Effects of 2,2’,4,4’,5-pentabromo diphenyl ether (PBDE 99), tetrabromobisphenol A (TBBPA) and ketamine (Ketalar

Effects of 2,2’,4,4’,5-pentabromo diphenyl ether (PBDE 99),

tetrabromobisphenol A (TBBPA) and ketamine (Ketalar ^® ) on caspase-3, -8, and -9 activity in

neonatal mouse brain

Anna Robertsson

Projektrapport från utbildningen i EKOTOXIKOLOGI

Ekotoxikologiska avdelningen

Nr 136

CONTENTS

ACKNOWLEDGMENTS ... 2

SUMMARY ... 3

INTRODUCTION... 4

V

ULNERABLE PERIODS AND BRAIN DEVELOPMENT

... 4

A

POPTOSIS

... 4

Caspase ... 6

Extrinsic pathway ... 6

Intrinsic pathway ... 7

Execution pathway... 9

B

ROMINATED FLAME RETARDANTS

... 9

Polybrominated diphenyl ethers (PBDEs)... 10

PBDEs and Neurotoxicity... 11

PBDEs and apoptosis ... 12

Tetrabromobisphenol A (TBBPA) ... 14

K

ETAMINE

... 15

AIMS ... 17

MATERIAL AND METHODS... 17

C

HEMICALS AND ANIMALS

... 17

E

XPOSURE TO

PBDE 99, TBBPA

AND

K

ETAMINE

... 18

A

NALYSIS OF CASPASE ACTIVITY

... 19

S

TATISTICAL ANALYSIS

... 20

RESULTS ... 20

O

PTIMIZATION OF THE CASPASE ACTIVITY ASSAYS

... 20

A

CTIVITY OF CASPASE

-9 ... 23

A

CTIVITY OF CASPASE

-3 ... 25

A

CTIVITY OF CASPASE

-8 ... 27

DISCUSSION ... 29

REFERENCES... 35

ACKNOWLEDGMENTS

First of all I wish to express my gratitude to my excellent supervisor, Dr. Henrik Viberg, for

his generosity with time for good and bad questions, exuberant enthusiasm and

encouragement. Moreover I wish to thank professor Per Eriksson for good advice and his

generosity of sharing knowledge and interesting information.

SUMMARY

Polybrominated diphenyl ethers (PBDE) are used as flame retardants and detected in environmental media, wildlife species and human tissues. Exposure to PBDEs during the neonatal development of the brain has been shown to affect behavior and learning in adult mice, while neonatal exposure to TBBPA (another brominated flame retardant) did not affect behavioral variables in the adult.

The sedative and anesthetic drug ketamine is used in human and veterinary medicine and the drug is known to induce apoptosis and neruodegeneration in the brain. In the present study the compound will be used as a positive control.

In this study, I hypothesized that the effects of these compounds could be reflected by

changes in caspase activity and thereby the apoptosis process. Caspases are cysteine aspartate- specific proteases important in the apoptotic pathways. I examined the activity of three caspases involved in the two major apoptotic pathways after a single oral exposure of 12 mg (21 μmol) PBDE 99/kg body weight or 11.5 mg (21 μmol) TBBPA/kg body weigh of or 50 mg ketamine/kg body weight of on postnatal day 10 (PND 10). The analysis of the caspase activity showed significant changes following the exposure to PBDE 99, TBBPA and ketamine in the neonatal brain. These results confirm that PBDE 99, TBBPA and ketamine can act as neurotoxicants and affect the caspase activity in the neonatal brain. These changes in caspase activity are interesting and further studies of the caspase activity and effects on the apoptotic pathway are needed to understand more about the mechanism behind the

developmental neonatal neurotoxicity of PBDEs.

INTRODUCTION

Vulnerable periods and brain development

The development of the mammalian brain and central nervous system is extremely complex and involve vulnerable processes, where small alterations can lead to malfunctions and disabilities. During the development there is a period of rapid growth. This period is called the brain growth spurt (BGS) (Dobbing and Sands 1979) and it is characterized by dendrite and axonal outgrowth, synaptogenesis, establishment of neural connections and other neurodevelopmental changes.

In rodents this period is postnatal and extends over the first 2-3 weeks after birth, with a peak around postnatal day (PND) 10. In humans this period starts already during the third trimester and continues during the first two years of life (Dobbing and Sands 1979). In recent years there have been several studies presented showing that the developing brain during this period is extremely sensitive and vulnerable to insults from xenobiotics and/or their metabolites (Eriksson et al. 1998, 2001; Eriksson et al. 2002; Fredriksson et al. 1993; Jevtovic-Todorovic and Olney 2008). In both rodents and humans parts of the BGS is occurring during the

lactation period and exposure of lipophilic compounds via the milk is of major concern due to the vulnerability of the developing brain during this time.

Apoptosis

Apoptosis, or the process of programmed cell death, is vital in a range of biological processes including embryonic development, proper development of the central nervous system,

function of the immune system, normal cell proliferation and homeostasis.

The process of apoptosis is complex and it consists of multiple steps and events, which are tightly regulated and there are numerous control mechanisms and checkpoints (Igney and Krammer 2002). Apoptosis is also an important defence mechanism when cells are damaged by harmful agents or disease (Norbury and Hickson, 2001) and defects in the apoptosis process is a causing factor in many human conditions including autoimmune disorders and certain types of cancer (Elmore 2007).

Apoptosis can be trigged by many different stimuli like cytokines, growth factors, calcium influx, toxins or oxidative stress (Mattson and Chan 2003; Nicotera et al. 1997). Some chemicals is known to induce apoptosis and neruodegeneration in the brain in mammalian species like rat, mice and guinea pig if administered at a time when the developing brain is especially sensitive and this may lead to permanent damage in the brain (Jevtovic-Todorovic et al. 2003; Rizzi et al. 2008).

