2009 Progress Report: Research Project C: Perinatal Environmental Exposure Disparity and Neonatal Respiratory Health

EPA Grant Number: R833293C003
Subproject: this is subproject number 003 , established and managed by the Center Director under grant R833293
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Southern Center on Environmentally Driven Disparities in Birth Outcomes
Center Director: Miranda , Marie Lynn
Title: Research Project C: Perinatal Environmental Exposure Disparity and Neonatal Respiratory Health
Investigators: Auten, Richard , Foster, W. Michael
Institution: Duke University
EPA Project Officer: Callan, Richard
Project Period: May 1, 2007 through April 30, 2012 (Extended to April 30, 2014)
Project Period Covered by this Report: May 1, 2009 through April 30,2010
RFA: Centers for Children’s Environmental Health and Disease Prevention Research (2005) RFA Text |  Recipients Lists
Research Category: Health Effects , Children's Health , Health


  1. To determine whether maternal exposure to airborne particulates (PM) and/or ozone (1st hit) restricts fetal growth and/or postnatal growth, and impairs lung development/function in newborn mice;
  2. To determine whether PM and/or ozone exposure ‘re-programs’ maternal inflammatory responses;
  3. To determine whether postnatal (2nd hit) ozone exposure further impairs postnatal somatic and lung development/function following maternal PM and/or ozone exposures;
  4. To determine whether genetic or developmental susceptibility to airway hyperreactivity exacerbates maternal and/or postnatal exposure effects on postnatal somatic and lung development/function.


Progress Summary:

  1. Repeated exposures using spontaneously inhaled diesel particles generated from an internal combustion engine at US EPA have demonstrated dose-dependent augmentation on ozone-induced airway hyperresponsiveness.
  2. This effect is sustained even after ceasing ozone exposure, since mice born to diesel exposed dams recovered for 4 weeks after neonatal ozone continue to demonstrate augmentation of airway hyperresponsiveness. This shows that prenatal diesel exposure has durable effects that persist to adulthood.
  3. The diesel exposure appears to mediating these effects in utero, since pro-inflammatory cytokines are elevated in fetal lung, brain, and placenta harvested from fetuses exposed in utero to maternal diesel inhalation. The precise pathway is not yet known, but we are currently conducting immunohistochemistry studies to identify the cellular compartments in the fetus that are affected first. This will allow us to design better studies to determine the path by which the pollutant affects fetal and neonatal development.
  4. We have repeated the studies described above using diesel particles collected in collaboration with US EPA, and then instilled by tracheal insufflation during pregnancy, and found identical effects on fetal inflammatory cytokines in lung, placenta, and brain, as well as juvenile lung inflammatory cytokines. Most important, we found the same augmentation of ozone-induced airway hyperreactivity. This allows us to quickly move to more detailed analysis of the mechanism for pollutant effects without having to rely on ambient inhalation exposures, which cannot be accomplished at the desired rate because so many other investigators use the facilities.
  5. Psychological stressors can contribute to the humoral and neural programming events in early life that would potentially affect a variety of the health outcomes we are studying. We have accordingly begun studies designed to mimic resource deprivation of nesting material during pregnancy and early postnatal rearing, as an analog to the stressors identified in Project R833293C001 and R833293C002. Our preliminary studies in collaboration with Staci Bilbo, Dept. of Psychology & Neuroscience at Duke, demonstrate significant effects on postnatal weight gain in mice, and the addition of postnatal ozone increased mortality of pups to nearly 45%. We are presently conducting additional exposures at lower “doses” to determine if combined exposures to stresses below the typical effect threshold combine to yield an effect not seen with individual stressors. This new effort is funded in part by a new pilot grant through P30 ES-011961-01A1. An additional application through the Duke Integrative Brain Sciences program is pending, and a letter of intent has been submitted to the Department of Defense.
  6. In order to determine whether the effects of combined pollutant exposure on airway hyperresponsiveness is mediated by changes in neural or airway smooth muscle programming, we evaluated the effects of immediate postnatal ozone exposure on airway mechanics in anesthetized mice, as well as in tracheal explants. We found that the airway hyperresponsiveness was not attributable to large effects on either airway smooth muscle bulk or on intrinsic airway smooth muscle responsiveness to neurotransmitter or electrical stimulation. There were some trends towards delayed relaxation in the ozone-exposed group. We next repeated the studies in animals that underwent cervical vagotomy at 6 weeks (2 weeks recovery after the 4 weeks of intermittent ozone exposure) just before measurement of airway mechanics. We found that vagotomy eliminated the ozone-induced airway hyperresponsiveness. This represents a paradigm shift of our understanding of the mechanisms by which ozone might affect asthma in children. 
Collaborations with other SCEDDBO Components
  1. Modification of study design:  As noted above, the effects of resource deprivation suggested by findings in Projects R833293C001 and R833293C002 prompted us to add the resource deprivation (nesting restriction) component to Project R833293C003 in order to test the proof-of-principle that the combination of multiple stressors/environmental contaminants may affect health even when the individual exposures do not.
  2. Because the main findings in Project R833293C002 pertain to fetal growth restriction and low birthweight, we are now designing studies in the animal model to mimic impaired uteroplacental insufficiency.


