i

The expression of p53, BAX and Bcl-2 proteins in cultured cells exposed

to zinc oxide nanoparticles.

Thesis by:

Anthony M. O‟Shea

May 2011

Supervisors: Drs. Orla Howe & Alan Casey

BSc Biomedical Science

Dublin Institute of Technology, Kevin Street

ii

Abstract

Detrimental effects to human health from ZnO nanoparticle exposure in industrial settings

has been well documented in epidemiological studies and cytotoxicity has been

documented in in-vitro studies yet nano zinc oxide is often an ingredient in sunscreens and

cosmetics.

In this study the effects of low dose ZnO nanoparticle exposure on expression of key

apoptotic proteins; p53, Bcl-2 and BAX will be studied in a dermal cell model using

immunocytochemical methods. Bcl-2 is a negative regulator of apoptosis and plays an

important role in cell survival. BAX or Bcl-2 associated protein X is a pro-apoptotic

protein of the same family as Bcl-2 that is antagonistic to Bcl-2 and promotes apoptosis by

mitochondrial outer membrane permeabilisation. p53 has numerous cellular functions as it

is a transcriptional regulator of over 4000 genes including those which regulate apoptosis

and enzymes that promote responses to cell stress, it also has several functional

interactions with already present pro- and anti-apoptotic proteins both in the cytosol and

associated with the mitochondria.

In this study it was found that Bax, p53 and Bcl-2 were all markedly increased in response

to zinc oxide nanoparticle exposure.

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Table of Contents:

Abstract ............................................................................................................. ii

Acknowledgements: .......................................................................................... v

1. Introduction ................................................................................................ 1

1.1. Background: .................................................................................................... 1

1.2. Properties of Nanoparticles: ............................................................................ 4

1.2.1. Physical characteristics: ......................................................................... 4

1.2.2. Chemical reactivity: ............................................................................... 6

1.2.3. Cellular interactions of ZnO nanoparticles ............................................ 7

1.3. Induction of cell stress ................................................................................... 11

1.4. Responses to cell stress ................................................................................. 13

1.4.1. BAX ..................................................................................................... 14

1.4.2. Bcl-2 .................................................................................................... 17

1.4.3. p53 ....................................................................................................... 19

1.5. Immunocytochemistry: .................................................................................. 21

1.6. Apoptosis and necrosis .................................................................................. 21

2. Materials and methods ............................................................................. 24

2.1. HaCaT cell line: ............................................................................................. 24

2.2. Cell culture: ................................................................................................... 24

2.2.1. Culture conditions: .............................................................................. 24

2.2.2. Resuscitating frozen cells: ................................................................... 25

2.2.3. Subculture: ........................................................................................... 25

2.2.4. Cell counting and seeding pre exposure .............................................. 25

2.2.5. Exposure to ZnO nanoparticles ........................................................... 26

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2.3. Immunohistochemistry, Imaging & evaluation ............................................. 27

2.3.1. Immunocytochemical staining ............................................................. 27

2.3.2. Imaging and cell counting methods ..................................................... 29

2.3.3. Recording percentage protein expression positivity ............................ 29

2.3.4. Necrosis and apoptosis: ....................................................................... 31

3. Results: ..................................................................................................... 32

3.1. Method validation for automated cell counting ............................................. 32

3.2. Apoptosis & necrosis: .................................................................................... 32

3.3. Bax protein expression .................................................................................... 36

3.4. Bcl-2: ............................................................................................................. 39

3.5. p53: ................................................................................................................ 41

4. Discussion ................................................................................................. 43

4.1. Apoptosis & necrosis ..................................................................................... 43

4.2. BAX ............................................................................................................... 44

4.3. Bcl-2 .............................................................................................................. 45

4.4. p53 ................................................................................................................. 45

5. Conclusion ................................................................................................ 47

6. Appendices: ............................................................................................. 48

7. References: .............................................................................................. 55

v

Acknowledgements:

I would like to extend my thanks to my supervisors Drs. Orla Howe and Alan Casey for

their help throughout the course of this research project as well as all the staff of the Focas

Institute who helped, particularly Jennifer Dorney and Niall O'Claonadh who were great

people to work with and went out of their way to accommodate my work. I would also like

to thank Karina Carey for the help and advice she gave for my numerous difficulties with

the project and Mark Bates for his advice from his previous experience with similar work.

As stressful as it was at times, I‟ll actually miss working in Nanolab.

I would also like to thank my all of my lecturers in DIT Kevin Street for their help over the

years and for providing me with a solid foundation of theory and practical knowledge

without which I could not have completed this thesis. In particular I would like to thank

Fabian McGrath who always made Transfusion Science a really interesting subject to study

and Dr. Helen Lambkin who provided the antibodies used in this project as well as for

lecturing me in my minor subject.

Finally I would sincerely like to thank my parents for providing me with an education

which I could not have even contemplated when I started four years ago. Realising all I

have learned since I started this degree is actually quite daunting and I wouldn‟t trade it for

the world.

Thanks a million everyone!

1

1. Introduction

1.1. Background:

Although Zinc oxide (ZnO) is normally regarded as being relatively benign to humans and

it has been included in the FDA database of substances generally regarded as safe (GRAS)

for use as food additives (U.S. FDA 2006) it has been known to have detrimental effects to

human health when in its nanoparticulate form (Kuschner et al, 1995).

Nanoparticles are particles with at least one dimension of less than 100 nm. They may

display size related properties that differ from the physiochemical and biological

characteristics of the same substance in bulk form (Nel et al, 2006).

Three main factors cause nanoparticles to differ in their reactivity from bulk material in

biological systems. The increase in surface area in relation to mass, quantum effects caused

by their minute size and altered interactions with physiological barriers such as cell

membranes, mitochondrial membranes and various interfaces between systems such as the

blood brain barrier (Buzea et al, 2007). Some proposed disease associations and exposure

pathways are outlined in Figure 1.1.

2

Figure 1.1: Schematic showing proposed disease outcomes and exposure pathways

(Buzea et al, 2007).

As the use of engineered nanoparticles is a relatively new phenomenon, much of the

information relating to the biological effects of nanoparticles comes from epidemiological

studies of occupational exposure to substances such as dust, fumes and asbestos which

contain nano-sized and ultra fine (<1µm) particles (Buzea et al, 2007; Schuler 2005; Nel et

al, 2006). A prime example of the harmful effects of ZnO nanoparticles is a form of

occupational asthma known as metal fume fever (MFF) which occurs in workers that weld

galvanised (zinc coated) steel (Blanc et al, 1993; Gordon & Fine, 1993). The condition has

been documented since the mid 1880‟s, then known as brass founders ague (brass being an

alloy of copper and zinc) (Kelleher et al, 2000) and is known to occur in up to 20% of

welders by age 30 (Gordon & Fine, 1993). In an in-vivo human study, purified ZnO fumes

3

were shown to increase inflammatory cytokine levels in bronchioalveolar lavage

specimens (Kuschner et al, 1995).

One of the main problems with the toxicity data currently available is the variation in the

methodology of testing and the substances being studied. In animal and in vitro models

different cell lines and animals are also often used. Some of the literature currently

available regarding nanotoxicology also involves relatively high dose exposures. In one in

vivo study on the inhalation of carbon nanotubes, some animals actually died from

asphyxiation on the material itself rather than associated toxicity (Warheit et al, 2004). In

this in vitro study however the effects of sub-lethal concentrations are studied in order to

reflect plausible real world exposure.

In contrast to the proposed negative effects to health, many positive applications have been

devised and are anticipated in the future which exploit the differing properties of

nanomaterials. The use of nanofibre scaffolds has been demonstrated to result in restored

sight in hamsters after severing of the optic nerve (Ellis-Behnke et al, 2006). Novel

antimicrobial nanopowders and coatings for surgical masks have been investigated (Buzea

et al, 2007). Due to their properties of increased cellular uptake, applications in gene

transfection, medical imaging and drug delivery are also being investigated (Buzea et al,

2007).

Zinc Oxide nanoparticles are beginning to replace traditional organic UV absorbers in

sunscreens as they do not scatter the visible wavelengths of light. This prevents whitening

of skin with topical application (Schuler, 2005). Studies investigating their cytotoxic

effects and potential for absorption into the skin are hampered however by the fact that the

particles are often coated with other materials to prevent cluster formation and allow for

light dispersion. The composition of such coatings and indeed their presence is often

4

difficult to elicit in commercial sunscreen products due to the need to retain trade secrets

(Schuler, 2005). Recent studies have been published describing novel transparent ZnO

containing nanoparticles that are not absorbed cutaneously (Cross et al, 2007).

