Hypochlorous acid (HOCl) is a powerful oxidant produced by activated phagocytes at sites
of inflammation to kill a wide range of pathogens. Yet, it may also damage and kill the
neighbouring host cells. The abundance of dead macrophages in atherosclerotic plaques
and their colocalization with HOCl-modified proteins implicate HOCl may play a role in
killing macrophages, contributing to disease progression. The first part of this research was
to investigate the cytotoxic effect and cell death mechanism(s) of HOCl on macrophages.
Macrophages require efficient defense mechanism(s) against HOCl to function properly at
inflammatory sites. The second part of the thesis was to examine the antioxidative effects
of glutathione (GSH) and 7,8-dihydroneopterin (7,8-NP) on HOCl-induced cellular
damage in macrophages. GSH is an efficient scavenger of HOCl and a major intracellular
antioxidant against oxidative stress, whereas 7,8-NP is secreted by human macrophages
upon interferon-γ (IFN-γ) induction during inflammation and can also scavenge HOCl.
HOCl caused concentration-dependent cell viability loss in human monocyte derived
macrophage (HMDM) cells above a specific concentration threshold. HOCl reacted with
HMDMs to cause viability loss within the first 10 minutes of treatment, and it posed no
latent effect on the cells afterwards regardless of the HOCl concentrations. The lack of
caspase-3 activation, rapid influx of propidium iodide (PI) dye, rapid loss of intracellular
ATP and cell morphological changes (cell swelling, cell membrane integrity loss and
rupture) were observed in HMDM cells treated with HOCl. These results indicate that
HOCl caused HMDM cells to undergo necrotic cell death. In addition to the loss of
intracellular ATP, HOCl also caused rapid loss of GAPDH enzymatic activity and
mitochondrial membrane potential, indicating impairment of the metabolic energy
production. Loss of the mitochondrial membrane potential was mediated by mitochondrial
permeability transition (MPT), as blocking MPT pore formation using cyclosporin A (CSA)
prevented mitochondrial membrane potential loss.
HOCl caused an increase in cytosolic calcium ion (Ca2+) level, which was due to both
intra- and extra-cellular sources. However, extracellular sources only contributed
significantly above a certain HOCl concentration. Preventing cytosolic Ca2+ increase
significantly inhibited HOCl-induced cell viability loss. This suggests that cytosolic Ca2+
increase was associated with HOCl-induced necrotic cell death in HMDM cells, possibly
via the activation of Ca2+-dependent calpain cysteine proteases. Calpain inhibitors
prevented HOCl-induced lysosomal destabilisation and cell viability loss in HMDM cells.
Calpains induced HOCl-induced necrotic cell death possibly by degrading cytoskeletal and
other cellular proteins, or causing the release of cathepsin proteases from ruptured
lysosomes that also degraded cellular components. The HOCl-induced cytosolic Ca2+
increase also caused mitochondrial Ca2+ accumulation and MPT activation-mediated
mitochondrial membrane potential loss. MPT activation, like calpain activation, was also
associated with the HOCl-induced necrotic cell death, as preventing MPT activation
completely inhibited HOCl-induced cell viability loss. The involvement of both calpain
activation and MPT activation in HOCl-induced necrotic cell death in HMDM cells
implies a cause and effect relationship between these two events.
HMDM cells depleted of intracellular GSH using diethyl maleate showed increased
susceptibility towards HOCl insult compared to HMDM cells with intact intracellular GSH
levels, indicating that intracellular GSH played an important role in protecting HMDM
cells against HOCl exposure. Intracellular GSH level in each HMDM cell preparation
directly correlated with HOCl concentration required to kill 50% of population for each
cell preparation, indicating intracellular GSH concentrations determine the efficiency of
GSH in preventing HOCl-induced damage to HMDM cells. Intracellular GSH and cell
viability loss induced by 400 μM HOCl were significantly prevented by 300 μM
extracellular 7,8-NP, indicating that added 7,8-NP is an efficient scavenger of HOCl and
out-competed intracellular GSH for HOCl. The amount of 7,8-NP synthesized by HMDM
cells upon IFN-γ induction was too low to efficiently prevent HOCl-mediated intracellular
GSH and cell viability loss.
HOCl clearly causes HMDM cells to undergo necrosis when the concentration exceeds the
intracellular GSH concentrations. Above this concentration HOCl causes oxidative damage
to the Ca2+ ion channels on cell and ER membranes, resulting in an influx of Ca2+ ions into
the cytosol and possibly the mitochondria. The rise in Ca2+ ions triggers calpain activation,
resulting in the MPT-mediated loss of mitochondrial membrane potential, lysosomal
instability and cellular necrosis.
| Identifer | oai:union.ndltd.org:canterbury.ac.nz/oai:ir.canterbury.ac.nz:10092/3662 |
| Date | January 2010 |
| Creators | Yang, Ya-ting (Tina) |
| Publisher | University of Canterbury. Biological Sciences |
| Source Sets | University of Canterbury |
| Language | English |
| Detected Language | English |
| Type | Electronic thesis or dissertation, Text |
| Rights | Copyright Ya-ting (Tina) Yang, http://library.canterbury.ac.nz/thesis/etheses_copyright.shtml |
| Relation | NZCU |
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