Superfund Research Program
Developmental Toxicity of non-Dioxin-like PCBs and Chemical Mixtures
The overall goal of this continuing research is to determine how enzymes that metabolize pollutant chemicals may be involved in the toxicity of those chemicals during development. The studies focus especially on the cytochrome P450 enzymes, which are the enzymes most responsible for metabolism and clearance of foreign chemicals from the body. The studies focus on the zebrafish (Danio rerio) as a research model species. The objectives of this project include identifying the entire complement of cytochrome P450 genes in this species, then determining the developmental patterns of expression of all of these genes and the impact of developmental toxicants on that expression. Subsequently, Dr. Stegeman and his research team will determine the involvement of specific genes in the toxicity of selected chemicals to the developing embryo. Generality of findings will be established by examining Fundulus heteroclitus, a marine species also used as a model in molecular toxicology. These studies have significance in several areas: First, the information generated will help the researchers to understand how chemicals may produce birth defects in animals, including humans. Second, the studies will enhance understanding of how pollutant chemicals affect fish, which are environmental sentinels and a critical source of protein in the world. Third, the research will provide information and tools for other researchers who wish to study mechanisms of chemical effect in this model species for biomedical research.
Project researchers have now identified all of the zebrafish CYP genes. Gene predictions for all of the predicted zebrafish CYPs in the current release of the zebrafish genome (Zv6) have been used to construct phylogenetic trees, and to design primers for use in a focused microarray. Primer design and gene cloning for the microarray is ongoing. The researchers have successfully cloned 50 of the 70 predicted zebrafish CYPs, and are actively cloning the rest. They are in the process of primer design and cloning of genes for the nuclear receptors and other associated genes, which will be represented on the focused microarray.
The research team has determined the expression and induction of the full set of cytochrome P450 genes in gene family 1 (CYP1A, CYP1B1, CYP1C1 and CYP1C2), in adult zebrafish and during development. They have also continued to test ideas regarding functional and evolutionary relationships among CYP1As in different species, identifying gene conversion as a factor in CYP1 gene evolution. They have also annotated all the CYP genes in another developmental model, the sea urchin, recently reported in the journal Science.
The project researchers have continued studies in collaboration with the Bioinformatics Core (Sandor Vajda), on molecular modeling and substrate docking to CYP1As. Distributions of the in silico predicted B[a]P oxidation product frequencies match quite closely the observed in vitro metabolite distributions, supporting the usefulness of this type of modeling. Models of zebrafish CYP1C have also been generated and are being used to predict the chemical features of possible substrates.