Quantification of p53-expressing cells andneurodegenerative cells in neonatal mousebrain after exposure to PBDE 99, TBBPA orketamineIwa Lee

Quantification of p53-expressing cells and neurodegenerative cells in neonatal mouse brain after exposure to PBDE 99, TBBPA or ketamine

Iwa Lee

Degree project in biology, Master of science (2 years), 2011

Examensarbete i biologi 30 hp till masterexamen, 2011

Table of Contents

Acknowledgments... 3

Abstract ... 4

Populärvetenskaplig sammanfattning... 5

Introduction ... 6

Brain development and critical periods ...6

Apoptosis...6

Caspases ...7

Extrinsic pathway ...7

Intrinsic pathway ...8

Execution ...8

Tumour suppressor protein 53 (p53) ...8

Brominated flame retardants...9

PBDEs...9

TBBPA ...10

Ketamine ...10

Objectives...11

Material and Methods... 11

Chemicals ...11

Animals and exposure...11

Histopathology...12

Sample preparation ...12

Screening ...12

Immunohistochemistry (IHC)...12

Fluoro-Jade B staining...13

Quantification...13

Statistical analysis ...14

Results ... 15

p53 expression ...15

Level P0-9...15

Level P0-34...15

Neurodegenerative cells ...16

Level P0-9...16

Level P0-34...16

Discussion... 17

References ... 20

Acknowledgments

First of all I would like to express my gratitude to my outstanding supervisors. To Henrik Viberg, my main supervisor, I want to say thanks for all the advice, guidance, time for questions and clarifications and your attempts to introduce me to “real music”. To Ulrika Bergström, my co-supervisor, thanks for the encouragement, positive cheers and tips, because

“why spend more time inventing the wheel again, when someone has already done it”.

Moreover I wish to say thanks to all the wonderful people at the Department of environmental

toxicology, for helping me with all the things I didn’t know, and for making my time there

fun and exciting. Lastly, I also want to thank Jan Örberg, for inspiring and opening my eyes

for ecotoxicology.

Abstract

Brominated flame retardants are commonly used in plastics, textiles and electronic

components to fight and limit the spreading of fires. These substances are ubiquitously found in the environment and increasing levels in humans and human breast milk have been

reported. Because of the flame retardants prevalence, they are of great concern, which makes it important to know and understand how these substances can affect the environment and humans. The brominated flame retardants consist of a large group of substances and amongst these are polybrominated diphenyl ethers (PBDEs) and tetrabromobisphenol A (TBBPA).

Due to PBDEs physico-chemical properties they are highly stable and lipophilic.

Exposure to PBDEs during the brains critical growth period has shown to induce permanent developmental neurotoxicity in adult mice. This has not been observed with TBBPA. The mechanism for how the neurotoxicity is induced is still unclear, however it is suggested that apoptosis play a major role. Apoptosis is triggered by various factors, although one contestant contributes to its activation in great deal. The p53 protein is a tumour suppressor protein, which regulates several cell survival functions such as DNA repair, differentiation and apoptosis.

The purpose of the present study was to investigate if there is a link between developmental neurotoxicity caused by PBDE 99 or ketamine and neuronal apoptosis induced via the p53- mediated pathway. In this study, the number of cells expressing p53 and the number of neurodegenerative cells were quantified 24 or 48 h after a single oral dose to 12 mg PBDE/kg bw, 11.5 mg TBBPA/kg bw or a single subcutaneous injection in the neck of 50 mg

ketamine/kg bw.

The result of the present study revealed no significant differences in that PBDE 99, TBBPA or ketamine elicited neuronal apoptosis. Since at least ketamine is known to cause

neuroapoptosis the explanation for lack of effects may be due to reasons such as insensitivity

of the method used, time points of analysis or that the apoptosis was not p53-mediated. Even

so, there is still compelling evidence that PBDEs and ketamine cause neurodevelopmental

toxicity, connected to impaired behaviour and cognitive function, when exposed during the

critical growth period of the brain.

Populärvetenskaplig sammanfattning

Flamskyddsmedel – De osynliga livräddarna eller de oförutsedda dråparna?

De finns utspridda världen över och nästintill dig. De är där för att förhindra uppkomst och spridning av bränder. De är där för att skydda dig. De är beständiga och stabila. Men är de enbart av godo? Flamskyddsmedel har bevisat ha hälso- och miljötoxiska effekter, men än så länge är mycket fortfarande okänt. Flamskyddsmedlen orsakar främst störningar i

nervsystemet, men mekanismen bakom deras effekt är ännu inte känd.

Flamskyddsmedel används som kemiska tillsatser bland annat i plaster, textiler, elektriska och elektroniska produkter. Det finns över hundra olika typer av flamskyddsmedel och de klassas generellt in i olika grupper. Bromerade flamskyddsmedel (BF) har använts och producerats i störst utsträckning, då deras egenskaper har visats mest lämpliga. BF är mycket stabila föreningar, på grund av deras kemiska struktur, och har även förmågan att ansamlas i fettvävnad. Detta gör att de kan stanna kvar och ackumuleras i kroppen.

De mesta kända BF är bland annat PBDE (polybromerade difenyletrar), TBBPA

(tetrabrombisfenol A), PBB (polybromerade bifenyler) och HBCD (hexabromcyklododekan).

