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Two roads diverged in a wood, and I -
I took the one less travelled by,
And that has made all the diff erence

-Robert Frost-

To those who love me

Supervisor
Professor Eva B Brittebo

Co-supervisor
Professor Nils Gunnar Lindquist
Doctor Anne-Lie Svensson

Faculty opponent
Professor Hans Tjälve

Members of the examining board

Professor Per Eriksson
Professor Margaretha Jägerstad
Doctor Leif Busk, Head of the Reasearch & Development Department of the
Swedish National Food Administration
PAPERS DISCUSSED

I Östergren A, Annas A, Skog K, Lindquist N G, Brittebo E B, Long-

term retention of `-carbolines in brain neuromelanin, Journal of Neural
Transmission (2004), 111: 141-157

II Östergren A, Fredriksson A, Brittebo E B, Norharman-induced motoric

impairment in mice: Neurodegeneration and glial activation in substan-
tia nigra, Journal of Neural Transmission (2005), In press

III Östergren A, Svensson A-L, Lindquist N G, Brittebo E B, Dopamine

melanin-loaded PC12 cells: A model for studies on pigmented neurons
of substantia nigra, Pigment Cell Research (2005), 18: 306-314

IV Östergren A, Lindquist N G, Brittebo E, Effects of dopamine melanin

on norharman-induced toxicity in PC12 cells, Manuscript

V Granberg L, Östergren A, Brandt I, Brittebo E B, CYP1A1 and CYP1B1

in blood-brain interfaces: CYP1A1-dependent bioactivation of 7,12-
dimethylbenz[a]anthracene in endothelial cells, Drug Metabolism and
Disposition (2003), 31: 259-265

Reprints were made with kind permissions from the publishers.

CONTENTS

INTRODUCTION ...................................................................................... 11
T MPTP- ................................................................................................12
`- .......................................................................................................13
Formation and occurrence .................................................................................13
Tissue distribution and levels .............................................................................14
Metabolism and bioactivation ...........................................................................14
In vivo eff ects ....................................................................................................15
In vitro eff ects ...................................................................................................16
N ....................................................................................................17
Melanogenesis and occurrence of melanin ...........................................................17
Melanin affinity................................................................................................19
N E P P ..............................................20
Mitochondrial dysfunction and oxidative stress .................................................. 20
ER stress .......................................................................................................... 20
Activation of glia cells........................................................................................21
Necrosis versus apoptosis ....................................................................................21
B-B I ...................................................................................23
Endothelial cells ................................................................................................23
7,12-Dimethylbenz[a]anthracene ......................................................................23
AIMS ............................................................................................................. 25
METHODS .................................................................................................. 26
RESULTS AND DISCUSSION ................................................................... 27
M-   `- ......................................27
N-     ...................................................29
N-       ......................33
I    -  ..............................35
N-     -
PC12  ...........................................................................................................38
Morphological changes .......................................................................................38
Mitochondrial activity ......................................................................................39
Oxidative stress .................................................................................................40
ER stress ...........................................................................................................40
Apoptosis ..........................................................................................................41
Necrosis ............................................................................................................43
CYP1-  ........................................................................ 44
`-carbolines ......................................................................................................45
7,12-Dimethylbenz[a]anthracene ......................................................................45
CONCLUDING REMARKS ....................................................................... 49
ACKNOWLEDGEMENTS ......................................................................... 51
REFERENCES ............................................................................................. 53
ABBREVIATIONS

ATP Adenosine 5’-triphosphate

Bmax Binding maximum
BNF ß-Naphthoflavone
CSF Cerebrospinal fluid
CYP Cytochrome P450
DAT Dopamine transporter
DMBA 7,12-Dimethylbenz[a]anthracene
DMSO Dimethylsulfoxide
EC Endothelial cells
ER Endoplasmic reticulum
GFAP Glial fibrillary acidic protein
Grp78 Glucose regulating protein 78
Harman 1-Methyl-9H-pyrido[3,4-b]-indole
HCA Heterocyclic amines
HUVEC Human umbilical vein endothelial cells
i.p. Intraperitoneal
i.v. Intravenous
Kd Equilibrium constant of dissociation
MPP+ 1-Methyl-4-phenylpyridinium ion
MPTP 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine
MTT 3-(4,5-dimethylthioazol-2-yl)2,5-diphenyl-tetrazolium
bromide
NGF Nerve growth factor
Norharman 9H-Pyrido[3,4-b]-indole
n.s. No statistical difference
6-OHDA 6-Hydroxydopamine
PC12 Cell line from rat pheochromocytoma
PCB126 3,3’,4,4’,5-Pentachlorobiphenyl
PD Parkinson’s disease
PAH Polycyclic aromatic hydrocarbon
ROS Reactive oxygen species
SMC Smooth muscle cells
s.c. Subcutaneous
TH Tyrosine hydroxylase
TNF Tumour necrosis factor
TUNEL Terminal deoxynucleotide transferase mediated deoxyuridine
triphosphate nick end labelling
UPR Unfolded protein response
 Role of Neuromelanin and Cytochrome P450 for Toxicity

INTRODUCTION

It is likely that several disorders of the brain such as parkinsonism and stroke
are results from multiple events and interactive mechanisms. These may include
genetic predisposition as well as environmental factors. Pathological mutations have
been identified in some genes (Ross and Farrer 2005). Nevertheless, twin studies
provide only a limited support for a genetic aetiology, but demonstrate a substantial
environmental component in most late onset cases of Parkinson’s disease (PD)
(Tanner et al. 1999). The risk factors for parkinsonism that have been identified
in epidemiological studies include well-water drinking, rural living and exposure
to pesticides, herbicides, industrial chemicals and metals such as manganese (as
reviewed by Di Monte et al. 2002).
One of the most commonly used model neurotoxicant is 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP) known for its ability to induce parkinsonism
in humans, primates and some laboratory animals (Langston et al. 1983; Heikkila
and Sonsalla 1992). Since MPTP is not normally present in the environment,
other compounds with structural resemblance to MPTP have been suggested to
be contributing factors for development of PD, among these compounds are some
heterocyclic amines (HCA) including the subgroup of `-carbolines. Other HCA
(i.e. a-carbolines) and some polycyclic aromatic hydrocarbons (PAHs) have been
identified for their ability to form adducts in blood vessels after bioactivation by
xenobiotic-metabolizing cytochrome P450 (CYP) enzymes (Annas and Brittebo
1998; Zhang et al. 1998; Granberg et al. 2000). Therefore it has been suggested
that blood vessels may be a target of PAH-induced toxicity, which is supported by
epidemiological studies indicating that exposure to PAHs could be of importance
for development of atherosclerosis and stroke (Hansen 1990; Bonita et al. 1999).
Despite that, there is limited knowledge about the uptake, biotransformation and
effects of HCA and PAHs in the brain.

11
Anna Östergren

T MPTP-

MPTP is one of the most commonly used model neurotoxicants for induction
of parkinsonism. In humans and primates low doses of MPTP cause irreversible
parkinsonism (Langston et al. 1983; D’Amato et al. 1987), whereas higher doses are
needed in rodents where the effects are often reversible (Petroske et al. 2001).
MPTP was first synthesized by Ziering and co-workers in 1947. It was ironically
examined clinically as a treatment for PD and depression (Lewin 1984). It was not
until 1982 when a large group of students in California developed symptoms of PD
within days after administration of MPTP-contaminated drugs that MPTP was
identified as a parkinsonism-inducing agent (Langston et al. 1983). Neuropathological
studies of some of the students revealed depletion of neuromelanin-containing
neurons in the substantia nigra and in one case large amounts of extraneuronal
neuromelanin was found (Langston et al. 1999). Gliosis and clustering of microglia
around neurons were also observed but no Lewy bodies.
After systemic administration, MPTP readily crosses the blood-brain barrier,
and is converted to a 1-methyl-4-phenylpyridinium ion (MPP+) by monoamine
oxidase B in astrocytes. MPP+ is then selectively accumulated in neuromelanin-
containing dopaminergic neurons of substantia nigra, where it inhibits the complex
I (ubiquinone oxidoreductase) of the mitochondrial respiratory chain. Cell death
results from a complex interplay among various phenomena such as impairment
of adenosine 5’-triphosphate (ATP)-production and oxidative stress (as reviewed
by Przedborski and Jackson-Lewis 1998). The neurons that are affected by MPTP
are preferentially dopaminergic neurons that contain neuromelanin (Herrero et al.
1993). MPTP has also been shown to have affinity for melanin and this has been
proposed to be a mechanism behind the selective vulnerability of the neuromelanin-
containing neurons (Lydén et al. 1983; Sokolowski et al. 1990). Pre-treatment with
chloroquine, that could block binding sites for MPTP on the neuromelanin, inhibits
MPTP-induced toxicity in primates, suggesting that the MPTP binding to melanin
is of importance for the ability of MPTP to cause neurotoxicity (D’Amato et al.
1987).
MPTP is not normally present in the environment but it has been suggested that
other compounds that are present in our environment may act in a way similar to
MPTP and thereby contribute to the development of PD. Particular interest has been
drawn to compounds that structurally resemble MPTP, such as the `-carbolines.

12
 Role of Neuromelanin and Cytochrome P450 for Toxicity

`-

Formation and occurrence

The `-carbolines norharman (9H-pyrido[3,4-b]-indole) and harman (1-methyl-
9H-pyrido[3,4-b]-indole) are formed by heating of amino acids such as tryptophan
and tryptamine during cooking of proteinaceous materials (Johansson et al. 1995;
Felton et al. 2000).

Figure 1. Chemical structures of the `-carbolines norharman (9H-pyrido[3,4-b]-indole) and

harman (1-methyl-9H-pyrido[3,4-b]-indole).

The `-carbolines are present in coffee, tobacco smoke and alcoholic beverages.
For an overview of the levels of norharman and harman in food products, see table
1. Certain plants can also biosynthesize norharman and harman (as reviewed by
Rommelspacher and Susilo 1985; Bourke et al. 1992).

