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With the present Master Thesis, the effects of oxidative stress, including reactive oxygen
species (ROS), on the Translocator Protein (TSPO), in correlation with cell viability and cell
death, including initiation of the mitochondrial apoptosis pathway were studied. The TSPO is
also known as the peripheral-type benzodiazepine receptor. The TSPO can be found in the
mitochondrial membrane, where it can be closely associated with the voltage dependent anion
channel and the adenine nucleotide transporter, which form the core components of the
mitochondrial permeability transition pore (MPTP). We suggested that the TSPO can modulate
the activity of the MPTP, affecting the mitochondrial membrane potential (Δψm). Some of the
putative functions of TSPO may include involvement of apoptotic processes induced by
oxidative stress. We found that application of 100 µM H2O2 to cells from the SHSY-5Y
neuroblastoma cell line led to cell death. This cell death appeared to be primarily necrotic,
while apoptosis appeared to play a minor part in cell death due to H2O2 exposure in our study.
In addition, we found that H2O2 exposure could lead to a collapse of the Δψm. Furthermore,
H2O2 exposure could lead to enhanced expression of TSPO as determined using [3H]PK 11195
binding studies and Western blot protein analysis. Application of the antioxidant glutathione
suggested that the effects of H2O2 on TSPO expression and cell viability were due to ROS.
Nonetheless, effects of H2O2 exposure on lipid peroxidation and protein carbonylization could
not be detected. Thus, our studies suggest that ROS generation by H 2O2, and possibly H2O2
itself, can affect TSPO expression and cause collapse of the Δψ m leading to cell death. Since
oxidative damage to cellular structure does not seem to play a major role in this form of cell
death, we suggest that ROS may affect the TSPO in a more direct way. This may be part of
TSPO's reported function as an oxygen sensor. The effect of H2O2 exposure on TSPO may
include activation of TSPO's putative modulatory role regarding the Δψm and lead to a
sustained collapse of the Δψm. This sustained collapse of the Δψm then may lead to necrotic cell
death, by-passing apoptotic mechanisms.

List of symbols and shortcuts
°c Degree centigrade
µg Microgram
µl Microliter
µM Micromolar
H Tritium
∆Ψm mitochondrial electrochemical transmembrane potential
ANT Adenine nucleotide transporter
ATP Adenosine tri-phosphate
B Bound ligand
B/F Ratio between bound and free ligand
Bmax Maximal number of binding sites
BSA Bovine serum albumin
BZ Benzodiazepine
CBR Central-type benzodiazepine receptor
cm Centimeter
CNS Central nervous system
CS Cigarette smoke
DBI Diazepam binding inhibitor
F Free ligand
fmol femtomole
FACS Fluorescent-activated cell sorting
H2O2 Hydrogen peroxide
IBP Isoquinoline binding protein
g g-force
GABA γ-aminobutiric acid
kDa KiloDalton
Kd Equilibrium dissociation constant
Ki Inhibition constant
MPTP Mitochondrial permeability transition pore
M Molar
mg Milligram
min Minutes
ml Milliliter
mM Millimolar
mmHg Millimeter of mercury
nM Nanomolar
nm Nanometer
nmole Nanomole
OMM Outer mitochondrial membrane
pmole Picomole
PBR Peripheral-type benzodiazepine receptor
PBS Phosphate buffered saline
PBS-T Phosphate buffered saline with Tween 20
PI Propidium iodide
ROS Reactive oxygen species
rpm Revolutions per minute
RT Room temperature
SDS Sodium dodecyl sulfate
TSPO Translocator protein
VDAC Voltage-dependent anion channel

Benzodiazepines, including diazepam also known as valium, form a class of drugs used
as sedatives, anxiolytics, muscle relaxants, and anticonvulsants (Zbinden and Randall, 1967).
In general, benzodiazepines act as hypnotics in high doses, anxiolytics in moderate doses, and
sedatives in low doses. Benzodiazepines are lypophilic molecules that have a characteristic
structure containing a benzene ring connected to a seven atom ring including nitrogens in the
positions 1 and 4 (Figure 1)

Figure 1. Benzodiazepine core structure

Benzodiazepines bind to the central-type benzodiazepine receptor (CBR) on the α subunit

of the GABA receptor which is localized on neurons in the central nervous system (CNS)
(Saano, 1988). Benzodiazepine binding enhances the GABA induced opening of the chloride
channels formed by the GABA receptor, which leads to hyperpolarization of cell membranes
and consequent inhibitory effects (Pritchett et al., 1989).
In 1977, Braestrup and Squires described that the mitochondrial fractions from rat kidney,
liver, and lung exhibit BZ specific binding. They termed these receptors the "peripheral
benzodiazepine receptor" (PBR). While the location of the CBRs is primarily on the cell
membranes of neurons, the Translocator Protein (TSPO) is particularly abundant on the
membranes of mitochondria in the various cell types that they are found (Anholt et al., 1986).

Functional characteristics of the TSPO

After being discovered in 1977, the peripheral benzodiazepine receptor was further
investigated in various directions, including its involvement in cellular processes such as
proliferation (Carmel et al., 1999; Papadopoulos, 2004), cellular respiration (Gavish et al.,
1999) steroidogenesis (Papadopoulos et al., 1990; Papadopoulos, 2004; Costa et al., 1994;
Lacapère et al., 2003) and apoptosis (Fischer et al., 2001; Fennell et al., 2001; Veenman et al.,
2007) and in pathological conditions such as neurodegenerative diseases, stress and behavioral
disorder, brain trauma and cancer (Gavish et al., 1999; Veenman and Gavish, 2000, 2006;
Veenman et al., 2007).

For the last three decades PBR was a popular and usable scientific name, but in 2007 it
was renamed translocator protein (TSPO) for three major reasons (Papadopoulos et al., 2006):
1. The receptor was found to bind not only benzodiazepines as ligands.
2. The receptor is not only peripheral but also found on neurons and microglial cells,
which are part of the CNS.
3. Many studies have shown that one of the major roles of the receptor is in the process
of transporting cholesterol to the mitochondria.

TSPO tissue localization:

Radioligand binding assays have detected various densities of TSPO in the majority of
tissues tested in rats (Figure 2). For example, the rat adrenal is particularly rich in TSPO,
while the liver and brain were found to express relatively low levels of TSPO (Anholt et al.,
1985). The tissue distribution of the protein can be inhomogeneous within a given organ. For
example, in the rat adrenal glands, the medulla is virtually devoid of TSPO, whereas cortical
density is high (Anholt et al., 1986). Similarly, the kidney shows a selective localization of
receptors in the distal convoluted tubules and the thick ascending limb of the loop of Henle
(Gehlert et al., 1985).

Figure 2. Tissue abundance of TSPO in

compare to adrenal. Ad, adrenal; Kd,
kidney; He, heart; Te, testis; Ov, Ovary;
Ut, uterus; Li, liver; Br, brain.
Gavish et al, 1999.

Subcellular TSPO location

TSPO is an 18-kDa isoquinoline binding protein, and is located primarily on the outer
mitochondrial membrane (Figure 3). The receptor interacts with several mitochondrial
proteins, in particular with the 32-kDa voltage-dependent anion channel (VDAC) in the outer

membrane and the 30-kDa adenine nucleotide translocase (ANT) in the inner membrane
(McEnery et al., 1992). VDAC and ANT form the backbone of a polyprotein complex
designated as the mitochondrial permeability transition pore complex (MPTP) (Castedo et al.,
TSPO is not exclusively expressed in the mitochondria but also found in the plasma
membranes, cytoplasm, nuclei membrane and other cell compartment's membrane (O’Beirne et
al., 1990; Oke et al., 1992; Olson et al., 1988a). In consistence with these observations, the
amino acid sequence of TSPO does not contain a typical mitochondrial targeting sequence
(Schatz, 1987).

The mitochondrial permeability transition pore

The mitochondrial permeability transition pore (MPTP) is a nonselective, high
conductance channel with multiple macromolecular components (Alano et al., 2002). It forms
at the contact sites between the inner and outer membranes of the mitochondria. The activation
of the MPTP by different stimuli increases mitochondrial membrane permeability and causes
mitochondria to become further depolarized, meaning that the Δψ m collapses. When the Δψm is
lost, protons and small molecules (< 1.5 kDa) are able to freely flow across the membrane
(Schinder et al., 1996; White and Reynolds, 1996). Loss of Δψ m interferes with the production
of adenosine triphosphate (ATP), the cell's main source of energy.
The collapse of the Δψm leads to massive swelling of the matrix (for a review, see
Halestrap 2006). This occurs as all small-molecular-mass solutes equilibrate across the inner
membrane, while proteins remain within the matrix at high concentrations and exert a colloidal
osmotic pressure. The resulting movement of water into the mitochondrial matrix causes
swelling of the matrix that can lead to an increase in volume by unfolding of the cristae.
However, this is not possible for the outer membrane, and as swelling proceeds the outer
membrane may rupture. This can lead to loss of intermembrane components to the cytosol
(Halestrap et al., 2007). These include cytochrome c and other apoptotic factors such as AIF
(apoptosis-inducing factor). The release of cytochrome c causes activation of caspase 9 and
then caspase 3, and this in turn leads to proteolytic cleavage of a range of proteins that mediate
the reorganization of the cell for apoptosis (Martinou and Green, 2001).
However, apoptosis is an ATP-dependent process, and if mitochondrial function remains
compromised by an open MPTP, cell death will proceed via necrosis even though caspase
activation may occur through cytochrome c release (Halestrap et al., 2000; Nicotera and
Melino, 2004).

The core components of the MPTP are formed by the 32kDa Voltage Dependent Anion
Channel and the adenine nucleotide transporter (ANT), which are closely associated with the
TSPO (Figure 3).
At physiological concentrations (nM range), the TSPO specific ligands, PK 11195 and
Ro5-4864, appear to be anti-apoptotic (for a review, see Veenman et al., 2007). Knockdown
of TSPO by genetic manipulation, resulting in reduction by more than 50% in [3H]PK 11195
binding, was reported to show anti-apoptotic effects in the C6 glioma cell line, suggesting a
potential pro-apoptotic function of TSPO (Levin et al., 2005). However, a reduction in TSPO
abundance in the MDA-MB-231 by siRNA was found to reduce cell proliferation, possibly due
to impairment of other essential cell functions (Li et al., 2007). We postulate that the pro-
apoptotic function of TSPO and some of its ligands may involve the modulation of the MPTP.

Cytochrome- c ,DBI, TTN, PLA2
release Protoporphyrin IX
TSPO Ligands


ANT cholesterol

apoptosis mitochondrial
permeability modulation of
respiration voltage dependent
calcium channels
steroidogenesis ischemia
microglial immune response
cell proliferation

Figure 3. The location and functions of TSPO. TSPO molecules are often found in groups and in
conjugation with VDAC and ANT. Note that TSPO can also function without interactions with VDAC and
ANT. Additional molecules, such as pk10, PRAX-1 and PAP7 can be linked to the TSPO, which is located
in the outer mitochondrial membrane. Furthermore, molecules of the Bcl-2 family and creatine kinase and
hexokinase can be attached to VDAC and ANT, which are located at the outer and inner mitochondrial
membranes, respectively. Various synthetic ligands that bind to TSPO have been developed. Well known
synthetic TSPO ligands include, PK 11195, Ro5-4864, FGIN-1-27, and SSR 180575. Endogenous ligands
that bind to TSPO include: protoporphyrin IX, DBI and its metabolite TTN, and PLA2. At the functional
end, TSPO and its ligands have been shown to be involved in mitochondrial permeability, apoptosis,
steroidogenesis, cell proliferation, mitochondrial respiration, modulation of voltage dependent calcium
channels, ischemia, microglial activation, and immune response. TSPO and its conjugated molecules
possess channel-like properties, including, but not restricted to, mitochondrial import of cholesterol, and
export of cytochrome c and ATP. Abbreviations: ANT, adenine nucleotide transporter; ATP, adenosine
triphosphate; DBI, diazepam binding inhibitor; IBP, isoquinoline binding protein; PAP7, PBR associated
protein 7; pk10, protein of 10 kiloDalton; PLA2, phospholipase A2; PRAX-1, PBR associated protein 1;
TSPO, 18 kDa translocator protein; TTN, triakontatetraneuropeptide; VDAC, voltage dependent anion
channel (from Veenman et al., 2007).