BF kan användas, antingen som en reaktiv tillsats, eller additiv tillsats. Reaktiv tillsats innebär att BF inkorporeras med plasten så att kemiska bindningar uppstår, till skillnad från additiv tillsats där BF blandas med materialet utan att några kemiska bindningar bildas. Därför anses reaktiva tillsatser mer ”säkera”, jämfört med additiva tillsatser, då de inte lika lätt kan läcka ut från plasten. Då skadliga effekter har observerats i djurförsök med PBDE och PBB har dessa blivit förbjudna och håller på att fasas ut. Man har ännu inte observerat att TBBPA orsakar skadliga toxiska effekter i försöksdjur, och är därför fortfarande i produktion.

Höga halter av BF har observerats i modersmjölk runt om i världen, vilket utgör en extrem hälsofara för nyfödda och spädbarn. Detta är för att amningsperioden, hos däggdjur, överlappar med en av hjärnans mest viktiga utvecklingsfaser. Exponering för främmande ämnen under denna känsliga fas har bland annat visats orsaka biokemiska förändringar i hjärnan, missbildningar, beteendestörningar och försämrad inlärnings- och minnesförmåga hos vuxna möss. Effekterna är permanenta, förvärras med åldern och ökar med ökande dos.

Hur BF orsakar dessa effekter är ännu oklart. Det har spekulerats att BF aktivera ”apoptos”, en typ av programmerad celldöd. Apoptos är en livsviktig mekanism för organismers utveckling och immunförsvar. Apoptos är en mycket reglerad, men även komplicerad och invecklad, process då den har flera aktiverings- och signaleringsmekanismer. Den mest kända molekylära mekanismen för verkställandet av apoptos är via enzymer som kallas caspaser.

I denna studie exponerades möss för en dos av BF under hjärnans kritiska utvecklingsfas.

Syftet var bland annat att se ifall det fanns en ökad mängd apoptos i vissa hjärnregioner i de djur som utsattes för BF jämfört med de som var kontroller. Detta gjordes genom att

analysera mängden av en specifik apoptosmarkör i försöksdjurens hjärnor. Min studie gav

inga statistiskt säkerställda resultat om att det fanns en ökad nivå av apoptos i de BF-utsatta

djuren. Det kan dock inte uteslutas att apoptos utgör mekanismen bakom de toxiska effekter,

då inte alla typer av apoptosmarkörer har analyserats.

Introduction

Brain development and critical periods

The brain and spinal cord development in mammals involves complex and intricate, as well as intrinsic processes. During the brain development the brain is most sensitive to insults such as xenobiotics, which may lead to severe disorders and malfunctions. The developmental

process can be divided into stages, each having its own critical period of vulnerability. When it comes to the development of the spinal cord and the central nervous system it can

somewhat be categorised into two stages. The first stage is comprised of organogenesis and neuronal multiplication i.e. the brain acquires its general shape and neuron precursors start to proliferate. The second stage is referred to as the “brain growth spurt”. It is distinguished by a maturation period of axonal and dendritic growth, synaptogenesis, glial multiplication and myelination (Dobbing and Sands, 1979).

The occurrence and duration of the BGS varies among different species. In humans, this period starts around the third trimester of pregnancy and continues on for the first two years of life. In rats and mice, the BGS takes place during the first four weeks of life, with a peak around postnatal day (PND) 10 (figure 1) (Dobbing and Sands, 1979).

Figure 1. Rate curves of brain growth in relation to birth between human and mouse. Values are calculated at different time intervals for each species (modified from Davison and Dobbing 1968).

The BGS coincide with the lactation period of humans, rats and mice. Therefore it is likely that offspring may be exposed to lipophilic xenobiotics via the milk, which can be retained and distributed in the brain. Because this period is very sensitive and vulnerable to insults (Dobbing and Sands, 1979), it composes a great concern to normal brain development. This concern have been proven valid, as studies have shown that exposure to xenobiotics during this critical period has lead to neuronal damage, consequently also to cognitive and

behavioural anomalies (Eriksson et al., 2001a; Jevtovic-Todorovic et al., 2003; Fredriksson et al., 2007).

Apoptosis

The process of cell death is critical for life as it is essential for normal development and

homeostasis (Meier and Vousden, 2007). Apoptosis can be regarded as programmed cell

death, a complicated process (figure 2), which is necessary to regulate excess and damaged

cells that are potentially dangerous (Hengartner, 2000). Unlike necrosis, apoptosis is a

controlled ATP-dependent process, which does not cause rupture of the cell membrane and

thus prevents cellular content to be released to the surrounding interstitial tissue. The apoptotic cell is rapidly phagocytosed by macrophages, or adjacent cells, and no

inflammatory reaction is initiated (Elmore, 2007). The main executioner of this complex cascade of events is a family of proteins named caspases.

Figure 2. Schematic overview of the two major apoptotic pathways in the mammalian cell (Igney and Krammer, 2002).

