Evaluation of neurotoxic properties of gliotoxin Stockholm University

Evaluation of neurotoxic properties
of gliotoxin
Viktoria Axelsson
Stockholm University
Doctoral dissertation, 2006
Department of Neurochemistry
Arrhenius Laboratories for Natural Sciences
Stockholm University, SE-106 91 Stockholm
Sweden
Cover: Bengt Ekberg, SVA, Sweden.
Growth of Aspergillus fumigatus on grain.
© Viktoria Axelsson, Stockholm 2006
ISBN 91-7155-286-3
Printed in Sweden by US-AB universitetsservice
Distributor: Stockholm University Library
Till Fredrik, Elias & Simon
List of original papers
This thesis is based on the following papers, which are enclosed in the end
and will be referred to in the text by their Roman numerals I-IV:
I
Glutathione intensifies gliotoxin-induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Axelsson V., Pikkarainen
K. and Forsby A. (2006) Cell Biol Toxicol. 22, 127-136
II
Gliotoxin induces caspase-dependent neurite degeneration
and calpain-mediated general cytotoxicity in differentiated
human neuroblastoma SH-SY5Y cells. Axelsson V., Holback
S., Sjögren M., Gustafsson H. and Forsby A. (2006) Biochem
Biophys Res Commun 345, 1068-1074
III
Effects of gliotoxin on mitochondrial function and protein
synthesis. Runesson J., Axelsson V. and Forsby A. (2006)
Manuscript
IV
Cytotoxic response of Aspergillus fumigatus-produced mycotoxins on growth medium, maize and commercial animal
feed substrates. Wenehed* V., Solyakov A., Thylin I.,
Häggblom P. and Forsby A. (2003) Food Chem Toxicol. 41, 395403
* Maiden name of V. Axelsson
Abstract
The occurrence of mould in food and animal feed is a severe problem due to
the secondary metabolites, called mycotoxins, which can possess toxic activity. Aspergillus fumigatus is a common fungus found in improperly stored
animal feed and the abundance of spores of the fungus is frequently spread
into the air. Gliotoxin has been identified as one of the most toxic second
metabolites produced by A. fumigatus. Although A. fumigatus is known to
produce mycotoxins that induce neurological syndromes, the neurotoxic
properties of gliotoxin have not previously been studied. In this thesis a neurotoxic activity of gliotoxin was demonstrated by using differentiated human
neuroblastoma SH-SY5Y cells as a surrogate for the nervous system. The
major findings were as follows:
i.
Gliotoxin is highly toxic to SH-SY5Y cells and there is a correlation between the toxicity and the cellular redox status.
ii.
Gliotoxin reduces the number of neurites, but does not affect the
cell bodies morphologically, at non-cytotoxic concentrations.
This indicates that the toxin may induce peripheral axonopathy
in vivo.
iii.
The intracellular free Ca2+ concentration is increased after exposure to gliotoxin, an effect that is the most ubiquitous feature of
neuronal cell death. Simultaneously, calpains and caspases, proteases known to be involved in neuronal death and axonal degeneration, are activated.
iv.
The observed irreversible neurite degenerative effects of gliotoxin are mainly dependent on caspase activation, whereas calpains are involved in the gliotoxin-induced cytotoxicity.
v.
Gliotoxin induces a decreased rate of protein synthesis at noncytotoxic concentration, which may contribute to the degeneration of neurites.
vi.
We did also succeed in developing an in vitro method for determination of toxic activity in animal feed. This study was done in
collaboration with National Veterinary Institute (SVA) in Uppsala, and the method is today established and in use at Department of Animal Feed, SVA.
Contents
1. Introduction .............................................................................................11
1.1 Aims and structure of the thesis ............................................................................12
1.2 Neurotoxicity .........................................................................................................12
1.2.1 Axonopathy...................................................................................................13
1.2.2 The neuronal cytoskeleton............................................................................13
1.2.3 Intracellular calcium homeostasis .................................................................16
1.2.4 Calpains........................................................................................................18
1.2.5 Mitochondria and cell injury...........................................................................20
1.2.6 Cell death, apoptosis vs. necrosis.................................................................23
1.2.7 Caspases......................................................................................................24
1.2.8 Cellular redox balance ..................................................................................29
1.3 Gliotoxin ................................................................................................................33
1.3.1 Proposed biosynthesis of gliotoxin ................................................................33
1.3.2 Biological activities of gliotoxin......................................................................34
1.3.3 Cellular uptake and metabolism....................................................................35
1.3.4 Toxic mechanisms ........................................................................................36
1.4 Aspergillus fumigatus ............................................................................................37
1.4.1 The pathogenecity of A. fumigatus................................................................37
1.4.2 Risk assessment of exposure to A. fumigatus and gliotoxin..........................38
1.5 Detection of mycotoxins in feed.............................................................................38
2. Methodological considerations .............................................................40
2.1 Cell system ...........................................................................................................40
2.1.1 Native SH-SY5Y cells (Paper IV) ..................................................................40
2.1.2 Differentiated SH-SY5Y cells (Paper I-IV).....................................................41
2.2 Preparation of A. fumigatus extracts (Paper IV) ....................................................41
2.3 Cell treatments ......................................................................................................42
2.3.1 Neurotoxic properties of gliotoxin (Paper I-IV)...............................................42
2.3.2 Toxic activity in animal feed (Paper IV) .........................................................43
2.4 Endpoints ..............................................................................................................43
2.4.1 General cytotoxicity (Paper I-IV) ...................................................................43
2.4.2 Neurite degeneration (Paper I, II and IV) ......................................................44
2.4.3 The cellular redox balance (Paper I) .............................................................44
2.4.4 Physiological changes (Paper II and III)........................................................45
2.4.5 Mitochondrial effects (Paper III) ....................................................................46
2.4.6 Changes in protein expression (Paper I and II) .............................................46
3. Results and discussion ..........................................................................48
3.1 Cellular redox status and toxicity of gliotoxin.........................................................48
3.2 Cytotoxic activity of gliotoxin .................................................................................49
3.2.1 Altered Ca2+-homeostasis and loss of cellular energy ...................................49
3.2.2 Impact of activated calpains..........................................................................50
3.3 Neurotoxic effects of gliotoxin ...............................................................................51
3.3.1 Caspase-dependent neurite degeneration ....................................................51
3.3.2 The aspect of decreased protein synthesis...................................................53
3.4 Sensitivity of the SH-SY5Y cells to gliotoxin..........................................................55
3.5 Bioassay for detection of toxic activity in feed .......................................................55
4. Conclusions.............................................................................................57
5. Sammanfattning på svenska .................................................................58
6. Acknowledgement ..................................................................................60
7. References...............................................................................................62
Abbreviations
AD
AIF
ANT
Apaf-1
ATP
BSO
[Ca2+]i
[Ca2+]m
calpeptin
CNS
CyP-D
CzDox broth
ER
ETC
ETP
FAD, FADH2
GCLC
GCLM
γ-GCS (or GCL)
GSH
GSSG
GST
HPLC
IF
IP3R
MAPs
MeHg
MF
MMP
MPT
MTG
MTR
NAD+, NADH
NADP+, NADPH
NF
NF-κB
PD
PMCA
PNS
RA
Alzheimer’s disease
apoptosis inducing factor
adenine nucleotide translocator
apoptosis activating factor-1
adenosine triphosphate
L-buthionine-sulfoxamine
intracellular free Ca2+ concentration
mitochondrial matrix Ca2+ concentration
benzyloxycarbonylleucyl-norleucinal
central nervous system
cyclophilin D
Czapek-Dox broth
endoplasmatic reticulum
electron transfer chain
epipolythiodioxopiperazine
oxidized and reduced flavin adenine dinucleotide
catalytic subunit of GCL
regulatory subunit of GCL
γ-glutamyl-cysteine-synthetase (or glutamate cysteine ligase)
reduced glutathione
glutathione disulfide (oxidized glutathione)
glutathione S-transferase
high-performance liquid chromatography
intermediate filament
inositol 1,4,5-triphosphate receptor
microtubule associated proteins
methyl mercury
microfilament
mitochondrial membrane potential
mitochondrial permeability transition
MitoTracker Green FM
MitoTracker Red
oxidized and reduced nicotinamide adenine dinucleotide
oxidized and reduced nicotinamide adenine dinucleotide
phosphate
neurofilament
nuclear factor-κB
Parkinson’s disease
plasma membrane Ca2+-ATPase
peripheral nervous system
retinoic acid
RAR
ROS
RXR
RyR
SBDP
SERCA
SOD
SVA
TNF
VDAC
Z-VAD-fmk
retinoic acid receptor
reactive oxygen species
retinoid X receptor
ryanodine receptor
spectrin breakdown product
sarco(endo)plasmatic reticulum Ca2+-ATPase
superoxide dismutase
National Veterinary Institute
(Statens Veterinärmedicinska Anstalt)
tumor necrosis factor
voltage dependent anion channel
carbobenzyloxy-Val-Ala-Asp-α-fluoromethylketone
1. Introduction
Say the word mould, and most people will wrinkle their nose (if they are not
thinking of a delicious cheese of course). But mould growth is not only an
unpalatable problem; it can also be a serious health problem due to the toxic
substances (mycotoxins) that can be produced. The name mycotoxin combines the Greek word for fungus “mykes” and the Latin word “toxicum”
meaning poison.
Most known mycotoxins are produced by species in the genera Aspergillus,
Penicillium and Fusarium (1). One mould genus can produce several different mycotoxins and one particular mycotoxin can be produced by several
mould species. Mycotoxins can display a diverse array of potent pharmacological effects for the exposed individuals; for example cause acute poisonings, be carcinogenic, immunosuppressive or neurotoxic (toxic to the nervous system) (2). Primarily, mould contaminates agricultural commodities
such as cereals, oilseeds and dried fruit, but also food, houses and timber.
The contaminations are known to be a problem occurring mostly under inadequate storage conditions and the single most important environmental
factor for controlling fungal development is moisture (1). However, the
presence of visible mould growth does not necessarily imply the presence of
mycotoxins, since formation of the toxins occurs only when toxigenic strains
are present and a secondary metabolite pathway is activated. On the other
hand, mycotoxins can contaminate feed or food even in the absence of visible mould (3).
The problem with mould and mycotoxin appearance has been known for
many years. However, it was the epoch-making discovery of the carcinogenic aflatoxins in 1960 (4), which led to the current international awareness
of problems associated with invasion of toxigenic fungi and mycotoxin formation in food- and feedstuffs. Today, there is a growing concern and
knowledge about the effects of this class of toxins and their impact on human and animal health. The Swedish Board of Agriculture (5) prescribes
hygiene guidelines for commercial animal feed, with the aim to avoid exposure to toxic compounds produced by microorganisms. Proposals of EUlegislation on food and feed safety have suggested the need for proper risk
assessment in relation to chemical and microbiological hazards, and in the
“White paper on food safety” (6) it is stated that the safety of animal feed
shall be equivalent to the safety of human foodstuffs. Nevertheless, there are
still big problems with mycotoxin-intoxications causing unnecessary suffering for animals and humans, economical losses because of decreased production as well as a possible transmission of toxins to humans via the foodstuffs.
11
1.1 Aims and structure of the thesis
Gliotoxin is a highly toxic mycotoxin produced by a number of fungi, for
instance Aspergillus fumigatus. Although A. fumigatus is known to produce
mycotoxins inducing neurological syndromes in animals and humans, the
neurotoxic properties of gliotoxin have not previously been evaluated.
This thesis is based on four separate scientific Papers (I-IV). Focus in the
thesis has been to evaluate the potential neurotoxic activity of gliotoxin, by
using differentiated human neuroblastoma (SH-SY5Y) cells as a neuronal
model. The mechanisms for general cytotoxicity and neurite degeneration
after gliotoxin-exposure were investigated by studying:
• The role of glutathione and an altered cellular redox balance
(Paper I)
• Involvement of calcium and activity of calpains and caspases
(Paper II)
• The protein synthesis rate and mitochondrial effects
(Paper III)
Furthermore, a special interest in the thesis was to investigate the possibility
to use SH-SY5Y cells as a bioassay for detecting cytotoxic activity in extracts from contaminated animal feed (Paper IV).
1.2 Neurotoxicity
The nerve cell (neuron) is among the morphologically most complex of all
cell types. It is composed of dendrites, which receive and mediate signals
from the surrounding environment into the cell, a cell body, and the axon
that leads impulses from the cell body and projects to other cells and/or muscles and glands. Not only the neuron is complex, the complexity of the
whole nervous system is immense. It is divided into the central nervous system (CNS), constituting of the brain and spinal cord and function as a central
of command, and the peripheral nervous system (PNS), consisting of nerves
sending information to or from the CNS. The brain consists of 100 billions
of cells and each cell has as many as 10,000 contacts with other cells. A
sensory input to the brain is able to start a million different chemical reactions and the combined length of all neuronal processes amounts to 500,000
km (7). These facts reflect the enormous number of possible targets for neurotoxicity.
12
Damage to neurons can arise as a consequence of chemical insult, injury,
inflammatory processes or genetic causes. Neurological lesions in brain and
the periphery are called central and peripheral neuropathies, respectively,
and are further classified as neuropathies (degeneration of the cell body),
axonopathies (retraction of the axon) (8), and myelinopathies (effects in the
myelin sheet) (9).
1.2.1 Axonopathy
Axonopathy is characterized by a selective degeneration of the axon, but
without any evident effects on the neuronal cell body (10, 11). Axonopathy
is a common indication of neurotoxicity and can occur both in the CNS and
PNS. However, in PNS a regeneration of the axon is possible whereas it is
rarely seen in the CNS (12). Axonopathy can be observed during toxic insult
of, for example, acrylamide (13) and organophosphorus compounds (10, 14),
and in diseases, such as diabetes (15). Wallerian degeneration (16) is the
classical example of axonal degeneration, and refers to a series of events that
occur in distal axons when they are severed from the cell body. After the
axon is transected, there is an accumulation of materials at the proximal end
of the distal axon stump, presumably owing to a block of retrograde axonal
transport (17). Next follows a rapid breakdown of the axonal cytoskeleton
and degeneration of mitochondria, which in turn triggers the responses from
the surrounding, including recruitment of microglia and macrophages. Another form of axonopathy is dying back degeneration. In this process, the
axon in an unhealthy neuron progressively degenerates, beginning distally
and progressing proximally (10). Pathologically, dying back degeneration is
quite similar to the Wallerian degeneration, and appropriately described as
“Wallerian-like” (18).
