Student Research: Melinda Vredevoogd

MS, Toxicology (Tox), 1998
Faculty Advisor: Elaine M. Faustman

Gene Expression Patterns as Potential Biomarkers of Heavy Metal Exposure in Terrestrial Ecosystems Faustman


Abstract

Biomarker have shown tremendous benefit in human health risk assessment but their potential has not yet been fully evaluated in ecological settings. Preliminary studies in aquatic ecosystems suggest biomarkers provide benefits, such as improved sensitivity, to these systems as well. This study furthers the investigation into the utility of biomarkers in ecotoxicology by focusing on terrestrial systems.

To study the utility of biomarkers in terrestrial ecosystems, a laboratory based study investigated the changes in gene expression of several genes (HSP70, MT, p21, p53, bax, bcl-2) in response to HgCl2 exposure. The hypothesis stated that these genes would be differentially expressed reflecting the dose trend, thus serving as a biomarker of exposure. The study design was based on the conditions found at the Hanford Nuclear Reservations which would allow the laboratory developed biomarker to be validated in the field where conditions are more variable and unpredictable.

Tenebrio molitor beetles were exposed to four different levels of HgCl2 (0, 0.5, 5.0, and 50 ppm) for 12 days. The RNA was extracted from each insect and used in a reverse Northern hybridization. The resultant mRNA expression patterns found no significant dose response pattern related to HgCl2 exposure of any of the genes investigated. Northern hybridizations, used to validate reverse Northern findings, showed no specific binding between the probes and beetle RNA (with the exception of action) suggesting a more specific probe was needed. Oligonucleotide probes were generated from the high homology regions of hsp70, MT and p21. Of these genes, p21 was the only probe that showed specific binding. Preliminary experiments suggest a possible dose response with HgCl2 exposure. A gene cloned from the Tenebrio molitor was also investigated, which also showed a possible dose response pattern with HgCl2. Further studies are needed to fully evaluate the relationship between p21 and dsp expression and exposure and to determine the utility of gene expression biomarkers to ecological risk assessment. Overall, these experiment suggest that oligonucleotides of high homology regions may prove the most useful biomarker probe in organisms where little is known about which genes are expressed or what their sequences are.