Caspases

Caspases are cysteine aspartyl-specific proteases that cleave substrates at specific aspartic residue bonds (Asp-xxx) (Thornberry et al., 1997). In apoptosis, caspases function mainly as effectors or initiators. Effector caspases (i.e. caspase-3, -6 and -7) are activated proteolytically by an upstream caspase, while initiator caspases (e.g. caspase-8 and -9) are activated by regulated protein-protein interactions (Thornberry and Lazebnik, 1998; Hengartner, 2000;

Elmore, 2007). Despite the vast diversity of cells in the body, the process of inducing cell suicide is remarkably similar. The two major pathways of apoptosis are the “intrinsic”

pathway and the “extrinsic” pathway. Both pathways are associated with an intricate cascade of caspases, which cleave cellular substrates and eventually gives rise to the morphological and biochemical characteristics of apoptosis (Igney and Krammer, 2002).

Extrinsic pathway

Apoptosis signalling via the extrinsic pathway involves triggering of certain members of the

tumour-necrosis factor (TNF) receptor superfamily known as death receptors (DRs) on the

cell surface (Hengartner, 2000). These DRs are similar in that they all share a common “death

domain”, which plays a crucial role in transmitting the death signal from the cell surface to

the intracellular signalling pathways (Elmore, 2007). Binding of the receptor/ligand causes

the recruitment of so-called “adapter proteins”. The adapter proteins in turn, will eventually

cause activation of caspase-8. The extrinsic pathway is also known as the death receptor

pathway (Igney and Krammer, 2002).

Intrinsic pathway

The intrinsic signalling pathway is mediated by the mitochondria, and is also often referred to as the mitochondrial pathway. The intrinsic pathway is used in response to extracellular signal and internal insults such as DNA damage, cellular stress, radiation and free radicals. The stimuli result in the release of cytochrome c and other pro-apoptotic proteins (Igney and Krammer, 2002). In the cytosol, the cytochrome c together with other proteins form a

complex known as the apoptosome. The association of the apoptosome leads to activation of caspase-9 (Igney and Krammer, 2002).

Execution

The intrinsic and extrinsic pathways converge at the activation of the effector caspases. The

“executioners”, for example caspase-3, cleave the DNA-strands in the nucleus leading to chromatin condensation, nuclear shrinkage, DNA-fragmentation and dismantling of the cytoskeleton. This eventually causes cell fragmentation, blebbing and formation of apoptotic bodies. The remaining cell then becomes engulfed by phagocytes (Igney and Krammer, 2002).

Tumour suppressor protein 53 (p53)

Apoptosis is triggered by various factors, however one particular protein has shown to play a key contribution to its activation. The p53 is a tumour suppressor protein, which regulates several responses, such as cell-cycle arrest, senescence, DNA repair, differentiation and apoptosis (figure 3) and is sometimes regarded as a “death star” or as the “Guardian of the genome” (Sigal and Rotter, 2000; Vousden and Lu, 2002). In humans, the TP53 gene encodes p53, which is the most commonly mutated gene in human cancers (Harms et al., 2004). In the majority of tumours the p53 is inactivated, thus resulting loss of apoptotic function (Sigal and Rotter, 2000).

Figure 3. p53 mediates the response of various stress signals. The choice of response depends on factors like cell type, cell environment or other alterations sustained by a cell (Vousden and Lu, 2002).

Regulation of p53 is achieved by an ubiquitn ligase, MDM2 (murine double minute 2) that inhibits p53 stabilisation and activation. MDM2 becomes inactivated by either

phosphorylation or by ARF (ADP ribosylation factors) proteins and when this occurs the p53

protein stabilises, which leads to increased p53 levels in the cell. The upregulation of p53

induce expression of pro-apototic proteins such as BAX (Bcl-2 (B-cell lymphoma 2) –

associated X protein), PUMA (p53 upregulated modulator of apoptosis) and p53AIP1 (p53-

regulated apoptosis inducing protein 1), which eventually in turn will activate the cascade of

caspases (Igney and Krammer, 2002; Vousden and Lu, 2002).

Brominated flame retardants

Flame retardants are a group of industrial chemicals that are widely used in materials such as plastics, textiles and electronic circuits to prevent and limit the spread of fires (de Wit, 2002).

There are more then 175 types of flame retardants and they are generally classed into four main groups; halogenated organic (e.g. brominated, chlorinated), inorganic,

organophosphorus and nitrogen containing (Birnbaum and Staskal, 2004). Brominated flame retardants (BFRs) have however, been shown to be more suitable for use, and the majority of production is made up of BFRs (Darnerud, 2003). The major BFRs consist of polybrominated diphenyl ethers (PBDEs), tetrabromobisphenol A (TBBPA), polybrominated biphenyls (PBBs) and hexabromocyclododecane (HBCD). The annual production in year 2000 exceeded thousands of tons per year, however since then PBDEs (penta- and

octabromdiphenyl composites) and PBBs have been banned and phased out of production.

The main commercial BFRs still in use are TBBPA, HBCD and decabromodiphenyl ether (BSEF, 2011). Several of these substances, and their metabolites, are persistent and lipophilic and have been shown to bioaccumulate (de Wit, 2002). PBDEs, PBBs and HBCDD are grouped as additive BFRs i.e. they are incorporated into the polymer prior to, during or following polymerisation without chemical binding. TBBPA can also be used as either an additive or reactive BFRs. Reactive BFRs are covalently bound to the polymer and are therefore considered to be “safer” then additives due to the reduced leaching risk (WHO, 1997).