1.2.2 The neuronal cytoskeleton
The cytoskeleton is an important part in axon and dendrite formation, allowing neurons to establish their asymmetrical and complex morphology essential for the neuronal physiology. The neuronal cytoskeleton network is composed of three types of protein filaments – microfilaments, microtubules and
intermediate filaments (19) (Fig. 1.1). The microtubules and microfilaments
are dynamic structures with a role in neuronal development and function,
e.g. establishment of neuronal cell shape, migratory processes, axonal outgrowth, synaptic plasticity and intracellular transport of proteins and organelles. Intermediate filament proteins are intermediates between the microtubules and microfilaments, and generally viewed as cytoskeleton components that confer stability to the cells.
13
1.2.2.1 Microtubule
The microtubules are assembled from tubulin and are approximately 25 nm
in diameter (20). They are structurally and functionally polar with a rapidly
growing (plus) and a slowly growing (minus) end (21). In axons, microtubules are found in densely packed bundles uniformly oriented with their
plus ends directed towards the distal parts. In dendrites, on the other hand,
their orientations are mixed with nearly equal proportions of the microtubules oriented with the plus ends directed towards the growth cone or the
cell body. Microtubule is a target for several neurotoxic compounds. For
instance, one mechanism of methyl mercury (MeHg)-induced damage in
neuronal cells has been proposed to be related to the impairment of microtubule assembly and disassembly, by binding to free thiol-groups on the
microtubule surface (22, 23).
A class of proteins shown to regulate the structure and function of microtubules are the microtubule-associated proteins (MAPs). In neurons, the
major MAPs include tau, MAP1a, MAP1b and MAP2 (24). Tau proteins are
the most important and extensively studied, and enriched in axons in both
the CNS and PNS (25). Tau is a phosphoprotein and its biological activity is
regulated by the phosphorylation state. To obtain the dynamics of the cytoskeleton, needed for appropriate neurite outgrowth, an increase in phosphorylation of tau within the microtubule binding domains is required, in
combination with a decrease in phosphorylation of tau in the flanking domains (26). Nevertheless, abnormally phosphorylated tau forms paired helical filaments upon hyperphosphorylation (27), which can impair the axonal
transport and eventually lead to neuronal cell death. These filaments are the
main components of the intracellular neurofibrillary tangles that can be
found in brain tissue from patients suffering from Alzheimer’s disease (AD)
(28).
1.2.2.2 Intermediate filament proteins
The intermediate filament (IF) proteins in neurons are composed of the neurofilament (NF) triplet proteins (NFTPs), peripherin and α-internexin (29).
NFTPs are the major neuronal IF proteins in the adult CNS and PNS, and
composed of NF-L (∼70 kDa), NF-M (∼160 kDa) and NF-H (∼200 kDa) for
low-, middle-, and high-molecular weight, respectively. Following synthesis
in the cell body, NF proteins are rapidly assembled into 10-nm filaments and
actively transported into the axon and along the microtubules towards the
distal part, at a slow rate of 0.2-1 mm/day (19). Assembly and transport of
NF proteins are regulated by phosphorylations, and their function, i.e. modifying the axonal diameter, is related to the phosphorylation state (30, 31).
NF-L is relatively poorly phosphorylated, while NF-M and NF-H are extensively phosphorylated (32). In axons and dendrites, NF proteins and micro-
14
tubule are aligned in parallel arrays and separated from one another by nonrandom distances. This distinctive organization has been attributed to crossbridges, formed by NF protein side arms or MAPs (see (33)). The side arms
of the NF proteins are rich in phosphorylation sites (32) and the extent of
NF-M and NF-H phosphorylation may have a functional role in regulating
the interactions between NF proteins and their surroundings.
Dysfunction of NF proteins has been linked to the etiology of various neurodegenerative disorders. Abnormal accumulations of NF proteins are detected in AD, Parkinson’s disease (PD), and amyotrophic lateral sclerosis,
among others (34). Accumulation of NF proteins has also been observed in
axonopathy induced by acrylamide (35), 2,5-hexandione (36), and organophosphates (37). A number of different mechanisms can lead to accumulation of NF proteins, including dysregulation of their synthesis, defective
axonal transport, abnormal phosphorylation and proteolysis (34), or direct
binding to (38), alternatively cross-linking of (39), the NF proteins.
Figure 1.1. Growth cone of a nerve fiber and the organization of the cytoskeleton
components. The rope-like intermediate neurofilaments are packed together in a
dense helical array. The microtubules are long, hollow cylinders made of the protein
tubulin, and actin filaments are two-stranded helical polymers of the protein actin.
Actin is coupled to cell surface proteins by spectrin and ankyrin. Forward movement
of the growth cone occurs by extension of filopodia, followed by formation of lamellar processes that fill in the space between the filopodia. The cytoskeleton proteins are not illustrated in proportional size.
1.2.2.3 Microfilaments
Microfilaments (MF) comprise actin subunits arranged like two intertwined
strings of pearls, forming fibrils with 4-6 nm in diameter (40). Actin MFs are
found throughout the neuron, but they are particularly concentrated in presynaptic terminals, dendritic spines and growth cones. In the growth cone
actin exists in two structural arrangements, lamellipodia and filopodia, which
are dynamic structures with high activity during axonal extension and cell
migration (41). In the axonal cytoplasm, MFs are most apparent in the vicin15
ity of microtubules and near the plasma membrane (40).
The major protein that provides structural basis for the cytoskeleton and
couples actin to cell surface proteins, is the actin-binding protein spectrin.
Spectrin (or fodrin; the brain-specific form of spectrin) is a tetramer consisting of two α and two β chains, with molecular weights of 240 and 220 kDa,
respectively. The spectrin-actin network requires accessory proteins to form
a membrane associated network and a major link is the protein ankyrin (42)
(Fig. 1.1).
1.2.3 Intracellular calcium homeostasis
Alterations in the intracellular calcium homeostasis play a major role in neurotoxicity. The calcium ions (Ca2+) are ubiquitous intracellular messengers
involved in a large number of cellular functions, such as control of cell
growth and differentiation, membrane excitability, synaptic activity and exocytosis of neurotransmitters and hormones (43). Hence, it is of importance
that the intracellular free Ca2+ concentration ([Ca2+]i) is strictly regulated.
The [Ca2+]i must remain at low levels (around 100 nM) as compared to the
extracellular concentration (1 mM), so that a relatively small or local increase in [Ca2+]i can be used to trigger physiological responses. For this,
neurons have evolved complex mechanisms to keep the equilibrium between
Ca2+ influxes, Ca2+ buffering, internal Ca2+ storage, and Ca2+ efflux, optimal
for the neuronal function (Fig. 1.2).
2+
1.2.3.1 Ca
influx
The main sources for Ca2+ entry through the plasma membrane are voltagedependent Ca2+ channels and ligand-operated Ca2+ channels (for example
glutamate receptors) (44, 45). Further possible routes of Ca2+ entry are gap
junctions between neurons and glia (46), or the electrogenic Na+/Ca2+ exchanger (47) that is activated in conditions of intracellular Na+ overload.
2+
1.2.3.2 Ca
sequestration/efflux
Once inside the cell, Ca2+ can either react with Ca2+ binding proteins, e.g.
calmodulin and calbindins (48), or become sequestered into intracellular
organelles. The uptake system with highest affinity for Ca2+ is the
sarco(endo)plasmatic reticulum Ca2+-ATPase (SERCA) pumps, located on
the endoplasmatic reticulum (ER) (49). ER is the main dynamic Ca2+ store in
cells, with a capacity to store Ca2+ up to millimolar concentrations. Also
mitochondria sequester Ca2+. That uptake is electrophoretically regulated
through a uniport transporter (50), which will be further discussed in section
1.2.5.1. Excessive amounts of Ca2+ may also be removed into the extracellular space by a plasma membrane Ca2+-ATPase (PMCA) (51) and by the
Na+/Ca2+-exchanger (52).
16
Figure 1.2. The regulation of Ca2+ influx and intracellular compartmentalization.
Cellular Ca2+ import through the plasma membrane occurs largely by ligandoperated and voltage dependent channels. Inside the cell, Ca2+ can either react with
Ca2+-binding proteins or become sequestered into ER or mitochondria. Ca2+ release
from ER is triggered by agonist stimulation of a G-protein coupled receptor at the
plasma membrane, with generation of IP3 and DAG, through hydrolysis of PIP2 by
PLC. Alternatively, Ca2+ stored in ER is released by ligand-binding to RyR. The
cytosolic Ca2+ concentration in unstimulated cells is kept around 100 nM by uptake
of Ca2+ into ER and extrusion out to the extracellular space with the ATP-driven
Ca2+-pumps, SERCA and PMCA, respectively. The mitochondrial Ca2+ uptake is
electrophoretically through a uniport transporter, and can be released again through
Na+/H+-dependent Ca2+exchange, or as a consequence of opening the MPT pore.
DAG, diacylglycerol; PIP2, phosphatidylinositol-4,5-bisphosphate; PLC, phospholipase C.
17
2+
1.2.3.3 Intracellular Ca
release and translocation
The [Ca ]i may rise not only because of influx from the extracellular space,
but also due to release from intracellular stores. A high percentage of the
raised [Ca2+]i is actually derived from intracellular pools, meaning that local
Ca2+ changes may regulate molecular processes, independently from Ca2+
influx through the plasma membrane (48). The Ca2+ stored in ER is released
by stimulation of two classes of receptors: the inositol 1,4,5-triphosphate
receptor (IP3R) and the ryanodine receptor (RyR), which are triggered by
activation of receptors on the plasma membrane and the subsequent generation of second messengers (49). The Ca2+ pool in ER is not static, but a
steady-state Ca2+ level is maintained by a constant leakage of Ca2+ into the
cytosol and a reimport of the ion via SERCA. A further source for increases
in [Ca2+]i is the mitochondria. The control of mitochondrial Ca2+ is discussed
in section 1.2.5.1.
2+
2+
1.2.3.4 Effects due to disturbed Ca - homeostasis
Neurotoxic actions of Ca2+ overload have been ascribed to the overstimulation of enzymes, including proteases, lipases, and endonucleases. One of the
major effects is activation of calpains, cleaving key cytoskeleton constituents
or proteins (53). Another consequence of Ca2+ overload might be mitochondrial dysfunction, activation of caspases and induction of apoptosis (see
(45)). Also, Ca2+-dependent nucleases involved in DNA degradation (54),
and phospholipases, in particular phospholipase A2, might be activated. Activation of PLA2 can cause generation of reactive oxygen species (ROS) and
disruption of the membrane integrity, facilitating further Ca2+ influx or Ca2+
release from intracellular stores (55).
1.2.4 Calpains
Calpains constitute a family of Ca2+-dependent cysteine proteases whose
members are expressed both ubiquitously and in a tissue-specific way. µand m-calpains are commonly referred to as the ubiquitous, conventional or
typical calpains (56); to distinguish from the tissue-specific calpains, p94
(skeletal muscle specific) and nCL-2 (stomach-specific) (57, 58). The terms
µ-calpain and m-calpain refer to the micromolar (µ-calpain) and millimolar
(m-calpain) Ca2+ concentration required for activation of the proteases (59).
However, the sensitivity of, in particular, m-calpains to [Ca2+]i that are out of
the range of cell normality, suggests the involvement of regulatory mechanisms dramatically reducing its Ca2+ requirements to be able to function at a
physiological Ca2+concentration (59).
18
Both µ-and m-calpains are heterodimers containing an identical 28-kDa subunit and a catalytic 80-kDa subunit that shares 55-60% sequence homology
between the two proteases. The large subunit is organized in four domains
(I-IV) and the small subunit in two (V-VI). Domain II is the cysteine protease domain containing the active triad of cysteine, histidine and asparagine
residues, which is characteristic for cysteine proteases. Domain IV and VI
are the Ca2+-binding domains with four and one binding site for Ca2+, respectively (see (56)).
1.2.4.1 Calpain substrates
A number of cellular targets have been identified as substrates for calpain
cleavage. Most documented is the cleavage of cytoskeleton proteins, especially those involved in cytoskeleton/plasma membrane interactions (Table
1.1).
Table 1.1. Examples of calpain substrates
Substrate
Reference
Cytoskeleton proteins
Actin-binding (ankyrin, αII- and βII-spectrin)
(60, 61)
Neurofilaments
(62)
MAPs (MAP2 and tau)
(63)
Tubulin
(63)
Membrane proteins
Epidermal growth factor receptor
2+
Ca -ATPase
(64)
(65)
Signal transduction enzymes
Kinases (protein kinase C)
(66)
Phosphatases (calcineurin)
(67)
Phospholipases (phospholipase C)
(68)
Others
Apoptotic regulatory factors (Bax, Apaf-1)
(69, 70)
Caspases
(71-73)
Transcription factors (c-Fos/c-Jun)
(74)
19
At physiological [Ca2+]i calpains regulate the normal dynamic turnover of
the cytoskeleton, and thus, they have a role in cell fusion (75) and changes in
cell shape, including extension of neurites (76, 77) and remodelling of synaptic terminals (78). Consequently, increased [Ca2+]i may lead to enhanced
activity of calpains and abnormal breakdown of the cytoskeleton and will
thereby damage cellular structure. As mentioned in section 1.2.2.2, the structural stability of NF proteins is suggested to be essential for neuronal survival, and a deterioration of NF proteins can contribute to nerve cell death
and axonopathy. Calpain-mediated degradation of NF proteins occurs in
traumatic spinal cord injury (79) and in glutamate-induced hippocampal
damage (80). In studies using hippocampal slices as a model system, the
neuronal survival was demonstrated to enhance when the degradation of NF
proteins was reduced with a calpain inhibitor (81). Calpain activation and
degradation of cytoskeleton components is also believed to be responsible
for Ca2+-mediated cell injury observed in ischaemic stroke (82) and Wallerian degeneration (83). Other substrates for calpain proteolysis include enzymes, membrane proteins, cytokines and transcription factors (Table 1.1).
In models of AD and PD, possible targets for calpain cleavage are the signal
transduction protein p35 (84), and the transcription factors c-Jun and p53
(85). As shown in Table 1.1, calpains may also have a role in apoptosis by
acting on caspases and regulatory factors for apoptosis. This will be further
discussed in section 1.2.7.3.
