2006 Progress Report: Clinical Sciences Project

EPA Grant Number: R829391C005
Subproject: this is subproject number 005 , 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: Clinical Sciences Project
Investigators: Lambert, George H. , Carmody, Dennis , Johnson, William , Mars, Audrey , Moreno, Rosanne , Seshadri, Kapila
Institution: University of Medicine and Dentistry of New Jersey
EPA Project Officer: Louie, Nica
Project Period: November 1, 2001 through October 31, 2006
Project Period Covered by this Report: November 1, 2005 through October 31, 2006
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 test the hypotheses that: (1) the regression of neurological function observed in some children with autism, which occurs at the time the children become more mobile, is a result of the exposure to high levels of neurotoxicants, reexposure to specific neurotoxicants, or unidentified neurotoxicants or caused by highly susceptible subjects secondary to specific genes or gene environment interaction; (2) in children with autism, high or unusual exposure to neurotoxicants alter regional brain growth or the combination of genes and environment can alter regional brain growth; and (3) susceptibility to develop autism and or regression can be influenced by the child or mother’s genotype involved in protecting the developing human from chemical-induced oxidative stress, regional brain growth, neurodevelopment, or genes associated with autoimmune susceptibilities.

Progress Summary:

This cohort represents the most comprehensively studied autism cohort ever described. The multifaceted evaluations and careful neurobehavioral assessment allows us to make careful comparisons between the neurobehavioral assessments, magnetic resonance imaging (MRI) and functional MRI (fMRI), chemical body burdens, metabolites, proteins, and genes.

Although 18 months remain on the grant, we have developed exciting and relevant findings regarding autism itself and autism and the environment. Even with just two-thirds of the children recruited and their data analyzed we probably have answered some of the hypothesis and revealed a critical and important future research pathway, a pathway that hopefully will have impact on the child with autism and their neurobehavioral function. The partial and preliminary answers to hypothesis will be discussed.

Recruitment

Currently we have 61 signed consent/parental permission forms and 60 children who have been seen. In addition, we have five consents mailed out for signatures from parents who have tentatively agreed to participate. All have met exclusion criteria. Because we waited for 1 year to begin recruitment, we are recruiting 26 subjects per year.

We have 34 subjects left to recruit from the 4 states where we are conducting the study. We and other investigators using our recruitment criteria find that only 2-3 percent of all children who are diagnosed with autism meet our criteria. Therefore, we are relaxing the strict recruitment criteria as of this month. We have increased the age of recruitment from 24-36 months to 24-42 months. In addition, we are changing the criteria to include families who have not moved since birth, rather than 1 year before birth, as currently written. These two changes will greatly increase greatly ease of recruitment.

For enhanced genetic analysis, we have expanded the family recruitment and gene analysis to include the child with autistic spectrum disorder (ASD), their parents, and starting this last year, both sets of grandparents when possible. This greatly expands the power of the genetic analysis.

Because the recruitment of the subjects did not begin until after Year 1 while the Exposure and Assessment Team developed and validated its methods, we are 2-and-a-half years into the recruitment of the study. The goal is the recruitment of 100 subjects and their parents, and now grandparents. The excitement of the New Jersey, Pennsylvania, New York, and Connecticut autism community for our study is high, and the commitment to research is stronger now than before as the data begin to point to critical findings and potential interventional strategies. The team remains overwhelmed by the parental support of our research.

Data

We closed all of the data for analysis for this report on May 15, 2005. Therefore, the data will be collected on 50 children with ASD and their families. Of the 50 children recruited, 88 percent are males and 12 percent are females.

Neurobehavioral Assessment

We have data from the psychological assessment of 44 children who have fully completed the initial psychological battery. Fifty-six percent have cognitive scores of 69 or below as measured by the Differential Ability Scales (DAS), 63 percent have scores below 69 as measured by adaptive functioning (Vineland Adaptive Behavior Scales; VABS), and 52 percent have cognitive and adaptive scores that are commensurate with a score of 69 or below. All children have diagnosis of ASD and 74 percent of the children have a diagnosis of autistic disorder (AD).

One-half of all the children have confirmed reports of social and/or language regression. There are no differences between the children without regression versus the children with regression in any of the neurobehavioral assessments listed above.

Seven participants have completed the process of the exit exam (Autism Diagnostic Observation Schedule, Autism Diagnostic Interview, DAS, VABS, Visual-Motor Integration, measures of self awareness, use of personal pronouns, theory of mind tasks, Child Behavior Checklist, Parenting Stress Index) at age 5 years. The variability is tremendous and the number of participants is too small to make any significant statement.