PBDEs

Theoretically there are 209 possible PBDE congeners (technical products predominately contained penta-, octa- and decabromodiphenyl ethers) due to the large number of positions where bromine can bind to the two phenyl rings (figure 4) (WHO, 1997). It is recognised that PBDEs are globally present in the environment and also in wildlife. PBDEs are lipophilic and due to their molecular structure very stable. Their structural resemblance to the well-known environmental contaminant PCBs (polychlorinated biphenyls) is one of the main reasons for environmental and health concern (de Wit, 2002; Birnbaum and Staskal, 2004).

Figure 4. Chemical structure of PBDEs.

The most critical endpoints concerning PBDEs are on neurotoxicity and to a lesser extent on thyroid hormone homeostasis (Darnerud, 2003). Several studies have shown that neonatal PBDE exposure (especially PBDE 99) alter adult spontaneous motor behaviour, habituation capability and cholinergic transmitter susceptibility in male mice (Eriksson et al., 2001b;

Eriksson et al., 2002; Viberg et al., 2002). Eriksson et al. (2002) were also able to define a

critical phase of sensitivity for neurodevelopmental toxicity for PBDEs. This critical phase

(postnatal day 10) overlap with the period of rapid brain growth i.e. the BGS. In addition the

results showed that the neonatally PBDE exposed mice displayed similar behaviour profile as

those exposed to PCBs (Eriksson and Fredriksson, 1996). These effects were observed in

adulthood and were also shown to worsen with age.

TBBPA

Tetrabromobisphenol A is the most commonly used BFR, mainly because its ability to be used as an additive or a reactive flame-retardant. TBBPA has a similar structure to PBDEs (figure 5) (WHO, 1997) and is highly lipophilic (log K

= 4.5) however, it is not very stable and partially breaks down under aerobic and anaerobic conditions at various degradation rates (Birnbaum and Staskal, 2004).

Figure 5. Chemical structure of TBBPA.

Unlike PBDEs, TBBPA has not been shown to cause adverse insults to experimental animals, which suggests that its toxicity is low (Darnerud, 2003). Previous studies by (Meerts et al., 2000; Meerts et al., 2001) showed that TBBPA potently inhibit binding of thyroxine (T

) to transthyretin (Mottram et al.) in vitro, by competition, which may disturb the thyroid

hormone homeostasis. Effects of this were not observed by Meerts et al. (Meerts et al., 2000) in an in vivo model with pregnant mice. Findings by (Saegusa et al., 2009) showed no

obvious toxic, histopathology, thyroid weight or serum T

-concentration changes in mice exposed to TBBPA. This suggests that evidence of developmental thyroid disturbances by TBBPA is still very limited.

Ketamine

Ketamine (Ketalar®) is a general anaesthetic used in surgical procedures in toddlers and newborns, as well as in veterinary medicine. It gives rise to a so-called “dissociative anaesthesia” by causing selective interruptions to association pathways in the brain (Kohrs and Durieux, 1998). Ketamine is a known non-competitive N-methyl-D-aspartate (NMDA)- glutamate receptor antagonist.

The mechanism for how ketamine induce apoptosis is still unclear, however it has been observed in several studies that blockage of NMDA receptors during neonatal development triggers apoptotic neurodegeneration, suggesting that NMDA receptors are crucial for neuronal survival (Ikonomidou et al., 1999; Olney et al., 2002; Jevtovic-Todorovic et al., 2003; Scallet et al., 2004; Slikker et al., 2007). The NMDA receptors are commonly found across the CNS and they increase in number during postnatal development. The NMDA receptors play an important role in the developing brain as glutamate, inter alia, promotes differentiation and proliferation of the CNS, migration of neuronal progenitors, synaptic plasticity, learning and memory.

The NMDA subtype of glutamate receptor goes through a period of hypersensitivity, causing neurons with NMDA receptors to be most sensitive to excitotoxic degeneration, which coincide with the BGS period and exposure to ketamine may very likely occur at this time (Collingridge et al., 1983; McDonald et al., 1988; D'Souza et al., 1993; Komuro and Rakic, 1993; Ikonomidou et al., 2001). The induced neuroapoptosis may be related to behavioural, learning and memory disorder in adult mice and rats, which seem to worsen with age

(Jevtovic-Todorovic et al., 2003; Fredriksson et al., 2004; Fredriksson et al., 2007; Viberg et

al., 2008). Viberg et al. (2008) exposed neonatal mice, on PND 10, to ketamine, which

resulted in changes in important biochemical substrates of neuronal development and

synaptogenesis. Also observed, was altered persistent spontaneous behaviour and habituation in a dose-response-related manner in adult mice (Viberg et al., 2008).

Objectives

The hypothesis of this study was that developmental neurotoxicity caused by PBDE 99 or ketamine is linked to neuronal apoptosis induced via the p53-mediated pathway, therefore the objectives of this study were to:

i) Develop an immunohistochemical model for detecting cells expressing p53 in mouse brain specimens,

ii) Quantify and evaluate cells expressing p53 in different brain regions after neonatal exposure to PBDE 99, TBBPA or ketamine,

iii) Assess if there is a higher frequency of degenerative neurons in specific brain regions with Fluoro-Jade B staining after neonatal exposure to PBDE 99, TBBPA or ketamine.