Table 1
Levels of norharman and harman in food products and cigarette smoke
Product Levels of Levels of Reference
norharman harman
(ng/g or ng/ml) (ng/g or ng/ml)

Beef 1.2-795.0 3.6-169.0 Totsuka et al. 1999

Pork 2.4-59.6 0.6-32.5 Totsuka et al. 1999
Chicken 0.1-6.9 0.3-7.5 Solyakov and Skog 2002
Fish 1.3-40.7 2.0-8.4 Herraiz 2000
Pan residue 52.6 14.0 Solyakov et al. 1999
Wine 0.3-0.7 0-22.5 Adachi et al. 2000
Beer 2.7-22.7 0.7-5.4 Rommelspacher et al. 1994
Coffee 450-91400 340-22380 Herraiz 2002
Cigarette smoke 133000-623000 478000- Totsuka et al. 1999
condensate 849000

13
Anna Östergren

In mammals norharman can also be biosynthesized endogenously with tryptamine

or tryptophan as precursors (Rommelspacher 1981; Fekkes et al. 2001a). Fekkes and
co-workers (2001a) reported that ingestion of tryptophan resulted in a low increase
of the plasma concentration of norharman, but concluded that the main norharman
content in the body came from exogenous sources.

Tissue distribution and levels

In rats the normal concentration of norharman is reported to be 0.0011 ± 0.0003 µM
in plasma and 12.3-22.5 ng/g tissue in heart, brain, liver, lung, spleen and kidney
(Fekkes and Bode 1993). After an intraperitoneal (i.p.) injection of norharman (2
mg/kg) the concentrations are increased in all the above tissues with the highest
level in spleen, followed by liver, brain, kidney, lung and heart. The level in the brain
after administration of norharman was more than four times higher than that in
plasma (Fekkes and Bode 1993).
Several binding sites for norharman have been identified in rat brain, including
substantia nigra (Pawlik et al. 1990). Since norharman also bound to nucleus
accumbens it was suggested that norharman was able to influence the dopamine
reward system and Baum and co-workers (1995) reported that injections of
norharman in rat caused an increased concentrations of dopamine in this brain
region. Norharman is also a potent ligand for benzodiazepine binding sites (Müller
et al. 1981; Rommelspacher et al. 1981).
In post-mortem brain from humans without signs of neurodegeneration, the
concentration of norharman and harman is significantly higher in the substantia
nigra (3.28 ± 1.64 respectively 0.21 ± 0.02 ng/g tissue) than in cortex (0.12 ± 0.02
respectively 0.05 ± 0.01 ng/g tissue) (Matsubara et al. 1993).
The levels of norharman and/or harman in humans are increased in some disorders.
For example chronic alcoholics have increased levels of norharman in plasma and
urine (Spies et al. 1995), although the results from other studies indicate that smoking
might be the main cause of the increased levels of `-carbolines in alcoholics (Breyer-
Pfaff et al. 1996; Spijkerman et al. 2002). The levels of norharman and harman have
also been reported to be increased in plasma and lumbar cerebrospinal fluid (CSF)
from individuals with PD (Kuhn et al. 1995; Matsubara et al. 1995) and patients
with essential tremors have increased plasma levels of harman (Louis et al. 2002).
High affinity binding sites for norharman and harman have also been identified
in the liver where norharman binds to the microsomal membrane fraction of rat
liver membranes while harman binds to mitochondrial membranes (May et al.
1994; Stawowy et al. 1999).

Metabolism and bioactivation

Norharman is mainly metabolized in the liver, where its half-life is 20 minutes
(Fekkes and Bode 1993). It has also been reported that norharman binding in
rat liver microsomes can be inhibited by CYP2E1 ligands and indole-3-carbinol

14
 Role of Neuromelanin and Cytochrome P450 for Toxicity

(Stawowy et al. 1999). Furthermore, norharman has been demonstrated to inhibit

the metabolism of the CYP1 substrate benzo[a]pyrene in rat liver microsomes
(Fujino et al. 1978). Norharman and harman are also reported to be methylated
by S-adenosyl-L-methionine-dependent N-methyltransferase on both the pyridyl
(2N-) and indole (9N-) nitrogens, forming a metabolite that resembles MPP+, as
shown in figure 2 (Gearhart et al. 1997). In the brain the N-methyltransferase
activity is normally highest in substantia nigra and this enzyme is increased in
post-mortem brains from PD-patients (Gearhart et al. 2000). As for the parental
compound norharman, the concentrations of the 2,9N-methylated norharman ions
are higher in substantia nigra than in cortex (Matsubara et al. 1993) and the levels
are also elevated in CSF of PD-patients (Matsubara et al. 1995).

Figure 2. Metabolic bioactivation of norharman leading to the 2,9N-methylated metabolite.

In vivo effects

Humans
Plants containing harmala alkaloids such as `-carbolines have been used as spices
and hallucinogens (reviewed by Rommelspacher 1981). After ingestion of these
plants humans fall into a contemplative mood accompanied with vivid fantasies.
Intake of higher doses leads to the sensations of flying or being transformed into a
bird (reviewed by Rommelspacher 1981). The doses of norharman in these reports
are, however, difficult to evaluate. In the pharmacokinetic study by Fekkes and co-
workers (2001b) neither a peroral administration of 7 or 65 µg/kg nor a sublingual
administration of 6.5 µg/kg caused any behavioural alterations.

Experimental animals
Several studies have reported that norharman and harman alter the behaviour
in rodents. As presented in table 2, the initial effects of norharman are mainly
convulsions and impairment of motor activity. Similar effects have also been
reported after administration of harman (Rommelspacher et al. 1981) and after
administration of 2N- and 9N-methylated norharman (Matsubara et al. 1998a).
Furthermore, norharman and harman (up to 54 mg/kg) are reported to induce
behavioural changes such as hypomotility, head tremor, limb paresis and mild
sedation in sheep (Bourke et al. 1992). All these behavioural studies have been
performed shortly after administration. In behavioural studies performed soon after

15
Anna Östergren

the last treatment it may be difficult to distinguish between persistent changes in

motor activity and temporal inactivity resulting from acute effects due to receptor
interactions. Additionally, the handling during administration of the neurotoxicant
may be stressful to the animal and influence the motor performance.

Table 2
Norharman-induced behavioural effects in rodents
Effects Dose Time Administration Reference
(mg/kg) after route
administra-
tion

Convulsions 24.5 0-1 hours i.v. Rommelspacher

et al. 1981
Dishminished move- 100 0-1 hours i.p. Fekkes and Bode
ments, exploratory 1993
behaviour, loss of
rightening reflexes,
total muscle relaxation
Bradykinesia 84x2 for 2 days i.p. Matsubara et al.
7 days 1998a
intravenous (i.v.)

Norharman, 2N-, 2,9N-methylated norharman and 2,9N-methylated harman

have furthermore been reported to reduce the striatal levels of dopamine after
direct injection into substantia nigra in rats. Nigral cell loss was also observed after
the intranigral administration (Neafsey et al. 1995). Reduction of dopamine in
striatum is also reported in mice after i.p. injections of norharman and its 2N- and
9N-methylated metabolites where the dopamine decrease was combined with loss
of the number of tyrosine hydroxylase (TH)-positive neurons in substantia nigra
(Matsubara et al. 1998a).
Selective uptake by dopamine transporters (DAT) has been suggested as a
mechanism behind selective toxicity on dopaminergic neurons. Norharman
and harman are, however, unfavourable substrates for the DAT whereas their
N-methylated metabolites could use DAT for uptake in dopaminergic neurons
(Drucker et al. 1990; Matsubara et al. 1998b).

In vitro effects
Exposure to norharman or harman (250-500 µM) for three hours has been reported
to cause DNA damage in a cell line from human neuroblastoma (SH-SY5Y cells)
(Uezono et al. 2001).

16
 Role of Neuromelanin and Cytochrome P450 for Toxicity

The 2,9N-methylated metabolites of norharman (500 µM) and both 2N- and
2,9N-methylated metabolites of harman (500 µM) have been reported to cause
increased lactate dehydrogenase release in a cell line from rat pheochromocytoma
(PC12 cells) after 48 hours of exposure (Collins et al. 1992; Cobuzzi et al. 1994). The
2,9N-methylated norharman metabolite has also been reported to have a selective
effect on dopaminergic neurons in primary mesencephalic cell cultures from rat.
After incubation with norharman (1 µM) for two days there was a 80% decrease in
the number of TH-positive neurons, whereas the concentration had to be increased
to more than 10 µM to affect TH-negative neurons (Matsubara et al. 1998a).
Metabolites of norharman and harman have been suggested to cause their effects
by inhibition of the mitochondrial respiratory chain, although only the 2,9N-
methylated norharman and harman are mitochondrial inhibitors whereas the parent
compounds and the 2N-methylated metabolites only display weak effects (Albores
et al. 1990; Collins et al. 1992).

