The Role of Base Excision Repair in Regulating Endotoxin

The Role of Base Excision Repair in Regulating Endotoxin Induced

Inflammation

A thesis submitted to The University of Manchester for the Degree of Doctor of Philosophy (PhD) in the Faculty

of Medical and Human Sciences

2012

Alan Carter

School of Medicine

Health Sciences Research Group Occupational and Environmental Health

Ph.D. Thesis 2012 Alan Carter

2

Table of contents……………………………………………………………………………….2 List of Figures…………………………………………………………………………………...5 List of Tables……………………………………………………………………………….….10 List of Abbreviations…………………………………………………………………………12 Abstract………………………………………………………………………………………....15 Declaration……………………………………………………………………………………..16 Copyright Statement………………………………………………………………………..16 Acknowledgements………………………………………………………………………….17 1. Introduction............................................................................. 18

1.1. Structure of Endotoxin .............................................................. 19

1.2. Endotoxin Induced Inflammation ............................................... 21

1.2.1. LPS Recognition ................................................................. 21

1.2.2. Activation of the inflammatory response .............................. 21

1.2.3. Inflammatory Signalling ...................................................... 22

1.2.3.1. Fibroblasts ................................................................... 24

1.2.3.2. Human Polymophonuclear Leukocytes ........................... 25

1.3. The Endotoxin Paradox ............................................................. 26

1.4. Factors affecting the inflammation response to endotoxin. .......... 27

1.5. BER Proteins and Endotoxin Induced Inflammation..................... 31

1.6. DNA Glycosylases and Endotoxin Induced Inflammation ............. 32

1.7. Oxidative Damage .................................................................... 34

1.7.1. Reactive Oxygen Species .................................................... 34

1.7.2. Oxidative Adenine Damage ................................................. 39

1.7.3. Oxidative Thymine Damage ................................................ 40

1.7.4. Oxidative Cytosine Damage ................................................ 41

1.7.5. Spontaneous Base Removal ................................................ 41

1.7.6. DNA strand breaks ............................................................. 42

1.7.7. Lipid peroxidation ............................................................... 43

1.8. DNA Repair .............................................................................. 44

1.8.1. Base Excision Repair ........................................................... 45

1.8.2. DNA Glycosylases ............................................................... 47

1.8.2.1. OGG1 .......................................................................... 49

1.8.2.2. NTH1 .......................................................................... 52

1.8.2.3. NEIL1 .......................................................................... 54

1.8.2.4. NEIL2 .......................................................................... 56

1.9. Hypothesis ............................................................................... 59

2. Materials and Methods ............................................................ 61

2.1. Materials .................................................................................. 61

2.1.1. Buffer composition ............................................................. 61

2.1.2. Tissue culture media .......................................................... 62

2.1.3. PCR primers ....................................................................... 62

2.1.4. Molecular biology reagents ................................................. 65

2.1.5. Protein analysis reagents .................................................... 65

2.1.6. Equipment ......................................................................... 66

2.1.7. Transgenic Mouse and MEF sources .................................... 66

2.2. Methods ................................................................................... 67

2.2.1. Mouse Colony Management ................................................ 67

2.2.2. Mouse Colony Characterisation ............................................ 67


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2.2.3. LPS Induced Organ Damage ............................................... 68

2.2.4. Induction of Immune Response ........................................... 68

2.2.5. Establishment of MEF cultures from mouse embryos ............ 68

2.2.6. MEF culture........................................................................ 70

2.2.7. Treatment of MEFs with LPS ............................................... 70

2.2.8. Genomic DNA extraction ..................................................... 71

2.2.9. Polymerase Chain Reaction (PCR) Genotyping ...................... 71

2.2.10. Agarose Gel Electrophoresis .............................................. 72

2.2.11. RNA extraction ................................................................. 72

2.2.12. Reverse transcription (RT) ................................................ 73

2.2.13. Protein Analysis ................................................................ 73

2.2.13.1. Protein extraction from mouse tissues ......................... 73

2.2.13.2. Protein quantification .................................................. 74

2.2.13.3. Western blot .............................................................. 74

2.2.13.4. ELISA ........................................................................ 75

2.2.13.5. Myeloperoxidase Assay ............................................... 76

2.2.13.6. Malondialdehyde Assay ............................................... 76

2.2.13.7. Glutathione Assay....................................................... 77

2.2.14. Statistical Analysis ............................................................ 78

3. Mouse models: Generation and characterisation .................... 79

3.1. Introduction ............................................................................. 79

3.1.1. Aims .................................................................................. 80

3.2. Results .................................................................................... 81

3.2.1. NEIL1 Mouse Colony........................................................... 81

3.2.1.1. Preparation of NEIL1 Knockout Mice. ............................. 81

3.2.1.2. Confirmation of Genotype. ............................................ 82

3.2.1.3. Viability and Mortality of NEIL1 mice. ............................ 84

3.2.1.4. NEIL1 Mouse Weights .................................................. 85

3.2.2. NEIL2 Mouse Colony........................................................... 91

3.2.2.1. Genotyping .................................................................. 91

3.2.2.2. Western and RT-PCR .................................................... 92

3.2.3. OGG1 Mouse Colony ........................................................... 94

3.2.3.1. OGG1 Mouse Genotyping .............................................. 94

3.3. Discussion ................................................................................ 95

4. Cytokine Output of DNA Glycosylase Disrupted Cells ........... 101

4.1. Introduction ............................................................................ 101

4.1.1. Aims ................................................................................. 102

4.2. Results ................................................................................... 102

4.2.1. TLR4 mRNA Transcription Analysis ..................................... 102

4.2.2. Cytokine Output from LPS Challenged MEF Cells ................. 103

4.3. Discussion ............................................................................... 108

5. Effects of NEIL1 Gene Knockout on Endotoxin Induced Inflammation ............................................................................. 113

5.1. Introduction ............................................................................ 113

5.1.1. Aims ................................................................................. 114

5.2. Results ................................................................................... 114

5.2.1. Cytokine Output in LPS Challenged NEIL1-/- Mice ................. 114

5.2.2. Myeloperoxidase activity in LPS challenged NEIL1-/- mice ..... 118


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5.2.3. Malondialdehyde content in LPS challenged NEIL1-/- mice .... 126

5.2.4. Glutathione levels in LPS challenged NEIL1-/- mice ............... 133

5.2.5. Age related cytokine output NEIL1-/- mice ........................... 141

5.3. Discussion ............................................................................... 141

6. Effects of OGG1 Gene Knockout on Endotoxin Induced Inflammation ............................................................................. 153

6.1. Introduction ............................................................................ 153

6.1.1. Aims ................................................................................. 154

6.2. Results ................................................................................... 154

6.2.1. Cytokine Output in LPS Challenged OGG1-/- Mice ................. 154

6.2.2. Myeloperoxidase Activity in LPS Challenged OGG1-/- Mice ..... 159

6.2.3. Malondialdehyde Content in LPS Challenged OGG1-/- Mice .... 167

6.2.1. Glutathione Activity in LPS Challenged OGG1-/- Mice ............ 174

6.3. Discussion ............................................................................... 182

7. Overall Discussion ................................................................. 190

8. References……………………………………………………………….202

Main text word count including figures and tables : 41,001


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Index of Figures

Figure 1.1: A schematic view of the structure of LPS 20

Figure 1.2: The Recognition of LPS and Activation of the Immune

Response in Macrophages 22

Figure 1.3: Generation of Antimicrobial Reactive Oxygen and Reactive

Nitrogen Species 26

Figure 1.4: Differences in Three Dimensional Shape of LPS from

Different Sources 28

Figure 1.5: Hypothesis linking Lipid A configuration to Cytokine Output

29

Figure 1.6: Comparison of Changes to Cytokine Production after LPS

Stimulation in PARP1 and OGG1 Knockout Mice 33

Figure 1.7: Production of Superoxide Radical and Hydrogen Peroxide by

the Electron Transport Chain 35

Figure 1.8: The Fenton Reaction 37

Figure 1.9: Formation of Oxidised Guanine Products 38

Figure 1.10: 8-oxoG paired with cytosine and mispaired with adenine 39

Figure 1.11: Formation of Oxidised Adenine Products 39

Figure 1.12: Formation of Oxidised Thymine Products 40

Figure 1.13: Formation of Oxidised Cytosine Products 41

Figure 1.14: Formation of an AP site 42

Figure 1.15: Lipid Peroxidation 43

Figure 1.16: Composition of malondialdehyde and 4-hydrooxyalkenal 44

Figure 1.17: Monofunctional DNA glycosylase BER (I), bifunctional

APE1 dependant BER (II) and independent (III) pathways

46

Figure 1.18: Sequence alignment of critical domains of NEIL1 and NEIL2

with E. coli Nei/Fpg 54

Figure 3.1: Diagram of the NEIL1 Knockout Construct and Genotyping

Results 81

Figure 3.2: Pedegree of NEIL1-/- Mice 82

Figure 3.3: Confirmation of a NEIL1 Null Phenotype 83


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Figure 3.4: The distribution of genotypes from HET x HET breeding

pairs of NEIL1 Transgenic Mice 84

Figure 3.5: Average litter composition from NEIL1 colony 85

Figure 3.6: Mortality rates of Male and Female Neil1 Transgenic Mice 86

Figure 3.7: NEIL1 Transgenic male and female mouse weights over 12

Months 87

Figure 3.8: Length and BMI of NEIL1 Transgenic Mice 88

Figure 3.9: Male NEIL1 transgenic mouse organ weight comparisons 89

Figure 3.10: Female NEIL1 transgenic mouse organ weight comparisons

90


Results 92

Figure 3.12: Confirmation of a NEIL2 Null Phenotype 93

Figure 3.13: Diagram of the OGG1 Knockout Construct and Genotyping

Results 94

Figure 3.14: The creation of transgenic mice can result in linkage

disequilibrium 97

Figure 4.1: RT-PCR for TLR4 102

Figure 4.2: IL-6 output of wildtype and knockout MEF cell lines 105

Figure 4.3: IL-10 output of wildtype and knockout MEF cell lines 106

Figure 4.4: MCP-1 output of wildtype and knockout MEF cell lines 107

Figure 5.1: IL-4 concentrations in blood serum taken from NEIL1

transgenic mice 115


transgenic mice 116


transgenic mice 117


transgenic mice 118

Figure 5.5: MPO activity in heart tissues of NEIL1 transgenic mice

exposed to LPS 121


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Figure 5.6: MPO activity levels in lung tissues of NEIL1 transgenic mice

exposed to LPS 122

Figure 5.7: MPO activity in liver tissues of NEIL1 transgenic mice exposed

to LPS 123

Figure 5.8: MPO activity in kidney tissues of NEIL1 transgenic mice

exposed to LPS 124

Figure 5.9: MPO activity in ileum tissues of NEIL1 transgenic mice

exposed to LPS 125

Figure 5.10: MDA levels in heart tissues of NEIL1 transgenic mice exposed

to LPS 128

Figure 5.11: MDA levels in lung tissues of NEIL1 transgenic mice exposed

to LPS 129

Figure 5.12: MDA levels in liver tissues of NEIL1 transgenic mice exposed

to LPS 130

Figure 5.13: MDA levels in kidney tissues of NEIL1 transgenic mice

exposed to LPS 131

Figure 5.14: MDA levels in ileum tissues of NEIL1 transgenic mice exposed

to LPS 132

Figure 5.15: GSH levels in heart tissues of NEIL1 transgenic mice exposed

to LPS 135

Figure 5.16: GSH levels in lung tissues of NEIL1 transgenic mice exposed

to LPS 136

Figure 5.17: GSH levels in liver tissues of NEIL1 transgenic mice exposed

to LPS 137

Figure 5.18: GSH levels in kidney tissues of NEIL1 transgenic mice

exposed to LPS 138

Figure 5.19: GSH levels in ileum tissues of NEIL1 transgenic mice exposed

to LPS 139


transgenic mice 142


transgenic mice 143


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transgenic mice 144


transgenic mice 145

Figure 6.1: IL-4 concentrations in blood serum taken from OGG1

transgenic mice 155


transgenic mice 156


transgenic mice 157


transgenic mice 158

Figure 6.5: MPO activity in heart tissues of OGG1 transgenic mice

exposed to LPS 161

Figure 6.6: MPO activity in lung tissues of OGG1 transgenic mice exposed

to LPS 162

Figure 6.7: MPO activity levels in liver tissues of OGG1 transgenic mice

exposed to LPS 163

Figure 6.8: MPO activity in kidney tissues of OGG1 transgenic mice

exposed to LPS 164

Figure 6.9: MPO activity in ileum tissues of OGG1 transgenic mice

exposed to LPS 165

Figure 6.10: MDA levels in heart tissues of OGG1 transgenic mice exposed

to LPS 169

Figure 6.11: MDA levels in lung tissues of OGG1 transgenic mice exposed

to LPS 170

Figure 6.12: MDA levels in liver tissues of OGG1 transgenic mice exposed

to LPS 171

Figure 6.13: MDA levels in kidney tissues of OGG1 transgenic mice

exposed to LPS 172

Figure 6.14: MDA levels in ileum tissues of OGG1 transgenic mice exposed

to LPS 173


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Figure 6.15: GSH levels in heart tissues of OGG1 transgenic mice exposed

to LPS 177

Figure 6.16: GSH levels in lung tissues of OGG1 transgenic mice exposed

to LPS 178

Figure 6.17: GSH levels in liver tissues of OGG1 transgenic mice exposed

to LPS 179

Figure 6.18: GSH levels in kidney tissues of OGG1 transgenic mice

exposed to LPS 180

Figure 6.19: GSH levels in ileum tissues of OGG1 transgenic mice exposed

to LPS 181

Figure 6.20: Pathways involved in the generation and degradation of

oxidants and the effects of OGG1 knockout at key

endpoints 184

Figure 6.21: Proposed model of PARP-1 inhibition by oestrogen 188

Figure 7.1: Alterations in organ activity due to adrenaline and observed

levels of GSH in NEIL1-/- mice 197


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Index of Tables

Table 1.1: The generation of major ROS and major avenues of protection

36

Table 1.2: Human DNA glycosylases located in the nuclei and the DNA

damage that they remove 48

Table 2.1: Molecular Biology Buffers 61

Table 2.2: Protein Analysis Buffers 62

Table 2.3: Tissue Culture Media 62

Table 2.4: PCR Genotyping Primers 63

Table 2.5: RT-PCR Primers 64

Table 3.1: Summary of Phenotypes of DNA Glycosylase Deficient Mice 100

Table 4.1: Comparison of IL-6 results between untreated and treated DNA

glycosylase deficient MEF cells 105

Table 4.2: Comparison of IL-10 results between untreated and treated

DNA glycosylase deficient MEF cells 106

Table 4.3: Comparison of MCP-1 results between untreated and treated

DNA glycosylase deficient MEF cells 107

Table 4.4 Summary of Results from cytokine assays 108

Table 5.1: MPO activity in tissues of NEIL1 transgenic mice exposed to LPS

120

Table 5.2: MDA levels in tissues of NEIL1 transgenic mice exposed to LPS

127

Table 5.3: GSH levels in tissues of NEIL1 transgenic mice exposed to LPS

134

Table 5.4: Summary of NEIL1 transgenic mouse blood serum cytokine

Results 146

Table 5.5: Summary of NEIL1 transgenic mouse organ damage results

147

Table 6.1: MPO activity in tissues of OGG1 transgenic mice exposed to LPS

160


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Table 6.2: MDA levels in tissues of OGG1 transgenic mice exposed to LPS

168

Table 6.3: GSH levels in tissues of OGG1 transgenic mice exposed to LPS

175

Table 6.4: Summary of OGG1 transgenic mouse cytokine Results 182

Table 6.5: Summary of OGG1 transgenic mouse organ damage results

183

Table 7.1: Summary and comparison of NEIL1 and OGG1 cytokine output

results when compared with those of WT animals 191

Table 7.2: Summary and comparison of NEIL1 and OGG1 organ damage

results 192


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List of Abbreviations

2-OH-A 2-hydroxyadenine

5′dRP 5′-deoxyribose-5-phosphate

5,6-DHU 5,6-dihydrouracil

5-FoU 5-formyluracil

5-FU 5-fluorouracil

5-HMU 5-hydroxymethyluracil

5-OHC 5-hydroxycystosine

5-OHU 5-hydroxyuracil

8-oxoA 7,8-dihydro-8-oxoadenine

8-oxoG 7,8-dihydro-8-oxoguanine

AP-1 Activator protein 1

APE1 Apurinic endonuclease 1

AP site Apurinic/apyrimidinic sites

BPI Bactericidal/permeability-increasing protein

Cg Cytosine glycol

DEP Diesel exhaust particles

ddH2O Double distilled water

DMSO Dimethyl sulfoxide

DSB Double strand breaks

DTNB 5,5’-dithio-bis(2-nitrobenzoic acid)

Egr-1 Early growth response 1

ERα Oestrogen receptor-α

FapyA 4,6-diamino-5-formamidopyrimidine

FapyG 6-diamino-4-hydroxy-5-formamidoguanine

GR Glutathione reductase

GPX Glutathione peroxidise

GSH Glutathione

H2O2 Hydrogen peroxide

HAE 4-hydroxyalkenals

HET Heterozygous


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HhH Helix-hairpin-helix

IFN-γ Interferon γ

IL-1 Interleukin 1

iNOS inducible nitric oxide synthase

IRAK Interleukin-1 receptor-associated kinase

KDO 2-keto-3-deoxyoctonic acid

KO Knockout

KPE Potassium phosphate EDTA

LBP LPS binding protein

LPS Lipopolysaccharide

MAC Membrane attack complex

MAPK1 Mitogen-activated protein kinase 1

MAPK8/JNK Mitogen-activated protein kinase 8

MDA Malondialdehyde

MIP1α Macrophage inflammatory protein 1

MMR Mismatch repair

MPG N-methylpurine-DNA glycosylase

MPO Myeloperoxidase

MyD88 Myeloid differentiation primary response gene

88

NADPH Nicotinamide adenine dinucleotide phosphate

NER Nucleotide excision repair

NEIL1/2 Nei endonuclease VIII-like 1/2

NFκβ Nuclear factor κβ

NO Nitric oxide

NOX NADPH oxidase

NTH1 Nth endonuclease III-like

O-2 Superoxide

OGG1 8-Oxoguanine glycosylase

PARP1 Poly (ADP-Ribose) Polymerase-1

PBS Phosphate buffered saline

PCNA Proliferating cell nuclear antigen


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PCR Polymerase chain reaction

PNK Polynucleotide kinase

Pol β DNA polymerase β

PUA 3′-phospho-α,β-unsaturated aldehyde

RPA Replication protein A

RT-PCR Reverse transcriptase - PCR

ROS Reactive oxygen species

SOD Superoxide dismutase

SMUG1 Single-strand selective Monofunctional Uracil

DNA Glycosylase

SSB Single strand breaks

TEMED N,N,N,N-tetramethyl-ethylenediamine

Tg Thymine glycol

Th1 T-helper 1

Th2 T helper 2

TLR4 Toll like receptor 4

TNF-α Tumour necrosis factor α

TRAF6 TNF receptor associated factor

Ug Uracil glycol

UNG2 Uracil-DNA glycosylase 2

WT Wildtype


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Abstract Endotoxins a component of the outer membrane of the cell wall of gram negative bacteria, stimulate the innate immune system to elicit an inflammatory response in mammals. Deletion of base excision repair (BER) genes has been reported to decrease the immune response to endotoxin in mouse models. It is currently unknown whether this role is limited to a few select proteins or a result of the general function of the BER pathway. The aim of this study was to identify if the loss of other BER proteins would trigger a similar response by measuring the levels of inflammatory cytokines produced and certain biomarkers of oxidative stress. To facilitate this, a new strain of NEIL1-/- mice was successfully created as well as a putative NEIL2-/- strain. A previous strain of NEIL1-/- mice displayed a sporadic obese phenotype, our NEIL1-/- mice showed no significant increase in bodyweight when compared to WT mice. Whilst there were significant differences in the serum content of cytokines IL-6, IL-12, IL-10 and IL-4 between wildtype, NEIL1-/- and OGG1-/- mice challenged with lipopolysaccharide (LPS, the active component of endotoxin). When compared to wildtype animals both NEIL1-/- and OGG1-/- mice produced lower levels of the Th1 cytokine IL-6 (♂ 1 h; p<0.05 and ♀ 24 h; p<0.01), and the Th2 IL-10 cytokine (♀ 6 and 24 h; p<0.01) along with other sex and genotype specific differences. When comparing LPS induced organ damage in NEIL1-/- and wildtype mice there were no significant differences in myeloperoxidase (MPO) activity or malondialdehyde (MDA) concentration due to genotype. However, there were significant differences observed in glutathione (GSH) levels in the heart (p=0.01), lung (p=0.05), liver (p=0.05) and ileum (p=0.05) that when considered alongside a significant increase in the weights of adrenal glands in NEIL1-/- knockout (♂ p=0.05, ♀ p=0.03) mice were suggestive of a raised level of adrenaline. The OGG1-/- mice displayed no significant genotype x treatment interaction in MPO activity, MDA levels or GSH levels. However, genotype x sex interactions were observed in the liver and lung tissues of OGG1-/- for MPO (lung p<0.01, liver p=0.02), MDA (lung p<0.01) and GSH (lung p=0.05, liver p=0.04) indicating that female OGG1-/- mice had greater protection from the oxidative effects of LPS induced inflammation. In conclusion whilst the knockout of OGG1 and NEIL1 genes had an effect on the inflammatory signalling response, this effect was not great enough to impact upon oxidative stress markers of inflammation within the tissues sampled. The mechanism of how this is accomplished is at present unclear and worthy of further study.


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Declaration “No portion of the work referred to in the thesis has been submitted in

support of an application for another degree or qualification of this or

any other university or other institute of learning.”

Copyright Statement

(i) The author of this thesis (including any appendices and/or schedules to

this thesis) owns any copyright in it (the “Copyright”) and s/he has given

TheUniversity of Manchester the right to use such Copyright for any

administrative, promotional, educational and/or teaching purposes.

(ii) Copies of this thesis, either in full or in extracts, may be made only in

accordance with the regulations of the John Rylands University Library of

Manchester. Details of these regulations may be obtained from the

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(iii) The ownership of any patents, designs, trade marks and any and all

other intellectual property rights except for the Copyright (the “Intellectual

Property Rights”) and any reproductions of copyright works, for example

graphs and tables (“Reproductions”), which may be described in this

thesis, may not be owned by the author and may be owned by third

parties. Such Intellectual Property Rights and Reproductions cannot and

must not be made available for use without the prior written permission of

the owner(s) of the relevant Intellectual Property Rights and/or

Reproductions.