Apoptosis and necrosis is two very different types of cell death. Necrosis, sometimes referred to as accidental cell death is not an active process and it does not require energy. It is often the type of cell death that occurs when the cell for some reason runs out of energy, often due to extensive mitochondrial damage. Apoptosis is organized modes of cell termination with no leakage to the surrounding cells and often without an inflammatory response, but apoptosis and necrosis can occur sequentially or simultaneously. The dose, intensity and/or duration of the stimulus can be the determining factor if the cell will die by apoptosis or necrosis (Elmore 2007; Nicotera et al. 1997).

There are two main apoptotic pathways: the extrinsic/death receptor pathway and the

intrinsic/mitochondrial pathway.

Caspase

In both the extrinsic and intrinsic pathway the cysteine aspartate-specific proteases (caspases) play important roles. Caspases are normally present in the cytosol and expressed in

catalytically inactive forms. Each pro-caspase consists of three domains, a pro-domain, a large subunit (ca 20kDa) and a small subunit (ca 10kDa) (Robertson et al. 2000). The activation is mediated by proteolysis processing at a specific aspartate residue that separates these three parts. The pro-domain is removed and the small and the large subunit heterodimerize resulting in active caspase (Cryns and Yuan 1998). Caspases cleave substrates, for example other caspases, at the carboxy-terminal to an aspartate residue and this often leads to that the activated caspase activates yet another pro-caspase into an active caspase (Cryns and Yuan 1998). The different caspases differ in their substrate specificity and also in their length and sequence of the pro-domain. Ten major caspases have been identified and they can be

classified in three groups: initiators (caspase-2, -8, 9, -10), effectors/executors (caspase -3, -6, -7) and inflammatory caspases (caspase-1, -4, and -5) (Elmore 2007).

Extrinsic pathway

The extrinsic pathway involves transmembrane death receptors and the best characterized ligand and death receptor include tumor necrosis factor-α, which binds to tumor necrosis factor receptor 1 (TNF-α/TNFR1) and Fas ligand (FasL) which is binding to Fas receptor (FasR) (Elmore 2007). After ligand binding to the death receptor there is a trimetric gathering of the receptors and the bound ligand in the cell membrane. Cytoplasmic adaptor proteins are then recruited and bind to the receptors/ligand complex. Adaptor protein coupled to

FasL/FasR is called Fas-associated death domain protein (FADD) and for TNF-α/TNFR1 the

adaptor protein TADD is recruited to the receptors after ligand binding. The outcome is the

formation of death-inducing signalling complex (DISC), which activates pro-caspase-8 to

active caspase-8. The active caspase-8 can now start the next part of the apoptosis pathway by activating pro-caspase-3 to active caspase-3 and this activation is the start of the execution pathway (Elmore 2007). See figure 1 for schematic overview of the apoptotic process.

Intrinsic pathway

The intrinsic pathway results in apoptosis without any receptor binding. The stimuli act on the cell directly and the mitochondria are the initiating part. Stimuli can act as positive or

negative factors, where the negative is for example the absence of hormones, cytokines or growth factors which results in loss of apoptotic suppression and the positive stimuli include for example toxins, free radicals and viral infections. Both the positive and the negative stimuli result in the formation of a mitochondrial permeability transition pore, loss of the mitochondrial membrane potential and the release of two main groups of pro-apoptotic proteins into the cytosol (Elmore 2007). The first group of proteins is released in the initial phase of the apoptosis process and consists of cytochrome c, Second Mitochondria-derived Activator of Caspases/Direct IAP Binding Protein with Low PI (Smac/DIABLO) and a serine protease named HtrA2/Omi. Cytochrome c binds apoptotic protease activating factor 1 (APAF1) resulting in the formation of an apoptosome. The apoptosome activates pro-caspase- 9 to active caspase-9, which then activates pro-caspase-3 to caspase-3 and the execution pathway begins (Elmore 2007). Smac/DIABLO promotes apoptosis by binding and inhibiting the inhibitors of apoptosis proteins (IAP), which are proteins capable of binding to and inhibit caspase even after it has been activated (Elmore 2007; Igney and Krammer 2002). See figure 1 for schematic overview of the apoptotic process.

The second group of pro-apoptotic proteins consists of apoptosis inducing factor (AIF),

endonucleases G and caspase activated DNase (CAD). These proteins are released from the

mitochondria during a later event during the apoptosis process. AIF translocates into the

nucleus and causes DNA fragmentation (Elmore 2007) and also endonucleases G translocates to the nucleus where it forms oligonucleosomal DNA. This implies that both endonucleases G and AIF induce apoptosis independent from caspases (Igney and Krammer 2002). The pro- apoptosis factor CAD is released from the mitochondria through the MPT pore and after cleavage by caspase-3 it translocate in to the nucleus and forms oligonucleosomal DNA fragmentation and chromatin condensation (Elmore 2007; Igney and Krammer 2002). These events are initiated in the intrinsic pathway, and they are part of the execution pathway.

Caspase 3

Nucleus

Degradation of chromosomal DNA Degradation of nuclear and cytoskeletal proteins

Chromatin and cytoplasmic condensation, nuclear fragmentation

Caspase 8 Caspase 9 Cytochrome C

Bid Bax

Extrinsic Pathway Intrinsic Pathway

Death receptor Death ligand

Adapotor proteins

Mitochondria

Apoptosis

APAF1 FLIPs

Bcl-2 Bcl-x

IAP

+ +

-

- -

-

Figure 1. Schematic overview of the apoptotic process in the cell.