N
“The neuromelanin granule may be the secret key to the understand-
ing of Parkinsonism. I don’t believe God put the melanin in the central
nervous system for nothing. It must be doing something. Something
big…”
G C Cotzias

Melanogenesis and occurrence of melanin

The origin of the name melanin, from the Greek word melanos (dark), is usually
attributed to the Swedish chemist Berzelius (reviewed by Fedorow et al. 2005).
Melanin is an extremely dense and insoluble chromophore of high molecular mass
(reviewed by Sarna 1992; Usunoff et al. 2002). The melanin granules are mixtures of
more or less similar polymers linked through heterogenous non-hydrolysable bonds
where covalent bonds are the most common (Bridelli et al. 1999).
In humans, melanin is formed in melanocytes, pigment epithelial cells and in
neurons of substantia nigra and locus coeruleus. The melanin in melanocytes and
epithelial cells is produced from L-tyrosine under the influence of tyrosinase in
melanosomes in the basal layer of the epidermis of the skin, the retinal pigment
epithelium and uveal tract of the eye, the hair matrix, the inner ear, in mucus
membranes and in leptomeninges of the central nervous system (as reviewed by
Sarna 1992; Fedorow et al. 2005).
In general, melanin has been suggested to protect tissues and cells. For example,
the main action of melanin in human skin is to absorb UV-light and attenuate UV-
penetration (reviewed by Fedorow et al. 2005). Melanin can also scavenge reactive
oxygen species (ROS) such as superoxide anions and hydrogen peroxide, formed
during biochemical and photochemical reactions (Sarna 1992). Morphologically, the

17
Anna Östergren

distribution of melanin gives further support for a protective role. Melanin has been
suggested to act as a filter that prevents noxious chemicals from reaching receptor
cells. This is supported by the localization of melanocytes in close proximity around
capillaries and that nutrients for the receptor cells in the eye and in the inner ear are
passing through the pigmented epithelia (Lindquist and Ullberg 1975).
In contrast to melanin in melanocytes and epithelia, neuromelanin is thought
to be formed by oxidative polymerization of dopamine or noradrenaline, with the
possible involvement of cysteinyl-derivatives (as reviewed by Usunoff et al. 2002;
Zucca et al. 2004). Neuromelanin is a granular, dark brown pigment, which is almost
exclusively found in dopaminergic neurons of substantia nigra and noradrenergic
neurons of locus coeruleus (Bazelon et al. 1967; Bogerts 1981). In heavily pigmented
neurons, neuromelanin granules are closely packed in clusters that are mainly found
in the axon hillock and initial portion of the axon (figure 3). Neuromelanin is less
abundant in the dendrites and is absent in the neuronal processes remote from the
cell bodies, the synaptic nerve endings and the glial cells (Fedorow et al. 2005).
Also the distribution of neuromelanin suggests that it has a protective role, since it
could protect the nucleus from catecholamines and their metabolites. The synthesis
of neuromelanin itself has been proposed to represent a mechanism where highly
reactive compounds such as quinones and semiquinones originating from excess of
cytosolic catecholamines are incorporated into inert neuromelanin polymers (Sulzer
et al. 2000; Zecca et al. 2003).

Figure 3. Schematic drawing of a neuromelanin-containing neuron with nucleus (n) and neu-
romelanin (nm) in the axon hillock and in the initial portion of the axon.

During life there is a gradual accumulation of neuromelanin in the dopaminergic

neurons with a maximum around 60 years of age, followed by a decline (Mann
and Yates 1983; Zecca et al. 2002a). The reason for the decrease in neuromelanin
with age is not clear although it could depend on a selective loss of dopaminergic
neurons.
A more pronounced selective loss of neuromelanin has been observed in
parkinsonism, as best described in patients with PD where the level of neuromelanin
is less than 50% of that seen in age matched controls (Zecca et al. 2002a). The
general view is that neuromelanin-containing neurons are the most vulnerable to
degeneration in parkinsonism (Mann and Yates 1983; Hirsch et al. 1988). Some
studies have indicated that the level of neuromelanin may also decrease in surviving
neurons (Mann and Yates 1983; Kastner et al. 1992).
After a cell loss of 80% of the dopaminergic neurons, the nigrostriatal pathway

18
 Role of Neuromelanin and Cytochrome P450 for Toxicity

is disrupted and symptoms of parkinsonism become appearant. The most noticeable

symptom in parkinsonism is impaired motor performance with symptoms such as
bradykinesia, akinesia, loss of postural reflexes, tremor at rest, rigidity and involuntary
movements (Fahn 2003). The diagnosis parkinsonism is defined by the presence of
two of the above motor symptoms, and at least one of them has to be tremor at rest
or bradykinesia (Fahn 2003). The most common form of parkinsonism is PD which
aetiology is incompletely understood.

Melanin affinity
Melanin contains a large number of free carboxylic acid residues and semiquinones,
which are responsible for the cation exchange properties observed for melanin and
are thought to provide most of the ionic binding sites. There are also other types of
melanin binding including hydrogen bonds and van der Waal’s attractions (Larsson
and Tjälve 1979).
In vivo there is an accumulation of different compounds to melanin. In studies
where neuromelanin has been analysed, several metals such as iron, zinc, copper,
manganese, chromium, cobalt, mercury, lead and cadmium were detected in the
polymer (Zecca et al. 2002b). Other compounds that has been reported to bind
to melanin include calcium (Liu et al. 2004), PAHs such as 7,12-dimethylbenz[a]-
anthracene (DMBA) and benzo[a]pyrene (Roberto et al. 1996), neurotoxicants such
as MPTP/MPP+ (Lyden et al. 1985; D’Amato et al. 1986), pesticides like paraquat
(Larsson and Tjälve 1979; Lindquist et al. 1988) and drugs such as chlorpromazine
and chloroquine (Lindquist 1973; Larsson and Tjälve 1979).
The binding of toxicants to melanin can probably protect tissues against toxic
insults after occasional exposures. The binding is, however, normally slowly
reversible and can furthermore be inhibited by pre-exposure of other compounds
with higher melanin affinity (Larsson and Tjälve 1979). Thereby melanin may serve
as a depot from which stored toxicants may be slowly released into the cytosol and
trigger oxidative stress or induce cell death. For example, long-term treatment with
chloroquine has been reported to cause oculotoxicity (Lindquist 1973; Dayhaw-
Barker 2002).
Neuromelanin can also act as a pro-oxidant, stimulating the production of
ROS due to its loading of metals and interaction with the cellular environment.
For example, when high concentrations of iron are bound to neuromelanin its
high-affinity binding sites are saturated. The excess metal binds to low-affinity iron
binding sites where ferrous iron may remain redox-active or alternatively be released
and stimulate hydroxyl radical production through the Fenton reaction (Youdim et
al. 1989; Faucheux et al. 2003). This is supported by results demonstrating that in
vitro incubations of iron with human neuromelanin or dopamine melanin stimulate
rather than decrease oxidative tissue damage (Mochizuki et al. 1993; Double et al.
1999).
On the other hand the melanin binding could also scavenge hydroxyl radicals
(Korytowski et al. 1995) and cells with melanin are protected against hydrogen

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Anna Östergren

peroxide-induced toxicity (Hoogduijn et al. 2004). This protection may not only
be limited to an antioxidant defence but may also be connected to the ability of
melanin to bind to calcium since exposure to hydrogen peroxide and other ROS has
been shown to increase the levels of intracellular calcium (Hoogduijn et al. 2004).

N   

Mitochondrial dysfunction and oxidative stress

Several neurotoxicants including MPTP and 6-hydroxydopamine (6-OHDA) induce
mitochondrial impairment by inhibiting mitochondrial complex I (as reviewed by
Schober 2004). These findings imply that inhibition of the mitochondrial respiratory
chain may cause a selective loss of dopaminergic neurons in the substantia nigra. A
selective decrease (30-40%) in activity of the mitochondrial respiratory chains from
substantia nigra of post-mortem brains from PD-patients compared to matched
patients without neurological or psychiatric diseases has also been reported (Schapira
et al. 1990).
Mitochondrial dysfunction often results in oxidative stress and MPTP, 6-OHDA
and rotenone produce ROS by inhibiting the mitochondrial respiratory chain.
The oxidative stress can damage the mitochondria and thereby induce a release
of proapoptotic factors such as cytochrome c that triggers caspase-9 (Liang et al.
2004). Oxidative stress has also been reported to activate the Fas ligand, which
in turn can activate the caspase-8/caspase-3 pathway. Furthermore, mitochondrial
ROS generation may promote calcium release from the endoplasmic reticulum (ER),
resulting in disturbed calcium homeostasis that may further promote production of
ROS (Jacobson and Duchen 2002; Gibson and Huang 2004).
Dopaminergic neurons have been reported to be particularly sensitive to oxidative
stress and some reports have suggested that this sensitivity is due to the fact that
dopamine easily transforms into dopamine adducts and quinones (Weingarten and
Zhou 2001). Others have suggested that neuromelanin has a prominent role for the
sensitivity since the melanin has an iron-content, which is suggested to promote
oxidative stress through the Fenton reaction (Youdim et al., 1989). However, in
humans it has been shown that when the neuromelanin content is reduced, the
cells become more susceptible to oxidative damages. Increased redox activity has for
example been detected in PD-patients (+69%) and was highest in patients with the
most severe neuronal loss (Faucheux et al. 2003).

ER stress
ER stress is triggered by the loss of calcium homeostasis or an accumulation of
unfolded/misfolded proteins in the ER lumen. ER stress induces a highly specific
“unfolded protein response” (UPR) (Kaufman 1999). Glucose regulating protein

20
 Role of Neuromelanin and Cytochrome P450 for Toxicity

78 (grp78) is considered to be a key regulator of ER stress transducers due to its

binding to the endoribonuclease, the serine/threonine kinase and the basic leucine-
zipper transcription factor in unstressed cells. During ER stress all three of these
transducers are released from grp78 and UPR target genes are activated (Lee 2005).
These reactions are attempts to reduce the load on the ER and prevent a subsequent
cellular damage. However, when ER stress is severe or long lasting there is an
induction of apoptosis.
ER stress is suggested to be a common denominator for neurodegenerative
diseases like PD and model neurotoxicants like MPTP/MPP+, 6-OHDA and
rotenone are reported cause ER stress by producing an increased amount of oxidized
proteins (Ryu et al. 2002; De Iuliis et al. 2005). ER stress may also contribute to the
inhibition of mitochondrial respiration by promoting cytochrome c release (Hori
et al. 2002). This indicates a mechanistic commonality of parkinsonism-inducing
neurotoxicants that is beyond their effect on the mitochondria.

Activation of glia cells

Astrocytes have important roles such as maintenance of ion homeostasis, provision
of energy substrate to neurons and uptake of neurotransmitters (Kandel et al. 1991).
Several studies have reported that astrocytes can confer neuronal protection through
the synthesis and release of glutathione (as reviewed by Teismann et al. 2003).
In response to any kind of injury in the central nervous system the astrocytes
become activated, proliferate and increase in size. The astrocytic processes also
become larger and more numerous and there is an increase of glial fibrillary acidic
protein (GFAP) content (Eng 1985; Pekny and Nilsson 2005). Activated astrocytes
are reported to produce inflammatory mediators such as tumour necrosis factor
(TNF)-_ (Johnstone et al. 1999), that can stimulate activation of astrocytes and
other glia cells and thereby amplify and promote the glial response and consequently
the glia-related injury to neurons. TNF-_ may also bind its receptor on dopaminergic
neurons and thereby trigger apoptotic signals (as reviewed by Teismann et al. 2003).
Furthermore, it has been proposed that astrocytes also can produce ROS and
nitric oxide and also thereby promote neuronal degeneration (Johnstone et al.
1999).

