# Presentation: 853328

## Studies on the Role of Environmental Factors on the Etiology of AutismChris Saint, Ph.D.1, Isaac Pessah, Ph.D.2, and George Lambert, M.D.3United States Environmental Protection Agency, Office of Research and Development, National Center for Environmental Research,2. University of California, Davis, 3. University of Medicine and Dentistry of New Jersey.

**Research Goals**

**Science Questions**

***What is the relationship between childhood exposure to environmental factors and the incidence and severity of ASD? ***
***What methods can be developed to determine how these exposures may influence normal social development?***
***What are the cellular and molecular mechanisms of ASD underlying abnormal development of brain regions which confer important determinants of social behavior?***

- The incidence of autism and its less-severe forms, which comprise autism spectrum disorder (ASD), appears to be increasing.  ORD’s research on ASD and the environment is designed to address the questions above. This research will help identify sub-populations that could be at risk for ASD, and the underlying environmental and biological basis for this increased vulnerability.

- In cooperation with the National Institute for Environmental Health Sciences (NIEHS), ORD supports research centers examining the relationships between environmental exposures and disease.  Two of these Centers, at the **University of Medicine and Dentistry of New Jersey **(UMDNJ) and the **University of California, Davis **(UC Davis), are conducting the first large-scale epidemiologic studies of possible links between exposure to environmental factors and ASD. The overall aim of these studies is to better understand patterns of dysfunction in ASD and to elucidate mechanisms by which environmental toxicants may contribute to abnormal development of social behavior in children so that new strategies for intervention, treatment and prevention can be developed.

**Methods/Approach**

**Impact and Outcomes**

**Results/Conclusions**

**Future Directions**

**References**

*“**Cherishing children is the mark of a civilized society”*
-       - Joan Ganz Cooney

- Although these studies are ongoing, they have provided new tools for investigating ASD and related neurobehavioral disorders including a number of new mouse and primate models for studying the effects of environmental toxins on brain development3,4.  They have also provided new insights into possible immunologic and neurologic mechanisms that may explain the role of environmental toxins in ASD.
- Studies have shown that blood mononuclear cells isolated from autistic children have different responses to antigen stimulations than those from non-autistic children and that cells from autistic children are more sensitive to the actions of polychlorinated biphenyls (PCBs) and organic mercurials.
- Other studies have provided new information on the importance of various molecular and cellular processes such as the impact of xenobiotics on neurogenesis,5,6 membrane metabolism,7  regulation of calcium transport across the microsome and alteration of dendritic cell function,8 alterations in the expression of adhesion and repulsion molecules,9 and the induction of neuroactive cytokines.  Researchers have also identified genetic factors that may increase the susceptibility of autistic children to the effects of environmental chemicals.
- Studies also indicate that heavy metals can cause rapid growth in certain brain regions, as is seen in autism, and in mice, the effects of sodium valproate appear to be prevented with co-administration of an antioxidant such as vitamin E.
- These findings are guiding the data collection activities in their epidemiologic and exposure data collection efforts and will help to interpret relationships between those data.

- Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, American Psychiatric Association, 1994. Website: http://www.appi.org/dsm.cfx.
- Kita, T, Paku, S, Takahashi, SA, Kubo, K, Wagner, GC, and Nakashima, T.  Methamphetamine-induced neurotoxicity in BALBc, DBA/2N and C57BL/6N mice.  Neuropharmacology 37: 1177-1184, 1998.
- Wagner, GC, Reuhl, KR, Cheh, M McRae, P and Halladay, AK. A new neurobehavioral model of autism in mice. (Submitted, 2005).
- Bauman, MD, Lavenex, P, Mason, WA, and Ameral, DG.  The development of social behavior following neonatal amygale lesion in rhesus monkeys. Cogn Neurosci.16:1388-411, 2004.
- Preliminary results presented at the Childhood Neurotoxicology Conference in Honolulu Hawaii. January, 2004.
-  Burke, K, Cheng, Y, Li, B, Caldiera, N, Petrov, A, Pushkar, J and DiCicco-Bloom, E.  Methylmercury (MeHg) alters forebrain neurogenisis through decreased precusor proliferation. (Submitted, 2005).
- Levitt JG, O'Neill J, Blanton RE, Smalley S, Fadale D, McCracken JT, Guthrie D, Toga AW, Alger JR.  Proton magnetic resonance spectroscopic imaging of the brain in childhood autism.  Biol Psychiatry. 54:1355-1366, 2003.
- Goth SR, Cherednichenko, G, Chu, RA, pessah, IN.  Expression of calcium channel proteins in murine dendritic cells: redox modulation of DC functions and apoptosis with nanomolar thimerosal.  J. Immunol. (Submitted).
- Wilson, DT, Polanus, MA, Zhou, R, Lowndesd, HE, Reuhl, KR. Methylmercury alters Eph and ephrin expression in P19 embryonic carcinoma cells and their neuronal derivatives.  Neurotoxicology (Submitted).

- This research effort is ongoing in the ORD-sponsored Children’s Environmental Health Research Centers.
- The Centers’ scientists are working closely with community groups to coordinate research efforts regarding ASD and the environment and are educating parents and other groups on the progress of research activities. They have also held an International Symposium on Autism Research (May 2004) to increase awareness and discussion of these issues in the scientific community.
- The expected impact is that a better understanding of the role that environmental toxins play in ASD will be utilized by US EPA program offices to improve exposure, dose-response and risk assessments.