(iv) Further information on the conditions under which disclosure,

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Intellectual Property Rights and/or Reproductions described in it may take

place is available from the Head of School of Medicine.


17

Acknowledgements

I would like to thank my supervisors Dr. Andrew Povey and Dr. Rhoderick

Elder, for their guidance and support throughout the course of this

research project, and my advisor Dr. Rachel Watson. I would also like to

thank the British Cotton Growing Association for providing the funding to

make this work possible.

I would also like to acknowledge the help I received from the members of

staff at the Transgenic Animals Facility at The University of Manchester,

especially Ian Townsend, Ruth Jones and Karen Fry. Also special thanks

should go to those who helped me learn some of the finer techniques Dr.

Mel Heeran and Brian A. Tefler.

A special mention should go to my colleagues in the Biomarkers Lab who

in addition to giving me moral support made the atmosphere we worked

in fun, a special thanks to W.M. Md. Saad whose work on the confirmation

of the NEIL2 knockout is found within. Finally I would like to thank all my

family and friends who supported me through their kind (and sometimes

less than kind - but needed) advice, caring words and thoughtful actions.


18

1. Introduction

Cotton workers, and other workers exposed to organic dusts, have been

observed to exhibit byssinosis (Mberikunashe et al., 2010; McKERROW et

al., 1958; ROACH and SCHILLING 1960). Symptoms include coughing,

shortness of breath and difficulty breathing. These were more pronounced

when the persons returned to work after the weekend, and then subsided

the following day leading to the moniker “Monday Asthma” (Liu, 2007).

Originally it was assumed that this was due to a build up of plant matter in

the airways of these workers (ROACH and SCHILLING 1960), but later it

was found to be due to the presence of endotoxins, potent inducers of

neutrophilic airway inflammation, carried on the dust (Cavagna et al.,

1969; Radon, 2006). Smokers can also exhibit similar symptoms due to

endotoxin carried with the smoke particles from tobacco (Hasday et al.,

1999). A person’s sensitivity to endotoxin induced inflammation has been

shown to be affected by their genetic makeup (Eder et al., 2004).

Endotoxin is composed of complexes of lipooligosaccharrides, lipoproteins

and lipopolysacharide (LPS), a substance found exclusively as part of the

outer membrane of the cell wall of gram negative bacteria; indeed 3-10%

of the dry weight of these bacteria is endotoxin (Fan and Cook 2004;

Vaara, 1999). All sources of gram negative phospholipid contain this

substance including blebs, vesicles and fragments of dead cells (Prins,

1996; Radon, 2006; Vaara, 1999). Hence humans can therefore be

exposed to LPS in several ways including working in the presence of

organic dust, smoking, exposure to invasive gram negative bacteria via

injury, and exposure to intestinal bacteria during surgery (Beutler and

Rietschel 2003; Hasday et al., 1999).

Endotoxin activates the innate immune system, that portion of the

immune system which defends the host from organisms in a generic


19

manner, in an attempt to destroy any bacterial invaders. Once bacteria are

killed, however, the endotoxins in their membrane continue to stimulate

the immune system fuelling further response (Prins, 1996). This escalating

response can lead to severe systemic inflammation, which manifests as

fever, increased heart and respiratory rates, and is described as endotoxic

(septic) shock (Beutler and Rietschel 2003). In order to understand why

this occurs, it is important to further discuss the structure of endotoxin

and the immune response to it, in further detail.

1.1. Structure of Endotoxin

Endotoxin refers to a mixture of bacterial cell surface molecules mainly

consisting of LPS but including lipooligosaccharrides and lipoproteins.

Alternatively, the term LPS refers to a pure substance which is not found

in nature. The structure of LPS is shown in Figure 1.1. The molecule

consists of four major domains, an O-antigenic polysaccharide, an outer

core oligosaccharide, an inner core oligosaccharide and an acylated

diglucosamine head group (lipid A) (Rietschel et al., 1994).

The O-antigenic polysaccharide consists of a repeating structure of one to

eight glycosyl residues, the structure of which varies between serotypes.

The size range of the O-antigenic polysaccharide chain is comparatively

specific to a bacterial species although there is some heterogeneity, even

within LPS from the same colony, in the length of these molecules. This is

seen as an evolutionary measure in order to protect the bacterium from

complement immune measures and phagocytosis by macrophages

(Aspinall et al., 1996; Beutler and Rietschel 2003; Rietschel et al., 1994).

The outer core oligosaccharide consists of the common hexoses D-

glucose, D-galactose, and N-acetyl-D-glucosamine. It is more uniform in

composition than the O-antigenic polysaccharide and indeed only five

different core types have been found in E. coli serotypes (Aspinall et al.,

1996; Rietschel et al., 1994). The inner core oligosaccharide is composed


20

of two unusual sugars (heptose and 2-keto-3-deoxyoctonoic acid (KDO)),

and it is important in the structure and functional viability of the outer

membrane, and hence the viability of the bacterium (Aspinall et al., 1996;

Rietschel et al., 1994).

Figure 1.1: A schematic view of the structure of LPS

Hep, L-glycerol-D-manno-heptose; Gal, galactose; Glc, glucose; KDO, 2-keto-3-deoxyoctonic acid; NGa, N-acetyl-galactosamine; NGc, N-acetyl-glucosamine. Adapted Magalhaes et al., 2007.

Lipid A is responsible for the toxic effects of the LPS chain and consists of

two glucosamine units with fatty acid chains attached, each of these fatty

acid chains normally contains a phosphate group (Raetz et al., 2009;

Wang and Quinn 2010). When purified lipid A, elicits the same cytokine

response as the whole molecule in animal models (Netea et al., 2002). All

four of the LPS components are required to maintain the virulence of the

bacterium, but only the inner core and lipid A are required for the viability

of the organism (Vaara, 1999).


21

1.2. Endotoxin Induced Inflammation

1.2.1. LPS Recognition

The inflammatory response is an innate response generally to invasive

pathogens, and occurs in two forms namely, an acute response which is

direct response to a stimuli, e.g. tissue damage, and a chronic response

which can lead to arthritis and the wasting associated with certain cancers

(Gupta et al., 2011; Matsukawa et al., 1997).

Endotoxin is recognised by host cells after binding to recognition sites on

their membranes, a process which requires the mediation of LPS-binding

protein (LBP). In vitro, without LBP, large concentrations (>1mg/ml) of

LPS are needed to activate macrophages. In vivo however, due to the

presence of LBP, only nanogram amounts of LPS are required for cellular

recognition, and an inflammatory response (Moore et al., 1976; Watson

and Riblet 1974; Watson and Riblet 1975).

LBP shares many features with other phospholipid binding proteins, and

works mainly as a transport protein. LBP first breaks down endotoxin from

large complexes so that separated LPS molecules can be transported to

target receptor, CD14/toll like receptor 4 (TLR4) as shown in Figure 1.2

(Paulos et al., 2007). Alternatively the LPS is transported by LBP to high-

density lipoproteins where it becomes unable to activate the immune

response and is removed from the system. Thus LBP is important for both

the recognition and removal of LPS (Beutler and Rietschel 2003; Martin,

2000).

1.2.2. Activation of the inflammatory response

One of the primary signalling cells in the LPS inflammatory response is the

monocyte which is present in all tissues (Butterfield et al., 2006). TLR4

having recognised LPS, then activates a number of proteins including

NADPH oxidase (NOX) proteins which catalyse the formation of superoxide


22

radicals. When considering the inflammation reaction the major protein

activator is nuclear factor κB (NF-κB) which is the key transcription factor

in the expression of several pro-inflammatory proteins including cytokines,

chemokines, inducible nitric oxide synthase (iNOS), the inducible form of

cyclooxygenase and adhesion molecules (Gloire et al., 2006).

Figure 1.2: The Recognition of LPS and Activation of the Immune Response in

Monocytes The LPS/LBP complex is recognised by CD14/TLR4 which activate a series of proteins within the cell, beginning with myeloid differentiation primary response gene 88 (MyD88), causing an inflammatory activation cascade to proceed via IL-1R-associated kinase

(IRAK) Adapted from (Buer and Balling 2003).

1.2.3. Inflammatory Signalling

Having been activated by LPB/LPS complexes, local cells (e.g. fibroblasts)

and the more mobile leukocytes proceed to release various signalling

proteins into the surrounding area including the primary inflammatory

cytokines Interleukin 1 (IL-1), IL-6 and Tumour Necrosis Factor-α (TNF-α).

These molecules act as stimulators of pro-inflammatory proteases (i.e.

collagenase and elastase) to aid in tissue remodelling as well as many

secondary messengers such as platelet activating factor (which induces


23

vasodilation and wound healing), prostaglandins (to induce vasodilation)

and reactive oxygen species (ROS; increasing proinflammatory cytokine

release) (Moller and Villiger 2006; Naik and Dixit 2011).

The release of the prostaglandins PGE2 and PGF2 influences the

contraction and relaxation of the blood vessels leading to greater vascular

permeability (vasodilation;(Williams, 1979)). The subsequent movement of

fluids influences several physiological changes including a raise in local

temperature, and the movement of neutrophils and macrophages from the

blood stream to the local areas. These cells are drawn by chemotaxis

(following concentrations of attractant proteins). Once at the source of the

chemotactic agents the neutrophils, macrophages and monocytes continue

releasing cytokoines and chemokines as well as beginning the process of

phagocytosis (DiStasi and Ley 2009).

IL-6 is often used as a measure of immune response as it is induced in

large quantities by LPS, and is released by fibroblasts (Van, 1990). Whilst

increased IL-6 levels are used to denote a greater level of inflammation, it

is in fact a pleiotropic cytokine as it acts as both a pro-inflammatory and

anti-inflammatory signal. It increases inflammation by directly increasing

macrophage activity. However, it also activates the release of

adrenocorticotrophic hormone which triggers the release of cortisol and

IL-10, both of which lead to a reduction in inflammation (Moller and

Villiger 2006).

IL-10 is also released from fibroblasts and activated macrophages, which

also release IL-12. These Th2 cytokines modulate the production of

cytokines from T lymphocytes and fibroblasts, including the

chemoattractants IL-8 and MIP-1α which recruit more neutrophils to the

surrounding area by chemotaxis (Maloney et al., 2005; Martinez et al.,

2004; Reed and Milton 2001; Wan et al., 2000; Wang et al., 2005). IL-12

increases the effectiveness of T helper 1 (Th1) cells, modulators of the


24

innate immune system, in the area of endotoxin challenge by further

promoting the production of lymphokines and interferon-γ, which trigger

the proliferation of cytotoxic T-cells and further activate the macrophages

so that they become more aggressive. These ‘angry’ macrophages also

release proinflammatory molecules, such as IL-1 and TNF-α, at a much

greater rate (Moller and Villiger 2006).

The differentiation of T helper 2 (Th2) cells is initiated by IL-4, and these

cells then create more IL-4 in a self promoting feedback loop (Takeda et

al., 1996). The release of IL-10 increases the activity of Th2 cells which in

turn activate B-cells increasing antibody production. IL-10 also inhibits the

production of IL-12 by macrophages, thus ensuring that antibody

production is kept at a maximum and that the Th1 cells are not over-

stimulated into an auto-immune reaction (Wan et al., 2000).

1.2.3.1. Fibroblasts Fibroblasts, connective tissue cells which secrete an extracellular matrix

rich in collagen and other macromolecules had previously not been

considered to produce inflammatory substances in significant amounts.

More recently they have been shown to aid in the initiation of

inflammatory cascades throught the release of proinflammatory cytokines

such as IL-6 (Van, 1990), but their major effects on the inflammatory

response come via the induction of bone marrow derived immune cells,

such as neutrophils and macrophages, to the area of infection through the

release of chemokines such as IL-8, MCP-1 and MIP-1α (Maloney et al.,

2005; Smith et al., 1997; Wang et al., 2005).

As fibroblasts can be cultured relatively easily when compared to other

immune cells, they have become a staple in in vitro experiments

measuring cytokine secretion. For instance they have been used in

experiments to define TLR activity and specificity (Kurt-Jones et al., 2004).


25

1.2.3.2. Human Polymophonuclear Leukocytes

Human polymorphonuclear leukocytes (neutrophils, basophils and

eosinophils) are important in the defence of the body from external

pathogens such as bacteria and fungi, and are attracted to the site of

invasion by macrophage release of IL-8, MCP-1 and Macrophage

inflammatory protein 1 (MIP-1α) (Martinez et al., 2004). Once at the site

of endotoxin challenge these cells attempt to remove bacterial invaders

directly by phagocytosis, then releasing many cytotoxic granules which

assist in the destruction and disposal of invasive organisms (Faurschou

and Borregaard 2003). Of the many granules released, two interact

directly with LPS; lysozyme which binds to LPS rendering it inactive, and

bactericidal/permeability-increasing protein (BPI) which binds to the lipid A

section of LPS, enabling other bactericides to reach the inner membrane

(Faurschou and Borregaard 2003). During the course of phagocytosis the

neutrophil produces ROS, including superoxide and hydrogen peroxide,

and hypochlorite which are highly effective at killing the invading organism

by directly damaging the cell (Figure 1.3) (Faurschou and Borregaard

2003).

As neutrophils are rapidly attracted to a site of bacterial invasion, great

numbers of them can accumulate and this can lead to problems when

they undergo necrotic lysis as this releases the ROS contained within the

cell (Figure 1.3). Cytotoxic compounds in one area can cause a variety of

problems including greater vascular permeability, oedema, oxidative stress

and eventually apoptosis (Vernooy et al., 2001). However, not all cellular

apoptosis is due to the action of neutrophils, as it has been shown that

LPS can stimulate lung cells to undergo apoptosis in the absence of an

inflammatory immune response. It has been proposed that the lipid A

portion of the LPS molecule can activate the apoptosis pathways due to its


26

similarities with ceramide which has long been associated with

programmed cell death (Pettus et al., 2002; Vernooy et al., 2001).

Figure 1.3: Generation of Antimicrobial Reactive Oxygen and Reactive Nitrogen Species

Within the neutrophil and macrophage several enzymes catalyse the formation of ROS from molecular oxygen. The superoxide anion can react with nitric oxide to form peroxynitrite or be oxidised into nitrogen dioxide. Adapted (Smith, 1994).

1.3. The Endotoxin Paradox

As early as 1966 it had been recognised that only mammals, and to a

lesser extent avian species, are sensitive to endotoxin (Berczi et al.,

1966). The question was asked, why if it causes such an exaggerated, and

sometimes harmful, immune response, is endotoxin sensitivity a beneficial

evolved characteristic? It has been shown that endotoxin insensitive mice

were more susceptible to inner ear and other infections and that they also

died at far lower doses of bacterial infection (Beutler, 2004). Sensitivity to

LPS may allow animals to elicit an immune response to minor bacterial

infections in order to prepare a response for times when the assault is

greater (Beutler, 2004).

In humans, studies have shown an inverse relationship between endotoxin

exposure and symptoms such as skin rash, shortness of breath and


27

coughing, and that in environments where endotoxin exposure occurs,

cases of asthma are reduced (Braun-Fahrlander et al., 2002). Exposure to

endotoxin and the subsequent release of cytokines may assist in the

maturation of Th1 modulated immunity reducing the risk of atopic

sensitisation (Braun-Fahrlander et al., 2002; Douwes et al., 2000;

McElvenny et al., 2011).

It has also been shown that cotton workers have less than the expected

level of lung cancers (Enterline et al., 1985; McElvenny et al., 2011). In

comparisons between animal farmers and crop farmers, it has been found

that those working with animals, who in theory are exposed to greater

quantities of endotoxin from bacteria in faeces, experience a reduced risk

of lung cancer (Lange et al., 2003). There is also epidemiological evidence

that endotoxin protects against the formation of cancer directly, those

who give up smoking experience a short period of increased lung cancer

risk (Lange et al., 2005), and it is believed that once smoking ceases this

protective effect of endotoxin no longer acts on cancers already initiated

by the carcinogenic compounds in cigarette smoke (Lange et al., 2005).

As a result has been hypothesised that the direct or indirect activity of

endotoxin reduces the ability of cancers to develop (Lange et al., 2005).

In two studies, rabbits and guinea pigs were exposed to airborne

endotoxins and the metastasis levels within the lungs were measured. In

both cases the animals exposed to endotoxin had significantly reduced

levels of cancer (Rylander, 2002).

1.4. Factors affecting the inflammation response to endotoxin. The structure of endotoxin can vary between bacterial species and the

inflammatory response does vary according to the strain of bacteria it

comes from. Purified endotoxin from E. coli and Salmonella typhosa

elicited the immune response similar to that detailed in section 1.2 at the

greatest levels, followed by that from Klebsiella pneumoniae (K.


28

pneumoniae) and Pseuudomonas aeruginosa (P. aeruginosa). Additionally,

the response over time was significantly different according to which

‘species’ of LPS was administered. Specifically, treatment with endotoxin

from K. pneumoiae caused a comparatively higher IL-1β, IL-10 and MCP-1

production, and a reduction in the amount of TNF-α secreted. More

extremely, endotoxin from P. aeruginosa, only stimulated the release of

MIP-1α and IL-1β after 4 and 8 hours respectively, whilst exhibiting almost

no response in the release of TNF-α, interferon γ (IFN-γ) or IL-10 at any

time point whilst all other LPSs tested initiated reactions after 4h (Mathiak

et al., 2003; Schromm et al., 1998).

It is thought that the three dimensional shape of the lipid A portion of the

LPS molecule could be responsible for this difference in response (Figure

1.4). By measuring the cytokine production in mice after treatment with

various forms of LPS it was hypothesised that those LPS molecules with a

more conical configuration were more readily detected by TLR4 whilst

those of an intermediate configuration were detected more readily by a

combination of TLR1/TLR2 and TLR4 although at a lesser efficiency, whilst

those with a conical configuration were not detected by either (Figure

1.5)(Netea et al., 2002; Schromm et al., 1998).

Figure 1.4: Differences in Three Dimensional Shape of LPS from Different Sources

The lipid A portion of E. Coli adopts a conical form, whilst that of P. Gingivalis has an intermediate form, and the artificially created lipid A precursor 1A is cylindrical (Netea et al., 2002).


29

Figure 1.5: Hypothesis linking Lipid A configuration to Cytokine Output

Lipid A in a conical configuration such as that from E. coli induces a strong proinflammatory response via TLR4, whilst those with a less conical form such as P. gingivalis bring about a smaller proinflammatory response via TLR2. Those with a cylindrical configuration activate neither, inducing minimal or no response.

As there is a smaller adaptive immune response to endotoxin as well as

the larger innate, response the number of times a particular strain of

bacterial endotoxin comes into contact with the immune system is also a

factor. Antibodies are used to opsinise LPS for disposal by phagocytosis,

and as the adaptive immune system comes into contact with specific

strains of LPS it can more efficiently remove them in the future (Chaby,

1999).

If the endotoxins eliciting the immune response are part of a bacterial

infection, the defence mechanisms of these invaders can play a part in

how well the body can respond, for example the E. coli strain CFT073 has

been found to release TIR domain containing–proteins which are taken in

by macrophages in vitro then disrupt TLR4 signaling via the MyD88

signaling pathway (Cirl et al., 2008).

The site of endotoxin exposure is also important factor in the immune

response. Membrane attack complex (MAC) proteins are a response to

bacterial invasion which destroy the invading organisms, releasing more

endotoxins from their cell membranes. It was reported that the production

of MAC proteins was reduced in lung tissues to reduce endotoxin exposure

to the lung cells (Bolger et al., 2007). It has also been reported that the


30

endotoxin induced response by lung wall epithelial cells produces a greater

ratio of IL-10 to IL-12 when compared to similarly stimulated blood borne

responses (Wan et al., 2000). This would suggest an effort from the

lungs, which are exposed to endotoxin more regularly, to reduce harmful

inflammation responses and promote the opsonisation of pathogens for

disposal by phagocytosis (Bolger et al., 2007).

The subjects sex has an effect on the inflammatory response to

endotoxin. It has been observed that females have a resistance to the

effects of acute inflammation, whilst their prognosis in more long term

inflammatory disorders is poorer (Lefévre et al., 2012).

In PARP-1 inhibited mice males responded less to endotoxin induced

inflammation when compared to WT animals (measured by TNFα output),

however the TNFα output of female WT mice began at a lower level and

the inhibition of PARP-1 did not alter this output. In order to identify if this

was due to the low output of TNFα in female mice further groups of WT

and PARP-1 inhibited mice were treated with a more robust amount of

LPS. Again no significant difference was observed between normal and

PARP-1 inhibited serum TNFα concentrations (Mabley et al., 2005b).

There are two proposed reasons for this sex difference in inflammation,

firstly that it is to do with hormones specifically oestrogen. Treatment of

LPS treated tissues with oestrogen has been shown to reduce the

production of MCP-1 from the brain, arteries and macrophages, IL-6 from

the arteries and MIP2-α from the brain when compared to those treated

with LPS alone (Nilsson, 2007). More recently it has also been described

that X chromosome linked genes such as CD99, which plays a role in

leukocyte diapedesis, may also contribute to the noted sex difference

(Lefévre et al., 2012).


31

Other genetic factors also have a role in determining the magnitude of the

endotoxin induced inflammatory response. It was shown that both

C3H/HeJ and C57BL/10ScCr mice which exhibit endotoxic

hyporesponsiveness both had mutations in the TLR4 gene (Qureshi et al.,

1999). It was later reported that similar mutations in the human TLR4

gene also contribute to a lessened inflammatory response to endotoxin

(Arbour et al., 2000). A host of gene deletions have also been recognised

to effect endotoxin induced inflammation including signal transducer and

activator of transcription 3 and Cyclooxygenase 1 and 2 (Kano et al.,

2003; Martin et al., 2006).

1.5. BER Proteins and Endotoxin Induced Inflammation

Proteins not initially thought to be involved in modulating the

inflammatory response have been discovered to have such a function.

