2005 Progress Report: Developmental Effects of PCBs and Methylmercury

EPA Grant Number: R829390C004
Subproject: this is subproject number 004 , established and managed by the Center Director under grant R829390
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: CECEHDPR - University of Illinois FRIENDS Children’s Environmental Health Center
Center Director: Schantz, Susan L.
Title: Developmental Effects of PCBs and Methylmercury
Investigators: Seegal, Richard F.
Institution: New York State Department of Health - Wadsworth Center
EPA Project Officer: Louie, Nica
Project Period: October 17, 2001 through October 16, 2002
Project Period Covered by this Report: October 17, 2004 through October 16, 2005
RFA: Centers for Children's Environmental Health and Disease Prevention Research (2001) RFA Text |  Recipients Lists
Research Category: Children's Health , Health Effects , Health

Objective:

The objective of this research project is to investigate the effects of exposure of organotypic striatal brain slices and synaptosomes to polychlorinated biphenyls (PCBs) and methylmercury (MeHg) on neuronal function at various developmental ages, including changes in dopamine (DA) neurochemistry and reactive oxygen species (ROS) formation. Because of the complexity of the biochemical pathways, we have examined: (1) the actions of each contaminant prior to examining the consequences of simultaneous exposure to PCBs and MeHg, and (2) the consequences and mechanisms of action of the contaminants in tissue derived from adult animals before using tissue from developing animals. We also are studying the effects of PCBs on synaptogenesis in organotypic co-cultures of embryonic substantia nigra and striatum. We have begun studies using in vivo microdialysis to examine the developmental neurochemical consequences of exposure to PCBs and MeHg following in utero exposure. In addition, we are planning collaborative studies with Dr. Schantz that will include in vivo microdialysis in animals developmentally exposed to PCBs and MeHg and have been tested behaviorally at the University of Illinois.

Progress Summary:

Effects of PCBs and MeHg on Striatal Synaptosomal Dopamine Function

We are studying the effects of PCBs and MeHg, alone or in combination, on striatal neuronal function, using purified striatal synaptosomes harvested from developing rats at postnatal days (PND) 7, 14, and 21. Previously, we have shown that both PCBs (the Fox River PCB Mix) and MeHg reduce synaptosomal DA content, with the greatest effects seen in tissue from PND 7 animals. These results demonstrate that, at least for ex vivo exposure, tissue derived from PND 7 animals is uniquely sensitive to these contaminants. The mechanisms responsible for this effect may involve mitochondrial damage leading to ROS formation. We also have shown that, following co-exposure of PND 7 striatal synaptosomes to PCBs and MeHg, MeHg antagonizes the reductions in synaptosomal DA seen following exposure to PCBs only.

In contrast, unlike the lack of interaction of PCBs and MeHg on media DA in PND 7 synaptosomes, we now report that co-exposure of striatal synaptosomes from PND 14 and 21 animals to PCBs and MeHg results in synergistic increases in media DA (Figures 1 and 2). These later results are similar to the synergism seen in adult striatal tissue following exposure to PCBs and MeHg (Bemis and Seegal, 1999), suggesting not only a very rapid maturation of the tissue, but a common mechanism of action—inhibition of the dopamine transporter.

Figure 1. PCBs and MeHg Synergistically Increase Media Dopamine at PND14

Figure 2. PCBs and MeHg Synergistically Increase Media Dopamine at PND 21

PCBs Decrease Striatal Dopamine, Reduce the Number of Tyrosine Hydroxylase Positive Neurons, and Increase Neuronal Cell Death in Organotypic Co-Cultures of Striatum and Substantia Nigra

We also are examining the biochemical consequences of PCB exposure using an organotypic co-culture model that allows two distinct regions of brain tissue to be cultured over an extended period of time. Here, embryonic (E) 21 rat striatum and E 14 rat substantia nigra (SN) are co-cultured to model the developing nigro-neostriatal DA system. In co-culture, DA neurons of the SN project to and innervate the striatal tissue in a manner similar to that observed in vivo. We now have demonstrated that exposure to low micromolar concentrations of the Fox River PCB Mix reduces the number of DA neurons in the SN (Figure 3), reduces striatal DA concentrations (Figure 4), and increases measures of neuronal cell death.

Figure 3. PCBs Reduce the Number of DA Neurons in the SN of Organotypic Co-Cultures of SN and Striatum

Figure 4. PCBs Reduce Striatal Dopamine Content of Organotypic Co-Cultures of SN and Striatum

Figure 5. PCBs Induce Neuronal Cell Death in Organotypic Co-Cultures of SN and Striatum as Indicated With Fluoro Jade B

Methylmercury Induces Reactive Oxygen Species Formation in Synaptosomes from Developing Animals and Reduces Mitochondrial Function

We are assessing the role that MeHg plays in inducing ROS formation in synaptosomes from adult rats as well as from PND 7, 14, and 21 rats. Previously, we have reported that the effects were greater in tissue from young rats than in the adult synaptosomes (Dreiem, et al., 2005). We now are investigating the effects of MeHg on ROS formation, mitochondrial membrane potential (ΔΨm), and mitochondrial metabolic function (assessed by reduction of methylthiazoletetrazolium [MTT]), starting with tissue from adult animals. MeHg increases ROS formation at MeHg concentrations of 5 µM or greater, an effect that was prevented by co-incubation with the antioxidant Trolox (Figure 6). MeHg also reduced mitochondrial membrane potential (Figure 7) and mitochondrial metabolic function (Table 1). These effects, however, were not prevented by co-incubation with Trolox (Table 1). Therefore, we conclude that ROS is not the cause of mitochondrial dysfunction and loss of mitochondrial membrane potential after MeHg exposure—rather, that mitochondrial membrane dysfunction is an early event in MeHg toxicity and that ROS formation may be an effect of that damage. These experiments will be expanded to include the Fox River PCB Mixture and combinations of PCBs and MeHg using tissue from animals of varying developmental ages.