For further information of the extrinsic pathway se page 11. For further explanation of the activity of APAF1,

IAP, AIF see page 12, intrinsic pathway. Bax, Bid, Bcl-x and Bcl-2 are members of the Bcl-2 protein family and

in this group there is both pro-apoptotic and anti-apoptotic members. Data from Elmore, 2007; Robertson, 2000

and Igney and Krammer 2002.

Execution pathway

Both the extrinsic pathway and the intrinsic pathway end up in the execution pathway. This is the final part of the process leading to apoptosis and it begins with the activation of the executor caspases, like caspase-3 (Igney and Krammer 2002). Caspase-3 is considered as one of the most important caspases in the apoptosis process and it has been suggested that almost 40 of the 70 identified caspase substrates can be cleaved and thereby be activated by caspase- 3 (Robertson et al. 2000).

The execution pathway continues with the activation of endonucleases, by executor caspases, and the endonucleases start to degrade nuclear material and cytoskeleton proteins. Actin and plectin are cleaved which leads to cell fragmentation, blebbing, shrinking and the formation of an apoptotic cell. See figure 1 for schematic overview of the apoptotic process.

The dying cell will then change surface sugars or expose phosphatidylserine on the surface which function as an opsonization and the cell is phagocytized and often no inflammation or spread of cell contents to the neighbouring cells are seen (Igney and Krammer 2002).

Brominated flame retardants

During the last 50 years there has been at tremendous increase in products made from

polymers on the market. Many of the products containing polymers that we come into contact

with, for example furniture, electronics and textiles, are based on petroleum products, which

make them highly flammable and to make them more fire safe and to reduce the risk of fire

flame retardants are added. Brominated flame retardants (BFRs) are a major group of

industrial chemicals used world wide to reduce fire-related injury and damage. There are

more than 17 different BFRs recognized and they are right now the largest group of flame

retardants used due to low production cost and high effectiveness (WHO 1994a, b).

In recent years many of the BFRs have been detected in the environment, mammals and human tissues, which have raised the concern for persistence, bioaccumulation, and possible toxicity and health effects toxicity of these compounds (Birnbaum and Staskal 2004; BSEF 2003; Darnerud 2003).

Polybrominated diphenyl ethers (PBDEs)

PBDEs belong to the group brominated flame retardants and are used as flame retardants in a variety of different devises like electrical apparatuses, building materials and textiles

(Darnerud et al. 2001).

The PBDEs consist of two phenyl rings with various numbers of bromine and hydrogen atoms and theoretically there are 209 different PBDEs. The IUPAC system used for numbering PBDEs was initially used for PCBs, which share many structural similarities with PBDEs (BSEF 2003; Darnerud et al. 2001). See figure 2 for general structure of PBDEs.

O

Br _1-10

2 2'

3 3'

4' 5' 6' 4

5 6

Figure 2. General structure of PBDEs

Because PBDEs are additive flame retardants it is believed that they are slowly released from the product during years and the compound end up in the food chain and in human population (Hutzinger et al. 1976). The dominating PBDEs in human and environmental samples are PBDE 47, PBDE 99 and PBDE 100 (Darnerud 2003; Darnerud et al. 2001). Because of the lipophilic characteristics of PBDEs they tend to accumulate in lipid rich tissue like adipose tissue. During nursing, when the body fat is used, the newborn baby is exposed to the

redistributed PBDEs that are excreted via the milk. In a study on human milk from Sweden it

was shown that the levels of PBDEs increased from 0.07 to 4.02 ng/g lipid weight milk between 1972 and 1997 (Meironyte et al. 1999). The highest detected concentration (85.7 ng/g lipid weight) comes from women in USA where levels are generally higher than in the rest of the world (Schecter et al. 2003). Exposure to PBDEs occur also via dust and indoor environment and PBDEs are present at relatively high concentrations within homes where people, and particularly young children, may be susceptible to exposure (Stapleton et al.

2005). These studies show that newborn children are exposed to PBDEs during the perinatal period.

PBDEs and Neurotoxicity

There are several studies reporting neurotoxicity in mice and rats exposed neonatally to different PBDEs. Previous work by Eriksson and Viberg show that neonatal exposure to several PBDEs, including PBDE 99, during a critical time during the postnatal brain development lead to disturbances in behavior, learning and memory, observed in the adult animal. Often these effects worsen with age (Eriksson et al. 2001; Eriksson et al. 2002;

Viberg et al. 2002). These studies also demonstrate that one period during the brain

development is extra sensitive to PBDE exposure and that this period occurs during the BGS, around postnatal day 10. The behavioral disturbances after exposure to PBDE 99 have been seen in both C57/Bl and NMRI mice of both sexes and are both dose and time dependent (Viberg et al. 2004a). PBDE 99 has also been shown to alter one of the major transmitter systems, the cholinergic transmitter system. Mice exposed neonatally to PBDE 99 have a decreased density of nicotinic cholinergic receptors and react differently to nicotine exposure as adults (Viberg et al. 2002; Viberg et al. 2004b).

In a recent study neonatal exposure to PBDE 99, but not TBBPA, significantly increased

Ca

²⁺

/calmodulin-dependent protein kinases (CaMKII) and synaptophysin levels in cortex and

hippocampus (Le Bescont et al. 2009) and this was also evident after exposure to the highly brominated congener PBDE 209, which also changes Growth Associated Protein 43 (GAP- 43) in the neonatal mice brain (Viberg et al. 2008a). Exposure to the anesthetic agent

ketamine also changes the levels of CaMKII, GAP-43 and synaptophysin in rat hippocampus (Viberg et al. 2008c).

In a study preformed by Branchi and co-workers, mice were exposed to different doses of PBDE 99, daily from gestational day (GD) 6 until postnatal day (PND) 21, and 6 mg/kg/day caused reduced numbers of pups and the dose 30 mg/kg/day delayed sensory motor

development and the pups were hypoactive as adults (Branchi et al. 2002).