Other protein interactions of the TSPO
In 1999, a yeast two-hybrid screening resulted in the identification of a novel protein that
specifically interacts with TSPO (Galiegue et al., 1999). This protein was named peripheral
benzodiazepine receptor-associated protein 1 (PRAX-1). PRAX-1 is a single 220-250-kDa
cytoplasmic protein that also partially colocalizes with TSPO in the mitochondria (Figure 3).
PRAX-1 interacts with the homodimer of TSPO and exhibits several motifs implicated in
protein–protein interaction. This may suggest PRAX-1 action as an adaptor protein recruiting
different targets to bring them in the vicinity of TSPO so as to modulate its function. Up-
regulation of PRAX-1 mRNA was shown in response to different antidepressant treatments,
with PRAX-1 mRNA expression being largely neuronal (Chardenot et al., 2002). This is in
contrast with the TSPO protein being mostly described to be glial in CNS. PRAX-1 is primarily
a brain-specific protein, raising the question whether the PRAX-1 protein implicates TSPO in a
specific function, which remains to be unraveled.
Additional proteins that interact with TSPO or with VDAC and ANT include the proteins
of the Bcl- 2 family, PBR associated protein 7 (PAP7), and different kinases such as creatine-
kinase (Veenman et al., 2007).

Pharmacological characteristics of TSPO

Synthetic ligands
The anticonvulsant, clonazepam, is one of the most potent benzodiazepines. It is a ligand
for both CBR and TSPO (Figure 4). It has a Ki (inhibition constant) value for CBR in the low
nanomolar range, but is several thousand-fold weaker at TSPO (Braestrup and Nielsen, 1978;
Braestrup and Squires, 1978). It was also found that the antianxiety drug, diazepam (Figure
4), binds with high (nanomolar) affinity to CBR as well as to TSPO (Braestrup and Squires,
1978). The putative convulsant 7-chloro-5-(4-chlorophenyl)-1, 3-dihydro-1-methyl-2H-1,4-
benzodiazepin-2-one (Ro5 4864), a benzodiazepine differing in structure from diazepam only
in the lack of a chlorine substituent on one of the rings (Figure 4), binds only weakly to CBR,
however strongly, in the nanomolar range, to TSPO (Weissman et al., 1983).
The most widely used TSPO specific ligand is the isoquinoline 1-(2-chlorophenyl)-N-
methyl-N-(1-methyl-propyl)-3-isoquinoline carboxamide (PK 11195) (Figure 4). PK 11195
interacts selectively and potently with TSPO (Verma and Snyder, 1989), and consistently show
high affinities across many species studied (Awad and Gavish, 1989)
There is evidence that the benzodiazepine Ro5-4864 and the isoquinoline carboxamide
PK 11195 bind to different conformational states of the transporter or that the benzodiazepine
binding site partially overlaps or is allosterically coupled to the isoquinoline carboxamide

binding domain (Papadopoulos 2007). One of the studies suggesting this hypothesis showed
that protoporphyrin IX (Figure 4) was unable to displace [3H]Ro5-4864 at micromolar
concentrations, whereas this compound could displace [3H]PK 11195 binding with an
inhibitory constant of 14 nM in rat (Escobedo et al., 1992), thus showing differential selectivity
recognition sites of both classes of compounds.
Imidazopyridines were another series of compounds developed for studying TSPO
(Figure 4). However, unlike the isoquinolines, imidazopyridines bind to both TSPO and
GABA receptors (Benavides et al., 1983). One of the imidazopyridines, alpidem, displayed one
of the highest affinity TSPO ligands thus far reported, exhibiting a dissociation constant in the
picomolar range.
More recently, derived from the chemical structure of imidazopyridines, a new series of
compounds were developed, the 2-aryl-3-indoleacetamides (Romeo et al., 1992). The
prototype of this series, FGIN-1-27, has an affinity close to that of alpidem. One more novel
TSPO ligand is SSR180575 (Ferzaz et al., 2002.). Both ligands possessing steroidogenic
properties, but while FGIN-1-27 is pro-apoptotic, SSR180575 is anti-apoptotic (Veenman and
Gavish, 2006).

Figure 4. Examples of compounds binding to the TSPO.
(From Papadopoulos, 1993).

Endogenous PBR ligands

Various studies have reported a number of putative endogenous TSPO ligands (Veenman
and Gavish, 2006). For example, Verma and colleagues (Verma et al., 1987; Verma and Snyder,
1988) found that porphyrins can act as endogenous TSPO ligands, while protoporphyrin IX in
particular displayed binding in the nM range.
Findings by Mantione et al., (1988) suggested that phospholipase A2 (PLA2) (Naja naja)
is also an endogenous TSPO ligand. In addition, in 1988, in situ hybridizations in rats revealed
high levels of diazepam binding inhibitor (DBI) mRNA in the peripheral tissues that are rich in
TSPO (Alho et al., 1988).
In human post-mortem tissues, a specific radioimmunoassay was used to describe the
distribution of DBI-like immunoreactivity [DBI-IR(51-70)] (Ball et al., 1989). This DBI-
IR(51-70) was unevenly distributed throughout the CNS (Ball et al., 1989). In human
peripheral tissues, highest concentrations of DBI-IR(51-70) were found in the liver and kidney
(Ball et al., 1989), reminiscent of the distribution of TSPO in living humans, where TSPO
density is also high in the liver (Versijpt et al., 2000). Chromatographic analysis revealed
several molecular forms of DBI-IR(51-70), the major form being of greater molecular weight
and hydrophobicity than the synthetic fragment peptide, DBI(51-70) (Ball et al., 1989). In
peripheral tissues, but not in the CNS, a small peak of immunoreactivity was indistinguishable
from the synthetic peptide. It was concluded that DBI-IR(51-70) is widespread, but that tissue
processing may be different (Ball et al., 1989). Bovolin et al. (1990) reported that DBI showed
competitive inhibition of binding of PK 11195 to the TSPO. This may suggest that DBI is an
endogenous ligand for these receptors. Interestingly, in rats, the highest DBI-like
immunoreactivity content was found in steroid-producing cells, such as the glomerulosa and
fasciculata cells of adrenal cortex and Leydig cells of testis (Bovolin et al., 1990). In addition,
high DBI expression in these tissues was shown to correlate with high maximal binding density
(Bmax) of TSPO (Bovolin et al., 1990).
Triakontatetraneuropeptide (TTN, DBI 17-50), a posttranslational product of DBI, was
found to be a specific TSPO ligand (Slobodyansky et al., 1989). These data suggest possible
roles for DBI and its posttranslational products as endogenous regulators of TSPO functions.

The involvement of TSPO in different pathologies
It has been found with many studies that TSPO are upregulated in a wide variety of
pathological conditions (Gavish et al., 1999; Veenman and Gavish, 2000, 2006; Veenman et al.,

Neurodegenerative Disorders
There are growing evidences indicating that mitochondrial dysfunction may play an
important role in the pathogenesis of many neurological disorders (Beal, 2007). Because
mitochondrial metabolism is not only the principal source of high energy intermediates, but
also of ROS, it has been suggested that inherited or acquired mitochondrial defects could be
the cause of neuronal degeneration as a consequence of energy defects and oxidative damage
(Schon and Manfredi, 2003). Considering the localization of TSPO in mitochondria and some
aspects of its functions, the question of its role in neurodegenerative pathologies is relevant.
In the CNS, the TSPO are located primarily in glial cells. Numerous studies show that
both astrocytes and microglia express TSPO, and that the density of TSPO can be increased
under conditions resulting in glial activation, including inflammation and metabolic stress
(Park et al., 1996; Banati et al., 1997). As an example for neurodegenerative diseases, in
Alzheimer patients, TSPO density was found to be increased in platelets taken from blood
samples, as well is in the brains of deceased patients (Owen et al., 1983; Diorio et al., 1991)
and (Bidder et al., 1990).

Neurological damage
The TSPO and its specific ligands have been found to participate in traumatic ischemic
brain damage (Veenman and Gavish, 2000). A time-course of the histopathological and
biochemical alterations resulting from mechanical brain injury in the rat showed a spatial
correlation between the histological alterations and TSPO binding (Toulmond et al., 1993).
The correlation reached highest values at 7 days after injury, at which time the lesion was
consolidated and astroglial cells replaced neuronal cells (Toulmond et al., 1993).

Response to stress
The involvement of TSPO in the regulation of basic biological processes that are related
to physiological response to stress, such as cellular metabolism, neuroendocrine activity, and
immune functioning, points to its possible role in the mediation of the organism’s adaptation to

It was found that exposure of rats to five inescapable tail shocks induced a significant
increase in renal TSPO density (Drugan et al., 1986). In contrast, 80 repeated tail shocks
resulted in a significant decrease of TSPO density in kidney, heart, pituitary gland, and cerebral
cortex. This biphasic effect of inescapable shock may indicate differences in TSPO response to
short- versus long-term exposure to stress, as has also been suggested by various other studies
(for a review see Veenman and Gavish, 2006).

As it was found that TSPO may be involved in cell proliferation, differentiation, and
apoptosis, many researchers focused on TSPO's role in cancer (Gavish et al., 1999). For
example, the next points indicate TSPO's potential functional involvement in the cancerous
1. The majority of malignant tumors have much higher level of TSPO in
comparison to the same healthy tissue (Table 1 from Gavish et al., 1999). In
particular, for various types of brain cancer TSPO is expressed in more than
one order of magnitude greater than for normal brain tissue (Table 1).
2. TSPO levels appear to correlate positively with the tumorigenicity of particular
types of cancer, for example in gliomas (Veenman et al., 2004)
3. Antisense knockdown of TSPO in mouse MA-10 Leydig cells, in rat C6 glioma
cells and in human HT29 colon cancer cells (Weisinger et al., 2004; Levin E.,
2005; Shoukrun Ph.D Thesis, Technion, 2004) resulted in:
a. reduced apoptotic rates
b. increased cell proliferation
c. enhanced tumorigenicity in vitro (anchorage independent cell growth)
d. blocked the pro-apoptotic effects of specific antitumor agents such as

Table 1. Alterations in TSPO binding characteristics in various hyperplastic and cancer tissues.

From Gavish et al. (1999)

Physiological functions of TSPO
As mentioned before, the TSPO plays an important role in cellular processes and
different pathologies (Figure 3). TSPO is involved in various functions (Veenman et al., 2007).
The functions that have attracted most attention are apoptosis and steroidogenesis (Gavish et
al., 1999; Veenman and Gavish, 2000, 2006; Veenman et al. 2007). These studies also
suggested that these TSPO functions may be part of a more general host defense response.
Furthermore, involvement of TSPO in mitochondrial respiration and TSPO function as an
oxygen sensor is also investigated (Yeliseev et al., 1997; Yeliseev and Kaplan, 1999).

TSPO's role in steroidogenesis

Evidence that brought researchers to study the involvement of TSPO in steroidogenesis is
that TSPO are quite abundant in many tissues but are extremely abundant in steroidogenic cells
(De Souza et al., 1985; Antkiewicz-Michaluk et al., 1988). This information suggested that
TSPO are likely to play a role in steroidogenesis.
Steroids are formed by several successive enzymatic transformations of cholesterol. The
primary point of control in the acute stimulation of steroidogenesis involves the first step in
this biosynthetic pathway in which cholesterol is converted to pregnenolone by the cholesterol
side-chain-cleavage (P-450SSC), an enzyme localized on the inner mitochondrial membrane,
which is dependent on an electron transport system comprised of adrenodoxin and adrenodoxin
reductase (Churchill and Kimura, 1979). Pregnenolone then leaves the mitochondrion towards
the endoplasmic reticulum where it undergoes further enzymatic transformations that give rise
to the final steroid products (Papadopoulos, 1993).
Looking for a specific TSPO role in steroid biosynthetic pathway, it has been shown that
cholesterol transport from outer to inner mitochondrial membrane is activated by TSPO ligands
(Krueger and Papadopoulos, 1990). Deletion mutations on recombinant TSPO have shown
that deletion of the C-terminus (Δ153–169 amino acids) drastically reduced cholesterol uptake
(70%), although it retained ability to bind PK 11195 ligand (Li and Papadopoulos, 1998). This
observation suggested that the cytosolic C-terminal domain of TSPO might be involved in
taking up and bringing cholesterol into the mitochondria in steroidogenic and also in bile acid
biosynthetic cells.

