Genetic Engineering of Plants to Improve Phytoremediation of Chlorinated Hydrocarbons in Groundwater
I. Mechanism of halogenated hydrocarbon oxidation We are using poplar culture cells to determine the pathway of TCE metabolism. In our earlier work, we found that trichloroethanol (TCEOH) is a major early intermediate. Our studies this year have focused on the steps that follow this toxic intermediate. We did several experiments to track the disappearance of TCEOH after the cells were removed from TCE. We could conclude that TCEOH is not an end-product but is rapidly degraded. Six flasks of poplar liquid suspension cells were exposed to a level of 50 {micro}g/ml TCE for three days. Three of the cultures were subjected to MTBE extractions to quantify the levels of TCEOH produced. The cells of the remaining three cultures were then pelleted and resuspended in fresh medium. After three more days, these were also subjected to MTBE extractions. The samples were analyzed by GC-ECD. After the three days of further metabolism, an average of 91% of the trichloroethanol was gone. When similar experiments were done with intact plants and both free and conjugated TCEOH were quantified, a similar rapid decline in both forms was seen (Shang, 2001). Therefore, it seems probable that similar mechanisms are taking place in both poplar suspension cells and whole poplar plants, so we continued to do our studies with the suspension cells. Metabolism of trichloroethanol may go through trichloroacetic acid (TCAA) prior to dehalogenation. To test this possibility, we exposed cells to TCE and analyzed for TCAA over time. The cultures were analyzed after 4, 5, 6, and 14 days from TCE exposure. We did not detect any significant amount of TCAA above the background in undosed cells. To determine if trichloroethanol itself is directly dehalogenated, we analyzed TCE-exposed cells for the presence of dichloroethanol. Undosed cells did not have any of the DCEOH peak but TCE-dosed cells that produced the highest levels of trichloroethanol did have a small DCEOH peak. Cultures that did not produce high levels of TCEOH did not have the DCEOH peak. This result repeated in two independent experiments. We decided to expose cells directly to TCEOH and look for DCEOH in the cell extracts. After one week of exposure, the culture cells produced consistent levels of DCEOH of approximately 0.02% of the TCEOH dose. However, when we did a control reaction with no cells, DCEOH was present, indicating that the TCEOH degrades in the absence of cells. We are currently conducting the same experiments with newly-purchased chemicals and in darkness (by wrapping the culture flasks in foil). We have had success using tribromoethanol as a surrogate for trichloroethanol in studying the dehalogenation reaction in poplar cells. We had previously shown that tribromoethanol is steadily metabolized over time in poplar culture cells, producing free bromide ion. TBEOH-dosed dead cells and no cell controls did not have any bromide ion production. We are currently using this system to test P450 inhibitors to determine if dehalogenation of TBEOH is through this mechanism. We have recently purchased tribromoethylene as a more easily monitored surrogate for TCE. We will conduct mass balance experiments to determine what percentage of the bromide is released from tribromoethylene.
- Research Organization:
- Univ. of Washington, Seattle, WA (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC)
- DOE Contract Number:
- FG02-03ER63663
- OSTI ID:
- 850327
- Report Number(s):
- EMSP-90170-2004; R&D Project: EMSP 90170; TRN: US200517%%485
- Country of Publication:
- United States
- Language:
- English
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