1.2.4.2 Regulation of calpain activity
The activity of calpains in cells is regulated by altered Ca2+ concentrations,
calpastatin, and probably also by their intracellular location (access to substrate). Calpastatin is a ubiquitously expressed protein that specifically and
reversibly inhibits the proteolytic activity of µ-and m-calpains by binding to
the proteases in a Ca2+ dependent way (86). Nevertheless, there is no evidence that calpastatin itself binds Ca2+, suggesting that the Ca2+ requirement
for inhibition of calpains must originate from the calpain molecule. One
hypothesis is that the bound Ca2+ induces a structural modification of calpain
that promotes complex formation with the inhibitor (86). The Ca2+ concentration required for calpastatin to inhibit calpains is significantly lower than
that needed to initiate the proteolytic activity of calpains, suggesting that
cells must possess some mechanism to allow calpain activity in the presence
of calpastatin (87). Otherwise, rising Ca2+ concentrations would cause calpastatin binding before the calpains could initiate their proteolytic activity.
1.2.5 Mitochondria and cell injury
Mitochondria are organelles central to the maintenance of many cellular
functions, and thereby also play an important role in both the survival and
death of neurons. The mitochondrion is surrounded by a double-membrane
20
system, consisting of an inner and an outer mitochondrial membrane that are
separated by the intermembrane space (Fig. 1.3).
Figure 1.3. Illustration of a mitochondrion showing the ETC, routes for influx and
efflux of metabolites, and the components of the MPT pore. When electrons are
transferred from complex I to IV, the MMP is established due to pumping of protons
into the intermembrane space. The MMP is used by ATP synthase to produce ATP.
Metabolites cross into the intermembrane space through VDAC and then through the
inner membrane to the matrix via their respective transporter systems. The basic unit
of the MPT pore (inserted) is the VDAC-ANT-CyP-D complex located at contact
sites between the mitochondrial inner and outer membrane. Other proteins associate
with the complex as indicated. cytc, cytochrome c; e-, electrons; H+, protons; PT, the
phosphate transporter; UQ, ubiquinone.
The organelles are powerful in the production of cellular energy, and the
ATP synthase-mediated synthesis of ATP (oxidative phosphorylation) is the
ultimate step in a reaction network called the electron transfer chain (ETC),
situated in the mitochondrial inner membrane (88) (Fig. 1.3). Electrons to
ETC are donated from the oxidation of NADH and FADH2 at complex I
(NADH-dehydrogenase) and complex II (succinate dehydrogenase), respectively. Thereafter, the electrons are shuttled to complex III (ubiquinolecytochrome c oxidoreductase) by ubiquinone, and to complex IV (cytochrome oxidase) by cytochrome c. In complex IV, four electrons are transferred to O2, which together with four protons yield water. During the transport of electrons through the ETC, protons are transferred from the matrix
side of the inner mitochondrial membrane to the intermembrane space, gen21
erating the mitochondrial membrane potential (MMP). The MMP is used as
a driving force by ATP synthase to produce ATP, but also for mitochondrial
uptake of Ca2+ to matrix (89).
2+
1.2.5.1 Mitochondrial Ca
regulation
2+
Mitochondrial Ca modulates the rate limiting enzymes of the citric acid
cycle; pyruvate dehydrogenase, 2-oxoglutarate and NAD+-isocitrate (90), but
also fatty acid oxidation and amino acid catabolism (91). Since the major
function of the citric acid cycle is to produce NADH, which donates electrons to the ETC, the rate of ATP synthesis may be influenced by the Ca2+
uptake (92). In turn, alterations in ATP-synthesis will affect the activity of
ATP-driven Ca2+ pumps responsible for removing Ca2+ from the cytosol.
When mitochondria are energized, i.e. respiring and translocating protons
over the inner mitochondrial membrane, Ca2+ will move down its electrochemical gradient and into matrix through the Ca2+ uniporter (89). The uniporter function in the absence of an immediate exchange of ions, and thus,
mitochondrial Ca2+ uptake is electrogenic leading to a depolarisation of the
mitochondrion. But, since accumulated Ca2+ also up-regulates the activity of
the citric acid cycle, the MMP will rapidly be re-established (93). As mitochondria utilizes the MMP to facilitate Ca2+ uptake, extrusion of Ca2+ is energetically uphill and requires either ATP hydrolysis or energetically favorable ion exchanges, like the Ca2+/Na+ antiporter together with the Na+/H+
antiporter, that re-establishes mitochondrial Na+ balance (50) (Fig. 1.3).
Uptake or release of Ca2+ by inner mitochondrial membrane transport systems, necessarily involves Ca2+ crossing the mitochondrial outer membrane
as well. To control MMP, ATP production and cell survival, the outer mitochondrial membrane is, however, not freely permeable to ions and metabolites. Except for a relatively few membrane-permeable compounds, like molecular oxygen, acetaldehyde and short chain fatty acids (94), most metabolites cross the outer mitochondrial membrane through the voltage-dependent
anion channel (VDAC). VDAC (also called mitochondrial porin) (95) is a
large channel that in the main conductance state is 2.5 nm in diameter (96)
and permeable to the ions Cl-, K+, Na+, and to large anions including succinate, pyruvate, ADP, ATP, and phosphate (97-99). It has also been demonstrated that VDAC is permeable to Ca2+ and possesses Ca2+ binding sites
(91). Hence, the VDAC provides the pathway for Ca2+ transport in and out of
mitochondria through the outer mitochondrial membrane.
The described mitochondrial transport systems for Ca2+ mediate a slow continuous Ca2+ cycling across the inner mitochondrial membrane, driven by the
respiratory-chain expulsion of protons. This enables slow or maintained
changes in cytosolic [Ca2+]i to be relayed into a change in the mitochondrial
matrix Ca2+, ([Ca2+]m). When [Ca2+]i is altered, the cycle establishes a steady22
state distribution of Ca2+ over the inner mitochondrial membrane, and each
increase in [Ca2+]i will give rise to a proportional greater increase in [Ca2+]m
until a “set point” is reached where [Ca2+]m is overloaded (100). Mitochondrial Ca2+ overload is a major feature of cell injury and may initiate a nonspecific increase in the permeability of the inner mitochondrial membrane
and opening of the mitochondrial permeability transition pore (92).
1.2.5.2 Mitochondrial Permeability Transition Pore
The mitochondrial permeability transition (MPT) pore is a non-specific
channel suggested to consist of a large protein complex formed by the
VDAC and the adenine nucleotide translocator (ANT), at contact sites between the inner and outer mitochondrial membrane, together with matrix
cyclophilin D (CyP-D) (see (92)) (Fig. 1.3). ANT is a carrier that is located
in the inner mitochondrial membrane and involved in the specific exchange
of ADP and ATP (101). CyP-D is suggested to interact with ANT and
thereby mediate a change in its conformation, which, when triggered by
Ca2+, induces pore opening (102). The ANT/VDAC complex can further
recruit a number of additional proteins like creatine kinase and hexokinases,
and members of the apoptosis regulating Bcl-2 family proteins (see further
section 1.2.7.1) (92), which will regulate the stability and activity of the
MPT pore.
Upon opening of the MPT pore, due to excess of [Ca2+]m but also oxidative
stress, thiol modification, ATP depletion, and high concentrations of inorganic phosphate (50, 102), a channel of approximately 2-3 nm in diameter is
generated, allowing molecules with a Mr of up to ~15 kDa to pass across.
The consequences of opening of the MPT pore are depolarization of the mitochondrial inner membrane, collapse of MMP, further ATP depletion and
release of mitochondrial matrix solutes (including Ca2+) to the cytosol. The
loss of osmotic control, normally maintained by the impermeable inner mitochondrial membrane, results in mitochondrial swelling until the outer mitochondrial membrane disrupts (50). Formation of the MPT pore results not
only in the release of Ca2+, but also in the release of pro-apoptotic proteins
including cytochrome c (103), Smac/DIABLO (104, 105) and apoptosis
inducing factor (AIF) (106), initiating activation of the caspase cascade (c.f.
Fig. 1.4).
1.2.6 Cell death, apoptosis vs. necrosis
Apoptosis and necrosis are two forms of cell death, which are clearly distinguished by morphological and biochemical features. Necrosis, or accidental
cell death, is a pathological process induced by physical or chemical stimuli
and is characterized by cellular disintegration and the release of toxic cellular constituents (107). The result of this process is usually an inflammatory
23
reaction that leads to cellular infiltration, vascular damage, injury to the surrounding tissue, and, in the most severe case, fibrosis. In contrast to necrosis,
apoptosis (programmed cell death) is an active, energy dependent process
that causes typical morphological cell changes, including cell shrinkage,
nuclear condensation, DNA fragmentation, and production of membraneenclosed particles, containing intracellular material known as “apoptotic
bodies” (108-110). In vivo, apoptotic cells are normally recognized by
phagocytes and ingested while the cell membrane is still intact, protecting
surrounding tissues from the harmful intracellular contents of the dying cell
(111).
Apoptosis is a genetically controlled and evolutional conserved form of cell
death that is of importance for normal embryonic development, and for the
maintenance of tissue homeostasis in the adult organism (112). However,
apoptosis does also occur in response to toxins, neurological injury and in
models of degenerative neurological diseases (see (113)). Apoptosis and
necrosis can occur simultaneously in tissues or cell cultures exposed to the
same stimuli (107). The decision of the cell to die by either necrosis or apoptosis is thought to depend largely on the nature and severity of the insult.
Consequently, the intracellular ATP concentration and the status of mitochondrial function are crucial factors that influence the mode of death (114).
1.2.7 Caspases
Caspases constitute a family of cysteine proteases playing a central role in
most cell death pathways leading to apoptosis. A total of 12 caspases have
been identified, and they play distinct roles in apoptosis and inflammation
(115) (Table 1.2). The enzymes are synthesized as inactive proenzymes
(procaspases), which are activated upon proteolysis. Caspases that are involved in apoptosis function either as upstream initiators (caspase-2, -8, -9
and -10) of the proteolytic cascade, activating other caspases, or as downstream effectors (caspase-3, -6 and -7) that cleave cellular targets.
Table 1.2. Subfamily members of the caspase family
Subfamily
24
Role
Members
1
Apoptotis activator
Caspase-2, 8 - 10
2
Apoptotis executioner
Caspase-3, 6 - 7
3
Inflammatory mediator
Caspase-1, 4-5, 11-12
1.2.7.1 Caspase activation
Generally, there are two pathways through which the caspases can be activated: one is the extrinsic, death receptor-mediated pathway; the other is the
intrinsic, mitochondrion-mediated pathway, also called the caspase-9dependent pathway (Fig. 1.4). The extrinsic pathway starts at the plasma
membrane where cell death signals, such as Fas ligand and tumor necrosis
factor (TNF)-2, are specifically recognized by their corresponding death
receptors, Fas or TNF receptor. Multiprotein complexes are then formed to
which procaspase-8/-10 are recruited by the adaptor molecules, Fas- or TNF
receptor associated death domains, forming the death inducing signaling
complex (116). Activated caspase-8/-10 then activate effector caspases,
which cleave target proteins leading to apoptosis. Caspase-8 can also activate the mitochondrion-mediated pathway by truncating Bid, a pro-apoptotic
Bcl-2 family member (see below), into its active form, tBid, which in turn
triggers the activation of the mitochondrion pathway (117). This crosstalk
between the apoptosis-inducing pathways results in amplification of the
death receptor-mediated pathway through mitochondria. In the mitochondrion-mediated pathway, cytochrome c is released from the mitochondria
upon cellular stress or activation of proapoptotic proteins. In the cytosol,
cytochrome c forms a cytosolic apoptosome complex with apoptosis activating factor-1 (Apaf-1) and pro-caspase-9, in the presence of dATP. This results in the activation of pro-caspase-9 and a subsequently activation of procaspase-3 (118) (Fig. 1.4). Furthermore, caspase-2-dependent (119), and
caspase-independent (120) signalling pathways leading to apoptosis in
mammalian cells have been reported.
Apoptosis is an irreversible event and numerous mechanisms exist to control
caspase activity. Bcl-2 family proteins are crucial regulatory factors in apoptosis, and they can be divided into two groups (121). Group I proteins are all
anti-apoptotic proteins, including e.g. Bcl-2 and Bcl-xL, and their main function is to inhibit pro-apoptoic proteins. The group II proteins are proapoptotic proteins, for example Bid, Bak and Bax. Bax and Bak are localized
in the cytoplasm, but in response to cellular damage or apoptosis signals,
they translocate to the mitochondrial outer membrane where they bind to and
accelerate the opening of the VDAC (122) (Fig. 1.4). This in turn changes
the mitochondrial membrane permeability leading to loss of MMP and release of cytochrome c (123, 124). In contrast, the anti-apoptotic Bcl-xL
closes VDAC by binding to the channel (122). The pro-apoptotic activity of
tBid involves triggering the Bax and Bak oligomerization (125, 126) and
interaction with mitochondria (127). On the contrary, it has also been reported that tBid, not Bax, regulates VDAC, by reducing metabolite exchange
leading to mitochondrial dysfunction (128). As compared to Ca2+-induced
stress opening of the MPT pore (see section 1.2.5.2), Bax/Bak-mediated
25
Figure 1.4. Pathways for caspase activation. Ligation of death receptors, such as Fas
or TNF receptor (FasR/TNFR), by their respective cell death signals, Fas ligand
(FasL) or TNF, is followed by activation of the receptors and aggregation of the Fasassociated death domain (FADD) or TNFR-associated death domain (TRADD). The
domains interact with the prodomain of procaspase-8 or procaspase-10, forming a
massive molecule complex known as the death-inducing signal complex that will
induce activation of the caspases. Activated caspase-8/-10 then activate effector
caspases, which cleave target proteins leading to apoptosis. Caspase-8 can also activate the mitochondrion-mediated pathway by cleaving Bid, which, in turn induces
translocation and insertion of Bax and/or Bak into the mitochondrial outer membrane. This is followed by the release of several proteins from the mitochondrial
intermembrane space, including cytochrome c, which forms a cytosolic apoptosome
complex with Apaf-1 and pro-caspase-9 in the presence of dATP. This results in the
activation of caspase-9 and subsequently activation of pro-caspase-3. Cellular stress
can induce release of cytochrome by induction of the MPT pore, either as a direct
affect or by activation of pro-apoptotic proteins in the cytosol. The activity of IAP is
inhibited by Smac/DIABLO. A caspase-independent mechanism involves the release
of AIF. The nuclear location of AIF is linked to chromatin condensation and DNA
fragmentation. DIABLO, direct IAP binding protein with low pI; IAP, inhibitor of
apoptosis protein.