In understanding nanoparticle interaction at a cellular and sub cellular context three levels

of interaction are considered. Firstly the physical and chemical properties of the

nanoparticles themselves and how they impact on factors such as surface functionalisation,

ion release, surface charge, and hydrophobicity and hydrophilicity. Secondly the

interaction of the nanoparticles with their surrounding medium (tissue culture media, or

serum/interstitial fluids and surfactants in vivo) and thirdly the interactions at the solid-

liquid interface at biological membranes (Nel et al, 2009)

1.2. Properties of Nanoparticles:

1.2.1. Physical characteristics:

For a given mass of a material the combined surface area of the particles is inversely

proportional to the size of the particles. For instance a carbon microparticle with a diameter

of 60 μm and a mass of 0.3μg has a surface area of 0.01 mm2, The same mass of carbon in

the form of nanoparticles with diameters of 60 nm would have a combined surface area of

11.3 mm2

(Buzea et al, 2007), (See Figure 1.2.a.) This increase in surface area results in a

greater proportion of molecules of the substance being expressed on the surface allowing

for much greater overall interaction between the molecules that makes up the particles and

their surrounding medium. Atoms at the surface of such particles will have fewer

neighbours, and as a result reduced binding energies, as demonstrated by the Gibbs-

Thompson equation. It has been shown that the melting temperature of gold can be reduced

by as much as 300°C due to this effect (See Figure. 1.2.b.).

5

Figure. 1.2: These graphs (a) demonstrate the inversely proportional relationship

between particle size and surface area and (b) the reduction in binding energy with

decreasing particle size (Adapted from: Buzea et al, 2007).

6

1.2.2. Chemical reactivity:

A key aspect to the cytotoxic properties of nanoparticles is their ability to create reactive

oxygen species (ROS). The small diameter of the zinc oxide nanoparticles may cause

discontinuity within the crystal lattice structures; these defects may alter the electron

configuration of molecules within the particle and create functionally reactive groups at the

surface (Nel et al, 2006). These reactive groups may lead to electron capture from

molecular oxygen (O2) forming the superoxide radical ( ) (Nel et al, 2006). This causes

further creation of ROS by mechanisms such as transition metals (including zinc) in

nanoparticles catalysing Fenton type reactions and the enzymatic action of superoxide

dismutase on the superoxide free radical (Buzea et al, 2007), (See Table 1.1.).

ROS such as O3 and H2O2 can also be carried on the surface of nanoparticles from

ambient air (Buzea et al, 2007). Because of their size nanoparticles may also lodge in the

mitochondria and interfere with the electron transport chain causing further generation of

ROS (Buzea et al, 2007; Nel et al, 2006).

Table 1.1: Description of propagation mechanisms of reactive oxygen species

Mechanism Reactions

Fenton type reaction:

• + H2O2

•

OH + • + O2

Superoxide Dismutase

Where M= Cu (n=1), Fe (n=2), Mn (n=2) and Ni (n=2)

7

Generation of electron hole pairs, causing free radical formation and bond breakages may

also occur, with exposure of the particles to UV light (Nel et al, 2006). This could be of

particular interest with the use of zinc oxide nanoparticles in sunscreens.

Zinc ion homeostasis may also be affected due to the ability of the nanoparticles to release

ions within cells after uptake (Wang et al, 2010). The increased release of zinc ions from

nanoparticulate ZnO as opposed to larger particles is presumably related to their increased

surface area and the oxygen vacancies left by the release of Zn2+

from ZnO could lead to

ROS generation (Nel et al, 2009). The mechanisms by which the Zn2+

ion mediates

toxicity are highly complex, as between 3 and 10% of the human genome codes for

proteins that interact with zinc (Loh, 2010).

1.2.3. Cellular interactions of ZnO nanoparticles

Due to the increased cellular uptake of nanoparticles, physiological barriers such as the

skin, gastric and pulmonary epithelia as well as interfaces between these points of entry

and various biological compartments such as the circulatory and lymphatic systems may be

permeable by nanoparticles (Buzea et al, 2007). Understanding of nanoparticle interaction

at a cellular and sub cellular level is therefore key to determining nanoparticle

biodistribution and possibly controlling it. Nanoparticle biodistribution is important in

areas investigating physiological toxicity as well as development of applications whereby

such improved biodistribution may be advantageous in areas such as drug delivery and

molecular biology (Buzea et al, 2007). In regard to zinc oxide nanoparticles this

intereraction and uptake by dermal epithelial cells is of particular interest.

As mentioned earlier in this section, to study the cellular interactions of nanomaterials,

their interactions with their suspending medium, their physical and chemical properties and

8

the interaction of nanoparticles and nanoparticle-protein complexes at the solid-liquid

interface of biological membranes must be considered.

The behaviour of nanoparticles in solution is affected by traditional colloidal forces i.e.

Zeta potential mediated repulsion between particles which can prevent agglomeration due

to the presence of double layer ionic clouds. This behaviour is largely dependent on the

properties of the surrounding medium, particularly ionic strength, pH and temperature as

well as the physical and chemical properties of the particles themselves (Nel et al, 2009).

Another key aspect to nanoparticle interaction with its suspending media is the formation

of a protein corona. The composition of such coating being dependant on the composition

of the surrounding fluid and the properties of the nanoparticle at its core. Depending on the

nanoparticle‟s composition, size and shape and the composition of the suspending

media/biological fluid, binding affinities for various proteins and therefore composition of

protein corona will vary (Nel et al, 2009). The composition of the protein corona is also

dynamic, for instance a particle may first be predominantly coated by a protein in high

abundance in the solution such as albumen, but later bind with proteins of higher affinity

which displace the transiently bound albumen. This dynamic media interaction may also

change the protein corona composition when the particle is moved from one biological

compartment to another (Nel et al, 2009).

Both the protein corona and its nanoparticle core may be affected by their mutual

interaction, as the coating of the nanoparticles causes release of free energy at the

nanoparticle surface. This may cause conformational changes in the protein corona,

revealing cryptic epitopes and altering protein structure and function (see Figure 1.3).

Such cryptic epitopes and altered proteins may the cause receptor mediated phagocytosis in

the professional phagocytes of the immune system or receptor mediated endocytosis in

other cells (Nel et al, 2009).

9

Figure 1.3. Schematic showing impact of the mutual interaction between the nanoparticle core and its protein corona on protein structure

and function.

10

Factors which affect internalisation into the cell include surface heterogeneity of the cell

membrane and particle size. Cell membranes are normally thought of as heterogeneous

structures with a vast array of proteins dotted around their surface, however areas of

homogenous lipid bi-layer exist between these proteins in patches of around 10-50 nm

across. Particles around this size therefore may interact non-specifically with these areas,

causing the membrane to fold in on itself and internalise the particle by endocytosis. As

such the size and surface chemistry of the nanoparticle and corona are also important in

determining its specific and non specific interaction with the cell membrane. A key

example of this is described by Nel et al, 2009 which states there is an optimal particle

diameter for receptor mediated endocytosis to occur. The invagination around the particle

must be large enough to induce the formation of a clathrin coated pit, however not so large

as that membrane elasticity or repulsion between transmembrane proteins would prevent

the particle from being engulfed into the cell.

Once inside the cell vesicles containing ZnO nanoparticles are thought to join with

lysosomes to form a single compartment. In this compartment the cationic surface of the

corona is capable of sequestering protons. This keeps up the action of the v-ATPase proton

pump, causing the retention of 1 Cl- ion and 1 water molecule per proton sequestered,

eventually causes the swelling and lysis of the lysosome with release of its highly reactive

contents and the ZnO nanoparticles into the cytoplasm. The acidic environment of the

lysosome also causes nanoparticle dissolution (Zn2+

release) (Nel et al, 2009) The free

ZnO nanoparticles in the cytoplasm may then lodge in the mitochondrial membrane,

Endoplasmic reticulum or nuclear membrane damaging the cell by various means and

releasing further reactive oxygen species as they do so (Buzea et al, 2007).