Material and Methods

Chemicals

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 Apoteket, Uppsala, Sweden.

PBDE 99 and TBBPA were dissolved in a mixture of egg lecithin (Merck, Darmstadt,

Germany) and peanut oil (Oleum arachidis). This was done to mimic mouse milk (fat content

~14%), so it could act as a carrier of the lipophilic substances. The substrates were 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. Ketamine was mixed with saline (0.9% NaCl) to yield a solution of 10 mg ketamine/ml. As a control, a 20% fat emulsion vehicle was used.

Animals and exposure

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

Sollentuna, Sweden. They were kept individually in plastic cages in a room with temperature of 22°C, a 12/12-h cycle of light and dark, free access to standard pellet food (Lactamin, Stockholm, Sweden) and tap water ad libitum. The day of birth of the litters was assigned PND 0. The size of the litters was kept intact during the whole experiment until the day of dissection. 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).

On PND 10, male and female pups were given 12 mg (21 µmol) PBDE 99/kg bw or 11.5 mg

(21 µmol) TBBPA/kg bw via a metal gastric tube as one single oral dose. Control mice

received 10 ml/kg bw of the 20% fat emulsion vehicle. On PND 10 male and female mice

were given a subcutaneous injection in the neck of 50 mg ketamine/kg bw. In a previous

study these doses have shown to have neurodevelopmental -and neurodegenerative effects

four or 48 h after exposure male mice from all exposure groups were sacrificed, the brains were taken out and incubated in formaldehyde for 48 h and thereafter stored in 70% EtOH.

In total forty brains were collected, twenty brains after 24 h of exposure and the same number after 48 h of exposure, which provided eight brains per treatment i.e. four brains for each of the five exposures and exposure times.

Histopathology

Sample preparation

All brains were prepared and handled in exactly the same manner. To prepare the brains for slicing they were dehydrated in ethanol (70%-99.5%) and xylene, and then embedded in low temperature paraffin (54°C). The slicing was carried out using a Microme HM 355 microtome making 4 µm thin slices that were fixated with water on super frost glass sections.

Screening

At first a total of 10 brains, one from each exposure group and exposure time, were screened through by making one section every 40 µm. Approximately two hundred sections were made for each brain. Out of these sections, 15-20 sections from various levels of the brain were analysed using immunohistochemistry for localisation of p53 in different part of the brain.

After analysing the results of the screening, two different levels of the brains were selected for further investigation. These two levels were named P0-9 and P0-34 (P0 stand for postnatal day 0) that refers to different brain levels in rats (Paxinos, 1990). Level P0-9 is situated at the anterior part of the brain (figure 6a and 6b), whilst level P0-34 is located at the posterior part of the brain close to the central sulcus (figure 7a and 7b).

Immunohistochemistry (IHC)

The brain sections were deparaffinised and rehydrated in xylene and ethanol (99.5-70%).

Before removing the paraffin, a circular carving was made around the brain sections to prevent dehydration. Subsequently, sections were washed in phosphate buffer saline (PBS, pH 7.4) and 0.3% PBS with Triton-100 (PBS-T) for 5 min respectively and rinsed with

distilled water (dH

O). Antigen retrieval was then performed by transferring the brain sections to a Tris-EDTA buffer (pH 9.0) bath in a steamer, which was heated to 97-98°C, and

incubated for 30 min. The sections were then allowed to cool down for 20 min at room temperature and then rinsed with dH

O for further cooling. Afterwards the sections were incubated in a 3% H

O

-solution (diluted in PBS) for 15 min in room temperature and then washed twice with PBS-T for 5 min. Next, the sections were blocked using a blocking

solution (BS) consisting of 5% normal goat serum (Invitrogen, PCN-5000) diluted in PBS for

60 min. The blocking solution was removed by gently tapping the sections, and then the

sections were treated with phosphor-p53 (Ser15) rabbit polyclonal antibody (Abcam, ab1413)

1:100 in BS. The sections were incubated over night at 4°C in a humidified chamber. The

following day, the primary antibody was removed and the sections washed once in PBS and

twice in PBS-T for 5 min, respectively. The brain sections were treated with a biotinylated

secondary antibody (Invitrogen, B2770) 1:100 in PBS-T for 30 min in a humidified chamber

and washed in PBS for 5 min. The sections were then exposed to Vectastain ABC-reagent

(Vector Laboratories, PK-4000) for 30 min in room temperature, washed in PBS for 5 min

and stained with a peroxidase substrate DAB (3,3'-diaminobenzidine)-solution (Vector

laboratories, SK-4100). Nickel chloride was added to the substrate solution to yield a gray-

black stain. Finally, the brain sections were counterstained with eosin and dehydrated in a

series of alcohol (70-99.5%) and xylene and mounted with Pertex®. The DAB staining was manually examined using a Leica DMRXE microscope.