26
conformational change of VDAC does not induce swelling of the mitochondrion and disruption of the outer mitochondrial membrane (124), suggesting
two distinct mechanisms for release of cytochrome c to the cytosol. The
activation and inactivation of caspases are also regulated by other factors,
such as inhibitor of apoptosis proteins (129) and calpains (see section
1.2.7.3).
1.2.7.2 Caspase substrates
Like calpains, caspases do also have a finite number of cellular protein substrates, including cytoskeleton proteins, enzymes involved in signal transduction, cell cycle proteins and nuclear DNA repairing proteins (Table 1.3).
The cleavage of cytoskeleton substrates by caspases is almost surely involved in the alterations in cell morphology that occur in cells undergoing
apoptosis, featuring cell rounding and membrane blebbing. However, there
is growing evidence that activated caspases are suggested to play an important role in axonal injury and degeneration. In an animal model of traumatic
brain injury, release of cytochrome c and activation of caspases have been
shown to participate in, and contribute to, axonal injury (130).
Table 1.3. Examples of caspase substrates
Substrate
Reference
Cytoskeleton proteins
Actin
(131)
Spectrin (αII and βII)
(132)
Tau
(133)
Signal transduction enzymes
Protein kinase C
(134)
Phospholipase A2
(135)
Stress-response enzymes
DNA-repair enzyme PARP*
(136)
Others
Apoptotic regulatory factors (Bcl-2, Bcl-xL)
(137, 138)
calpastatin
(139)
NF-κB
(140)
*PARP=poly(ADP-ribose) polymerase
27
The involvement of caspases has also been reported for hippocampal neurons undergoing β-amyloid-induced neurite degeneration, before cell bodies
show any sign of toxicity (141). Furthermore, caspase-induced cleavage
products, and the active form of caspase-3, are expressed in degenerative
axons of dopamine neurons following axotomy (142), and in a model of
HIV-associated peripheral neuropathy (143), the axonal degeneration of
sensory fibers is proposed to be dependent on local activation of caspases in
the axon.
1.2.7.3 Cross talk between calpains and caspases
Several features indicate the existence of calpain and caspase interactions.
The two families of cysteine proteases have resembling substrate specificity
(see Tables 1.1 and 1.3), and αII-spectrin, for example, is a well-known substrate for both the proteases. Calpains produce two breakdown products of
145 and 150 kDa (SBDP 145 and 150), (144), while caspases produce a different fragment of 150 kDa (SBDP 150) and a distinct fragment of 120 kDa
(SBDP 120), (132) (Fig. 1.5A).
Figure 1.5. Crosstalk between calpains and caspases. Schematic illustration of caspase- and calpain-induced cleavage of αII-spectrin generating their respective spectrin breakdown products (SBDP) (A). Cleavage of the endogenous calpain inhibitor
calpastatin leads to activation of calpains. Activated calpains, in turn, can cleave
several pro-caspases, which can either activate or inactivate their function (B).
28
Both calpains (69) and caspases (137, 138) can also cleave the protective
anti-apoptotic proteins Bcl-xL and Bcl-2, and convert them to lethal proapoptotic proteins capable of causing release of cytochrome c from mitochondria. Further, calpains can cleave pro-caspases, resulting in either caspase inhibition (71, 145) or activation (72, 73) (Fig. 1.5B). Conversely, caspases might regulate calpain activity by mediate degradation of calpastatin,
the endogenous specific inhibitor of calpains, in cells (139, 146). Calpains
can also down-regulate the mitochondrion-mediated cell death pathway by
cleavage Apaf-1 (70).
1.2.8 Cellular redox balance
It is generally assumed that oxidative stress and an imbalanced redox homeostasis are functionally the same and occur when there is either an overproduction of ROS or a deficiency in the total sum of antioxidant defense.
Depending on the severity, oxidative stress can lead to cell damage, apoptosis or necrosis.
1.2.8.1 Reactive oxygen species and antioxidant systems
Oxidation is the loss of electrons from an atom or molecule, and free radicals
are species that contain one or more unpaired electrons. Hence, reactive
oxygen species (ROS) are free radical derivatives of molecular oxygen (O2).
However, also the non-radical hydrogen peroxide (H2O2) belongs to the
ROS. Free radicals are generated in vivo as byproducts of normal metabolism, but are also produced when an organism is exposed to ionizing radiation and drugs capable of redox cycling, or to xenobiotics (substances that
are foreign to the body) that can form free radical metabolites in situ (147).
When ROS are generated in living systems, a wide variety of antioxidant
mechanisms come into play (Fig. 1.6). The superoxide anion (O2• ) is converted to H2O2 by superoxide dismutase (SOD) (148). There are two types of
SOD in eukaryotic cells; the manganese-containing SOD (MnSOD) located
in mitochondria, and the copper and zinc SOD (Cu/ZnSOD) occurring primarily in the cytosol. Both O2• and H2O2 can damage cellular targets, but
most of their damage is thought to be due to their conversion into more reactive species, including the hydroxyl radical (•OH) (149, 150). Generation of
O2• and H2O2 in close proximity to active redox pools of copper and iron
(Cu+ and Fe2+) provide the requisite conditions for the Fenton chemistry
leading to •OH formation. •OH is able to diffuse from its site of formation
and to attack and harm most cellular molecules. Therefore, in order to avoid
the formation of •OH in the first place, H2O2 is either quickly converted to
H2O by GSSG peroxidase, a reaction where reduced glutathione (GSH) is
used as an electron donor and oxidized to glutathione disulfide (GSSG), or
detoxified by catalases (151).
29
Additional members of free radicals are derived from the interaction of carbon- and nitrogen-based radicals with O2. These include organic peroxides,
alkoxyl (RO•) and peroxyl (RO2•) radicals, as well as peroxynitrite (ONOO )
and nitric oxide (•ON) (151). Cells contain multiple sites for ROS production
and major cellular sources of ROS are the mitochondria (152), which produce significant amounts of O2• from the ETC. Other sources of ROS production identified in the CNS are the P450 oxidases, monoamine oxidases,
nitric oxide synthase, and NADPH oxidase of macrophages and microglia
(151). NADPH oxidase catalyzes the reduction of O2 to O2• , by using
NADPH as the electron donor.
Figure 1.6. Elimination of O2•- by conversion to H2O2 by SOD. H2O2 is detoxified to
H2O and O2 by GSH peroxidase or catalase. Alternatively, H2O2 is degraded to •OH
and OH- in the Fenton reaction.
1.2.8.2 Glutathione
Glutathione (L-γ-glutamyl-L-cysteinyl-glycine; GSH) is a tripeptide derived
from glutamic acid, cysteine and glycine (Fig. 1.7A). Due to the characteristic structural features of GSH, a γ-glutamyl linkage and a sulfhydryl group,
the tripeptide is involved in a number of cellular redox functions (153). Its
low oxidation potential and thus high reducing activity, makes the GSH thiol
a potent antioxidant (151) (c.f. Section 1.2.8.1). Besides being an antioxidant, GSH also function as an intracellular reducing agent for maintaining
thiol groups of other molecules, and participates in the detoxification of
various xenobiotics, by forming conjugates in a reaction catalyzed by glutathione S-transferase (153) (Fig. 1.7B).
GSH is expressed ubiquitously and found in millimolar concentrations (1-10
mM) intracellularly (154). In cells, total GSH can be free or bound to pro30
teins, leading to the formation of glutathionylated proteins (see (153)). In
normal conditions, GSH is the predominating form over the GSSG form,
with an approximate 1:100 relationship. However, if stress levels in the cell
increase, or if other factors limit the reduction of GSSG back to GSH, the
GSSG form may accumulate and the thiol redox status of the cell will shift
(155). Thus, the cellular redox status depends on the relative amounts of
GSH and GSSG ([GSH]/[GSSG]) and appears to be a critical determinant in
cells. The utilization of GSH in different essential cellular functions can lead
to depletion of GSH pools that ultimately must be replenished. This is done
through two major routes: (i) reduction of GSSG via GSSG reductase, and
(ii) de novo synthesis of GSH.
1.2.8.3 Synthesis of glutathione
GSH is synthesized from its constituent amino acids by the sequential action
of two ATP-dependent enzymes (Fig. 1.7B). The first and rate-limiting enzyme, glutamate cysteine ligase (GCL, also called γ-glutamyl-cysteinesynthetase, γ-GCS), is a heterodimeric protein composed of a catalytic
(GCLC) and a regulatory (GCLM) subunit (156). γ-GCS combines glutamic
acid and cysteine, through condensation of the γ-carboxyl group of glutamic
acid with the α-amino group of cysteine, forming the dipeptide γ-Glu-Cys
(154). The second enzyme, GSH synthetase, catalyzes the final step in the
synthesis, by activation and condensation of the α-carboxyl of cysteine with
glycine. Both enzymes are exclusively cytosolic and the rate of GSH synthesis is controlled by the level of γ-GCS, by the availability of L-cysteine
(which is often limiting), and by feedback inhibition of γ-GCS by GSH (see
(157)).
1.2.8.4 Mitochondrial glutathione
The mitochondria do not contain enzymes for GSH synthesis (157) and all
mitochondrial GSH needed for the antioxidant defense, i.e. destruction of
H2O2, must be translocated from the cytosol. A rapid exchange occurs between mitochondrial and cytosolic GSH, and the mitochondrial GSH homeostasis is affected by a multicomponent transport system that includes a
high- and a low-affinity component for uptake of GSH (158). Mitochondrial
GSH can also be conserved during periods of cytosolic depletion (159).
1.2.8.5 Transport of glutathione
Cellular export of GSH may provide reducing compounds to the immediate
environment of the cell membrane, and thereby protect it from oxidative
damage. But the transport may also facilitate transport of amino acids (especially cysteine), peptides and amines out from the cell (153). The major
transported form is GSH, rather than GSSG. However, small amounts of
GSSG may be transported, and such a transport increases when the intracellular GSSG level is increased (159). When GSH synthesis is inhibited, the
31
cellular level of GSH is decreased because the export of GSH continues even
in the absence of a significant re-synthesis. The fact that the rate of decreased cellular GSH is similar to the rate of export, indicates that there is
usually little intracellular degradation of GSH. The GSH breakdown occurs
extracellularly and is catalyzed by γ-glutamyl transpeptidase and dipeptidases, bound to the external surface of the cell membrane (159). The reason
why GSH is protected from intracellular protease digestion might be the γglutamyl linkage between glutamic acid and cysteine, instead of a regular
peptide bond at the α-carbon (154).
Figure 1.7. Structure of GSH (A). Biosynthesis and the utilization of GSH in detoxification, antioxidant and reduction reactions (B). The latter generating the glutathione redox cycling. The re-conversion of GSSG back to GSH by GSSG reductase is dependent on NADPH as a cofactor.
1.2.8.6 Glutathione S-transferase
Glutathione S-transferase (GST) is a family of Phase II detoxification enzymes that catalyzes the conjugation of GSH to a broad variety of physiologic and xenobiotic substances (Fig. 1.7B). In most cases, this conjugation
step inactivates a reactive and potentially dangerous metabolite, enables its
excretion and thereby protects cells from chemical insult and oxidative stress
(160). The enzyme is most abundant in the liver, and human GSTs are divided into three main families; cytosolic, mitochondrial and membranebound microsomal (161). The mammalian cytosolic family of GSTs exists as
monomers and is further divided into seven classes based on 60% or higher
32
identity within a class, focused mainly on the conserved N-terminal domain.
The GSTs are active as homo- or heterodimers, and they interact with the
thiol group of GSH via the catalytically active tyrosine, cysteine or serine
residue in the N-terminal (162).
1.3 Gliotoxin
Gliotoxin (Fig. 1.8) is a highly toxic secondary metabolite produced by several species of filamentous fungi (Table 1.4). Gliotoxin belongs to a class of
fungal metabolites called epipolythiodioxopiperazin (ETP) (163), which are
cyclic dipeptides characterized by the presence of an internal disulfide
bridge that imparts all known toxicity of these molecules (164). Gliotoxin
was the first ETP to be reported and is today one of the best characterized
members of this group of toxins. The name gliotoxin is derived from its
identification as a metabolite of Gliocladium fimbriatum (165, 166), although this fungus was later discussed to possibly be a strain of Trichoderma
spp. (167, 168).
Table 1.4. Gliotoxin producing fungi
Producing organism
Reference
Aspergillus chevalieri
(169)
Aspergillus fumigatus
(170)
Candida albicans
(171)
Gliocladium fimbriatum
(172)
Penicillium obscurum
(173)
Trichoderma virens
(168)
1.3.1 Proposed biosynthesis of gliotoxin
In spite of almost 50 years of research since the structure of gliotoxin was
first described (174), very little is known about the biosynthesis of ETPs, or
even their primary role in the biology of the organisms that produce them.
All natural ETPs isolated to date contain at least one aromatic amino acid
and the diketopiperazine ring is derived from a cyclic dipeptid (175). In the
predicted biosynthesis of gliotoxin (Fig. 1.8), only one intermediate has been
isolated, the cyclo-L-phenylalanyl-L-serine. It is produced in the first reaction between the amino acids phenylalanine and serine, which is catalyzed
by the non-ribosomal dioxopiperazine synthase (176). Subsequently, a series
of sulfurisation, oxidations, and methylation are predicted. Introduction of
33
the sulfur atoms into the core ETP moiety is poorly understood. Methionine,
cysteine and sodium sulfur can all act as sources of sulfur, although cysteine
is thought to be the direct donor (177).
The genes that encode for enzymes in biosynthesis of fungal secondary metabolites are usually clustered in the genome (178). In 2004, the first report
of the genes responsible for biosynthesis of an ETP was published (179), and
recently, a cluster of 12 genes involved in biosynthesis of gliotoxin was
identified in A. fumigatus (176).
O
OH
O
NH2
+
A
O
B, C
NH
O
HN
OH
OH
HO
O
NH2
O
O
HN
O
NH
S S
HN
OH
S S
O
O
O
D
NH
OH
E
NH
S S
N
OH
OH
O
N
CH3
S S
N
OH
OH
O
Gliotoxin
Figure 1.8. Proposed pathway for gliotoxin biosynthesis. The only known intermediate is cyclo-L-phenylalanyl-L-serine. It is produced in the first step, which is likely
to involve condensation of phenylalanine and serine, and catalyzed by dioxopiperazine synthase (A). Subsequently, a series of sulfurisation (B), oxidations
(C, D) and methylation (E) are predicted. The bracketed compounds are suggested
and have not been isolated.
