Access Type

Open Access Dissertation

Date of Award

January 2017

Degree Type


Degree Name



Biological Sciences

First Advisor

David Njus


Oxidation of dopamine to toxic metabolites is considered to be one of the prime factors involved in the death of dopaminergic neurons in Parkinson’s disease. Some dopamine oxidation products have the capability to redox cycle in the presence of molecular oxygen, further contributing to oxidative stress. Therefore, our aim here was to study the redox cycling of dopamine oxidized metabolites and elucidate the underlying mechanism by which they cause oxidative stress.

Redox reactions involve transfer of one or more electrons between two compounds

resulting in either oxidation or reduction. In redox cycling, a compound undergoes

alternate oxidation and reduction, transferring electrons from a reductant to molecular

oxygen. Therefore, we began by investigating different modes of redox cycling by

measuring the rate of oxygen consumption using a Clark-type oxygen electrode in the

presence of different reductants. We compared chemically synthesized redox cyclers

such as menadione, 6-hydroxydopamine (6-OHDA), 3-methyl-5-anilino-1,2-

benzoquinone (3-MAQ) and 9,10-phenanthrenequinone, using ascorbic acid and

dithiothreitol (DTT) as reductants. Addition of superoxide dismutase diminished DTT dependent redox cycling activity (except in the case of menadione) but had no effect on

the ascorbate-dependent redox cycling activity. This suggests that DTT drives a two electron reduction whereas ascorbate causes a one-electron reduction. NADHdependent

redox cycling mediated by mitochondria was also studied using 3-MAQ. This mitochondrially mediated redox cycling activity was inhibited by mersalyl acid, thereby

suggesting the involvement of the outer-mitochondrial membrane protein, NADH dependent cytochrome b5 reductase, in the redox cycling mechanism.

We identified hypochlorite-oxidized cysteinyl-dopamine (HOCD) as a redox cycling

product and a potential candidate for dopaminergic neuron toxicity in the progression of

Parkinson’s disease. The dopamine oxidation product cysteinyl-dopamine has attracted

attention as a contributor to the death of dopaminergic neurons in Parkinson’s disease.

Treatment of cysteinyl-dopamine with hypochlorite yields an even more cytotoxic product.

This product, HOCD, has potent redox-cycling activity and initiates production of superoxide in PC12 cells. Taurine, which scavenges hypochlorite, protects PC12 cells

from cysteinyl-dopamine but not from HOCD, suggesting that HOCD, not cysteinyl-dopamine itself, is toxic. Furthermore, rotenone, which enhances expression of the

hypochlorite-producing enzyme myeloperoxidase, increases the cytotoxicity of cysteinyl-dopamine but not of HOCD. This suggests that dopamine oxidation to cysteinyl-dopamine followed by hypochlorite-dependent conversion to a cytotoxic redox-cycling product HOCD, leads to the generation of reactive oxygen species and oxidative stress and may contribute to the death of dopaminergic neurons.

Our findings of HOCD toxicity in PC12 cells was followed by our study to determine

the mode of cell death. The morphological changes in the cell such as membrane

blebbing and appearance of biochemical markers such as cleaved poly-ADP ribose

polymerase and active caspase-9 suggested cell death by apoptosis. Moreover,

increased expression of tumor suppressor protein p53, indicated mitochondrial mediated

apoptotic cell death. Our observations have raised an unappreciated possibility that may

link dopamine oxidation, microglial inflammation, oxidative stress and the rotenone model

of Parkinson’s disease. Furthermore, it offers a promising new approach in the search for

a therapeutic cure for Parkinson’s disease.