11

1.3. Induction of cell stress

The build-up of ROS induces cell stress when the natural antioxidative mechanisms such

as glutathione oxidation and the antioxidative enzymes such as superoxide dismutase and

hydrogen peroxidise become overwhelmed. This oxidative stress results in either

adaptation to the altered redox state of the cell or damage to the cell depending on the level

of oxidative stress undergone (Nel et al, 2006). The cell‟s response occurs in three

different levels (Figure 1.2) depending on the level of oxidative stress, which can be

classified by the ratio of glutathione (GSH) to glutathione disulphide (oxidised glutathione,

GSSH)

Figure 1.3. Model of mechanisms of oxidative stress (Nel et al, 2006)

12

At lower levels of oxidative stress antioxidant enzymes are upregulated by activation of

antioxidant response elements (ARE) via the Nrf-2 signalling pathway (Nel et al, 2006).

This antioxidant response attempts to compensate for the increase in ROS and restore the

redox state of the cell. It is also thought that this upregulation of antioxidant enzymes

responsible for a phenomenon known as ischemic preconditioning where resistance to

ischemic injury (which causes ROS and free radical formation) may develop in tissues that

have been repeatedly or chronically deprived of oxygen (Haliwell & Gutteridge 2003; Nel

et al, 2006; Nel et al, 2009). At intermediate levels of oxidative stress pro-inflammatory

cascades such as the Nf-kB pathway are upregulated, and transcription of precursors of

apoptotic proteins increased. Whilst at higher levels of oxidative stress, apoptosis is

induced by mitochondrial membrane permeabilisation causing the release of pro-apoptotic

substances. Once these substances are released the apoptotic response takes place

independently of the original stimulus (Buzea et al, 2007; Nel et al, 2006)

13

1.4. Responses to cell stress

Injured or damaged cells need to be either allowed time to repair themselves by cell cycle

arrest or be set along a pathway for their controlled removal in order to prevent

proliferation of damaged cells (Lodish et al, 2008). Apoptosis is initiated by one of two

different pathways; the intrinsic pathway where apoptosis is initiated from intracellular

signals such as oxidative stress and DNA damage, and the extrinsic pathway where

apoptosis is initiated by interaction of membrane receptors with extracellular ligands (Tait

& Green, 2010). The process of apoptosis culminates in the activation of the caspase

(cysteine-dependent aspartate-directed protease) cascade which results in the cleavage of

numerous proteins. The caspase proteins consist of two broad categories; (a) imitator

caspases, with a narrow range of substrates including self cleavage, Bcl-2 homology

domain cleavage and pro-caspase cleavage. (b) The executioner caspases with a more

diverse range of substrates, these cause widespread degradation of cellular components

causing the typical apoptotic phenotype which subsequently lead to the cell‟s recognition

and removal by the immune system (Tait & Green 2010).

The regulation of the intrinsic pathway and crosstalk with the extrinsic pathway is chiefly

achieved by members of the Bcl-2 family. The Bcl-2 family proteins are defined by the

presence of between 1 and 4 Bcl-2 homology domains named BH1-4 (Chipuk et al, .

2010). It was originally thought that BAX as well as the other Bcl-2 effector Bak (Bcl-2

Antagonist Killer-1) lacked a BH-4 domain, however this domain has recently been shown

to be partly conserved in BAX (Chipuk et al, 2010).

p53 acts in the regulation of apoptosis in its capacity as a transcription factor and a post

transcriptional regulator of apoptosis related proteins including the Bcl-2 family, in

particular through the activation of the BH3 only “activator/effector” proteins such as

PUMA Bax and Bak (Letai, 2009). p53 also induces the expression of p21, a cyclin

14

inhibitor which causes cell cycle arrest inducing cellular senescence, a step which is

thought to be involved in the process of apoptosis in conjunction with caspase mediated

protein cleavage. p21 overexpression on its own however may prevent apoptosis (Brady &

Attardi, 2010). p53 also targets genes related to cell survival such as Gdf-15 (growth and

differentiation factor 15) and Plk-3 (Polio like kinase 3) (Han et al, , 2008).

1.4.1. BAX

BAX (Bcl-2 Associated Protein-X) is a pro-apoptotic effector protein of the Bcl-2 family.

The Bcl-2 family of proteins contains both pro- and anti-apoptotic proteins which when

activated by other members of the same family plays a key role in mitochondrial outer

membrane permeabilisation (MOMP). This is an absolute requirement for intrinsically

activated apoptosis in mammalian cells (Tait & Green, 2010). Several competing models

exist as to how exactly BAX is involved in MOMP. Such models include its

oligomerisation and insertion into the outer mitochondrial membrane in a manner similar

to the formation of a membrane attack complex. Another hypothesis is the possible

interaction of oligomerised BAX with outer membrane lipids forming transient lipid pores

or inverted micelles (Tait & Green, 2010). See Figure 1.4.

15

Figure 1.4: The formation of a pore in the mitochondrial outer membrane leading to

release of cytochrome c from the IMS. (Tait & Green 2010; Chipuk et al, 2010).

Whatever the exact mechanism of MOMP may be, contents of the intermembrane space

become released into the cytoplasm. When cytochrome c is free in the cytoplasm it

complexes with APAF1 (apoptotic protease activating factor 1) and recruits and activates

procaspase 9 to form an oligomeric structure known as the apoptosome. This platform in

turn activates the executioner caspases 3 and 7 (Tait & Green, 2010). Other substances

released from the intermembrane space such as SMAC (Second Mitochondria-derived

Activator of Caspase, also known as DIABLO) and OMI (also known as HRTA2) serve to

inhibit XIAP (X-linked inhibitor of apoptosis) which inhibits caspases 3, 7 and 9

(Deveraux & J. C. Reed, 1999). This antagonistic mechanism is also a requirement for

FAS ligand mediated apoptosis in type II mammalian cells such as hepatocytes (Tait &

Green, 2010). See Figure. 1.5.

16

Figure 1.5.: Schematic showing the role of MOMP in

apoptosis (Tait & Green, 2010):

A: The intrinsic pathway involves activation of BAX, MOMP

and neutralisation of XIAP by SMAC and OMI allowing for

apoptosis via the apoptosome and caspases 3 and 7

B: The extrinsic pathway is initiated by membrane receptors,

leading to the dimerisation and activation of caspase 8, which

can then directly cleave and activate caspase 3 and caspase 7,

leading to apoptosis. Crosstalk between the extrinsic and

intrinsic pathways occurs through caspase 8 cleavage and

cleavage of BID to truncated BID; (tBID) is required in some

cell types for extrinsic apoptosis.

17

Activation of BAX takes place via conformational changes induced by its interaction with

BH3 only proteins such as BIM, and BID as well as PUMA (p53 Upregulated Mediator of

Apoptosis) and possibly p53 itself and others leading to its dimerisation, higher order

oligomerisation and translocation from the cytosol to the mitochondrial membrane

(Gavathiotis et al, , 2008; Letai, 2009). A specific activation site has not yet been

identified; however SAHBs (Stabilised Alpha Helix domains of Bcl-2) complexed with

BIM (a known BAX activator) have been shown to activate BAX in vitro (Gavathiotis et

al, 2008). The C terminal alpha helix of BAX is amphiphatic, allowing it to interact closely

with a hydrophobic groove on the protein surface or to be dislodged from this groove and

be exposed to the cytosol. In order for translocation to the mitochondrial membrane to take

place this α-helix is dislodged due to conformational changes induced by the interaction at

the SAHB/BIM interaction site which was identified by (Gavathiotis et al, 2008) in

conjunction with other yet to be identified conformational change(s). Mutations of this

novel interaction site were also shown to prevent MOMP induced apoptosis.