1.3.2 Biological activities of gliotoxin
Gliotoxin possesses a spectrum of biological activities and did early attract
attention because of its antimicrobial (antibacterial, antifungal and antiviral)
properties, by inhibiting proliferation of some bacteria (180), fungi (168) and
viruses (181-183). A renewed interest in the biological activity of gliotoxin
followed by the observation that the toxin displayed immunosuppressive
activity, both in vitro (184) and in vivo (185), by its induction of apoptotic
cell death (186-188) and the inhibition of activation and proliferation of T
and B cells (189). Due to the antimicrobial and immunosuppressive properties, gliotoxin was early suggested to be useful clinically, but the systemic
toxicity of the compound later showed it unsuitable for that purpose (190).
34
Gliotoxin is reported to be cytotoxic to mouse L929 fibroblast cells (191)
and hog renal LLC-PK1 cells (192), as well as to be genotoxic both in bacterial test systems and in mammalian cells (193). Furthermore, gliotoxin affects rat liver cells (194), and the liver was also the primary target in an
acute toxicity study with hamsters (195).
Gliotoxin has also been found to be associated with some diseases attributed
directly or indirectly to fungal infections. Gliotoxin has been isolated from
lung tissues from turkeys with “airsacculitis” (196), detected in women with
vaginal Candida infection (197), and suggested to play an important role in
the pathogenesis of invasive aspergillosis (198), a disease that will be further
discussed in section 1.4.1.
1.3.3 Cellular uptake and metabolism
Gliotoxin is rapidly taken up into cells (199), and its unique structural features confer the ability of a reversible cellular accumulation by a redox
mechanism that results in very high intracellular concentrations and thus,
efficacy in inducing cell death (200). The uptake of gliotoxin requires the
bridged disulfide bond, demonstrated by abrogated cellular uptake after extracellular reduction of gliotoxin. Toxin entry into cells is followed by a
rapid reduction to the cell-impermeable dithiol form by GSH. Accumulation
of gliotoxin as the reduced form in cells suggests a mechanism of rapid
metabolic conversion of the toxin, driving accumulation down a concentration gradient until equilibrium is reached (Fig. 1.9). Hence, cellular uptake of
gliotoxin is also GSH-dependent and loss in cellular GSH parallels decreased uptake (200). In conclusion, the active concentration of gliotoxin in
cells is dependent on an external oxidizing environment and the intracellular
reducing milieu maintained by GSH. This redox uptake mechanism of gliotoxin may also explain the effect of cell density on efficacy of killing by
ETP toxins. As cell density decreases, more toxin per cell is at equilibrium
between reduced and oxidized form (175).
Figure 1.9. Scheme for the proposed redox mechanism for cellular uptake and accumulation of gliotoxin. Uptake of gliotoxin requires the oxidized disulfide form,
(GliotoxinOx), whereas the intracellular concentration of gliotoxin is dependent on
the intracellular reducing milieu, driving accumulation of gliotoxin as the reduced
dithiol form (GliotoxinRed) down a concentration gradient until equilibrium is
reached.
35
Despite the subversion from a protective function of GSH, to facilitate the
uptake and accumulation of gliotoxin, GSH is suggested to be involved in
the possible detoxification of gliotoxin. Hepatocytes, a liver cell type with
high GSH levels, has been reported to rapidly and extensively metabolize
gliotoxin, and proposed metabolites are the reduced dithiol form of gliotoxin
and gliotoxin-GSH conjugates (194, 201). GSH forms adducts with gliotoxin
via a thiol disulfide exchange system with the cyclic disulfide moiety of
gliotoxin (202).
1.3.4 Toxic mechanisms
ETPs do not have exclusive protein targets but their molecular structure with
the disulfide bond makes them very reactive and they can therefore affect
numerous cellular functions. At least two mechanisms are suggested for the
biological activities of gliotoxin, and they will be further described below.
1.3.4.1 Covalent interactions
As a disulfide, gliotoxin is able to form conjugates with other molecules with
free thiol groups, and thereby alter their functions. Putative targets for induction of apoptosis by gliotoxin include thiol-dependent enzymes such as
creatine kinase (203) and Ras farnesylating enzyme (204, 205), but also mitochondria, presumably by thiol-modification of the MPT pore (206-208).
Another target for gliotoxin is the transcription factor nuclear factor-κB (NFκB) (189) that has an important function in the activation of the immune
system. Gliotoxin inhibits the activation of NF-κB, probably by the formation of mixed disulfides with 20S proteasomes (209) that are central for degradation of the inhibitory subunit IκB-α, a key feature during NF-κB activation (210).
1.3.4.2 Oxidative effects
The property of ETP compounds to go through a redox cycle in the presence
of an appropriate reducing agent has led to an array of suggested mechanisms of gliotoxin-induced toxicity. These involve the production of O2• and
H2O2, when gliotoxin is reoxidized to the disulfide form in the presence of
molecular oxygen. ROS generated by gliotoxin during redox cycling have
been shown to damage plasmid and cellular DNA (211), as well as to induce
cell death in LLC-PK1 cells (192), macrophages (212), and human hepatic
stellate cells (207). In contrast, one study has shown that gliotoxin inhibits
production of O2• in human neutrophils by target NADPH oxidase (213).
Gliotoxin has also been reported to increase [Ca2+]i by oxidation of cysteine
residues in skeletal muscle RyR (214), and by affecting a redox-sensitive
Ca2+ channel in the plasma membrane in thymocytes (215).
36
1.4 Aspergillus fumigatus
The fungal genus Aspergillus is a group of moulds, also called filamentous
fungi, which are found worldwide. These kinds of fungi can grow over a
wide range of water contents, but storage fungi such as Aspergillus fumigatus grow if the moisture content is higher than 15% (1). A. fumigatus is a
thermophilic mould that grows on soil, hay, rice, maize, cereal grain, compost, and wood, to mention some substrates, (1, 216-218). The properties of
A. fumigatus have been extensively studied, and recently, the complete genome sequence of A. fumigatus was identified (219).
Gliotoxin has been detected as one of the most toxic second metabolites
produced by A. fumigatus. Other toxins that can be produced include verruculogen and fumitremorgins, which have been reported to be neurotoxic
by causing tremor (220). Tremorgenic mycotoxins constitute a group of
lipophilic molecules that cross the blood-brain barrier and can thus act on the
CNS, causing sustained tremors, convulsions and even death (221).
1.4.1 The pathogenecity of A. fumigatus
Diseases caused by Aspergillus species, and A. fumigatus in particular, are
called aspergillosis and vary from an allergy-type of illness to lifethreatening generalized infections. A. fumigatus can infect and germinate in
animals and humans and is today the most prevalent airborne fungal pathogen. The respiratory tract is regarded as the main entrance for A. fumigatus
to the human or animal body, and the airborne conidia are small enough (2-3
µm) to reach the alveoli in the lung (222). Then, if the conidia can overcome
the immune defense mechanisms in the lung, they germinate and produce a
branched vegetative mycelium that invades the lung tissues. The severity of
aspergillosis is determined by various factors, but one of the most important
is the state of the immune system of the person (223). As a product of A.
fumigatus, it is possible that in situ production of an immmunosupressive
agent such as gliotoxin, may further compromise an infected host and thus,
play an important role in the etiology of diseases such as aspergillosis. Indeed, gliotoxin has been detected in lungs and sera from mice with experimentally induced invasive aspergillosis, and also in sera from patients with
documented invasive aspergillosis (224). Invasive aspergillosis is mainly
restricted to the respiratory system, but some Aspergillus genera, including
A. fumigatus, can also invade the CNS, causing lethal cerebral aspergillosis
(225, 226). The cerebral conidia may reach the brain from the nasal sinus, or
are blood borne from the original infection in the lungs or gastrointestinal
tract.
37
1.4.2 Risk assessment of exposure to A. fumigatus and
gliotoxin
A. fumigatus is a common contaminant in food- and feedstuffs and thus, the
risk of exposure is high for humans and animals. A. fumigatus is one of the
major contaminating moulds in silos (227) and does also belong to the group
of indicator microorganisms typical of moisture-damaged buildings (228).
Furthermore, it occurs in indoor air in sawmills, where the temperature in the
drying chambers varies between 55°C and 60°C, and the relative humidity is
high (216). Since A. fumigatus has high sporulating activity, which can result
in high concentrations of conidia in the air indoors and outdoors (222), livestock and humans are faced with the dual hazard of high levels of conidia in
the atmosphere, but also the exposure to potent mycotoxins (including gliotoxin) in contaminated material.
Isolated A. fumigatus strains from moldy silage (229), building materials
(228) and blue mussel (230), have all been reported to produce gliotoxin.
Gliotoxin has also been isolated from the udder of a cow that was naturally
infected with A. fumigatus (231), and detected in hay samples associated
with cases of intoxication in camels (232). Furthermore, neurological disorders have been observed in humans and animals that have been exposed to
material contaminated with gliotoxin-producing species of fungi (216, 233).
Since there is a high risk that both humans and animals are exposed to gliotoxin, it is most important that the neurotoxic properties of the toxin are
evaluated.
1.5 Detection of mycotoxins in feed
In line with increasing demands on proper food and feed safety, the needs
for monitoring contamination of mycotoxins have increased. Fungi produce
a huge amount of mycotoxins, which together constitute a heterogenic group
concerning structure and biological effects (2). This phenomenon can make a
problem when analyzing samples that are suspected to be of poor hygiene
quality. The analyses of feed most commonly used today are mainly focused
on microbiological investigations, considering the occurrence of bacteria,
yeast or fungus. However, the analyses do not give any information about
occurrence of toxic metabolites or other toxic properties. An alternative is to
use chemical detection methods, like high-performance liquid chromatography (HPLC) and/or thin-layer chromatography (234, 235), and analyze for
specific mycotoxins in a sample. However, these methods are expensive, the
sensitivity of detection between mycotoxins varies, and they are not practical
to use for screening for all known mycotoxins. Instead, a strategy would be
to use bioassays, i.e. assays based on a biological activity, as a first screening
38
step. This approach would make it possible to sort out potentially harmful
samples which may deteriorate the health of animals or humans. The possibility of detecting toxicity of mycotoxins by the use of cellular in vitro methods has been confirmed in a variety of cell lines of animal and human origin
(236-240). However, most cytotoxicity studies have been carried out using
pure mycotoxins, and only few have been performed using complex
samples, like inoculated substrates or contaminated animal feed (241-243).
This is a necessary and important step in the evaluation of bioassay applicability for toxicity screening of feed samples.
39
2. Methodological considerations
The methods used in the thesis are described in detail in each paper. In this
part, theoretical aspects of the methods will be discussed.
2.1 Cell system
Throughout this study, native or differentiated human neuroblastoma SHSY5Y cells have been used. Neuroblastoma cell lines provide a convenient
model for studies on neuronal functions including differentiation, neurodegeneration and neurotoxicity.
2.1.1 Native SH-SY5Y cells (Paper IV)
The human neuroblastoma SH-SY5Y cell line is a subclone of the SK-N-SH
cell line that in 1971 was established in cell culture from a bone marrow
aspirate of a metastatic neuroblastoma from a 4-year old girl (244). A neuroblastoma is most commonly a childhood solid tumor, composed of primitive
cells derived from precursors of the autonomic nervous system (245). In
their native form (Fig. 2.1A), SH-SY5Y cells express adrenergic (catecholamine) neurotransmitter release enzymes (246), neuron-specific enolase
(247), the major calpain isoforms, as well as calpastatin (248), and they have
a low resting membrane potential (249).
Figure 2.1. Human neuroblastoma SH-SY5Y cells, in their native form (A), and
differentiated with 1 µM RA for 72 h (B).
40
2.1.2 Differentiated SH-SY5Y cells (Paper I-IV)
The native SH-SY5Y cells can be further differentiated into a mature neuronal phenotype by incubation with all-trans retinoic acid, a naturally occurring hormone (250). Retinoic acid (RA) is a vitamin A derivative and belongs to a family of small hydrophobic molecules (retinoids) that play essential roles in processes such as vision and cellular differentiation (251). RA
binds to two families of nuclear receptors; the RA receptors (RARs) family,
binding both all-trans RA and 9-cis RA, and the retinoid X receptors (RXRs)
family, preferentially binding 9-cis RA (252, 253). RARs appear to function
predominantly as heterodimers with RXRs (254) and RAR/RXR hetrodimers
bind to retinoic acid-responsive elements on the target genes, resulting in
transcription activity. SH-SY5Y cells express all three subtypes of RARs (α,
β, and γ) (255). The most commonly measured characteristic of differentiation is neurite extension, defined as a process whose length equals or exceeds the cell body diameter (256) (Fig. 2.1B). Neurites are similar to the
axons and dendrites of fully differentiated neurons (245), and RA promotes
SH-SY5Y cells to establish a neurite network expressing a mature morphology with respect to cytoskeleton organization (257). In addition, RA-induced
differentiation of SH-SY5Y cells favours muscarinic acetylcholine receptor
expression (258), membrane excitability (259), and calpain activity (248).
2.2 Preparation of A. fumigatus extracts (Paper IV)
In Paper IV the possibility of using SH-SY5Y cells for detecting cytotoxic
response of A. fumigatus-produced mycotoxins in feed samples was investigated. For that purpose, conidia suspensions of A. fumigatus spores (CCUG
17460) were inoculated on different substrates. First, to confirm that the A.
fumigatus strain was a potent mycotoxin producer, the growth medium
Czapek-Dox broth (CzDox-broth) was used, since it has been reported to be
an optimal substrate for the production of gliotoxin in vitro by A. fumigatus
(260). Indeed, HPLC analyses of purified samples from the inoculated substrate demonstrated to contain gliotoxin. Next, the more complex substrates,
maize and animal feed grains, were inoculated. Here we also wanted to examine the significance of moisture content and incubation time for the appearance of mycotoxin production, i.e. cytotoxicity of contaminated material. The water content in the maize samples was adjusted to 13, 22 or 32%
and thereafter incubated with conidia for 2 weeks. While for the feed grain
samples, the water content was set to 22% in all samples, but instead inoculated for 3 days, 2 or 4 weeks. The water content in samples was controlled
every two days and ultrapure deionised Super-Q water was added to adjust
the water content to the selected parameters. Following extraction and filtration of the maize and feed samples, the filtrates were further purified for
41
gliotoxin analyses with solid-phase extraction using ISOLUTE silica columns SI (Sorbent AB, V. Frölunda, Sweden). Re-dissolved filtrates were
transferred to pre-conditioned columns, in which gliotoxin was thought to
bind to the sorbent. The washed and eluted extracts were then analyzed in
accordance to gliotoxin, however, it was discovered that the HPLC system
not was optimized for such complex samples.