Fluoro-Jade B staining

Fluoro-Jade is anionic flourochrome capable of selectively staining neurodegenerate cells in brain slices. It is still a novel method for histochemical localisation, however it has been shown to be reliable and simple as well as equally sensitive as older routine histochemical stain types as Nissl or HandE (hematoxylin and eosin) (Schmued et al., 1997). Additionally, the Fluoro-Jade is compatible with other labelling techniques as immunofluorescence and fluorescent Nissl counterstaining (Schmued et al., 1997; Scallet et al., 2004). The method has been controversial as the mechanism by how Fluoro-Jade labels degenerative neurons is still unknown. In recent years new Fluoro-Jade derivatives (e.g. Fluoro-Jade B) has been

developed with greater affinity to detect neurodegenerative cells as well as distal dendrites, axons and axon terminals (Schmued and Hopkins, 2000a; Schmued and Hopkins, 2000b).

The staining was principally conducted according to the method described by Schmued et al.

1997. The brain sections were deparaffinised in as series of xylene and ethanols, as mentioned in previous passage, and then rinsed for 2 min in dH

O. The sections were then transferred to 0.06% potassium permanganate solution for 10 min whilst gently shaking. The sections were washed in dH

O for 2 min afterwards and incubated in a staining solution of 0.0004% Fluoro- Jade B (Millipore, AG310) and 0.0002% 4´,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma, D9542).The staining solution was made by firstly preparing a 0.01% Fluoro- Jade B stock solution by adding 10 mg to 100 ml dH

O. Secondly, 4 ml of the stock solution was added to 96 ml of 0.1% acetic acid to make up 100 ml of 0.0004% staining solution. To be able to counterstain the Fluoro-Jade B, DAPI was added to the staining solution. The mixture solution was prepared by taking 2 ml of 0.01% DAPI stock solution (10 mg to 100 ml dH

O) and added to 98 ml Fluoro-Jade B staining solution. After 20 min the sections were rinsed in three 1 min changes of dH

O, excess water was dried of and the sections were put on a slide warmer (40-50°C) for 10 min to become completely dry. The sections were then cleared by wash immersing them twice in xylene baths for 3 min and lastly mounted with Pertex

The Fluoro-Jade B staining was examined manually using a Leica DMRXE microscope equipped with a fluorescence filter (450nm).

Quantification

The brain sections were pre-screened to determine which structural regions were of most interest. Based on the pre-screening, focus were put on the diagonal band of broca (DB) and cingulated cortex (Cg) regions for the P0-9 level sections (figure 6a and 6b), and the

retrosplenial granular cortex (Lunell et al.), the occipital cortex (Oc), the Cornus Ammonis (CA3) field of the hippocampus and the DB for the P0-34 level sections (figure 7a and 7b).

The same brain regions were analysed both from sections stained with Fluoro-Jade B as those

for the IHC-model for p53 detection.

A B

Figure 6. A) Coronal section of a mouse brain (gestation day 18) representative for P0-9. The analysed brain regions, the diagonal band of broca (DB) and cingulated cortex (Cg), are indicated by the labelled circles, opposite corresponding right side regions where evaluated similarly. B) Sagittal section view of A, the line indicates the plane orientation of level P0-9 (Schambra 1992).

A B

Figure 7. A) Coronal section of a mouse brain (gestation day 18) representative for P0-34. The analysed brain regions (retrosplenial granular cortex (RGS), occipital cortex (Oc), diagonal band of broca (DB) and Cornus Ammonis (CA3) field of the hippocampus) are indicated by the labelled shapes, corresponding opposite right side regions where evaluated similarly. B) Sagittal section view of A, the line indicates the plane orientation of level P0-34 (Schambra 1992).

Statistical analysis

The number of cells that expressed p53 as well as the number of degenerative neurons was

statistically analysed in four different brain regions; the Cg- and DB-regions on level P0-9

and the RGS-, Oc-, CA3- and DB- regions on level P0-34 were evaluated. The difference in

the number of cells expressing p53/number of degenerative neurons between different brain

regions were analysed for all exposures using one-way ANOVA (GraphPad Prism 3.03).

Results

p53 expression

Level P0-9

There was no significant difference in the number of p53-expressing cells 24 or 48 h after exposure to a single oral dose of either 20% fat emulsion vehicle, 12 mg PBDE 99/kg bw, 11.5 mg TBBPA/kg bw (10 ml/kg bw), single subcutaneous injection of 0.9% saline or 50 mg ketamine/kg bw (5 ml/kg bw) on PND 10 in any of the Cg- and DB-regions of level P0-9 (figure 8a and 8b).

A B

Figure 8. A, B) The number of p53-expressing (mean ± SD, n = 3) cells, on level P0-9, in neonatal mice in the structural brain regions of the cingulated cortex (Cg) and the diagonal band of broca (DB) 24h or 48h after exposure of single oral dose of either 20% fat emulsion vehicle, 12 mg PBDE 99/kg bw, 11.5 mg TBBPA/kg bw (10 ml/kg bw), single subcutaneous injection of 0.9% saline or 50 mg ketamine/kg bw (5 ml/kg bw) on PND 10.

Each brain region of the two brain halves was evaluated separately. One-way ANOVA revealed no significant difference between the control and PBDE 99, TBBPA or ketamine treated animals.