1.4.2. Bcl-2

Bcl-2 is the founding member of the Bcl-2 family of proteins and acts as a negative

regulator of apoptosis (Chipuk et al, 2010; Tait & Green 2010; Zinkel et al, 2006). The

role of Bcl-2 in cell survival was first identified when translocations causing Bcl-2 over

expression were found in B cell lymphomas alongside the c-Myc gene. When Bcl-2 was

overexpressed in the absence of c-Myc overexpression proliferation was not observed but

resistance to apoptosis was maintained (Chipuk et al, 2010). Along with the other pro-

apoptotic members of its family the Bcl-2 protein possesses all four BH1-4 domains and

the protein is typically associated with the mitochondrial membrane, although it may also

be present on the endoplasmic reticulum membrane as well as the in the cytosol (Chipuk et

al, 2010). The Bcl-2 protein inhibits the overall progression of the MOMP pathway

18

although the mechanism by which it achieves this is unclear. In vitro models suggest the

binding of pro-apoptotic members of the Bcl-2 family including Bcl-2 and Bcl-XL to

APAF1, may act as a checkpoint in mitochondrial mediated apoptosis, by preventing the

activation of pro caspase 9 and inhibiting apoptosome function (Hu et al, 1998). The role

of Bcl-2 in the regulation of BAX and BAK activity has also been extensively studied with

two prevalent models of this interaction being described in literature today. Firstly the

“direct activation” model where “sensitizer” or “inactivator” BH3 only proteins

(BAD,BIK, Hrk and Noxa) cause the release of “activator” BH3 only proteins (BIM, BID

and possibly PUMA) which are normally sequestered by Bcl-2 anti apoptotic subfamily

proteins (Chipuk et al, 2010; Ku et al, 2010). The second “indirect activation” model

proposes that the anti-apoptotic Bcl-2 proteins sequester activated BAX and BAK in

healthy cells and release it in dying cells due to interaction with BH3 only “activator”

proteins. The indirect model is less widely accepted due to the fact that BAX and BAK

normally are present in the cytosol whereas the anti-apoptotic Bcl-2 proteins are

predominantly located on or associated with the outer mitochondrial membrane. Also the

binding between BAX, BAK and the Bcl-2 anti-apoptotic proteins demonstrated in vitro is

quite weak (Ku et al, 2010). Contrary to these arguments against the indirect model, it is

proposed that the activation of BAX may directly lead to its localisation to the

mitochondrial membrane (via dislodgement of C-terminal α helix allowing for membrane

insertion), and that conformational changes induced by its activation affect its binding

affinity (Gavathiotis et al, 2008). It is therefore possible that either or both models of Bcl-2

mediated BAX and BAK regulation may be relevant in the process of how exactly Bcl-2

regulates MOMP.

Activity and transcription of Bcl-2 itself is regulated at several different levels. IL-3

mediated phosphorylation at Serine 70 in the FLD (flexible loop domain) between BH3-4

19

promotes its anti-apoptotic functions, whereas binding of p53 to amino acids 32-68 of this

domain reduces Bcl-2-BAX interaction and increases apoptosis (Deng et al, 2006).

1.4.3. p53

p53 is the product of the tumour suppressor gene TP53 which is known to be mutated in

over 50% of human cancers, and a mutant allele causing the multiple tumour type

reoccurring Li-Fraumeni cancer syndrome (Brady & Attardi, 2010). The protein itself acts

as a transcription factor which promotes or inhibits a multitude of different target genes,

with over 4000 possible target genes having been identified in the human genome to date

(Loh, 2010). Major targets for p53 include genes involved in DNA repair, cell cycle

regulation, apoptosis and angiogenesis (Ellis-Behnke et al, 2006) as well as the production

and control of reactive oxygen species (Méplan et al, 2000). Regulation of p53 itself

occurs mostly at a post transcriptional level by both control of its degradation (stabilisation

and accumulation) and qualitative modifications which regulate its activity (Loh, 2010;

Méplan et al, 2000). In normal cells the turnover of p53 is quite rapid due to its

antagonistic interaction with MDM2 family proteins (p53 upregulated), which promote its

ubiquination and destruction by the proteosome as well as inhibits its DNA binding

capacity (Loh, 2010).

The fate of the cell (temporary senescence, apoptosis, or upregulation of antioxidant

response genes) in response to p53 expression depends largely on the cell type, since

epigenetic differences affect gene transcription differently in cells of different phenotypes

even in the same organism (Orphanides & Reinberg, 2002). The environmental conditions

of the cell and the nature of the inducing stress (ATP depletion, Nucleotide depletion, ROS

ect.) also affect the cells ultimate fate. (Brady & Attardi, 2010).

20

p53 can promote gene expression via its interactions with the transcription complex and

also repress the transcription of several genes by binding to response elements and

recruiting co-repressors (such as histone de-acetylases and Sin3a) or by simply blocking

the binding of transcriptional factors to DNA (Brady & Attardi, 2010).

In its capacity as a transcription factor, p53‟s positive regulation of transcription of BAX,

APAF1, PUMA NOXA and caspase 9 are key to its role in apoptosis as is its translational

suppression of pro-survival proteins such as Bcl-2 (Brady & Attardi, 2010; Tait & Green,

2010).

p53 in addition to its role as a transcription factor also has cytosolic roles in apoptotic

regulation. This has been determined by p53 induced apoptosis in the absence of protein

synthesis or with transactivation deficient p53. (Brady & Attardi, 2010; Deng et al, , 2006).

p53-Bcl-2 binding has been shown to decrease Bcl-2-BAX interaction and promote

apoptosis via the binding of p53 to two sites on the FLD (flexible loop domain) between

BH3-4 on Bcl-2. This FLD contains two distinct regulation sites one for p53 interaction

which causes inhibition of Bcl-2‟s anti-apoptotic function and the other being a

phosphorylation site, which when blocked the survival function of Bcl-2 is also diminished

(Deng et al, 2006).

Although cytoplasmic p53 has also been shown to mediate BAX/BAK oligomerisation and

Bcl-2 inactivation it can also severely disrupt the stability of both inner and outer

mitochondrial membranes independently of BAX or BAK pores, and its localisation to the

mitochondrial membrane occurs independently of BAX, BAK or PUMA (Wolff et al,

2008).

21

1.5. Immunocytochemistry:

Immunocytochemical staining is a technique which allows for the identification of highly

specific cellular epitopes. The technique involves the ligation of the epitope in question

with a specific primary antibody and the labelling of this complex with a secondary

antibody raised against the species of the primary antibody. For example, a rabbit anti

mouse P53 primary antibody would be used with an goat anti rabbit secondary antibody.

Secondary antibodies for Immunocytochemistry typically contain a label used with in

conjunction with a detection system for localisation of staining. Such labels include

flourescins, biotin conjugated enzymes and other labels such as colloidal metals and

radioisotopes. In this study, Vectastain elite rabbit and mouse ABC kits were used (Vector

Laboratories, USA), so that only anti rabbit secondary antibodies were used for Bax and

anti mouse secondary antibodies were used for p53 and Bcl-2. These secondary antibodies

were conjugated to a horseradish peroxidise (HRP) tag so that Diaminobenzidine (DAB)

staining could be used to detect the antigen-antibody signal from the positive cells

expressing the test protein (P53, Bax and Bcl2) (Fig 1.6.)

1.6. Apoptosis and necrosis

Necrosis is uncontrolled cell death characterised by the rupture of cell membranes,

cytoplasmic swelling and the random destruction of cellular components. This type of cell

death is characteristic of sudden or severe cellular injury and occurs spontaneously after

exposure to stimuli such as exposure to strongly toxic chemicals or ischemia. Necrosis

usually occurs in groups of cells exposed to the same pathogenic stimulus. Release of

cytoplasmic contents can also trigger an inflammatory response in-vivo (Wyllie, 1997).

Conversely apoptosis is a highly regulated process that tends to occur in single cells rather

than in large groups, and the morphology of apoptotic cells is characteristic of their more

22

controlled destruction. Instead of membrane rupture or cellular swelling seen in necrotic

cells, the membranes of apoptotic cells break away more gradually in “blebs” without

releasing cytoplasmic contents into the extracellular environment. Chromatin condenses

and the nucleus may begin to fragment. Vesicles may also begin to appear. Apoptosis tends

to occur in single cells which may separate away from -surrounding cells during the

process. The membrane of apoptotic cells may also express more phosphatidylserene on its

outer surface or display other extracellular signals to encourage its engulfment by cells of

the immune system (Savill & Fadok, 2000).

Apoptosis is not entirely distinct from necrosis, as it is both a process that requires energy

and causes the metabolic wind-down of the cell, in the final stages of apoptosis may not be

quite as ordered and its effects may begin to overlap with those of necrosis (Savill &

Fadok, 2000).

Figure 1.6: Detection of antigen with the ABC immunostaining technique. The ABC

reagent contains avidin conjugated horseradish peroxidise. The avidin-HRP complex binds

with the biotinalyted secondary antibody localising the enzyme. The action of this enzyme

causes 3’3-diaminobenzidine to form an insoluble brown compound.