These include certain proteins linked with base excision repair. PARP1, a

DNA damage sensor for single and double stranded breaks, was the first

BER protein to be implicated in the inflammatory response. This was

discovered through the use of PARP1-/- knockout mice which showed a

marked decrease in inflammation induced by caecal ligation and puncture,

which results in the extrusion of gut content into the peritoneal cavity,

when compared to wildtype mice. There was also a significant reduction in

TNF-α (1059 ± 166 pg/ml vs. 1529 ± 122 pg/ml), IL-6 (approx. 28 ± 2

ng/ml vs. 40 ± 4 ng/ml) and IL-10 (4750 ± 2274 pg/ml vs. 6875 ± 2076

pg/ml) plasma concentrations when compared with wildtype mice 24 h

after caecal ligation. Similarly MPO activity was also reduced in PARP1-/-

gut (approx. 3.2 ± 0.5 mU/m protein vs. 5.8 ± 0.4 mU/mg protein) and

lung (approx. 180 ± 15 mU/m protein vs. 370 ± 30 mU/mg protein)

tissues after 24 h. Survival rates of PARP1-/- mice after caecal ligation were

also significantly higher compared to PARP+/+ (20% vs. 4% survival)

(Soriano et al., 2002; Virag and Szabo 2002).


32

This is probably due to the regulatory control PARP1 has on certain

proteins, including iNOS. PARP1 also has been found to be a co-factor

with NF-κB and AP-1, key inflammation transcription factors (Kiefmann et

al., 2004). A suggested model for the role of PARP1 has been described by

Virag and Szabo. The invasion of microbial particles activates neutrophils

in the local area, leading to the release of ROS from these monocytes in

order to damage, and kill the invaders. ROS release leads to DNA damage

and causes the activation of PARP1, which in turn triggers chemokine and

inflammatory cytokine production via NF-κB and AP-1 leading to the

further recruitment of neutrophils to the area of invasion (Virag and Szabo

2002).

APE1 is involved in redox reactions throughout the cell and is important in

the regulation of many systems. Indeed APE1-/- mouse foetuses are

unable to develop beyond implantation (Xanthoudakis et al., 1996).

Expression of APE1 is activated by the presence of ROS (Yang et al.,

2007). APE1 activates transcription factors NF-κΒ and AP-1 by the

reduction of active cystine residues (Ando et al., 2008). Indeed the down

regulation of APE1, via antisense cDNA and siRNA, attenuates NF-κΒ and

AP-1 activation (Daily et al., 2001; Fung and Demple 2005). Therefore it

can be hypothesised that a reduction in APE1 activity within the cell would

reduce cytokine and chemokine output from leukocytes, reducing the

inflammation reaction in a similar fashion to that of PARP1.

1.6. DNA Glycosylases and Endotoxin Induced Inflammation More specific base excision repair proteins, such as DNA glycosylases

which remove modified DNA bases, have also been shown to alter the

inflammatory response. It has been reported that 8-Oxoguanine

glycosylase-/- (OGG1-/-) mice are resistant to the damage caused by

endotoxin induced inflammation, indicating that this protein may also have

a role in the regulation of inflammation (Mabley et al., 2005a). When

given intraperitoneal injections of LPS (80 mg/kg) OGG1-/- mice showed a


33

significant decrease in the output of the chemokine MIP1-α (~60%

decrease), and the cytokines TNFα (~31%) and IL-12 (~50%) when

compared to WT controls, but a large increase in the Th2 cytokines IL-4

(~4.7 fold) and IL-10 (~2.4 fold). Th2 cytokines reduce IL-12 production

and also signal for an increase in antibody production. Additionally, the

MPO activity measured in lung (~60%) and heart (~50%) tissues was

significantly lower in OGG1-/- mice than their OGG+/+ counterparts: no

difference was observed, however, in the liver, kidney or gut. Finally MDA

content was significantly lower in the lung (~58%), heart (~55%) and

liver (~72%) tissues of LPS treated OGG1-/- mice than those of OGG1+/+

mice, whilst there was no difference observed in kidney or gut (Mabley et

al., 2005a). A comparison of OGG1 and PARP1 responses to LPS induced

inflammation are found in Figure 1.6.

Figure 1.6: Comparison of Changes to Cytokine Production after LPS

Stimulation in PARP1 and OGG1 Knockout Mice

The macrophage is activated when LBP binds with LPS and is presented to TLR4 creating a inflammatory signal cascade via MAL and MyD88 and ultimately nuclear factors such as NF-κΒ and AP-1. The down regulation of APE1 down regulates the activity of these nuclear factors as does the knockout of PARP-1 which has been shown to have an attenuating effect on IL-6, IL-10 and TNFα production. OGG1-/- mice have been shown to be protected against the negative effects of LPS induced inflammation, the probable mechanism of these differences is unknown but an increase in IL-4 and IL-10 production were observed whilst IL-12, MIP-1α and TNFα were reduced.


34

It has also been reported that the inflammatory response to Helicobacter

pylori, a gram negative bacterium, was reduced in OGG1 deficient mice.

OGG1-/- mice displayed less gut inflammation and histological lesions

compared to WT mice, as well as a lowered tendancy to recruit

polymorphonuclear cells (neutrophils, basophils and eosinophils) to the

gut tissue (OGG1-/- 33% vs. OGG1+/+ 100%). In these mice a reduction in

the mRNA expression of iNOS was also observed (Touati et al., 2006).

These results then suggest that OGG1 has a role in the expression of pro-

inflammatory proteins in response to Gram-negative bacteria (Mabley et

al., 2005a; Touati et al., 2006). In contrast it has been reported, that

when exposed to diesel exhaust particles (DEP; inhaled 20 mg/m3) the

difference between the macrophage and neutrophil population in BAL fluid

between wildtype and OGG1-/- mice was negligible, although there was a

~58% reduction in IL-6 mRNA production in OGG1-/- lung tissue when

compared to that of OGG1+/+ (Risom et al., 2007).

1.7. Oxidative Damage

The number of neutrophils and macrophages at the site of injury during

inflammation increase dramatically. When these cells die they are subject

to necrotic lysis and release reactive oxygen species into the area. The

original purpose of these molecules was bactericidal, but they are also

damaging to the host tissues and can damage anything they come into

contact with (Smith, 1994). This includes DNA which has its own repair

systems in place, the one most associated with oxidised bases is base

exscision repair (Slupphaug et al., 2003).

1.7.1. Reactive Oxygen Species

DNA damage is classified as any alteration to DNA structure or chemistry.

The agents that react with DNA to cause this damage come from two

sources, exogenous sources (i.e outside of the body) including ionizing

radiation, solar radiation and environmental carcinogens such as tobacco


35

smoke, and endogenous sources (i.e. inside the body) such as the by

products of aerobic respiration (Droge, 2002). Perhaps the most important

of these is oxidative stress due to both the metabolism of xenobiotic

compounds and the ubiquitous nature of endogenous ROS (Hazra et al.,

2002a).

Whilst each of us is dependent on oxygen for respiration, the reduction of

oxygen in normal aerobic respiration causes ROS to be formed as energy

is produced by the electron transport chain in the inner mitochondrial

membrane (Figure 1.7) (Turrens, 2003). Additionally, as previously

mentioned, ROS are also released in mammalian cells as part of the

inflammatory response (Beutler, 2004).

There are many methods of reducing the amount of ROS within the cell,

for example the enzymes catalase, superoxide dismutase and the

glutathione S-transferase family of enzymes, as well as reducing agents

like vitamins A, C and E (Oakley, 2005; Slupphaug et al., 2003).

Figure 1.7: Production of Superoxide Radical and Hydrogen Peroxide by the

Electron Transport Chain Complexes I, II and III of the electron transport chain can leak electrons creating superoxide radical (O-

2). This can then aid in the production of hydrogen peroxide which can also be leaked into the cytosol of the cell (Turrens, 2003).


36

An overview of the main steps of generating and reducing ROS are found

in Table 1.1. The formation of hydrogen peroxide (H2O2) is catalysed by

iron (Figure 1.8), and to a lesser extent copper and nickel, into superoxide

radicals via the Fenton reaction (Imlay et al., 1988; Lloyd and Phillips

1999). As a result there are defence mechanisms within the cell that

sequester Fe2+ ions to protect the the cell from superoxide damage

(Tenopoulou et al., 2005).

Some ROS such as the superoxide radical (O2-), and H2O2 are also key

secondary messengers, important to the function of the cell and thus a

balance must be struck between reducing oxidative damage, yet allowing

cellular signalling to continue (Dalton et al., 2000). If the levels of these

damaging molecules overwhelm the protections within the cell it can result

in various types of damage including base damage and single- and

double-strand breaks.

Table 1.1: The generation of major ROS and major avenues of protectiona

Generation of ROS

H2O → H2O+ + e-

H2O+ + H2O → OH● + H3O

OH● + OH● → H2O2

eaq + O2 → O2●-

2O2●- + 2H+ → O2 + H2O2

Protection against ROS

2O2●- + 2H+ → O2 + H2O2 – catalysed by superoxide

dismutase

2H2O2 → 2H2O + O2 – catalysed by catalase

aAdapted from Slupphaug et al., 2003


37

H2O2 + Fe2+ → Fe3- + OH● + OH-

Figure 1.8: The Fenton Reaction

H2O2 is formed by endogenous metabolism (Table 1.1) and is catalysed by Fe2+, Cu2+ and Ni2+ to generate reactive oxygen species capable of damaging DNA.

DNA structural alterations that include the hydrolysis of the base to leave

apurinic/apyrimidinic sites (AP sites), the formation of single-strand breaks

and the addition of oxygen or alkyl groups can be termed minor damage

as they do not halt the transcription process. This damage, however, if left

unrepaired, can result in base mis-pairing during DNA synthesis leading to

mutations, carcinogenesis and cell death. This damage is ongoing with in

the cell and it has been calculated that DNA sustains approximately 800

alterations per hour (Vilenchik and Knudson, Jr. 2000).

Damage which if unrepaired can halt the transcription process can be

termed major damage, and may cause apoptosis and premature aging.

These types of damage include double-strand breaks, that are lethal to

the cell if unrepaired and single-strand breaks which are converted to

double-strand breaks during the process of DNA replication (Slupphaug et

al., 2003).

Base lesions are chemically altered forms of the four DNA bases (adenine,

thymine, guanine and cytosine) produced by reactive oxygen species. If

the base lesion halts transcription by blocking the progress of RNA

polymerase II, then this a potentially lethal lesion (Wallace, 2002).

Oxidative Guanine Damage

1.7.2. Oxidative Guanine Damage

Guanine is the most sensitive base target for ROS in DNA. The majority of

the lesions formed are 7,8-dihydro-8-oxoguanine (8-oxoG) and 6-diamino-

4-hydroxy-5-formamidoguanine (FapyG) (Bruskov et al., 2002; Fromme


38

and Verdine 2004). Both are created when ROS react with carbon position

8, forming a double-bond by reducing or oxidising the bond with the

adjacent nitrogen to a single-bond (Figure 1.9). Whilst 8-oxoG does not

block DNA replication the resulting lesion has a high potential to be

mutagenic as it can mispair with adenine (see Figure 1.10) and if left

unrepaired will cause a GC → TA transversion following DNA replication

leading to mutation (Fromme et al., 2004). As 8-OxoG is such a common

lesion many studies use it as a marker of DNA damage (De Bont and van

Larebeke 2004). FapyG in contrast can halt the replication process and is

potentially mutagenic (Krishnamurthy et al., 2008; Wallace, 2002).

Figure 1.9: Formation of Oxidised Guanine Products

The reaction of ROS with Guanosine produces the unstable radical intermediate that can

either be oxidized to 8-OxoGua or reduced to FapyG (adapted from (Kalam et al., 2006)).


39

Figure 1.10: 8-oxoGua paired with cytosine and mispaired with adenine

adapted from Fromme et al., 2004

1.7.3. Oxidative Adenine Damage

Similar to the oxidative products of guanine adenine is vunerable to

oxidation at carbon position 8, resulting in 7,8-dihydro-8-oxoadenine (8-

oxoA) and 4,6-diamino-5-formamidopyrimidine (FapyA) (Figure 1.11;

(Kalam et al., 2006)). 8-oxoA does not block the replication process but it

can pair with either thymine or guanine, which can result in AT → CG

transversions but this happens comparatively rarely and upon most

occasions it will pair with thymine. FapyA can halt the replication of DNA

(Girard et al., 1998; Wallace, 2002).

Figure 1.11: Formation of Oxidised Adenine Products.

ROS react with adenine to produce 8-oxoA or FapyA


40

1.7.4. Oxidative Thymine Damage

The major stable product of thymine oxidation in vitro is 5,6-dihydroxy-

5,6-dihydrothymine or thymine glycol (Tg). It is created in two forms but

the cis isomer is produced more often (Figure 1.12; (Brown et al., 2008)).

It is formed when the double-bond between the fifth and sixth carbon is

broken by ROS and an OH group is added to each carbon (Hazra et al.,

2002b). Tg causes significant extrahelical distortions in a double DNA

strand, and can block the progress of repair or replicative DNA

polymerases along the DNA strand, and when left unrepaired this has

been found to be lethal in vivo (Aller et al., 2007).

It has been demonstrated that oxidation of thymine can lead to TA→CG

transversions in vivo. Thymine glycol can be bypassed by DNA polymerase

I in vitro and in vivo, and the efficiency of this bypass depends on whether

the cis or trans isoform is created (Hayes and LeClerc 1986). This

suggests that the cis form may be considered more mutagenic to the cell,

and that mutagenesis is preferred to apoptosis (Wallace, 2002).

Figure 1.12: Formation of Oxidised Thymine Products When thymine reacts with ROS both isomers of thymine glycol are created, although the cis version is more common (adapted (Miller et al., 2004)).


41

1.7.5. Oxidative Cytosine Damage

Cytosine can form an oxidation product similar to thymine, cytosine glycol

(Cg). However, Cg is unstable and will swiftly degrad in vivo to either 5-

hydroxycytosine (5-OHC) (by dehydration) or uracil glycol (by

deamination) which will further be dehydrated to 5-hydroxyuracil (5-OHU)

(Figure 1.13; (Tremblay et al., 2007)). 5-OHU will preferentially pair with

adenine leading to CG → TA transitions. In vitro it has been shown that 5-

OHC has a chance of mispairing with cytosine resulting in CG → GC

transversions (Purmal et al., 1994).

Figure 1.13: Formation of Oxidised Cytosine Products

The reaction of ROS with cytosine leads to cytosine glycol, which in turn can be dehydrated to 5-hydroxycytosine, deaminated to uracil glycol or deaminated and dehydrated to 5-hydroxyuracil (adapted (Tremblay et al., 2007)).

1.7.6. Spontaneous Base Removal Spontaneous depurination occurs when a purine is excised from the

deoxyribose sugar by hydrolysis of the N-glycosidic link by ROS. It is

estimated that 5000 purine bases are removed per cell, per day from DNA

due to this process. The same process occurs with pyridimines but at a

much lower rate (Maynard et al., 2009). In double stranded DNA


42

Apurinic/apyrimidinic sites (AP sites) are efficiently repaired by BER, but in

single strand DNA the missing base is replaced at random potentially

leading to transitions and transversions. AP sites have also been shown to

be cytotoxic as they block the activities of replicative DNA polymerases

(Maynard et al., 2009).

Figure 1.14: Formation of an AP site.

The guanine is removed from the sugar phosphate backbone of the DNA strand by hydrolysis, leaving an AP site. Adapted from (Sheppard et al., 2000).

1.7.7. DNA strand breaks Single-strand breaks (SSB) are caused by oxidative damage to the carbon

bonds on the sugar phosphate backbone of the DNA strand. When these

breaks are found opposite each other double-stranded breaks (DSB) are

formed. DSBs are also formed when the replication fork encounters

blocking lesions, including those formed by ROS, leading a strand break

(Shrivastav et al., 2008)

Single strand breaks can be repaired by BER, but DSBs and SSBs in single

stranded DNA are considered the most severe type of DNA damage

(Shrivastav et al., 2008). Failure to repair, or misrepair of DSBs can lead

to deletions, translocations, and chromosome fusions that enhance

genome instability. These breaks can lead to mutations when breaks are


43

incorrectly repaired, and trigger apoptosis in order to prevent the

propagation of mutant or cancerous cells (Shrivastav et al., 2008).

1.7.8. Lipid peroxidation Lipids are readily oxidised by ROS, creating unstable molecules which after

a series of reactions form lipid peroxides (Figure 1.15). These are a well

established cause of cellular damage including that of DNA (Marnett,

1999).

Polyunsaturated fatty acid peroxides decompose to produce α,β-

unsaturated aldehydes including malondialdehyde (MDA) and 4-

hydroxyalkenals (HAE) which react with DNA to form DNA adducts which

are used as markers of lipid peroxidation (Marnett, 1999).

Figure 1.15: Lipid Peroxidation

A lipid peroxide is formed in three stages. During the initiation stage a free radical interacts with a hydrogen atom to form a lipid radical and water. In the propagation stage the unstable lipid radicals interact with other lipids at which point their radical interacts with a molecular oxygen to create a different fatty acid and a lipid peroxide. The process terminates when the radical interacts with another radical to produce a non-radical. Adapted from (Imlay, 2003).


44

Figure 1.16: Composition of malondialdehyde and 4-hydrooxyalkenal

1.8. DNA Repair

Damage to bases which cause a disruption of DNA replication and RNA

creation and/or RNA synthesis may result in cell death or base mis-pairing

(mutation) (Michell et al., 2003). But as there are so many DNA damage

causing agents within the cell it is important that mechanisms are in place

to repair any damage that occurs. There are over 130 known genes

involved in DNA repair and many of these are shared common co-factors

from processes such as cell cycle regulation, transcription and DNA

replication including Proliferating Cell Nuclear Antigen (PCNA) and

replication protein A (RPA) (Slupphaug et al., 2003; Wood et al., 2001).

Several DNA repair mechanisms have been identified including those that

use the undamaged strand of the DNA molecule as a template for repair.

These cut and patch type mechanisms include BER (Slupphaug et al.,

2003), nucleotide excision repair (NER) (Mitchell et al., 2003) and

mismatch repair (MMR) (Golyasnaya and Tsvetkova 2006). These

pathways vary widely in the type of damage removed and the size of the

excision made.

The importance of DNA repair systems is illustrated by the variety of

diseases which are associated with their disruption. The first such disease

was identified in 1968 when Cleaver established a link between NER and

the skin disease xeroderma pigmentosum (Cleaver, 1968). Since this

discovery connections have been formed between a lack of DNA repair

and other diseases. Cockayne’s syndrome, characterised by a sensitivity to


45

light and the appearance of premature aging, and trichothiodystrophy,

which causes mental and physical retardation, are also associated with

NER (Lehmann, 2003) and Hereditary nonpolyposis colorectal cancer is

associated with defects in MMR (Abdel-Rahman et al., 2005). Until 2003 it

was thought that base excision repair was not linked to any inherited

disease due to the large overlap in the function of BER proteins with other

mechanisms (Krokan et al., 2000; Slupphaug et al., 2003). However, links

between MUTYH and an autosomal recessive syndrome of adenomatous

colorectal polyposis and very high colorectal cancer risk have been

observed (Cheadle and Sampson 2003). Additionally, the single-strand

breaks that are created as intermediates in the BER process have been

implicated in the progression of neurodegenerative diseases such as

spinocerebellar ataxia (Caldecott, 2003). Links have also been made

between cancer (e.g. lung, skin, leukaemia and breast) and

polymorphisms in BER proteins although these tend to be weak, such as

that for OGG1 which has shown no consistant conections with any form of

cancer (Hung et al., 2005). Polβ, however, has shown a strong connection

with cancers, as ~30% of human tumours express Polβ variants (Starcevic

et al., 2004).

1.8.1. Base Excision Repair

BER is the main mechanism used to remove non-helix distorting DNA base

lesions (Slupphaug et al., 2003). However BER can also repair the

consequences of base deamination, spontaneous hydrolysis of the N-

glycosidic bond and SSBs (Fromme and Verdine 2004). Repair is initiated

by one of a group of enzymes named DNA glycosylases, which remove

damaged bases from DNA leaving the sugar phosphate backbone intact

(Hazra et al., 2002a). Many different DNA glycosylases have been

identified, and as a result there are several methods of excising damaged

bases and repairing the subsequent AP site. These can be split into three

major mechanisms of action: (i) monofunctional glycosylase APE1

dependent pathway, (ii) bifunctional glycosylase APE1 dependent pathway


46

and the third pathway is the (iii) APE1 independent pathway and is

initiated by the NEIL1 and NEIL2 proteins (Figure 1.17) (Hazra et al.,

2007).

There are two forms of BER, namely short- and long-patch repair. Short

patch repair results in the removal of only a single damaged base whilst

long patch repair as its name suggests repairs a longer section of DNA (2-

15 nucleotides) and recruits many proteins active in DNA synthesis

(Robertson et al., 2009).

Figure 1.17: Monofunctional DNA glycosylase BER (I), bifunctional APE1 dependant BER (II) and independent (III) pathways

* represents oxidised base. Adapted from Hazra et al., 2007.

All DNA glycosylases begin the reaction in a common manner (see Figure

1.17). The damaged base is located and the DNA is bent around the DNA

glycosylase protein. The base is drawn into the active site of the protein

where it is excised by a nucleophilic amino acid at the active site resulting

in the formation of an AP site (Stivers and Jiang 2003). Monofunctional

DNA glycosylases, such as Single-strand selective Monofunctional Uracil


47

DNA Glycosylase (SMUG1), then require APE1 to cleave the DNA strand 5′

to the AP site leaving a 5′-deoxyribose-5-phosphate (5′dRP) and 3′-OH

(Christmann et al., 2003). This configuration blocks normal DNA

polymerase activity. Poly (ADP-Ribose) Polymerase-1 (PARP1) then

mediates the attachment of DNA polymerase β (Pol β) to the DNA strand,

which adds a single nucleotide to the 5' end of the nick. The dRP lyase

activity of Pol β removes the sugar phosphate moiety and the process is

completed by either DNA Ligases I or III, which repairs the sugar

phosphate backbone of the DNA.

DNA glycosylases such as OGG1 and NTH1 act like monofunctional

glycosylases but also have an AP lyase activity that incises the AP site via

β-elimination, thus the term bifunctional DNA glycosylases. This process

leaves a 5′-dRP and a 3′-phospho-α,β-unsaturated aldehyde (PUA). This is

a substrate for APE1 which cleaves the 3' PUA leaving a 3′-OH group

(Figure 1.17) (Dianov et al., 2003; Izumi et al., 2003). Pol β and DNA

ligase III/XRCC1 can seal the nick and the repair process is complete

(Dianov et al., 2003).

The APE independent pathway is proposed to be used by bifunctional DNA

glycosylases such as NEIL1 and 2. This takes advantage of the 5' and 3'

phosphates produced during β,δ-elimination to utilise polynucleotide

kinase (PNK) instead of APE1, to generate the 3′ OH group (Figure 1.17)

(Hazra et al., 2007).