PBDEs share structural similarities to the hormone thyroxin and can bind with high affinity to the thyroxin plasma transporter transthyretin (TTR) and even displace the thyroid hormones and thereby induce a disturbance in thyroid hormone balance (McDonald 2002; Meerts et al.

2000). This disturbance may impact the developing brain and contribute to the neurotoxicity associated with PBDE exposure because the prenatal thyroid hormone homeostasis is very important for normal brain development (Brouwer et al. 1998).

PBDEs and apoptosis

The mechanisms behind the disturbances seen in the above mentioned studies are not known,

but there are studies indicating that apoptosis may be involved in the neurotoxic mechanism

of PBDEs. Madia and co-workers found that there was an increase in apoptotic cells in a

human astrocytoma cell culture after exposure to PBDE 99 and that there was an increase in

expression of the tumour suppressor protein p53. They suggest that the neurotoxicity seen

may be due to apoptosis via p53 over-expression. The dose of 100 μM leads to a significant

increase in apoptosis, but not necrosis (Madia et al. 2004).

Reactive oxygen species (ROS) can induce apoptosis in many different cell systems (Simon et al. 2000) and increased levels of ROS were found in human hematoma cells upon exposure to

10–100 μM PBDE 209 (Hu et al. 2007). Also in a study by Reistad and Mariussen it was shown that human neutrophil granulocytes exposed to DE-71 (a commercial polyprominated diphenyl ether mixture that consist of tetra- and penta-bromodiphenyl ethers), as well as to PBDE 47 enhanced the production of ROS (Reistad and Mariussen 2005). Yu and co-workers exposed human neroblastoma cells to DE-71 and investigated frequency of apoptotic cells, ROS formation, Ca

²⁺

levels and the activity of caspase-3, caspase-8 and caspase-9. They found an increase in cell death due to mostly apoptosis and not necrosis. Also an increase in intracellular Ca

²⁺

concentration was seen. The activities of the investigated caspases were elevated compared to the control cells and the amount of cytochrome c released from the mitochondria to the cytosol and the level of Bax protein translocated from the mitochondria were also elevated compared to the controls. The conclusion is that the release of cytochrome c from the mitochondria activates the caspase cascade that in turns triggers apoptosis (Yu et al. 2008).

He and co-workers exposed neuroblastoma cells to PBDE 47 and found a higher frequency of cells dying by apoptosis and increased intracellular concentration of Ca

²⁺

and there was a positive correlation between the apoptosis levels and the Ca

²⁺

levels (He et al. 2009).

Increased intracellular levels of Ca

²⁺

trigger apoptosis via the mitochondria and the release of

cytochrome c into the cytosol (Mattson and Chan 2003). He and co-workers also found an

increase in caspase-3 and cytochrome c mRNA levels after exposure to PBDE 47 compared to

the control (He et al. 2009).

Tetrabromobisphenol A (TBBPA)

TBBPA is the most widely used brominated flame retardant in the world (BSEF 2004) and it has structural similarities to PBDEs (Darnerud 2003). See figure 3 for chemical structure of TBBPA.

Figure 3. Chemical structure of TBBPA.

The molecular formula of TBBPA is C

15

H

12

Br

4

O

2.

Like the PBDEs, TBBPA can be used as an additive flame retardant in polymers, which leads to release into the environment, but TBBPA can also be used as a reactive component and be incorporated in the polymer product resulting in a more stabile interaction (Darnerud 2003).

TBBPA have, like PBDEs, been detected in environment, human plasma samples and mother’s milk, but the compound is generally not detected in water (BSEF 2003, 2004;

Darnerud 2003). In contrast to PBDEs TBBPA has not shown the same functional neurotoxic effects and does not seem to affect learning, memory or spontaneous behavior after neonatal exposure (Eriksson et al. 1998, 2001).

There are many studies investigating possible effects of TBBPA on the developing brain. In a study by Saegusa and co-workers exposure to TBBPA, via food during gestation, did not cause any toxic effects in the offspring or the mother and there was no evidence of disturbances in the brain development (Saegusa et al. 2009). Lilienthal and co-workers

showed that, the calculated daily intake of TBBPA throughout life did not induce neurological

damage, but an increase in brainstem auditory evoked potential (BAEP) threshold and this effect on the auditory system is proposed to be due to effect on thyroid hormone system (Lilienthal et al. 2008). In a recent study where humans and rats where exposed to a single dose of TBBPA it was shown that the compound was rapidly conjugated and excreted from the body, which indicates ittle likely-hood to cause serious toxicity (Schauer et al. 2006).

Ketamine

Ketamine is an N-methyl d-aspartate receptor (NMDAR) blocking drug used in human and veterinary medicine and the drug is especially used in obstetric and pediatric medicine as a sedative and anesthetic (Ikonomidou et al. 1999).

In many studies in rat and mice (Ikonomidou et al. 2000; Ikonomidou et al. 1999; Jevtovic- Todorovic et al. 2003) it have beeen shown that neonatal treatment with drugs that blocks the NMDA receptor during the synaptogenesis cause apoptosis and neurodegeneration in the developing brain. This neruodegeneration is probably one of the reason for the learning, behavorial and memory deficiencies seen in the adult rats and mice exposed ketamine during the BGS (Fredriksson et al. 2004; Fredriksson et al. 2007; Jevtovic-Todorovic 2005; Jevtovic- Todorovic et al. 2003).