23

1.7. Project aims

In this project the HaCaT skin cell line will be used as a model to investigate changes of

expression of the apoptotic regulatory proteins; BAX, Bcl-2and p53 in response to

exposure of ZnO nanoparticles at different concentrations for different timepoints. The

concentrations of ZnO nanoparticles used in this study were low (< 150mg/L) in order to

reflect plausible real world exposure to ZnO nanoparticles. The most extensively studied

pathways for nanoparticle exposure in humans are models of the respiratory tract,

gastrointestinal tract and skin, as ZnO nanoparticles are currently being used in cosmetics

and sunscreen (Schuler, 2005). Therefore, using human keratinocyte models (such as

HaCaT cells) in this study are particularly relevant and currently topical.

24

2. Materials and methods

2.1. HaCaT cell line:

The HaCaT cell line is an aneuploid non tumorigenic cell line derived from normal adult

skin keratinocytes. The cell line was first isolated in 1988 by Boukamp et al from cells

taken from an excised melanoma specifically from a region containing no histological

abnormalities. Therefore, the cell line spontaneously transformed in vitro and became

immortal without transformation methods. As such its phenotype is better preserved than

in vitro transformed cell lines. The cell line has been tested for tumorigenicity by

transplantation into immunodeficiant mice and found negative. This cell line was the first

example of spontaneous transformation in human keratinocyte cells (Boukamp et al,

1988).

2.2. Cell culture:

2.2.1. Culture conditions:

HaCaT cells were cultured in commercially prepared DMEM F-12 medium (Dulbecco‟s

Modified Eagles Medium) with HAMs F12 modification containing: L- glutamine,

pyridoxine, NaCHO3 and phenol red indicator (Sigma, Dublin). This culture media was

supplemented with 10% v/v foetal bovine serum, Penicillin-Streptomycin antibiotics

(100IU penicillin and 100 µg streptomycin to 500 ml of media) (Biosciences, Dublin).

Media supplementation was carried out under aseptic conditions. All cell cultures were

incubated at 37°C in a 5% CO2 atmosphere in a humidified incubator. Cells were checked

daily for contamination and to observe growth patterns. Cells were grown to between 70-

95% confluence so that they were always in the logarithmic phase of growth for

experimentation

25

2.2.2. Resuscitating frozen cells:

When cells were required for experiments, they were resuscitated from frozen stocks.

Cryovials containing 1ml of cells in 10% FBS and 1% DSMO (Sigma, Dublin)

cryoprotectant were taken from vapour phase liquid nitrogen and thawed at room

temperature. The cells were then aseptically pipetted into a T75 cm2 flask (Thermo,

Dublin) containing approximately 20 ml of fresh medium with supplements. Cells were

then incubated for 24 hours and medium changed to remove any unattached or dead cells.

2.2.3. Subculture:

10 ml of versene (0.17M EDTA in PBS) was added to a 10ml volume of trypsin (Gibco),

inverted and left to stand. The culture media was discarded from the flask of cells so that

the monolayer of cells was exposed. The flask was then rinsed with sterile PBS once to

remove any remaining media or dead cells. Approximately 10 ml of the trypsin/versene

mixture was used to briefly wash the flask again before letting the remainder of the

mixture incubate in the flask for 5 to 10 minutes at 37°C to dislodge the adherent cells

from the base of the flask. The flask was checked periodically under an inverted

microscope to observe the cells detaching from the base of the flask and prevent over

trypisinisation. The trypsinised cells were then poured into a sterile universal containing an

equal volume of fresh media to neutralise and halt the action of trypsin. This cell

suspension was then used for subsequent exposure studies or used to seed new stock flasks

and maintain the cells in culture.

2.2.4. Cell counting and seeding pre exposure

A volume of 1 ml of the cell suspension was added to 25 ml of isoton solution and a cell

count was performed using a coulter counter model Z2 (Beckman Coulter, USA). The

count obtained was taken to be equal to the number of cells per ml in the cell suspension.

A volume of the cell suspension containing 3x105 cells was then seeded into 10 ml of fresh

26

medium in 25cm3 culture flasks (Biosciences, Dublin). Three flasks were seeded for each

exposure point and exposure control for each protein along with 5-6 extra flasks to be used

as negative staining controls (at least 180 flasks in total). These flasks were incubated for

48 hours until they were in the logarithmic phase of growth (approx 70%).

2.2.5. Exposure to ZnO nanoparticles

A stock solution of 50mg/L of ZnO nanoparticles (Size charachteristis, Appendix 1.4) was

prepared by adding 5mg of autoclaved ZnO nanoparticles aseptically to 100 ml of sterile

culture media. The solution was inverted and sonicated for 30 minutes before being diluted

according to Table 2.1

Table 2.1: Preparation of ZnO nanoparticle solutions.

1 mg/L 5 mg/L 10 mg/L 15 mg/L

Volume of ZnO

stock solution

1.6 8 16 24

Volume of cell

culture media

78.4 72 64 56

Total Volume 80 80 80 80

All volumes in ml.

27

The media was poured off and 80 ml volumes of the prepared suspensions of 1, 5, 10 and

15 mg/ml of ZnO nanoparticles were made up in fresh media and 5 ml aliquots of these

solutions were added to each 25 cm3 flask and incubated for 1, 3, 6 or 24 hours. For the

control concentration 0 mg/ml, 5 ml of fresh media was added to the triplicate cultures.

After each time point, the cultures were briefly rinsed in PBS (Appendix 1.1.) and fixed in

10% neutral buffered formalin (Appendix 1.2.) for at least 24 hours until ready for

staining. Each exposure time and dose was performed in triplicate for each of the three

proteins under investigation. A single extra flask of cells was also prepared for each

triplicate set for use as an immunostaining negative control; these flasks were to receive a

change of fresh media without nanoparticles for the same duration as the test flasks.

2.3. Immunocytochemistry, Imaging & evaluation

2.3.1. Immunocytochemical staining

Flasks were rinsed in PBS and cracked using pliers to expose the monolayer of formalin

fixed cells at the base of the flask. A 3% solution of hydrogen peroxide in methanol was

applied for 5 minutes to block endogenous peroxidise activity. The cells were then rinsed

in distilled water. An area of the slide was circled with a hydrophobic Dakopen® marker to

conserve reagents in a particular area on the flask. PBS was applied for at least 2 minutes

to re-hydrate the slides. The slides were then drained and covered with 2-3 drops of normal

serum made up in 10ml of PBS (Vectastain Elite Rabbit or mouse ABC kit) for 5 minutes.

The slides were drained and the appropriate dilution of primary antibody (see Table 2.1)

was applied for 30 minutes.

28

Table 2.1: Optimal primary antibody dilutions

Primary antibody Species Clone # Manufacturer Optimal

dilution

BAX Rabbit N-20 Santa Cruz Biotechnology

Inc.

1:250

Bcl-2 Mouse 124 Dako 1:25

p53 Mouse DO-7 Dako 1:25

Slides were then rinsed for 1 minute three times in PBS and 1 drop of the appropriate

biotinalyted secondary antibody made up in 10ml of PBS (Vectastain kit) was applied for

30 minutes. Slides were rinsed again three times with PBS and then the final ABC reagent

(Vectastain kit) was prepared by adding 2 drops of A and B to 10mls of PBS and applied

for 10 minutes. Slides were rinsed again three times in PBS. Diaminobenzidine (DAB)

chromagen (Dako kit) prepared according to manufacturer‟s instructions was applied for

10 minutes. (Notes: DAB is carcinogenic; therefore all precautions were taken to ensure

safety when using this chemical. DAB is also light sensitive so immunostaining trays were

covered in aluminium foil during incubation times). Slides were then rinsed with tap water

and counterstained in Mayer‟s Haematoxylin for 20 seconds, blued in warm tap water for

1-2 minutes and mounted in glycergel aqueous mounting medium (See Appendix 1.3.)

which was pre-heated and melted in a microwave at approximately 60°C.

29

2.3.2. Imaging and cell counting methods

Photographs from five sequential fields of view representative of each specimen were

taken with a camera attached to a light microscope using the 20x objective lens Each

image was saved as a jpeg image and labelled with an appropriate file name identifying the

antibody used, dose and exposure time.

