Superfund Research Program
Developmental Toxicity of non-Dioxin-like PCBs and Chemical Mixtures
The overall goal of this project is to determine how enzymes that metabolize pollutant chemicals, especially the cytochrome P450 enzymes, may be involved in toxicity of those chemicals during development. The studies use fish as models, as fish are known to be extremely sensitive to the developmental effects of some chemicals, and they are sentinels in aquatic systems. Dr. Stegeman's team uses zebrafish, a vertebrate model for development, and Fundulus heteroclitus, a marine fish model that is found at marine superfund sites and in which resistance to toxicity has evolved.
Several new target cytochrome P450 genes were cloned and their functions are being characterized. Novel CYP1 family genes, defining a new category (CYP1C) were identified in zebrafish and Fundulus as well as in other fish species. CYP1 genes are induced by dioxins and selected toxic planar PCBs and PAH, and CYP1 proteins may be involved in the toxicity of these inducers. Novel CYP2 family genes were also identified, by cloning from zebrafish. These new genes, designated CYP2AA, have been expressed in mammalian cell lines and functional characterization begun. These genes, which have eluded discovery in fish for decades, may be the counterparts of human P450 genes that are inducible by non-planar PCBs. This possibility is under study, and could provide new markers for chemical exposure in fish. Other genes newly cloned from Fundulus and/or zebrafish include a cytochrome P450 (termed CYP51) that may have a role in reproduction, and also a receptor (termed the PXR) that may regulate the expression of several P450 genes during development.
Planar PCBs could be toxic because they 'uncouple' the catalytic cycle of CYP1A, a process these investigators identified and further defined with SBRP support. Uncoupling results in about 120 moles of toxic reactive oxygen per mole of CYP1A before the enzyme is inactivated by that reactive oxygen. Together with the Bioinformatics Core, Dr. Stegeman is modeling the structure of fish CYP1As and examining the molecular features of chemicals that cause uncoupling to predict which chemicals could be toxic by this mechanism.
Chemical effects on gene expression were studied with zebrafish cDNA microarrays (developed in collaboration with Mark Fishman). This confirmed that CYP1A is the single most responsive gene in embryos exposed to TCDD. TCDD caused strong changes in expression of other known and unknown genes. In collaboration with H. Teraoka (Japan) it was found that in developing zebrafish, CYP1A expression in the vasculature is linked to changes in blood flow and to apoptosis in the developing brain (mesencephalon). This project is leading to new understanding of how some Superfund chemicals may cause defects during development, and providing information that may lead to new markers of exposure and effect.