Superfund Research Program
Developmental Toxicity of non-Dioxin-like PCBs and Chemical Mixtures
Aim 1. Behavioral phenotypes caused by ortho-PCBs have been examined. Increased activity was greater with PCB52 and PCB95 than with PCB153, an intriguing difference.
Transcriptomic effects after short term (6 hr) and longer-term (24 hr) exposures to an effective dose of PCB153 are being analyzed. A surprisingly large number of genes were down regulated, more after the short exposure (> 900 genes down regulated) than after the longer exposure (ca. 175 down). Analyses of the genes and implications are underway. Exposure to additional ortho-PCBs for transcriptomic effects also is underway.
Aim 2. Zebrafish transcriptomics (RNA-seq) is identifying transcriptional changes in embryos treated with PN, in which functional PXR was greatly reduced by morpholino knockdown. The hypothesis is that PXR is important in controlling gene expression, for steroidogenesis, and for some detoxification enzymes. The results show both agonist-dependent and independent effects. The PN exposure resulted in 347 genes up-regulated and 115 down-regulated. Among the genes up-regulated were cytochrome P450 genes not previously recognized, especially CYP2K6. Further analyses of the responses and implications are underway.
Additional PXR agonists and antagonists in human were tested in zebrafish embryos, to identify responses in vivo, against which to compare effects of PCBs. Stegeman and his researchers have examined pregnenolone (PN), pregnenolone-16a-carbonitrile, PN-sulfate, clotrimazole, nifedipine, hyperforin, bexarotene, triclosan and others. With those compounds that caused increased expression of the target genes pxr and CYP3A65, the response in vivo was substantially less than predicted based on activation of ligand-binding domain in cell-based reporter assays. Several chemicals tested, notably hyperforin and bexarotene, decreased expression of PXR target genes. The possibility that these are antagonists was studied further using combined exposures with the demonstrated zebrafish PXR agonist PN. The expression of CYP3A65 and CYP2K6 was decreased with addition of hyperforin or bexarotene. The suggestion that these are antagonists is being studied further, including with PXR modeling and ligand docking studies.
The possibility that differing responses to PXR agonists in different groups of embryos could involved PXR allelic variation has been examined further. Exposure groups selected based on different responses of CYP3A65 also showed differences in pxr and CYP2K6 expression. Cloning and sequencing of the PXR from those groups revealed sequence differences in the translated protein that further indicate zebrafish that PXR alleles function differently in vivo. These studies are continuing.
The research team found earlier that expression of the orphan P450 CYP20 was increased by PCB153 and PCB52. In this year researchers have found that this effect does not involve PXR. Researchers also observed that an ortho-PCB that strongly affects ryanodine receptors (PCB95) did not induce CYP20. The mechanism of CYP20 induction is not yet known. The team also reported that morpholino knock down of CYP20 expression caused increased activity and decreased visual responsiveness in zebrafish larvae. The relevance of CYP20 to developmental neurobehavioral disorders was further indicated by suggestions that deletion of CYP20 in human is associated with hyperactivity disorders. Genes syntenic with CYP20 also are induced by PCB153. A grant application has been submitted to explore CYP20 involvement in developmental neurobehavioral changes.
Aim 4. The ryanodine receptor studies in collaboration with the UC-Davis SRP are ongoing; a paper has been published on RyRs in New Bedford Harbor killifish, and studies of RyRs in zebrafish are being developed.