1.8.2. DNA Glycosylases

The major DNA glycosylases specialised for removal of oxidised base

lesions in BER can be split into three families. First are monofunctional

glycosylases such as; N-methylpurine-DNA glycosylase (MPG) which

removes alkylated bases from DNA, Uracil-DNA glycosylase 2 (UNG2) and

SMUG1 which remove uracil residues from DNA. Second are bifunctional


48

DNA glycosylases, such as the mammalian DNA glycosylases OGG1 and

NTH1 (Dou et al., 2003), that share features and methods of reaction with

the Escherichia coli gene nth. Proteins of this family contain a helix-

hairpin-helix motif and have an internal Lys residue that acts as an active

site, which carries out β-elimination cleaving the DNA strand 3' to the

oxidised base and leaving 5'-phosphate and 3' PUA termini (Hazra et al.,

2002a). The third major class of DNA glycosylases share homology with

the E. coli Nei (endonuclease VIII) including helix-2turn-helix motifs and a

catalytic pro2 residue, and are described as NEI Like enzymes 1, 2 and 3

(NEIL1, NEIL2 and NEIL3).

Table 1.2: Human DNA glycosylases located in the nuclei and the DNA damage

that they removea

FaPyG, 2,6-diamino-4-hydroxy-5-formamidopyrimidine; 8-oxoG, 7,8-dihydro-8-oxoguanine; 5-OHC, 5-hydroxycytosine; Tg, thymine glycol; 5-FoU, 5-formyluracil; 5,6-DHU, 5,6-dihydrouracil; FaPyA, 4,6-diamino-5-formamidopyrimidine; 5-OHU, 5-hydroxyuracil; 5-HMU, 5-hydroxymethyluracil; 5-FU, 5-fluorouracil; 2-OH-A, 2-hydroxyadenine; ss, single-stranded. aadapted from Slupphaug et al., 2003

Name Lyase Activity Known Substrates

α-OGG1 Yes FaPyG:C > 8-oxoG:C > 8-oxoG:T, 8-oxoA:T

NTH1 Yes 5-OHC, Tg:A > Tg:G, 5-OHU, 5,6-DHU,

FaPyG:A/G/T, 5-FoU

NEIL1 Yes FaPyA, FaPyG, 8-oxoG:C > 8-oxoG:G > 8-oxoG:T,

Tg, 5-OHC, 5,6-DHU

NEIL2 Yes 5-OHU, 5,6-DHU, 5-OHC NEIL3 Yes FaPyA,FaPyG

SMUG1 No ssU > U:G > ss5-HMU:G > U:A > 5-HMU:A > eC:G

> 5-FU:A

UNG-2 No ssU > U:G > U:A > 5-FU

MUTYH No A:G, A:8-oxoG > 2-OH-A:G >> C:A

MTH1 No Cpg:T, CpG:U

MPG No 3-methyladenine>Adenine residues

TDG No U:G, T:G

MBD4 No U:G, T:G


49

1.8.2.1. OGG1

OGG1 is the mammalian homologue of the MUTM gene in E. Coli (Arai et

al., 1997). The human DNA glycosylases α-OGG1 and β-OGG1 are

produced by alternative splicing of the same DNA transcript found on

chromosome 3p26. The full code for OGG1 consists of 8 exons, and α-

Ogg1 is a result of the transcription of exons 1-7 which results in the

formation of a protein of weight 39 KDa, whilst β-Ogg1 is formed when

exons 1-6 and 8 are transcribed and produce a protein 47 KDa in weight.

The structure indicates that OGG1 contains both a zinc finger and a helix-

hairpin-helix (HhH) DNA binding site (Arai et al., 1997).The OGG1 proteins

are found ubiquitously throughout the body in the nucleus and

mitochondrion. It is transcribed in greater amounts in thymus, testis,

kidneys, intestine and brain tissues (Radicella et al., 1997).

In its human form α-OGG1 is located in the nucleus and excises 8-OxoA

opposite T, 8-OxoG opposite C and T and both FapyG and meFapyG

opposite C (Table 1.2), whereas the mouse form only excises 8-oxoG

lesions opposite C and T (Audebert et al., 2000; Campalans et al., 2007;

Dherin et al., 1999). The structure of the hOGG1 protein suggests that it

makes contact with the base opposite 8-oxoG through hydrogen bonds to

hydrogen acceptors of the paired base. This may explain the different

levels of affinity it has for certain pairings (Banerjee and Verdine 2006).

Human β-OGG1 is located in the mitochondrial matrix, but its role in the

repair of mtDNA is unknown as it does not have any detectable DNA

glycosylase or AP lyase activity (Hashiguchi et al., 2004). Indeed it has

been suggested that while 8-oxoG glycosylase activity has been detected

in the mitochondrial matrix, this is the result of α-OGG1 activity in the

mitochondria rather than that of β-OGG1 (Bohr, 2002; Hashiguchi et al.,

2004). β-OGG1, however, is thought to play a role in the regulation of

apoptosis within the cell, as an over expression of the protein was shown

to have a protective effect when the cells were challenged with xanthine


50

oxidase induced ROS (Dobson et al., 2002; Ruchko et al., 2005). Further

studies have linked β-OGG1 with oxidant induced apoptosis, noting that its

disruption increases programmed cell death (Panduri et al., 2009).

Whilst Ogg1 expression is not affected by the cell cycle, it has been

proposed that Ogg1 expression can be induced by certain exogenous

signals such as oxidative stress (Risom et al., 2007). There are many

layers of regulation on the activity of OGG1 including product inhibition

(Sidorenko et al., 2008), reversible phosphorylation and nitric oxide (NO)

inactivation (Jaiswal et al., 2001). In the presence of APE1 the efficiency

of OGG1 can improve up to 5 fold (Hill et al., 2001) and APE1 may thus

have a role in facilitating product dissociation of OGG1. Phosphorylation

has also been shown to regulate OGG1 activity and cellular localisation.

The exact location of the phosphorylation site is yet to be confirmed, four

have been identified, but preliminary in silico analysis has identified

serine326 as a likely target for protein kinase A (Smart et al., 2006; Svilar

et al., 2011). The adjacent cysteine is able to form a thiolate anion that is

susceptible to oxidation. The secondary messenger NO has been shown to

disrupt the zinc finger motif through the process of thiol nitrosylation,

which causes the ejection of the zinc ion and irreversible loss of catalytic

activity (Jaiswal et al., 2001).

When there are mutations in the OGG1 gene, incidence of cancer and

other age related diseases thought to have links with DNA damage are not

clearly increased, but this thought to be due to the presence of other BER

proteins acting as backups, or that the mutation has no functional effect

(Osterod et al., 2001). When considering specific polymorphisms

alterations at codon 326 (Ser/Cys and Cys/Cys) are the ones most

documented. It has been shown in MEFs that these polymorphisms lead to

the accumulation of FaPyG within the cell (Bravard et al., 2009; Smart et

al., 2006). A higher risk of several cancers has been linked with this


51

polymorphism including lung (Kohno et al., 2006), gastric (Farinati et al.,

2008), prostate (Chen et al., 2003), and orolaryngeal (Elahi et al., 2002).

Conversely there have been several studies that have shown no links with

this polymorphism and the risk of specific cancers including breast cancer

(Vogel et al., 2003), colorectal (Hansen et al., 2005) and Stomach

(Hanaoka et al., 2001).

The mouse OGG1 gene is found on chromosome 6 and shares 83% amino

identity with the human α-OGG1, evidence for OGG1 activity has been

identified in mouse mitochondria. It has been suggested that mOGG1 is

distributed throughout the cell, to where it is needed via active transport

involving the cells microtubules (Conlon et al., 2004). Again OGG1 is found

ubiquitously in the mouse but is at its highest levels in tissue extracted

from the testes (Rosenquist et al., 1997).

In OGG1 knockout mice reported thus far no significant difference has

been observed in the viability, appearance or survival when compared to

wildtype mice. In the 12 month period that these mice were studied there

was no increased incidence of cancer in the mice (Klungland et al., 1999).

However, it has been reported, in a different strain of OGG1-/- mice, that

the incidence of lung cancer increased after 19 months of observation

(Sakumi et al., 2003) It was also reported that the accumulation of 8-oxoG

residues did increase when compared with wildtype mice. In nuclear DNA

there was a tissue specific increase of 10 – 100 % (testes and liver

respectively) in 8-oxoG residues. The removal of oxidised guanines from

the DNA was not halted completely though, and there was significantly

slower repair in the knockout line. In the mitochondria disruption of the

OGG1 gene can cause 8-oxoG to accumulate to 20 times the amount

found in normal cells it has been shown that this has no significant effect

on the respiratory function of the mitochondria (Stuart et al. 2004, de

Souza-Pinto et al., 2001).


52

1.8.2.2. NTH1

The mammalian homologue of E. coli nth, or endonuclease III, is NTH1.

The gene encoding the human form of this protein is found on

chromosome 16p13 and comprises 6 exons (Martin et al., 2004). The

NTH1 protein has a mass of 34 KDa and is expressed throughout the body

within the nucleus of cells, although it is more highly expressed in the

heart, liver and brain tissues, and least expressed in muscle tissue (Gros

et al., 2002; Hazra et al., 2002b; Ikeda et al., 2002). As well as the

conserved HhH DNA binding motif present in other nth like DNA

glycosylases, it also has a 4Fe-4S cluster loop motif which helps to locate

the enzyme more accurately onto the DNA molecule (Gros et al., 2002;

Izumi et al., 2003). The two iron-sulphur clusters form a pocket with the

catalytically active Lys120 and Asp138 at its mouth.

NTH1 expression is cell cycle dependant, with expression low during the

G0-G1 phases, but increased at the start of S-phase where it reached its

maximum level. This suggests a link between NTH1 expression and DNA

synthesis (Luna et al., 2000). Like OGG1, NTH1 is also substrate inhibited

and the addition of APE increases its efficiency at removing Tg from DNA

~3 fold (Marenstein et al., 2003). NTH1 activity has been shown to be

modulated by XPG, a structure-specific endonuclease involved in NER, and

P53, thus connecting BER with NER and p53-dependent damage response

pathways. Both of these interactions stimulate NTH1 activity; XPG at the

RNA transcription complex and p53 during DNA damage sensing (Oyama

et al., 2004). The transcription factor YB-1 has also been shown to

increase the rate of lyase activity in vitro (Marenstein et al., 2003). NTH1

was shown to bind to PCNA, but was not stimulated as a result of such

binding (Dou et al., 2008). However it has been theorised that the

interaction with PCNA, could play a coordinating role during S phase

(Oyama et al., 2004) and that PCNA in the replication machinery recruits


53

NTH1 to Tg lesion when the DNA replication machinery stalls (Oyama et

al., 2004).

As a bifunctional DNA glycosylase NTH1 can remove specific base lesions

via β-elimination (Dizdaroglu et al., 1999). The substrates that NTH1

removes most efficiently are the oxidised pyrimidines shown in Table 1.2.

Of these products thymine glycol (Tg) is toxic, unlike 8-oxoG which is

mutagenic. Tg halts the progress of replicative DNA polymerases and, if

not removed, can lead to cell death (Izumi et al., 2003).

The mouse NTH1 gene is located on chromosome 17 and comprises 6

exons. The resulting protein has a mass of 37 kDa and shares 84% amino

acid identity with hNTH1 and shares similar substrate specificity (Sarker et

al., 1998). Whilst human NTH1 protein is exclusively transported to the

nucleus, mouse NTH1 protein is transported to the mitochondria (Ikeda et

al., 2002).

NTH1-/- mice do not show any phenotypical difference to wildtype mice in

terms of viability, growth rate, age related differences and cancer risk

(Parsons and Elder 2003). In the genomic DNA of NTH1-/- mice Tg seemed

to be removed at normal rates suggesting a functional backup that repairs

oxidised pyridimines is involved. However, Tg was not observed to be

removed at all from mitochondrial extracts (Karahalil et al., 2003; Parsons

and Elder 2003). NTH1-/- cells showed no increased sensitivity to

menadione, H2O2 or X-ray treatments, and still removed Tg lesions,

although it was reported that they did so at a slower rate with substantial

amounts of BER intermediates detected after treatment when none were

detected in WT cells (Parsons and Elder 2003). In NTH1-/- mouse thymus,

removal of Tg was observed, interestingly this cleavage was greater

against Tg:G than Tg:A base pairs (Ocampo et al., 2002). However there

was no Tg excision activity observed in liver mitochondrial extracts from a

different strain of NTH1-/- mice (Karahalil et al., 2003).


54

1.8.2.3. NEIL1

The fact that NTH1 knockout mice appear phenotypically normal was

crucial in the discovery of the NEIL1 and NEIL2 proteins, as it was

discovered that in the absence of NTH1 other as yet unknown proteins

were repairing Tg (Hazra et al., 2002a). Of these proteins NEIL1 was

identified as a backup for NTH1, as the substrates it excises are similar

(Takao et al., 2002a).

The NEIL1 gene is located on chromosome 15 at location q24.2 and

comprises 10 exons, the subsequent protein has a mass of 44 KDa and is

present in both the nucleus and the mitochondria of the cell (Hazra et al.,

2002a; Vartanian et al., 2006; Zody et al., 2006).

As an APE independent enzyme (Figure 1.17), NEIL1 performs β,δ-

elimination. Whilst it contains a DNA binding motif (a helix-2turn-helix), it

does not contain the expected zinc finger DNA binding motif, although it

does contain a ‘zincless finger’ which has been proposed to fill a similar

role (Doublie et al., 2004). The suggested active site is a conserved Pro2

at the N-terminal (Figure 1.18) (Hazra et al., 2002a).

Figure 1.18: Sequence alignment of critical domains of NEIL1 andNEIL2 with

E. coli Nei andFpg adapted from Hazra et al., 2007

NEIL1 is a bi-functional DNA glycosylase/AP lyase protein that removes a

wide range of oxidised bases from DNA (Table 1.2) (Shinmura et al.,

2004; Takao et al., 2002a). It has been suggested that as NEIL1 can

excise the same substrates as OGG1 that it could be a functional back up.


55

However, it excises 8-oxoG from double stranded DNA at a much slower

rate than OGG1 (Hazra et al., 2002a; Morland et al., 2002).

Expression of human NEIL1 varies throughout the body with levels higher

in the pancreas, liver and thymus than the testis and muscle (Hazra et al.,

2002b). Significantly it is expressed 6-7 times more abundantly during the

S-phase of the cell cycle than during the G0-G1 phase. This contrasts with

that of other DNA glycosylases such as OGG1 whose expression is not

significantly affected by the cell cycle (Hazra et al., 2002a). This co-

ordinated change in expression and the wide range of lesions repaired,

suggest that NEIL1 is involved in the repair of damaged bases at the

replication fork (Takao et al., 2002a). This is further supported by the

work of Dou et al. who showed that NEIL1 (and NEIL2) can function as

DNA glycosylases in a bubble structure of DNA and on single- and double-

stranded DNA, and indeed the catalytic activity of NEIL1 is greater when

repairing single-stranded DNA (Dou et al., 2003).

FEN1, PCNA and CSB proteins have been shown to increase the activity of

NEIL1 in vitro (Dou et al., 2003; Hegde et al., 2008; Muftuoglu et al.,

2009). NEIL1 has also been shown to interact with DNA polβ and DNA

ligase IIIα. This suggests that the enzyme may have a role in DNA repair

co-ordination rather than being a less efficient backup for OGG1 (Dou et

al., 2003). Interestingly, whilst no direct interaction has been observed

between NEIL1 and OGG1, the presence of NEIL1 has been shown to

increase the efficiency of OGG1 in vitro, and is due to the NEIL1 protein

higher affinity for AP sites. It is also suggested that NEIL1 is acting as a

backup for APE1 to stimulate 8-oxoG repair (Hazra et al., 2007; Mokkapati

et al., 2004b). A study of various hNEIL1 polymorphisms as shown that a

reduction in the efficiency of the NEIL1 protein may be involved in the

pathogenesis of gastric cancers (Shinmura et al., 2004).


56

The mouse NEIL1 gene is located on chromosome 9 at location 9c and

comprises 9 exons. The subsequent protein has a mass of 44 KDa and is

present in the nucleus of the cell (Morland et al., 2002). Expression levels

for NEIL1 vary between mouse tissues with levels highest in the prostate

ovary brain and spleen. Again the expression of the mouse NEIL1 protein

is cell cycle dependant, with the greatest expression in S-phase (Hazra et

al., 2002a). When compared with WT controls, the mortality rate of Neil1-

deficient cell lines high in response to low doses of γ-irradiation (3 min in

a Gammacell-40 Cesium irradiator at a dose rate of 0.75 Gy/mim)

(Rosenquist et al., 2003).

Whilst NEIL1 knockout mice do not show any immediate effects on weight

and viability, Vartanian et al. (2006) reported that a high proportion of

mice within the colony had been found to exhibited symptoms similar to

the human metabolic syndrome. This occurred in all of the male knockout

mice in the study and a large proportion of the female mice, with the

males being much more seriously obese than the females after around 8

months. Additionally serum levels of leptin were siginificantly higher in

NEIL1-/- mice. It was suggested that the symptoms associated the with

metabolic syndrome may have been caused by a build up of DNA damage

in the mitochondria disrupting metabolic processes (Vartanian et al.,

2006). A further breeding pair from this colony was housed in a different

animal facility but the resulting offspring did not display any of the

symptoms previously documented (Chan et al., 2009). However more

recently the link between NEIL1 and obesity has been renewed due to

data showing a significantly greater susceptability to obesity and reduction

in voluntary exercise, and it is now thought that the disruption of the

NEIL1 protein is a predisposing factor to obesity (Sampath et al., 2011).

1.8.2.4. NEIL2

The NEIL2 gene is located on chromosome 8 at position p23.1, it contains

five exons and codes for a protein with a mass of 37 KDa and is located


57

mainly in the nucleus (Nusbaum et al., 2006). The protein sequence

indicates several conserved sequences that are also found in the NEIL1

protein including a catalytic proline terminal, the catalytic lysine residue,

the H2TH binding motif and additionally a zinc finger motif (Figure 1.18)

(Hazra et al., 2007). Like NEIL1, NEIL2 can perform APE independant β/δ

elimination and acts primarily on 5-OHU, as well as other oxidised

cytosines (see Table 1.2) (Dou et al., 2003; Hazra et al., 2002b).

NEIL2 mRNA is found ubiquitously throughout the body, but at different

levels depending on the tissues examined. High gene expression has been

reported in skeletal muscle and testis, and low expression levels in lung,

liver, spleen, thymus, prostate, and ovaries (Morland et al., 2002). The

tissue specificity of this enzyme seems to compensate for NEIL1 and NTH1

as the latter two enzymes are expressed less in muscle and testis,

whereas NEIL2 levels are higher in these two tissues. NEIL2 has a greater

affinity with the replication bubble structure of DNA than NEIL1, but unlike

NEIL1 its expression is not connected to that of the cell cycle. This

suggests that NEIL2 may be involved in global base excision repair, but it

can be inferred that it is connected with DNA replication and transcription

due to its ability to bind with single- and double-stranded DNA (Dou et al.,

2003; Hazra et al., 2002b; Takao et al., 2002b).

NEIL2 activity can be increased by interactions with YB-1, or decreased by

interacting with pyridoxal-5′-phosphate (Das et al., 2007b; Grin et al.,

2010). NEIL2 has also been shown to stably interact with the p300

transcriptional cofactor which acetylates Lys sites of proteins. The

acetylation of Lys49 of NEIL2 is the most effective target which leads to

inhibition of DNA glycosylase and AP lyase activity. Acetylation of other

Lys residues does not produce an inhibitory effect, indicating that

reversible modification of Lys49 could regulate the activity of NEIL2

(Bhakat et al., 2004).


58

The mouse NEIL2 gene is located on chromosome 14 D1. It contains five

exons and codes for a protein with a mass of 37 KDa and is located mainly

in the nucleus (Church et al., 2009). To date no NEIL2-/- mice have been

reported.


59

1.9. Hypothesis “If DNA glcosylases, specifically NEIL1, NEIL2, OGG1 and NTH1, are

involved in the regulation of endotoxin induced inflammation and then the

knockout of these genes, in murine models, will result in a reduction in the

inflammatory response to endotoxin.”

In order to test this hypothesis the following objectives will be carried out:

I. The molecular characterisation of new NEIL1-/- and NEIL2-/-

knockout mouse strains.

II. The identification of any phenotypes in the gross physiology of

NEIL1-/- and NEIL2-/- knockout mouse strains.

III. The maintainance of colonies of NEIL1-/-, OGG1-/- and NEIL2-/- mice

(Chapter 3).

IV. Creating murine embryonic fibroblasts (MEFs) from WT, OGG1-/-

NEIL1-/- and NEIL2-/- mouse strains.

V. Using the resulting MEF strains in conjunction with pre-existing MEF

cultures (NTH1-/- and [OGG1/NTH1] -/-) as models of inflammation

to identify the magnitude and the nature of this reduction in

inflammatory response due to the disruption of base excision repair

enzymes by measuring the levels of IL-6, IL-10 and MIP-1α

released from the MEFs after exposure to LPS (Chapter 4).

VI. Identifying the extent to which the knockout of NEIL1 and OGG1

reduces the immune response to LPS induced inflammation in

whole animal models by:-

a. Establishing whether levels of cytokines IL-6, IL-12, IL-10

and IL-4 differ between NEIL1-/-, OGG1-/- and WT mouse

serum, at basal and LPS treated levels.


60

b. Establishing whether levels of MPO, MDA and GSH are

altered between NEIL1-/-, OGG1-/- and WT mouse tissues, at

basal and LPS treated levels (Chapters 5&6).


61

2. Materials and Methods

2.1. Materials

2.1.1. Buffer composition

The compositions of the various buffers and solutions used are contained

in Tables 2.1 and 2.2. All buffers were made in double distilled water

(ddH2O).

Table 2.1: Molecular Biology Buffers

Use Name Compostion

DNA electrophoresis

5 x TBE 0.45 M Tris-HCL pH 8.2, 0.45 M boric acid, 7.9nM

EDTA. 6 x

loading buffer

3.6 mM bromophenol blue, 1.2 M sucrose, 120mM EDTA.

Agarose gel

0.8 - 1.5% (w/v) agarose in 0.5 TBE, 0.8mM ethidium bromide.

Protein extraction

RIPA buffer

150 mM NaCl, 50 mM Tris-HCl pH 8.0, 12mM sodium deoxycholate, 0.1% (w/v) SDS, 1% (v/v) Triton x-

100. Cell lysis buffer

50mM Tris-HCl pH 8.0, 200mM NaCl, 1 mM EDTA, 1 mM DTT, 1/1,000 (v/v) protease inhibitor cocktail.