TSPO and apoptosis
Organisms possess mechanisms to eliminate cells that are redundant, damaged, or
infected, including programmed cell death, also known as apoptosis (Kerr et al. 1972). This
mechanism is vital for normal development, for maintenance of tissue homeostasis, and for an
effective immune system. Because of the role of apoptosis in cellular homeostasis, disorders
of apoptosis result in either the accumulation of abnormal cells (leading to cancer and
autoimmunity), or the loss of cells (leading to immunodeficiency and neurodegenerative
disorders) (Cory and Adams, 2002; Cory et al., 2003).
Two principal pathways to apoptosis activation have been recognized. One is triggered
by engagement of so called “death receptors” on the cell surface (Strasser et al., 2000;
Ashkenazi, 2002). The other is the mitochondrial pathway that is provoked by various forms of
stress, including inadequate cytokine support and diverse types of intracellular damage (Gross
et al., 1999; Cory and Adams, 2002; Cory et al., 2003). There are many studies that support
the participation of TSPO in the apoptosis process, among them:
1. Its association with the MPTP complex, which is known to be involved in apoptosis
(McEnery MW 1992).
2. TSPO levels in glioma cancer cells were found to be correlated with tumorigenicity
(Veenman et al., 2004).
3. Genetic manipulation of TSPO affects apoptosis level. For example, antisense
knockdown of the TSPO resulted in a 60% reduction in the number of cells in the
apoptosis phase (Levin et al, 2005).
4. The relatively high levels of TSPO expression in skin, as compared to TSPO levels in
skin cancer, was found to correlate with relatively low levels of apoptosis as induced
by oxygen radicals or ultraviolet light (Stoebner et al., 1999, Casellas, 2002).
5. The myxoma poxvirus M11L protein, which interacts physically and functionally
with TSPO, inhibits host cell apoptosis (Everett, 2002).
6. TSPO ligands with nanomolar affinity for the receptor, such as Ro5-4864 and PK
11195, modulate cancer cell response to apoptosis inducing signals (Hirsch, 1998;
Miyazawa et al., 1998; Carmel et al., 1999; Gavish et al., 1999; Maaser, 2001;
Decaudin et al., 2002; Strohmeier et al., 2002, Veenman et al., 2007).

It can therefore be speculated that binding of ligands to TSPO induces a conformational

change of the MPTP that may sensitize the cell to an apoptotic message (Veenman et al., 2004;
Levin et al., 2005). Potentially the TSPO could modulate apoptosis via direct molecular

interactions with the MPTP components, VDAC and ANT, and/or antiapoptotic Bcl-2- and pro-
apoptotic Bax-like proteins (Decaudin, 2004).

Mitochondrial respiration and oxygen control

Because of their high level of expression in the mitochondria, initial studies of TSPO
function revolved around its effect on mitochondrial respiration (Hirsch et al., 1989). Drugs
such as deuteroporphyrin IX, DBP, and UHDBT that bound to mitochondrial TSPO of the rat
kidney produced several effects on mitochondrial respiration. For example, the drugs increased
state IV and decreased state III respiration rates, which resulted in a significant decrease in the
respiratory control ratio (Hirsch et al., 1989). These results suggest that ligand binding to
mitochondrial TSPO results in inhibition of mitochondrial respiratory control.

Reactive Oxygen species (ROS)
Evidence for the participation of the TSPO in oxidative processes was indicated by the
involvement of the TSPO in the protection of hematopoietic cells against apoptosis following
H2O2 treatment (Carayon et al., 1996). In this study, levels of TSPO expression and resistance
to H2O2 toxicity of hematopoietic cell lines were correlated. Moreover, the resistance of cells
to H2O2 could be significantly increased by transfection with a TSPO expression vector.
The mitochondria are the greatest source, as well as a target, of ROS (Lee and Wei,
2000). The electron transport chain associated with mitochondrial respiration is the major
source of ROS production under normal conditions (Bradyet al., 2006). ROS are “activated”
oxygen molecules that can readily react with organic substances by noncatalytic means (Figure
5) (Ryter et al., 2007). The most common forms of ROS include superoxide anion (O 2•),
hydrogen peroxide (H2O2), and the highly reactive hydroxyl radical (OH•) (Figure 5).

Figure 5. Reactive Oxygen Species (ROS).

ROS that interact with biological molecules give rise to secondary reactive products such
as lipid peroxides and protein carbonyls (Hwang et al., 2007). Thus, ROS can induce damage
to cellular macromolecules such as lipids, protein, and DNA. In this context, ROS have been
implicated in the regulation of diverse cellular functions including defense against pathogens,
intracellular signaling, transcriptional activation, proliferation, and apoptosis (Devasagayam et
al., 2004; Datta et al., 2000). However, ROS have also been increasingly implicated in the
ageing process and in different diseases such as cancer, Alzheimer’s disease, Parkinson’s

disease, ischemia/reperfusion injury, etc. (Datta et al., 2000.). Under pathological conditions,
ROS may overwhelm a cell's antioxidant capacity and damage to cellular macromolecules such
as lipids, protein, and DNA may occur. Under normal conditions, antioxidant activity is in a
tight biological balance with ROS production (Devasagayam et al., 2004.).
One of the cellular antioxidants is a tripeptide molecule known as Glutathione (GSH).
GSH contains an unusual peptide linkage between the amine group of the cysteine and the
carboxyl group of the glutamate side chain. GSH protects cells from toxins such as free
radicals (Karihtala and Soini, 2007). Thiol groups of the GSH are kept in a reduced state within
~5 mM in animal cells. In effect, GSH reduces any disulfide bonds formed within cytoplasmic
proteins to cysteines by acting as an electron donor. GSH is found almost exclusively in its
reduced form, since the enzyme which reverts it from its oxidized form (GSSG), GSH
reductase, is constitutively active and inducible upon oxidative stress. In fact, the ratio of
reduced to oxidized GSH within cells is often used scientifically as a measure of cellular
toxicity. For our study, we applied GSH to be able to make inferences regarding the oxidative
activity of H2O2.
H.J.H Fenton discovered (Fenton, 1894) that iron has a special oxygen transfer property
which improves the use of hydrogen peroxide. Actually, some metals have a strong catalytic
power to generate the highly reactive hydroxyl radicals (OH•). Since this discovery, the iron
catalyzed hydrogen peroxide has been called Fenton's reaction (Liu, 1993; Firek and
Beresewicz , 1990; Kaiserová H et al., 2006).
• Fe2+ + H2O2 ----> Fe3+ + .OH + OH-
• Fe3+ + H2O2----> Fe2+ + .OOH + H+
Nowadays, Fenton's reaction is used to treat a large variety of water pollutant such as
phenols, formaldehyde, BTEX, pesticides, rubber chemicals and more. For our application, we
tried to apply Fenton's reaction in order to use H2O2 efficiently, and to minimize the H2O2
concentration needed.
We also used ultraviolet (UV) light as a ROS inducer. UV is electromagnetic radiation
with a wavelength shorter than that of visible light, but longer than soft X-rays. UV light can
cause reactive oxygen species such as H2O2 and including free radicals such as (OH·), resulting
in oxidative stress (Kohli et al., 1990; Konishi et al., 1991; Jurkiewicz and Buettner, 1994).
For our studies we used a skin cancer cell line, MeWo, to study the effects of UV light, as
the relationship between UV vs. skin cancer may be most significant. For studies of the ROS,
H2O2, we used the neuroblastoma cell line, SHSY-5Y, since this type of ROS may be relevant
for neuropathological conditions, such as brain cancer and neurodegeneration (Hirai et al.,

The involvement of the TSPO preventing mitochondria from ROS damage is supported
by studies of the cellular components of the skin. TSPO are preferentially expressed in the
superficial keratinocytes of the differentiated layers of normal epidermis (Stoebner et al.,
1999). It was suggested that TSPO reduces production of ROS and apoptosis induced by UV
(Stoebner et al., 2001).
The TSPO may be involved in at least two aspects of mitochondrial oxidative processes.
As mentioned above, the TSPO may be a significant candidate for the cellular oxygen sensor,
acting to transduce an oxygen-triggered signal leading to an adaptative cellular response. In
addition, the TSPO can mediate protective effects against ROS damage.
The present Master thesis aims to elucidate some of the aspects of oxidative stress the
TSPO may be involved in.

Research aims
We set out to determine the effects of oxidative stress, including reactive oxygen species
(ROS), on the Translocator Protein (TSPO), in correlation with cell viability and cell death,
focusing on the mitochondrial apoptosis pathway. For this purpose we pursued the following
1. To determine whether TSPO are present in the human melanoma MeWo cell line and
in the human neuroblastoma SHSY-5Y cell lines, including characterization of the
binding characteristics of the radiolabelled TSPO specific ligand [3H]PK 11195.
2. To examine and characterize cellular damage due to the induction of ROS in these
cell lines.
3. To study whether TSPO may actually play a role in the initiation of apoptosis by
ROS. In particular, the effects of ROS on the mitochondrial membrane potential

Materials and methods
Cells of the MeWo human melanoma cell line were cultured and maintained in RPMI-
1640 (Roswell Park Memorial Institute) with the addition of bovine calf serum (FBS) (10%,
v/v), 200 mM L-glutamine saline solution (2%, v/v), Na-pyruvate (1% v/v), and penicillin–
streptomycin solution (10,000 units/ml penicillin sodium salt and 10 mg/ml streptomycin
sulfate) (1%, v/v).
Cells of the SHSY-5Y human neuroblastoma cell line were cultured and maintained in
Dulbecco modified eagle medium (with glucose 4500 mg/l, without Na-pyruvate and L-
glutamine) (DMEM) supplemented with the same ingredients without additional Na-pyruvate.
Cells of both cell lines were grown at 37°C in a humidified atmosphere containing 5%

Materials and equipment

 Biocell PC test (protein carbonyl enzyme immuno-assay kit) was purchased from
Biocell Corporation, (Papatoetoe, New Zealand).
 Bovine Serum Albumin (BSA) was purchased from Sigma (St Louis, USA).
 Bradford solution for determination of protein concentration was obtained from Bio-
rad (Munich, Germany).
 Cell Death ELISAPLUS Kit (Cell Death Kit) was obtained from Roche Diagnostics
GmbH (Mannheim, Germany).
 Culture medium ingredients were obtained from Biological Industries (Beit HaEmek,
 EZ- ECL Chemiluminescence Detection Kit from Biological industries (Beit
Haemek, Israel).
 FALCON ™ FACS tubes- FALCON 352052 obtained from BD Biosciences (San
Jose, CA).
 Flow cytometer for Fluorescence Assisted Cell Sorting: FACS Calibur of Becton
Dickinson - Biosciences (Mountain View, CA).
 JC-1 cation (5,5` ,6,6` -tetrachloro-1,1` ,3,3` -tetraethyl- benzimidazolylcarbocyanine
chloride) used for mitochondrial potential evaluation was purchased from
Calbiochem® (Darmstadt, Germany).
 Kinematica ® Polytron of Brinkmann Instruments (Westbury, NY)
 KODAC BioMax MR Film, Scientific Imaging Film (Chalon-sur-Saone, France).