All preparation of substrates, inoculations, and work up with extractions
were performed at the Department of Animal Feed, National Veterinary
Institute (SVA) in Uppsala, and thereafter transported to the Department of
Neurochemistry at Stockholm University, for cellular analyses.
2.3 Cell treatments
2.3.1 Neurotoxic properties of gliotoxin (Paper I-IV)
In all four papers, the effects of gliotoxin (CAS No. 67-99-2) on differentiated SH-SY5Y cells have been studied. Native cells were differentiated for
72 h with 1 µM all-trans retinoic acid (RA) in chemically defined medium
without serum (N2), (N2/RA), to express neurospecific properties as described above. Thereafter, the exposure to N2/RA without (control) or with
gliotoxin was initiated and proceeded for another 72 h. This is an exposure
period that mimics a subchronic exposure situation in vivo, needed for specific functional and pathological signs of axonopathy (262). The use of serum-free N2 medium is advantageous in both to achieve increased differentiation and to avoid unwanted interference with serum proteins, which may
affect the results (261).
2.3.1.1 Co-incubation with inhibitors (Paper I and II)
Cells were incubated with gliotoxin in the presence of L-buthioninesulfoxamine (BSO) to study how a reduction in total cellular GSH parallels
the toxicity of gliotoxin, as suggested by Bernardo and co-workers (200),
(Paper I). BSO hinders the first step in the synthesis of new GSH, by binding
its S-butyl group to the site at γ-GCS normally occupied by L-cysteine (263),
and thereby decreases the GSH levels.
The role of calpains and caspases in gliotoxin-induced toxicity was also examined by the use of inhibitors (Paper II). The specific calpain inhibitor
benzyloxycarbonylleucyl-norleucinal (calpeptin) is a short, hydrophobic Nblocked dipeptidyl aldehyde lacking charged residues, and is thus able to
penetrate membranes by passive diffusion (264, 265). Since aldehyde inhibitors are reversible (266), calpeptin had to be added daily to the cells during
42
exposure. On the other hand, the broad-spectrum caspase inhibitor carbobenzyloxy-Val-Ala-Asp-α-fluoromethylketone (Z-VAD-fmk) is irreversible
(266), and was added to the cells only at the start of gliotoxin exposure. Cysteine protease inhibitors, like calpeptin and Z-VAD-fmk, arrest the activity
of their substrates by covalent interaction between the active-site cysteine
thiol group and an electrophilic center of the inhibitor (265, 267).
2.3.2 Toxic activity in animal feed (Paper IV)
In the attempt to use the cell model as a bioassay for cytotoxic evaluation of
mouldy feed substrates, native (undifferentiated) SH-SY5Y cells were used.
Differentiated cells are not applicable for rapid screening assays since they
have to undergo the differentiation procedure of 72 h before exposure. In
addition, the cells were plated in 96-well microtiter plates, allowing direct
reading of the samples in a multiscan spectrophotometer.
HPLC analyses of the inoculated CzDox-broth samples showed that one ml
filtrate from the sample that had been inoculated for 87 h was equal to 320
ng (0.98 nmol) gliotoxin. From that filtrate, a dilution series was performed
covering cytotoxic (>0.1 µM) as well as non-cytotoxic concentrations of
gliotoxin in SH-SY5Y cells, and from which all CzDox-extracts as well as
pure gliotoxin were diluted. The content of gliotoxin was not possible to
determine in the maize and animal feed samples, and these extracts were
dissolved to highest possible concentration according to solubility.
2.4 Endpoints
2.4.1 General cytotoxicity (Paper I-IV)
Basal cytotoxicity is the sum of more subtle effects, and may be reflected as
effects on the energy status, inhibition of proliferation, or cell death. Since
this in vitro endpoint has been shown to correlate well to human lethal blood
peak concentration, it may reflect the toxic potency for most chemicals
(268). One method that may indicate acute systemic toxicity is the determination of the total cellular protein content after 24-96 h of exposure to a test
substance, as compared to unexposed cells. Here, the protein content levels
reflect the amount of cells per well after treatment. For the protein determinations we used a colorimetric assay of Lowry and co-workers (269) with
some minor modifications (270). The method relies on the mixture of protein
lysates with CuSO4-solution and folin reagent, in which copper ions (Cu2+)
bind to the peptide bonds, resulting in a reduction to the Cu+ form. Cu+ then
catalyzes oxidation of aromatic amino acids in the samples, which changes
43
the Folin-Ciocalteu reagent to a blue colored compound that is measurable at
690 nm.
2.4.2 Neurite degeneration (Paper I, II and IV)
Effect on the neurites may be a more sensitive indication of neurotoxicity
than neuronal cell death. Quantitative and qualitative morphological investigations of neurites, including number, length and diameter, as well as cell
swelling and structural alterations, have been proven to be valuable in vitro
methods for the assessment of neurotoxicity, and used for classification of
compounds generating axonopathy in vivo, for example organophosphorus
compounds (271, 272), MeHg (273), acrylamide and triethyltin chloride
(274). To reveal if gliotoxin is an axonopathy-inducing toxin, the neurite
degenerative effects of gliotoxin were estimated. The morphological method
used for that purpose was to compare the ratio of neurites per cell body in
exposed cultures, in contrast to unexposed control cells (270, 274), an endpoint shown to correlate well with peripheral axonopathy in vivo (275).
2.4.3 The cellular redox balance (Paper I)
The cellular levels of GSH were measured by the DTNB-GSSG reductase
recycling assay for GSH and GSSG (276). This is a sensitive and specific
enzymatic procedure for assaying total glutathione (GSH and GSSG, in GSH
equivalents). As indicated in the reactions below, it is based on the oxidation
of GSH by DTNB (5,5´-dithiobis(2-nitrobenzoic acid)) to give GSSG with
stochiometric formation of TNB (5-thio-2-nitrobenzoic acid), and the conversion back to GSH by the action of the highly specific GSSG reductase
and NADPH. The rate of TNB formation in the samples is followed spectrophotometrically at 412 nm and is proportional to the sum of GSH and GSSG
present.
2GSH + DTNB → GSSG + TNB
GSSG + NADPH + H
+
    
→ 2GSH + NADP +
GSSG reductase
(1)
(2)
The determination of GSSG in biological samples can be more difficult because GSSG is normally present in very low levels as compared to GSH
(ratio about 1:100). Since GSH is essential for maintenance of intracellular
redox balance, oxidation must be kept at a minimum. The assay described
above for total glutathione may be used also for GSSG, but only after treating samples with agents that protect thiol groups and prevent disulfide formation. For this purpose we used 2-vinyl pyridine, which, at the concentration used, does not inhibit GSSG reductase and interfere with the assay.
44
One unit of GSSG reductase activity is defined as the amount of enzyme
catalyzing the oxidation of 1 µmol NADPH per minute (277), and hence, the
activity of GSSG reductase was calculated by measuring the rate at which
NADPH was oxidized, according to reaction (2), followed by a decrease in
absorbance at 340 nm (277).
2.4.4 Physiological changes (Paper II and III)
2+
2.4.3.1 Intracellular Ca concentration
In Paper II, [Ca2+]i was measured by the Fura-2 fluorescence technique
(270). Fura-2 is a Ca2+ complex-binder (278) that is taken up into cells by
passive diffusion as a Fura-2-acetoximethylester (Fura-2/AM) (279). Inside
the cell, Fura-2/AM is hydrolyzed to Fura-2, which cannot pass cellular
membranes and is therefore accumulated in the cytoplasm. The fluorescence
emission at 510 nm, after excitation at 340 nm, is used as an assessment of
[Ca2+]i. The [Ca2+]i was calculated according to (280), by using consecutive
additions of ionomycin and manganese and the approximate dissociation
constant, Kd = 224 nM for Ca2+ and Fura-2 (278).
[Ca2+]i
Q
∆FI
= Kd / (1/Q-1)
= ((FI-FImin ) – (0.35∆FI)) / (0.65∆FI)
= FImax - FImin
2.4.3.2 Rate of protein synthesis
One way to measure the rate of protein synthesis is to determine the amount
of [4,5-3H]leucine ([3H]leu) incorporated in cells during two hours (Paper
III). The proteins are then precipitated with trichloracetic acid (TCA) to release [3H]leu that has not been incorporated into proteins. The TCA solution
is transferred to a scintillation vial, and the precipitated proteins remaining in
the well are dissolved in NaOH. The protein fraction is then transferred to
another scintillation vial, and both fractions are radioactively measured in a
beta-scintillation recorder. By co-measuring the cellular proteins levels in
the cultures, the rate of protein synthesis (PS) can be calculated by using the
equation (281):
PS =
total protein (mg) × cpm in NaOH fraction
cpm in TCA fraction
45
2.4.5 Mitochondrial effects (Paper III)
2.4.5.1 Level of cellular ATP
To examine quantitative cellular ATP levels, a bioluminescence assay with
recombinant firefly luciferase and its substrate D-luciferin was used (282).
As shown below, firefly luciferase catalyses the oxidation of D-luciferin in
the presence of ATP, Mg2+ and O2 to generate oxyluciferin and light, which
can be detected by measuring the luminescene and thereby the amount of
ATP in the samples can be calculated indirectly.
luciferase + Mg 2+
luciferin + ATP + O 2     → oxyluciferin + AMP + CO 2 + light
2.4.5.2 Mitochondrial membrane potential
The MMP was determined by selectively staining of cells with two different
cell-permeable and mitochondrion selective probes. Mito Tracker Red
(MTR) is a red-fluorescent potential-sensitive dye that stains mitochondria
dependent upon the membrane potential. The green-fluorescent mitochondrial dye, Mito Tracker Green (MTG), interacts with mitochondrial lipids
regardless of mitochondrial membrane potential (283). After labelling of
mitochondria, i.e. incubation in nanomolar concentrations of Mito Tracker
probes and consecutive washings, the MTR and MTG fluorescence was
determined at 585/615 nm (ex/em) and 480/510 nm (ex/em) respectively,
and the MMP (MTR fluorescene) is then related to the amount of
mitochondria (MTG fluorescence).
2.4.6 Changes in protein expression (Paper I and II)
Western blotting, also known as “protein blotting” or “immunoblotting”, is a
well-established method used to distinguish a target protein from a mixture
of proteins. We utilized the assay in order to investigate the protein expression of γ-GCS (Paper I) and αII-spectrin (Paper II) in SH-SY5Y cells after
exposure to gliotoxin. First, extracted proteins were separated by their molecular weight on polyacrylamide gels. Sodium dodecyl sulfate was added to
protein samples and buffer, to confer a negatively charge to all proteins and
ensure they would migrate towards the positively charged anode. Thereafter,
western blotting involves the transfer and immobilization of proteins from
the gel to a solid support membrane of nitrocellulose or polyvinylidene difluoride, PVDF. We used PVDF membranes because they have highest binding capacity, and do not allow the smaller proteins to pass through and end
up in the transfer buffer. Next, non-specific binding of antibodies was reduced by blocking membranes with non-fat, dried milk in solution, followed
46
by incubation with the primary antibody directed against the target protein.
In Paper I we used polyclonal antisera raised against ovalbumin-conjugated
peptides from GCLC and GCLM of the γ-GCS. The peptides were selected
for their homology between rat, mouse, rabbit, and human GCLC and
GCLM, and developed in the laboratories of Terrance J. Kavanagh at the
University of Washington, and Gary L. Schieven of Bristol-Myers Squibb,
USA. The primary antibody selected for Paper II (MAB1622, Chemicon),
was specific for the 240 kDa αII-spectrin as well as the spectrin breakdown
products (SBDPs) of 120 and 150 kDa caused by caspases (SBDP 120 and
SBDP 150), and the fragments of 145 and 150 kDa (SBDP 145 and SBDP
150) produced by calpains (Fig.1.5).
The secondary antibodies, directed against the primary IgG antibodies were
conjugated with horseradish peroxidase, which enables detection with enhanced chemiluminescence. Chemiluminescence is the most popular labelling system for western blotting and its principle relies on the enzymatic
conversion of a luminal-like molecule to a reactive molecule by HRP, generating light at the site of antibody binding which can be detected on an
autoradiography film.
47
3. Results and discussion
In the very first part of this project, which started in 1999, we wanted to
investigate the possibility to use native human neuroblastoma SH-SY5Y
cells as a bioassay for detecting cytotoxic activity of mycotoxins in feed
samples. One study included substrates that were inoculated with a gliotoxin-producing strain of A. fumigatus, and pure gliotoxin was used as a
reference metabolite in the system (Paper IV). Since A. fumigatus is known
to be able to produce neurotoxic metabolites, gliotoxin was also exposed to
differentiated SH-SY5Y cells. It was observed that gliotoxin induced a concentration-dependent decrease in the number of neurites per cell in differentiated SH-SY5Y cells, and this was the first time any toxic activity of gliotoxin was reported in cells of neuronal origin (Paper IV). To further evaluate
the possible neurotoxic properties of gliotoxin, general and specific cellular
functions were investigated in differentiated SH-SY5Y cells (Paper I-III). In
this section of the thesis, the major findings of Paper I-IV are discussed.
3.1 Cellular redox status and toxicity of gliotoxin
With the knowledge that gliotoxin, with its typical disulfide ring, is a redox
active toxin, the role of GSH in gliotoxin-induced toxicity was studied in
Paper I. The results demonstrated that the intracellular concentration of GSH
played an important role in the toxic activity of gliotoxin. Reduction of the
total intracellular levels of GSH by approximately 40%, by inhibition of γGCS, significantly attenuated the cytotoxic effects of gliotoxin. The cytotoxic effects were completely reduced at 0.1 µM gliotoxin, and at 0.5 µM the
protein content was reduced by 20% after addition of BSO, as compared to
50% in cultures without BSO. Accumulation of gliotoxin in SH-SY5Y cells
might be GSH-dependent, and a loss in total cellular GSH, parallels decreased uptake, in agreement with the redox mechanism for cellular uptake
of gliotoxin suggested by Bernardo and co-workers (200). The utilization of
GSH was demonstrated by a concentration-dependent increase in the level of
GSSG, with higher concentrations of gliotoxin. The ratio of [GSSG]/[GSH]
increased from 0.14 ± 0.01 in control cultures, to 0.25 ± 0.08 in cells exposed to 1.0 µM (mean ± S.E. from five individual experiments). However,
the GSH level was not depleted due to a GSH-gliotoxin redox reaction. Efforts to compensate for increased GSH oxidation was indicated by a slight
increase in the activity of GSSG reductase, as well as increased protein levels of the enzyme γ-GCS, involved in GSH synthesis (Fig. 3.2).