Protein electrophoresis

Loading gel

17% (v/v) Protogel, 125mM Tris-HCl pH 6.8, 0.1% (w/v) SDS, 2mM ammonium persulphate, 0.85mM N,N,N,N-tetramethyl-ethylenediamine (TEMED).

Resolving gel

40% (v/v) Protogel, 375mM Tris-HCl pH 8.8, 0.1% (w/v) SDS, 2mM ammonium persulphate, 0.85mM

TEMED. 5 x SDS loading buffer

0.3 M Tris-HCl pH 6.8, 0.25 M DTT, 20% (v/v) glycerol, 10% (w/v) SDS, 0.05% (w/v) bromophenol

blue. 10 x SDS running buffer

0.25 M Tris Base, 1.92 M glycine, 1% w/v SDS.

Western blot

Transfer buffer

25 mM Tris Base, 0.192 M Glycine, 20% (v/v) methanol.

10 x TBS 0.5 M Tris-HCl pH 7.5, 1.5 M NaCl.

TBS(T) 1 x TBS, 0.1% (v/v) Tween-20

Blocking solution

5% (w/v) skimmed milk powder (Marvel) in TBS(T).

Coomassie Blue stain

0.25% (w/v) Coomassie Blue R250, 40% (v/v) methanol, 10% glacial acetic acid.

Gel destain

20% (v/v) methanol, 5% (v/v) glacial acetic acid.


62

Table 2.2: Protein Analysis Buffers

Use Name Composition

IL-6 ELISA Carbonate buffer

0.2 M sodium carbonate, 0.2 M sodium bicarbonate pH 9.6.

Amplex Red mastermix 25 µl of Amplex Red (25 mg/ml), 5.7 µl

H2O2, ddH2O to 10 ml.

MPO assay MPO Working buffer

0.5% (w/v) hexadecyltrimethylammonium bromide, 10 mM 3-N-

morpholinopropanesulfonic acid.

MPO Detection buffer 1.6 mM 3,3’,5,5’-Tetramethylbenzidine, 1

mM H2O2.

MDA assay MDA Working buffer 5 mM Tris pH 7.4, 1.15% (w/v) KCl.

GSH assay

Potassium phosphate EDTA (KPE) buffer

solution A 1.36% (w/v) KH2PO4

KPE buffer solution B 1.7% (w/v) K2HPO4

KPE buffer 16% solution A, 84% solution B, 0.33%

EDTA pH 7.5.

2.1.2. Tissue culture media The compositions of the tissue culture media used are detailed in Table

2.3. All media was filter sterilised (0.22µm; Millipore, UK) before use.

Table 2.3: Tissue Culture Media

Name Composition

MEF Media A

Dulbecco's Modified Eagle Medium : F12 Nutrient mixture (DMEM:F12), 10% (v/v) heat-inactivated fetal calf serum (PAA Biotechnology, UK), 2 mM L-glutamine, 0.11 M NaHCO3, 1x Penicillin-streptomycin.

MEF Media B

Dulbecco's Modified Eagle Medium : F12 Nutrient mixture (DMEM:F12), 10% (v/v) heat-inactivated fetal calf serum, 2 mM L-glutamine, 0.11 M NaHCO3.

2.1.3. PCR primers All primers were obtained from MWG, Germany.


63

Table 2.4: PCR Genotyping Primers

Shaded areas indicate combinations of primers that were not used.

Target

Gene Name Position Sequence (5' - 3')

WT Size

(bp)

KO Size

(bp)

Annealing

Temp (oC)

NEIL1

1907f Exon 2 CAA CTA TCT GCG GGC AGA GAT 1108 57

3015r Intron between exons 5 and 6 GGA GAC AAC TCT GGA GTC AAC

807f Intron between exons 1 and 2 GTT TAA ACT TTC ACC ACA TTG ATG ACG TGC

GG 1226 57 nNEOr Within Neo-TK cassette GCA GCC TCT GTT CCA CAT ACA C

NEIL2

410f Intron between exons 1 and 2 CTT GGA CCA GAG ATA CTT CTC AG 553 57

963r Intron between exons 1 and 2 GGG TTA GTT AGA CTA CAG ACT

-871f Intron before exon 1 GCG AGT ACT CTC CCT TAT ACT G 1341 57

nNEOr Within Neo-TK cassette GCA GCC TCT GTT CCA CAT ACA C

OGG1

6333f Intron between exons 4 and 5 GTG GCT GAC TGC ATC TGC TT 323 58

6656r Intron between exons 5 and 6 GCA TAA GGT CCC CAC AGA TTC

2372f Exon 3 GCT TCC CAA ACC TCC ATG C ~1000 58

oNEOr Within Neo-Pgk cassette GCC GAA TAG CCT CTC CAC CCA AGC

NTH1

2956f Exon 4 TGA GCT GGT AGC CTT GCC AGG TG 597

60

3530f Intron between exons 4 and 5 GCA CAG TCA AGC ATG ATT ATA AG ~1800 60

ntNEOr Within Neo-Pgk cassette GCT CTG ATG CCG CCG TGT TCC G


64

Table 2.5: RT-PCR Primers

Target Gene

Name Position Sequence (5' - 3') Size (bp)

Annealing Temp

(oC)

NEIL1 328f Exon 1 CTT GCC CTT TGC TTC GTA GAC ATC

217 57 545r Exon 2 GGA TCT CTG CCC GCA GAT AGT T

GAPDH 534f Exon 4 GTT TAA ACT TTC ACC ACA TTG ATG ACG TGC GG

222 58 765r Exon 5 GCA GCC TCT GTT CCA CAT ACA C

TLR4 12926 Exon 2 CAG TGG GTC AAG GAA CAG AAG C

540 60 13443 Exon 2 GAC AAT GAA GAT GAT GCC AGA GC


65

2.1.4. Molecular biology reagents

All reagents obtained from Sigma-Aldrich except:-

- AMV reverse transcriptase (10 U/µl): Roche Applied Biosciences, UK.

- Reverse transcription random primers, RNasin (40 U/µl): Promega, UK.

- Restriction endonucleases Xmn1 (10 U/Xl) and associated buffers: New

England Biolabs, UK.

- BIOTaq polymerase (10 U/µl) and associated buffer, Bio-X-Act long DNA

polymerase (10 U/µl) and associated buffer, dNTPs and HyperLadder I:

Bioline,UK.

- Direct PCR (cell) and Direct PCR (ear) lysis buffer: Viagen Biotech, US.

- RNeasy minikit, DNeasy blood and tissue kit: Qiagen, UK.

2.1.5. Protein analysis reagents

All reagents obtained from Sigma-Aldrich except:-

- Precision plus protein-all blue standards and DC protein assay:

Bio-Rad, UK.

- Protogel (30% Acrylamide / 0.8% bisacrylamide) solution: National

Diagnostics, US.

- Enhanced chemiluminescent (ECL) advanced western blot detection kit,

and HiBond 0.45 Xm ECL nitrocellulose membrane: Amersham, UK.

- Antibodies, Abcam, UK.

- Horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary

antibodies: Dakocytomation, Denmark.

- Nylon Membranes, positively charged: Roche Applied Sciences, UK.

- Labeling and Detection - DIG system for in situ hybridiasation: Roche

Applied Sciences, UK.


66

- ELISA Kits – eBioscience, UK

- Heparin Sodium (with preservative) 25,000 i.u./5ml, Wockhardt UK Ltd.

- Amersham Hyperfilm – Amersham, UK

2.1.6. Equipment

The following equipment was used:

- Multimode scanner: TyphoonTM 9400 (accompanying software for image

quantification: ImageQuant TL TM): GE Healthcare, UK.

- Spectrophotometric plate reader: SPECTRAmax PLUS 384 (software:

SOFTmax PRO): Molecular Devices, US.

- NanoDrop 1000 spectrophotometer: Thermo Fisher Scientific, UK.

- PAGE electrophoresis and western-blot: Mini-Protean 3 cell vertical

electrophoresis system and Mini Trans-Blot cell blotting system: Bio-Rad,

UK.

- Sonicator: Sonopuls HD2070 MS72 sonicator, Bandelin electronics,

GmbH&Co., Germany.

- Fluorescent plate reader: FluoroSkan Ascent (accompanying software:

Ascent SoftwareTM version 2.4): Thermo Scientific, UK.

- PCR Machines: Techne TC-3000G, Geneflow; DNA Thermal Cycler 480,

Perkin Elmer. Thermal Cycler, Hybaid PCR, OmniGene.

2.1.7. Transgenic Mouse and MEF sources NEIL1-/- and NEIL2-/- knockout mice were generated by Dr. Rhod Elder using

published protocols (Perez-Campo et al., 2007). OGG1-/- mice had been

designed previously (Klungland et al., 1999) and were obtained from

embryos stored at the Patterson Institute. All MEFs used of these genotype


67

were derived from these mice and in addition from from NTH1-/- (Elder and

Dianov 2002) and [NTH1/OGG1]-/- (Karahalil et al., 2003) mice.

2.2. Methods

2.2.1. Mouse Colony Management

Heterozygous (HET) NEIL1, putative NEIL2 and OGG1 female mice were

backcrossed to a C57Bl/6J male. A HET male from the resulting offspring was

selected at random to breed to further C57B1/6J females for a further five

generations. Various HET mice were intercrossed and produced mice for

initial phenotypic characterisation. Knockout x knockout (KO x KO) and

wildtype x wildtype (WT x WT) mice were interbred to produce mice for

experimental purposes such as the creation of MEFs. All mice were housed in

solid floored cages where food and water were provided ad libitum.

2.2.2. Mouse Colony Characterisation

The offspring of HET X HET crosses were housed in boxes separated by sex

and litter, and these became the characterisation group. Mouse body weight

was then measured at monthly intervals and mice were also examined to

detect outward phenotypic changes. After 12 months the NEIL1 mice were

sacrificed by cervical dislocation and scalp to tail length of the mouse

measured. Major organs were also taken and the weights of the heart, lungs,

liver, kidney, spleen, omentum fat, reproductive organs (testes and both

ovaries and uteris in male and female mice respectivly) and adrenal glands

measured. Six male and six female mice of each genotype had blood samples

taken by cardiac puncture whilst under terminal anaesthetic (isoflurane) to be

used with cytokine ELISA’s.


68

2.2.3. LPS Induced Organ Damage

For MPO, MDA and GSH assays, the wildtype, NEIL1-/- and OGG1-/- mice to be

treated with LPS were first weighed individually, then injected i.p. with the

appropriate amount of LPS (20 mg/kg) in phosphate buffered saline (PBS; pH

7.4). After 12 h the mice were sacrificed by cervical dislocation and the heart,

lungs, liver, kidneys, and a section of ileum were removed and snap frozen in

liquid nitrogen. These were then stored at -80oC.

2.2.4. Induction of Immune Response

For cytokine assays the wildtype, NEIL1-/- and OGG1-/- mice to be treated

with LPS were first weighed individually, then injected i.p. with the

appropriate amount of LPS (2 mg/kg) in PBS. After 1, 6 or 24 h the mice

were placed under terminal anaesthetic (isoflurane) and exsanguinated by

cardiac puncture after which they were killed by cervical dislocation. The

blood was stored on ice with heparin (0.05 ml/ml blood). Blood samples were

then centrifuged for 15 min at 8000 g and the supernatant removed to a

separate Eppendorf. Both cells and supernatant were than stored at -80oC.

2.2.5. Establishment of MEF cultures from mouse embryos

In order to establish wildtype, NEIL1-/- and OGG1-/- MEF cell lines WT x WT

and KO x KO pairings were set up in order to produce the required offspring.

The females of these pairings were checked daily for vaginal plugging that

would indicate that mating had occurred. Thirteen days after plugging was

observed the pregnant mouse was killed by cervical dislocation, the uterus

removed, and placed in filtered phosphate buffered saline (PBS).

The individual embryos were then removed from the uterus and separated

from the placenta. The head was cut off and stored for genotyping.


69

Additionally the umbilical chord and internal organs (heart, liver, kidney &

gut) were excised from the embryo, and the embryo was then washed in PBS

to remove any remaining debris. Using a scalpel blade the remaining tissue

was minced finely, and 1 ml of 0.5 µg/ml trypsin added. To ensure sufficient

primary breakdown of the embryo, the trypsin/embryo mixture was aspirated

through a 1000 µl pipette tip. The mixture was then incubated in a water

bath for 30 min at 37°C, mixing at 10 min intervals.

In order to wash out the trypsin the mixture was diluted in 6 ml of MEF

media A, and was then centrifuged for 5 min at 126 g. At this point a jelly like

substance may have formed in the supernatant and this was kept and the

rest of the supernatant discarded, and cells from the pellet and jelly

resuspended in 1 ml MEF media A. The cells were then plated onto 25 cm2

(T25) vented cell culture flasks and incubated with 3 ml of MEF media A in a

humidified atmosphere containing 5% CO2 and 3% O2. After a day, when the

cells had reached confluence, they were washed with PBS, 250 µl of Trypsin

added and the cells incubated at 37°C. When all cells had detached from the

flask, 2 ml of MEF media A was added and the mixture transferred to a sterile

centrifuge tube. Cells were centrifuged for 5 min at 126 g, the supernatant

discarded and the pellet was resuspended in 1 ml of MEF media A. This was

then placed in a new T25 and 3 ml of primary MEF culture medium A added.

When the cells in this flask had grown to confluence they were removed from

the flask by trypsinisation and after centrifugation in MEF media A, the cells

were resuspended in media containing 10% dimethyl sulfoxide (DMSO),

transferred to a labelled cryotube and placed on ice for 30 min before being

placed in storage in a -80oC freezer.


70

2.2.6. MEF culture

NTH1-/- and [OGG1/NTH]-/- MEFs were taken from stocks held in liquid N2.

Cells were thawed rapidly in a 37°C water bath, and 6 ml of MEF media B,

held at room temperature, added slowly. To wash out the DMSO, the cells

were centrifuged at 126 g for 5 min, the supernatant discarded and the cells

resuspended in MEF media B. All MEFs were incubated at 37°C in 75 cm2

(T75) vented flasks in a humidified atmosphere containing 5% CO2 and 3%

O2.

Cells were passaged upon reaching confluence by washing cells in PBS,

trypsinising them with 750 µl of Trypsin-EDTA and incubating at 37°C. When

all the cells had been detached from the flask, 6 ml of MEF media B was

added and the mixture transferred to a sterile centrifuge tube. Cells were

centrifuged for 5 min at 126 g, the supernatant discarded and the pellet was

resuspended in 1 ml of MEF media B. The MEFs were then counted using a

haemocytometer and 6 x 104 cells plated in a new T75 flask containing 10 ml

of medium B.

At regular stages, stocks of MEFs were frozen for future use; cells were

pelleted by centrifugation and the supernatant discarded. The cells were then

resuspended in 1 ml of MEF media B containing 10% DMSO, and transferred

to a labelled cryotube, which was placed on ice for 30 min before storage in a

-80oC freezer.

2.2.7. Treatment of MEFs with LPS

Duplicate samples of 2 x 105 MEF cells of each genotype WT, OGG1-/-,

NTH1-/-, [OGG1/NTH1]-/- and NEIL1-/-) were plated in separate wells in a 24-

well plate. Controls were incubated with 1 ml of MEF media B only, while the


71

experimental samples were treated with 1 ml of MEF media B containing 0.25

µg/ml purified LPS from Escherichia coli serotype 127:B8. Plates were then

incubated for 18 h before the supernatant was aspirated into clean Eppendorf

tubes and frozen.

2.2.8. Genomic DNA extraction

DNA was extracted from a variety of sources, including earpunches, heads of

embryos and MEF cells. Such material was placed into a clean 0.5 ml

Eppendorf tube. One hundred and fifty microlitres of the appropriate

DirectPCR Lysis Reagent was added (Direct PCR ear for earpunch and head

pieces, Direct PCR cell for cells). The samples were then incubated for 5 – 6 h

at 55°C with occasional agitation (~ every 2 h) to ensure all the material

came into contact with the lysis solution. At the end of the incubation,

samples were examined visually to ensure that complete lysis of the material

had occurred. If not the mixture was then left until lysis had been completed

(up to overnight). After lysis, the temperature was increased to 85°C for 45

min to inactivate the Proteinase K. DNA concentration was determined prior

to storage by measuring the absorbance of the solution at 260 nm using a

Nanodrop spectrophotometer. For quality control purposes, the ratio of

absorbance at λ = 260 and 280 nm and at λ = 260 and 230 nm were

monitored and values from 1.8 to 2.2 were accepted as good quality

indicators.

2.2.9. Polymerase Chain Reaction (PCR) Genotyping For each 50 µl reaction, 1.5 µl of the appropriate DNA solution (section

2.2.8) was used, to which was added; 5µl of 10x Taq buffer, 0.5 µl of dNTPs

(25 mM), 1 µl of forward primer (10 pmol/µl), 1 µl of reverse primer (10

pmol/µl), 0.2 µl of Taq DNA polymerase and 38.8 µl of H2O. The PCR


72

amplification cycle was as follows; 95oC for 1 min; 95oC for 30 s

(denaturing), ~60oC (varies according to primer used; Table 2.4) for 30 s

(annealing), and 72oC for 1 min (elongation) for 30 cycles; 72oC for 5 min

and 25oC for 1 s.

2.2.10. Agarose Gel Electrophoresis Agarose gels were prepared by melting agarose in 0.5 x TBE in a microwave.

6 µl of loading buffer was added to each 50 µl PCR reaction and 15 µl of this

was then added into each well. In order to estimate fragment size, one well

was loaded with 3 µl of a DNA marker (Hyper Ladder I). For DNA fragments

below 1 kb a 1.5% agarose gel was used whilst for those fragments above 1

kb a 0.8% agarose gel was required. Electrophoresis was performed in 0.5x

TBE at 90 V for approximately 45 min. Results were visualised by using a

TyphoonTM laser, scanning at 532 nm.

2.2.11. RNA extraction

For total RNA extraction, 2 x 105 MEF cells were trypsinised (section 2.2.6)

and pelleted in a centrifuge at 8000 g for 10 min. A RNeasy mini prep kit was

used according to manufacturer’s instructions. Cells were disrupted by adding

600 µl of buffer RLT and the mixture homogenised by passing the lysate at

least 5 times through a blunt 20–gauge needle attached to a syringe. 600 µl

of 70% ethanol was added to the homogenised lysate which was mixed by

pipetting and 700 µl of the resulting mixture added to an RNeasy spin column

placed in a 2 ml collection tube. This was centrifuged at 8000 g for 15 s and

the flow through discarded. Seven hundred microlitres of buffer RW1 was

added to the spin column, the mixture centrifuged at 8000 g for 15 s and the

flow through discarded. The column membrane was washed by adding

500 µl of buffer RPE and centrifuging at 8000 g for 15 s and discarding the

flow through; this step was repeated but with this centrifugation step lasting


73

2 min in order to dry the membrane. The spin column was placed in a clean

1.5 ml collection tube and 30 µl of RNase free water was added to the

column. This was centrifuged at 8000 g for 2 min to elute the RNA. Total RNA

concentration was determined by measuring the optical density of the

solutions at λ= 260 nm using an ND-1000 spectrophotometer (NanoDrop

Technologies).

2.2.12. Reverse transcription (RT)

1.5 µg of RNA was dissolved in 11.75 µl of RNase free water and incubated

for 10 min at 70°C. The mRNA was reverse-transcribed into first strand cDNA

by adding 4 µl MgCl2 (25 mM), 2 µl 10x reverse transcriptase buffer, 1 µl

dNTPs (10 mM), 1 µl random primers and 0.25 µl AMV reverse transcriptase.

The samples were then incubated at the following temperatures: 25oC for 10

min, 42oC for 1 h, 95oC for 5 min and 0oC for 10 mins. The PCR step was

performed with Taq DNA polymerase as previously described (section 2.2.9)

using 1 µl of cDNA with an annealing temperature of 60oC.

2.2.13. Protein Analysis

2.2.13.1. Protein extraction from mouse tissues

Approximately 5 mg of mouse tissue was added into a clean mortar, which

was kept cool over ice, 300 µl cold RIPA buffer added and the organ was

disrupted using a pestel. The solution was transferred to a 1.5 ml Eppendorf

tube and agitated for 2 h on ice. The sample was centrifuged at 8000 g for

20 min at 4°C, and the supernatant was removed. 20 µl of the supernatant

was used for protein quantification (see 2.2.13.2). An additional supernatant

aliquot, to be used for the western blot, was diluted in 1 x SDS-PAGE loading

buffer and denatured by heating to 95°C for 5 min.


74

2.2.13.2. Protein quantification

Diluted protein samples (10 µl, 1/5 - 1/25 dilution) and a range of bovine

serum albumin (BSA) protein standards (10 µl, 0 – 2.0 µg/µl) were mixed

with 200 µl of Bio-Rad DC protein assay reagent, in triplicate, in a 96-well

plate. The absorbance at 595 nm was measured in each well using a plate

reader. Protein concentration in the supernatant was determined using the

standard curve.

2.2.13.3. Western blot

Up to 50 µg of cell lysate (see 2.2.13.1) in 1 x SDS buffer was separated on a

12 or 16 % polyacrylamide gel (Protogel) by electrophoresis for 1 h at 200 V,

in SDS running buffer. A blue pre-stained molecular weight marker was

included in each gel. A positive control in the form of protein from a WT

animal was also included. The proteins were then transferred onto a

nitrocellulose membrane by electro-blotting for 1 h at 100 V, in ice cold

western blotting transfer buffer. The membrane was then incubated in

blocking solution for 1 h at RT and then incubated with primary antibody in

blocking solution (dilution: 1/2000) for 1 h at room temperature. After three

washes in TBS(T), the membrane was incubated with HRP conjugated

secondary antibody in blocking solution (dilution: 1/1,000) for 1 h at RT. The

blot was then washed three times in TBS(T) prior to ECL detection according

to the manufacturer’s instructions. Briefly, the membrane was drained and 1

ml of a 50:50 mixture of reagent A and reagent B was poured over the

membrane and incubated for 5 min at room temperature. The membrane

was drained of the excess ECL reagent and exposed to ECL-sensitive film.