Level P0-34

There was no significant difference in p53-expressing cells 24 or 48 h after exposure to a single oral dose of either 20% fat emulsion vehicle, 12 mg PBDE 99/kg bw, 11.5 mg

TBBPA/kg bw (10 ml/kg bw), single subcutaneous injection of 0.9% saline (5 ml/kg bw) or

50 mg ketamine/kg bw on PND 10 in any of the RGS-, Oc-, CA3- and DB-regions of level

P0-34 (figure 9a and 9b).

Figure 9. A, B) The number of p53-expressing (mean ± SD, n = 3) cells, on level P0-34, in neonatal mice in the structural brain regions of the retrosplenial granular cortex (Lunell et al.), Occipital cortex (Oc), Cornus Ammonis (CA3) field of the hippocampus and the diagonal band of broca (DB) 24h or 48h after exposure of single oral dose of either 20% fat emulsion vehicle 12 mg PBDE 99/kg bw, 11.5 mg TBBPA/kg bw (10 ml/kg bw), single subcutaneous injection of 0.9% saline or 50 mg ketamine/kg bw (5 ml/kg bw) on PND 10. Each brain region of the two brain halves was evaluated separately. One-way ANOVA revealed no significant difference between the control and PBDE 99, TBBPA or ketamine treated animals.

Neurodegenerative cells

Level P0-9

There was no significant difference in the number neurodegenerative cells 24 or 48 h after exposure to a single oral dose of either 20% fat emulsion vehicle, 12 mg PBDE 99/kg bw, 11.5 mg TBBPA/kg bw (10 ml/kg bw), single subcutaneous injection of 0.9% saline or 50 mg ketamine/kg bw (5 ml/kg bw) on PND 10 in any of the Cg- and DB-regions of level P0-9 (figure 10a and 10b).

A B

Figure 10. A, B) The number of neurodegenerative cells (mean ± SD, n = 3), on level P0-9, in neonatal mice in the structural brain regions of the cingulated cortex (Cg) and the diagonal band of broca (DB) 24h or 48h after exposure of single oral dose of either 20% fat emulsion vehicle, 12 mg PBDE 99/kg bw, 11.5 mg TBBPA/kg bw (10 ml/kg bw), single subcutaneous injection of 0.9% saline or 50 mg ketamine/kg bw (5 ml/kg bw) on PND 10.

Each brain region of the two brain halves was evaluated separately. One-way ANOVA revealed no significant difference between the control and PBDE 99, TBBPA or ketamine treated animals.

Level P0-34

There was no significant difference in the number neurodegenerative cells 24 or 48 h after

exposure to a single oral dose of either 20% fat emulsion vehicle, 12 mg PBDE 99/kg bw,

11.5 mg TBBPA/kg bw (10 ml/kg bw), single subcutaneous injection of 0.9% saline or 50 mg

ketamine/kg bw (5 ml/kg bw) on PND 10 in any of the RGS-, Oc-, CA3-, and DB-regions of

level P0-34 (figure 11a and 11b).

A B

Figure 11. A, B) The number neurodegenerative (mean ± SD, n = 3) cells, on level P0-34, in neonatal mice in the structural brain regions of the retrosplenial granular cortex (Lunell et al.), Occipital cortex (Oc), Cornus Ammonis (CA3) field of the hippocampus and the diagonal band of broca (DB) 24h or 48h after exposure of single oral dose of either 20% fat emulsion vehicle, 12 mg PBDE 99/kg bw, 11.5 mg TBBPA/kg bw (10 ml/kg bw), single subcutaneous injection of 0.9% saline or 50 mg ketamine/kg bw (5 ml/kg bw) on PND 10. Each brain region of the two brain halves was evaluated separately. One-way ANOVA revealed no significant difference between the control and PBDE 99, TBBPA or ketamine treated animals.

Discussion

In recent years the increasing presence of brominated flame retardants (BFRs), especially polybrominated diphenyls (PBDEs) and tetrabrombisphenol A (TBBPA), in the environment have become a growing concern. The underlying cause, stem from the still ambiguous effects BFRs have on the environment and in humans (Darnerud, 2003). As more studies have been performed it has been shown that PBDEs induce persistent developmental neurotoxic effects in mice and rats, when exposed during the critical “brain growth spurt” period. The toxic effects are dose-related and appear to worsen with age, and the neurotoxic effects can cause neurobehavioral alterations and impair cognitive functions (Eriksson et al., 2002; Viberg et al., 2003; Viberg, 2004). The mechanism of action behind the neurotoxic effects is still not resolved. The in vitro study by Madia et al. (Madia et al., 2004) detected apoptosis in human astrocytoma cells after exposure to PBDE 99, also studies by He et al. (2008) show signs of apoptosis after exposure to PBDE-47 in cultured hippocampal cells of the brain. It has been suggested that the neurodegeneration result from apoptosis via the classical apoptotic pathways (He et al., 2009).

In the present study, no significant difference was observed in the number of p53-expressing cells, 24 or 48 h after exposure to PBDE 99, TBBPA or ketamine in any of the brain regions analysed on level P0-9 or level P0-34. Similarly, no significant difference was observed in the number of degenerative neurons, 24 or 48 h after exposure to PBDE 99, TBBPA or ketamine in any of the brain regions analysed on level P0-9 or level P0-34.

