Brugia Malayi - Clark Science Center - Smith College

Dopamine Cytotocicity and its Implications Pertaining to Parkinson’s
Disease
Rosa Drummond
Parkinson’s Disease is a neurodegenerative disease characterized by the loss of fine motor control, dementia, and the death of
dopaminergic cells in the substantia nigra. This disease is very rarely genetic, but its overall cause is unknown as well as the cause of
cell death. We investigated the role of dopamine itself in dopaminergic cell death. When dopamine (DA) is oxidized to DOPAC,
a precursor to norepinephrine, a byproduct is hydrogen peroxide (H 2
O 2
), which in turns oxidizes DOPAC into cytotoxic hydroxyl
radicals. Ultimately, these hydroxyl radicals may play an important role in, or be the cause of, cell death. We focused on the role
of H 2
O 2
and how best to measure the actual concentration of H 2
O 2
in the affected cells (NIE-115 Neuroblastoma cell line). Our
ultimate goal was to see how much dopamine, and in turn H 2
O 2
, the cells could handle, and what concentration of dopamine was
lethal to the cells.
We used chemiluminescence as an indicator of H 2
O 2
levels. Luminol, when catalyzed by horseradish peroxidase (HRP), emits
luminescence, which we measured with the SpectraMax M5/M5e Microplate Reader in the Center for Molecular Biology in Ford
Hall. Using a 96-well plate, we silvered clear microwells via the Tollens’ Reaction in order to decrease the luminescence absorbance
observed in black wells and reduce the cross talk observed in white wells. We trialed a variety of concentrations of luminol and
HRP in cell culture, as well as several different other chemicals relating to hydrogen peroxide, including catalase, aminotriazole,
plasma amine oxidase, dopamine, and menadione. Our best results divulged a very smooth, concave up graph, indicating H 2
O 2
levels began at relatively high levels and then decreased at an exponential-like level due to HRP. We also ran these tests over longer
periods of time (i.e. one-two hours) and found similar results.
The SpectraMax gives levels of luminescence as Relative Luminescence Units (RLUs), and although we did not run enough
trials to decisively say the exact concentration of H 2
O 2
each RLU measured, our best estimate was 100RLU/nM H 2
O 2
.
Perhaps one of our most important findings was that the neuroblastoma could easily survive long-term exposure (two days
were tested) to luminol and HRP. If we had more time, our eventual experiment was to measure luminescence from brain slice
preparations, using an entire DA pathway (the tuberoinfundibular pathway is the shortest DA pathway, and connects the arcuate
nucleus of the hypothalamus to the median eminence and the pituitary gland). We would measure a baseline level of H 2
O 2
by way
of luminescence, and continue adding DA until the brain slice was no longer alive, theoretically meaning we reached our peak in
healthy DA concentration and also the threshold of toxic DA concentration. We would hopefully also be able to gain some insight
into DA production relating to circadian rhythms, by continuously monitoring the changes in dopamine release over extended
intervals. (Supported by the Howard Hughes Medical Institute)
Advisor: David Bickar
References:
http://www.carolina.com/category/teacher%20resources/classroom%20activities/luminol%20the%20glowing%20reaction.do.
2012
71