MRI and fMRI

Seven MRI exams have been completed. Although many parents are anxious to have an MRI on their child, about 25 percent of the parents decline to have their children undergo conscious sedation for the MRI. We do not expect sleep deprivation MRIs will be successful in this population. Our MRI data has been examined by the National Institute of Child Health and Human Development (NICHD) Multi-Center Brain MRI group lead by Washington University. We provided our MRIs to them to evaluate the MRI quality and exact procedural methods, as we will compare regional brain volumetrics from our MRIs of children with autism to their hundreds of MRIs of children who are completely healthy. All of our MRI studies met their high technical standards and motion was not apparent in any of the MRIs as compared to more than 50 percent of the NICHD’s MRIs. We are able to obtain complete motionless MRIs because of the fact that we now use conscious sedation with continuous IV propophol. To maximize safety of the subject, propophol is administered and monitored throughout the study by a pediatric anesthesiologist. Dr. Lambert, who is a board certified pediatrician and intensive care neonatologist, also is present for each MRI study. We have not had any adverse events.

The fMRI of speech and language skills have also been conducted on each subject with great success and interest. Children with AD demonstrated that on fMRI using taped voice of their mother that the children did not respond to names but did respond to numbers. This reflects our neurobehavioral assessments in these children. These novel findings currently are being written up for publications as demonstration of success of proof of principle using the fMRI in consciously sedated children with autism and clinical correlates.

With the assistance of the NICHD National MRI study methods and their rapidly increasing database of MRI volumetric analysis of normal children, we have begun to compare the regional brain volumetric findings of the MRIs in our children to their normal children. We also will compare the MRIs from our autistic children with regression versus none regression, in addition we will compare the regional brain volumes to specific brain growth genes such as ENGRAILED-2, to speech development genes with the volumetric and fMRIs, to ROS genes, and to biological levels of specific environmental chemicals.

In addition, in cooperation with Siemens Corp Research Institute Division of Intelligent Vision and Reasoning, we are developing better methods for higher resolution of the MRI three-dimensional volumetric analysis via segmentation and quantitative analysis; and the development of new spectral mass spectrometer analysis of MRIs in an effort to detect specific environmental chemicals in the brain.

Biological Levels

The biological levels of the children have been discussed in the Exposure Assessment and Intervention section of the clinical study. We have compared the levels of lead and mercury in the children with regression versus without regression. This comparison did not reveal any significant differences between the two groups. One group of investigators from a highly respected research institute reported last week at a conference on autism and oxidative stress that they found differences in the environmental chemicals levels of children with and without regression and autism. This report was based on very few children who were not very well characterized and was not in agreement with our preliminary data. Our final conclusions will have to await completion of our study. In answer to one hypothesis, however, we do not believe that regression is a result of the exposure of these children to higher levels of the standard environmental chemicals.

Metabolomics – Urinary Markers for Oxidative Stress

Dr. Ming (who is our Center’s identified faculty member to bring into the field of environmental pediatrics) currently is studying the children with autism and also has data from other children with autism. She has identified that the children with autism have markedly increased urinary isoprostane levels in comparison to controls. Other urinary markers of oxidative stress, however, were not different in the children with autism as compared to children without autism. These data have been accepted for publication. Some of the autism children in her report were taking over- the-counter (OTC) drugs, vitamins, and other drugs for autism. Therefore the further study of the Center’s children will be important because almost all of these children are naïve subjects having never been exposed to OTCs nor any drugs for autism. In addition the Center children’s urinary metabolites are being compared to their specific genotypes of related oxidative stress proteins.

In addressing the hypothesis, these data would suggest strongly that certain children with autism have increased susceptibility to oxidative stress and therefore most likely to select environmental chemicals.

Neurogenetics – Oxidative Stress Genes and Cellular Regulatory Genes

The genetic studies of the cohort and other children with autism will be some of the most important findings of the Center. Dr. Johnson and his neurogenetics laboratory have identified three new genes associated with autism. Two of the genes are involved in oxidative stress, and the other gene is a cell regulatory gene that also is involved to a degree in oxidative stress. The two oxidative stress genes reports have been submitted for publication and the manuscripts were initially turned down by Lancet. Data on the third gene are being increased by additional subjects in the study and will be submitted shortly. Currently, all of these genes have been found to be statistically (P < 0.05) identified at a higher frequency in children and the families with autism. The identification of these genes was enhanced greatly by the availability of the trios and grandparent’s blood for analysis and by the careful screening of the subjects to assure that they in fact had ASD. Specific identification of the specific gene polymorphisms will be discussed after the papers are accepted.