The underlaying mechanism causing the apoptosis is not known. Slikker and co-workers suggest that it may be the up-regulation of the NMDA receptor NR1 that causes the induction of apoptosis. This up-regulation will lead to more Ca

²⁺

influx in the mitochondria, resulting in reduced membrane potential and an impaired electron transport k in the electron transport, which will lead to the production of reactive oxygen species and initiation of apoptosis (Slikker et al. 2007).

Jevtovic-Todorovic and co-workers show that both the major apoptotic pathways are trigged

after exposure to ketamine, the intrinsic pathway during the initial face of exposure and the

extrinsic pathway later in time. A peak in caspase-9 activity was seen after 4 h of exposure

and the peak in caspase-8 activity was seen after 6 h of exposure and apoptotic cells were

distributed widely throughout the forebrain, midbrain, cerebellum and brainstem (Jevtovic-

Todorovic and Olney 2008).

AIMS

The objectives of this study were to: 1) develop a model to quantitatively measure caspase activity in mice brain tissue homogenates 2) investigate if exposure to PBDE 99, TBBPA or ketamine, in mice during critical time of brain development, alter caspase activity and apoptosis in hippocampus, frontal cortex and/or parietal cortex.

MATERIAL AND METHODS

Chemicals and animals

2,2´,4,4´,5-pentabromo diphenyl ether (PBDE 99) and tetrabromobisphenol A (TBBPA) were provided by Johan Eriksson at Wallenberg Laboratory (Stockholm University, Sweden).

Ketamine, (Ketalar

^®

50 mg/ml Pfizer Inc. New York, USA) was bought from Pfizer Inc.

PBDE 99 and TBBPA were dissolved in a mixture of egg lecithin (Merck, Darmstadt, Germany) and peanut oil (Oleum arachidis) and then sonicated with water to yield a 20%

weight water:fat emulsion vehicle containing 1.2 mg PBDE 99/ml or 1.15 mg TBBPA/ml. To obtain absorption of the chemicals that resembles mouse milk (fat content around 14%) the 20% fat emulsion vehicle was used in this study.

Ketamine was mixed with saline (0.9% NaCl) to yield a solution of 10 mg ketamine/ml.

Pregnant Naval Medical Research Institute ( NMRI) mice were purchased from B&K,

Sollentuna, Sweden. The mice were kept individually in plastic cages in a room with

temperature of 22°C and a 12/12-h cycle of light and dark. They had free access to standard

pellet food (Lactamin, Stockholm, Sweden) and tap water ad libitum. The day of birth was

assigned postnatal day (PND) 0 and the cages contained both male and female pups. The size

of the litters was adjusted to 10-12 pups within the first 48 h after birth by killing excess female pups. This was done in the litter exposed to PBDE 99 or TBBPA but not in the litters exposed to ketamine.

Animal experiments were conducted in accordance with and after approval from the local ethical committee (Uppsala University and Agricultural Research Council) and by the Swedish Animal Welfare Agency (license C185/9).

Exposure to PBDE 99, TBBPA and Ketamine

On PND 10 both male and female mice were given 10 ml/kg body weight of 12 mg (21 μmol) PBDE 99/kg body weight or 11.5 mg (21 μmol) TBBPA/kg body weight of via a metal gastric tube as one single dose. Control mice received 10 ml/kg body weight of the 20% fat emulsion vehicle. Each treatment group comprised mice from 13 different litters. Only male mice were used in the experiment in order to compare result from this study with earlier developmental neruotoxicological studies with PBDEs (Eriksson et al. 2001; Eriksson et al. 2002; Viberg et al. 2002; Viberg et al. 2004b; Viberg et al. 2008a).

On PND 11, 24 hours after exposure, the mice were killed and hippocampus, frontal cortex and parietal cortex were dissected out on dry ice and frozen in liquid nitrogen and stored in -80°C until further processing. The entire procedure from sacrifice to freezing of the brain was completed within 3 min for each mouse.

The ketamine study was conducted in the same way as the PBDE/TBBPA study but on PND 10 both male and female mice was given a subcutaneous injection (5 ml/kg bw) in the neck of 50 mg ketamine/kg bw. The dose is known from earlier studies to affect the

neurodevelopment (Fredriksson et al. 2004). The control animals received the same volume

of 0.9% saline. On PND 11, 24 h after exposure, the mice were killed and the hippocampus,

frontal cortex and parietal cortex was dissected out on ice and frozen in liquid nitrogen and stored in -80°C.

Analysis of caspase activity

Homogenization of the brain structures where done with a Potter-Elvehjem homogenizator and extraction buffer (25 mM HEPES, pH 7, 5 mM MgCl

2

, 1 mM EGTA, 1 mM Pefablock, 1 μg/ml pepstatin, 1 μg/ml leupeptin and 1 μg/ml aprotinin). After homogenization the samples were incubated for 30 minutes in 4°C. The amount of extraction buffer added to each tissue sample were 60 mg brain tissue/ml extraction buffer and the work were preformed in a room with ambient temperature of 4°C. After the incubation the samples were centrifuged (15 minutes, 4°C, 18000 g) and the supernatants were stored in -80°C until assayed. The protein concentration was measured using the BCA method (Pierce kit) and spectrophotometric analysis (Wallace Viktor3 1420Multilabel counter).

The activity of caspase-3, caspase-8 and caspase-9 in the homogenizsed brain tissue were measured using Caspase-Glo assay kit (Promega) with a modified protocol.

