2004 Progress Report: Neurotoxicant Effects on Cell Cycle Regulation of NeurogenesisEPA Grant Number: R829391C001
Subproject: this is subproject number 001 , established and managed by the Center Director under grant R829391
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: CECEHDPR - University of Medicine and Dentistry of New Jersey Center for Childhood Neurotoxicology and Assessment
Center Director: Lambert, George H.
Title: Neurotoxicant Effects on Cell Cycle Regulation of Neurogenesis
Investigators: DiCicco-Bloom, Emanuel
Institution: University of Medicine and Dentistry of New Jersey , University of Medicine and Dentistry of New Jersey
Current Institution: University of Medicine and Dentistry of New Jersey , University of Medicine and Dentistry of New Jersey
EPA Project Officer: Fields, Nigel
Project Period: November 1, 2001 through October 31, 2006
Project Period Covered by this Report: November 1, 2003 through October 31, 2004
RFA: Centers for Children's Environmental Health and Disease Prevention Research (2001) RFA Text | Recipients Lists
Research Category: Children's Health , Health Effects , Health
The objective of this research project is to investigate the hypothesis that neurotoxic metals and teratogens disrupt neurogenesis in developing forebrain and hindbrain systems in vitro and in vivo, acting to inhibit proliferation by altering mitogenic growth factor receptors and cell cycle and signaling pathways.
We have made progress this past year on four of our research aims for this project. Indeed, we have found evidence supporting our hypothesis that neurotoxic metals and teratogens disrupt neurogenesis in developing forebrain and hindbrain systems, altering precursor mitosis, survival, and differentiation. We now are beginning to characterize changes in specific neuronal populations and define underlying cell cycle mechanisms.
Methylmercury (MeHg) Decreases Cortical Precursor Mitosis and Survival in Culture
Using embryonic day 14.5 (E14.5) cerebral cortical precursors in 24-hour cultures, we found that MeHg (0.01-3.0 µM) elicited complex effects on DNA synthesis and cell survival: there was a modest 20 percent stimulation at 0.3 µM and progressive reduction thereafter, with approximately 50 percent decreases at 1.0-1.5 µM and 90 percent inhibition at 2.0 µM. The reduction in DNA synthesis occurred in parallel with decreased cell numbers. At lower doses at which the metal elicited stimulation (0.3 µM), however, there was still modest cell loss, suggesting MeHg induced both cell death in some cells as well as premature cell cycle entry into S phase.
Following a Modest Early Reduction in Cell Survival and DNA Synthesis, Lead Acetate (Pb) Enhances Cortical Neuron Survival and Process Outgrowth
Over a range of concentrations studied (0.03-100 µg/ml), Pb had little effect on DNA synthesis at 24 hours, except at the higher range. At 30 and 100 µg/ml, there was approximately 20-30 percent reduction in DNA synthesis, which appeared to correlate with reduced cell survival. As cultures aged and cells were lost in the controls, however, there were marked differences; at 4 days there were 4- to 5-fold more cells in the Pb-treated cultures than in the controls. In addition, process outgrowth markedly increased, exhibiting complex branching never observed in control cultures. Moreover, there appears to be differential actions of Pb, with lower doses, such as1 µg/ml, eliciting enhanced cell survival without effects on process elaboration, suggesting differential pathway activation, issues currently under study. Furthermore, because it is outside the current proposal, an Environmental and Occupational Health Sciences Institute pilot project grant has been awarded to define changes in gene expression induced by lead at early times, using an Affymetrix-based analysis in collaboration with the W.M. Keck Center for Collaborative Neuroscience.
Methyl Mercury (MeHg) Exposure of Newborn Rats Differentially Inhibits Neurogenesis in Hippocampus and Cerebellum
To examine acute effects of MeHg on precursor proliferation, we injected postnatal day 7 (PND7) rats with MeHg (0.01-30 µg/gbw) and [3H]thymidine 6 hours later, and assessed incorporation into whole regions 2 hours later. At 8 hours, DNA synthesis was reduced 16 percent at 0.1 µg, 50 percent at 3 µg, and 80 percent at 30 µg/gbw in hippocampus, indicating rapid effects on cell cycle progression, suggesting G1-S block. In contrast, MeHg did not affect cerebellum, suggesting differential vulnerability, because blood flow and mercury content were similar to hippocampus. To directly define cerebellar precursor sensitivity, we examined granule cells in vitro: MeHg elicited inhibition at 24 hours, reducing DNA synthesis 50 percent at 1 µM and 90 percent at 3 µM, paralleling effects on survival. At 6 hours, however, MeHg inhibited DNA synthesis 25 percent at 3 µM and 90 percent at 10 µM without altering survival, suggesting effects on G1 progression. In contrast, after in vivo MeHg and [3H]thymidine injection, DNA synthesis was unchanged in isolated granule cells, indicating their resistance to MeHg when in situ. Therefore, MeHg alters neurogenesis in the developing brain in a region-specific fashion.
