortho-substituted BDE may have a stringent structure-activity relationship that may parallel that of PCBs. Both the mechanism by which PCB and BDE structures alter Ca2+ signaling in DC and their mechanism(s) will be explored in detail.
We are actively pursuing research on how xenobiotics influence murine glia in term of growth, morphology and secretion of neuroactive cytokines. The approach is focuses on the mechanisms underlying observed changes in cell signaling, especially Ca2+ signaling. In collaboration with colleagues at NICHHD, we recently showed that oligodendrocyte progenitors express both IP 3 and ryanodine receptors in a spatially segregated pattern. Importantly these microsomal channels emit elemental units of Ca2+ release that are distinct in morphology yet exhibit a significant amount of cross-talk. This discovery provides a basis on which we will define exactly how thimerosal and halogenated organics (PCB and BDE) of interest to the Center alter these intracellular signals.
Future Activities:
We will define the mechanism by which thimerosal, PCBs and BDEs alter the function of dendritic cells and their ability to activate T cells. The neurotoxicity of these agent are being explored in primary neuronal/glial cultures and hippocampal slices. In year two, work will begin defining the responses of PMBCs collected from the CHARGE study.
Journal Articles:
No journal articles submitted with this report: View all 23 publications for this subprojectSupplemental Keywords:
Autism, thimerosal, halogenated organics, cell signaling, immunotoxicology, neurotoxicology,, RFA, Health, PHYSICAL ASPECTS, Scientific Discipline, ENVIRONMENTAL MANAGEMENT, Health Risk Assessment, Chemistry, Risk Assessments, Susceptibility/Sensitive Population/Genetic Susceptibility, Disease & Cumulative Effects, Physical Processes, Children's Health, genetic susceptability, Biology, Risk Assessment, chemical exposure, neurotoxic, xenobiotics, biomarkers, neurodevelopment, gene-environment interaction, pesticides, exposure, halogenated aromatics, neurobehavioral, neurodevelopmental, neurotoxicity, children, susceptibility, etiology, neurobehavioral effects, autism, mechanisms, biological markers, exposure assessment, neurological development, biomarker, synergistic interactionsProgress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R829388 UC Davis Center for Children's Environmental Health and Disease Prevention Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R829388C001 Environmental Factors in the Etiology of Autism; Analytic Biomakers (xenobiotic) Core
R829388C002 Environmental Factors in the Etiology of Autism; Cell Activation/Signaling Core
R829388C003 Environmental Factors in the Etiology of Autism; Molecular Biomakers Core
R829388C004 Environmental Factors in the Etiology of Autism; Childhood Autism Risks from Genetics and the Environment (The CHARGE Study)
R829388C005 Environmental Factors in the Etiology of Autism; Animal Models of Autism
R829388C006 Environmental Factors in the Etiology of Autism; Molecular and Cellular Mechanisms of Autism
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
21 journal articles for this subproject
Main Center: R829388
175 publications for this center
157 journal articles for this center