Superfund Research Program
Developmental Toxicity of non-Dioxin-like PCBs and Chemical Mixtures
Cytochrome P450 enzymes are most responsible for metabolism and clearance of foreign chemicals from the body. The overall goal of this project is to determine how enzymes that metabolize pollutant chemicals, especially the cytochrome P450 enzymes, may be involved in pollutant toxicity during development. The studies use fish as models, as fish are known to be extremely sensitive to the developmental effects of some chemicals. Fish also are commonly used as sentinels in assessing chemical exposure in aquatic systems. Dr. Stegeman and his research team use zebrafish (Danio rerio), a vertebrate model for development, and Fundulus heteroclitus, a marine species increasingly used as a model in marine toxicology. Fundulus also are found at marine Superfund sites where they have developed (evolved) resistance to toxicity of polychlorinated biphenyls. Progress was made in identifying and analyzing the function of cytochrome P450s from several species. The researchers highlight two areas of advance for this year.
Collaborative studies with the BU-SBRP Bioinformatics core on the construction and refinement of homology models of Fundulus cytochrome P450 1A (CYP1A) and human CYP1A1 have continued to yield beautiful results. “Docking” of substrates 3,3',4,4'-tetrachlorobiphenyl (PCB-77) and benzo[a]pyrene (B[a]P), was carried out to address questions of how the architecture of these fish and mammalian proteins evolved from a common ancestor, differ from each other. The observations are consistent with the researcher’s hypothesis that the fish CYP1A have more latitude of different substrate shapes than do CYP1As in mammals. It appears that the chlorine substituents may act physically to restrict the access of the pollutant PCB-77 into the fish CYP1A substrate access channel or binding site to a greater degree than in human CYP1A1. A more restricted substrate acceptability for fish CYP1As than mammalian CYP1A1 is suggested also by the more pronounced regio-specificity of the fish CYP1As for oxidation of the benzo-ring of BaP. Significance of these finding: these findings indicate that fish and mammalian CYP1A proteins differ in ways that may be related to their roles in toxicity of the chemicals. The models also will be useful in studies to evaluate unstudied chemicals for their potential metabolism by these enzymes.
A second area of progress highlighted involves the formation of toxic reactive oxygen species by the CYP enzymes. In the current year BU researchers examined the formation of reactive oxygen species by liver microsomes from Fundulus heteroclitus obtained from New Bedford Harbor (where they are resistant to PCB toxicity) and from Scorton Creek (where they are susceptible to PCB toxicity). Studies showed that fish from the two populations differ in the amounts of reactive oxygen released. Studies with CYP inhibitors indicated that the formation of reactive oxygen in this species involves prominently a form of P450 that is a CYP2E-like protein, as well as a CYP1A. Identifying the sources of reactive oxygen may point to the roles of P450 enzymes in the toxicity of the PCBs that are present in the New Bedford Superfund site. The mechanisms of that toxicity are still largely unknown.