No generation of ROS during redox cycling of gliotoxin could be observed
and we, hence, concluded that increased ROS production is not likely the
48
primary mechanism of gliotoxin-induced toxicity in SH-SY5Y cells. In addition, the cytotoxic effect of gliotoxin was attenuated when the antioxidant
defense of GSH was reduced, by inhibition of GSH synthesis with BSO.
Nevertheless, vitamin C indeed decreased the cytotoxic activity of gliotoxin,
but this may be due to maintenance of the GSH pool and thereby suppression
of the GSH-gliotoxin reaction, further confirming the redox theory.
3.2 Cytotoxic activity of gliotoxin
3.2.1 Altered Ca2+-homeostasis and loss of cellular energy
We found in our first study (Paper IV) that gliotoxin generated a decrease in
the total amount of cellular proteins in SH-SY5Y cells with 20% (EC20) at
0.12 µM. This classifies gliotoxin to be highly toxic to these neuron-like
cells. Since a disturbed Ca2+-homeostasis plays a critical role in neurotoxicity, the [Ca2+]i was measured in the cells after exposure to different concentrations of gliotoxin (Paper II). No changes in [Ca2+]i could be detected after
acute exposure to gliotoxin, in contrast to other studies with gliotoxin (206,
215). On the other hand, after 72 h of exposure to 0.25 µM, a significant
effect in the Ca2+-homeostasis was observed by an increase in [Ca2+]i from
157 ± 38 to 216 ± 33 nM (mean ± S.E. of four independent experiments).
This indicated that the disturbed Ca2+-homeostasis was a delayed effect in
the cells, and most likely, not mediated via direct interactions between gliotoxin and Ca2+-permeable receptors or ion channels in the plasma membrane.
Gliotoxin is reported to release Ca2+ from intracellular Ca2+ pools like mitochondria (206), and putative targets for gliotoxin may be the cysteine residues on ANT, one component of the MPT pore (284, 285). If gliotoxin
stimulates the pore to open, mitochondrial Ca2+, but also pro-apoptotic proteins, would be released to the cytosol (see section 1.2.5.2). Another way for
gliotoxin to alter the Ca2+-homeostasis can be by inhibition of creatine
kinase. Creatine kinase catalyzes the formation of ATP from creatine phosphate and ADP (286), and is sensitive to inactivation of thiol-specific agents,
like gliotoxin (203). Inhibition of creatine kinase that is associated with
PMCA or SERCA would disable this route of Ca2+ extrusion and allow
uncontrolled Ca2+ increases (Fig. 3.2).
Sustained elevations of Ca2+ can be deleterious for cells in many ways, and
one is the depletion of energy reserves. Because of the 10,000 times lower
concentration of Ca2+ in the cytoplasm, as compared to extracellular concentration or levels in ER and mitochondria, Ca2+ entry to the cytosol occurs
with no input of cellular energy. On the other hand, restoration of the Ca2+homeostasis, which is necessary for cell survival, is an energy requiring
49
process due to activation of ATP-coupled Ca2+-pumps. In Paper III, a significant decrease of the cellular ATP level was observed at gliotoxin concentrations higher than 0.25 µM. This can be an effect of the highly energy demanding restoration of the Ca2+-homeostasis, also shown to be disturbed at
0.25 µM gliotoxin. The hypothesis that the reduced cellular ATP level is a
result of increased consumption of energy, rather than a decrease in ATP
synthesis, was indicated by the maintained MMP in the cells during gliotoxin exposure (Paper III).
3.2.2 Impact of activated calpains
In addition to depletion of energy stores, a major effect of extensive increase
in [Ca2+]i is the activation of calpains. This was also true for gliotoxinexposed SH-SY5Y cells (Paper II). Addition of the calpain inhibitor
calpeptin to the cells during exposure, attenuated the cytotoxic effect of gliotoxin significantly. The protein content was reduced by only approximately
14% after addition of calpeptin, as compared to 42% in cultures exposed to
0.25 µM without calpeptin. The involvement of calpains was noted at the
same concentration where [Ca2+]i was significantly raised, indicating that
gliotoxin-induced cytotoxicity may be a Ca2+-dependent process (Fig. 3.2).
The implication of calpains has not previously been reported for the proposed mechanisms underlying the toxicity of gliotoxin. Activated calpains
have a preference for cytoskeleton proteins (c.f. Table 1.1.), and hence, the
breakdown of αII-spectrin was studied (Paper II). αII-spectrin is also a wellknown substrate for caspases (see Fig. 1.5), and, indeed, by studying the
breakdown pattern of this protein, a gliotoxin-dependent activation of both
the proteases was confirmed. However, no alteration in calpain-specific
breakdown products could be detected in the cells, unless the activated caspases were inhibited. This suggests that αII-spectrin is not the primary target
for “gliotoxin-activated” calpains in these cells.
Gliotoxin significantly increased cleavage of αII-spectrin into 120 kDa
fragments, which in SH-SY5Y cells has been demonstrated to be a specific
and characteristic marker for caspase-3 activity (132). Gliotoxin-mediated
activation of caspase-3 and induction of apoptosis have been reported for
several cell lines (192, 207, 212). However, caspase-induced apoptosis is not
likely the mechanism for gliotoxin-induced cytotoxicity in SH-SY5Y cells,
since inhibition of caspases did not protect the cells from cytotoxic effects
mediated by gliotoxin (Paper II). In conclusion, the cytotoxic mechanism of
gliotoxin, reflected as inhibition of cell growth at moderate concentrations, is
related to calpain activity but independent of caspases. Furthermore, the
calpains probably act on substrate(s) that are essential for cell
growth/survival, but are not targets for caspases.
50
3.3 Neurotoxic effects of gliotoxin
3.3.1 Caspase-dependent neurite degeneration
Criterion for a substance to be revealed as potentially axonpathy-inducing is
that the neurite degeneration is induced at a significantly lower concentration
than the cytotoxic concentration (274). Neurite degeneration is morphologically visualized as a reduction of number of neurites, but without evident
effects on the cell body. Gliotoxin induced a 20% degeneration of neurites
(ND20) at 0.06 µM (Paper IV), which is significantly lower than the EC20
value (0.12 µM). This indicates that gliotoxin is potentially neurotoxic and
might induce axonopathy in vivo (Fig. 3.1A,B). The ability of the cells to
recover from these effects was investigated by exchanging the exposure medium for non-gliotoxin-containing medium, subsequent to the 72 h of gliotoxin exposure. The recovery was studied over 72 h and no reversible effects
on the neurite degeneration could be observed, which shows that the effect
on the neurites is irreversible (Fig. 3.1C,D). If this also is true for an in vivo
situation, it implies that gliotoxin might cause severe damage in the nervous
system.
As for the cytotoxicity studies, the possible involvement of activated calpains and caspases in the neurite degenerative activity of gliotoxin was
evaluated (Paper II). No significant implication of the calpains on the
amount of neurites could be observed, but we found that caspase activity is a
key mechanism in gliotoxin-induced neurite degeneration. Inhibition of caspases with the general caspase inhibitor, Z-VAD-fmk, reduced the gliotoxininduced neurite degeneration almost completely at 0.25 µM, as compared to
cultures exposed to gliotoxin without Z-VAD-fmk. Axonal degeneration
mediated by local activation of caspases in the axon, but independent of cell
body apoptosis, is a phenomenon that has been shown in models of AD
(141), and HIV-associated axonopathy (143). A caspase-3 dependent degradation of αII-spectrin was proven in Paper II, and that can be the major factor contributing to gliotoxin-induced neurite degeneration (Fig. 3.2).
Mitochondria play a key role in caspase activation, through the release of
cytochrome c (103) and AIF (106) (c.f. Fig. 1.4). Most theories about cytochrome c release include opening of the MPT pore with a subsequent mitochondrial depolarization (287). This event has for example been demonstrated in human hepatic stellate cells after gliotoxin treatment (207).
51
Figure 3.1. Differentiated SH-SY5Y cells exposed for 72 h to, no gliotoxin (A), and
0.25 µM gliotoxin (B). Control cells (C) and cells exposed to 0.25 µM (D), after a
recovery period of 72 h to non-gliotoxin-containing medium
Whether cytochrome c is released in the SH-SY5Y cells has not yet been
investigated in these studies. There is a possibility that gliotoxin induces
modulation of the MPT pore due to cellular stress, like Ca2+ overload and
ATP depletion, or by a direct thiol modification, as described above in section 3.2.1. In Paper III, no changes of the MMP in the cells could be detected, which demonstrates that activation of caspases, implicated in mechanisms of gliotoxin-induced neurite degeneration, does not require mitochondrial depolarization. However, the lack of MMP depolarization does not
necessarily exclude the release of cytochrome c, since its translocation from
mitochondria can proceed independently of a change in MMP (124, 288).
Indeed, and in contrast to the observation by Kweon and co-workers (207),
gliotoxin activates caspases in macrophages by the release of cytochrome c,
but without attenuation of the MMP (212).
52
3.3.2 The aspect of decreased protein synthesis
A number of factors can promote axonal degeneration. Even though various
studies have suggested that protein synthesis can occur in axons as well as
axonal synapses (289, 290), the axon and neurites are largely dependent on
the synthesis of macromolecules and organelles in the cell body, and a
proper translocation of these to the periphery by anterograde transport (291).
A study with distal axonal segments of rat PNS and CNS, has shown that
Wallerian degeneration is accelerated if the protein synthesis is inhibited
(292). The effect of gliotoxin on protein synthesis in SH-SY5Y cells after
subchronic exposure was investigated in Paper III. It was shown that 72 h of
exposure to 0.25 µM gliotoxin significantly decreased the protein synthesis
rate with 56%. This observation was made at a non-cytotoxic concentration,
indicating a specific neurotoxic effect, which differed from the general cytotoxicity. Reduced synthesis of proteins, due to or together with the effects on
ATP levels that were observed (Paper III), may be a limiting factor for the
maintenance of neurites in differentiated SH-SY5Y cells after exposure to
gliotoxin. Previously, acrylamide that is classified to generate axonopathy in
vivo has been demonstrated to decrease the rate of protein synthesis as a
specific neurotoxic effect in SH-SY5Y cells (270).
Taken together, gliotoxin induces a concentration-dependent and irreversible
degeneration of the neurites in differentiated SH-SY5Y cells after 72 h of
exposure. The effects are probably dependent on caspase-cleavage of the
cytoskeletal component αII-spectrin. Gliotoxin indeed also attenuates protein synthesis, which may contribute to the degeneration of neurites (Fig.
3.2). A third possible mechanism by which gliotoxin might execute the neurite degenerative effects can be through direct binding to the free thiolgroups on the microtubule surface. However, the role of that mechanism has
not been studied in this thesis.
53
Figure 3.2. Proposed mechanisms by which gliotoxin exerts its cytotoxic and neurite
degenerative effects in SH-SY5Y cells. Following diffusion into cells in its oxidized
form, gliotoxin is rapidly reduced to the dithiol form by GSH. Continuous reduction
of gliotoxin leads to a shift in the GSH redox balance towards GSSG, but also to
increased uptake of gliotoxin. To compensate for increased oxidation of GSH and to
maintain the cellular redox balance, the activity of GSSG reductase as well as γ-GCS
is increased. Both the reduced and oxidized form of gliotoxin can inactivate and
inhibit thiol-dependent enzymes and proteins. This conjugation can be cytotoxic to
the cells, but also cause an increase in [Ca2+]i. Due to thiol-modification of the MPT
pore, Ca2+ and cytochrome c, can be released from mitochondria. Simultaneously to
the increased [Ca2+]i, calpains and caspases are activated. Calpains mediate cytotoxic effects of gliotoxin, whereas neurite degeneration is caspase-dependent. Caspase-3 dependent degradation of αII-spectrin can be the major factor contributing to
gliotoxin-induced neurite degeneration. A sustained elevation of Ca2+ depletes the
energy reserves, which in turn affects the protein synthesis and the ability of the
cells to maintain the neurites. An additional mechanism by which gliotoxin might
execute the neurite degenerative effects can be through binding to free thiol-groups
on the cytoskeleton.
54
3.4 Sensitivity of the SH-SY5Y cells to gliotoxin
The ND20 and EC20 values calculated for pure gliotoxin in Paper IV, are
comparable with 0.0196 µg/g and 0.0392 µg/g (errata in Paper IV where is
says 0.0326 µg/g), respectively. These values are well below observed concentrations of gliotoxin in tissues from animals naturally infected with A.
fumigatus (Table 3.1), and indicate that our cell model can detect pathological concentrations of gliotoxin. The values are also below concentrations of
gliotoxin in samples of feedstuff associated with intoxication of camels.
However, it should be noted that EC20 and ND20 values are simulated to
correlate with the concentration of a substance in the target tissue and,
hence, differ from a dose taken orally.
Table 3.1. Observed cytotoxic and neurite degenerative concentrations of gliotoxin
(µg/g) in the SH-SY5Y cell model, and pathological concentrations of gliotoxin in
tissues and feedstuffs contaminated with A.fumigatus.