- The epidemiological studies will continue to recruit and collect data from their cohorts during 2005.  They are also beginning analysis of the biomarker and outcome data.  The mechanistic and exposure studies are using newly developed animal models to further elucidate possible mechanisms linking neurotoxicant exposure, genetics and autism and autism spectrum disorders.

- These studies are evaluating the role played by a broad of array of factors in autism including:
**Environmental exposures**: these include industrial chemicals and consumer products, medications, vaccinations and diet.
**Physiologic factors:** these include factors that may affect brain and immune system development, including the mother’s health during pregnancy and the child’s after birth; lipids; biological chemicals involved in the immune and nervous systems that govern how cells communicate, how chemicals are handled by the body and specific genotypes and phenotypes in both the child and the mother.

**UC DAVIS**

- Researchers at UMDNJ are recruiting children with ASD from community groups, and these children are also participants in an exposure assessment and intervention project.  Children in the study undergo a detailed medical, developmental, and environmental history, psychological testing and have regional brain growth studies by magnetic resonance imagery (MRI).
- Neurological function and regional brain growth are analyzed with respect to exposure to neurotoxicants in the local environment.  Researchers are also examining the frequency of genes coding for proteins that may influence susceptibility to environmental chemical-induced toxicities. Researchers are also investigating whether children’s susceptibility to ASD can be influenced by the child or maternal genotype for enzymes involved in protecting the developing human from chemically induced oxidative stress.

**UMDNJ**

- Animal models are being developed at both Centers to determine how early exposure to known immunotoxicants and neurotoxicants influences development of social behavior.  While neurotoxicants such as organic mercury and PCBs can alter cognitive function in humans and animals, little is known about their impact on the development of social behavior.
- Mouse models are being used to study mechanisms of action of suspected immune and neurotoxicants on social behavior, and permit genetic manipulation of expression of candidate genes.  Once animals are tested for social functioning, tissue samples are tested to determine distribution and levels of xenobiotics in the central nervous system, together with autoantigen (cytokines) profiling of the nervous system and measurements of alterations in gene expression.
- Non-human primates are being used to assess social behavior in response to neonatal exposure to thimerosal and a mixture of PCBs, permitting detailed analysis of  behaviors that are most closely related to those observed in developing children.

- Researchers at UMDNJ are quantifying the exposure and body burden of neurotoxicants in children with neurological disorders and deficits.  The goals of this community-based study are to obtain the distribution of exposure to neurotoxicants including heavy metals, volatile organics and pesticides in the child’s local ambient and residential and personal environment, to uncover links between exposure and behavior in neurologically impaired children, and to see what effective interventions can be developed.
- Methodologies include geographical information system (GIS) mapping to qualitatively identify personal pathways or environmental sources and a video assessment of each child’s routine activities.  Researchers are developing intervention strategies and educational programs to reduce environmental residential exposures to neurotoxicants within the affected population and measurements will be taken after the intervention has been effective.  The project will also provide valuable exposure information for toxicological studies and for comparison with health related data from epidemiologic studies.

**ANIMAL MODELS**

**EPIDEMIOLOGIC STUDIES**

**CELLULAR AND MOLECULAR MECHANISMS**

**EXPOSURE ASSESSMENT AND INTERVENTION**

- Pre- and post-natal exposure to VPA and MeHg produces behavioral deficits similar to those seen in autistic individuals, including retardation in development.
- Prenatally exposed mice were retarded in the development of mid-air righting reflexes.  They were also delayed in acquisition of a spatial learning task.
- Co-exposure to vitamin E appears to prevent the measured effects of VPA.

- Researchers at UC Davis are conducting the **Childhood Autism Risks from Genetics and the Environment (CHARGE) Study, **a case-control study comparing 3 groups: children with autism, children with mental retardation but not autism, and children with typical or expected development.  These children undergo a clinical evaluation including cognitive and adaptive assessments, medical history, environmental exposure to toxins, diet, genetic background, chemical and cellular markers.
- Researchers will characterize phenotypic variation in the autistic case group, including the pattern of clustering of symptoms, whether the child regressed after gaining language or other skills and presence or absence of mental retardation, with regard to specific exposure and genetic patterns.

- These studies are looking to determine the underlying mechanisms conferring susceptibility to immunotoxic and neurotoxic agents and their relationship to ASD.   These studies will:

- Characterize cytokine secretion profiles for peripheral blood mononuclear cells (PBMCs) to and determine autistic children exhibit significant differences in sensitivity and/or cell activation and cytokine secretion when challenged with antigens.
- Investigate the underlying signaling mechanisms which may contribute to the effects caused by certain xenobiotics.
- Test an immunological and neurological mechanism by which mercurials and halogenated organics (such as PCBs and PBDEs) influence glia/neural cell interactions and how they may influence neuronal growth and development.
- Investigate the functional and biochemical changes in mice with social behavioral deficits after exposure to antigen and xenobiotic combinations.

- Investigate the impact of neurotoxic metals on morphoregulatory (adhesion and repulsion) molecules during development and adulthood, and correlate perturbations of these molecules with functional alterations within the CNS of mice.
- Examine the effects of neurotoxicants on cognitive, sensory and motor skills of developing mice including impacts on mid-air righting reflex, locomotor coordination, water maze activity and a model for intrusive stereotypic and self-injurious behavior2.
- Define the effects of methylmercury, lead and the teratogen valproic acid (VPA) on the proliferation and differentiation of forebrain cortical precursor cells in cell culture derived from rat brain..
- Evaluate the effects of neurotoxicants and teratogens on fetal forebrain and hindbrain cell proliferation, suvival, and neurogenesis, comparing direct transuterine injection to indirect material exposure.