 Nitrocellulose membranes Hybond ECL, Amersham Biosciences (Buckinghamshire,
 Opti-Fluor for binding assay was obtained from Zunsser Analytic (Frankfurt,
 Phosphate buffered saline (PBS). For all our assays we used Dulbecco's PBS, with
CaCl2 and MgCl2, from Sigma (St Louis, USA), unless indicated otherwise.
 Precision Plus Protein™ Standards obtained from Bio-Rad laboratories Inc.
(Hercules, CA).
 Propidium Iodide (PI) for determining cell viability was purchased from Invitrogen
(Carlsbad, CA).
 Protease inhibitor (Complete) was obtained from Roche Diagnostics (Manheim,
 TBARS kit was assay from Cayman Chemical (Ann Arbor, MI).
 The CellQuest software of Becton Dickinson - Biosciences (Mountain View, CA).
 The Spectrophotometer Zenyth 200 (ELISA reader) was from Anthos, (Eugendorf,
 Unlabeled PK 11195 was purchased from Sigma-Aldrich Israel (Rehovot, Israel).
 Vibra-Cell VCX 750 Sonicator of Sonics and Materials (Newtown, Connecticut).
 [3H]PK 11195 was obtained from New England Nuclear (Boston, MA).
 1600CA Tri-Carb liquid scintillation analyzer Packard - Global Medical
Instrumentation, (Ramsey, Minnesota,).

UV exposure
MeWo cells were exposed to UVC emitted from a UV light source equipped with a UVC
8W/G8T5 bulb (kindly provided by Prof. A. Aharonheim, Technion). For all the UV light
exposures, cells were grown in Petri dishes. The cells were allowed to proliferate until they
were confluent after which they were subjected to irradiation. Immediately prior to exposure
the medium and dish cover were removed. In order to reach a certain dose of radiation two

parameters were considered: 1) the UVC intensity of radiation in ; was measured by the
cm 2
UV LIGHT METER, 2) the duration of the radiation was determined. The total amount of
radiation reaching the cells is the radiation intensity multiplied by the duration of radiation,

according to the equation:

mJ watt joule
amount _ of _ radiation( ) = int ensity ( 2 ) = ( ) • time(sec) .
cm 2
cm sec⋅ cm 2
Since the cells were exposed to the radiation without medium, the duration of radiation
was limited to a maximum of 2 minutes.

H2O2 treatments protocols

For H2O2 exposure, SHSY-5Y cells were grown on 6-well plates. One set of experiments
was done with a short exposure (SE) protocol in which H2O2 was added to the medium in
various concentrations ranging from 100 µM to 1000 µM for 4 hours. After the exposure time,
the cells were immediately collected for the various assays (Figure 6).
Since the SE protocol did not result in changes in TSPO expression we decided to modify
the H2O2 treatment protocol. As part of the calibration for the alternative, long exposure (LE)
protocol, one experiment was done using a 5 h treatment and 72 h recovery. Since all the cells
died with this experiment, we shortened the duration of the treatment and the recovery, and
decided to use lower concentrations of H2O2 for the present study, as described below. For this
standard LE protocol the cells were subjected to H2O2 exposure for two hours and then the
medium was replaced by fresh medium without H2O2. After a recovery of 24 hrs in the
incubator, the cells were collected for the various assays of this study. The range of H 2O2
concentrations in this protocol was from 50 µM to 250 µM. The SE and LE H 2O2 exposure
protocols are presented in Figure 6.

Treatment Treatment Collection of

starts ends cells for
SE protocol:
4 hrs H2O2 treatment
4 hrs 24 hrs
LE protocol: 2 hrs
2 hrs H2O2 treatment
+ 24 hrs recovery Collection
of cells
Treatment Treatment for assays
starts ends

Figure 6. H2O2 SE and LE treatment protocols compared.

Cell viability
In order to estimate viability versus cell death, several methods were used as described
Trypan blue
Using an inverted microscope, inclusion of trypan blue (0.025% final concentration)
indicative of cell death was studied and the numbers of living and dead cells were counted with
the aid of a hemocytometer (Levin et al., 2005).
Propidium Iodide
Cell death levels were determined by PI inclusion using the flow cytometer. PI does not
cross intact membrane and is generally excluded from viable cells. PI binds to DNA by
intercalating between the bases with little or no sequence preference and with a stoichiometry
of one dye molecule per 4–5 base pairs of DNA. PI also binds to RNA, necessitating treatment
with nucleases to distinguish between RNA and DNA staining. Once the dye is bound to
nucleic acids, its fluorescence is enhanced 20- to 30-fold, the fluorescence excitation maximum
is shifted ~30–40 nm to the red and the fluorescence emission maximum is shifted ~15 nm to
the blue. The emission characteristics can be used for measurements using FACS. For cell
viability determination in our assays, confluent cells were collected from 6-well plates and
centrifuged (1,000 × g, 10 min, 4°C), and suspended in 0.5 ml PBS containing, 10 µg/ml of PI
and 10 µg/ml RNase. After 10 min of incubation, routinely 30,000 cells were collected per
sample and analyzed with the flow cytometer.
Annexin V combined with PI
In this assay the Apoptosis Detection Kit of Calbiochem® was used. With this kit, a
fluorescein isothiocyanate (FITC) conjugate of Annexin V, combined with PI, is used allowing
the distinction of populations of cells displaying various viability effects. FACS scan with the
flow cytometer was used to detect the cells of these various popultations. Annexin-V reacts
with Phosphotidyl-Serine (PS), which already in the early stages of apoptosis translocates to
the outer surface of the membrane (Figure 7). Since membrane permeabilization is observed
in necrosis, necrotic cells can also bind Annexin-V. Nonetheless, we found that not all necrotic
cells show Annexin V binding. Application of PI is used to further distinguish various
populations, including: (1) viable (V); (2) early apoptotic (EA); (3) necrotic (N); and (4)
necrotic plus late apoptotic cells (NLA) (Figure 8). PI is excluded from both viable FITC
negative cells (1) and from early apoptotic FITC positive cells (2). Necrotic cells without
labeling with FITC (3) or with labeling with FITC (4) stain with PI. In the absence of
phagocytosis, final stages of apoptosis involve necrotic-like disintegration of cells (Majno and

Joris, 1995.) Thus, similar to what happens with necrotic cells, late apoptotic cells will be
labeled with both FITC and propidium iodide (4).

Figure 7. Phosphotidyl-Serine (PS) in

early apoptosis. With apoptosis, the
phospholipids Phosphotidyl-Serine (PS)
flip out to the outer surface of the plasma
membrane and can react with annexin
V.The arrows indicate the location of the

Figure 8. An example of annexin-PI quadrant. The

x-axis indicates the level of Annexin V labeling. The
y-axis indicates the level of PI labeling. Relatively
high levels of PI labeling indicate necrotic effects.
Relatively high levels of Annexin V labeling indicate
N NLA apoptotic effects. Viable cells (V) show low levels of
Annexin V and PI labeling (lower left quadrant).
Early apoptotic cells (E) show high levels of Annexin
V labeling (lower right quadrant). Necrotic cells (N)
show high levels of PI labeling (upper left quadrant).
Necrotic plus late apoptotic cells (NLA) show
V E labeling with Annexin V and PI (upper right

For our assays, confluent cells were collected and the cell suspension was adjusted to a
concentration of approximately 1 × 106 cells/ml. Then, 0.5 ml of the cell suspension (i.e. 5 ×
105 cells) was transferred to a microvial. Then the Apoptosis Detection Kit was used according
to the instructions of the manufacturer. Briefly, 10 μl Media Binding Reagent and 1.25 μl
Annexin V-FITC (i.e. a final concentration of 0.25 μg/mL) were added and the cell suspension
was incubated for 15 min at room temperature (18–24oC) in the dark. The suspension was then
centrifuged at 1000 × g for 5 min at room temperature and the medium was removed. The cell
pellet was resuspended in 0.5 ml cold 1 × Binding Buffer (as provided with the Apoptosis

Detection Kit) and 10 μl PI was added (at final concentration of 0.6 μg/mL) and the samples
were placed on ice and analyzed with the flow cytometer.

DNA fragmentation
The Cell Death Kit was used to determine the effects of UV light and H2O2 on DNA
fragmentation as an indication of apoptotic levels. For this purpose a photometric enzyme-
immunoassay for the qualitative and quantitative in vitro determination of cytoplasmic histone-
associated-DNA-fragments (mono- and oligonucleosomes) was used. Briefly, samples of
confluent cells were lysed with lysis buffer according to the manual provided by the
manufacturer (Roche, Mannheim, Germany ). The lysate was centrifuged at 200 × g for 10
min. A fraction of the supernatant was transferred to the streptavidin-coated microtiter plate
modules. Immunoreagent was added (anti-histone-biotin and anti-DNA-peroxidase in the
incubation buffer), and after incubation with gentle shaking for 2 hrs, the modules were rinsed
three times with the incubation buffer. Then the signal for apoptosis (DNA fragments bound to
the bottom of the wells of the streptavidin-coated microtiter plate) was labeled with 2,2’-azino-
bis-[3-ethylbenzothiazoline]-6-sulfonic acid (ABTS) solution, as provided with the Cell Death
Kit, for 10-20 min. The resulting opacity of the ABTS solution was determined with an ELISA
reader, at the 405 nm wavelength, using the reference wavelength of 490 nm. ABTS solution
by itself was used as a blank. The positive control was provided with the Cell Death Kit.

Mitochondrial transmembrane potential analysis

The specific stain JC-1 (5,5` ,6,6` -tetrachloro-1,1` ,3,3` -tetraethyl-
benzimidazolylcarbocyanine chloride) was used to assay changes in the Δψm, as described
previously (Chelli et al., 2004). The negative charge established by the intact Δψm allows the
lipophilic dye JC-1, bearing a delocalized positive charge, to enter the mitochondrial matrix.
In healthy cells, the JC-1 molecules form J-aggregates in the mitochondria. When the
mitochondrial membrane potential collapses, the JC-1 does not accumulate within the
mitochondria and remains in the cytoplasm in the monomeric form. When excited by the 575
± 26 nm wave length, J-aggregates emit at 590 nm (orange-red fluorescence) and the
monomers form emits at 527 nm (green fluorescence). The ratio red/green represents the
incidence of the depolarization of the mitochondria. As a positive control the proton ionophor
Carbonyl Cyanide m-Chlorophenyl Hydrazone (CCCP), which causes depolarization of the
Δψm, was used (Chelli et al., 2004). Briefly, for our assays, samples of confluent cells were
collected and centrifuged at 200 × g for 7 min. Cell pellets were resuspended in 0.5 ml of a 5
µM JC-1 solution in PBS and incubated in the incubator at 37˚c for 15-20 min. After
incubation, the cells were centrifuged at 200 × g for 5 min, and resuspended in 0.5 ml PBS.
Then, the cell suspensions were transferred into a 5 ml FALCON® FACS tube and analyzed
with the flow cytometer using CellQuest software.

Protein concentration assays
Protein concentrations were determined by the method of Bradford [1976] as required for
binding assays, Western blot analysis, and the carbonyl test. BSA was used to establish the
standard curves.