Target
Species
Gliotoxin (µg/g)
Reference
Protein content
SH-SY5Y cells
0.0392
Paper IV
Neurites
SH-SY5Y cells
0.0196
Paper IV
Udder tissue
cattle
9.2
(231)
Lung tissue
turkeys
16.5-126.3
(196)
Feedstuff
camel
0.495
(232)
(i.e. general cytotoxicity)
3.5 Bioassay for detection of toxic activity in feed
To elucidate if native SH-SY5Y cells are applicable for detection of mycotoxins in animal feed, the cytotoxic effects of extracts inoculated with a
gliotoxin-producing strain of A. fumigatus, as well as from pure gliotoxin
were investigated. Gliotoxin was chosen as a well known reference metabolite from A. fumigatus, and because it has been proven to be cytotoxic in
other cell systems (191, 192). The strain of A. fumigatus used was confirmed
to produce gliotoxin when inoculated on the growth medium CzDox-broth
with 30% glucose, which is reported to be the optimal substrate for production of gliotoxin in vitro by A. fumigatus (260). The cytotoxicity of the extracts from inoculated CzDox-broth was similar to that from pure gliotoxin,
and the effects correlated well with the amount of gliotoxin in the extracts,
as analyzed with HPLC. Despite the fact that a slight effect on total cellular
protein content could be observed from uninoculated and gliotoxin-free
55
CzDox-broth, it was from this preparatory part of the study verified that
toxic activities of mycotoxins can be detected with SH-SY5Y cells.
Extracts from two different feed substrates, maize and commercial feed
grain, inoculated with the same strain of A. fumigatus as above were also
cytotoxic to the cells. These effects were most probably due to produced
metabolites, but due to the complexity of these two substrates it was not
possible to detect gliotoxin by HPLC analyses. Hence, it can not be ruled out
that mycotoxins, other than gliotoxin, were present in the samples. Nevertheless, the purpose was to investigate the use of SH-SY5Y cells for cytotoxic
evaluation of mouldy feed substrates, which was successful. The results for
the maize extracts, where an increase in toxicity could be seen with increasing water content during inoculation, illustrated the importance of proper
feed and food products storage, i.e. at a low humidity, to avoid mould
growth. For the feed extracts, there was a difference in cytotoxicity due to
inoculation time. The extract from the substrate that had been inoculated for
4 weeks, which was the maximum time of inoculation, was the most toxic to
the cells. Just like for CzDox- broth, both maize and commercial feed grain
displayed matrix effects, but the cytotoxic effects of the inoculated substrates
were significantly higher.
This study was done in collaboration with SVA in Uppsala. From the time
since the work was published in 2003, the cell model has been established at
the Department of Animal Feed at SVA, and is in use for cytotoxicity studies
of different mould contaminated feed samples.
56
4. Conclusions
Gliotoxin is highly cytotoxic and causes neurite degeneration to human neuronal SH-SY5Y cells. There is a correlation between the cytotoxicity and the
cellular redox status, indicated by a decrease in the cytotoxic effects following reduction of the cellular GSH levels. The basal [Ca2+]i is elevated, and
simultaneously, both calpains and caspases are activated and demonstrated
to underlie gliotoxin-induced cytotoxicity and neurite degeneration, respectively. The major factor contributing to gliotoxin-induced neurite degeneration might be attributed caspase dependent degradation of αII-spectrin. Also
the rate of protein synthesis is decreased in differentiated SH-SY5Y cells
after exposure to gliotoxin. This effect, due to or together with depletion of
the cellular energy levels, may be one limiting factor for the maintenance of
neurites. This is the first study where the possible neurotoxic properties of
gliotoxin have been evaluated. I conclude so far, that gliotoxin has the potential to be an axonopathy-inducing compound, but also that the toxic mechanisms have to be fully revealed before a risk assessment of exposure for A.
fumigatus and gliotoxin can be done.
In collaboration with SVA, we succeeded in developing an in vitro method
for determination of the general cytotoxicity of mycotoxins in animal feed.
The method is today established and in use at Department of Animal Feed,
SVA.
57
5. Sammanfattning på svenska
Förekomst av mögelsvampar i livsmedel och djurfoder är ett problem som
kan påverka kvaliteten på produkterna. Alla jordbruksprodukter, inklusive
fodermedel som är producerade av dessa, kan vara angripna (kontaminerade)
av mögelsvampar. Svamparna infekterar materialet innan skörd, under skörd,
vid förvaring eller under bearbetning. Kontamineringarna är ett stort problem för alla som producerar djurfoder och livsmedel, inte bara på grund av
obehaget med mögelväxt utan också på grund av de gifter (toxiner) som kan
bildas, s.k. mykotoxiner. En mycket vanlig mögelsvamp i fodermedel, livsmedel, komposter, växthus och s.k. sjuka hus är Aspergillus fumigatus. Dess
sporer sprids lätt i luften, och därmed är risken för exponering stor, både för
djur och för människa. Gliotoxin har identifierats som en av de giftigaste
toxinerna som produceras av A. fumigatus. Andra mykotoxiner som kan
produceras av denna svamp är bl.a. verruculogen och fumitremorgener, vilka
är kända för att orsaka ataxi (gångsvårigheter och oförmåga att styra benrörelser), vilket kan orsakas av att perifera nerver degenererar, s.k. axonopati.
Dock saknas det studier som har utrett gliotoxins effekter på nervsystemet.
Därför har jag i denna avhandling, som bygger på fyra delarbeten, studerat
om gliotoxin har några toxiska egenskaper som kan vara skadliga för nervsystemet, dvs. vara neurotoxiskt.
Vi har i vår forskningsgrupp utvecklat en in vitro-metod (från latin ”i
glas[kärl]” till skillnad från in vivo, ”i [den] levande kroppen”) för detektion
och studier av substanser som kan orsaka axonopati. Metoden baseras på att
mänskliga celler, SH-SY5Y celler, stimuleras att utveckla nervcellstrådar,
neuriter, varefter de exponeras för testsubstansen under 72 timmar. Antalet
neuriter per cellkropp kvantifieras därefter och resultatet relateras till oexponerade celler. En substans bedöms som potentiellt axonopatiskt om koncentrationen som orsakar effekten på neuriterna är signifikant lägre än den allmänt toxiska effekten i cellerna, vilken erhålls genom att jämföra minskningen av den totala proteinmängden i exponerade celler jämfört med ickeexponerade.
Vi fann att gliotoxin genererar en minskning av det totala cellulära proteininnehållet i SH-SY5Y celler med 20% vid 0,12 µM, vilket klassas som
högtoxiskt. Gliotoxin har också en effekt på neuriterna och inducerar en 20%
degenerering av neuriterna vid 0,06 µM, dvs. signifikant lägre än den generella toxiciteten. Detta ger indikationer på att gliotoxin kan vara ett axonopatiskt toxin. Effekterna kan bero på den cellulära stress som induceras av den
långvariga förhöjda koncentrationen av kalcium i cellerna efter exponering
för gliotoxin. En ökad kalcium nivå kan leda till aktivering av calpainer och
caspaser, vilka är två specifika cellnedbrytande enzymer som ofta är inblan58
dade i neuronal celldöd och axonal degenerering. Dessa proteaser observerades att ha olika roller i gliotoxins toxiska mekanismer i SH-SY5Y cellerna.
Den oåterkalleliga degenereringen av neuriterna som orsakas av gliotoxin är
i huvudsak beroende på aktiverade caspaser, som troligtvis bryter ned αIIspektrin, en komponent som är viktig för neuriternas struktur och stabilitet.
Den generella toxiciteten visades vara medierad av calpainerna. Gliotoxin
reducerar också cellernas förmåga att nybilda proteiner, vilket är nödvändigt
för att cellerna ska fungera optimalt.
Detta är den första studie där de eventuella neurotoxiska egenskaperna av
gliotoxin har studerats. Från de resultat som har erhållits så här långt kan
slutsatsen dras att gliotoxin uppvisar en potential att vara ett axonopatiskt
mykotoxin. De toxiska mekanismerna måste dock identifieras fullt ut innan
det är möjligt att göra korrekta riskbedömningar för exponering av A. fumigatus och gliotoxin.
Ett annat syfte i avhandlingen har varit att i samarbete med Statens Veterinärmedicinska Anstalt (SVA) i Uppsala utveckla en cellbaserad metod för
översiktstestning (screening) av fodermedel, för att påvisa toxiska egenskaper. Den foderhygieniska analys som används idag utgörs till största delen
av mikrobiologiska undersökningar med avseende på förekomst av bakterier,
jästsvampar och mögelsvampar. Analyserna ger dock inte svar på den eventuella närvaron av toxiska substanser eller andra toxiska egenskaper hos
fodret. Ett alternativ är att kemiskt analysera för specifika mykotoxiner i ett
foderprov, men dessa analyser är dyra och det är praktiskt omöjligt täcka in
alla kända mykotoxiner, ca 400 stycken. En screeningmetod med avseende
på allmän toxicitet skulle därför kunna möjliggöra en första utsortering av
foderprover där hög sannolikhet föreligger att det kan försämra djurets hälsa,
och därmed ange vilka prover som det eventuellt är angeläget att analysera
vidare.
Studien visade att mykotoxiner kan ge kraftiga utslag i vårt in vitro-system
med SH-SY5Y celler, och att vi även kan detektera och bestämma en generell toxisk aktivitet av mykotoxiner i foderprover. Metoden är idag etablerad
på Avdelningen för foder på SVA och används för testning av olika mögelskadade fodersorter.
59
6. Acknowledgement
Det är med blandade känslor som jag har närmat mig det här sista kapitlet. Lättnad
och glädje över att snart vara klar, men samtidigt med en känsla av tomhet och lite
saknad över att en lång period faktiskt snart är slut. Jag har ju varit här på institutionen för Neurokemi sedan 1998... Ni är många som jag har träffat under den här
tiden, och som alla på sitt sätt har bidragit till att jag har trivts så bra. Tack!
Det största av tack går till min handledare, Anna Forsby. Tack för att du inte har
låtit mig tappa taget och ge upp när det har känts som mest jobbigt. Du har en enorm
förmåga att alltid se lösningar och att kunna ge den inspiration som behövs för att
man ska orka bita ihop och försöka igen. Tack för att du alltid är så glad och stolt
över vad vi gör i våra projekt och för att du även påminner oss om att man måste ha
roligt också!
Ett stort tack till alla som är, eller har varit, på SVA för ett roligt och givande samarbete. Tack Per Häggblom för att du tillsammans med Emma var med och drog
igång allting 1999. Tack till Alexey, Eva, Anna, Ingrid, Ann-Christine, och Anja
- för att du tappert pendlade till Stockholm för att lära dig odla SH-SY5Y celler och
sedan ta med dig dessa tillbaka till SVA.
Tack till alla lärare och handledare på institutionen för Neurokemi, Ülo, Anders,
Kerstin, Tiit och Mattias för allt jobb ni lägger ner på föreläsningar, kurser och att
hålla verksamheten igång. Det är tungrott mellan varven, vi vet, men ni kämpar på.
Ett extra tack till Kerstin för att du läste igenom manuskriptet till avhandlingen.
Det har sagts förr, men är ju så sant, att institutionen över huvudtaget fungerar är
tack vare Birgitta, Siv och Ulla. Ni är så hjälpsamma och har verkligen stenkoll på
det mesta, och om inte, så tvekar ni inte att hjälpa till att ta reda på det. Dessutom är
ni väldigt måna om oss och frågar ofta hur vi mår. Det uppskattas.
Alla doktorander (räknade ut att jag har lärt känna fyra ”generationer”; två äldre,
min egen och en yngre) och post-docs. Ni har varit fantastiska arbetskamrater allihop! Maria, Pernilla, Linda F (synd att du inte kan räkna bowlingpoäng ordentligt), Yang, Sandra (du har nog en poäng i att en avhandling ska man inte skriva på
för länge..), Linda A, Katarina, Mats (no, pink figures are not presentable. Thanks
for the help with the pdf. files), Malin, Sofia (tack för att du kämpade med spektrinstudien), Veronica, Karolina, Maarja (you are great and your questions were never
disturbing me!), Ulla, Caroline, Helena M (din spontanitet är härlig), Jonas, Peter,
Henrik och Samir (tror att världens alla ämnen kan avhandlas på en dag inne hos
er. Intressant, och oundvikligt, att lyssna på ibland!)
60
Helene - för många år sedan gick vi ”ronden” här på morgonen. Nu försöker vi
hinna med ronder i lekparken och på gymmet! Ursel - it is so nice to see you here
again.
Världens bästa rumskamrater, nu och då. Pontus (min adept) - alltid glad, go′ och
omtänksam. Linda L - du är så okomplicerad och bra. Vi har hjälpt och stöttat varandra och jag kommer att sakna dig. Tina - tack för att du har stått ut här inne de
sista månaderna och för att du är så otroligt positiv. En liten tanke går till Doris
också. De gamla 409:orna, vars vänskap har flyttat ut från rummets fyra väggar,
Anna - du har ett skratt och en humor som är oslagbar, och jag hoppas det blir tid
till att ses mera nu. Daniel - du är en klippa. Inte bara på att göra syntesfigurer (tack
för fig. 1.8), utan även på att lyssna, ge råd eller mest bara snacka.
En speciell tacksamhet känner jag till alla i vår grupp. Den har varit dynamisk i
storlek, men alltid med en viss skånsk dominans. Marika - tack för att du hjälpte
mig i början när jag var ny. Helena - vi har följts åt sedan ”starten” och det har varit
otroligt roligt. Minns många fester och konferenser… Nu är det min tur att skriva att
jag tänker på dig och dina pojkar! Johanna - snacka om girl power. Du är starkast.
Tack för ditt stöd och din uppmuntran. Johan - du gjorde en strålande insats på
projektet i våras och har varit mitt bollplank det sista halvåret. Tack för all hjälp.
”Kvinnor kan” och speciellt om de börjar läsa i Sundvall tillsammans. När jag tänker
på vad vi har presterat och gått i mål med i år, så blir jag både häpen och stolt! Karin - du och din fantastiska familj ger oss den bästa av vänskaper. Erika - vem hade
kunnat tro allt det här när vi satt och småfrös i bilen på Radiogatan och helt plötsligt
fick en oväntad körning?
Stort tack till ALLA våra vänner som det är så trevligt och kul att umgås med!
Tack till mina familjer;
Staffan, Inger, Caroline, Patrik och Totte – Stor Kram till er.
Fantastiska Åsa - allt du gör för oss är ovärderligt. Tack snälla du.
Allra käraste Karl, Cecilia och Maria - det är en otrolig styrka och veta att vi alltid
finns för varandra. Tack även till Eva-Marie, Daniel, Viktor och Dan, som gör er
så glada.
Mamma och Pappa - ni är otroliga och bäst. Tack för allt ni gör för alla oss, men
framför allt för varandra. Det värmer att se er två tillsammans.
Simon och Elias - mina två hjärtan. Jag är så vansinnigt lycklig och stolt över er.
Fredrik - vår kärlek till varandra och det vi skapar ihop står över allting. Tack för
din tålmodighet. Älskar dig så.
61
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