# Presentation: 699373

## Health-Related Methods Development Projects for the National Children’s Study

**Health-Related Methods Development Projects for the National Children’s Study**
- Pauline Mendola1, Jane Gallagher1, John Rockett1, Suzanne Fenton1, Danelle Lobdell1, Suzanne McMaster1, Robert MacPhail1, Stan Barone2, Tara Lyons-Darden1
- 1U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory
- 2U.S. Environmental Protection Agency, Office of Research and Development, National Center for Environmental Assessment

- Can we identify and validate comparable biologic markers in both test animals and humans that can be used to investigate the risk and mechanism of environmental exposures associated with functional impairments of the reproductive and nervous systems;
- Can we determine the key genetic and physiologic factors that modify associations between exposures and outcomes in a similar fashion in both animal and humans?

**Research Goals**

**Methods/Approach**

**Impact and Outcomes**

**Results/Conclusions**

**Future Directions**

**Science Question**

- Maximize human-animal extrapolation by identifying and validating common biomarkers from observational human studies and experimental animal studies;  and
- Focus on samples that could be obtained with minimal invasiveness from human subjects, including children, infants and pregnant women.

-  Whole blood and hair follicles yield sufficient quantity and quality of RNA to conduct microarray analysis, suggesting that hair samples may be used as a surrogate tissue for gene expression profiling studies
-  Human sperm contain a common set of RNAs reflective of spermatogenesis and male fertility, with variation in gene expression suggesting the utility of sperm  RNA as a biomarker of reproductive exposures.
-  Uroepithelial cells are of insufficient quality (and in some cases quantity) to be a useful source of RNA for gene expression profiling studies.
-  Our studies on finger nails suggest we can evaluate exposure and susceptibility in infants because the first nail clippings form *in utero* and reflect critical windows of exposure and development that are otherwise difficult to measure.
-  We were able to use peripheral whole blood as a source of neurodevelopmental markers including neurotrophins (nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4), cytokines (interleukin-6 and tumor necrosis factor alpha), and neuropeptide Y.

- The results of this research will influence the selection of biomarkers and methods to be used for assessment in the NCS.  Evaluating the utility of non-invasive sample matrices is essential to the long term success of this study, especially if we hope to make use of developing fields of science such as gene expression.  Understanding data needs relevant to risk assessment implementation, such as comparable animal and human biological markers, provides important guidance for the measurement of NCS outcome.  EPA will gain valuable information on methods to measure children’s environmental health risks and data that can improve risk assessment for susceptible populations.

- Data collection for ongoing studies of infant motor activity and breast milk constituents will be completed in 2005.  These measurements will be evaluated for inclusion in the NCS.
- This work has provided the foundation for a successful  computational toxicology proposal headed by Dr. Jane Gallagher.
- We plan to continue our biomarker development work with a focus on parallel animal and human studies to advance risk assessment in children’s environmental health.

_**Behavioral Indicators – Animal and Human Studies**_
-  Developmental disorders in humans are linked to fundamental processes that are common to humans and laboratory animals.
-  Exposure scenarios that are suspected to affect development in human infants can be replicated, amplified and/or disaggregated in the laboratory. Animals can be exposed to the same environmental conditions observed in homes and day care settings. This will provide the bridge needed to link environmental exposures to clinical outcomes.
-  Working with the SHR, a popular model for ADHD, we are studying responses selected to evaluate a common characteristic of children with ADHD – failure to habituate to a familiar environment.

_**Method Development Studies**_
**MALDI-TOF Analyses of Fingernails**
- Obtaining biological specimens from newborn humans is a particular challenge. We developed methods to use non-invasive samples that can reflect *in utero* exposures, such as fingernail specimens.  We validated the usefulness of fingernail DNA for assessing relevant metabolic and detoxification genetic polymorphisms, comparing nail results to blood and buccal cell samples and found that a MALDI TOF high throughput technique could be used in place of standard genetic analysis.
**Collection and Storage of Breastmilk**
- Work is also being conducted to develop fractionation and freezing protocols for breast milk samples.  This will facilitate the development of reliable assays to measure endogenous and exogenous constituents of fresh and frozen breast milk, and to evaluate whether blood, saliva, and urine are useful surrogate mediums for the analysis of some constituents of milk.  Preliminary analyses have determined the appropriate sample collection and preservation techniques and subjects began giving samples in December 2004.
**Neurodevelopmental Markers**
- We have evaluated peripheral whole blood as a source of neurodevelopmental markers including neurotrophins (nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4), cytokines (interleukin-6 and tumor necrosis factor alpha), and neuropeptide Y.  We were able to measure all of the proteins of interest using Enzyme-Linked ImmunoSorbent Assay (ELISA) analysis.

_**Mercury in Human Nails and Various Animal Tissues**_
- We have extended our work to evaluate whether nails are an appropriate dosimeter of exposure relative to blood and target tissue brain following in utero exposure to methyl mercury.

- Levels of mercury observed in blood and fingernail samples from human subjects.

- Methyl mercury levels in various rodent tissues

- Levels of motor activity for SHR (N=18) and WKY (N=18) rats from two experiments.  Each symbol represents mean ± SEM total activity for each of four or  five 30-min consecutive test sessions.