Necrosis versus apoptosis

Necrosis and apoptosis are two distinct forms of cell deaths. Necrosis is always a
pathological process where a large number of cells die. It is characterized by cellular
swelling due to loss of ionic homeostasis, damage to organelles and cell lysis.
The term apoptosis from the Greek term for “dropping off ” of leaves from a
healthy plant, was coined by Kerr and co-workers (Kerr et al. 1972). In apoptosis,
the plasma membrane stays intact while the nucleus fragmentizes, the chromatin
condensates and the cytoplasm shrink. Subsequently fragments of the cells are
packed into apoptotic bodies, which on their surface membranes express ligands

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Anna Östergren

that are recognized by phagocytes (Kerr et al. 1972). Thereby apoptosis may affect
only selective cells.
There are two pathways through which apoptosis is induced. One is the extrinsic
pathway that involves death receptors and is exemplified by Fas-mediated caspase-8
activation. The other is the intrinsic pathways that involves the mitochondrial and/
or the ER pathway that activate caspase-9 and caspase-12 respectively (Ferri and
Kroemer 2001; Ueda et al. 2002). As shown in the schematic overview of apoptosis
in figure 4, these pathways converge on caspase-3 activation resulting in nuclear
degradation and cellular morphological changes (Ferri and Kroemer 2001; Ueda
et al. 2002).
There are several reports of apoptosis in parkinsonism. In post-mortem brains
of PD-patients as well as in brains of MPTP- or 6-OHDA-treated rats the levels
of pro-apoptotic cytokines such as TNF-_ are increased while the levels of anti-
apototic neurotrophins are decreased (reviewed by Teismann et al. 2003; Nagatsu
and Sawada 2005).

Figure 4. Schematic overview of some apoptotic pathways.

Apoptosis induced by MPTP/MPP+ is considered to mainly be induced by events

in the mitochondria, including release of cytochrome c, caspase-9 and caspase-3
(Kaul et al. 2003). Apoptosis can also be preceded by ER stress that can be induced
by 6-OHDA (Ryu et al. 2002). ER stress can activate caspase-12, which in turn
can activate caspase-3 and/or caspase-9 (as reviewed by Groenendyk and Michalak
2005).

22
 Role of Neuromelanin and Cytochrome P450 for Toxicity

Although apoptosis is the preferential cell death after exposure to MPTP and
6-OHDA, high concentrations of these neurotoxicants induce necrosis (Hartley
et al. 1994). Since apoptosis is an energy dependent (ATP-requiring) process, the
deviation from apoptosis to necrosis may depend upon the lack of ATP (Eguchi
et al. 1999). This may explain why high concentrations of neuroxicants preferably
induce necrosis.

B- 

Endothelial cells
Xenobiotics are transported via the blood and have to pass the blood-brain barrier to
reach the brain. The blood-brain barrier consists of capillary endothelial cells (EC)
that are tightly joined together and glia cells that provide a mechanical and physical
support (Kandel et al. 1991).
Toxic damage to EC and to the vascular smooth muscle cells (SMC) surrounding
the blood vessels have been proposed as key events in the development of atherosclerosis
and stroke (Ross 1993).
Similar to other cell types, both cerebral EC and choroidal epithelial cells
contain a variety of drug metabolizing enzymes, including CYP enzymes.
Many are inducible and capable of converting xenobiotics to more water-soluble
species, thereby preventing them from entering the brain. The role of CYP1A1 in
endothelial linings of the brain has, however, not been clarified although studies
have shown a strong correlation between the induction of CYP1A1 activities in
EC and endothelial dysfunction such as impaired barrier function, oedema and
cardiovascular dysfunction (Guiney et al. 1997).

7,12-Dimethylbenz[a]anthracene

Formation and occurence

DMBA is a synthetic compound that often is used as model compound for PAHs.
PAHs are formed by the incomplete combustion or pyrolysis of organic material
during certain industrial processes such as work with machines that produce sparks,
combustion in engines and oil heating. Occupational exposures also occur during
work with coal tar, asphalt and chimney sweeping (as reviewed by Boffetta et al.
1997). Another major source for PAH exposure is tobacco smoke (Lodovici et al.
2004).

Tissue distribution
DMBA has been demonstrated to bind to the liver and to melanin in pigmented
mice (Roberto et al. 1996). In albino mice, DMBA binding was primarily observed

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Anna Östergren

in the liver (Granberg et al. 2000). After pre-treatment with the CYP1-inducer
ß-naphthoflavone (BNF) or 3,3’,4,4’,5-pentachlorobiphenyl (PCB126) there was,
however, a selective irreversible binding of radioactivity in EC of arteries and veins
in albino mice (Granberg et al. 2000).

Metabolism and bioactivation by CYP1 enzymes

CYP enzymes are heme-containing proteins that oxidize, hydrolyze or reduce
compounds. Based on the sequence similarity, CYP enzymes are categorized into
families and subfamilies. The mammalian CYP1 family consists of CYP1A1,
CYP1A2 and CYP1B1. CYP1A1 and CYP1B1 have been detected in various
extrahepatic tissues, whereas CYP1A2 is mainly localized in the liver (as reviewed
by Nebert et al. 2004). The CYP1 enzymes are regulated by the aryl hydrocarbon
receptor. Although the purpose of the enzymatic activity is to facilitate the
elimination of xenobiotics, CYP1 enzymes have also been reported to bioactivate
numerous PAHs including DMBA into reactive metabolites such as epoxides and
diolepoxides (figure 5), metabolites that can form DNA-, RNA- or protein adducts.
For DMBA, this bioactivation is primarily performed by CYP1A1/2 enzymes, but
also by CYP1B1 (Shimada et al. 1996).

Figure 5. Metabolic bioactivation of DMBA leading to the ultimate toxicants (+)-DMBA-4R,3S-

diol-2S,1R-epoxide.

Vascular effects
Epidemiological studies have reported an increased risk for stroke and atherosclerosis
among persons exposed to PAHs through direct and indirect tobacco smoking
(Bonita et al. 1999).
There is also evidence that PAHs can induce atherosclerotic lesions in experimental
animals (Penn 1990; Wakabayashi 1990) and induce apoptosis in human umbilical
venous endothelial cells (HUVEC) (Wang et al. 2001). The mechanism behind these
events are complex and not completely understood (Powell 1998). In concordance
with results from benzo[a]pyrene (Ramos 1999) it is likely that the electrophilic
dihydrodiols of DMBA bind covalently to bases of DNA, while mono-hydroxylated
metabolites of DMBA form quinones that can participate in redox cycles and thereby
generate ROS.

24
 Role of Neuromelanin and Cytochrome P450 for Toxicity

AIMS

The overall objective of this thesis has been to study the uptake and distribution
of `-carbolines and DMBA in the brain and to identify mechanisms for selective
retention and toxicity. Furthermore the potential of `-carbolines for induction of
parkinsonism was examined.

The specific aims of the present thesis were:

Paper I
To investigate the role of melanin for uptake and distribution of the `-carbolines
norharman and harman in the brain of mice and frogs, and to study melanin-
binding of these `-carbolines in vitro.

Paper II
To investigate if norharman can induce neurodegeneration in substantia nigra and/
or induce behavioural impairment in C57BL/6 mice.

Paper III
To develop an in vitro system of PC12 cells with and without melanin that could be
used for studies of the role of melanin in chemical-induced toxicity.

Paper IV
To compare the effects of norharman in conventional PC12 cells without melanin
and in melanin-loaded PC12 cells.

Paper V
To examine the expression of CYP1A1 and CYP1B1 in the brain and to investigate
the role of these enzymes for the distribution of DMBA in the brain.

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Anna Östergren

METHODS

The methods that have been used in this thesis are listed in table 3. The reader is
referred to “Material and Methods” in the specific papers for details.
Table 3
Methods used in this thesis
Method Paper(s)

Histological techniques
Tape-section autoradiography I and V
Light-microscopic autoradiography I and V
Fluoro-jade II
Immunohistochemistry I, II and V
Immunocytochemistry II, III and IV
Transmission electron microscopy III
Fontana-Masson silver staining III
Behavioural studies
Spontaneous motor activity II
Radial arm maze II
Morris water maze II
Cell viability assays
MTT-assay II, III and IV
Trypanblue exclusion assay II, III and IV
TUNEL-staining II
Western immunoblot III
Melanin-studies
Dopamine melanin synthesis I, II and IV
Melanin binding in vitro I
Melanin-loading of PC12 cells III and IV
Determination of oxidative stress
Carboxy-H2DCFDA oxidation IV
CYP1-activity assay
7-Ethoxyresorufin O-deethylase activity assay V

26
 Role of Neuromelanin and Cytochrome P450 for Toxicity

RESULTS AND DISCUSSION

M-   `-

In parkinsonism there is a selective degeneration of neuromelanin-containing
dopaminergic neurons. Earlier studies have demonstrated that norharman and
harman are unfavourable substrates for the DAT (Drucker et al. 1990; Matsubara
et al. 1998b), therefore it was of interest to study if other factors such as melanin-
binding could be of importance for a selective distribution of norharman and
harman in the brain.
As presented in paper I there was a selective localization of radioactivity in
pigmented tissues and in the liver of C57BL/6 mice injected with 3H-norharman or
3
H-harman (figure 6).

Figure 6. Autoradiograms of pigmented

C57BL/6 mice 30 minutes (A), four hours
(B), 24 hours (C) and five days (D) after an
i.v. injection with 3H-harman. A selective
localization of radioactivity (as demonstrated
by white areas) was observed in melanin-con-
taining tissues.