75

2.2.13.4. ELISA

To each well of a Maxisorp 96-well plate, 50 µl of carbonate buffer containing

25 µg/ml rat monoclonal anti-(mouse)interleukin-6 IgG was added, and

incubated overnight at 4oC. Each well was washed twice with PBS and

blocked with 100 µl of PBS containing 5% (w/v) non-fat milk (NFM) and

incubated at room temperature for a further 2 h. Each well was washed twice

with PBS before triplicate samples of 50 µl of either, increasing

concentrations of IL-6 in tissue culture medium, or each sample was added to

a well. Following incubation for 2 h at room temperature, the plate was

washed three times in PBS and 50 µl of rabbit polyclonal anti-(mouse)

interleukin-6 IgG diluted 1:2000 in PBS containing 0.5% (w/v) NFM was then

added to each well. After incubation at room temperature for 2 h, the plate

was washed 3 times with PBS-Tween 20, 50 µl of goat anti-rabbit IgG

conjugated with horseradish peroxidase diluted 1:2000 in PBS containing

0.5% (w/v) NFM added, and the plate incubated at 4°C for a further 16 h.

Plates were then washed a further 3 times with PBS containing 1% Tween 20

and 50 µl of Amplex Red mastermix was added. After 5 min the plate was

transferred to a TyphoonTM laser system, and fluorescence visualised by

scanning at 580 nm. A standard curve was produced using the results from

the known levels of IL-6 and this was used to determine the levels of IL-6 in

the cell supernatants.

All other ELISAs were obtained from eBioscience: the is described in brief. 50

µl of sample was placed into each well of a 96 well plate along with a

standard curve of the protein to be examined, and incubated overnight at

4oC. The wells were then emptied and washed 3 times in PBS-T, 100 µl of

blocking solution added, and incubated for 1 h at RT. Again the wells were

emptied, washed 5 times with PBS-T, 50 µl of primary antibody solution

added and incubated for 2 h at RT. After another 5 washes with PBS-T, 50 µl


76

of the detection antibody solution was added to the wells and incubated for a

further 30 min. Again the wells were washed 5 times with PBS-T and 50 µl of

the developer solution was added. The development was halted after 10 min

by the addition of 25 µl of 2N H2SO4. The absorbance was then read at 650

nm and compared with the standard curve to identify the protein content of

the original samples.

2.2.13.5. Myeloperoxidase Assay

The protocol was adapted from Graff et al., 1998. ~25 mg tissue samples

were weighed and placed in 1.5 ml Eppendorf tubes containing 5 µl/mg MPO

working buffer. This was sonicated over ice for 2 s and the resulting solution

centrifuged at 8000 g for 15 min. After discarding the supernatant the pellet

was resuspended in 500 µl of MPO working buffer (Graff et al., 1998).

The resulting mixture was sonicated over ice for 20 s and then snap frozen,

after which it was thawed. This procedure was repeated twice. The resulting

solution was centrifuged at 8000 g for 10 min and stored on ice.

An aliquot of the supernatant was diluted 1:2 in working buffer and a DC

protein assay was performed (Section 2.2.13.2). To 10 µl of diluted

supernatant 190 µl of detection buffer was added and the absorbance read at

650 nm every 30 s for 2 mins. This was compared to the protein result from

the DC assay to give a result measured in mU(MPO)/mg protein.

2.2.13.6. Malondialdehyde Assay

The protocol was adapted from Mabley et al., 2005a. Tissue samples were

weighed (~20 mg) and placed in MDA working buffer (100mg/ml) inside a

1.5 ml Eppendorf tube. This was then sonicated for 5 s. In a separate 1.5 ml

Eppendorf, 75 µl of homogenate was placed to which was added 546 µl 0.8%


77

(w/v) thiobarbutic acid, 75 µl 8.1% (v/v) SDS, 546 µl 20% (v/v) acetic acid

(pH 3.5) and 225 µl distilled H2O. An external standard was created using

1,1,3,3-tetramethoxypropane (0 – 14 mmol). All solutions were heated to

90oC for 45 min and the absorbance measured on a microplate reader at 532

nm. Protein in each sample was measured using a DC protein assay (see

2.2.12.2). MDA were adjusted to give a result measured in nMol(MDA)/mg

protein (Mabley et al., 2005a).

2.2.13.7. Glutathione Assay

The protocol was adapted from Rahman et al., 2006. Tissue samples (~20

mg) were weighed and placed in 1.5 ml Eppendorf tubes containing 5 µl/ml

0.6% (w/v) sulfosalcylic acid solution (if assaying liver 5% (w/v)

metaphosphoric acid was added as well). The mixture was sonicated over ice

for 5 s and centrifuged at 3000 g at 4oC for 10 min. The supernatant was

then removed and stored at 4oC up to 1 h before use. Samples were diluted

1/20 in ddH2O immediately before use (Rahman et al., 2006).

20 µl of the diluted samples were added to wells on a 96 well plate. 2 mg of

both 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB) and Nicotinamide adenine

dinucleotide phosphate (NADPH), and 40 µl of glutathione reductase (GR)

were each added to separate vials of 3 ml KPE buffer. DTNB and GR solutions

were mixed together equally and 120 µl added to each well. The samples

were incubated at room temperature for 30 s and 60 µl of NADPH solution

was added. Absorbance was read at 412 nm immediately and every 30 s for

2 min. Results were compared to a standard curve of glutathione (GSH).

Protein content was measured using a DC protein assay (2.2.13.2) and GSH

levels reported as nmol(GSH)/mg protein.


78

2.2.14. Statistical Analysis

In order to compare numbers of pups of each genotype in each litter a chi

squared test was performed. To compare survival rates of each genotype a

log-rank analysis was performed.

When comparing cell cytokine production and mouse cytokine production

one-way ANOVAs were performed. ANOVAs were performed when comparing

weights, and as there were 3 groups of mice (WT, HET and KO) a LDS post

hoc test was performed. When comparing organ damage results (MPO, MDA

and GSH) factorial analyses were performed using a 3-way ANOVA. Sample

size was calculated using a statistical power of 0.8. A p<0.05 was considered

statistically significant.


79

3. Mouse models: Generation and characterisation

3.1. Introduction

The first NEIL1-/- mice were reported in 2006; there was no immediate

difference observed in weight or viability of the mice when compared to

NEIL1+/+ mice. However, after 6-10 months all the male mice in the third

generation became significantly overweight when compared to the WT mice

(mean weight 50 g vs. 28 g), and the female mice were also overweight, but

to a less severe degree (mean 32 g vs. 28 g). The abdominal cavity of both

male and female NEIL1-/- mice was found to contain extensive fat deposits,

and their livers were significantly larger due to fat storage. The plasma of the

NEIL1-/- mice also contained elevated concentrations of leptin, which is

usually noted with elevated fat deposits, but not insulin (at fasting and fed

conditions) which would also be expected (Hoffler et al., 2009). Increased fat

deposits and hyperleptinemia were consistent with the human disease

metabolic syndrome. It is suggested by Vartanian et al. that the build up of

DNA damage in the mitochondria of the NEIL1-/- mice was disrupting the

metabolic processes. Other intermittent phenotypes observed included

reduced subcutaneous fat deposits, skin ulcerations, joint inflammation,

infertility and cancers (Vartanian et al., 2006). However when mice taken

from this colony were bred in a different animal facility, no obese phenotype

was observed in any of the mice bred there despite several years of

continuous breeding (Chan et al., 2009). This suggested that there may have

been another reason for the increased fat content in this strain of NEIL1-/-

mice. In a follow up paper the same group, at Oregon Health and Science

University, have shown that with an increase in oxidative damage (caused by

a high fat diet) a greater percentage of male and female mice exhibit markers

the metabolic syndrome, such as increased weight and hyperleptinemia

(Sampath et al., 2011). This supports the mitochondrial damage model


80

previously mentioned which suggested that the metabolic syndrome

phenotype is not caused wholly by the absence of the NEIL1 gene, but its

removal does increase the mouse’s susceptibility to the symptom. The

removal of any protein which repairs oxidative damage in the mitochondria

could then contribute to the condition (Sampath et al., 2011). Indeed, in one

strain of OGG1-/- mice it has been reported that male OGG1-/- mice were

significantly heavier than OGG1+/+ mice after 24 -28 weeks (Arai et al.,

2006).

More work has been done to identify the effects of the knockout of OGG1 in

vivo. An OGG1-/- mouse was first documented in 1999, and there were no

significant differences in viability, and they were indistinguishable from

OGG1+/- and OGG1+/+ mice up to 18 months of age (Klungland et al., 1999).

In a different strain of OGG1-/- mice it was noted that after 19 months the

incidence of cancer in the animals increased (Sakumi et al., 2003).

In contrast to this work on NEIL1 and OGG1, very little work has been done

on the effects of NEIL2 in vivo, and whilst a few binding partners have been

identified to this date no NEIL2-/- mice have been reported (Bhakat et al.,

2004; Das et al., 2007b; Grin et al., 2010).

3.1.1. Aims

The objectives of this chapter are to:

I. Confirm that the disruption of the NEIL1 and NEIL2 genes have

resulted in the absence of NEIL1 and NEIL2 proteins respectively in

newly developed strains of mice.

II. Identify any phenotype in the gross physiology of these NEIL1-/- and

NEIL2-/- mice, especially pertaining to symptoms similar to the human

metabolic sydrome.


81

III. Confirm the presence of the OGG1 knockout construct in the genomic

DNA of OGG1-/- mice.

3.2. Results

3.2.1. NEIL1 Mouse Colony

3.2.1.1. Preparation of NEIL1 Knockout Mice.

The NEIL1 mouse colony was created from two NEIL1+/- chimeras created

from ES cells prepared by Dr. Rhod Elder (Perez-Campo et al., 2007). Animals

in this colony were genotyped by PCR (Figure 3.1) and so identified as either

wildtype (NEIL1+/+, WT), heterozygous (NEIL1+/-, HET) and knockout

(NEIL1-/-, KO) mice. The appropriate mice identified from these PCRs were

then used in the selective breeding of the NEIL1-/- line and backcrossing to a

C57/B16J background (Figure 3.2).

Figure 3.1: Pedegree of NEIL1 Transgenic Mice

♂ Chimera 83 was crossed with a ♀ C57/B16J mouse to produce a litter of WT and HET animals. Then a ♂ C57/B16J and HET ♀ 29 were bred together to produce a backcross, from the resulting litter ♂ 130 was used to further backcross the animals. HET male 20 was crossed with HET ♀ (22 and 25) to produce a litter containing WT, HET and KO animals. These mice were then be used to produce further WT (140 x 121), KO (139 x 146) and HET (138 x 119 & 120) litters.


82

3.2.1.2. Confirmation of Genotype.

In order to confirm the disruption of the NEIL1 gene at the transcription level

RT-PCR was performed on cDNA isolated from liver tissue excised from

NEIL1-/- (KO) and NEIL1+/+ (wildtype) mice (Figure 3.3A). The PCR primers

were designed to amplify a section of DNA 449bp in length spanning exons 1

– 3. This amplicon was only observed in the PCR using the NEIL1+/+ sample

for its source cDNA. This confirmed that there was no complete NEIL1 cDNA

in samples created from RNA extracted from tissues taken from NEIL1-/- mice,

whilst it was detected in the cDNA created from WT animals. A PCR spanning

exons 4 and 5 of the GAPDH gene was used as a control, this fragment was

predicted to be 222 bp in length. Its presence in both WT and KO samples

confirmed that the samples contained viable cDNA.


Results

(A) To test for the WT gene, the primers 1907 – 3015 were used, giving rise to a band of 1108 bp. Primer 1907 was designed to anneal to the region in the NEIL1 gene to be deleted. For the KO allele the primers used were 201 – 807 (1226 bp) where the 201 primer is complementary to a sequence in the TK/Neo cassette. (B) The PCR genotyping results for offspring of NEIL1 HET x HET crosses. For mice 2 and 3, only the 1108 bp bands (left lane) are obtained and it is thus a WT, whilst for mouse 4 only the 1226 bp band (right lane) is obtained identifying it as a KO mouse. However, mouse 1 shows both bands and is therefore a HET.


83

A Western blot was then used to show the presence or absence of NEIL1

protein in samples taken from NEIL1 WT and KO mice. A band indicating a

protein ~44 KDa (NEIL1 weighs 43.7 KDa) was detected in the samples taken

from male and female NEIL1 WT animals. In the samples taken from NEIL1

KO animals no band was observed (Figure 3.3).

(A)

(Bi) (Bii)

Figure 3.3: Confirmation of a NEIL1 Null Phenotype

(A) RT-PCR of RNA extracted from liver of NEIL1 WT and KO mice. PCR was performed using primers 328 – 545 (exon 1-2, 217 bp) for NEIL1 and primers 534-765 (exon 4-5, 222 bp) for GAPDH (picture courtesy of Dumax-Vorzet). The absence of a band in the NEIL1 lane of the NEIL1 KO indicates that NEIL1 RNA is not being produced in the liver of these mice. (Bii) 10 µg of liver protein from male and female Wt and Neil1-/- were separated on a 12% SDS-PAGE gel. (Bi) The proteins were transferred onto nitrocellulose membrane and the membrane probed with Neil1-/- anti-serum. A band corresponding to NEIL1 (43.7 kDa) is evident in the WT lane of males and females only, indicating that the NEIL1 protein is not produced in the knockout animal.


84

3.2.1.3. Viability and Mortality of NEIL1 mice.

The number of pups produced from NEIL1 HET X HET crosses were produced

in a ratio of 31:47:20 (WT:HET:KO). This was not significantly different from

the expected ratio of 1:2:1 (p=0.27 by Chi-squared test; Figure 3.4).

31

47

20

0

10

20

30

40

50

WT HET KO

Genotype

Nu

mb

er o

f A

nim

als

Figure 3.4: The distribution of genotypes from HET x HET breeding pairs of NEIL1

Transgenic Mice.

There was no difference between the genotype distribution (1:1.5:0.6) and the expected result (1:2:1; p=0.27).

To further examine the viability of the mice a comparison of the litter sizes

from WT X WT, HET X HET and KO X KO pairs was performed. No significant

difference in litter size was observed (p=0.18). Neither were there any

significant differences in the number of males or female mice produced per

litter (p=0.28 and 0.54 respectively; Figure 3.5).

To compare mortality rates HET X HET litters were separated into males and

females and fed ad libitum, dates of death were recorded and used to create

the Kaplan-Meier survival curves. Of the mice only 5 males died during the

course of the experiment (2 WT, 1 HET, 2 KO), and 3 females (2 WT, 0 HET,

1 KO), and all deceased mice were found dead in their cages. No significant


85

01

23

45

67

WT HET KO

Genotype

Ave

rag

e M

ales

per

Lit

ter

01234567

WT HET KO

Genotype

Ave

rag

e F

emal

es p

er

Lit

ter

0

2

4

6

8

10

12

WT HET KO

Genotype

Ave

rag

e L

itte

r S

ize

difference in mortality was observed between WT, HET and KO mice in either

male (p=0.92) or female (p=0.20) animals (Figure 3.6).

(A) (p=0.18)

(B) (C)

(p=0.54) (p=0.28)

Figure 3.5: Average litter composition from NEIL1 colony

(A) Combined litter sizes, (B) amount of females per litter, (C) amount of males per litter. Litter size and composition did not differ significantly with genotype (n=16 (WT), 10 (HET), 13 (KO)).

3.2.1.4. NEIL1 Mouse Weights

Mice from HET X HET crosses were housed by sex in their original litters and

fed ad libitum. Their weights were measured every month for twelve months.

There was no evidence that the weight of the animals varied by genotype at

any time point (Figure 3.7).

After twelve months the animals were sacrificed, measured from scalp to tail

and organs collected and weighed. When examined several mice had


86

symptoms of disease. A single male NEIL1+/+ mouse had a splenic growth

and a NEIL1-/- male mouse had a cyst on its gut, that was not connected to

any abnormal growth. One NEIL1-/- female mouse had cataracts over each

eye, one female NEIL1+/- mouse had a large cyst connected to a tumour on

its stomach, and a NEIL1+/+ mouse had many smaller cancers along the

length of its intestines.

(A) Male

(B) Female

Figure 3.6: Mortality rates of (A) Male and (B) Female Transgenic Mice

Survival curves for NEIL1 mice over the course of 370 days, Log-Rank analysis indicates no significant difference in the mortality rate of male (p=0.92; n=53) or female (p=0.20; n=43) WT, HET and KO mice.


87

(A) Male

05

101520253035404550

2 3 4 5 6 7 8 9 10 11 12

Age (month)

Wei

gh

t (g

)

WT

HET

KO

(B) Female

05

101520253035404550

2 3 4 5 6 7 8 9 10 11 12Age (Month)

Wei

gh

t (g

)

WT

HET

KO

Figure 3.7: NEIL1 transgenic male and female mouse weights over 12 months

Mean values and standard error of mean for 54 mice split between (A) male (n=30) and (B) female mice (n=48); two way ANOVA indicates no significant difference at any time point (Male p>0.06, Female p>0.35)

The length, BMI, and organ weights of the mice are shown in Figures 3.8-10.

No significant differences were observed due to genotype in BMI or scalp to

tail lengths (Figure 3.8).

In male mice, a significant difference was observed in the total weight of the

sex organs between NEIL1-/- and NEIL1+/- mice (KO 209.6 ±13.7 mg; HET

244.9 ±7.9 mg; p=0.03). This was also shown when adjusted as a proportion

of total bodyweight (KO 0.5 ±0.0 %; HET 0.6 ±0.0 %; p=0.06). In female


88

75

80

85

90

95

100

WT HET KO

Genotype

Len

gth

(m

m)

75

80

85

90

95

100

WT HET KO

Genotype

Len

gth

(m

m)

0

0.005

0.01

0.015

0.02

0.025

0.03

WT HET KO

Genotype

BM

I

0

0.005

0.01

0.015

0.02

0.025

0.03

WT HET KO

Genotype

BM

I

mice, evidence of a significant difference in the weight of the sex organs was

observed between HET and KO when adjusted for length (KO 5.8 ±0.9

mg/mm; HET 4.4 ±0.4 mg/mm; p=0.08).

(A) ♂ scalp to tail length

(C) ♂ BMI

(B) ♀ scalp to tail length

(D) ♀ BMI

Figure 3.8: Length and BMI of NEIL1 Transgenic Mice Mice were sacrificed at 12 months and weight and length measured (male n=19 WT, 22 HET, 7 KO; female n=9 WT, 19 HET, 9 KO). A – B show mean weights and standard error and C – D shows mean organ size (± SEM) as a percentage of bodyweight. No significant difference due to genotype was detected in scalp to tail measurement (male p=0.51; female p=0.07) or BMI (male p=0.27; female p=0.41).

There was a significant difference in male mouse ommentum weight with KO

mice (1124.3 ±102.7 mg) having significantly less ommentum fat than WT

mice (1537.5 ±56.6 mg; p=0.03). This was also the case when considering

the spleen as a percentage of total bodyweight (KO 2.7 ±0.2 %; WT 3.4

±0.1 %; p=0.02) and the scalp to tail length of the animal (KO 20.5 ±1.6

mg/mm; WT 17.2 ±0.6 mg/mm; p=0.02; Figure 3.9).


89

0

500

1000

1500

2000

2500

3000

3500

Heart Lungs Spleen Liver Kidneys Sex Organs Abdominal Fat OmmentumFatTissue

Wei

ght (

mg)

WT

HET

KO

0

1

2

3

4

5

6

7

8

Heart Lungs Spleen Liver Kidneys Sex Organs Abdominal Fat Ommentum Fat

Tissue

% T

otal

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ght

WT

HET

KO

0

20

40

AdrenalsWei

gh

t (m

g)

00.050.1

Adrenals

% T

ota

l B

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ywei

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t

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15

20

25

30

35

Heart Lungs Spleen Liver Kidneys Sex Organs Abdominal Fat Ommentum Fat

Tissue

mg/

mm WT

HET

KO

(A) Organ weight

(p=0.05)

(p=0.03) (p=0.07) (p=0.03) (B) Organ weight as a percentage of body weight

(p=0.02) (p=0.06)

(C) Organ weight per mm length (p=0.02)

Figure 3.9: Male NEIL1 transgenic mouse organ weight comparisons

Mice were sacrificed at 12 months and their organs removed and weighed (male n=19 WT, 22 HET, 7 KO). Panel A shows total tissue weights by genotype, panel B tissue weight as a percentage of body weight by genotype and panel C organ weight to length by genotype. There was evidence of a difference between WT and KO in the weight of adrenal glands and kidneys (p=0.07). There was a significant difference between HET and KO in the weights of the mouse sex organs (p=0.03) and evidence of a difference when seen as a percentage of bodyweight (p=0.06). There was a significant difference between WT and KO for ommentum fat in each case (p<0.03).

00.20.4

Adrenals

mg

/mm


90

0

500

1000

1500

2000

2500

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Heart Lungs Spleen Liver Kidneys Sex Organs AbdominalFat

OmmentumFat

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Heart Lungs Spleen Liver Kidneys Sex Organs Abdominal Fat OmmentumFat

Tissue

mg

/mm

len

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WT

HET

KO

0

20

40

AdrenalsW

eig

ht

(mg

)

00.050.1

0.15

Adrenals

% T

ota

l B

od

ywei

gh

t

00.20.40.6

Adrenals

mg

/mm

(A) Organ weight (p=0.03)

(p=0.08) (B) Organ weight as a percentage of body weight

(p=0.01)

(p=0.08)

(C) Organ weight per mm length

(p=0.02)

(p=0.09) (p=0.08)

Figure 3.10: Female NEIL1 transgenic mouse organ weight comparisons

Mice were sacrificed at 12 months and their organs removed and weighed (female n=9 WT, 19 HET, 9 KO). Panel A shows total tissue weights by genotype, panel B tissue weight as a percentage of body weight by genotype and panel C organ weight to length by genotype. In each case there were significant differences between HET and KO for adrenal glands (p<0.03). There was also evidence of a difference in spleen weights between WT and KO in each case (p<0.09). Evidence of a significant difference was observed between HET and KO in the weight of the sex organs when considering length (p=0.08).


91

In female mice the adrenal glands were significantly heavier in NEIL1-/- mice

(27.8 ±7.2 mg) than in NEIL1+/- mice (17.3 ±1.4 mg; p=0.03), this was born

out when considering the weight as a proportion of body weight (KO 0.8 ±0.2

%; HET 0.5 ±0.1 %; p=0.01) and when compared to length (KO 0.3 ±0.1

mg/mm; HET 0.2 ±0.0 mg/mm; p=0.02). There was also evidence of a

difference in male adrenal gland weight, where the NEIL1-/- mice (27.8 ±7.2

mg) were heavier than the NEIL1+/+ mice (17.3 ±1.4 mg; p=0.03).