To define the long term consequences of acutel MeHg mitotic inhibition, PND7 rats were injected once with MeHg 3 µg/gbw and total cell number was assessed at PND21 by quantifying DNA: there was 17 percent reduction in hippocampal cell number, whereas there was no change in cerebellum, indicating differential inhibition of postnatal neurogenesis results in sustained region-specific effects on brain composition. Ongoing studies are assessing the number of BrdU-labeled cells in hippocampal dentate gyrus (DG) after Hg exposure, measuring Hg content at both 6 hours and at PND21, and finally, counting total granule neurons in the DG. These studies will define how a single exposure can initially reduce developmental neurogenesis in hippocampus, and have sustained effects on cell composition. This work is contained in a submitted abstract to the Society for Neuroscience, 2003.
Valproic Acid (VPA) Increases Cortical Precursor Cell Proliferation In Vitro and In Vivo
Using E14.4 cortical precursors we have found that VPA can elicit increased DNA synthesis at 0.3 to 1.5 mM concentration, with cell death induction thereafter. Significantly, the increased DNA synthesis is caused by more cells entering S phase, as indicated by increased BrdU labeling, as well as more cells remaining in the precursor state, identified by nestin expression. In contrast, less cells express cytoskeletal molecule, beta III tubulin, and fewer cells elaborate neurites. These observations suggest that VPA retains cells in a proliferative condition. In turn, we have begun to define in vivo models to examine whether the drug has similar effects during prenatal cortical development. Indeed, preliminary studies indicate that VPA treatment of pregnant rats elicits region-specific stimulation of cortex, whereas there is no change in the hindbrain. This model suggests that at certain doses, VPA may promote a cortical overgrowth, leading to mismatched neuronal populations during development. Significantly, recent neuroimaging studies indicate abnormal control of brain growth in autism. Furthermore, these studies may be relevant to spina bifida, where neural tube closure is prevented as a result of excess precursor proliferation. We currently are defining the cellular and cell cycle mechanisms underlying these changes in developing cortex.
Pb Elicits Inhibition of DNA Synthesis in P7 Hippocampus In Vivo
Although Pb had small effects on DNA synthesis in cultures of cerebral cortex, we examined potential effects on G1/S-phase transition in vivo, using the same 8-hour paradigms as for MeHg. Significantly, 8 hours after Pb injection, there was a 30 percent inhibition of hippocampal DNA synthesis, suggesting that the metal affects ongoing neurogenesis, perhaps more effectively than observed in cortical precursors. Further studies in both cerebellum and hippocampus are planned.
Our future studies will characterize the changes in specific neuronal populations induced in the developing animals by metals and VPA. Furthermore, we will define underlying cell cycle mechanisms and alterations in gene expression to identify target molecules and pathways.
Journal Articles:No journal articles submitted with this report: View all 8 publications for this subproject
Supplemental Keywords:children’s health, disease and cumulative effects, ecological risk assessment, environmental chemistry, health risk assessment, risk assessments, susceptibility/sensitive population/genetic susceptibility, toxicology, genetic susceptibility, assessment of exposure, assessment technology, autism, behavioral assessment, behavioral deficits, childhood learning, children, developmental disorders, developmental effects, environmental health hazard, environmental toxicant, exposure assessment, gene-environment interaction, neurodevelopmental, neurological development, neuropathological damage, neurotoxic, neurotoxicity, outreach and education, public health,, RFA, Health, Scientific Discipline, Health Risk Assessment, Biochemistry, Children's Health, developmental neurotoxicity, biological response, neurodevelopmental toxicity, environmental toxicant, environmental health hazard, children's environmental health, growth & development
Progress and Final Reports:Original Abstract
2005 Progress Report
2006 Progress Report
Main Center Abstract and Reports:R829391 CECEHDPR - University of Medicine and Dentistry of New Jersey Center for Childhood Neurotoxicology and Assessment
Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R829391C001 Neurotoxicant Effects on Cell Cycle Regulation of Neurogenesis
R829391C002 Adhesion and Repulsion Molecules in Developmental Neurotoxic Injury
R829391C003 Disruption of Ontogenic Development of Cognitive and Sensory Motor Skills
R829391C004 Exposure Assessment and Intervention Project (EAIP)
R829391C005 Clinical Sciences Project