27
Anna Östergren

The high level of radioactivity remained in the melanin-containing tissues up

to five days after injections (the longest survival time examined), indicating both
a selective distribution and a long-time retention of radioactivity in melanin-
containing tissues such as the skin and the eyes. In albino NMRI mice no such
distribution or retention of `-carbolines was observed.
Both pigmented and albino mice are known to lack neuromelanin. In order to
examine whether neuromelanin can mediate an uptake in discrete brain regions,
the distribution of radioactivity after injection of 3H-norharman and 3H-harman
was also examined in frogs. Amphibians such as frogs are known to accommodate
neuromelanin. In this species there was a selective localization and retention of
radioactivity in pigmented parts of the brain still after 30 days post injection (the
longest survival time examined). The radioactivity in the pigmented brain regions
could not be removed by extensive extractions with organic solvents suggesting that
the `-carbolines were not bound to the lipid component of neuromelanin but to the
actual melanin. These results were confirmed by light-microscopic autoradiography,
which revealed that there was a distinct and selective distribution of silver grains in
pigmented neurons (figure 7).

Figure 7. Light-microscopic autoradiograms of frogs 30 days after an i.p. injection with 3H-norhar-
man (A) and 3H-harman (B). A selective binding of radioactivity (as demonstrated by black silver
grains) was observed in melanin-containing neurons.

Neuromelanin in the mammalian brain is strictly accumulated in the

catecholaminergic neurons, whilst the neuromelanin in frogs has a more general
distribution (reviewed by Usunoff et al. 2002). In paper I the frog melanin-containing
neurons with silver grains were also TH-positive in an immunohistochemical
staining. These results demonstrate that norharman and harman were bound to
dopaminergic melanin-containing neurons.
In post-mortem brains of humans the levels of norharman and harman are
reported to be higher in substantia nigra than in cortex (Matsubara et al. 1993).
The reason for this selective distribution may be melanin-binding, as demonstrated
in paper I.
In paper I both norharman and harman were also demonstrated to bind to
dopamine melanin in vitro. Scatchard plots revealed two binding sites for norharman
respectively harman, with binding parameters presented in table 4.

28
 Role of Neuromelanin and Cytochrome P450 for Toxicity

Table 4
Binding of `-carbolines to dopamine melanin in vitro
Norharman Harman

Kd high affinity site (µM) 5.0 3.2

Kd low affinity site (µM) 1057.0 2364.0
Bmax high affinity site (µmol/mg melanin) 1.6 0.8
Bmax low affinity site (µmol/mg melanin) 51.3 130.7
Equilibrium constant of dissociation (Kd), binding maximum (Bmax)

Dopamine melanin is often used as a model for neuromelanin since it exhibits

several structural similarities to this pigment (Youdim et al. 1994; Bridelli et al.
1999; Double et al. 2000), but although dopamine melanin is the main precursor of
neuromelanin the latter also contains a peptide component (eg cystein) and metals
such as iron (Zecca et al. 1992,Zecca et al. 1994; Zucca et al. 2004). The binding
of norharman and harman to dopamine melanin was in the same range as MPTP,
which is reported to have three binding sites with Kd-values of 1.7 µM, 82.0 µM and
1302.1 µM (Lydén et al. 1983).
Norharman and harman were also demonstrated to bind to melanin granules
from Sepia officinalis. The binding to the melanin granules was in the same range
as other compounds with well-known melanin affinity, for example chloroquine
(Larsson and Tjälve 1979).
The present results suggest that neuromelanin is an intracellular binder of
norharman and harman. The binding of toxicants to neuromelanin has usually
been considered as a way of protection. A progressive release of toxicants that have
been accumulated on the neuromelanin granules may, however, lead to a sustained
exposure of these compounds at this site.

N-    

Neuromelanin-containing neurons of the substantia nigra are selective targets for
neurotoxicants like MPTP (Herrero et al. 1993). High doses of norharman (>100
mg/kg twice daily for 7 days) have been reported to induce neurodegeneration in
this specific cell population (Matsubara et al. 1998a). In paper II lower doses (3
mg/kg or 10 mg/kg) were injected subcutaneuous (s.c.) twice daily for five days.
Eighteen hours after the last injection the animals were killed and the brains were
subjected to immunohistochemical staining against TH (figure 8).

29
Anna Östergren

Figure 8. Immunohistochemical staining against TH in sections of substantia nigra from C57BL/6

mice injected s.c. with vehicle (A), 3 mg/kg norharman (B) or 10 mg/kg norharman (C) twice daily
for five consecutive days and thereafter killed 18 hours after the last injection.

The number of TH-positive neurons was 95.6 ± 28.3% (3 mg/kg), and 99.0 ±
42.3% (10 mg/kg) of the vehicle-treated control. The lack of norharman-induced
effect on the number of TH-positive neurons is in concordance with some reports
on subacute and acute treatment with MPTP (Petroske et al. 2001). Although
several reports demonstrate an acute loss of TH-positive neurons in substantia nigra
pars compacta after MPTP-treatment (as reviewed by Schober 2004), others report
that the number of TH-positive neurons do not change during the first month post
MPTP lesioning (Jakowec et al. 2004).
Fluoro-jade was used as another histological marker for neurotoxicity. Fluoro-
jade is an anionic fluorescein derivative that can be used for localization of neuronal
degeneration in brain tissue sections. Detection of neuronal damage using fluoro-
jade has been validated by a variety of well-characterized neurotoxicants, such as
MPTP (Schmued et al. 1997; Schmued and Hopkins 2000). Degenerating neurons
stained with fluoro-jade appear bright yellow-green against a dark background
(Schmued et al. 1997; Schmued and Hopkins 2000).
In paper II the fluoro-jade staining revealed that repeated norharman treatment
induced degenerative changes in the substantia nigra (figure 9).
Since fluoro-jade stains all types of degenerating neurons and glial cells (Schmued
and Hopkins 2000), the norharman-induced degeneration in substantia nigra could
be related to changes both in neurons and glia cells. Glia cells are activated during
all types of damage in the central nervous system and activated astrocytes synthesize
increased amount of GFAP (Eng 1985; Pekny and Nilsson 2005). In paper II
norharman induced increased number of GFAP-stained astrocytes in substantia
nigra of C57BL/6 mice (figure 10).

30
 Role of Neuromelanin and Cytochrome P450 for Toxicity

Figure 9. Degeneration as demonstrated by fluoro-jade staining (green) in substantia nigra of

C57BL/6 mice injected s.c. with vehicle (A), 3 mg/kg norharman (B) or 10 mg/kg norharman (C)
twice daily for five consecutive days and thereafter killed 18 hours after the last injection. A semi-
quantative measurement of fluoro-jade stained area (D) where each point represent three sections
from one animal, n=5-6 animals/treatment, and the straight line indicates the mean value. Statisti-
cal analysis (ANOVA) indicated a significant difference between the groups (p<0.05). Post-hoc test-
ing (GraphPad QuickCalcs) indicated a higher value for animals receiving norharman (10 mg/kg)
compared to vehicle-treated animals, * p<0.05.

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Anna Östergren

Figure 10. Immunohistochemical staining against GFAP in sections of substantia nigra from
C57BL/6 mice injected s.c. with vehicle (A), 3 mg/kg norharman (B) or 10 mg/kg norharman (C)
twice daily for five consecutive days and thereafter killed 18 hours after the last injection. A semi-
quantiative measurement of GFAP stained area (D) where each value represent two to six sections
from one animal, n=2-3 animals/treatment, and the straight line indicates the mean value. Statisti-
cal analysis (ANOVA) indicated a significant difference between the groups, p<0.05. Post-hoc test-
ing (GraphPad QuickCalcs) indicated a higher value for the mice treated with 10 mg/kg norhar-
man compared to vehicle, *p<0.05.

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 Role of Neuromelanin and Cytochrome P450 for Toxicity

Astrocytes are considered to be able to protect neurons against fluctuations in

ion homeostasis and excess of released neurotransmitters under normal conditions
(Kandel et al. 1991). Nevertheless, all forms of brain injury induce activation of
astrocytes. When the astrocytes become reactive, their ability to carry out normal
support functions is reduced (Pekny and Nilsson 2005). Activation of astrocytes
is a common feature of both acute and chronic neurodegenerative conditions (as
reviewed by Teismann et al. 2003). For example can astrocyte activation be observed
after exposure to neurotoxicants such as MPTP and 6-OHDA (Strömberg et al.
1986). However, neither the process by which astrocytes become activated nor the
functional consequences of this activated phenotype are well understood, though it
has been proposed that activated astrocytes may be involved in oxidative stress and
apoptosis (as reviewed by Teismann et al. 2003).

N-     


Earlier studies have shown that norharman causes motor impairments in rodents
and sheep (Bourke et al. 1992; Fekkes and Bode 1993; Matsubara et al. 1998a).
All these studies were, however, performed shortly after the administration of
norharman. Considering that `-carbolines such as norharman have been proposed
as neurotoxicants contributing to parkinsonism it was of interest to examine if
norharman could cause sustained behavioural impairments such as hypoactivity
that is a noticeable finding in parkinsonism.
In paper II the behavioural study demonstrated that repeated injections of
norharman (10 mg/kg; s.c.) in C57BL/6 mice during five days caused a significant
decrease in motor activity two weeks later. This study included three different
parameters for measuring motor activity: total activity, locomotion and rearing. As
shown in figure 11 A, the highest dose of norharman (10 mg/kg) caused a reduction
of total activity in all measured time periods. Locomotion and rearing were decreased
in the first and second period, but not during the last 20 minutes (figure 11 B and
C).
A seeking behaviour is normally induced when mice are put into a new
environment. When the mice have examined the cage, a reduced motor activity can
be observed. Mice treated with norharman (10 mg/kg) were hypoactive compared
to control mice at all time points. However, in the group of norharman-treated mice
(10 mg/kg) no reduction of total activity and locomotion was observed between the
first and second 20-minute time period indicating that these mice might also have
reduced acclimatisation behaviour.
The behavioural study in paper II also included measurements on spatial learning
and memory, as evaluated by the radial arm maze and Morris swim maze. Spatial
learning and memory have been associated with the cholinergic system especially
in hippocampus. The key feature of spatial learning may be the need to integrate
information from multiple stimuli. Lesions of the hippocampal region disrupt

33
Anna Östergren

Figure 11. Spontaneous motor activity represented by total activity (A), locomotion (B) and rear-
ing (C) in C57BL/6 mice two weeks after s.c. injections of norharman or vehicle twice daily for five
consecutive days. The values represents mean ± SD, n=12 animals/treatment. Statistical analysis
(ANOVA) indicated significant difference between the groups, p<0.001 for total activity and loco-
motion, p<0.01 for rearing. Post-hoc testing (Tukey) indicated a reduced level of activity in norhar-
man-treated animals compared to vehicle-treated animals, **p<0.01, *p<0.05.