In a study by Robertsson (2010), induction of caspases (-3 and -9) activity was seen in

hippocampus homogenates from neonatal mice exposed the same doses of PBDE 99, TBBPA

or ketamine, as in this study. The presence of caspase activity supports the hypothesis that

apoptosis is involved in the mechanism behind developmental neurotoxic effects caused by

PBDEs. In the same study caspase-8 activity was also investigated, but since there were no

significant difference in caspase-8 activity in the hippocampus after exposure to ketamine, it

can be activated via the p53-mediated pathway in response to external or internal stress

signals, which in turn activates caspase-9 and eventually caspase-3 (figure 2) (Elmore, 2007).

The investigated time frame, of this study, was 24h or 48h after exposure. Since no significant difference in cells expressing p53 was observed during this time period, it is likely that p53 is expressed either before 24h or after 48h of exposure. Although, as seen in the study by Södervall (2010) caspase-3 did increase during this time interval, and because caspase-3 is activated later on in the apoptotic cascade it appears more likely that the apoptosis is induced much earlier after the exposure. Caspase-3 activation occurs within minutes after cytochrome c release, which may vary from 1 to 24 hours (Huai et al., 2010) and p53 is expressed in the onset of the caspase cascade.

Another explanation for the lack of significant difference of cells expressing p53 is that apoptosis can be induced independently of p53. Another family of transcription factors, the E2f, are known to play a part in controlling cell cycle progression. A particular member of this family, E2F1, is able to induce apoptosis via both a p53-dependent and p53-independent pathway. E2f regulates p53 levels and activity indirectly, by regulating the expression of genes that encode proteins e.g. ARF and caspases, which interfere with p53. E2f is also able to regulate p73, another member of the p53 family, which serves as a pro-apoptotic cofactor of p53. Additionally, p73 can regulate the expression of pro-apoptotic genes, like p53, and it have been suggested that p73 play a central role in E2F1-induced p53-independent pathway (Polager and Ginsberg, 2010).

Furthermore, Chen et al. (2010) suggested that alterations in second messenger signalling and oxidative stress play a role behind the mechanism of developmental neurotoxicity. What was observed, after exposing neonatal hippocampal neurons to different concentrations of PBDE 209, was an elevated Ca

concentration, increased levels of reactive oxygen species (ROS), increased nitrous oxide content and increased expression of p38 MAPK (p38 mitogen-

activated protein kinase). The observed effects were also dose-dependent (Chen et al., 2010).

This could also be a possible explanation to the lack of significant difference in the present study.

In addition, a study by Slikker et al. (2007) indicated that the mode of action for

neurodegeneration of ketamine is caused by up-regulation of NMDA receptor NR1-subunit mRNA. The study showed that increased numbers of the NR1-subunit was present in brain areas where enhanced cell death was apparent. It is postulated that an increase of activated NMDA receptors cause a higher degree of Ca

influx. The Ca

loading reduces the membrane potential in the mitochondria resulting in disrupted electron transport and

increased production of ROS, which in turn eventually induce apoptosis (Slikker et al., 2007).

If this is the case i.e. if the underlying the reason for apoptosis, caused by exposure to PBDE 99 or ketamine, are mediated by Ca

and ROS, it would be unlikely that the p53 would be expressed in elevated levels.

The results of TBBPA show no indication of eliciting a changed apoptotic response, which supports the findings of the studies by (Eriksson et al., 2001a; Eriksson et al., 2002) that TBBPA does not cause developmental neurotoxicological effects.

The Fluoro-Jade B staining, in the current study, showed no significant difference between

any of the treatments 24 or 48 h after exposure. This is consistent with results of the p53

response after exposure of PBDE 99, TBBPA or ketamine. However, in an earlier study by

Fredriksson et al. (2004) similar Fluoro-Jade staining in neonatal mouse brains after ketamine exposure, showed increased neruodegeneration, which is a major discrepancy to the present results. Dose (50 mg/kg bw) and time of exposure (PND 10) were the same for both studies.

The discrepancy may be explained by that the present study had fewer numbers of test animals (n = 3 compared to n = 6), and that different brain regions were investigated.

The aims of the present study i.a. were to develop an immunohistochemical model for detecting and quantifying p53 expression in neurons. The results show that only a limited number of neurons were detected, with a high sporadic degree of variation. This suggests that this method for detecting p53 expressing cells is not very sensitive. Possible reason for this is that the method is not yet fully optimised e.g. the antibodies used for detecting p53 is not suitable, or even that the method itself is not appropriate for this type of apoptotic

quantification. A similar method was used in a previous study by Södervall (2010), with caspases-3, where the cells also gave a weak signal. Due to the insensitivity of the method, it may have affected the results of the study. No significant differences between the different groups were seen in the Fluoro-Jade B stained sections either, however the signal was a lot stronger and the degree of variance was much lower compared to the immunohistochemical model, which suggest that the method is more stabile.

In conclusion, this study showed no significant difference in cells expressing p53 or cell

degeneration in neurons, which might indicate that the mechanism of action for PBDE 99 and

ketamine is not mediated through apoptosis and neurodegeneration. Therefore, further studies

are necessary to understand the mechanism behind the neurotoxic effects and the relation to

neurodeficits disorders.

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