Grantee Research Project Results
2004 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, 2004 through May 7, 2005
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 freshwater mussel as the model organism. These are compl ementary objectives because solvated electron reductions in Na/NH3 produce a complex variety of reduced products from PAH mixtures. The PAHs themselves are totally converted 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 has 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 work on solvated electron reduction chemistry this year concentrated on comparing sodium versus calcium utilization when a PAH mixture containing eight compounds was reduced in the presence of water in liquid ammonia. Also, the same PAH mixture was used in a purposely contaminated clay soil and then it was reduced by sodium (Na) versus calcium (Ca) in liquid ammonia. The PAH mixture used was an equimolar mixture of naphthalene, anthracene, phenanthrene, acenaphthylene, fluoranthene, pyrene, 1,2-benzanthracene, and chrysene. In all cases, sodium was more efficient than calcium. This was confirmed in pure ammonia and when either 2 percent or 7 percent of water was present. In all cases, a larger molar amount of Ca than Na was required to achieve complete disappearance of the PAH mixture.
The same higher efficiency of sodium was found when purposely contaminated soils were employed. For both the soil slurry and the solution solvated electron reductions, the advantage of sodium over calcium use became more pronounced as the amount of water present was increased. An economic analysis of these results shows that it is more economical to use sodium than calcium in all of the remediation processes that we have considered to date. These results all further confirm the conclusions we reached with a variety of samples from Superfund sites and other contaminated sites in our previous publications (see for example, Getman, et al., 2003) .
The use of a PAH complex mixture is a much more realistic test of process efficacy than the use of a single PAH, because each PAH undergoes a complex reduction pattern where the rates vary for each separate reduction step. Thus, the kinetics of all the reduction steps that occur for a total of eight sample PAHs covers a wide rate range. The ultimate efficacy achieved depends on the relative consumption of solvated electron by the PAHs versus the consumption by water. The faster the PAHs undergo reduction, the less water competes for the solvated electron. We have firmly established that PAH remediation requires higher consumptions of Na or Ca than the remediation of PCBs. This is the result of the higher rate of PCB versus PAH reduction, so competition by water is more serious in PAH remediations.
To develop a suite of molecular biomarkers of chemical stress response, experiments were conducted to isolate genes differentially expressed in the bivalve Corbicula fluminea. A normalized cDNA library was constructed by subtractive hybridization, and 2,304 randomly selected cDNA clones were used in four microarray experiments. Phenanthrene was used as the model compound, and fluorescently labeled cDNA synthesized from mRNA of exposed and control bivalves were used as probes. Although there was significant statistical correlation among the four experiments, the level of correlation was only 0.16. More disturbing was the lack of genes that showed large changes in expression between control bivalves and bivalves exposed to phenanthrene. More than 100 genes showed elevated levels of expression in each experiment, but only 26 were common in all experiments at a threshold of 1.5-fold and none at a threshold of 2-fold. Similarly, only six cDNAs had reduced levels of expression in all experiments at a threshold of 1.5-fold but none at a threshold of 2-fold.
Fifty-four cDNAs whose expression level appeared different between exposed and control C. fluminea were selected for detailed analysis. Some of the clones contained the same gene. This was not unexpected because genes that are expressed at higher levels have higher representation in the library. Among the 54 clones sequenced, 14 were unique sequences. Among the 14 sequences, primers for 10 worked well for real-time RT-PCR. Unfortunately, the expression analysis results were not consistent. In one experiment, the expression level of the 10 genes were not different between exposed and control bivalves. In the second, one gene designated B11E showed a 2.7 -fold reduction in expression in exposed bivalves. In the third experiment, four genes B11E, D6A, G9H, and X8E showed 1.99 -, 3.93 -, 4.30 -, and 2.09 -fold reduction among exposed C. fluminea, respectively.
