Grantee Research Project Results
Final 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. , 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 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 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 complementary 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.
Summary/Accomplishments (Outputs/Outcomes):
We completed work on comparing the toxicity of PAH before and after treatment with solvated electron reduction technology. First, we identified genetic markers of PAH exposure using DNA microarrays, and then we compared the effects of exposure to neat and reduced PAH using gene expression analysis with quantitative realtime PCR. We used two species, the freshwater bivalve Corbicula fluminea and the nematode worm Caenorhabditis elegans. Results obtained using the bivalve model were reported in the previous report, and thus will not be included in this report. In general, the problems we encountered using a bivalve as a model were overcome by switching to the worm C. elegans as a model. The use of C. elegans allowed us to overcome problems with inconsistent results because we were able to carefully control the lifecycle of the worms and use worms that were synchronized in terms of both age and physiology. Another problem that we overcame was the large number of unidentified genes when we tried to use bivalve cDNA clones. C. elegans has a well characterized genome and most of the genes targeted for analysis in the present study were of known identity.
We screened the worm genome using microarrays and found 75 genes that were consistently up-regulated in worms exposed to phenanthrene in three separate experiments. Among these 75 up-regulated genes, members of three gene families showed up multiple times. Seven were cytochrome P-450 genes, six were UDP-glucuronosyl and UDP-glucosyl transferase genes, and four were glutathione S-transferase genes. Using members of these three gene families as molecular markers, we found that C. elegans are sensitive to phenanthrene exposure with significant up-regulation in worms exposed to 40 ppb, the lowest concentration used. Exposure to higher doses (120 ppb) resulted in further increases in the expression of cytochrome P450 and UDP-glucosyl transferase, but exposures to 360 and 720 ppb did not cause their expression level to increase further. The expression of glutathione S-transferase was not as sensitive to phenanthrene exposure compared to the other two genes, but the positive correlation between expression level and exposure concentration was significant up to 360 ppb.
Maximum increase in the level of mRNA occurred after 2 hours of phenanthrene exposure for glutathione S-transferase and after 6 hours for cytochrome P450 and UDP-glucosyl transferase. Up-regulation as a result of phenanthrene exposure occurred promptly for all three genes as an increase in mRNA levels could be measured as soon as 15 minutes after exposure.
Results from the expression analyses of 15 up-regulated genes indicate that exposure to the products of treating phenanthrene with solvated electron reduction reduces but does not eliminate the toxicity found with phenanthrene. Our assumption that the expression of each of these genes was up-regulated by worms in an effort to detoxify xenobiotics is supported by the inclusion of members in the cytochrome P450, UDP-glucosyl transferase, and glutathione S-transferase superfamilies. They encode enzymes that catalyze either oxidation of xenobiotics or conjugation reactions to help rid the body of potentially harmful substances by excretion. For example, members in the UDP-glucosyl transferase gene family encode microsomal enzymes, which catalyze the transfer of glucuronic acid to a wide variety of lipophilic substrates and are of major importance in the detoxification and subsequent elimination of xenobiotics. We note, however, that the expression level of these genes in worms exposed to reduced phenanthrene were still much higher than that in control worms, suggesting that the toxicity of phenanthrene was not completely eliminated.
In terms of relevance to the U.S. Environmental Protection Agency’s mission and potential practical applications, our results suggest that chemical reduction by solvated electron reduction technology reduces but does not completely eliminate the toxicity exhibited by PAHs. C. elegans exposed to reduced phenanthrene exhibited a decrease in their stress response compared to worms exposed to neat PAH. Genes turned on in response to PAH exposure were turned on to a smaller degree in worms exposed to reduced PAH.
Why might C. elegans exhibit any elevated stress response (enhanced gene expression) when exposed to the products of solvated electron-reduced phenanthrene? If complete reduction of phenanthrene, 1, to saturated hydrocarbon, 2, occurred, there might be a response of C. elegans to this compound. However, complete reduction does not occur. Instead small amounts of dihydro- and tetrahydrophenanthrenes are formed under many conditions (see Figure 1).
Figure 1.
Over a period of time, dissolved oxygen oxidizes 4 back to phenanthrene and 5 may undergo such a change. Therefore, phenanthrene may be reformed in small amounts after its initial complete disappearance during Na/NH3 reductions. The presence of this phenanthrene may cause the weaker response that was seen. A second possibility is that products such as 4 or 5 might reoxidize to phenanthrene inside C. elegans. Thus, oxidative enzymes could generate small amounts of phenanthrene as early metabolic products of partially reduced phenanthrene.
Comparing the three test organisms used in the study, the nematode C. elegans turned out to be superior to the fish Fundulus grandis and bivalve C. fluminea. There were three main reasons. The first is that commercially available DNA chips are available for C. elegans. Not only do commercial DNA chips save cost and time, the genome coverage is far greater on a commercial chip. For example, the commercial C. elegans chip we used contained more than 22,000 features, whereas the bivalve chip we made contained only 2,304 features. The second reason that C. elegans turned out to be a better model is that its genome is already well characterized. Therefore, few of the genes identified as being differentially expressed were unidentified. In contrast, most of the bivalve cDNAs identified as being differentially expressed were of unknown genes. Without knowing their identity, it is not possible to ascertain their physiological significance. The third reason is that C. elegans is much smaller in size, has a short generation time, and is much easier to work with in the lab. This helped us increase the reliability of the results by allowing us to use age synchronized worms and more replicates in each experiment.
One of the advantages of using C. elegans as a model is that they can be used in both aquatic and terrestrial environments. They were used in an aquatic experimental system in the present study because the original proposal was concerned with a scenario in which material leached from either a PAH-contaminated or remediated site entered freshwater streams. In the future, we recommend that C. elegans be included in a terrestrial assay system to test some of the higher molecular weight PAHs such as pyrene and chrysene, which are more toxic but not very soluble in water. Solvated electron reduction technology is an effective method to destroy PAH in contaminated soil. C. elegans would be an ideal model for further tests on the efficacy of solvated electron reduction technology as a soil decontamination strategy.
One of the most pressing matters is to publish results from the study. Currently, we are completing some physiological experiments comparing reproductive performance between worms exposed to phenanthrene and reduced phenanthrene. We decided to add this component at the end of the project period because we thought it was important to confirm our conclusions concerning the reduced toxicity of remediated PAHs with physiological performance data.
The project period ended, but we plan to continue work comparing the toxicity of higher molecular weight PAHs before and after remediation efforts using C. elegans in a terrestrial exposure system. The extent of the work will depend on the availability of research funds but we now have the experimental techniques worked out and know C. elegans well as an experimental model organism. We also plan to complete a related project that was initiated without research funding. The goal was to create a sentinel organism that can be used as a reporter of pollution. We wanted to create a system that is both easy to use and easy to interpret. We recently have created several strains of transgenic worm that fluoresce when exposed to PAH. During 2007, we plan to characterize their response to PAH. For example, we plan to determine the sensitivity and specificity of the fluorescence response, the time course of the response, and how to make it a user friendly and inexpensive method to monitor pollution.
Journal Articles:
No journal articles submitted with this report: View all 6 publications for this projectSupplemental 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.