Grantee Research Project Results
2003 Progress Report: The Use of Mussel Gene Expression Profiles to Determine the Pollutant Decontamination Efficacy of Solvated Electron Reduction Technology - An Interdisciplinary Collaboration
EPA Grant Number: R829421E01Title: The Use of Mussel Gene Expression Profiles to Determine the Pollutant Decontamination Efficacy of Solvated Electron Reduction Technology - An Interdisciplinary Collaboration
Investigators: Wang, Shiao Y. , Biesiot, Patricia M. , Pittman, Charles U.
Current Investigators: Wang, Shiao Y. , Pittman, Charles U. , Biesiot, Patricia M.
Institution: University of Southern Mississippi , Mississippi State University
EPA Project Officer: Chung, Serena
Project Period: May 8, 2002 through May 7, 2004 (Extended to May 7, 2006)
Project Period Covered by this Report: May 8, 2003 through May 7, 2004
Project Amount: $228,750
RFA: EPSCoR (Experimental Program to Stimulate Competitive Research) (2001) RFA Text | Recipients Lists
Research Category: EPSCoR (The Experimental Program to Stimulate Competitive Research)
Objective:
The first objective of this research project is to improve and better understand the use of solvated electron reduction chemistry using Na/NH3 to remediate polynuclear aromatic hydrocarbon (PAH)-contaminated soils and sludge. The second objective is to develop a suite of molecular biomarkers of chemical stress response using the freshwater mussel as the model organism. These are complimentary objectives because solvated electron reductions in Na/NH3 produce a complex variety of reduced products from PAH mixtures. The PAHs themselves are converted totally to these products and are absent from the remediated soils. The question becomes: Is it acceptable to leave these byproducts in the soil? Because specific genes are activated to meet metabolic needs, the expression pattern of genes in mussels in a healthy environment differs from that of genes in mussels in a polluted environment. By comparing the gene expression profile of mussels exposed to treated and untreated PAHs to that of control mussels, it will be possible to determine whether the toxicity of PAHs have been reduced or eliminated as a result of solvated electron reduction. Thus, gene expression is being studied as an early warning system to evaluate the efficacy of the Na/NH3 remediation of PAHs.
Progress Summary:
The reduction of several neat PAHs was studied in Na/NH3 solvated electron solutions at room temperature under the autogeneous pressure of NH3. This was done in a stainless steel reactor and in thick-walled glass tubes. In each reaction, 0.1g of the PAH was dissolved in 10 mL of NH3 and excess sodium (0.5 to 1 g) was added. These solutions were left for 30 minutes and the NH3 was evaporated. The residual salts were washed out with distilled water and the organic residues were dissolved in benzene/cyclohexane and dried over anhydrous MgSO4. The MgSO4 was filtered and the residues were analyzed by gas chromatography (GC) to detect any unreacted starting PAH. The PAH destruction efficiency was defined as:
[detected PAH after reaction] ÷ [PAH originally charged to NH3] x 100
The PAHs each were removed to below detectable limits (by the GC procedure used).
PAH |
Destruction Efficiency |
| Phenanthrene | 99.9 + |
| Benzo[a]anthracene | 99.9 + |
| Fluorene | 99.9 + |
| Pyrene | 99.9 + |
The product analysis for the phenanthrene system produced a mixture of dihydro-, tetrahydro-, and hexahydro-phenanthrenes with higher amounts of hydrogenation occurring when longer times and larger amounts of sodium were added. Complex product distributions were obtained in the other three cases.
The results for the treatments of soils contaminated with various PAHs now have been published and a summary is available (Getman and Pittman, 2003).
To study the biological response of exposure to PAH and reduced PAH, phenanthrene (PHN) was used as the model compound. In addition to mussels, a fish (the Gulf killifish Fundulus grandis) was included to expand the scope of the study. Research on the effects of PHN exposure on fish gene expression has been completed. The study of effects of PHN exposure on mussel gene expression currently is in progress.