In female mice there was evidence that the spleens in NEIL1 KO (139.3 ±14.4

mg) mice were significantly lighter than the NEIL1+/+ mice (230.4 ±43.7 mg;

p=0.08). This was also the case when considering the spleen as a percentage

of total bodyweight (KO 0.4 ±0.0 %; WT 0.7 ±0.2 %; p=0.08) and the scalp

to tail length of the animal (KO 1.7 ±0.2 mg/mm; WT 2.8 ±0.6 mg/mm;

p=0.09; Figure 3.10).

3.2.2. NEIL2 Mouse Colony

3.2.2.1. Genotyping The NEIL2 mouse colony was bred from NEIL2+/- chimeras created from

constructed ES cells prepared by Dr. Rhod Elder (Perez-Campo et al., 2007).

Animals in this colony were genotyped by PCR (Figure 3.11) and so identified

as either wildtype (NEIL2+/+, WT), heterozygous (NEIL2+/-, HET) and knockout

(NEIL2-/-, KO) mice. The appropriate mice identified from these PCRs were

then be used in the selective breeding of the NEIL2-/- line and backcrossing to

a C57/B16J background.


92

(A)

(B)

Figure 3.11: The NEIL2 Knockout Construct and Genotyping Results.

(A) Diagram of NEIL2 construct. To test for the WT gene, the primers 410 – 963 were used, giving rise to a band of 553 bp. Primer 410 was designed to anneal to the region to be deleted in the NEIL2 gene. For the KO allele the primers used were -871 – 201 (1.3 kb) where the 201 primer is complementary to a sequence in the TK/Neo cassette. (B) PCR genotyping results for offspring of NEIL2 HET x HET crosses. For mouse 3, only the 553 bp band (left lane) is obtained and it is thus a WT, while for mouse 1 only the 1.2 kb band (right lane) is obtained and is thus a KO mice. However, mouse 2 shows both bands and is therefore a HET.

3.2.2.2. Western and RT-PCR The confirmation of genotype was performed by a colleague (Saad, W M), the

results of which are given in brief below.

In order to confirm the disruption of the NEIL2 gene at the transcription level

RT-PCR was performed on cDNA produced from mRA obtained from liver,

testes and skeletal muscle tissue excised from NEIL2-/- (KO) and NEIL2+/+

(wildtype) mice (Figure 3.12A). The PCR primers were designed to amplify a

section of DNA ~600bp in length. This amplicon was observed in all the PCRs

using animals identified as NEIL2+/+ and NEIL2-/- by genotyping when

compared to a positive contro, although there were variances in the strength

of the band produced. Specifically the bands for WT skeletal muscle and KO

Tk/Neo 3.8 kb 3.28 kb 1 kb

-812 476 269

963

Pmel Fsel Pacl AscI

201

4248

410

-871 4343


93

liver were much lighter than others produced from the same amount of source

cDNA. This indicated that NEIL2 mRNA was still being produced in the putative

NEIL2-/- mouse.

(A) RT-PCR

(Bi) (Bii)

Figure 3.12: Confirmation of a NEIL2 Null Phenotype (A) RT-PCR of RNA extracted from NEIL2 WT and putative KO mice. PCR was performed on tissues taken from liver, testes and skeletal muscle. There is a band present in each lane at ~600 bp indicating the presence of cDNA corresponding to NEIL2 mRNA. (Bi) 10µg of protein extracts from WT and Neil2-/- livers (male) and protein control (+ve) were separated on a 12% SDS-PAGE gel. (Bii) The protein was transfer onto a nitrocellulose membrane and the membrane were probed with Neil2-/- primary antibody, a band corresponding to NEIL1 (36.9 kDa) is evident in the WT, KO and positive control lanes, indicating that the NEIL1 protein is produced in the putative knockout animal (data courtesy of Saad W M).

A Western blot was used to identify the absence of the NEIL2 protein in

samples taken from NEIL2 WT and KO mice. A band indicating a protein ~37

KDa (NEIL2 weighs 36.9 KDa) was shown in the samples taken from NEIL2+/+

and NEIL2-/- mice as well as the positive control. This indicated that the NEIL2

protein was still being produced in its undisrupted form in the putative NEIL2-/-

mice (Figure 3.12).


94

3.2.3. OGG1 Mouse Colony

3.2.3.1. OGG1 Mouse Genotyping OGG1-/- embryos were removed from storage in liquid nitrogen and thawed

and implanted into a female mouse to be brought to term. These animals were

then bred with WT (C57/B16J) mice to produce OGG1+/- mice. Animals from

these litters were interbred to produce wildtype (OGG1+/+, WT), heterozygous

(OGG1+/-, HET) and knockout (OGG1-/-, KO) mice. Animals in this colony were

genotyped by PCR (Figure 3.17) and so identified as WT, HET and KO mice.

The appropriate mice identified from these PCRs were then used in the

selective breeding of OGG1+/+ and OGG1-/- for experimentation.

(A)

(B)

Figure 3.13: Diagram of the OGG1 Knockout Construct and Genotyping Results

(A) Diagram of OGG1 knockout construct. To test for the WT gene, the primers 2327 – 663 were used, giving rise to a band of 323 bp. Primer 1907 was designed to anneal to the deleted region in the NEIL1 gene. For the KO allele the primers used were NEO – 2327 (~1000 bp) where the NEO primer is complementary to a sequence in the TK/Neo cassette. (B) PCR genotyping results for offspring of NEIL1 HET x HET crosses. For animal 2, only the 323 bp band is obtained and it is thus a WT, whilst for animal 3 only the 1000 bp band is obtained identifying it as a KO mouse. However, animal 1 shows both bands and is therefore a HET.

Pgk-Neo

3.1 kb

1.8 kb

4.7 kb

-100 3033

7620

11976

HindIIl

HindlII

XhoI XhoI

NEO 6632327


95

3.3. Discussion

In order to identify phenotypes displayed by DNA glycosylase deficient mice, a

new strain of NEIL1-/- mice has been created. PCR, RT-PCR and western blot

procedures indicate that disruption of the NEIL1 gene has proved a success.

The PCR test to identify changes in genomic mouse DNA of the mice has

proved the existence of both the WT gene and the correctly placed KO

cassette within the colony (Figure 3.1). RT-PCR has also indicated the lack of

NEIL1 mRNA production within the tested animals (Figure 3.3).

Furthermore, the western blotting indicates that the NEIL1 protein, whilst

present in NEIL1+/+ animals, is not detected in the tissues of the NEIL1-/- mice.

The band produced from samples taken from WT animals is at the expected

position on the blot to be identified as the NEIL1 protein (Figure 3.3; 43.7

kDa), but there is also a lighter band at ~28 kDa. When the NEIL1 protein was

first reported the western blot identifying it had several bands one of which

was around 25 kDa (Takao et al., 2002a).

When considering the mean weights of other C57BL/6 mice colonies in which

the mouse weights rarley exceed 35 g (males) and 30 g (females) (Arai et al.,

2006; Goodrick et al., 1990), compared to this the weights of the mice in this

colony are very high, especially those of WT males who at 12 months have a

mean weight of 47.7 g. So whilst this does not directly support the theory that

the lack of the NEIL1 protein leads to the onset of metabolic syndrome, these

animals are already very large, a factor that perhaps eclipses any differences

that would be shown from the knockout of the NEIL1 gene.

The NEIL1-/- mice reported here did not display a comparatively obese

phenotype when compared to their WT siblings, nor was the extensive

abdominal fat described by Vartanian et al., (2006) found in the NEIL1-/- mice.

Indeed, the ommentum fat content of the NEIL1-/- was significantly lighter

than that of the NEIL1+/+ mice. Previous observations of NEIL1-/- mice have

also described enlarged livers and kidneys due to fatty deposits within them


96

(Vartanian et al., 2006). This was not observed in the newly developed

NEIL1-/- mice. Indeed there was a suggestion that the kidneys of the male

NEIL1-/- mice were lighter than those of the NEIL1+/+ mice although this was

found to be non-significant after adjustment for weight and length.

Significant differences were observed between weights of reproductive tissues

and adrenal glands. Changes in the weights of reproductive tissues are often

synonymous with changes in the output of the hormones associated with

them, (eg. testosterone in males) or a sign of greater hormone output from

other sources (eg. FSH in females; (Hewitt and Korach 2003; Jin et al., 2008)).

The increase in the size of the adrenal glands is suggestive of an increase in

adrenalin production. All of these hormones affect behaviour, and in the recent

paper by Sampath et al. (2011) small changes in behaviour were noted to the

mouse circadian rhythms. The mice seemed to be active for more of the light

dark cycle, but they did not engage in much exercise during this time,

preferring to eat.

One reason for the apparent differences in the two NEIL1-/- strains could be

the genes foreign to C57BL/6 mice flanking the NEIL1 gene. This problem

stems from the fact that the ES cells that are used to create the chimera for

the knockout are usually from a different strain of mice (Tc-1) to the

background that the KO gene will be backcrossed onto (C57BL/6). Thus, not

only the disrupted gene will be transmitted to their progenitors but any genes

that are on the same allele. When comparing the knockout animals created

with this method with wild-type animals, most of these transmitted genes will

not pose a problem, due to their independence from the knockout gene

(Wolfer et al., 2002).

However, for any genes linked to the targeted gene a “linkage disequilibrium”

will be created, because the null mutation will be flanked by two foreign

genes, and the wild-type locus flanked by two C57BL/6 genes. This could

cause an effect that would be confused for an overt phenotype. This can be


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overcome by using C57BL/6 to create the ES cells, or by breeding an

extensively backcrossed knockout (10 generations) with the strain the ES cells

were created from and comparing the phenotype of the resulting mice to that

of those being investigated (Wolfer et al., 2002). The ES cells for our NEIL1-/-

mice were taken from TC-1 mice, and those from the previously reported

NEIL1-/- colony used ES cells from CJ7 mice which could account for the

difference in phenotype.

Figure 3.14: The creation of transgenic mice can result in linkage disequilibrium The cloned disrupted C57BL/6 gene of interest is inserted into the ES cell DNA (A), and the resulting ES cells are combined with blastocysts to create chimeras containing the disrupted gene. The resulting chimera is bred with a C57BL/6 mouse (B) and through this and successive backcrosses the allele containing the disrupted gene is integrated into the C57BL/6 DNA (C). However, the flanking genes on the Tc-1 allele will always be associated with the knockout.

The success of the production of the NEIL1 knockout model was tempered by

the conflicting results in the NEIL2 genotyping, RT-PCR and western blot.

Indeed, both the NEIL1 and NEIL2 strains were created by the same method

outlined by Perez-Campo et al., (2007) and as one of the strains has achieved

the knockout of its target gene it is unlikely to be a flaw in the technique. As

the gene was inherited normally (i.e. the ratio of WT:HET:KO mice was within

expected amounts, data not shown) it indicates that at whatever point the

Neo/Tk insert has been placed in the genome it has not caused reduced

viability in the “knockout” mice, which is further supported by the lack of

(A)

(B)

(C)


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significant difference observed in the mortality of NEIL2+/+, NEIL2+/- or the

putative NEIL2-/- mice.

Several reasons for the failure of the knockout can be postulated: (i) the

Tk/Neo insert is located in the wrong position, (ii) there could be a duplicate

NEIL2 gene, (iii) conserved portions of the NEIL2 gene are being spliced from

another region of the genomic DNA. None of these explain fully why PCR

genotyping gives the expected sized bands for the primers used and why

apparently KO, HET and WT NEIL2 animals are created. In order to determine

the actual positioning of the Tk/Neo insert the technique fluorescent in situ

hybridization could be used which would at least confirm that the insert was on

the correct chromosome. A further assay that could be performed would be an

activity assay on purified “NEIL2” protein obtained from putative NEIL2-/- mice.

This would confirm that the protein identified on the western blot was a

functional NEIL2, rather than a similar sized, and shaped protein that the

antibody recognized.

The lack of a strong observable phenotype in the NEIL1 knockout animals is

hardly surprising, as in most cases the deletion of a DNA glycosylase results in

very little overt phenotypic change (Table 3.1). This is theorized to be due to

the overlapping functions of the DNA glycosylases meaning that they act as

functional backups for each other (Parsons and Elder 2003). These backups

mean that BER is available to the cell even if there are dysfunctional DNA

glycosylases, as knockouts of more downstream BER proteins which cannot be

functionally backed up, such as APE1, Pol β, DNA ligase I & III and XRCC1 are

lethal to the cell (Larsen et al., 2007). There is one exception to this trend,

TDG, which has been found to be embryonic lethal when disrupted in the

mouse genome. This is suggested to be due to TDG’s activity in maintaining

chromatin throughout cell development, and that the knockout of this gene

results in the loss of epigenetic control (Cortazar et al., 2011). In most cases it

takes a double knockout of two DNA glycosylases which excise similar lesions

to cause a major response such as that of [OGG1/MUTYH]-/- which causes the


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50% survival age to drop to 10.3 months, when compared to OGG1-/-,

MUTYH-/- and WT animals whose 50% survival age was never below 18 m.

Increased tumor incidence in the [OGG1/MUTYH]-/- mice from two months of

age was suggested to be due to the build up in mutations in the codon 12

region of the K-Ras oncogene (Xie et al., 2004).

This implies the question which two DNA repair genes would be best to knock

out in a mouse model. Future work could be best served by considering the

properties of NEIL1, in order to test the hypothesis that it is damage within the

mitochondria that causes the symptoms of metabolic syndrome, another DNA

glycosylase that operates in the mitochondria, such as OGG1 in which one

strain of knockout mice has also shown increased weight of KO mice (Arai et

al., 2006), could be knocked out. When a working NEIL2 model is presented it

would be good to create a [NEIL1/NEIL2]-/- to study the effects of the loss of

single strand BER within the cell.


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Table 3.1: Summary of Phenotypes of DNA Glycosylase Deficient Mice.

DNA

Glycosylase Type Gross Phenotype Molecular Charachterisics Reference

MTH1 Monofunctional Increased levels of lung, liver and stomach cancers (16.1% vs. WT 4.4%)

Non-signficant increase in 8-OxoG (Sakumi et al., 2003; Tsuzuki et al., 2001)

MPG Monofunctional No overt phenotype at 24 months Increased alkylation sensitivity (Elder et al., 1998;

Engelward et al., 1997)

UNG Monofunctional After 24 months increased disposition to B-cell lymphoma (28% vs. 1.3%)

12 months 1.5 fold increase in C:G → T:A transitions

(Nilsen et al., 2003; Nilsen et al., 2000)

MUTYH Monofunctional After 18 months increased lung, liver and stomach cancers (59.5 % vs. 34.9%)

Increased 8-oxo-dGTP in the nucleotide pool (Tsuzuki et al., 2001)

SMUG1 Monofunctional No overt phenotype Increased mutation rate in ES cells (Hirano et al., 2003)

TDG Monofunctional Embryonic lethal Lethal (Cortazar et al., 2011)

MBD4 Monofunctional No overt Phenotype Increased Mutation frequency (Millar et al., 2002)

Ogg1 Bifunctional/monofunctional

No overt Phenotype to 18 months Increased levels of 8-OxoG in genomic and mtDNA (Klungland et al., 1999)

Greater incidence of lung cancers after 19 months (48% vs. 11.1%)

(Arai et al., 2006; Sakumi et al., 2003)

OGG1/MTH1 Bifunctional Reduced tumorgenesis compared to OGG1-/- mice (0% vs. 14.3%)

Increased 8-OxoG in genomic DNA (Sakumi et al., 2003)

OGG1/MUTYH Bifunctional/monofunctional Increased tumour incidence after 2 months (31.4% vs. 4.3%)

No significant difference in repair efficincy noted (Xie et al., 2004)

NTH1 Bifunctional No overt phenotype No significant difference in repair

efficincy noted (Elder and Dianov 2002; Ocampo et al., 2002; Takao et al., 2002b)

OGG1/NTH1 Bifunctional/bifunctional No overt phenotype No significant differences noted (Elder et al., 2004)

NEIL1 Bifunctional Sporadic symptoms of human metabolic syndrome Increased mtDNA Damage

and Deletions (Vartanian et al., 2006)

NEIL1/NTH1 Bifunctional/bifunctional Increase in tumours, especially lung (74%) and liver (43%) after 24 months

Similar DNA damage patterns as NEIL1 (Chan et al., 2009)

NEIL2 Bifunctional No known knockout available n/a

NEIL3 Bifunctional No known knockout available n/a


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4. Cytokine Output of DNA Glycosylase Disrupted Cells

4.1. Introduction The creation of a new strain of NEIL1-/- mice has been described and no overt

phenotype was found in unchallenged animals (Chapter 3). Other mouse

knockout models of the bifunctional DNA glycosylases OGG1 and NTH1 have

already been created and no observable overt phenotype was also observed

in these animals (Elder and Dianov 2002; Klungland et al., 1999). Neither was

there any overt phenotype described in the double knockout of

[OGG1/NTH1] (Parsons and Elder 2003).

Several proteins linked to BER have been identified as having links with the

inflammatory response including PARP-1, APE-1, OGG1 and MUTYH. PARP-1

and APE-1 knockouts reduce the inflammatory response via an attenuation of

the redox sensitive transcription factor NF-κβ transcription factor (Ando et al.,

2008; Virag and Szabo 2002). There is also a reduction in inflammation

noted in the OGG1-/- models of LPS induced inflammation and Helicobacter

Pylori induced gut inflammation (Mabley et al., 2005b; Touati et al., 2006).

Previously MEF’s have been used in the study of the DNA repair/damage

response of KO mice, although no in vitro studies have been carried out to

identify any alterations in MEF response to LPS in any DNA glycosylase

deficient models. (Elder and Dianov 2002; Le Page F. et al., 2000).

Additionally fibroblasts are a good model when studying the inflammatory

response, as they participate in the inflammatory and wound healing

processes by producing various cytokines and immune mediators in response

to LPS and other antigens (Ivor, 1994). The inflammatory response to LPS is

initiated through the TLR4 receptor, the cellular response includes the release

of various signalling molecules including IL-1, IL-6, IL-8, IL-10, MCP-1, MIP-


102

1α, TNF-α and NO (Kurt-Jones et al., 2004; Shirota et al., 2006; Tominaga et

al., 1997).

4.1.1. Aims To identify if the disruption of BER enzymes (specifically the bifunctional DNA

glycosylases OGG1, NTH1 and NEIL1), affects the release of inflammatory

cytokines and chemokines from MEF’s by measuring the levels of IL-6, IL-10

and MIP-1α.

4.2. Results

4.2.1. TLR4 mRNA Transcription Analysis

To confirm that the MEFs would be responsive to LPS challenge via the

TLR4/MyD88 pathway, total mRNA was extracted and an RT-PCR reaction

was performed to test for TLR4 gene expression, using the primers designed

by Kurt-Jones et al. (2004) both of which are found in exon 2 and give rise to

a band of 540 bp (Figure 4.1). This band is present in each cell line indicating

the presence of TLR4 transcription.

Figure 4.1: RT-PCR for TLR4

The 540 bp fragment indicating TLR4 transcription was identified in WT, NTH1-/-, OGG1-/-, NTH/OGG1-/- and NEIL1-/- MEFs.


103

4.2.2. Cytokine Output from LPS Challenged MEF Cells Figures 4.2 – 4.4 show the concentration of the cytokines IL-6, IL-10 and

MIP-1α in the supernatant taken from MEFs cultured with and without LPS.

WT (324.2 ± 31.7 pg/ml) cells have the greatest basal IL-6 output (Figure

4.2A). NTH1-/- and NEIL1-/- MEFs showed significantly lower levels of IL-6

within the supernatant, with NEIL1-/- having the lowest level (71.6 ± 16.5

pg/ml, p=0.01), 22% that of WT MEFs, followed by NTH1-/- (194.3 ± 34.4

pg/ml, p=0.03). The supernatant of OGG1-/- MEFs (232.6 ± 59.5 pg/ml) and

[OGG1/NTH1]-/- MEFs (257.4 ± 64.9 pg/ml) had lower IL-6 content but this

was not significantly different to that of the WT cells (p=0.12 and p=0.25

respectively). There were no significant differences in treated IL-6 levels

between WT and KO MEFs (Figure 4.2B; p>0.29), although all but one result

is between 43 - 53% (NEIL1-/- 1407.9 ± 488.4 pg/ml - OGG1-/- 1726.3 ±

827.8 pg/ml) of the WT (3267.6 ± 606.7 pg/ml). Levels using

[OGG1/NTH1]-/- MEFs were higher (4563.3 ± 1681.9 pg/ml) and were 139%

of the WT result. The increase in IL-6 levels following LPS treatment was

approximately 10 fold using WT MEFs (10.1 fold), slightly lower using OGG1-/-

(7.4 fold) and NTH1-/- MEFs (8.5 fold), and was much greater using

[OGG1/NTH1]-/- (17.7 fold) and NEIL1-/- (19.7 fold) cell lines (Table 4.1).

Basal IL-10 levels differed significantly from WT (131.7 ± 22.1 pg/ml) only

for OGG1-/- MEFs (203.7 ± 44.8 pg/ml; Figure 4.3A; p<0.01). For the other

cell lines, the mean IL-10 concentration was 65 - 93% of the wildtype

concentration ([OGG1/NTH1]-/-, 85.3 ± 16.0 mg/ml - NTH1-/-, 123.0 ± 21.6

pg/ml respectively; p>0.30). IL-10 levels after LPS treatment were similar in

the knockout cell lines when compared to the WT cells. OGG1-/- MEFs

produced more IL-10 than WT cells and each of the other cell lines was 60 -

87% lower. The increases in IL-10 after LPS treatment can be split between

two general categories (Table 4.2), WT (10.1 fold), OGG1-/- and NEIL1-/- (7.1


104

and 7.2 fold respectively), and NTH1-/- (4.8 fold) and [OGG1/NTH1]-/- (4.4

fold).

Basal MIP-1α levels in knockout MEFSs were significantly lower than those in

the spernatant of WT MEFs (262.95 ± 21.7 pg/ml; Figure 4.4A). Of the KO

MEFs, the cell line with the highest output was [OGG1/NTH1]-/- (77.1 ± 21.1

pg/ml), followed by NTH1-/- (53.7 ± 15.3 pg/ml), NEIL1-/- (29.9 ± 6.3 pg/ml)

and OGG1-/- cells (25.3 ± 5.4 pg/ml; p<0.02). After LPS treatment, MIP-1α

levels in the supernatant of KO MEFs were significantly different from WT.

NTH1-/- MEFs (1651.1 ± 346.8 pg/ml) had the highest concentration of MIP-

1α followed by [OGG1/NTH1]-/- (1455.7 ± 466.4 pg/ml), OGG1-/- (1223.4 ±

561.7 pg/ml) and NEIL1-/- cells (360.5 ± 180.9 pg/ml; p<0.03; Figure 4.4B).