34
 Role of Neuromelanin and Cytochrome P450 for Toxicity

memories of visited arms in a radial arm maze. Mice could still learn to avoid arms
that were never baited. No norharman-induced changes were observed in these
mazes.
Since norharman only caused impairment of motor activity, but not of altered
learning or memory capacities, it is tempting to suggest that norharman could cause
a selective damage to brain areas of importance for motor activity.

I    - 

A major problem for the study of the significance of neuromelanin in development of
parkinsonism is that common experimental animals such as mouse, rat and guinea
pig lack neuromelanin (Marsden 1961; Barden and Levine 1983). Primates and
amphibians do have neuromelanin (Marsden 1961), but these species lack available
non-pigmented variants making comparative studies impossible.
PC12 cells are commonly used for studies of neurotoxicity in vitro. These
cells are comparable to other cells derived from pheocromocytomas in that they
synthesize, store and secrete large quantities of catecholamines (Greene and Tischler
1982). In PC12 cells these catecholamines predominantly consist of dopamine and
noradrenaline (Greene and Tischler 1982).
PC12 cells do not normally contain melanin. Nevertheless, long-time exposure
to D- or L-dihydroxyphenylalanine has been reported to induce biosynthesis of
neuromelanin in PC12 cells and in primary mesencephalic cells (Sulzer et al. 2000).
Furthermore, Offen and co-workers (1997, 1999) have reported that PC12 cells are
able to phagocytize dopamine melanin, although the internalized melanin decreased
the cell viability.
In paper III, PC12 cells were cultured in presence of L-dihydroxyphenylalanine
for 54 days, as a pilot study. No melanin was, however, observed by light-microscopic
examination or after staining against melanin with the Fontana-Masson silver
method. PC12 cells were therefore instead exposed to dopamine melanin. In contrast
to the results from Offen and co-workers (1997, 1999) exposure to dopamine melanin
up to 0.5 mg/ml for 48 hours did not cause any marked cytotoxic effect. There were,
however, several differences between the dopamine melanin used in these studies.
The dopamine melanin used in paper III was synthesized by a longer oxidation
step and the melanin suspension was heated in an autoclave (121°C for 45 minutes)
before used in cell cultures. It was also observed that a four-week storage period
of the dopamine melanin suspension after synthesis could increase the number of
melanin-loaded cells (figure 12).

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Anna Östergren

Figure 12. Number of melanin-loaded PC12 cells after 48 hours of incubation with dopamine
melanin, that been freshly synthesized or stored for four weeks. The values represent mean ± SD,
n=4 for freshly synthesized dopamine melanin and n=3 for stored dopamine melanin. Statistical
analysis (Student’s t-test) indicated that there was a higher number of melanin-loaded cells after
incubation with 0.25 and 0.5 mg/ml stored dopamine melanin compared to cells exposed to freshly
synthesized dopamine melanin, *p<0.05.

After exposure to dopamine melanin (0.1-0.5 mg/ml) for 48 hours black dopamine
melanin granules were preferentially localized in the cytoplasm close to the neurite
hillock and into the proximal part of the neurites (figure 13). This corresponds to
the in vivo localization of neuromelanin granules in pigmented neurons, suggesting
that the intracellular handling of the synthetic dopamine melanin in PC12 cells is
similar to that of neuromelanin in neurons.

Figure 13. Representative light-microscopic image of undifferentiated PC12 cells incubated with
dopamine melanin for 48 hours.

36
 Role of Neuromelanin and Cytochrome P450 for Toxicity

The effects of dopamine melanin (0.5 mg/ml) in PC12 cells were studied by
transmission electron microscopy, trypanblue exclusion assay, immunocytochemistry
against grp78 and cleaved caspase-3, and Western immunoblot against cleaved
caspase-3. Neither of these assays demonstrated any cytotoxic effect of dopamine
melanin. When the concentration of dopamine melanin was increased to 1.0
mg/ml there was, however, a statistically significant increase of necrotic cells
as demonstrated by the trypanblue exclusion assay. Also several morphological
changes were observed, including marked cell membrane blebbings, swelling and
disappearance of neurites.
When grown in the presence of nerve growth factor (NGF), PC12 cells extend
neurites, become electrically excitable, have increased numbers of calcium channels
and increase their synthesis of several neurotransmitters (Greene and Tischler 1982).
Generally NGF-differentiated PC12 cells are less sensitive to toxicants (Ohyashiki
et al. 2002; Salinas et al. 2003), but the opposite has also been reported (Chung and
Hong 1998). In this study, the effects of dopamine melanin were similar in NGF-
differentiated and undifferentiated cells. In addition, cleaved caspase-3 could not be
detected in either undifferentiated or NGF-differentiated PC12 cells after exposure
to dopamine melanin (0.25, 0.5 or 1.0 mg/ml) for 48 hours. In cells exposed to
staurosporine (positive control) cleaved caspase-3 was present (as shown in figure
14).

Figure 14. Representative Western immunoblot analysis (a) of cleaved caspase-3 expression in
NGF-differentiated PC12 cells exposed to vehicle (lane 1) or dopamine melanin (0.25 mg/ml, lane
2; 0.5 mg/ml, lane 3; 1.0 mg/ml, lane 4) for 48 hours, or staurosporine (0.5 µM) used as positive
controls, lane 5. No expression of cleaved caspase-3 was observed in melanin-loaded cells, while
there were caspase-3 positive bands in cells treated with staurosporine.

There was furthermore no difference between undifferentiated and NGF-

differentiated cells in their response to the cytotoxic peptide amyloid-ß.
The results from paper III demonstrated that dopamine melanin (≤ 0.5 mg/ml)
did not induce cytotoxicity and suggest that dopamine melanin-loaded PC12 cells
may be used as an in vitro model for neuromelanin-containing neurons.

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Anna Östergren

N-   

 - PC12 

Morphological changes
In paper IV conventional PC12 cells without melanin and melanin-loaded PC12
cells were cultured on collagen-coated glass slides and exposed to norharman (50
and 500 µM) or vehicle for 24 hours. In conventional cells without melanin there
was a decreased number of neurites after exposure to norharman and the neurite
terminals became swollen (figure 15 B, C)

Figure 15. Representative images of conventional PC12 cells without melanin (A-C) and melanin-
loaded PC12 cells (D-F) exposed to vehicle (A, D), 50 µM (B, E) or 500 µM (C, F) norharman for
24 hours.

There was also a decreased number of neurites and appearance of swollen neurite
terminals after exposure to norharman in melanin-loaded cells (figure 15 E, F),
although these changes were less severe than those in conventional cells. Furthermore,
the melanin did not only appear as large clusters in the neurite hillocks (15 D),
but also as uniformly dispersed granules throughout the cell body (15 E, F). In
75% of the cells dispersed melanin granules were also detected in swollen neurite
terminals.

38
 Role of Neuromelanin and Cytochrome P450 for Toxicity

Mitochondrial activity
Mitochondrial activity could be measured by adding yellow tetrazolium salt
(3-(4,5-dimethylthioazol-2-yl)2,5-diphenyl-tetrazolium bromide; MTT) to cells.
MTT is taken up by cells and yield a purple formazan product when reduced
by a mitochondrial succinate dehydrogenase. Accumulation of formazan thereby
reflects the actitivity of mitochondria directly and the cell viability or cell number
indirectly.
In paper II and IV the MTT-assay was used to study the ability of norharman
to cause a decrease of mitochondrial activity. Conventional PC12 cells without
melanin and melanin-loaded PC12 cells were exposed to norharman (50, 100, 250
and 500 µM) for 24 or 96 hours. In the conventional cells there was a concentration-
dependent decrease of mitochondrial activity. In melanin-loaded cells there was a
similar concentration-dependent decrease. The melanin-loaded cells were, however,
more sensitive to 50 and 100 µM norharman after 96 hours of exposure, although
melanin was observed to protect against 500 µM norharman after both 24 and 96
hours of exposure (table 5). The shift of melanin-induced sensitivity/protection has
not been fully understood.
Table 5
MTT-reduction in PC12 cells exposed to norharman
Concentration of Conventional cells Melanin-loaded Statistics
norharman without melanin Cells (Student’s t-test)
(% of control (% of control
without treatment) without treatment)

24 hours exposure n=14 wells

Control 100.1 ± 0.1 99.9 ± 0.1 n.s.
Vehicle 100.5 ± 3.2 103.3 ± 1.5 n.s.
50 µM 98.9 ± 10.1 102.6 ± 0.5 n.s.
100 µM 97.1 ± 5.8 100.8 ± 1.4 n.s.
250 µM 84.9 ± 11.4 90.6 ± 2.7 n.s.
500 µM 84.0 ± 6.9 94.3 ± 6.9 p<0.001

96 hours exposure n=20 wells

Control 100.0 ± 6.7 100.0 ± 6.6 n.s.
Vehicle 107.0 ± 10.6 104.6 ± 10.0 n.s.
50 µM 94.4 ± 6.2 85.7 ± 7.0 p<0.001
100 µM 79.9 ± 4.3 73.2 ± 7.4 p<0.05
250 µM 46.1 ± 5.4 47.7 ± 4.3 n.s.
500 µM 29.9 ± 3.3 34.6 ± 3.9 p<0.001
no statistical difference (n.s.)

