Superfund Research Program
Developmental Toxicity of non-Dioxin-like PCBs and Chemical Mixtures
This project is addressing the hypothesis that species differences in metabolism and effects of planar halogenated aromatic hydrocarbons reflect differences in CYP1A active site structure. Teleost CYPIB will be simiIarly examined, and compared to CYP1A and to mammalian CYPlBl. Project investigators have cloned full length CYP3A30 from the killifish Fundulus heteroclitus, and carried out a full phylogenetic analysis of the CYP3A subfamily. Heterologous expression of the killifish CYP3A30 is underway. Similarly, the heterologous expression of killifish CYP1A is underway, in Hepa?1 cells and in HEK cells. Modeling efforts have been initiated in collaboration with Sandor Vajda at BU Department of Biomedical Engineering (BUSBRP Research Core). The killifish CYP1A (as well as the scup CYP1A) have been realigned with bacterial CYP sequences. The alignments will be used to generate new models for testing docking programs.
Expression of CYP isoforms will be examined from cleavage through post-hatching and larval periods, emphasizing blastula and gastrula stages, when there are critical windows for effects of some chemicals. The project investigators elected to follow a path to assessing gene expression that should yield greater information than the procedures originally proposed. They are developing DNA microarrays for determination of expression profiles of zebrafish genes. The initial arrays showed a good signal with 10 mg of total RNA from a zebrafish heart. Upwards of 5,000 sequences have been produced, including approximately 2,500 known genes. The known genes include all the CYP genes originally specified, which we had already cloned from zebrafish, as well as additional CYP genes that were identified from published sequences. The known genes also include as many nuclear receptors as could be identified in zebrafish, as well as other genes for proteins involved in xenobiotic interactions. Hybridization conditions are being optimized.
Additionally, the project investigators are examining the effects of chemicals on CYP expression during development. Chemicals will be selected for known/suspected interaction with key receptors and/or effects on development. Zebrafish embryos have been treated with TCDD in anticipation of extracting RNA for hybridization. The exposure conditions and internal doses have been established carefully, using tritiated TCDD to confirm the internal dose in embryos. Groups of embryos were exposed to doses on either side of a previously determined LD50. The RNAs from these embryos will be hybridized with the arrayed cDNAs in the coming weeks. Eventually these arrays will be used in analysis of different classes of xenobiotics for effects on gene expression profiles, and may be used as well to meet similar objectives of collaborating investigators in the BUSBRP.
Fundulus resistant to toxicity of planar halogenated aromatic hydrocarbons also have suppressed CYP 1A induction, implicating the AHR pathway in the resistance. Establishing the patterns of regulation of CYPlB, putatively AHR regulated, could amplify the understanding of the mechanisms of resistance. This aspect represents the principal area of continuation, and includes completion of the analysis of CYP1A induction in additional species from the locations where the Fundulus have acquired resistance to CYP1A induction. The studies in eel show that not all species have acquired resistance. The researchers have also progressed on the expression of Fundulus CYPlB in HEK cells.