Areas to be photographed were selected by photographing a single random field of view,

then photographing the two fields of view immediately above and below this image and the

two to its immediate left and right as shown in diagram below (Figure 2.1):

Figure 2.1: Selection of areas for imaging

2.3.3. Recording percentage protein expression positivity

Images were then opened in the ImageJ image analysis software and the formatting was -

changed to 32 bit greyscale. The apply threshold command was run and the automatically

selected level was applied. This changed the image into a binary format, with coloured

30

objects being displayed in total black, and background areas being displayed as completely

white. The apply watershed command was then applied to apply separation of touching

stained areas which would otherwise be counted as a single cell.

The analyse particles command was used to count the number cells. To eliminate

background noise a counting range of objects 440 pixels2-infinity was applied.

For Bcl-2 and p53 stained slides the number of positive cells was counted manually in each

photograph in each 5 image set due to the more faint DAB staining with these antibodies.

For BAX stained slides the DAB staining was stronger, therefore an automated plug-in for

stain separation was used as follows:

The original image used for counting the total number of cells was opened in ImageJ and

the colour deconvolution plug-in by Gabrielle Landini was run (available from:

http://www.dentistry.bham.ac.uk/landinig/software/cdeconv/cdeconv.html). This plug-in

was evaluated by Ruifrok & Johnston in 2001 and found to: “[provide] a robust and

flexible method for objective immunohistochemical analysis of samples stained with up to

three different stains, using a laboratory microscope and standard RGB camera setup, and

the public domain program NIH image”. The option for DAB-Haematoxylin (H-DAB) was

selected from the drop down menu. The image was split into 3 different channels,

haematoxylin stained cells, DAB stained cells and a monochrome composite of other

colour ranges known as the zero channel. The second channel window containing the

image of DAB stained cells was analysed in the same manner as described above to count

the number of positive cells. The results from each image in the 5 image set were added

together.

A positivity ratio was calculated by dividing the number of positive cells across the 5 fields

of view by the total number of cells across the same 5 fields of view

31

2.3.4. Necrosis and apoptosis:

Necrosis was identified based on microscopic necrosis morphology of (a) cellular

swelling/membrane rupture and (b) membrane blebbing. In general, necrotic cells are

distinct because they display severely disordered morphology and a „bubbly‟ appearance

within the cell.

Apoptosis was identified by any two of the following morphological characteristics; (a)

cellular shrinking, (b) membrane blebbing, (c) vacuole formation, (d) chromatin

condensation or (e) nuclear fragmentation. A group of apoptotic bodies in the one area,

which seemed to originate from a single cell, would also be also be counted as single

apoptotic cell.

Percentage apoptosis and necrosis levels in cells were calculated by dividing the number of

apoptotic or necrotic cells in each five image set by the total number of cells across the

same five images as counted by the ImageJ software.

32

3. Results:

3.1. Method validation for automated cell counting:

The methods for automated cell counting in immunostained slides in described in Section

2.3.3. were found using a χ2 test (Microsoft Excel) to provide cell count results that were

not significantly different to manual cell counting. (p= 0.391, 2 tailed p value). The data

used in this calculation may be found in Appendix 2.1.

3.2. Apoptosis & necrosis:

Apoptotic cells were identified based on presence of the morphological characteristics

described in section 2.4 ((a) cellular shrinking, (b) membrane blebbing, (c) vacuole

formation, (d) chromatin condensation or (e) nuclear fragmentation). Examples are shown

in Figure 3.1.

Apoptotic cells were counted manually in each five set for each exposure concentration

and time point and compared with the control. The total number of apoptotic cells in each

set was divided by the total number of cells to give ratios of apoptotic cells. The results are

shown in Table 3.1.

Necrotic cells were counted manually in each five image set for each exposure

concentration and time point and assessed in the same manner as apoptotic cells. Necrotic

cells were identified by cellular swelling or membrane rupture with severe abnormalities to

morphology such as those shown in Figure 3.2. Results shown in Table 3.2.

33

Figure.3.1. Morphological characteristics of apoptosis: (A) Shows nuclear

fragmentation, scale bar= 50 µm. (B) Shows cell shrinkage, note the cytoplasm has

decreased in size to the point where the membrane is almost wrapping the nucleus. Scale

bar = 50 µm. (C) Demonstrates membrane blebbing from an apoptotic body, note there is

also positive DAB staining for BAX in this image. Scale bar 20 µm. (D) Demonstrates an

example of both nuclear shrinkage (chromatin condensation) and shrinkage of the

cytoplasm. Note that this cell is weakly positive for BAX also. Scale bar 20 µm. values

rounded to 2 significant figures.

34

[nano ZnO] /

mg/dl

Exposure time

% Apoptosis

1 hour 3 hours 6 hours 24 hours

0 mg/dl 2.69 % 7.90 % 8.32 % 10.17 %

1 mg/dl 6.85 % 6.71 % 8.76 % 4.81 %

5 mg/dl 4.06 % 8.06 % 8.40 % 8.88 %

10 mg/dl 4.40 % 6.86 % 9.44 % 5.89 %

15 mg/dl 5.39 % 11.6 % 8.52 % 13.16 %

Table 3.1: Percentage of apoptotic cells at various exposures to nano ZnO. Apoptosis

was seen to increase with time from 2.69% at 1 hour to 10.17% at 24 hours in unexposed

controls. In the 15 mg/dl exposed flasks apoptosis increased from 5.39% to 13.16%

although this increase was non linear in nature. No general trend can be seen in the 1, 5,

or 10 mg/dl exposures. Raw data shown in Appendix 2.2.

35

Figure 3.2: Examples of necrotic cells displaying (A) nuclear and cytoplasmic

membrane rupture and (B) swelling of cytoplasmic membrane with vacoulation. (Scale

bars = 20µm)

[nano ZnO] /

mg/dl

Exposure time & % Necrosis

1 hour 3 hours 6 hours 24 hours

0 mg/dl 0.06 % <0.01 % 0 % <0.01 %

1 mg/dl 0.01 % <0.01 % <0.01 % <0.01 %

5 mg/dl <0.01 % <0.01 % <0.01 % <0.01 %

10 mg/dl 0.01 % <0.01 % 0 % 0 %

15 mg/dl 0.01 % 0 % 0 % 0.01 %

Table 3.2: Shows percentages of necrotic cells. Necrosis was uncommon with only 89

necrotic cells out of 27,700 cells counted overall. Many of the image sets contained

absolutely no necrotic cells. When observed, necrotic cells were often grouped. The largest

group seen was of 18 cells and the average size of groups was 3.412 cells. Raw data can

be found in Appendix 2.2., results rounded to 2 significant figures.

36

3.3. Bax protein expression

Positive BAX staining was clearly demonstrated in the cytoplasm of positive cells, it

appeared in numerous uniformly sized clusters dispersed evenly around the cytoplasm

when viewed under oil immersion (100x), this reflects the mitochondrial localisation of

activated BAX. When counting using the 20x objective lens staining presented as light

brown to dark brown depending on level of expression Figure. 3.3. BAX positive cells

were counted using the ImageJ colour deconvolution plug-in in each five image set for

each exposure and time point and divided by the total number of cells (also counted with

assistance of ImageJ software) for that image set to give the ratios (converted to

percentages) shown in Table 3.3. and Figure 3.4.

37

Figure. 3.3. Immunostaining for BAX: (A) Demonstrates granular cytoplasmic staining

for BAX under oil immersion, the staining pattern is characteristic of the localisation of

activated BAX to the mitochondria, scale bar = 20 µm (B) Demonstrates examples of

strong and weak positivity for BAX at 3 hours exposure to 15 mg/dl of nano ZnO, scale bar

= 50 µm. (C) Shows negative staining for BAX at 1 hour, not exposed to nano ZnO, scale

bar = 50 µm.

38

Figure. 3.4.

Table 3.3.

[nano ZnO] /

mg/dl

Exposure time

1 hour 3 hours 6 hours 24 hours

0 mg/dl 0 % 0.62 % 1.16 % 1.55 %

1 mg/dl 0.38 % 4.03 % 10.76 % 7.67 %

5 mg/dl 0 % 11.66 % 12.78 % 16.63 %

10 mg/dl 0 % 9.64 % 11.75 % 15.38 %

15 mg/dl 0 % 18.20 % 18.23 % 23.49 %

Figure 3.4. & Table 3.3: BAX positivity increased sharply with increasing

concentration of zinc oxide nanoparticles at exposures times longer than one hour. Explain

results a bit more. Add one or two more lines explaining that Bax expression was most

evident at 24 hours etc. There was also a slight yet regular increase (from 0-1.5%) in BAX

expression in cells that were not exposed to zinc oxide nanoparticles over the course of 24

hours incubation. Data rounded to 2 significant figures. Raw data in Appendix 2.2.