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Anna Östergren

Oxidative stress
In paper IV the ability of norharman to induce oxidative stress was determined in
conventional PC12 cells without melanin and melanin-loaded PC12 cells.
The cells were preincubated with carboxy H2DCFDA, a probe that becomes
fluorescent in the presence of ROS. Thereafter the cells were exposed to norharman
(5, 50 and 500 µM) for up to 24 hours.
The results indicated, nevertheless, that norharman does not induce ROS. This
result is consistent with studies on other `-carbolines such as tetrahydrocarbolines
and a-carbolines, which are not considered as pro-oxidants but have been reported to
be efficient antioxidants (Stolc 1999). When PC12 cells were exposed to the positive
control (hydrogen peroxide, 4 mM) there was a rapid increase of oxidative stress.
The induction of ROS was lower in melanin-loaded cells. These results are consistent
with previous reports where Hoogduijn and co-workers (2004) demonstrated that
highly melanized melanocytes are protected against hydrogen peroxide-induced
DNA damage. This protection was suggested to be dependent on the ability of
melanin to bind calcium. Increased intracellular levels of calcium are known to
activate calcium-dependent nucleases. Both natural and synthetic dopamine melanin
can interact with ROS and scavenge superoxid radical anions and hydroxyl radicals
(Korytowski et al. 1986). The degree of pigment aggregation has been suggested to
influence the interaction of melanin with radicals and it has been suggested that
intracellular aggregates of melanin granules might be less effective than a suspension
of melanin particles in test tubes (Korytowski et al. 1995). Natural neuromelanin
appears in highly aggregated form and clusters in vivo. In melanin-loaded PC12 cells
most of the aggregates were localized in the neurite hillocks and showed a similar
intracellular localization as neuromelanin granules in dopaminergic neurons in vivo.
Korytowski and co-workers (1995) have suggested that the aggregation of natural
melanins may affect the radical scavenging efficiency and that the effective units
of melanin decrease with increasing aggregation. They propose that the scavenging
of radicals plays a minor role in vivo for the protection of pigmented cells against
oxidative stress and damage. Since melanin-loaded PC12 cells were protected against
hydrogen peroxide, it seems reasonable that also melanin aggregates protect cells
against oxidative stress induced by hydrogen peroxide.

ER stress
In paper IV the ability of norharman to induce ER stress was determined.
Conventional PC12 cells without melanin and melanin-loaded PC12 cells were
exposed to norharman (5, 50 or 500 µM) for 24 hours. In the conventional cells
without melanin, norharman (up to 50 µM) induced an increased number of grp78-
positive cells. Melanin-loaded cells were less sensitive to 5 µM norharman than
the conventional cells, equally sensitive to 50 µM and more sensitive to be grp78-
positive after 500 µM norharman (figure 16).

40
 Role of Neuromelanin and Cytochrome P450 for Toxicity

Figure 16. Immunocytochemical staining against grp78 in conventional PC12 cells without mela-
nin and in melanin-loaded PC12 cells exposed to norharman for 24 hours. Values represent mean
± SD, n=5-6 sections, including ≥150 cells/concentration. Statistical analysis (Student’s t-test)
indicated difference between cells without melanin compared to melanin-loaded cells, ***p<0.001,
**p<0.01.

These results indicate that melanin may have protective effects after exposure
to lower concentrations of norharman. After exposure to high concentrations of
norharman the melanin may become saturated and does not protect against ER
stress.
A large amount of work has established that specific induction of grp78 is
indicative of ER stress and that UPR is triggered (Lee 2005). These events are
probably induced to promote cell survival. The ER has, however, been pointed out
as a site of convergence of both pro- and anti-apoptotic molecules and may therefore
represent a novel focal point for the regulation of apoptosis (Lee 2005).

Apoptosis
In paper II conventional PC12 cells without melanin were exposed to norharman (5,
50 and 500 µM) for 24 hours. Thereafter the cells were stained against cleaved caspase-
3 or terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate
nick end labelling (TUNEL). The results demonstrated that norharman (5 and 50
µM) caused an increased number of both caspase-3- and TUNEL-positive cells
(figure 17), although the number of caspase-3- and TUNEL-positive cells decreased

41
Anna Östergren

after exposure to 500 µM norharman. Similar results have been reported for other
neurotoxicants such as MPTP and 6-OHDA, and it has been proposed that high
concentrations of these neurotoxicants may severely decrease rates of ATP synthesis
and result in an inability of the cells to maintain normal cellular function and so
result in necrosis (Hartley et al. 1994).

Figure 17. TUNEL-staining (inverted) of conventional PC12 cells without melanin exposed to
vehicle (A) or norharman 5 (B), 50 (C) or 500 µM (D) for 24 hours. Values represent mean ± SD,
n=2-3 sections, including >450 cells/concentration.

In paper IV, melanin-loaded PC12 cells were also stained against cleaved
caspase-3 after exposure to norharman (5, 50 and 500 µM) for 24 hours. The
results from this study demonstrated that melanin could protect against caspase-
3 activation after exposure to 5 and 50 µM. It was also demonstrated that there
was an increased number of caspase-3-positive melanin-loaded cells after exposure
of 500 µM norharman. These results may be interpreted as a protective effect of
melanin against necrosis, since melanin-loaded cells were able to undergo apoptosis
even after exposure to 500 µM norharman while cells without melanin lost this
ability. However, the number of cells stained against cleaved caspase-3 increased in
the melanin-loaded cells after exposure to 500 µM norharman, demonstrating that
melanin does not protect against apoptosis (figure 18).

42
 Role of Neuromelanin and Cytochrome P450 for Toxicity

Figure 18. Immunocytochemical staining against cleaved caspase-3 in conventional PC12 cells
without melanin and in melanin-loaded PC12 cells exposed to norharman for 24 hours. Values rep-
resent mean ± SD, n=6 sections, including ≥750 cells/concentration. Statistical analysis (Student’s
t-test) indicated a significant difference in the number of caspase-3 positive cells when comparing
melanin-loaded cells and conventional cells, ***p<0.001.

Necrosis
In paper II conventional PC12 cells without melanin were exposed to norharman
(5, 50 or 500 µM). After 24 or 96 hours the cells were trypsinated, resuspended
in medium and the number of necrotic cells was determined by the trypan blue
exclusion assay. The results demonstrated that there was a low but statistically
significant increase of necrotic conventional cells without melanin after exposure
to 500 µM norharman for 24 hours and after exposure for 50 µM and 500 µM for
96 hours.
In paper IV also melanin-loaded PC12 cells were exposed to norharman. The
melanin had a protective effect on induction of necrosis after exposure to all
concentrations of norharman for 24 hours and to 5 µM norharman for 96 hours, as
shown in figure 19. These results further support that melanin could protect PC12
cells against norharman-induced toxicity.

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Anna Östergren

Figure 19. Trypanblue staining of conventional PC12 cells without melanin and melanin-loaded
PC12 cells exposed to norharman for 24 or 96 hours. The values represent mean ± SD, n=5, includ-
ing ≥1300 cells/concentration exposed for 24 hours, and n=4, including ≥100 cells/concentration.
Statistical analysis (Student’s t-test) indicated a significantly lower level of norharman-induced
necrosis in melanin-loaded cells as compared to cells without melanin, ** p<0.01, *p<0.05.

Altogether, the results from paper I-IV demonstrate that neuromelanin may be
of importance for a selective retention of the `-carbolines norharman and harman.
Furthermore, norharman was demonstrated to induce degenerative effects in vivo
and in vitro and to cause motor impairment in C57BL/6 mice. The in vitro effects
could be attenuated by the presence of melanin. This is in concordance with earlier
suggestions that melanin has protective effect during short-term exposures. Melanin
binding is, however, slowly reversible, therefore the long-term effects of melanin
binding may be a continuous release of the neurotoxicant. This may possibly promote
rather than protect against toxicity.

CYP1- 
Selective expression of CYP enzymes in certain tissues and cell types has been
associated with tissue or cell specific toxicity of bioactivated toxicants. CYP1
metabolizes many toxicants, including benzo[a]pyrene and other PAHs, as well
as HCA, into reactive metabolites, which become bound to protein and nucleic
acids (Shimada et al. 1996). The expression of CYP1 enzymes can be induced by
treatment with aryl hydrocarbon receptor agonists such as BNF and PCB126.

44
 Role of Neuromelanin and Cytochrome P450 for Toxicity

`-carbolines
Previous studies have demonstrated that several HCA could be metabolized by
CYP enzymes (as reviewed by Yamazoe and Nagata 2000). Selective expression
of CYP1A1 have also been demonstrated to be of importance for localization of
some HCA (Annas and Brittebo 1998). Since norharman has been reported to
inhibit the metabolism of the CYP1 substrate benzo[a]pyrene in rat liver (Fujino
et al. 1978) it was of interest to study if induction of CYP1 could influence the
distribution of norharman and harman in mouse brain. 3H-Norharman and 3H-
harman were therefore injected i.v. in BNF-pretreated mice. The distribution was
evaluated by tape-section autoradiography and light-microscopic autoradiography.
The distribution of radioactivity after in the brain after injections of 3H-norharman
or 3H-harman was similar in BNF-pretreated mice compared to vehicle-treated mice
and the expression of CYP1 enzymes is not suggested to play a significant role for
localization of norharman and harman in the brain of mice.

7,12-Dimethylbenz[a]anthracene
Paper V revealed a selective distribution of radioactivity in blood-brain interfaces
after injection of 3H-DMBA in rodents pretreated with either BNF or PCB126. A
cellular localization of radioactivity in endothelial cells was further demonstrated by
light-microscopic autoradiography in vivo (figure 20) and in vitro with brain slices
of BNF- and PCB126-pretreated rodents. In vehicle-pretreated animals no such
selective distribution of radioactivity was observed in vivo or in vitro.

Figure 20. Light-microscopic auto-

radiography of BNF-pretreated mice
24 hours after an i.v. injection with
3
H-DMBA. A selective localization
of radioactivity (as demonstrated by
white areas) was observed in the capil-
lary loop EC of the choroid plexus (A)
and in EC of cerebral veins (B).

45
Anna Östergren

Induction of CYP1 in small intact tissue preparations of leptomeninges

and choroid plexus from PCB126-pretreated rats was also determined by the 7-
ethoxyresorufin O-deethylase activity assay.
Since BNF and PCB126 are known for their ability to induce CYP1 enzymes an
immunohistochemical study was performed to study if the selective distribution of
3
H-DMBA could be related to the expression of CYP1 enzymes. It was found that
CYP1A1 was selectively expressed in EC in cerebral veins, veins in the leptomeninges
and in capillary loops of choroid plexus of BNF- and PCB 126-treated mice,
whereas no similar expression of CYP1A1 was detected in vehicle-treated mice
(figure 21). Based on these observations it was concluded that EC in veins and in the
leptomeninges and capillaries of the choroid plexus of rodents express a constitutively
low but highly inducible and catalytically active CYP1A1 enzyme.