Although all of the genes associated with autism identified to date are important, we are most interested in our two genes associated with oxidative stress in the children with autism and in addition to the cell regulatory genes associated with oxidative stress. One oxidative stress gene is a deletion polymorphism found in the father and the child with autism at much higher frequency than the general population, and the other gene is a polymorphism of the same gene family but found more frequently in the mothers of children with autism as compared to the general population. These data support the hypothesis that the children with autism are more susceptible to environmental chemicals because of their genes and their mothers and to somewhat a surprise, the father’s genes. We believe that these results indicate a prevention intervention strategy for autism and environmental chemicals may be possible. The work of Dr. Wagner on antioxidant protection from environmental chemicals and the work of Dr. Ming on isoprostane enhances the excitement of our Translational Research Group. These leads also have been used to develop new funded research and others to follow on these aspects of gene environment interaction in animal models, clinical trials, and as we discover every week, translational synergism and studies.

The translational research team and the Center have been joined by world’s experts on oxidative stress in the perinatal period (Dr. Stein and his colleagues) and cell regulatory experts (Dr. Levine). The research group and the parents easily feel the academic excitement. We expect that studies will begin shortly that take data from the genes of the human and develop animal data and animal data findings to develop clinical trials.

Translational Research – New Animal Models and Knockouts

The Translational Research Group is very excited about these finding in association with our genetic and animal models as discussed above. In collaboration with Dr. Oleg Mirochnitchenko, we have ordered knockout mice from the University of Connecticut, with the oxidative stress gene deletion found by Dr. Johnson in the children and their fathers. After the mice are received and characterized by Dr. Mirochnitchenko, Dr. Wagner will begin his studies on animal models of regress, intrusion, and retardation on the mice. In particular, focusing on levels of sensitivity of the mice to neurotoxicants and pharmacological methods to ameliorate or prevent the neurotoxicant effects on neurodevelopment. We then will cross the oxidative stress knockouts with Engrailed-2 knockouts to study the interaction of the brain developmental gene and the oxidative stress deletion in sensitivity to neurotoxicants and neuroprotective effect of antioxidants in the backcrosses.

The Engrailed-2 gene is being analyzed in the cohort and compared to its MRIs. If the gene does in the human what it does in the animal model we should be able to show an association with Engrailed-2 and specific regional brain growth as determined by MRI. This also may be true for our newly discovered other regulatory gene, which we have not submitted for publication. From the preliminary work of Dr. Wagner, the mice with the Engrailed-2 gene do have delayed and abnormal neurodevelopment, and they currently are assessing the mouse’s sensitivities to neurotoxicants.

These genes may then explain why children with autism may be at higher risks from exposure to environmental chemicals. The findings will go a long way to addressing our hypothesis and, what we hope most, in developing interventional strategies for autism. Researchers from the Translational Research Groups have patents on this approach.

Summary

The Translational Research Group finds the pharmacological animals studies, the clinical studies including the gene studies, and the oxidative stress urinary metabolites studies very, very exciting. The data points to an important and possibly critical link between susceptibility to oxidative stress (endogenous and exogenous) and autism. Other investigators also have inferential data of the importance of this mode of action pathway to autism and possibly other neurodevelopmental disorders and other environmental disorders such as asthma as shown by the Asthma Children Center in California.

We have the correct distribution of expertise among the Center members and the natural formation of Translational Research Group. The dedication and excitement of this Center’s researchers are most rewarding and far beyond our expectations of potential outcome of the Center award. With the animal modelers, the development of specific new gene knockout mice, the genetics team, the oxidative stress team including our Center-supported new faculty (Dr. Ming) and the new members of the Center (Drs. Stein, Mirochnitchenko, Zhang, and Levine), the clinical expertise, and the expertise in experimental pediatric clinical pharmacology; the Center is poised to exploit some of these findings and hopefully change the lives and hopes of the children with autism and their families. This hope is years off, but the path appears to be exciting, clear, and holds great promise.

Future Activities:

Indirect markers are consistent with greater oxidative stress in autism. They include greater free-radical production, impaired energetics and cholinergics, and higher excitotoxic markers. Brain and gut, both abnormal in autism, are particularly sensitive to oxidative injury. Higher red-cell lipid peroxides and urinary isoprostanes in autism signify greater oxidative damage to biomolecules. A preliminary study found accelerated lipofuscin deposition—consistent with oxidative injury to autistic brain in cortical areas serving language and communication. Double blind, placebo-controlled trials of potent antioxidants—vitamin C or carnosine—significantly improved autistic behavior. Benefits from these and other nutritional interventions may be a result of reduction of oxidative stress. To understand possible relationship between genetic polymorphism of one of the antioxidant enzyme associated in humans with autism, namely glutathione S-transferase M1 (GSTM1), the role of oxidative stress in the pathophysiology and abnormal behavior will be studied in mice deficient in corresponding mouse enzyme. For this purpose GstM1(-/+) and GstM1(-/-) animals are being developed and characterized.