The kit is based on a proluminicent substrate containing the caspase-3 tetrapeptide substrate (Z-DEVD-aminoluciferin), caspase-8 tetrapeptide substrate (Z-LETD-aminoluciferin) or the caspase-9 tetrapeptide substrate (Z-LEHD-aminoluciferin). Upon cleavage of the substrates by the respective active caspase, aminoluciferin is formed and contributes to the generation of light in a luminescence reaction. The amount of light is proportional to the caspase activity in the sample. Optimization of the caspase activities assays were done through evaluations and analysis of different caspase activities in increasing concentrations of total proteins in the homogenate. An equal volume (100 µl) of reagents and sample diluted to a protein

concentration of 40 μg/ml were added to a white–walled 96-well plate. To detect background

activity a blank comprised of reagent and extraction buffer was used. After incubation at

room temperature for 2 hours the luminescence of each sample was measured in a plate- reading luminometer (Wallace Viktor3 1420Multilabel counter).

Statistical analysis

The activity of the caspases were analyzed in hippocampus, frontal cortex and parietal cortex in PBDE 99 treated, TBBPA treated, ketamine treated or control mice. Difference in caspase activity between control and PBDE 99 or TBBPA exposed animals were analysed using one- way ANOVA with Newman-Keuls post-hoc test (GraphPad Prism 3.03). Difference in caspase activity between control and ketamine exposed animals were analysed using Student’s t-test.

RESULTS

There were no visual signs of toxicity in the PBDE 99-, TBBPA-, or ketamine-treated mice at any given time during the experimental period, nor were there any significant differences in the body weight in the treated mice, compared with the control mice treated with vehicle or saline.

Optimization of the caspase activity assays

To demonstrate the linear response of the assay, increasing concentrations of total proteins in the homogenate were analyzed for caspase activity. The material used in this part of the study where brain from male mice exposed to saline on PND 10 and killed 24 hours later. Figure 4, figure 5 and figure 6 show the caspase activity (luminometry units) vs. protein concentration.

The analyzed samples show higher luminescence, indirectly meaning higher caspase activity,

in relation to higher protein concentrations and the protein concentration and luminescence shows first order proportionality. The protein concentration 40 μg/ml is in the middle of the curve for all the three different caspases and there is a possibility for higher and lower levels of caspase activity without reaching the top or the bottom of the luminescent curve. The concentration of 40 μg/ml was therefore used for all the caspase assays.

Figure 4. A plot of caspase-9 activity (luminometry units) vs. protein concentration (μg/ml) in homogenates

from male mouse hippocampus 24 h after a single subcutaneous injection of 0.9% saline (5 mg/kg bw) on

postnatal day 10. The results are presented as mean from 6 animals at each protein concentration.

Figure 5. A plot of caspase-3 activity (luminometry units) vs. protein concentration (μg/ml) in homogenates from male mouse parietal cortex 24 h after a single subcutaneous injection of 0.9% saline (5 mg/kg bw) on postnatal day 10. The results are presented as mean from 4-6 animals at each protein concentration.

Figure 6. A plot of caspase-8 activity (luminometry units) vs. protein concentration (μg/ml) in homogenates

from male mouse parietal cortex 24 h after a single subcutaneous injection of 0.9% saline (5 mg/kg bw) on

postnatal day 10. The results are presented as mean from 6 animals at each protein concentration.

Activity of caspase-9

After administration of PBDE 99 (12 mg/kg bw) to mice on PND 10 the activity of caspase-9 was measured 24 hours later (Fig. 7) and the activity in frontal cortex was significantly higher (p≤ 0.01) compared to controls exposed to the 20% fat emulsion vehicle. Mice exposed to TBBPA (11.5 mg/kg bw) did not show any significant changes in caspase-9 activity in frontal cortex compared to control animals. In parietal cortex no differences in caspase-9 activity were detected for PBDE 99 or TBBPA exposed animals compared to controls. In the

hippocampus from PBDE 99 and TBBPA exposed animals significantly lower (p≤ 0.01 and p≤ 0.001, respectively) caspase-9 activity was detected compared to the control animals.

Figure 7. Caspase-9 activity (luminometry units) in male mouse brain 24 h after a single oral dose of either 20%

fat emulsion vehicle (10 ml/ kg bw), PBDE 99 (12 mg/kg bw) or TBBPA (11.5 mg/kg bw) to mice on postnatal day 10. The results are presented as the mean ± SD from 19-20 animals. The statistical evaluations were made using one-way ANOVA and significant difference between control and exposed are indicated as follows:

**p≤ 0.01; ***p≤ 0.001

After administration of ketamine (50 mg/kg bw) to mice on PND 10 the activity of caspase-9 was measured 24 hours later (Fig. 8) and the activity was not significantly different in frontal cortex or parietal cortex compared to controls. In the hippocampus from ketamine exposed animals significantly lower (p≤ 0.05) caspase-9 activity was detected compared to the control animals.

Figure 8. Caspase-9 activity (luminometry units) in female mouse brain 24 h after a single subcutaneous injection of 0.9 % saline (5 ml/kg bw) or ketamine (50mg/kg bw) on postnatal day 10. The results are presented as the mean ± SD from 10-12 animals. The statistical evaluations were made using Student’s t-test and

significant differences between control and exposed are indicated as follows: *p≤ 0.05

Activity of caspase-3

After administration of PBDE 99 (12 mg/kg bw) or TBBPA (11.5 mg/kg bw) to mice on PND 10 the activity of caspase-3 was measured 24 hours later (Fig. 9) and there were no

significantly differences in caspase-3 activity in frontal cortex or parietal cortex compared to controls. In the hippocampus from PBDE 99 or TBBPA exposed animals significantly lower (p≤ 0.01 and p≤ 0.001, respectively) caspase-9 activity was detected compared to the control animals.