Figure 21. Immunohistochemical staining against CYP1A1 in brain sections including EC in

cerebral veins (A), veins in leptomeninges (B), cerebral arteries of the leptomeninges (C, D), capil-
lary loops of the choroid plexus (E, F) and vasa vasorum (G, H) from NMRI mice after an i.p.
injection with 0.25 mg/kg PCB126 (A, B, C, E, G) or vehicle (D, F, H), and a survival time of five
days post-injection.

46
 Role of Neuromelanin and Cytochrome P450 for Toxicity

An immunohistochemical study against CYP1B1 was also performed in paper

V. In contrast to CYP1A1, CYP1B1 expression was mainly found in smooth muscle
cells (SMC) in arteries of the leptomeninges, cerebral arteries, arterioles and in
choroid plexus arterioles, whereas the expression of CYP1B1 in EC was negligible.
The CYP1B1-staining was observed in mice pretreated with BNF or PCB126 but
also in mice pretreated with vehicle (figure 22).

Figure 22. Immunohistochemical staining against CYP1B1 in brain sections including arteries (A,
B) and veins (C) from the leptomeninges and capillary loops of choroid plexus from NMRI mice
after an i.p. injection with vehicle (A) or 0.25 mg/kg PCB126 (B,C and D) and a survival time of
five days post-injection.

Although DMBA is known to be metabolized by CYP1B1 (Gonzalez 2001),

selective localization of 3H-DMBA adducts was not observed in SMC of arteries
in the leptomeninges or cerebral arteries, neither in vehicle-treated or in BNF- or
PCB126-treated rodents.
There are several possible explanations for the differential bioactivation and
binding of 3H-DMBA in EC and SMC: CYP1B1 could be less catalytically active
than CYP1A1 toward DMBA or its metabolites; the two enzymes could form
different metabolites; or other enzymes, differentially expressed in EC and in SMC
could compete for DMBA or its reactive intermediates.
Injury to the blood vessel endothelium is often reported as an early event in
cardiovascular disease. Earlier studies have shown that there is an induction of
CYP1A1 and irreversible binding of 3H-DMBA also in HUVEC following pre-
treatment with BNF (Annas et al. 2000b). Interestingly shear stress has been
demonstrated to induce CYP1A1 and CYP1B1 in HUVEC (McCormick et al. 2001).
The induction of these enzymes in human EC may be an important determinant

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Anna Östergren

for the susceptibility of EC towards protoxicants and procarcinogens. The formation

of reactive intermediates and subsequent reaction with vital macromolecules may
result in a changed or impaired function leading to detrimental consequences for
the cell.
Both tobacco smoking and combustion effluents are important sources for PAH
exposure. Epidemiological studies indicate exposure to PAHs to be a common risk
factor to be involved in the aetiology of stroke and atherosclerosis (Bonita et al.
1999). Smokers are also known to have an increased expression of CYP1 enzymes.
The results from the presents study demonstrating the presence of 3H-DMBA
adduct in EC of some blood-brain interfaces of CYP450-induced rodents combined
with results from others demonstrating PAH-adducts and benzo[a]pyrene-induced
DNA damage in HUVEC (Annas et al. 2000a, Annas et al. 2000b,), suggest
that PAH exposure should be considered as a potential risk factor with regard to
cerebrovascular disease.

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 Role of Neuromelanin and Cytochrome P450 for Toxicity

CONCLUDING REMARKS

`-carbolines have been suggested to be involved in parkinsonism due to their

structural resemblance to MPTP. High levels of the `-carbolines norharman and
harman have also been reported in human post-mortem brains (Matsubara et al.
1993) and the levels of these compounds are increased in plasma and CSF of PD-
patients (Kuhn et al. 1995; Matsubara et al. 1995).
A general problem in studies of parkinsonism is that conventional model toxicants
generates a rapid neurotoxicity. Although this is practical for the researcher these
models may not be relevant to the slow, progressive nature of human parkinsonism
or mimic the long and latent neurodegenerative symptoms associated with chronic
exposure to environmental compounds. It is furthermore reasonable to believe that
co-exposure to several environmental agents may lead to synergistic toxic effects.
In this thesis, norharman was demonstrated to induce neurodegeneration and
sustained motor impairment in mice, although the results indicate that glia cells and
not dopaminergic neurons, may have been the primary target for the neurotoxicity.
It was also demonstrated that norharman could cause ER stress in PC12 cells.
However, these results were achieved after rather high doses and concentrations,
whereas humans normally are only exposed to low doses of these compounds.
Nevertheless, humans are likely to be exposed to `-carbolines and other similar
compounds over a lifetime. The results in this thesis demonstrated that there is a
long-term retention of `-carbolines in melanin-containing tissues. Therefore it is
likely that an accumulation of high concentrations of `-carbolines may be built
up in melanin-containing tissues although the exposure level is moderate. To be
able to study the effects of melanin on neurotoxicity induced by `-carbolines, an
in vitro model was developed. In this model, dopamine melanin was demonstrated
to be able to attenuate toxicity induced by low concentrations of norharman. This
is in concordance with that melanin has a protective function for single exposure
of toxicants. Melanin binding is, however, normally slowly reversible. Therefore,
a melanin binding that initially protects can later lead to a situation where the
melanin may function as a depot for neurotoxicants that are slowly released into the
cytosol and thereby promote a continuous exposure that may induce toxicity. It is
not possible to perform in vitro experiments over a long time, therefore it would be
of interest in to study if pre- or co-exposure with melanin-binding compounds could
reduce the protective effect of melanin on norharman-induced toxicity. Such studies
may include co-exposure to `-carbolines and calcium since others have reported
that melanin and calcium interact and disruption of the calcium homeostasis are
reported in several neurodegenerative disorders (Hoogduijn et al. 2004; Gibson

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Anna Östergren

and Huang 2004). When melanin becomes saturated it may not longer protects
against cytoxic events. Indeed, when the PC12 cells were exposed to the highest
concentration of norharman melanin was still able to attenuate necrosis, but the
intracellular stress or apoptosis increased.
PAHs have been suggested to contribute to disorders such as stroke and
atherosclerosis that may be caused by toxic effects on the endothelial cells. Earlier
studies have reported that DMBA could be metabolized and bioactivated by CYP1A1
as well as CYP1B1. In this thesis it was shown that there was a differential expression
of these enzymes; CYP1A1 was inducible and expressed in some EC whereas CYP1B1
was constitutively expressed in SMC. Furthermore, the distribution of DMBA was
in concordance with the expression of CYP1A1 suggesting that this enzyme may be
important for bioactivation and adduct formation of DMBA in the brain.

50
 Role of Neuromelanin and Cytochrome P450 for Toxicity

ACKNOWLEDGEMENTS

This work was carried out at the Department of Pharmaceutical Biosciences, Division
of Toxicology, Faculty of Pharmacy, Uppsala University, Sweden
The studies were founded by by the Swedish Council for Forestry and Agricultural
Research (SJFR) and the Swedish Research Council for Environment, Agricultural
Sciences and Spatial Planning (Formas). Grants were also received from Facias grant
2000-A-6 and from IF stiftelse.

I wish to express my gratitude to everyone who made this work possible,

especially:
Eva Brittebo, my main supervisor, who offered me the opportunity to become
a PhD-student. Thank you for your excellent scientific guidance, constructive
criticism and support in both personal and professional matters.
Nils Gunnar Lindquist, my co-supervisor who has been my melanin mentor,
always taking the time to share his great knowledge. Thank you for your enthusiasm
and all the inspiring discussions.
Anne-Lie Svensson, my second co-supervisor, thank you for all scientific as well
as unscientific discussion and for your never failing support.
My co-authors, Anita Annas, Kerstin Skog, Anders Fredriksson, Lizette
Granberg and Ingvar Brandt for engagement and valuable critisism.
I would also like to thank the people at “toxen”: Lennart Dencker, head of the
division of Toxicology for creating a pleasant and stimulating atmosphere in the
workplace, Raili Engdahl and Lena Norgren for all technical assistance, personal
interest and support, Anita Annas for being my mentor in the beginning and
introducing me to the world of cell culturing, Anna Franzén who shared my life
as a PhD-student from start to end, Elena Piras for friendship, support and a lot of
unscientific discussions, Maria Andersson, Camilla Svensson, Paulina Tuvendal,
Estibaliz Lopéz for time spent with delicious food and interesting discussions,
Henrik Alm and Mats Nilsson for nice lunch company, Simon Haile and Måns
Jergil for being good room-mates, Per Stenqvist for taking care of my endothelial
cells and for being the master of the plate-reader, Birger Scholz for keeping me
company at the lab during weekends, and Kim Kultima for fun time at PhD-
student parties. Michel Stigsson, Anne-Lee Gustavsson, and Björn Hellman for
valuable critism during the years.
All the students who have been working with me, especially Alexandra
Trifunovski, Anna Vikerfors, Anna Milbrink, Sara Sörensen and Fredrik
Jenerén for your contributions to this thesis.

51
Anna Östergren

I would also like to thank all my friends, better friends than you are not to be
found anywhere! Some special thanks go to MalinII my oldest friend who still likes
to play with me, Martin, for always being there when I needed you, Jenny, for your
excellent taste in everything and “La famiglia”: MalinI and Ulrika, for sharing
the PhD-student life with me, Clara and Sigrid for fun time spent with exercise,
Liselott for explaining jurisprudence and for the canoe vacation, Elisabeth for
sensible love advise and lots of parties, Kristina and Anders for your overwhelming
belief in how many publications I am going to have eventually, Peter for caring and
not tickling me, Gunnar, Johan T and Frederik for being my “flirt-friends.
Mum and dad (Inger and Anders Östergren) thank you for raising me to be
a self-dependent person not afraid of taking chances and finding my own way,
giving me endless of love and always being ready to help me with everything I could
possibly ask for.
Last but not least, Johan, my love for encouragement, patience, sharing life with
me and keeping me warm in heart and soul. I am looking forward to spending more
time with you

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 Role of Neuromelanin and Cytochrome P450 for Toxicity

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