Superfund Research Program
Developmental Toxicity of non-Dioxin-like PCBs and Chemical Mixtures
These studies are examining the role of cytochrome P450 (CYP) enzymes involved with detoxifying contaminants in the body. Project investigators are looking at several kids of cytochrome P450, specifically CYP1A and CYP1B, induced by aryl hydrocarbon receptor agonists, and CYP5, which catalyzes a key step in the synthesis of cholesterol. We have also made advances in the past year identifying patterns of gene expression in fish embryos exposed to toxicants, including the highly teratogenic TCDD, commonly known as dioxin.
The studies with CYP51 included complete sequencing of the CYP51 coding regions from zebrafish and from killifish (Fundulus heteroclitus). The expression of CYP51 was determined for 10 organs in killifish, both reproductively active and reproductively inactive. The expression of CYP51 in gonads as well as several other organs showed a pronounced difference. This is likely due to changing need for cholesterol associated with developing gametes.
The zebrafish CYP51 gene was put into bacteria, E. coli, and the bacteria synthesized quantities of the protein. This enzyme was purified and catalytic properties examined. These are the first such studies of CYP51 of a lower vertebrate.
The studies with CYP1A and CYP1B have continued in zebrafish, and the researchers are examining the role of CYP1A in the toxicity of TCDD. Prior project studies showed that fish CYP1A was a source of free radicals (highly reactive and unstable molecules) when the enzyme bound to planar PCBs. In the past year, project investigators found that TCDD also causes the enzyme to release large amounts of oxygen radicals (a specific type of free radical). The binding of TCDD and planar PCBs to the CYP1As from fish and humans are suggesting a molecular basis for the greater ability of the fish CYP1A to produce radicals. In a continuing collaborative effort with a group in Japan, these researchers have examined the toxicity of TCDD in zebrafish embryos in which active CYP1A protein was not made. The studies showed that in the embryos in which CYP1A was knocked-down, some toxicities of TCDD were reduced or eliminated. This advances our understanding of the mechanisms of dioxin toxicity.
Analysis of gene expression in zebrafish embryos exposed to TCDD continued, using the cDNA microarrays (gene chips) that they developed. These studies have now identified suites of responsive genes, both some that were expected and some that were unexpected. The profiling showed that induction of one gene, CYP1A, still is the strongest molecular response. Among the new genes identified in these studies is a retroelement, or a remnant, of a retrovirus that is induced by TCDD and highly expressed in the heart. The other gene expression changes provided molecular support for known phenotypic effects, including a dilated cardiomyopathy (a subset of congestive heart failure). At different doses, TCDD-responsive genes in zebrafish embryos differed, in part, from those genes affected in some mammalian systems, indicating a complicated nature of the effects of dioxin. Gene expression also is being examined in zebrafish treated with other types of chemicals.