Results from the bivalve study indicated that an alternative model organism should be used. There were three significant problems. First, the difference in the level of gene expression between control C. fluminea and those exposed to phenanthrene was small. This is likely a reflection of their survival strategy. Because they are immobile and have relatively low metabolic rates, they “clam up” when exposed to pollutants to limit their exposure. Second, few bivalve genes have been identified. The identity of most of the cDNA sequenced remained unknown after exhaustive BLAST searches. Among the 54 cDNA clones sequenced, those that could be identified encode 18 and 28S rRNA and a ribosomal protein. Without unknowing the identity of the other sequenced cDNAs, it is not possible to ascertain their physiological significance.
The third problem was that results from different exposure experiments were not consistent. This may have been the result of using bivalves that differed in their physiological condition in the different experiments. Bivalves were collected from the wild and then acclimated in the laboratory for 1 week prior to use in experiments. Because each exposure and microarray experiment took up to 4 weeks to complete, C. fluminea collected at different times were used. The possibility of this creating a problem was anticipated, but a decision was made early during the study to limit the duration over which C. fluminea are kept in the laboratory. Bivalves have a hard shell, which prevents one from knowing their health status through observation, and it is difficult to tell whether they are feeding in the laboratory. We decided that freshly collected C. fluminea that have been acclimated to the laboratory provided us the best chance of using healthy bivalves.
To overcome the problems listed above, we decided to switch to the nematode Caenorhabditis elegans as the model organism. It offered several important advantages. First, its entire genome has been sequenced, which greatly enhances our chance of identifying the function of differentially expressed genes. Second, it can be cultured in the laboratory using standardized protocols. This provides us with the assurance that test subjects used in all experiments are of the same physiological condition. In fact, we use synchronized worms so that worms used in each experiment are of the same age. Third, commercial microarrays can be purchased. Not only does this provide us the opportunity to examine much larger numbers of genes, but also the DNA chips are of higher quality and uniformity than those we make in the laboratory. We now are able to analyze the expression levels of more than 22,000 genes simultaneously as compared to 2,304 with clam microarrays.
Initial results from using C. elegans are in stark contrast to those obtained using C. fluminea. The identity of almost all of the genes selected for detailed expression analysis is known. The results are highly consistent from one exposure experiment to another. For most of the differentially expressed genes, the difference in expression level between worms exposed to PAH and control worms w as much larger as compared to bivalves. The expression level of some upregulated genes increase by as high as a thousand fold in worms exposed to PAH. Experiments currently are in progress, and details will be provided in the next progress report.
Future Activities:
Expression analysis of differentially expressed genes will be conducted using C. elegans exposed to different concentrations of PAHs to determine their sensitivity. Analysis also will be conducted to determine the gene expression response of C. elegans to different types of PAH and most importantly, whether the gene expression response is different between C. elegans exposed to PAH and those exposed to reduced-PAH. 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. The eventual goal is to determine efficacy of solvated electron reduction technology in eliminating the toxic nature of PAHs. Significant efforts will be made to present the results at conferences and to publish the results.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 6 publications | 4 publications in selected types | All 2 journal articles |
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Type | Citation | ||
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Pittman,C.U.. Detoxification and destruction of PCBs, CAHs, CFCs, and halogenated biocides in soils, sludges, and other matrices using Na/NH3. Pesticide Decontamination and Detoxification. 2004; (863) : 181- 198 |
R829421E01 (2004) |
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Supplemental Keywords:
PAHs, PCBs, Caenorhabditis elegans, PCR, subtractive cloning, cDNA library, genetic biomarkers, environmental chemistry, remediation, solvated electron reduction, gulf coast, water, groundwater, soil, estuary, leachate, stressor,, Scientific Discipline, Water, Waste, Ecosystem Protection/Environmental Exposure & Risk, Environmental Chemistry, Geochemistry, Contaminated Sediments, Analytical Chemistry, Fate & Transport, Ecology and Ecosystems, electrokinetics, fate and transport, contaminant transport, bioavailability, contaminated sediment, desorption kinetics, PAH, electron reduction technology, nitrate compounds, bioremediation of soils, chemical kinetics, munitions residues, mussel gene expression profiles, water quality, sorption experiments, TNT, contaminant transport models, munitionsProgress 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.