Assessment of Mice. We will examine heterozygote and homozygote (if viable) knockout mice as follows: anatomical and pathological changes in brain and other organs, developmental and behavioral changes, alterations in GSTs and other antioxidant enzymes expression, levels of oxidative stress (variety of markers, including level of lipid peroxidation, protein carbonyl content, DNA oxidative adducts, GSH, and GSH/GSSG ratios).

Role of GSTM1 in Chemically Induced Toxicity. Several model systems will be employed to evaluate detoxifying capabilities of knockout mice. We will initiate studies by evaluating animal sensitivity to paracetamol-induced hepatotoxicity and paraquat-induced brain and lung injury. All of these models are well known for the active role of oxidative stress in initiating and propagating corresponding cell and tissue damage. Immune-related properties of knockout mice will be studied in lipopolysaccharide-induced model of septic shock.

Effect of GSTM1 Deletion on Brain Redox/Sensitive Signaling Pathways. We will investigate function of neuronal and glial signaling pathways mediating brain cells proliferation, differentiation and programmed cell death in cell cultures as well as in vivo experiments under the normal and stress conditions.

The GSTs represent a major group of detoxification enzymes. All eukaryotic species possess multiple cytosolic and membrane-bound GST isoenzymes, each of which displays distinct catalytic as well as noncatalytic binding properties: the cytosolic enzymes are encoded by at least five distantly related gene families (designated class alpha, mu, pi, sigma, and theta GST). Evidence suggests that the level of expression of GST is a crucial factor in determining the sensitivity of cells to a broad spectrum of toxic chemicals.


Journal Articles on this Report : 7 Displayed | Download in RIS Format

Other subproject views: All 9 publications 9 publications in selected types All 8 journal articles
Other center views: All 86 publications 50 publications in selected types All 49 journal articles
Type Citation Sub Project Document Sources
Journal Article Ayotte P, Dewailly E, Lambert GH, Perkins SL, Poon R, Feeley M, Larochelle C, Pereg D. Biomarker measurements in a coastal fish-eating population environmentally exposed to organochlorines. Environmental Health Perspectives 2005;113(10):1318-1324. R829391 (2004)
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  • Journal Article Calafat AM, Needham LL, Silva MJ, Lambert G. Exposure to di-(2-ethylhexyl) phthalate among premature neonates in a neonatal intensive care unit. Pediatrics 2004;113(5):e429-e434. R829391 (2004)
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  • Journal Article Carmody DP, Dunn SM, Boddie-Willis AS, DeMarco JK, Lewis M. A quantitative measure of myelination development in infants, using MR images. Neuroradiology 2004;46(9):781-786. R829391 (2004)
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  • Journal Article Fitzgerald EF, Hwang SA, Lambert G, Gomez M, Tarbell A. PCB exposure and in vivo CYP1A2 activity among Native Americans. Environmental Health Perspectives 2005;113(3):272-277. R829391 (2004)
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  • Journal Article Israel BA, Parker EA, Rowe Z, Salvatore A, Minkler M, Lopez J, Butz A, Mosley A, Coates L, Lambert G, Potito PA, Brenner B, Rivera M, Romero H, Thompson B, Coronado G, Halstead S. Community-based participatory research:lessons learned from the Centers for Children's Environmental Health and Disease Prevention Research. Environmental Health Perspectives 2005;113(10):1463-1471. R829391 (2004)
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  • Journal Article Johnson WG, Scholl TO, Spychala JR, Buyske S, Stenroos ES, Chen X. Common dihydrofolate reductase 19-base pair deletion allele: a novel risk factor for preterm delivery. American Journal of Clinical Nutrition 2005;81(3):664-668. R829391 (2004)
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  • Journal Article Yang C-Y, Yu M-L, Guo H-R, Lai T-J, Hsu C-C, Lambert G, Guo YL. The endocrine and reproductive function of the female Yucheng adolescents prenatally exposed to PCBs/PCDFs. Chemosphere 2005;61(3):355-360. R829391 (2004)
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  • 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, ENVIRONMENTAL MANAGEMENT, Environmental Chemistry, Health Risk Assessment, Risk Assessments, Susceptibility/Sensitive Population/Genetic Susceptibility, Children's Health, genetic susceptability, Biology, Risk Assessment, childhood learning, neurotoxic, gene-environment interaction, developmental effects, children, neurotoxicity, assessment of exposure, public health, behavioral deficits, environmental health hazard, autism, outreach and education, assessment technology, developmental disorders, exposure assessment, neurological development

    Progress and Final Reports:

    Original Abstract
  • 2002
  • 2003
  • 2004 Progress Report
  • 2005 Progress Report
  • Final

  • 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