0

5

10

15

20

25

0 mg/dl 1 mg/dl 5 mg/dl 10 mg/dl 15 mg/dl

% P

osi

tive

[nano ZnO] mg/dl

Bax Expression

1 hour

3 hours

6 hours

24 hours

39

3.4. Bcl-2:

Bcl-2 staining appeared diffuse in the cytoplasm and was difficult to image clearly with the

RGB camera however it could be seen more clearly directly through the microscope. As

such counts were preformed manually on the microscope counting at least 1000 cells,

approximately 5-10 fields of view under the 20x objective. In the images taken this greyish

brown stain appeared more clearly against the darkened background of the nucleus (Figure

3.5). Positivity ratios are given in Table 3.4.

Figure. 3.5.: (A) shows non exposed control, scale bar 100 µm. (B) Shows Bcl-2 staining

in cells exposed to 10 mg/dl for one hour scale bar 100 µm.

40

Table 3.4: Bcl-2 expression increased sharply in exposed cells compared with the

control. Bcl-2 also showed a general trend of decreasing with time in response to

nanoparticle exposure however this decrease was non linear, for example Bcl-2 expression

decreased from 98.4% at 1 hour to 93.4% at 24 hours when exposed to 15 mg/dl.

[nano ZnO] /

mg/dl

Exposure time

1 hour 5 hours 10 hours 15 hours

0 mg/dl - - - 7

1 mg/dl 97.8% 99.2% 98.2% 83.1%

5 mg/dl 98.6% 99.3% 95.4% 95.3%

10 mg/dl 99% - 95% -

15 mg/dl 98.4% 96.3% 97% 93.4%

41

3.5. p53:

p53 staining was noted in the nuclei of positive cells and to a lesser extent in the

cytoplasm. Like Bcl-2, p53 staining was diffuse and not as strong in colour as

demonstrated with anti-BAX (Figure 3.6) therefore counts had to be preformed manually.

Table 3.5. Shows the percentage positivity for p53 in exposed cells and a control specimen

Figure 3.6: (A) Shows a non exposed control (B) shows strong nuclear staining and weak

cytoplasmic staining for p53. Scale bars 100 µm.

42

[nano ZnO] /

mg/dl

Exposure time

1 hour 5 hours 10 hours 15 hours

0 mg/dl - - - 10%

1 mg/dl 89.4% 85.7% 82.9% -

5 mg/dl 92.5% 82.5% 76.2% 79.3%

10 mg/dl 83.8% 91.7% 74.6% 74.2%

15 mg/dl 89.6% 94.4% 81% 80%

Table 3.5: Shows the percentages for p53 positivity. Expression of p53 increased

sharply in all exposures to nano ZnO when compared to the non exposed controls. In the

1mg/dl flasks expression decreased from 89.4% after 1 hour to 82.9 after 10 hours, the

data from the 15 hour exposure was not available due to problems with contamination.

Overall there was a general trend in exposed cells that p53 expression was initially

increased after one hour and decreased in a non linear manner over 15 hours.

43

4. Discussion

4.1. Apoptosis & necrosis

Necrosis was quite a rare finding with only 86 necrotic cells found amongst the 27,700

counted. No discernable pattern emerged between percentage necrosis and either exposure

time or concentration of zinc oxide nanoparticles used. This was an expected and desirable

finding as the aim of the study was to examine the effects of low concentrations of zinc

oxide nanoparticles that would not be immediately lethal to cells and cause necrosis.

Apoptosis was observed to occur in between 2.69% and 13% of cells with the incidence of

apoptosis increasing with time in unexposed controls. The reason for this is that apoptosis

is a normal cellular response. Cells are programmed to die via apoptosis when they have

reached the end of their life cycle. Therefore increased apoptosis levels over time would be

expected in normal unexposed controls. A general but non linear increase in apoptosis with

time was also noted in cells that were exposed to the higher concentration of zinc oxide (15

mg/dl) nanoparticles. Levels of apoptosis in cells exposed to higher concentrations of ZnO

nanoparticles would have been analysed if there had been more time allocated to the

project. However, due to time constraints this could not be done. It would be interesting to

observe would apoptosis levels have increased if higher concentrations of ZnO

nanoparticles were used. Furthermore, these levels could be compared to other types of cell

lines as it is possible that HaCaT cells may be more robust than other cells and less

sensitive to damage from ZnO nanoparticle exposure.

In a study conducted by Dechsakulthorn et al, in 2007 it was found using an MTS cell

viability assay (assay based on colorimetric measurement of enzyme activity) that

exposure to at least 18.98 mg/dl ± 0.113 mg/dl nanoparticulate ZnO was necessary before

44

adverse effects to cells could be observed at 4 hours, and exposure of concentrations

greater than 0.113 mg/dl ±0.45 produced adverse effects after 24 hour exposure.

In contrast, this study did not find such a sharp response in cells exposed to similar or

even lesser concentrations of ZnO nanoparticles for 24 hours, however the authors of that

study used freshly cultured fibroblasts taken from in house pathology specimens as

opposed to the HaCaT (ATCC) cell line and they did not state any size characterisation

data for the nanoparticles used (ZnO nanoparticles used in this study outlined in Appendix

1.4.). This makes direct comparison problematic, since ZnO nanoparticle toxicity occurs in

a cell type dependant manner and the ROS generating capability of ZnO nanoparticles is

dependent on particle size (Hanley et al, 2009). The methodology used by Dechsakulthorn

et al in determining concentrations at which adverse effects are apparent is however

superior to subjectively counting cells in a monolayer, as apoptotic cells often detach from

the flasks, become suspended in the media and therefore cannot be stained and counted.

4.2. BAX

BAX positivity increased significantly in a time and dose dependant manner indicating that

the process of mitochondrial outer membrane permeabilisation was taking place in

response to nanoparticle exposure.

Staining occurred in a granular pattern elucidating the localisation of activated BAX to the

mitochondrial outer membrane. Very strong positive staining was observed in apoptotic

bodies and cells in later stages of apoptosis, many of which were in the process of

“rounding up” and about to detach. Milder BAX positivity could also be seen in cells

which appeared morphologically normal; this demonstrated a clear relationship between

the amount of activated BAX in a cell and its stage in the gradual process of apoptosis.

45

The continuous increase in BAX expression is in agreement with the theory that

membrane outer membrane permeabilisation is a point of no return, and once it takes place,

it is outside the control of its regulators (Nel et al, 2006), (See Figure 1.3). BAX is

therefore is therefore a good indicator of transient cellular damage and a marker for early

stages of apoptosis which are not visible morphologically.

In later stages of apoptosis however quantification may become unreliable due to the

tendency of dying cells to detach from the flasks and be washed away with the media

before staining. Techniques such as immunoprecipitation of cell lysate could be

advantageous when evaluating quantitative BAX expression.

4.3. Bcl-2

Bcl-2 expression did not show significant variation in response to the various

concentrations of ZnO nanoparticles used. However its expression was markedly greater in

exposed cells than the non exposed control. This could be due to the action of negative

feedback mechanisms promoting cell survival in the face of cellular stress. Bcl-2

expression did decrease slightly with time in the flasks which were exposed suggesting that

although Bcl-2 may have been upregulated initially its expression gradually reduced,

suggesting that the balance between pro-apoptotic and anti-apoptotic regulators had gone

in favour of apoptosis. It could also be a consequence of a possible genotoxic effect

4.4. p53

p53 expression like that of Bcl-2 showed a marked increase in the exposed cells when

compared with the control however no general pattern became apparent in relation to

nanoparticle concentration or exposure time. This could because of the interaction with one

of p53‟s main target genes for upregulation, MDM2 which is an E3 ubiquitin ligase that

targets p53 for destruction in the proteosome (Brady & Attardi, 2010). This labile nature of

46

p53 is a common property of transcription factors and the tight control via a negative

feedback mechanism is often necessary as a one fold increase in the expression of a

transcription factor may result in a multiple fold increase in expression of its targets

(Orphanides & Reinberg, 2002). Coupled with this is the complex nature of the

consequences of p53 expression, as p53 is involved in the regulation of expression of over

4000 different proteins (Loh, 2010). This observed data in conjunction with the nature of

p53 supports the hypothesis that p53 expression, although instrumental in the response of

the cells to nanoparticle exposure is not proportional to the observed end point of Bax

expression.