Protein Carbonyl assay

To determine whether the effects on viability by our H2O2 treatment were correlated with
oxidative stress we assayed carbonylization and lipid peroxidation (see below). For example,
carbonyls are formed through oxidation of proteins and are sensitive markers of oxidative
injury. For our carbonyl assessment we used the Protein Carbonyl Enzyme Immuno-assay kit
from Biocell. In this assay, carbonyl groups are allowed to react with dinitrophenylhydrazine
(DNPH) and an anti-dinitrophenyl antibody is used to quantify the bound DNPH molecules.
Briefly, confluent cells were collected, centrifuged at 1000 × g at 4°C for 5 min and lysed
with a buffer containing: [45 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES), 0.4 M KCl, 1 mM Ethylenedinitrilotetraacetic acid (EDTA), and glycerol (10%
v/v)], pH 7.8, for 30 min on ice. After incubation, the lysate was centrifuged (11,000 × g for
10 min at 4°C) and the supernatant was stored at -20°C. On the day of carbonyl analysis, the
supernatant was thawed and protein amount was determined. All samples contained 20 µg
protein and were brought to the same volume of 100 µl by adding DDW. Then we added 0.8
volumes of ice cold 28% trichloroacetic acid (TCA), mixed well, and after 10 min incubation
on ice the tubes were centrifuged (10,000 × g, 3 min 4°C). Supernatant was carefully
aspirated. Then 5 µl of enzyme immunoassay (EIA buffer) (1 M phosphate solution containing
BSA (1% v/v), 4 M NaCl, 10 mM EDTA and 0.1% sodium azide) and 15 µl of diluted DNP
solution (DNPH solution diluted 1:10 with guanidine hydrochloride, as provided with the kit)
was added to samples. After 45 min incubation at room temperature, 5 µl was taken to a
complimentary set of 1.5 ml microvials containing 1 ml EIA buffer. The solutions were mixed
well and 200 µl of each sample was added to assigned ELISA-plate wells provided with the
kit. The plate was covered and left overnight at 4°C. The next day, the plate was washed with
EIA buffer (3 ×250 µl per well) and 250 µl of diluted blocking solution was added per well.
After 30 min incubation at room temperature, the wells were washed with EIA buffer as
described above and 200 µl of diluted anti-DNP-biotin-antibody was then added per well and
for 1 hr incubated at 37°C. The wells were washed with EIA as described above and then 200
µl of diluted streptavidin-horseradish peroxidase (HRP) was added per well. After 1 hr of
incubation at room temperature the plate washed with EIA as described above. In order to

obtain color development we added 200 µl of chromatin reagent per well (according to the
manufacturer’s instructions) and after 5 min we added 100 µl of stopping reagent per well.
Absorbance of the samples was measured at the wavelength of 450 nm directly after addition
of the stopping reagent as provided with the kit. The same procedure was done on standards
and controls provided by the manufacturer in order to create standard curves. Carbonyl content
of the samples (nmole/mg protein) was derived from comparison with the standard curve.

2-thiobarbituric acid (TBA) -reactive substances (TBARS assay).

To assay lipid peroxidation due to our treatments, malondialdehyde (MDA), which is a
secondary lipid oxidation product served as a marker. To do this we used the TBARS assay
from Cayman. Lipid peroxides were detected by reacting malondialdehyde (MDA) with
thiobarbituric acid (TBA) to form a 1:2 adduct measurable by spectrofluorometric analysis.
Briefly, following H2O2 treatment, confluent cells (2×107) were collected by centrifugation at
300 × g at 4°C for 5 min, and resuspended in 1 ml of PBS. This cell suspension was sonicated
with a Vibra-Cell VCX 750 on ice for 3 × 5 sec intervals at setting 7. Then 100 µl samples of
the cell homogenates were used to quantify lipid peroxides using the TBARS assay. Cell
homogenates or MDA standard (0 µM to 50 µM) were mixed with 100 μl of SDS in test tubes
and swirled to mix. Then, 4 ml of a TBA reagent mix was added by carefully pouring down
the side of each tube. The mixtures were incubated in a boiling water bath for 60 min. Covers
were placed on the tubes during the incubation period to avoid excessive evaporation of the
reaction mixture. After cooling the tubes for 10 min on ice, the reaction mixture was
centrifuged at 1600 × g at 4°C for 10 min. The fluorescence in the supernatants was read by
ELISA reader with an excitation wavelength of 530 nm and an emission wavelength of 550
nm. The concentrations of TBARS were calculated using MDA as a reference standard. The
quantities of TBARS were expressed as µmol/mg protein calculated from the standard curve as
provided from the MDA standards, according to the instruction provided by the manufacture.

We also assayed oxidative stress induced by H2O2 treatment with the aid of the anti-
oxidant glutathione (GSH). We dissolved GSH to create a 100 mM stock solution. On the day
of experiments, 20 µl of the solution was added to 2 ml medium (final concentration 1 mM),
unless mentioned otherwise.

TSPO binding

Assays of the binding capacity (Bmax) of the TSPO specific tritiated ligand [3H]PK 11195
were conducted as described before [Awad and Gavish, 1987; Kelly-Hershkovitz et al., 1988;
Carmel I et al., 1999]. Briefly, after they had become confluent, the cells were scraped from
the flasks, collected in their culture medium, and centrifuged (1000 × g, 10 min, 4°C).
Following centrifugation, the cell pellets were snap frozen in liquid nitrogen and stored at
-70ºC until further use. On the day of the assay, the pellet was thawed and homogenized in 1
ml of PBS using a Kinematika Polytron (setting 6) for 10 seconds. Then the protein content
was determined. The [3H]PK 11195 binding was assayed in 50 mM potassium phosphate
buffer, pH 7.4, at 4°C in a final volume of 500 µl. The reaction mixture contained 400 µl of
the homogenized membranes in question (40 µg protein) and 25 µl of [3H]PK 11195 solution
(0.18-6.0 nM final concentration) in the absence (total binding) or in the presence (non-specific
binding) of unlabeled PK 11195 (10 µM final concentration). After incubation of the reaction
mixture for 80 min at 4°C, the samples were filtered under vacuum through Whatman GF/C
filters, washed three times with 4 ml of ice cold phosphate buffer 5 mM, pH 7.4 and the filters
placed in vials containing 4 ml of Opti-Fluor. Radioactivity was counted after 12 hrs with a
1600CA Tri-Carb liquid scintillation analyzer. The Bmax and equilibrium dissociation constant
(Kd) were calculated with Scatchard analysis of saturation curves as described previously
(Awad and Gavish, 1987).

Western Blot
Western blot analysis was used to assay the relative protein levels of the TSPO, and of the
MPTP component VDAC, using β-actin signal as a loading reference. Briefly, cells were
washed once with PBS and lysed with a buffer containing: sodium dodecyl sulfate (SDS)
(0.1% w/v), 1% Triton X-100 and 1 tablet/10 ml of Complete Protease Inhibitor. After 30 min
of lysis at room temperature the lysate was centrifuged (10,000 × g 10 min at 4°C) and the
supernatant stored at -20°C. On the day of Western blot assay, protein concentrations were
determined according to Bradford (1976). Samples with equal amount of protein were
prepared in 2 × sample buffer (0.125 M 2-amino-2-(hydroxymethyl) propane-1,3-diol (Tris)-
HCl, pH 6.8, glycerol (20% v/v), SDS (4% w/v), 0.14 M β-mercaptoethanol and bromophenol
blue (0.005% w/v). The samples were boiled for 10 min at 100˚C and subjected to
electrophoresis through a 12% SDS-polyacrylamide gel (10-33 µg protein/lane). The protein
extracts were then electrophoretically transferred to a nitrocellulose membrane in 20 mM Tris-
HCl, pH 6.8, 150 mM Glycine and 20% (v/v) methanol for 90 min at 90 V, followed by 1 hr of
blocking of the membrane in 5% dried milk in 62.5 mM sodium phosphate buffer, pH 7.4,
containing 100 mM NaCl and 0.1% (v/v) Tween-20 (PBS-T). After 3 washes (5 min each), the

membrane was incubated overnight at 4˚C with the primary antisera of choice, including
human anti-TSPO prepared in our laboratory based on a method described previously (Golani
et al., 2001), anti-VDAC (Calbiochem, Darmstadt, Germany), and monoclonal anti-β-actin
(Sigma-Aldrich, Rehovot, Israel). Following the incubation with the primary antibody, the
membrane was washed 3 times with PBS-T (5 min each time) and incubated for 1 hr with a
horse radish peroxidase-linked secondary antibody directed against the host of the primary
antiserum i.e. either anti-rabbit IgG or anti-mouse IgG. After 3 washes with PBS-T the
membrane was incubated 1 min with ECL detection reagent and exposed to Imaging Film for
10-120 sec. Precision Protein Standards loaded together with the samples on the gel was used
as a reference for the MW of the TSPO, VDAC, and β-actin bands.

Analysis of data
Results are expressed as means ± SD. The appropriate analysis of variance and post-hoc
tests were used to analyze the data. Bartlett's test for homogeneity of variance was used to
determine the appropriate model i.e. parametric or non-parametric. Statistical significance was
defined at p<0.05.

UVC exposure
For this part of the study the human melanoma cell line MeWo was used, as UVC is
considered as a cause for human skin cancer. Thus, we considered the use of human skin cell
derivatives to provide the most relevant results in the context of UVC exposure.

TSPO characteristics
As we wanted to determine whether the TSPO may play a role on the effects of UVC
exposure on MeWo melanoma cells, we examined this cell line for the presence of TSPO. For
this purpose we used binding assays with [3H]PK 11195. The average Bmax was calculated
from saturation curves with the application of Scatchard analysis to 6 independent experiments
and was found to be 4,713 ± 2,383 fmol/mg protein. The Kd value was 13.0 ± 7.0 nM (Table
2). Based on those calculated saturation cures, we suspected that the MeWo cell line may
posses two population of TSPO, one of which is high affinity and the other is low affinity
binding sites for [3H]PK 11195. To further investigate this possibility, we applied the use of
lower concentrations of [3H]PK 11195 (0.185-1.5 nM) (Figure 9). With this approach we
found a Bmax of 365 fmol/mg protein and a Kd value of 0.6 nM.

Bmax Kd (nM) Table 2. Binding

(fmol/mg protein) characteristics of untreated
Average 4,713 13.0 MeWo cell line. The average
of 6 independent experiments
SD 2,383 7.0 is presented with the SD for
each parameter.


0.2 0.6
fmol/ mg protein


Figure 9. [3H]PK 11195 binding of the high affinity TSPO population in MeWo
melanoma cell line. Binding assay obtained with concentrations from 0.18 nM –
1.5 nM 0
0 of [ H]PK 11195. B = Bound (fmol/mg protein). F= Free ligand.

0 0.25 0.5 0.75 1 1.25 1.5 1.75 0 0.1 0.2 0.3 0.4
[3H]PK 11195 [nM] B

As an additional assay for examining the presence of TSPO, Western blotting was
performed. The 18 kDa protein TSPO was detected using our polyclonal antibody against
TSPO (Figure 10). β-actin was used as a standard. Only one band of TSPO antibody labeling

was detected (at the 18 kDa level), suggesting that TSPO is only represented in the monomeric

kDa 42 β-actin Figure 10. Western

Blot showing the
presence of TSPO in
TSPO the MeWo cell line.

TSPO binding and its protein level in response to UVC radiation.

We assumed that TSPO binding characteristics would be affected by UV C light and would
be involved in the response to the oxidative stress created. Therefore, we assayed changes in
[3H]PK 11195 binding measured at 6 nM of [3H]PK 11195 in response to increasing doses of
UVC radiation. We also measured protein levels of TSPO with Western blot analysis.
We found that the [3H]PK 11195 binding after UVC radiation did not change compared to
control (Figure 11). Regarding protein levels, Western blot analysis indicated that TSPO level
did not change either (Figure 12). The measurements were done in duplicates. The two
measurements of the control suggest a large variability. This, however, was not the case for
the TSPO protein levels detected with the increasing UVC doses. Furthermore, no UVC dose
related changes in TSPO levels could be detected. Thus, we conclude, as can also be derived
from the [3H]PK 11195 binding assay, that UVC exposure in our study does not affect TSPO

Binding of [3H]PK 11195 ( 6nM final conc.) after UVc Figure 11: Binding of
exposure [3H]PK 11195 was
1600 measured at 6 nM after
Figure doses
increasing 12: of UV
B (fmol/mg protein)

1200 irradiation. blot

Western No
analysis after
differences between UVCthe
control groupradiation
and the
in MeWo
experimental groups
(measurements are in
200 A- TSPOthe
duplicate, bends
0 against β-actin
are presented).
control 1.2 7.2 8.4 16.8 seems to reveal
UVC no
exposure is expressed
dose related
UVc [mJ/cm2] as mJ/cm2.
response in
protein levels of
β-actin B- Normalization
of TSPO levels
against β-actin
UVC exposure
TSPO expressed in
34 mJ/cm2.
Control 1.2 7.2 8.4 16.8
[UVc [mJ/cm

Normalized TSPO level in response to UVc

10 raddiation
TSPO/ β-actin signal

0 1.2 7.2 8.4 16.8
B UVc [m J/Cm 2]

DNA fragmentation
Irradiation of cells with UVC is reported to cause cell death and apoptosis (Fan et. al.,
2007). Thus we studied apoptosis levels after exposure of MeWo cells to UVC radiation. We
tested the level of DNA fragmentation as a measure of apoptosis levels and found that in
response to 1-16.8 mJ/cm2 dose of radiation there were only slight non-significant increases in
DNA fragmentation (Figure 13). Moreover, no UVC dose dependence was detected. Also no
increases in necrotic cell death were observed (data not shown). Thus, our experimental
conditions regarding UVC exposure did not induce appreciable effects on TSPO characteristics,
TSPO protein levels and cell death, including apoptosis.