Figure 6. Trolox Reduces MeHg-Induced Ros Formation in Striatal Synaptosomes

Figure 7. MeHg Decreases Mitochondrial Membrane Potential (ΔΨm) in Striatal Synaptosomes

Table 1. Trolox Does Not Prevent MeHg-Induced Loss of Mitochondrial Function

 

6 µM Trolox

MeHg1

MeHg1 + 6 µM Trolox

MTT assay2 (% of control)

106.4 ± 1.8

71.4 ± 2.3ca

70.3 ± 3.4a,b

ΔΨm3 (% of control)

98.7 ± 5.0

57.5 ± 10.4a

61.7 ± 11.0a,b

1 MeHg concentration was 5 µM in the
2 methylthiazoletetrazolium (MTT) assay and 0.5 µM MeHg for
3 mitochondrial membrane potential (ΔΨm ) measurements.
a Significantly different from control (p≤0.001).
b Not significantly different from MeHg alone.

Future Activities:

We will continue to investigate the effects of exposure of synaptosomes to PCBs and MeHg on DA neuronal function at various developmental ages, including studies utilizing tissue from early postweaning animals. Parallel studies will be undertaken in tissue derived from animals exposed in utero to these contaminants either alone or in combination.

We will continue to examine the role that PCBs, MeHg, and the two toxicants in combination play in inducing ROS formation using synaptosomes and organotypic co-cultures exposed to the above contaminants. These studies also will be expanded to include the determination of ROS formation in tissue from rats exposed in utero.

We will continue to examine the effects of PCBs and MeHg on synaptogenesis in organotypic culture of embryonic co-cultures of substantia nigra and striatum. The use of tissue exposed in utero from either substantia nigra or striatum will allow us to determine whether toxicant-induced alterations in the “target” (striatum) or the substantia nigra influence axonal growth, synaptogenesis, and contaminant-induced apoptotic cell death.

We will continue studies using in vivo microdialysis to examine the developmental neurochemical consequences of exposure to PCBs and MeHg following in utero exposure in postweaning animals, expanding the studies to include animals that were exposed developmentally and tested behaviorally at the University of Illinois.


Journal Articles on this Report : 2 Displayed | Download in RIS Format

Other subproject views: All 7 publications 3 publications in selected types All 3 journal articles
Other center views: All 38 publications 22 publications in selected types All 21 journal articles
Type Citation Sub Project Document Sources
Journal Article Dreiem A, Gertz CC, Seegal RF. The effects of methylmercury on mitochondrial function and reactive oxygen species formation in rat striatal synaptosomes are age-dependent. Toxicological Sciences 2005;87(1):156-162. R829390 (2005)
R829390 (Final)
R829390C004 (2005)
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  • Journal Article Seegal RF, Brosch KO, Okoniewski RJ. Coplanar PCB congeners increase uterine weight and frontal cortical dopamine in the developing rat: implications for developmental neurotoxicity. Toxicological Sciences 2005;86(1):125-131. R829390 (2005)
    R829390 (Final)
    R829390C004 (2005)
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  • Supplemental Keywords:

    children’s health, disease and cumulative effects, ecological risk assessment, susceptibility, sensitive population, toxicology, Fox River, PCBs, exposure assessment, heavy metals, methylmercury, pesticides, fish consumption,, RFA, Health, Scientific Discipline, PHYSICAL ASPECTS, ENVIRONMENTAL MANAGEMENT, Toxicology, Health Risk Assessment, Risk Assessments, Susceptibility/Sensitive Population/Genetic Susceptibility, Physical Processes, Children's Health, genetic susceptability, Molecular Biology/Genetics, Risk Assessment, developmental neurotoxicology, neurotoxic, sensitive populations, childhood cancer, biomarkers, PCBs, animal model, developmental effects, exposure, Human Health Risk Assessment, children, methylmercury, assessment of exposure, children's vulnerablity, residential populations, PCB, neurodevelopmental toxicity, neurobehavioral effects, biological markers, toxics

    Progress and Final Reports:

    Original Abstract
  • Final

  • Main Center Abstract and Reports:

    R829390    CECEHDPR - University of Illinois FRIENDS Children’s Environmental Health Center

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R829390C001 Neurobehavioral Effects of PCBs and Methylmercury in Rats
    R829390C002 Perinatal PCB Exposure and Neuropsychological/Auditory Function
    R829390C003 FRIENDS Analytical Toxicology Core Facility
    R829390C004 Developmental Effects of PCBs and Methylmercury