Figure 9. Caspase-3 activity (luminometry units) in ale mouse brain 24 h after a single oral dose of either 20%

fat emulsion vehicle (10 ml/ kg bw), PBDE 99 (12 mg/kg bw) or TBBPA (11.5 mg/kg bw) to mice on postnatal day 10. The results are presented as the mean ± SD from 19-20 animals. The statistical evaluations were made using one-way ANOVA and significant difference between control and exposed are indicated as follows:

**p≤ 0.01; ***p≤ 0.001

After administration of ketamine (50 mg/kg bw) to mice on PND 10 the activity of caspase-3 was measured 24 hours later (Fig. 10) and the activity was not significantly difference in frontal cortex or parietal cortex compared to controls. In the hippocampus from ketamine exposed animals significantly lower (p≤ 0.05) caspase-3 activity was detected compared to the control animals.

Figure 10. Caspase-3 activity (luminometry units) in female mouse brain 24 h after a single subcutaneous

injection of 0.9 % saline (5 ml/kg bw) or ketamine (50 mg/kg bw) on postnatal day 10. The results are presented

as the mean ± SD from 10-12 animals. The statistical evaluations made using Student’s t-test and significant

difference between control and exposed are indicated as follows: *p≤ 0.05

Activity of caspase-8

After administration of PBDE 99 (12 mg/kg bw) or TBBPA (11.5 mg/kg bw) to mice on PND 10 the activity of caspase-8 was measured 24 hours later (Fig. 11) and there were no

significantly differences in caspase-8 activity in frontal cortex or parietal cortex compared to controls. In the hippocampus from PBDE 99 or TBBPA exposed animals significantly lower (p≤ 0.01 and p≤ 0.001, respectively) caspase-8 activity was detected compared to the control animals.

Figure 11. Caspase-8 activity (luminometry units) in male mouse brain 24 h after a single oral dose of either 20% fat emulsion vehicle (10 ml/ kg bw), PBDE 99 (12 mg/kg bw) or TBBPA (11.5 mg/kg bw) to mice on postnatal day 10. The results are presented as the mean ± SD from 20 animals. The statistical evaluations were made using one-way ANOVA and significant difference between control and exposed are indicated as follows:

**p≤ 0.01; ***p≤ 0.001

After administration of ketamine (50 mg/kg bw) to mice on PND 10 the activity of caspase-8 was measured 24 hours later (Fig. 12) and the activity was not significantly different in frontal cortex, parietal cortex or hippocampus compared to controls.

Figure 12. Caspase-8 activity (luminometry units in female mouse brain 24 h after a single subcutaneous

injection of 0.9 % saline (5 ml/kg bw) or ketamine (50 mg/kg bw) on postnatal day 10. The results are presented

as the mean ± SD from 12 animals. The statistical evaluations were made using Student’s t-test.

DISCUSSION

Exposure to PBDE 99, during the brain growth spurt, has in earlier studies been shown to result in changes in adult spontaneous behavior, learning and memory. The effects worsen with age and were also dose-related (Eriksson et al. 1998, 2001; Eriksson et al. 2002; Viberg et al. 2004b). The mechanism behind this neurotoxicity is not understood and there may be

different causes to the disturbances.

Nerve cell apoptosis have been detected in human astrocytoma cells (Madia et al. 2004) after exposure to PBDE 99 and a study by Reistad and co-workers (Reistad and Mariussen 2005) indicates that DE-71 causes cell death by apoptosis. These studies suggest that apoptosis may be a part of the neurotoxicity seen after exposure to PBDEs.

One objective of the present study was to develop a model to quantitatively measure caspase activity in brain tissue homogenates. The results show that caspase activity was proportional to the protein concentration in the samples. The results showed linearity and it is possible to quantitatively measure the activity of the different caspases in the brain homogenates. The model seemed stabile but the sensibility of the assay is not evaluated, which may affect the result of the present study.

In the present study caspase-9 activity was elevated in frontal cortex in mice treated

neonatally with PBDE 99. The activity of caspase-9 shows that the intrinsic pathway has been

activated and this activation is initiated by the mitochondria and often due to an increase in

intracellular Ca

²⁺

and the generation of ROS. The intrinsic pathway and caspase-9 can be

activated not just from increase in intracellular Ca

²⁺

and the generation of ROS, but the

activation may also be derived from a p53 dependent mechanism in response to different

signals (Vousden 2000). The present study indicates that PBDE 99 may activate caspase-9 via the intrinsic pathway via one or several of the different factors activating caspase-9. Madia and co-workers showed in an earlier study that PBDE 99 causes an increase of the

transcription factor p53 (Madia et al. 2004) and this increase in p53 may be the reason for the caspase-9 activity seen in the present study.

On the contrary mice exposed to ketamine did not show a significantly higher caspase-9 activity in the frontal cortex compared to untreated mice, even though ketamine is a known inducer of apoptosis/neurodegeneration in the brain, according to several studies (Fredriksson et al. 2004; Ikonomidou et al. 1999; Jevtovic-Todorovic and Olney 2008). Furthermore, there

was no effect on caspase-9 activity in the parietal cortex or frontal cortex. The reason for this may be that the analyzing method used in the present study is not sensitive enough. Earlier studies where ketamine have been shown to induce apoptosis and neruodegeneration is studies (Fredriksson et al. 2004; Fredriksson et al. 2007; Jevtovic-Todorovic 2005; Jevtovic- Todorovic et al. 2003) where sections of the brain, and not homogenate, have been used and it is possible that the method of using tissue sections with antibodies to detect apoptosis is much more sensitive. Also repeated dosing have been used in these earlier studies (Jevtovic-

Todorovic 2005; Jevtovic-Todorovic et al. 2003; Slikker et al. 2007) and the higher

concentration of the drug in the body may affect the apoptosis process more than one single dose used in the present study.

