Superfund Research Program
Epigenetic Effects of Pre- and Post-Remediated Environmental Toxicants
Project Leader: James E. Trosko
Grant Number: P42ES004911
Funding Period: 1995 - 2006
Final Progress Reports
Year: 2004 1999
Specific Aim 1: In preparation to apply various toxicants to the developed in vitro human neuronal stem cells, the team has characterized a number of PCB’s and PAH compounds for their ability to (a) induce various signal transduction mechanisms and (b) inhibit gap junctional intercellular communication regulating cell proliferation, differentiation, and apoptosis. Specifically, working on normal rat liver and human pancreatic cells, in vitro, they have shown that there exists a clear structure/function relationship between those classes of environmental toxicants, their ability to induce specific signaling molecules that correlate with their ability to inhibit gap junctional intercellular communication (GJIC). The significance of these studies include: (a) the continued validation of the in vitro GJIC-assay to detect “epigenetic toxicants”; (b) the possibility that small molecular weight PAHs, which have been generally ignored as significant environmental toxicants, might be as important, if not more so, than the more extensively studied large molecular weight PAHs; and (c) effects on GJIC depend on specific structural requirements of both PAHs and PCBs; (d) the upstream molecular events controlling GJIC in response to both PAHs and PCBs are very similar; (e) induction of oxidative stress by organic peroxides are mechanistically different for each compound, but tumor promoting peroxides nevertheless inhibit GJIC and activate MAPK, while the non-promoting peroxide does not affect these endpoints indicating the fundamental importance of these biomarkers in tumor promotion; (f) the red wine and peanut butter antioxidant, resveratrol, reversed the effects of the promoting peroxides on either GJIC or MAPK, indicating the potential chemopreventative properties of this dietary antioxidant.
Key Results (2004):
• The structural specific inhibition of GJIC by PAHs is the same in several cell types including rat liver epithelial cells, rat cardiac myocytes, and human neuronal, pancreatic and human liver stem cells.
• The structural specific inhibition of GJIC by PAHs and PCBs involves the activation phosphatidylcholine phospholipases resulting in the release of arachidonic acid.
• Arachidonic acid activates mitogen activated protein kinases (MAPK) and inhibits GJIC in mammalian cell cultures, thus offering a potential connection to phospholipase activation by PAHs and PCBs.
• Prevention of GJIC activation in a human pancreatic stem cell line by PAHs blocks the differentiation of this cell line.
• Proteomic analyses of cells treated with bioactive PAHs results in the up-regulation of tubulin dependent proteins and the downregulation of calmodulin.
• Initiation of oxidative stress by two organic peroxides, benzoylperoxide and dicumylperoxide, which are tumor promoters, results in the activation of MAPK and inhibition of GJIC, whereas, the non-promoting peroxide, t-butylperoxide, had no effect on the two endpoints.
• Only benzoylperoxide caused a decrease of reduced glutathione (GSH) and caused cytotoxicity.
• N-acetylcysteine, protected the cells from GSH depletion and cytotoxicity by benzoylperoxide but did not prevent inhibition of GJIC and activation of MAPK. Similarly, NAC did not prevent the effects of dicumylperoxide on GJIC and MAPK.
• Resveratrol, a prevalent antioxidant in red wine and peanut butter, significantly prevented dicumylperoxide effects on GJIC and MAPK, while it had no effect on benzoylperoxide induced inhibition of GJIC, but in contrast had a very large effect in preventing MAPK activation.
Specific Aim 2: Toxicogenomic in vitro investigations attempt to classify compounds based on gene expression profiles. In preparation to determine whether human stem cells are appropriate to use in toxicogenomic investigations of arylhydrocarbon receptor (AhR) and estrogen receptor (ER) ligand mediated effects on human cells Dr. Trosko’s team (1) determined that mRNA for the receptors of interest are present in the stem cells and, (2) have obtained preliminary microarray data detecting transcriptional changes upon exposure to prototypical ligands, TCDD and 17β-estradiol (E2), for the AhR and ER, respectively. Specifically, they have shown that normal human HL1-1 liver and 905K-1 kidney stem cells transcriptionally express the receptors of interest and demonstrate transcriptional changes upon exposure to each ligand.
Results of this study will: (1) determine whether these cell lines are transcriptionally responsive to TCDD and 17β-estradiol, (2) establish baseline gene expression profiles of prototypical ligands which other ligands may be compared to and, (3) be used to assess whether the gene expression profiling is comparable to what has been seen in other cancerous in vitro models.
Key Results (2004):
• HL1-1 and 905K-1 cell transcriptionally express AhR and ER mRNA
• Lists of approximately 2000 differentially expressed features for each experiment may be summarized as follows:
Cell Line HL1-1 905K-1
Ligand TCDD E2 TCDD E2
Time point examined (hours) 24 8 24 8
Number Annotated Up-regulated Genes 65 244 1548 1373
Number Annotated Down-regulated Genes 1462 902 2 24
Range of Differential Expression 6.90 – 0.39 2.55 – 0.62 3.17 – 0.74 1.65 – 0.59
• Greatest Up-regulated Genes:
1. HL1-1 TCDD: cytochrome P450, family 1, subfamily B, polypeptide 1
2. 905K-1 TCDD: cytochrome P450, family 1, subfamily B, polypeptide 1
3. HL1-1 E2: matrix metalloproteinase 17 (membrane-inserted)
4. 905K-1 E2: SMT3 suppressor of mif two 3 homologue 1 (yeast)
• Greatest Down-regulated Genes:
1. HL1-1 TCDD: PDGFA associated protein 1
2. 905K-1 TCDD: nuclear transcription factor Y, alpha
3. HL1-1 E2: arylacetamide deacetylase (esterase)\
4. 905K-1 E2: protein inhibitor of activated STAT,