Undergraduate Research at Virginia Tech

Honors Biology Student Studies Impact of Mercury-Based Compound on Cells

By Jessica Cooper, marketing management major and honors student

Now a senior biology major, Angela Barker delved into the world of research while in pursuit of an honors degree. Fascinated by animals, Barker approached the Virginia-Maryland Regional College of Veterinary Medicine. However, not wanting to test directly on animals. Barker chose instead to conduct cell-based research. Under the direction of Damani Parran in the college's Laboratories for Neurotoxicity Studies, Barker spent two semesters examining the effects of the mercury-based compound, thimerosal, on developmental signaling in human neuroblastoma (SY5Y) cells. In the presence of nerve growth factor (NGF), the SY5Y cells can be used as a model of developing neurons.

Thimerosal was a widely used preservative in pediatric vaccines from the 1930s until 1999. Now some researchers suspect that the use of thimerosal may be linked to such developmental disorders as autism and attention deficit hyperactivity disorder. Approximately 50 percent ethylmercury by weight, thimerosal is believed to have profound negative effects on developmental signaling of the SY5Y cells. With this in mind, Barker and Parran tested the levels of thimerosal toxicity on three different aspects of these neuroblastoma cells.

First, Barker sought to determine if there was a concentration effect of thimerosal on the NGF-induced activation of signaling proteins. Specifically she looked at the TrkA receptor and the MAPK signaling cascade, which initiate the branching (neurite) necessary for nerve cell communication, Minutes after the thimerosal was applied to the cells, the results showed that as the single concentration of thimerosal applied to each cell culture increased, the phosphorylation (activation) of the signaling proteins in the cascade decreased. This may ultimately lead to effects on neuronal development.

Next, Barker tested the effects of thimerosal on lactate dehydrogenase (LDH), an enzyme related to energy production in cells. LDH is released when cells die and was measured as a proportion of the number of cells that had died resulting from exposure to increasing concentrations of thimerosal. In this step, the effects of thimerosal were tested on two different groups of cells. The first group of cells was in the presence of a (NGF) while the second group lacked this neurotrophin. Results after 24 and 48 hours showed that increasing concentrations of thimerosal had less detrimental effects on the cells in the presence of NGF than on the cells not exposed to NGF.

These results led Barker to conclude that the presence of the NGF had a protective effect against thimerosal-induced toxicity. Additionally, there was a concentration and time-dependent increase in cell death (LDH release) following exposure to thimerosal whether in the presence or absence of NGF.

Finally, Barker used the Enzyme-Linked ImmunoSorbent Assay (ELISA) to determine the effects of thimerosal on the type of cell death. Studies were conducted on the two types of cell death, apoptosis and necrosis, which occur as a result of different levels of thimerosal toxicity. Apoptosis is programmed cell death or naturally occurring cell death that induces DNA fragmentation, whereas necrosis is the inducing of cell lysis, or essentially the bursting of a cell, and is usually a result of highly toxic exposures.

Barker's studies showed that there was a higher level of DNA fragmentation, hence apoptosis, when the cells were exposed to a lower level of thimerosal, versus the lower levels of DNA fragmentation as a result of higher thimerosal concentrations, implying necrosis.

Without any background knowledge of neuroblastoma cells or hands-on research experience involving cell cultures, Barker felt as though she may have inhibited the process. "I was just getting acquainted with the research myself," when she dived right in, she said. However, not knowing exactly what she was doing at all times turned out to be advantageous, allowing the entire research process to be an excellent learning experience.

Just weeks away from crossing the stage and receiving her diploma, Barker is turning her focus back towards animals. "I would like to go into animal behavior," she said. While she is still deciding if going to graduate school or getting a job will be her next step, Barker is certain of one thing. Whether it is doing research or becoming a professor, she wants to continue to add knowledge to the field of biology and to the general public as well.