The next step in the apoptotic pathway, when caspase-9 is activated, is the activation of pro-

caspase-3 to active caspase-3, which will lead to activation of endonucleases and apoptosis of

the cell. If apoptosis is induced in the mice exposed to PBDE 99 during the time when the

developing brain is undergoing rapid development with the characteristic axonal and dendrite

outgrowth and formation of neural connections among other events, there is a great risk that

these processes is disturbed by the induction of caspase-9 activity, and consequently apoptosis.

The higher levels of caspase-9 activity in the PBDE 99 exposed animals indicate that also the activity of caspase-3 may be elevated in the PBDE 99 exposed animals, but in parietal cortex and frontal cortex there were no changes in the caspase-3 activity 24 hours after exposure to PBDE 99 compared to the unexposed control. The apoptotic pathway is trigged by different activation steps and the activation of the executor caspase-3 is a late event in the chain. In order to activate caspase-3, other caspases, like caspase-8 and caspase-9, have to be activated first and the time of 24 hours may be too short for detectable activation of caspase-3 in vivo.

In a study by Alm and co-workers where cerebellar cells (GD17) from rat were exposed to PBDE 99 for 24 hours and there was no evidence for increased caspase-3 activity (Alm et al.

2008). Furthermore mice treated with ketamine did not show an increased activity of caspase- 3 in the cortex. Since ketamine is a known inducer of apoptosis/neurodegeneration, these findings also support the hypothesis that the activation of caspase-3 demands a longer time of exposure. But due to the fact that we see no changes in caspase-9 activity after the exposure to ketamine it is also likely that no changes of caspase-3 activity are seen. And that, and not the time aspect, is the reason to way no caspase-3 activity is detected in the brain from mice exposed to ketamine.

In the present study there was no significant change in the levels of caspase-8 activity in

frontal cortex or parietal cortex in mice exposed to any of the three tested compounds. This

indicates that if apoptosis is initiated it is by the intrinsic pathway and not by the extrinsic

pathway. Caspase-8 is activated by the binding of ligand to the death receptor and the result

of the present study indicates that no such binding have occurred in cells of parietal cortex or

frontal cortex after the exposure to PBDE 99, TBBPA or ketamine. This is interesting and

puzzling since the known neurodegenerative agent ketamine has been shown to elevate caspase-8 and caspase-9 activity in brain (Jevtovic-Todorovic and Olney 2008). The reason to way no change in caspase-8 activity was detected after the exposure to ketamine may be the same reason to way no changes in caspase-9 activity was detected after the exposure of the known inducer of apoptosis: less sensitivity in the model used in present study or repeated dosing used in earlier studies.

In the present study a decrease in caspase-3, -8 and -9 activity was detected in hippocampus after exposure to PBDE 99. This decrease in activity was also seen in hippocampus for all three caspases after exposure to TBBPA. When looking at ketamine exposed animals the activity of caspase-3 and caspase-9 were also decreased in hippocampus, but not caspase-8.

More than half of the initially-formed neurons are deleted in certain brain regions during normal development (Blomgren et al. 2007) and the homeostasis in the process is complex and important. Disturbances to the apoptosis homeostasis or apoptotic pattern may be one explanation, or one part of the explanation, to the disturbances in impaired working memory in hippocampus-related behaviour tests in adult mice exposed to PBDE 99 on PND10

(Eriksson et al. 2001). Furthermore it has earlier been shown that neonatal PBDE 99 exposure is associated with a 20% decrease in the density of cholinergic nicotinic receptors in the hippocampus of adult mice exposed as pups (Viberg et al. 2004b). Levels of proteins

important in normal brain development, namely CaMKII, GAP-43 and synaptophysin is also

affected in hippocampus after exposure to PBDE 99 or PBDE 209 or ketamine (Le Bescont et

al. 2009; Viberg et al. 2008a; Viberg et al. 2008b). Taken together the results from these

studies indicate that the developing hippocampus is sensitive to disturbances from different

xenobiotics.

Like PBDE 99, TBBPA decreased the caspase activity in the hippocampus in the present study. TBBPA has in earlier studies (Birnbaum and Staskal 2004; Fredriksson et al. 2004) not shown the same neurotoxic potential as the PBDEs but there are some studies indicating that TBBPA can cause apoptosis in different cell lines (Ogunbayo et al. 2008; Ogunbayo and Michelangeli 2007; Reistad and Mariussen 2005). This raises an interesting question concerning the ability of both these brominated flame retardants to affect the apoptosis homeostasis or apoptotic pattern in the developing hippocampus, even though just one of the compounds causes neurotoxic effect shown as changes in adult behavior and memory.

In this study, it is also interesting to note the differences in response to PBDE 99 between the cortex and the hippocampus. Regional differences in the effects of neonatal exposure could result from the underlying heterogeneity between the different brain structures and cell

populations and/or that the developmental processes occur at different times and differ in their susceptibility.

The control and regulation of the apoptosis pathway are complex and involves several

different factors. The regulation of the intrinsic pathway is mediated by the Bcl-2 proteins and in this group there are both pro-apoptotic and anti-apoptotic members, which might be

affected to enable the lower activity of caspase-9 and via lower activity of caspase-9, decrease the activity of caspase-3. Further studies are needed to evaluate effects on the regulation of apoptosis after exposure to the compounds used in the present study.

In conclusion the results from the present study indicate that apoptosis may be involved in the neurotoxicity seen after exposure to PBDE 99 during a critical time of brain development.

The present study also shows that exposure to PBDE 99 can increase the caspase-9 activity in

frontal cortex and that exposure to PBDE 99 and TBBPA decrease the activity of multiple

caspases in hippocampus in the developing brain. This might impair the developing neurons

in the neonatal brain. Further studies are required in order to understand the mechanism and

the consequences of the apoptosis or changes of the apoptosis homeostasis or apoptotic

pattern after exposure to PBDE 99 or TBBPA during a critical time of development.

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