DNA Fragmentation level after UV exposure Figure 13. DNA

fragmentation in
3.5 response to UVC
3.0 radiation in MeWo
DNA Fragmentation [OD]

melanoma cell line.

The X axis represents
2.0 increasing doses of UVC
1.5 radiation (expressed in
mJ/cm2), and the Y
axis is the level of DNA
0.0 (measurements are in
Hduplicate, the averages
control 1.2 7.2 8.4 16.8
2O2 exposure
UVc [mJ/cm2] are presented).
Since our UVC exposure
did not seem to suggest an appreciable load of oxidative stress, we decided to apply one type of

ROS to cultured cells (H2O2). For this part of the study the human neuroblastoma cell line the
SHSY-5Y cell line was used.

TSPO binding characteristics

In order to test the presence of TSPO in the SHSY-5Y cell line, binding assays were
performed using the TSPO specific ligand [3H]PK 11195. The Bmax and Kd value were
calculated using Scatchard analysis. The Bmax was calculated as an average of 7 independent
experiments and was 1,880 ± 910 fmol/mg protein and the Kd was 3.4 ± 1.6 nM. In Figure 14
a representative saturation curve is presented
Western blot analysis also demonstrated the presence of the 18 kDa TSPO, as shown later
for our assays of the effects of H2O2 exposure.

2 1.6
B (fmol/ mg protein)

1.5 1.2

1 0.8
0.5 0.4
0 0
0 2 4 6 0 0.5 1 1.5 2 2.5

[3H]PK 11195 [nM] B

Figure 14. A representative example of a saturation curve for [3H]PK 11195 binding
and the corresponding Scatchard analysis for the SHSY-5Y cell line. B = Bound
(fmol/mg protein). F = Free ligand.

Cell viability
The first parameter to evaluate cellular damage due to H2O2 treatment was cell viability.
This was done by using trypan blue or PI exclusion analysis. Since trypan blue exclusion is the
“gold standard” of non-fluorescent vital dyes, we wanted to compare the percentage of viable
cells determined with trypan blue to those determined with PI. Both dyes are not absorbed by
viable cells, while dead cells are stained since their membrane is damaged and the dye is
absorbed. In Figure 15 the comparison between PI and trypan blue is presented with the
application of the SE protocol. A good correlation between the two methods was discerned and
for that reason all the viability assays were subsequently done with PI. To obtain a more

detailed dose response curve of the H2O2 dose dependent decrease in viability, we applied H2O2
at smaller increments with the SE protocol as presented in Figure 16.

Cell viability of SHSY-5Y using Trypan Blue Figure

and 15.
Propidium Iodide Comparison of
Trypan blue
and PI
100 Propidium Iodide
Viability tests. Cells
were treated using
viability [%]

80 2
R (TB) = 0.9007
70 R (PI) the SE protocol and
= 0.9186
60 then viability was
tested. A good
correlation was
found between cell
10 death determined
0 with trypan blue
control 200µM 400µM 600µM 800µM
PI. (Each point
H2 O2 concentration [µM] was measured in

Cell viability of SHSY-5Y using the SE protocol

Figure 16. Viability
of SHSY-5Y cells
100 after SE protocol
80 tested with PI. The
R2 = 0.9527
portion of the living
viability [%] (PI)

60 cells decreases as
40 H2O2 concentration
30 increases
(measurements are
0 in duplicate, the
averages are













H2O2 concentration [µM]

Viability was also tested using the LE protocol in which the cells were found to be
sensitive to H2O2 already at concentrations of 100 µM and higher. The range of concentrations
for LE protocol used was smaller than for the SE protocol by almost one order of magnitude .
The viability test, with the LE protocol, resulted in a similar dose dependence curve as with SE
protocol in response to increasing concentrations of H2O2 (Figure 17).

Cell viability of SHSY-5Y cells using the LE protocol
Figure 17.
Viability of SHSY-
5Y cells with the
80 2LE protocol tested
R = 0.9196
with PI. The
viability [%] (PI)

50 percentage of the
40 living cells
30 decreases as H2O2
20 concentration is
10 increased
0 (measurements
are in duplicate,


the averages are





H2O2 concentration [µM]

One method we used to determine the main process contributing to cell death due to H2O2
exposure was application of the Apoptosis Detection Kit (Calbiochem) which allowed us to
distinguish between forms of apoptosis and necrosis, as described in the methods' section
(Figure 18).
In response to the SE protocol, the amount of viable cells appeared to be reduced in an
H2O2 dose dependent fashion (Figure 18). We also observed that the amount of necrotic cells
appeared to be increased by increasing doses of H2O2. Regarding apoptosis, the amount of
early apoptotic cells appeared to decrease in a dose dependent manner. The population of cells
positive for both PI and annexin indicative for both necrotic and late apoptotic cells appeared
to stay stable (Figure 18). These data indicate that cell death observed in response to H 2O2
treatment in the SE protocol is primarily due to necrosis and less to apoptosis.

v iable cellsFigure 18: Annexin-V - PI

Annexin FITC-PI necrosis results using the SE
80 protocol results in a
early apoptosis
70 decrease
late apoptosis of the
and necrosis
percentage of living cells.
Living cells (%)

60 The percentage of
50 necrotic cells is
40 increased. The percentage
of early apoptotic cells is
decreased. The
20 percentage of the
10 population combining late
0 apoptotic and necrotic
control 400 cells appears
650 to stay
stable (preliminary
H2O2 concentraion [µM] results).

DNA fragmentation
An additional analysis, using DNA fragmentation as determined with the Cell Death Kit
(Roche), was done to measure apoptosis levels. In response to the LE protocol the absolute
level of DNA fragmentation appeared to decrease in a dose dependent manner (Figure 19).
Since the OD, measured in 405nm, decreased below 1.00, it is incorrect to clam that the level
of apoptosis decreased as the concentration of H2O2 rose. Instead, it is possible to conclude that
the level of necrosis increased.
Thus, H2O2 causes cell death primarily by way of necrosis, and apparently not by means
of apoptosis (Figures 15,16,17,18).

DNA fragmentation value after H2O2 treatment Figure 19. DNA

1.40 fragmentation as an
indication of
1.20 apoptosis in
response to the LE
DNA fragmentation

[OD 405nm ]

0.80 The level of

apoptosis in the
form of ABTS
0.40 solution opacity
(OD). (The
measurements are
0.00 in duplicate, and
0 40 80 120 160 200 240 280 320 the averages ± SD
H2O2 concentration [µM] are presented).

Mitochondrial potential (Δψm)

B As discussed in the Introduction, collapse of the Δψm may lead to apoptosis as well as
necrosis. As we observed a decrease in viability in response to H2O2 we wanted to examine
whether the Δψm was affected by H2O2 exposure. In response to the SE protocol the Δψm
decreased. In particular 1000 µM H2O2 causes a dramatic and significant decrease of almost
10 fold in the Δψm (p < 0.01) (Figure 20). Thus, the decrease in cell viability appears to be
accompanied by a drop in the Δψm.

Mitochondrial potential Figure 20.

Incidence of Δψm
0.35 collapse in
0.30 response to the SE
red/green ratio

** P < 0.01 vs.
control (n=3).

0.10 The effect of the

0.05 1000 µM of H2O2 is
0.00 comparable to that
Control 300 650 1000 cccp of the positive
H2O2 concentration [µM] control CCCP.

Induction of Fenton reaction
As we found only weak apoptotic responses to H2O2 we attempted to enhance the H2O2
effects. H2O2 is considered a weak radical, but in the presence of transition metals such as iron,
the Fenton reaction occurs resulting in the very aggressive hydroxyl radical (OH·) as one of the
end products, causing most of the cellular damage (for a review see Crichton et al., 2002).
Therefore we applied FeCl2 to our H2O2 protocols in attempt to enhance the H2O2 effects.
Surprisingly, when viability and mitochondrial potential were tested with the SE protocol, iron
exhibit protective activity at the higher concentrations of H2O2, whereas at low concentrations
of H2O2 iron slightly enlarged the damage (Figure 21), but still well less below damage levels
caused by higher H2O2 concentrations. The dose of FeCl2 did not seem to be relevant. Thus,
FeCl2 did not appear to enhance H2O2 effects. Maybe FeCl2 deactivated H2O2 even before it
reached the cells.

Viability of SHSY cell line after treatment with H2O2 and iron for 15h Figure 21: The effect
H2O2 alone of FeCl2 and H2O2
H2O2+ FeCl2 50uM combined on cellular
H2O2 alone
H2O2+ FeCl2 100uM damage using the SE
H2O2+ FeCl2 100uM protocol.
70 A- Viability- Two
viability [%] (PI)

H2O2+ FeCl2 50uM concentrations of


FeCl2 did not
appreciably enhance
H2O2 effects and even
appeared to protect
20 at higher
10 concentrations of
0 H2O2. (n=2)
0 100 200 300 400 500 600 700 800 900 1000 B- Mitochondrial
H 2O2 concentration [µM] potential- FeCl2
prevented the
decrease in red/green
Mitochondrial potential H2O2 alone
ratio caused by H2O2
Fe 50uM
0.4 at concentration of
Fe 100uM
650 µM and 1000 µM
0.3 H2O2 alone 100uM
ratio (red/green)

0.25 50uM 100uM

100uM 50uM
H2O2 alone

H2O2 alone
0 300 650 1000
H2O2 concentration [µM]

The effects of glutathione (GSH)
One way to assay whether the effects of H2O2 on cell viability may be due to free radical
damage to the cells is by repairing the phenotype with the aid of an anti-oxidant. GSH is an
anti-oxidant that protects cells by neutralizing free radicals such as H2O2 by acting as an
electron donor. In Figure 22 the percentage of dead cells in response to the SE protocol was
analyzed with PI. 4 mM GSH protected the cells from the cell death caused by the addition of
H2O2 to the culture. GSH by itself had no effects. Thus, this experiment suggests that cell
death caused by the addition of H2O2 is due to free radical activity.

The effect of GSH on viability Figure 22. The effect

of GSH on cell
25 viability with
application of the SE
20 protocol. 4 nM of GSH
Dead cells [%] (PI)

appeared to protect
15 cells from the lethal
effects of H2O2,
10 keeping the death
level at the level of
5 control
(measurements were
0 done in duplicate).
cont 100 200 300 400 Cell viability
500 is
presented in the form
H2O2 concentration [µM] of dead cells.

On one occasion we applied the LE protocol, using the same H2O2 concentrations as were
used in the SE protocol, to study the effects of GSH (Figure 23). We found that at all
concentrations used, H2O2 caused death to all of the cells (100%). On the other hand, 4 mM
glutathione kept the percentage of the dead cells at control levels. GSH by itself had no effects.
This indicated that the effects of H2O2 on cell viability indeed are ROS induced, both in the SE
and LE protocols. This experiment also led us to reduce the concentration of H2O2 in the new
LE protocol.

Figure 23. Viability test

Cell death in response to H2O2 exposure and
after 5h treatment and
72h recovery. Cell 72 hrs recovery GSH+
viability is presented in GSH -
the form of dead cells.
All the cells died in
Cell Deaeth [%]

response to H2O2 applied

at the concentrations
listed, but 4mM GSH 60.0
protected the cells40.0
the free radical and the
level of dead cells 20.0
stayed as in control 0.0
(measurements were
cont 200 400 600 800
done in duplicate).
H2O2 concentration [µM]

We also determined GSH protective effects at lower concentrations of GSH (1 mM and 2
mM) in the LE protocol. In Figure 24 the percentage of dead cells is presented. GSH at both
concentrations tested protected the cells from H2O2 at concentration of 100 µM and 200 µM.
Since at 1 mM GSH there was still good protection against cell death, most of the experiments
with GSH presented next were done with a concentration of 1 mM of GSH. Furthermore, we
restricted ourselves to the application of a concentration of 100 µM H2O2 to the LE protocol.

