Superfund Research Program
Developmental Toxicity of non-Dioxin-like PCBs and Chemical Mixtures
The overall goal of this research is to determine how enzymes that metabolize pollutant chemicals may be involved in 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. Specific aims include identifying the complete 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. John 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 be help to understand how chemicals may produce birth defects in animals, including in humans. Secondly, 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.
With repeated searching of new genome releases, the full complement of predicted zebrafish cytochrome P450 genes has now grown to 84. Gene predictions have been refined from last year. The focused microarray has been replaced with a commercial microarray platform to which oligonucleotide probes for all of the CYP genes, phase II genes, and nuclear receptors have been added. This new array is being tested at the present time.
Studies of new genes in the cytochrome P450 1 gene family members have continued. The team showed that zebrafish CYP1C1 and CYP1C2 are both induced by planar PCBs as well as by dioxin (TCDD). As with CYP1A and CYP1B1, the induction of the CYP1C1 and CYP1C2 was shown to be dependant on the aryl hydrocarbon receptor in studies with embryos in which the expression of the receptor was knocked down. They also cloned a fifth new CYP1 gene, CYP1D1, and showed that it is not under the regulation of the AHR, even though it is closely related to the CYP1A. Molecular modeling of these new CYPs is underway. They also have now expressed all five of the zebrafish CYP1 genes in yeast and are beginning to characterize these new CYP1s directly. The study of these new CYP1 genes is important to understanding the mechanisms by which chemicals act in development.
The researchers also are studying the occurrence of homologues of these diverse CYP1 genes in other species, including mammals. The influence of the high level contamination by PCBs on the expression of CYP1A, 1B and 1C genes is also being examined in the killifish Fundulus heteroclitus from New Bedford Harbor.