The peak expression in this experiment would have been at some point between 1 and 5

hours post exposure. Its pattern of expression during this time would have contributed

largely towards the regular dose/response relationship seen between the concentration of

zinc oxide nanoparticles and percentage Bax positivity but due to the labile nature of p53

immunocytochemical staining, particularly when limited to four time points may not be

able to model this pattern of expression. Micro-arrays or RT-PCR techniques

simultaneously measuring p53 and Bax on the same cells may therefore be advantageous.

47

5. Conclusion:

The use of nanomaterials in consumer products such as sunscreens and cosmetics is

becoming more and more commonplace and this trend is expected to continue into the

future, therefore it is important that the safety of these materials in relation to human health

be determined accurately. Although the results obtained showed a clear response to

nanoparticulate ZnO at doses less than 15 mg/dl, further study is necessary to determine

the safety of this material. As cultured cells are normally under some level of oxidative

stress when grown under standard conditions (Han et al, 2008), in-vivo investigations may

also be needed to accompany in vitro observations.

In vitro studies such as this one are however useful as initial experiments for marker

development and developing future experimental design. If the methodology or the

markers used for assessing low level toxicity were standardised, experiments such as this

could be repeated easily using different sizes and types of nanomaterials as well as

different cell lines of varied sensitivities. This is of importance since nanoparticle toxicity

is largely dependent on cell type as well as nanoparticle size (Hanley et al, 2009).

Since BAX was found to respond in a regular dose dependant manner at low doses it may

be a suitable marker for future studies. However, further studies investigating Bax

expression at higher and lower doses of ZnO nanoparticle exposure in cells lines of

different sensitivities would need to be conducted to validate this hypothesis.

48

6. Appendices:

Appendix 1: General laboratory reagents

Appendix 1.1. PBS:

1.95 g Sodium dihydrogen orthophosphate (NaH2PO4 2H2O), 5.35 g Disodium

orthophosphate anhydrous (Na2 HPO4), 42.5 g Sodium chloride (NaCl), Made up to 5

litres with deionised water

Appendix 1.2. 10% neutral buffered formalin

100 ml 40% formaldehyde, 4 g Sodium dihydrogen phosphate monohydrate, 6.5 g

Disodium hydrogen phosphate anhydrous, Added to 900 ml of deionised water

Appendix 1.3 Glycergel aqueous mounting medium:

10g Gelatine, 70 ml Glycerol, 0.25 g Phenol, 60 ml deionised water

Method:

Dissolve gelatine in distilled water, Tare balance with 100 ml universal, and pour in the

viscous glycerol until balance reads 88.2 g (density of glycerol is 1.26 g/ml, therefore 88.2

g = 70 ml). Add dissolved gelatine to this container. Weigh out phenol, taking appropriate

precautions since phenol is bio hazardous. add to the rest of the ingredients, microwave to

melt glycerine mix well and allow to set.

Appendix 1.4:

ZnO nanoparticles (Sigma, Dublin)

Characherised in earlier unpublished work by A. Casey and M. Bates, Nanolab, Focas,

DIT; Mean diamaters: Transmission Electron Microscopy: 43 ± 9.3 nm, Atomic Force

Microscopy: 51 ± 11.9 nm , Dynamic Light Scatter: 49 ± 3.2 nm

49

Appendix 2: Microscopy:

2.1. Method validation

Image 1 2 3 4 5

Manual 249 197 166 210 257

Automated 238 194 167 199 286

χ2 test: p= (2-tail sig.) 0.3914049

0

50

100

150

200

250

300

350

1 2 3 4 5

Cells

Image

Manual and automated HaCaT cell counts

manual

automated

50

Appendix 2.2. Raw microscopy data:

The following table shows the cell counts for each image used in determining % apoptosis,

necrosis and Bax positivity. Each exposure time and concentration is represented by 5

images, the file names of which are displayed in the “image” column the file names are in

the format A h B mg C, where A is exposure time in hours B is nano zinc oxide

concentration in mg/dl and C is the image number within the 5 image set.

Image name

Total

number

of cells

Number

of Bax

positive

cells

Number

of

apoptotic

cells

Number of

necrotic cells

1h0mg 1 411 0 9 1

1h0mg 2 324 0 10 0

1h0mg 3 296 0 7 0

1h0mg 4 304 0 12 0

1h0mg 5 335 0 7 0

1h1mg 1 305 0 16 0

1h1mg 2 204 0 16 5

1h1mg 3 179 3 13 0

1h1mg 4 150 1 15 1

1h1mg 5 227 0 13 3

1h5mg 1 259 0 8 2

1h5mg 2 236 0 11 2

1h5mg 3 333 0 13 0

1h5mg 4 290 0 11 0

51

1h5mg 5 285 0 14 0

1h10mg 1 317 0 14 1

1h10mg 2 247 0 4 6

1h10mg 3 286 0 8 4

1h10mg 4 300 0 21 0

1h10mg 5 259 0 15 2

1h15mg 1 311 0 12 0

1h15mg 2 299 0 12 0

1h15mg 3 268 0 12 0

1h15mg 4 211 0 17 0

1h15mg 5 340 0 24 8

3h0mg 1 201 1 16 0

3h0mg 2 168 1 14 0

3h0mg 3 167 0 13 0

3h0mg 4 206 3 16 1

3h0mg 5 220 1 17 0

3h1mg 1 324 7 19 0

3h1mg 2 256 5 24 0

3h1mg 3 303 9 21 0

3h1mg 4 346 17 17 0

3h1mg 5 334 25 24 6

3h5mg 1 247 22 22 0

3h5mg 2 232 23 16 0

3h5mg 3 232 31 12 6

52

3h5mg 4 200 25 20 0

3h5mg 5 230 32 22 1

3h10mg 1 255 17 11 1

3h10mg 2 292 36 19 0

3h10mg 3 262 22 12 0

3h10mg 4 257 26 21 0

3h10mg 5 231 24 26 0

3h15mg 1 132 21 14 0

3h15mg 2 234 63 20 0

3h15mg 3 174 20 37 0

3h15mg 4 287 57 18 0

3h15mg 5 173 21 27 0

6h0mgr 1 195 5 16 0

6h0mgr 2 166 1 9 0

6h0mgr 3 114 0 15 0

6h0mgr 4 188 2 17 0

6h0mgr 5 202 2 15 0

6h1mg 1 184 10 12 0

6h1mg 2 182 11 21 1

6h1mg 3 202 13 13 0

6h1mg 4 208 9 22 0

6h1mg 5 172 59 15 0

6h5mg 1 172 10 14 3

6h5mg 2 189 25 19 0

53

6h5mg 3 216 14 15 0

6h5mg 4 257 67 22 0

6h5mg 5 238 21 20 0

6h10mg 1 264 33 22 0

6h10mg 2 153 44 19 0

6h10mg 3 233 23 15 0

6h10mg 4 211 17 33 0

6h10mg 5 347 25 25 0

6h15mg 1 230 73 22 0

6h15mg 2 210 46 17 0

6h15mg 3 279 44 18 0

6h15mg 4 265 33 28 0

6h15mg 5 272 33 22 0

24h0mgr 1 327 2 25 0

24h0mgr 2 265 11 36 0

24h0mgr 3 256 4 32 0

24h0mgr 4 363 3 37 1

24h0mgr 5 342 4 28 1

24h 1mg 1 537 86 22 0

24h 1mg 2 388 57 23 1

24h 1mg 3 493 17 21 0

24h 1mg 4 559 2 28 6

24h 1mg 5 434 23 22 0

24h5mg 1 340 29 29 0

54

24h5mg 2 370 29 25 1

24h5mg 3 370 36 35 0

24h5mg 4 238 119 33 0

24h5mg 5 372 68 28 0

24h 10mg 1 415 124 16 0

24h 10mg 2 474 64 27 0

24h 10mg 3 444 69 33 0

24h 10mg 4 361 33 20 0

24h 10mg 5 478 44 32 0

24h15mg 1 306 173 45 0

24h15mg 2 374 51 48 0

24h15mg 3 336 37 34 0

24h15mg 4 312 34 50 0

24h15mg 5 328 94 41 18

55

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