70 FigureGSH
The effect of GSH on viability
Percentage of dead
cells with different
concentration of
Cell Death[%] (PI)

40 concentrations of 1
mM and 2 mM
30 applied in the LE
protocol prevented
the increase in cell
10 death in response to
H2O2 treatment
0 (measurements
control 100 were
200 done in
H2O2 concentration [µM] duplicate).

We next wanted to examine the effect GSH has on apoptosis. We found that although
GSH has a protective effect against cell death created by H2O2, GSH by it self can cause an
small increase in DNA fragmentation (Figure 25). GSH in combination with H2O2 prevented
the decrease in DNA fragmentation caused by H2O2 alone (Figure 25). We assume that this
rescue includes the reduction in necrotic cell death observed before (Figures 18,19).

The effect of GSH and H2O2 on DNA Figure 25. GSH effect
fragmentation on apoptosis levels.
GSH - The amount of
GSH + apoptosis in the form
of ABTS solution
DNA fragmentation

opacity. GSH
1.20 increases

1.00 substantially the level

0.80 of DNA fragmentation
0.60 in the presence of
0.40 H2O2. (Measurements
0.20 are in duplicate, the
0.00 averages are
0 160 320 presented).
H2O2 concentration [µM]

Since we found that GSH by itself causes an increase in the apoptosis levels, we wanted
to test whether potential toxic effects of GSH may be expressed in the oxidative stress
parameters we applied. We found that 2 mM GSH causes an increase in lipid peroxidation as
determined with the TBARS kit from Cayman. In combination with 100 µM H2O2 the lipid
peroxidation levels may increase to almost 6 fold of the lipid peroxidation level induced by 100
µM H2O2 by itself (Figure 26). GSH had no effects on protein cabonylization (see Figure 27).
Thus, these data may suggest that GSH has a toxic effect on lipids and by itself may induce a
form of oxidative stress that could lead to apoptosis as presented above.

Figure 26. GSH

The effect of GSH on lipid peroxidation effects on lipid
peroxidation levels.
Lipid peroxidation [µmole/mg]

GSH by itself
3 increases lipid
peroxidation levels
and in combination
2 with H2O2
peroxidation levels
1.5 are 6 fold higher
1 than the H2O2
effects by
0.5 themselves.
(n=2 for control and
GSH, and n=1 for
control GSH 2mM 100uM H2O2 GSH + H2O2 100µM H2O2 and GSH
+ H2O2).

H2O2 effects on protein carbonylization

Carbonyl groups on proteins are the result of protein oxidation, and the level of protein
carbonylization is indicative of oxidative stress intensity created in the cell. We used the
Protein Carbonyls test in order to evaluate the oxidative stress damage created by H2O2. The
Protein Carbonyl test showed that the amount of basal carbonylization, utilizing the LE
protocol, was around 1 nmol/mg protein, and that GSH by itself did not cause any change in
the amount of carbonyls. H2O2 slightly increased the level of carbonyl groups, and when GSH
was added to H2O2 it seemed that this carbonylization was reduced a little (Figure 27). Thus, it
appears that the effect of H2O2 on protein carbonylization is not as strong as the H2O2 effect on
cell viability.

Figure 27. Carbonyl
Protein Carbonylization GSH - formation in response
Carbonyl level [nmol/mg protein] 2.50 GSH + to the LE protocol.
GSH by itself had no
2.00 effect compared to
untreated control cells.
1.50 H2O2 cased a small
increase in carbonyl
1.00 formation. GSH seemed
to slightly reduce the
0.50 carbonylization level
cased by 100 H2O2 alone
0.00 (measurement were
control 100 µM H2O2 done in duplicate).

H2O2 effects on lipid peroxidation

Another measure for oxidative stress is the quantification of lipid peroxidation measured
with the aid of TBARS KIT (Cayman chemical). The TBARS test presented in Figure 26
shows that the amount of the basal lipid peroxidation was around 0.5 µmol/mg, and that H 2O2
alone did not cause an increase in the damage to the lipids as expected. Thus, lipid
peroxidation does not appear to be a contributing factor on the H2O2 effect on cell viability.

TSPO characteristics in response to H2O2 exposure:

TSPO characteristics in the SE protocol
In order to determine whether there may potentially be a correlation between cell
viability, changes in the Δψm and TSPO characteristics, we studied TSPO binding levels and
affinity as well as protein levels. In the SE protocol, TSPO binding capacity (at 6 nM
radioactive ligand) was as in untreated cells in response to low concentration of H2O2 (≤ 250
µM). At higher concentrations of H2O2 (≥ 500 µM) TSPO binding showed a non significant
trend of increase (Figure 28). Thus, this suggests that in the SE protocol changes in TSPO are
not required for the effects seen on cell viability.
After we noticed that TSPO binding was not significantly affected by the SE protocol, we
also examined TSPO protein levels in the SE protocol and we found that TSPO protein levels
did not show differences compared to control (data not shown).

Binding of [3H]PK 11195 (6nM final conc.) using the Figure 28. Binding
SE protocol of [3H]PK 11195 in
response to the SE
protocol. No
B [fmol/mg protein]

2500 increase in the
2000 binding level was
1500 found in response
1000 to H2O2 exposure.
500 (Measurements
0 are in duplicate,
0 100 250 500 1000 the averages are
H2O2 concentration [µM] presented).

TSPO binding characteristics in the LE protocol

The LE protocol showed unambiguous results regarding TSPO binding. TSPO binding,
as shown in Figure 29, increased significantly (p < 0.01) in response to 100 µM H2O2. GSH
by itself did not affect the TSPO ligand binding, as detected with 6 nM [3H]PK 11195, but in
the presence of H2O2 it appeared to prevent the increase observed with H2O2 alone. Also
Western blotting analysis after application of LE protocol did not show any change in the
protein levels of TSPO (data not shown).

Binding of [3H]PK 11195 (6 nM final conc.) to TSPO Figure 29. Binding

using the LE protocol of 6 nM [3H]PK
11195 in response
20000 to LE protocol. H2O2
caused an increase
B [fmol/mg protein]

16000 in TSPO binding

levels whereas GSH
12000 prevented this
8000 GSH by itself had no
effect on TSPO
4000 bindinf levels.
**- p < 0.01 control
vs. H2O2,
control H2O2 100uM GSH 1 mM GSH + H2O2

Interestingly, in one experiment Western blot analysis in response to very long exposure
period (14h) with 1000 µM H2O2 without recovery caused a conspicuous increase in response
(Figure 30). Both the levels of TSPO and VDAC can be seen to be raised whereas the levels
of the housekeeping protein β-actin is decreased (Figure 30).

β- actin Figure 30. Western blot in

42kDa response to 14 hrs exposure
kDa 32 VDAC with 1000 µM H2O2. TSPO
and VDAC protein levels
increased whereas β-actin
kDa 18 TSPO decreased.
Control 1000µM

The effects of unlabeled PK 11195 on [3H]PK 111195 binding and cell

viability due to H2O2 exposure.
As mentioned, by applying the LE protocol we found that application of 100 µM H2O2
significantly increased [3H]PK 11195 binding levels (p < 0.01). Unlabeled PK 11195 by itself
did not change [3H]PK 11195 binding compared to control, and when added to the H2O2
treatment, the ligand prevented the increase observed in response to H2O2 alone (Figure 31).

The effect of unlabled PK 11195 on [3H]PK 11195
Binding to TSPO
20000 **
B [fmol/mg protein]



4000 n=6 n=6

control H2O2 100µM PK 11195 1 µM PK + H2O2

Figure 31. Binding of 6 nM [3H]PK 11195 in response to LE protocol.

H2O2 caused an increase in TSPO binding levels whereas 1µM PK 11195
prevented this increase.
** p < 0.01 control vs. H2O2.

Cell viability
After we found that PK 11195 apparently prevented TSPO ligand binding increases by
H2O2, we wanted to check whether cell viability would also be affected by PK 11195. The
effects of different concentrations of PK 11195 on changes in viability in response to the LE
protocol are shown (Figure 32). In this experiment H2O2 at 100 µM causes almost total cell
death (96%). PK 11195 at different concentrations did not affect this level of cell death caused
by H2O2. Note, however that in this experiment the level of cell death is much higher than
determined with the standard curve in Figure 17.

Figure 32. The effect

The effect of unlabled PK 11195 and H2O2 on cell viability
of 100 µM H2O2 and
PK 11195 various concentrations
PK + H2O2 of unlabeled PK 11195
100 in on cell viability. PK
90 11195 by itself at the
80 various concentrations
Cel Death [%]

70 did not cause a

change in the level of
cell death. In
30 combination with
20 H2O2, PK 11195 did not
10 prevent cell death
0 induced by 100 µM
0 10 100 1000 H2O2. (n=6).
PK 11195 concentration [nM]

With this thesis we wanted to study whether TSPO may play a role in the potential effects
of ROS on cell viability and related parameters. The subjects we studied are present in Figure

ROS → lipid peroxidation and protein carbonylization → TSPO → Δψm → cell death

Figure 33. Assumed involvement of TSPO in cell death caused by ROS. We hypothesized that
ROS can activate the TSPO, which in turn affects the Δψm, finally leading to cell death.

In particular, we wanted to determine whether effects of ROS generation on cell viability

may correlate with modulations of TSPO expression. Furthermore, we want to assay whether
such processes may be associated with changes in the Δψm. We also wanted to know whether
such changes may correlate with oxidative stress, including protein carbonylization and lipid
peroxidation. For this purpose, we also applied the anti-oxidant GSH. As these processes are
known to affect cell viability, we were interested to know whether the mechanisms modulated
by our ROS inducing treatments could be associated with a particular form of cell death i.e.
apoptosis and necrosis.
We at first applied UVC radiation to the human melanoma MeWo cell line to induce ROS
generation. To assay whether the TSPO may actually play a role in such responses, we first
wanted to check whether TSPO are actually present in the melanoma MeWo cell line. Here we
report the presence of TSPO in this cell line. We expected to find TSPO in this cell line since
previous studies showed that other melanoma cell lines also possess TSPO molecules
(Matthew et al., 1981, Landau et al., 1998). However, it has been reported that
malignant skin tumors can show decreased TSPO expression compared with normal skin (Han
et al., 2003). This is uncommon, since TSPO usually is upregulated in various cancerous
tissues such as ovary, colon and brain (Black et al., 1990; Katz et al., 1990a,b; Cornu et al.,
1992). However, also for kidney cancer, reductions in TSPO levels were reported (Katz et al.,
1989). The potential discrepancies reported between various forms of skin cancer regarding
TSPO may be partly related to different populations of TSPO, some showing high affinity for
their ligands and other showing low affinity, as has been reported for other tissues (Woods et
al., 1996).
Our present results may also suggest that the MeWo cell line may possess two
populations of TSPO, one of which with high affinity and the other with low affinity to [3H]PK
11195. A possible explanation for the presence of two populations of TSPO may be the
cellular location of TSPO (Woods et al., 1996). There is also evidence that, in addition to the

mitochondrial membranes, TSPO are also located in the cell nucleus, golgi, lysosomes, rough
endoplasmic reticulum, microsomes, peroxisomes and plasma membranes (O'Beirne et al.,
1990, Woods et al., 1996; Hardwick et al., 1999; Berkovich et al., 1993). Another potential
explanation for more than one population of TSPO may be polymerization of the receptor.
Delavoie et al (2003) have shown that in response to oxidative stress TSPO may undergo
polymerization, and that this dynamic process might modulate the functions of TSPO,
including its ligand binding properties. Based on the Delavoie et al. (2003) study, we
considered the possibility that skin cells, from which the MeWo cell line is derived, may have
developed a mechanism by which TSPO polymers deal with the oxidative stress. We consider
the possibility that, since skin cells are often subject to oxidative stress, such cells may readily
form TSPO polymers. However, with our Western blot assays we did not find evidence that
this actually occurs in the MeWo cell line. Potentially this may be due to the short interval
between the UVC exposure, and the moment we collected the cells for our assays (2 min) i.e.
not sufficient time had elapsed for observable increase in TSPO polymers. Another
explanation may be, for example, derived from the study by Woods et al. (1996), which
suggests that different affinities of TSPO do not depend on the molecular weight of the TSPO.
It would be worthwhile to further investigate the characteristics of the two potential TSPO
populations showing different affinities for [3H]PK 11195.
As mentioned, in this study we wanted to investigate a possible relationship between
TSPO and ROS. In order to do so we exposed the MeWo melanoma cell line to UVC light that
is known to induce oxidative stress, including cellular ROS (Gomes et al., 2005). Since UV
light is known to cause cellular death and apoptosis (for review- Ryter et al., 2007), we
expected to find increasing levels of apoptosis in response to our UVC exposure.
With this study, we did not find a significant increase in the level of apoptosis and there
may be two major reasons for that:
1. We could not use intensity of radiation that caused cell death in previous studies
(Stoebner et al., 2001), due to the UV source we had available. We also did not
think that extending the UV exposure time would be acceptable, since the exposure
included a removal of the medium from the cells and we did not want to confound our
experiment by longer periods of medium deprivation.
2. The cells were collected immediately after a 2 min UV exposure and it is possible that
detectable apoptosis and other processes would have taken place if a recovery time of
several hours would have been given. Indeed, Stoebner et al (2001) have shown
that the amount of apoptotic cells increased when a recovery period (4 hours or more)
was applied.