To accomplish the fish gene expression study, two cDNA libraries were constructed using subtractive hybridization technology. Forty-two clones of genes that appeared to be differentially expressed were selected for DNA sequencing. Eventually, 12 unique genes of interest were identified. Three of the genes (vitellogenin and choriogenins L and H) are involved in egg production, four (serotransferrin, fibronectin, serum amyloid A, and α-1-antiproteinase-like protein) in routine maintenance, three (complement component C3, C-type lysozyme, and complement regulatory protein) in defense and two (trypsinogen 1 and cytochrome C oxidase I) in digestion and energy metabolism. Polymerase chain reaction (PCR) primers were designed for each gene to examine the difference in the expression among control fish and fish exposed to PHN and reduced PHN using real-time quantitative reverse transcription PCR.
The expression of transferrin, fibronectin, and α-1-antiproteinase-like protein were lower in fish exposed to both PHN and reduced PHN relative to control fish, indicating their potential usefulness as biomarkers of pollutant exposure. The expression of genes involved in egg production differed in fish exposed to PHN and reduced PHN and are of interest because of the endocrine-disrupting potential of PAHs. The expression of choriogenin H and L were similar between control fish and PHN-exposed fish, but significantly higher in fish exposed to reduced PHN. On the other hand, the expression of vitellogenin was similar between control fish and fish exposed to reduced PHN, but significantly lower in fish exposed to PHN. These results suggest that reduced PHN, but not PHN itself, is estrogenic in terms of promoting egg membrane protein synthesis. PHN, on the other hand, may function as an estrogenic antagonist by blocking estrogen receptors, thereby reducing vitellogenesis. One of the main objectives of the research project is to determine whether reducing PAHs alters their toxicity. These results suggest that PHN and reduced PHN are pharmacologically different in their effects on fish reproduction.
The proportion of cDNA clones containing differentially expressed genes relative to all clones in the two subtraction libraries was approximately 4 percent, which is much lower than anticipated. Combined with the large amount of labor required to screen clones to check their expression levels, we concluded that the subtractive hybridization approach was an inefficient approach to isolating differentially expressed genes. Currently, we are using microarray technology as a screening tool to isolate differentially expressed genes in freshwater bivalves. We developed a method to produce a normalized cDNA library to reduce the over-representation of abundantly transcribed genes in the library. Approximately 2,400 gene inserts have been synthesized by PCR and then purified. DNA chips based on these cDNA clones currently are being made and will be used in the coming months to examine the effects of PAH and reduced PAH exposure on gene expression in bivalves.
Future Activities:
Microarrays containing mussel cDNA will be used as a screening tool to examine differences in gene expression between bivalves exposed to PAH and reduced PAH. Because of its greater solubility and lower volatility, most of the work will use PHN. The Mississippi State University group will prepare soil samples containing PAHs as well as remediated soil samples for leaching so that effluent waters can be tested on freshwater bivalves. Work will continue as planned on the development of molecular biomarkers and no change in the project schedule is anticipated. The expression pattern of these cDNAs in bivalves exposed to treated and untreated PAHs and effluents leached from PAH-contaminated soil before and after treatment will be compared to determine the efficacy of solvated electron reduction technology in eliminating the toxic nature of PAHs.
Journal Articles:
No journal articles submitted with this report: View all 6 publications for this projectSupplemental Keywords:
polynuclear aromatic hydrocarbons, PAH, PCBs, fish, mussels, PCR, subtractive cloning, cDNA library, genetic biomarkers, environmental chemistry, remediation, solvated electron reduction, southeast, Gulf coast, water, groundwater, soil, estuary, leachate, stressor,, Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, Water, Waste, Fate & Transport, Environmental Chemistry, Analytical Chemistry, Contaminated Sediments, Geochemistry, Ecology and Ecosystems, bioavailability, contaminant transport, sorption experiments, munitions, bioremediation of soils, contaminated sediment, desorption kinetics, water quality, electrokinetics, electron reduction technology, munitions residues, mussel gene expression profiles, fate and transport, TNT, nitrate compounds, chemical kinetics, contaminant transport models, PAHProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.