Future Activities:

  1. Determine whether the combination of ozone/diesel exposure with resource restriction impairs postnatal airway hyperresponse and postnatal cognition. Because the fetal brain cytokine elevations following environmentally relevant diesel exposures were significant, we think it will be important to determine if there are neurobehavioral effects from the combined early-life contaminant exposures.
  2. We will begin to develop a refinement of our multi-agent rodent models by adding uterine artery flow restriction in a rat model, or thromboxane infusion to the mouse model. Although all of our previous work has been done with the mouse models, the addition of fetal growth restriction is a critical component to achieving synergy across the projects. Fetal growth restriction and low birthweight are major outcome variables for Projects R833293C001 and R833293C002. The mechanisms for these effects in humans more likely involve uteroplacental blood flow rather than caloric restriction.
  3. Inflammation induced via the innate immune response has been previously linked to the development of ozone-induced AHR in adult mice. Inflammation is certainly present early in the ozone exposures in our juvenile exposure model as well, but we do not know if the innate immune system is important to the initial maternal inflammatory responses or the fetal inflammatory responses that may trigger the events leading to AHR in offspring. Accordingly we have begun a collaboration with John Hollingsworth, Dept. of Pulmonary and Critical Care Medicine at Duke, to conduct studies using Toll-like receptor 4 knockout (Tlr4-/-) mice to delineate this pathway. Our first studies, which have just begun, involve breeding null dams with wild-type sires in order to isolate the effects on Tlr4. We will determine whether maternal Tlr4 activation is required for the fetal inflammatory response independent of manipulation of Tlr4 in the fetuses, since all embryos will be phenotypically wild-type (Tlr4+/-heterozygote).
  4. Since epigenetic pathways are particularly likely biochemical transducers of environmental exposures on human disease, we have also begun a collaboration with Robert H. Lane, Professor of Pediatrics, University of Utah, who is a leader in this field. Our future studies will evaluate specific methylation targets relevant to air pollutant exposure using our mouse models. Since these pathways are also being explored in new collaborations begun in Project R833293C002, we expect these investigations to be mutually informative. In particular, low birth weight and fetal growth restriction appear to have epigenetic effects on brain glucocorticoid receptor gene which may link growth restriction (a focus of Project R833293C002) with some of the exaggerated stress responses in the pilot studies of the mouse models (Project R833293C003).


Journal Articles:

No journal articles submitted with this report: View all 18 publications for this subproject

Supplemental Keywords:

Airway hyperreactivity, diesel exhaust particles, air pollution, lung function

Progress and Final Reports:

Original Abstract
  • 2007
  • 2008
  • 2010 Progress Report
  • 2011 Progress Report
  • 2012
  • Final Report

  • Main Center Abstract and Reports:

    R833293    Southern Center on Environmentally Driven Disparities in Birth Outcomes

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R833293C001 Research Project A: Mapping Disparities in Birth Outcomes
    R833293C002 Research Project B: Healthy Pregnancy, Healthy Baby: Studying Racial Disparities in Birth Outcomes
    R833293C003 Research Project C: Perinatal Environmental Exposure Disparity and Neonatal Respiratory Health
    R833293C004 Community Outreach and Translation Core
    R833293C005 Geographic Information System and Statistical Analysis Core