3. We tested only one UV wavelength from the hole UVC range, and it is possible that
we did not manage to cause an insult to the desirably molecules (i.e. proteins) with
the aid of this wavelength.

Thus, for potential future studies regarding the effects of TSPO activation due to UV
exposure, a more suitable UV source would be required, and it would be advisable to include a
"recovery" period for the cells in culture after the UV exposure.
Prior to the experiments, we hypothesized that TSPO levels would be affected by UV
exposure since others have reported an increase in TSPO binding levels (Delavoie F et al.,
2003). However, we did not see any significant effect on TSPO binding and protein levels,
probably due to the relatively low UV exposure intensity, and the lack of a post-exposure
response time (as also suggested for the lack of effects on TSPO polymerization and
Since we had run into technical problems regarding our paradigm of ROS induction by
UVC radiation, we decided to apply exogenous ROS immediately on a cell culture. In this way
we hoped to be able to see stronger effects on TSPO levels, as well as on the Δψ m, cellular
damage, including protein carbonylization and lipid peroxidation, and cell death. We chose to
use the ROS H2O2 since it is known that H2O2 causes cell death by inducing oxidative stress in
the cell (for review- Ryter et al., 2007). We wanted to choose a cell line for which ROS
generation can be considered relevant. Therefore, we used the human SHSY-5Y cell line of
neuronal origin, since for neuronal damage, as is also observed with neurodegeneration, ROS
play an important role in the processes of cell damage and cell death (Cui et al., 2004)
In our assay, the SHSY-5Y displayed one population of TSPO (Bmax= 2,278 fmol/mg
protein) with relatively high affinity (Kd = 3.4 nM). This Bmax is higher than what is typically
found for normal brain tissue (Gavish et al., 1999; Veenman et al., 2002). In particular, in
comparison to different tissues throughout the body, TSPO are less abundant in brain tissue
(see Figure 3). Our data, thus, are in accord with other studies demonstrating upregulation of
TSPO in different forms of brain cancer (Cornu et al., 1992, Black et al., 1990).
In our system we exposed the SHSY-5Y cells to two different treatment protocols: The
first was a short H2O2 exposure (4 hrs of acute treatment, SE). The second protocol was longer
since we added a recovery time for 24 hrs after 2 hrs of H2O2 treatment (LE). Application of
the SE protocol showed no changes in TSPO binding levels and TSPO protein expression, but
did result in reductions in the Δψm, decreases in cell viability, and increases in necrotic levels,
accompanied by decreases in apoptotic levels. On the one hand, this may suggest that TSPO
are not needed for the effects of H2O2 treatment on cell death, and the processes leading to cell

death. On the other hand, it may suggest that it is not necessary to upregulate TSPO expression
to induce such effects, but pharmacological activation of TSPO may be sufficient to induce
such effects.
Considering the possibility that H2O2 used with the SE protocol was not potent enough to
have effects on TSPO expression, we decided to apply a technique to enhance the effects of
H2O2. Thus, we applied induction of the Fenton Reaction by Fe (II) and H 2O2 in order to
produce the highly aggressive hydroxyl radical. This reaction was used for similar purposes by
others (Liu, 1993; Firek and Beresewicz , 1990; Kaiserová H et al., 2006). Surprisingly, we
did not succeed. Instead, the opposite seemed to happen; Fe (II) seemed to protect the cells
from the destructive effects at least at higher concentrations of H2O2. At lower concentrations,
the application Fe(II) did result in a reduction in cell viability but not more than achieved with
higher concentrations of H2O2. We did not find reports in the literature describing this effect.
In fact, with application of Fe(II) we found a constant effect on cell viability, unrelated to the
concentration of the H2O2 co-applied with the Fe(II). Since H2O2 did not appear to contribute
to changes in levels of viability, it may be that H2O2 actually was inactive in this set up. We
consider the possibility that application of Fe(II) may have induced the Fenton reaction already
in the culture medium and effectively reduced the concentration of H2O2 reaching the cells.
Maybe it is advisable to pre-treat cell culture with Fe(II) and then apply in H 2O2 in fresh
On another track, we assumed that the lack of a recovery period after the H 2O2 treatment
in the SE protocol prevented the occurrence of changes in TSPO levels. Therefore we applied
the LE protocol. With the LE protocol we did observe changes in the TSPO levels.
Furthermore, for effects regarding cell viability, as desired, lower concentrations of H2O2 were
needed with the LE protocol than for the SE protocol. Thus, we decided to apply the LE
protocol for most experiments of our study.
Interestingly, for both the SE and LE protocol we found that the majority of cell death
was due to necrosis and not apoptosis. Halestrap (2006) suggests that if the mitochondria
receive a severe insult, the MPTP remain open, potentially causing and irreversible collapse of
the Δψm, and necrosis is the consequence, even though intracellular apoptosis signals and
activation of caspases occur. Indeed we found a significant decrease in the Δψ m with the SE
protocol, indicating that H2O2 treatment caused the MPTP to open.
For this thesis we used three different assays to test the occurrence of oxidative stress: 1)
the use of the anti-oxidant GSH which protects against the cell death caused by H2O2 (Ryter et
al., 2007); 2) detection of carbonyls formation, which is a measure of oxidative damage to

proteins ((Hwang et al., 2007); and 3) lipid peroxidation, which is a measure of oxidative
damage to lipids, including lipid membranes ((Hwang et al., 2007).
Applying GSH verified that our SE and LE protocols induced oxidative stress. As
reported in many other studies, GSH reduced the effects of H2O2 on cell death (for review-
Ryter et al., 2007). With the LE protocol, we found little effect regarding carbonyl formation.
Comparable to our results, Grant et al. (2005) also found with a similar treatment as ours on the
SHSY-5Y cell line, that only little increases in carbonyl formation occurred with application of
100 µM H2O2. A possible explanation is that the low carbonyl formation observed may reflect
early removal of oxidized proteins by the 20S proteasome, which predominantly removes and
degrades oxidized proteins (Grune et al., 2001, Davies, 2001). Furthermore, it has been
demonstrated that H2O2 increases protein degradation by the 20S proteasome (Grune et al.,
2001, Davies, 2001).
Regarding lipid peroxidation, we also did not find an effect induced by the LE protocol.
Surprisingly, we found that GSH by itself caused an increase in lipid peroxidation, and
unexpectedly GSH and H2O2 seemed to have a synergistic effect on lipids damage. To our
knowledge such effects have not been reported previously (Gueorguieva et al., 2006).
In any case, it appears that ROS generation plays a role in cell death induced by H2O2, as
deduced from the effects of the anti-oxidant GSH. However, lipid peroxidation and protein
carbonylization do not appear to play a role in cell death induced by H2O2. This may imply
that ROS due to H2O2 application by themselves may provide a signal to induce cell death. We
suggest that ROS may activate the TSPO, which in turn may increase the opening time of the
MPTP. TSPO is considered to be an oxygen sensor, and to be activated by oxidative stress
(Yeliseev et al., 1997). We suggest the possibility that TSPO may also function as a sensor for
oxidative stress, and consequently be involved in activation of cell death mechanisms due to
ROS. It has been reported that TSPO may be involved in apoptotic and necrotic cell death of
RPTEC cells due to activation by H2O2 (Kunuzova et al., 2004).
To study whether oxidative stress may activate TSPO in our paradigm, we wanted to find
whether oxidative stress, as induced by H2O2, would have an effect on TSPO expression. In
our study, TSPO binding appeared to increase in response to H 2O2 treatment. While such
changes induced by the SE protocol were insignificant, with the LE protocol dramatic and
significant increases in TSPO binding were observed (Figure 31, p > 0.01). We assume that
the differences seen between the SE and LE protocols regarding TSPO binding are due to the
recovery time provided with the LE protocol, as discussed above in relation to UVC exposure.
Protein translation could be accomplished during this recovery time, and TSPO responses
thereby would become noticeable.

Since our study suggests that TSPO appears to be involved in cell death induced by H 2O2
treatment, including collapse of the Δψm, we considered the possibility that such TSPO
involvement could be regulated by pharmacological agents. Li et al. (2007) have reported that
the TSPO ligand Ro5-4864 can maintain mitochondrial cytochrome c content and also the Δψm,
both of which otherwise decrease during oxidative stress (Li et al., 2007). Also the TSPO
ligand, SSR180575, significantly decreased post-reperfusion oxidative stress and tubular
apoptosis and necrosis in the kidney (Kunduzova et al., 2004). In vitro, the protective effect of
TSPO ligands on a model of H2O2-induced oxidative stress on human lymphoblastoid cell line
U937 was also shown (Bono et al., 1999). Thus, it appears to be possible to affect TSPO with
pharmacological means and thereby protect the mitochondria from dysfunction due oxidative
stress, and prevent collapse of the mitochondrial potential that may lead to cell death.
Based on our study, and this information from the literature, we applied the TSPO ligand
PK 11195. However, we did not see a protective effect of PK 11195 on cell death induced by
H2O2. This, we believe, was caused by a technical problem, since the level of cell death
induced in this particular experiment (96%) was much higher than otherwise induced by the
same concentration of H2O2 used in our study (45%). At present we cannot decide, which is
the exact cause of the apparent enhanced effect of H2O2 in this last experiment (Figure 32).
Thus, it is advisable to repeat this experiment.

Table 3. Effects of H2O2 exposure.

Lipid's and
TSPO Protein's Antioxidant effect
ROS induction Δψm Apoptosis Necrosis
expression oxidative (GSH)

H2O2 SE No effect Decrease Decrease Increase Not available Protects against cell death

H2O2 LE increase Not available Decrease Increase No effect Protects against cell death

In conclusions, our results indicate that oxidative stress as induced by H2O2 can increase
TSPO levels. This may lead to a collapse of the mitochondrial membrane potential. In turn
this may lead to cell death. The majority of cell death appeared to be necrotic. We suggest that
in our paradigm the H2O2 exposure led to extended opening of the MPTP, potentially induced
by TSPO activation, and subsequently leading to necrotic cell death. The H 2O2 exposure did
not appear to affect lipid peroxidation and protein carbonylization. However, application of the
antioxidant GSH indicated that the effects of H2O2 exposure on cell death were due to

generation of ROS. We suggest that the ROS may be activators of the TSPO, which in turn
may lead to a collapse of the mitochondrial membrane potential leading to cell death.


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