Superfund Research Program
First Glimpse of the Human Fetal Proteome Signals Early Effects from i in utero Toxic Exposures
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Background: Environmental and occupational exposures of pregnant women to toxic materials may lead to negative health effects in their children. Documented effects range from low birth weight to developmental defects, and researchers believe that some chronic diseases, such as diabetes and heart disease, may have their basis in fetal and early childhood exposures. Prenatal exposures are difficult to quantify, as are their temporal and enduring effects, particularly when exposure occurs during the early stages of pregnancy.
With funding from the SBRP, Dr. Rolf Halden (Biodesign Institute, Arizona State University) is working with a team of researchers to study the effects of environmental exposures to toxicants. The group applies the latest analytical tools in proteomic mass spectrometry using output from high-speed DNA sequencing and bioinformatics to identify biomarkers of early effects from prenatal exposure to toxicants. Their goal is to use these biomarkers to detect adverse effects of toxic exposures before the onset of adult disease, thereby providing opportunities to reduce the occurrence and severity of disease in exposed populations.
Advances: Using cigarette smoke as a model toxicant, the researchers completed an initial global assessment of the human cord blood proteome, i.e., the proteins detectable in fetal serum, and identified biomarkers whose expression levels were modulated by prenatal toxic exposures.
Dr. Halden and his collaborators, Drs. Goldman and Witter of Hopkins' Tracking Health Related Environmental Exposures (THREE) study, collected umbilical cord blood samples from more than 300 full-term single-birth babies born in the Johns Hopkins Hospital (Baltimore, MD) over a five-month period. Samples were assigned as either "control" or "exposed" based on the levels of the nicotine metabolite, cotinine, found in cord blood sera. To eliminate potential confounders, the researchers considered parameters such as gender of the baby, race of the baby, method of delivery, exposure to alcohol or drugs, and pre- and neo-natal complications in both mother and child.
From the possible participants, six active smokers and six non-smokers were selected for inclusion in the study. Within the control and exposed groups, researchers blended the cord blood sera into two pools for analysis. The sera were first selectively labeled with isobaric tags for relative and absolute quantification (iTRAQ), and then analyzed in a "shotgun sequencing" approach employing two-dimensional liquid chromatography coupled with nanospray tandem mass spectrometry (2D-LC-MS/MS).
Dr. Halden's team identified over 200 unique proteins with >95% confidence. The researchers then looked for proteins or protein levels that differed between the smoking and non-smoking groups. They identified statistically significant differences between the control and exposed groups in 17 proteins. They then determined the biological roles of the 17 proteins that were significantly up- or down-regulated in babies born to smoking mothers and found that 14 of these candidate biomarkers have previously been associated with cigarette smoking. Thus, the data demonstrate the feasibility of biomarker identification via global screening and also show that babies exposed to cigarette smoke in utero exhibit alterations similar to those previously observed in adult smoke-exposed subjects and in animal models.
There were no unique biomarkers — the researchers did not identify a single protein unique to either the smoking or non-smoking group. This finding illustrates the necessity of using quantitative approaches in biomarker discovery and validation.
Significance: This research provides the first global map of the human cord blood serum proteome. The analytical approach validated here could be an important tool to monitor exposure to a wide range of toxicants that may have an impact on the growth and development of a child and on disease later in life. The group hopes to use the same techniques to examine a wide range of environmental exposures and their effects on human health.
This work also is significant in that it serves as a starting point for developing low-cost assays for biomarkers of antenatal exposure and disease. Further areas of investigation include a refinement of the method using western hybridization or high-throughput mass spectrometry processes, and the analysis of sera on a person-by-person basis to explore the variability between individuals.
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