In WT cells, the increase from basal rate to treated level of cytokine was 12.9

fold (Table 4.3) but all of the other increases, except for that of NEIL1, was

much higher, the greatest of which was produced by OGG1-/- MEFs (48.5

fold), followed by NTH1-/- (18.9 fold), [OGG1/NTH1]-/- (18.9 fold) and NEIL1-/-

(12.1 fold).


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(A) IL-6 released from untreated cells

(B) IL-6 released from LPS treated cells

Figure 4.2: IL-6 output of wildtype and knockout MEF cell lines Cells were treated with 0.1mg/ml LPS and incubated for 18 h, and the supernatant was then assayed for IL-6 by ELISA. Panels A-B show levels of IL-6 released by the different cell lines tested (A) without LPS treatment and (B) with LPS treatment (+/- SEM, N=5). Significant results are noted for NEIL1-/- (p<0.01) and NTH1-/- (p=0.03) basal results when compared to wildtype.

Table 4.1: Comparison of IL-6 results between untreated and treated DNA glycosylase deficient MEF cellsa

Cell Type IL-6 (pg/ml) Mean

increase Untreated Treated

WT 324.1 ± 31.7

3267.6 ± 606.8

10.1

OGG1-/- 232.6 ± 59.5

1726.3 ± 827.8

7.4

NTH1-/- 194.3 ± 34.1

1657.9 ± 969.2

8.5

[OGG1/NTH1]-/- 257.4 ± 64.9

4563.3 ± 1681.9

17.7

NEIL1-/- 71.6 ± 16.5

1407.9 ± 488.4

19.7

aMean +/- SEM, N=5.

(p>0.29)

(p<0.01)

(p=0.03)

(p=0.12) (p=0.25)


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(A) IL-10 released from untreated cells

(B) IL-10 released from LPS treated cells (p>0.20)

Figure 4.3: IL-10 output of wildtype and knockout MEF cell lines Cells were incubated with 0.1mg/ml LPS for 18 h, and the supernatant was then assayed for IL-10 by ELISA. Panels A-B show levels of IL-10 released by the different cell lines tested (A) without LPS treatment and (B) with LPS treatment (+/- SEM, N=5). There was a significant difference in basal results between WT and OGG1-/- (p<0.01). Table 4.2: Comparison of IL-10 results between untreated and treated DNA glycosylase

deficient MEF cellsa

Cell Type IL-10 (pg/ml) Mean

Increase Untreated Treated

WT 89.6 ± 14.8

906.7 ± 192.5

10.1

OGG1-/- 185.2 ± 33.2

1319.0 ± 432.9

7.1

NTH1-/- 163.2 ± 33.8

789.9 ± 315.7

4.8

[OGG1/NTH1]-/- 122.3 ± 28.8

541.6 ± 239.0

4.4

NEIL1-/- 100.4 ± 24.8

724.0 ± 374.8

7.2

a Mean +/- SEM, N=5.

(p=0.40) (p=0.75) (p=0.34)

(P<0.01)


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(A) MIP-1α released from untreated cells

(B) MIP-1α released from LPS treated cells

Figure 4.4: MIP-1α output of wildtype and knockout MEF cell lines

Cells were incubated with 0.1mg/ml LPS for 18 h, and the supernatant was then assayed for MIP-1α by ELISA. Panels A-B show levels of MIP-1α released by the different cell lines tested (A) without LPS treatment and (B) with LPS treatment (+/- SEM, N=5). Basal MIP-1α levels were reduced when compared to WT in each case (p<0.02). In treated levels MIP-1α was significantly lower in each case (p<0.03). Table 4.3: Comparison of MIP-1α results between untreated and treated DNA glycosylase

deficient MEF cellsa

Cell Type MIP-1α (pg/ml) Mean

Increase Untreated Treated

WT 263 ± 21.7 3400 ± 556.4

12.9

OGG1-/- 25.3 ± 5.4 1223.4 ± 561.7

48.5

NTH1-/- 53.7 ± 15.3#

1651.1 ± 346.8

30.7

OGG1/NTH1-/- 77.1 ± 21.1

1455.7 ± 466.4

18.9

NEIL1-/- 29.9 ± 6.3 360.5 ± 180.9

12.1

aMean +/- SEM, N=5.

(p=0.03)

(p=0.03)

(p<0.01)

(p<0.01)

(p<0.02)


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4.3. Discussion Fibroblast cells are good indicators of level of inflammatory response,

although they are only one cell type and as such are limited. Disruption of

bifunctional DNA glycosylases reduces the basal and LPS treated output of

MIP-1α from all KO MEFs when compared to WT MEFs. Additionally, OGG1-/-

cells had higher basal IL-10 levels, NTH1-/- cells had lower basal IL-6 levels as

did NEIL1-/- cells (Table 4.4).

Table 4.4 Summary of Results from cytokine assays

IL-6 IL-10 MIP-1α

Cell type Untreated LPS Untreated LPS Untreated LPS

OGG1-/- - - ↑ - ↓ ↓

NTH1-/- ↓ - - - ↓ ↓

[OGG1/NTH1] -/- - - - - ↓ ↓

NEIL1-/- ↓ - - - ↓ ↓

↑ = Significant increase compared to WT ↓ = Significant Decrease compared to WT

To explore possible causes for the IL-6 results it is best to identify possible

connections between those MEFS which showed marked reductions in output

of IL-6 namely NTH1-/- and NEIL1-/-. NEIL1 and NTH1 both remove oxidised

cytosines and Tg from DNA, but in experiments to detect these oxidative

compounds in liver tissue, there was no significant difference between either

NTH1-/-, NEIL1-/- or [NTH1/NEIL1]-/- and the control mice (Chan et al., 2009).

It was suggested that this might have been due to a complemntary enzyme,

namely NEIL2, removing the oxidised cytosine products. Additionally, as the

NER system has been shown in vivo to remove Tg at a biologically significant

rate, it may compensate for the lack of BER Tg removal (Reardon et al.,

1997). Even with these backup repair systems in place [NTH1/NEIL1]-/- mice

had a higher incidence of both lung and liver tumours, which was seen as

evidence of a previously unknown base damage type that was not being

excised due to the absence of these genes (Chan et al., 2009). It was also


109

surmised that this unidentified damage type would be highly damaging as it

caused the initiation of cancers in the normally resistant liver tissues (Chan et

al., 2009). If there is such an oxidised base it may be that its reduced

excision from the DNA causes a reduction in the basal release of the

proinflamatory cytokine IL-6. Both NTH1 and NEIL1 are capable of β-

elimination in the BER process, although NEIL1 is also thought to be capable

of APE1 independent β,δ-elimination (Elder and Dianov 2002; Takao et al.,

2002b), but there are no protein intermediates that would be specific to

these two proteins in the BER system to account for why only cells from

these strains produced a reduced basal IL-6 level.

PCNA is a protein which binds to both NEIL1, increasing NEIL1 activity, and

NTH1, the activity of which remains unchanged (Dou et al., 2008; Oyama et

al., 2004). This protein links both NEIL1 and NTH1 to the S-phase as PCNA

plays a role in DNA replication at this point (Kisielewska et al., 2005).

Additionally, due to the positive correlation between PCNA concentration and

severity of inflammation, PCNA has been used as a marker for inflammation

in inflammatory bowel disease and several types of cancer including liver

(Ding et al., 2005; Harrison et al., 1993). As basal IL-6 is also linked to

embryonic development and organ regeneration (Chan et al., 2009; Lee,

1992), this could indicate that a disruption in the NTH1 and NEIL1 genes may

have a deleterious effect at this stage, although our own and previous work

with NTH1 and NEIL1 mice does not suggest one (Chapter 3) (Chan et al.,

2009; Elder and Dianov 2002; Ocampo et al., 2002; Takao et al., 2002a;

Vartanian et al., 2006).

When considering the output of IL-6 it is intersting that the double knockout

of [OGG1/NTH1]-/- showed a greater fold increase (17.5) in output than both

OGG1-/- (8.5) and NTH1-/- (7.4) cells (Table 4.1). It would be logical to

conclude that as the OGG1-/- and NTH1-/- cells both displayed reductions in


110

IL-6 output compared to WT cells, that the double knockout of these proteins

would show a similar, if not addative effect. However there is a precedent for

double knockouts of DNA glycosylases leading to unexpected results. A strain

of OGG1-/- mice was reported to exhibit increased carcinogenesis after 19

months; the double [OGG1/MTH1] knockout was expected to increase the

mouse susceptibility to cancer. However, whilst the double knockout did

increase the occurrence of oxidised base lesions significantly no cancers

found in the animals (Sakumi et al., 2003). The reasoning for this unexpected

result was suspected to be either (i) a build up of oxidised purines

suppressing tumourgenesis by interfering with oncogenic processes or that

(ii) a tumour suppressor gene was transferred from the ES cells on the same

allele as the disrupted MTH1 gene, and was not removed through

backcrossing ((Sakumi et al., 2003); See comments on linkage disequilibrium,

section 3.5)).

As both single knockout cell types used in this study showed a decreased IL-6

response it would suggest that the unexpected result may be due to

increased oxidised bases in the DNA. It has already been reported that an

increase in oxidised lipids in an area can stimulate an immune response of

increased IL-8 and MIP-1α (Berliner and Watson 2005). However, if a similar

effect was in effect here it would most probably also result in a significant

increase in MIP-1α output which is not shown here. It would still be

interesting however, to identify if there was a change in immune response

from cells with greater levels of DNA base oxidation.

Another potential reason for this unexpected result may be is due to the

difference in passage number between the [OGG1/NTH1]-/- stock that were

thawed from stocks of immortalised cells and the WT, OGG1-/- and NEIL1-/-

MEFs which were all created from primary cells; and the NTH1-/- which were

thawed from stocks at passage 10. The immortalised cells may have


111

spontaneously mutated to increase IL-6 production, especially as they are

from a model that is already prone to mutation due to the disruption of BER

processes.

The increased output of basal IL-10 from the OGG1-/- mice is interesting as

IL-10 is most often seen as an anti-inflammatory cytokine, it is a sign of a

concerted anti-inflammatory response from any of the cells rather than a

blanket effect reduction seen in other cell lines such as the treated NTH1-/-

cells.

The reduction in MIP-1α outputs of both basal and treated DNA glycosylase

knockout cells was reduced significantly in all cases. A reason for the

seemingly reduced levels of MIP-1α could be that the WT cells that the DNA

glycosylase knockout cells are compared to are producing an abnormally

large amount of MIP-1α. However, other work with LPS as a ligand has

shown similar levels of MIP-1α being produced from WT MEF’s (Kurt-Jones et

al., 2004). A reduction in MIP-1α would indicate that less neutrophils would

be recruited to the area of inflammation. This would cause a net reduction in

the amount of ROS accumulated in the region and thus less MPO, MDA and

more GSH to be observed indicating, less DNA damage occurring.

The IL-6 released from OGG1-/-, NTH1-/- and NEIL1-/- cells after stimulation

also seemed less than that of WT cells, although not significantly. This could

be explained by a suppression of NF-κβ, but, this would result in a global

suppression of cytokine production (Donadelli et al., 2000). The rise in IL-10

output in OGG1-/- cells would suggest a controlled change in inflammatory

response.

NEIL1-/- cells had the lowest output of MIP-1α both at basal and LPS treated

levels, indicating that the neutrophil response and thus ROS levels in the


112

inflammatory response would be lower than in other strains of animals.

Whilst the effectiveness of MEF’s as a model of immune reactions has already

been discussed (section 4.1), the fact remains that they are colonies of a

single cell type and that as such only the accumulation of the measured

cytokines can be known, there are no mechanisms in place for the dispersal

and clearing of unwanted cytokines. In vivo experiments will be able to give

more detail, on the cytokine response in the body at specific time points and

give more details on how this response relates to neutrophil infiltration and

oxidative damage.


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5. Effects of NEIL1 Gene Knockout on Endotoxin Induced Inflammation

5.1. Introduction

The first protein linking BER to the inflammatory response was PARP-1 in a

model of septic shock. Septic shock was induced via. caecal ligation and

puncture, which resulted in the extrusion of gut contents into the peritoneal

cavity inducing a multi-bacterial response (Soriano et al., 2002; Virag and

Szabo 2002). In PARP1-/- mice there was a reduction in the plasma

concentration of TNF-α, IL-6 and IL-10 cytokines when compared to that of

PARP-1+/+ mice. There was also a reduction in MPO activity and MDA levels in

PARP-1-/- mice indicating that there was a protective effect against the

deleterious effects of inflammation in the PARP1 knockout (Soriano et al.,

2002). OGG1-/- mice have also shown resistance to the deleterious effects of

inflammation induced by LPS and Helicobacter Pylori, with animals displaying

decreased mortality and reduced gut inflammation respectively (Mabley et al.,

2005a; Touati et al., 2006).

NEIL1-/- mice have been described as having symptoms of metabolic

syndrome, a human disease which has been associated with raised serum

levels of the inflammatory markers IL-6, TNFα and fibrinogen (Mabley et al.,

2005a; Touati et al., 2006). However in later studies this phenotype has been

shown to be inconsistent and it has been suggested that the absence of

NEIL1 increases susceptibility to increased weight (metabolic syndrome;

(Chan et al., 2009; Sampath et al., 2011)). A new NEIL1-/- mouse strain has

recently been developed (Chapter 3), and these animals will thus be used to

identify if the NEIL1 protein modifies LPS induced inflammation in order to

further study the links between LPS induced inflammation and the BER

pathway.


114

5.1.1. Aims

To identify the extent to which NEIL1 determines the immune response to

LPS by:

1) Establishing whether seum levels of IL-6, IL-12, IL-10 and IL-4 differ

between NEIL1-/- and NEIL1+/+ mice, at baseline or after LPS

treatment.

2) Identifying whether any LPS induced changes in MPO activity, GSH

levels and MDA levels differ between NEIL1-/- and NEIL1+/+ mice.

3) Determining if the effects of oxidative damage caused by age are

altered in NEIL1-/- mice when compared to NEIL1+/+ mice.

5.2. Results

5.2.1. Cytokine Output in LPS Challenged NEIL1-/- Mice

Figures 5.1 – 5.4 present interleukin concentrations present in the blood

serum of mice injected with LPS (2 mg/kg i.p.) after 0, 1, 6 and 24 h (mean

± SEM; n=6). IL-4 concentrations were not significantly different at any time

point when comparing male NEIL1+/+ and NEIL1-/- mice (Figure 5.1A). There

was, however, a significant difference between WT and KO female mice at 6

h; here the NEIL1+/+ mice had more IL-4 within the serum than NEIL1-/- mice

(216.9 ± 37.0 pg/ml vs. 63.9 ± 15.9 pg/ml; Figure 5.1B; p<0.01).

IL-6 concentrations were significantly different between male WT and KO

mice at baseline (WT 340.8 ± 12.0 pg/ml vs. KO 7.3 ± 5.6 pg/ml; p<0.01), 1

h (WT 5383.5 ± 378.2 pg/ml vs. KO 6690.0 ± 492.7 pg/ml; p=0.04) and 24

h (WT 390.6 ± 458.6 pg/ml vs. KO 29.0 ± 17.7 pg/ml; p<0.01). At 0 and 24

h the WT concentration of IL-6 was higher than KO levels, whilst at 6h the

KO levels were higher than WT levels (Figure 5.2A). There were significant


115

differences between concentrations of IL-6 in the serum of female WT and

KO mice at 1 (WT 7143.1 ± 605.3 pg/ml vs. KO 5518.7 ± 504.0 pg/ml;

p=0.05) and 24 h (WT 2684.6 ± 819.4 pg/ml vs. KO 72.4 ± 16.4 pg/ml;

p<0.01). In both of these cases IL-6 levels were greater in WT serum than

KO serum (Figure 5.2B)

(A) Male IL-4 levels

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Figure 5.1: IL-4 concentrations in blood serum taken from NEIL1 transgenic mice Mice were treated with LPS (2 mg/kg i.p.), sacrificed at the appropriate time and blood removed by cardiac puncture for analysis by ELISA (mean ± SEM; n=6). Panels A and B show serum IL-4 levels at 0, 1, 6 and 24 h in male and female mice respectively. (* - p≤0.05; ** - p≤0.01). (A) No significant difference was observed between WT and KO ♂ mice (p>0.14). (B) There was a significant difference in IL-4 levels between ♀ WT and KO mice at 6 h (p<0.01).

**


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(A) Male IL-6 Levels

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Figure 5.2: IL-6 concentrations in blood serum taken from NEIL1 transgenic mice Mice were treated with LPS (2 mg/kg i.p.), sacrificed at the appropriate time and blood removed by cardiac puncture for analysis by ELISA (mean ± SEM; n=6). Panels A and B show serum IL-6 levels at 0, 1, 6 and 24 h in male and female mice respectively. (* - p≤0.05; ** - p≤0.01). (A) A significant difference in IL-6 levels was observed between WT and KO ♂ mice at 0 (p<0.01), 1 h (p=0.04) and 24 h (p<0.01). (B) There was a significant difference in IL-6 levels between ♀ WT and KO mice at 1 (p=0.05) and 24 h (p<0.01).

IL-10 concentrations did not differ significantly between male WT and KO

mice (p>0.11). There were significant differences between concentrations of

IL-10 in the serum of female WT and KO mice at 6 h (WT 446.2 ± 185.4

pg/ml vs. KO 138.2 ± 57.7 pg/ml; p=0.02) and 24 h (WT 34.3 ± 9.3 pg/ml

vs. KO 24.7 ± 1.6 pg/ml; p<0.01). In both of these cases IL-10 levels were

greater in WT serum than KO serum (Figure 5.3B).

*

**

**

**

*


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Figure 5.3: IL-10 concentrations in blood serum taken from OGG1 transgenic mice Mice were treated with LPS (2 mg/kg i.p.), sacrificed at the appropriate time and blood removed by cardiac puncture for analysis by ELISA (mean ± SEM; n=6). Panels A and B show serum IL-10 levels at 0, 1, 6 and 24 h in male and female mice respectively. (* - p≤0.05; ** - p≤0.01). (A) No significant difference was observed between WT and KO ♂ mice (p>0.11). (B) There was a significant difference in IL-10 levels between ♀ WT and KO mice at 6 (p=0.02) and 24 h (p<0.01).

IL-12 concentrations were significantly different between male WT and KO

mice at 6 h (WT 23.8 ± 3.8 pg/ml vs. KO 36.0 ± 0.7 pg/ml; p<0.01). At this

time point the KO concentration of IL-12 was higher than WT levels (Figure

5.4A). There was no significant difference between NEIL1+/+ and NEIL1-/- in

IL-12 levels at any time point in female mice (p>0.67).

*

**


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Figure 5.4: IL-12 concentrations in blood serum taken from NEIL1 transgenic

mice Mice were treated with LPS (2 mg/kg i.p.), sacrificed at the appropriate time and blood removed by cardiac puncture for analysis by ELISA (mean ± SEM; n=6). Panels A and B show serum IL-12 levels at 0, 1, 6 and 24 h in male and female mice respectively. (* - p≤0.05; ** - p≤0.01). (A) A significant difference was observed between WT and KO ♂ mice at 6 h (p<0.01). (B) There was no evidence of a significant difference in IL-12 levels between ♀ WT and KO mice (p>0.67).

5.2.2. Myeloperoxidase activity in LPS challenged NEIL1-/- mice

The MPO activity in various tissues of male and female WT and NEIL1 KO

mice following treatment with a vehicle or LPS is shown in Table 5.1. Figures

5.5 – 5.9 present MPO activity in the heart, lung, liver, kidney and ileum

**


119

respectively. Panel A of these figures shows the main effects of comparing

treatment (vehicle vs. LPS), genotype (WT vs. NEIL1 KO) and sex (male vs.

female). Panels B - D show the interactions of (B) treatment and genotype,

(C) treatment and sex and (D) genotype and sex. Panel E shows the three

way interaction between treatment, genotype and sex.

In the heart there were no three- or two-way interactions in MPO activity.

There was an overall sex difference with the tissues from female mice (2.62

±0.58 mU/mg protein) having much lower MPO activity than those from the

males (12.06 ±1.16 mU/mg protein; Figure 5.5A; p=0.01).

Similarly in the lung tissues there were no three- or two-way interactions.

Treated animals (118.08 ±17.08 mU/mg protein) had greater MPO activity

than those of the vehicle treated animals (70.84 ±11.09 mU/mg protein;

Figure 5.6A; p=0.02).

Whilst there was no three-way interaction observed in the liver tissues, there

was a significant treatment x sex interaction. Both male and female mouse

liver tissues have similar levels of MPO activity when challenged with LPS

there were radically different levels of MPO activity at basal levels meaning

that whilst activity increased in female mice (5.14 fold), levels actually

decreased in male mice (0.87 fold; Figure 5.7C; p=0.05). Evidence of a

significant treatment x genotype interaction was observed showing that while

treatment caused an increase in MPO activity, this increase was greater in

NEIL1+/+ mice (1.9 fold) than NEIL1-/- mice (1.5 fold; Figure 5.7B; p=0.08).

There were no significant differences observed in kidney tissues (Figure 5.8).

The three-way interaction in ileum tissues was significant. In vehicle treated

female animals and vehicle treated and LPS treated male animals


120

Table 5.1: MPO activity in tissues of NEIL1 transgenic mice exposed to LPS Mice were treated with LPS (20 mg/kg i.p.) or a vehicle (PBS), sacrificed after 12 hours and tissues removed for analysis. Results are shown as tissue MPO activity (mU/mg protein; mean ± SEM; n=6).

Vehicle Treated Treatment x Tissue Male Female Male Female Sex x

WT KO WT KO WT KO WT KO Genotype

Heart 10.28 (±0.80)

12.20 (±0.72)

1.31 (±0.41)

1.99 (±0.97

10.73 (±1.10)

15.03 (±4.77)

3.03 (±1.67)

4.12 (±1.45) p=0.70

Lung 76.41 (±23.19)

130.70 (±24.68)

35.67 (±3.91)

40.60 (±7.55)

119.11 (±33.10)

121.52 (±35.03)

139.77 (±56.43)

91.91 (±30.24) p=0.99

Liver 17.00 (±2.23)

18.85 (±5.21)

1.76 (±0.40)

2.69 (±2.21)

16.85 (±3.39)

14.25 (±2.42)

10.17 (±2.58)

3.62 (±0.98) p=0.30

Kidney 16.18 (±4.84)

13.47 (±3.10)

8.49 (±2.94)

7.64 (±2.15)

25.26 (±15.90)

16.44 (±3.84)

9.77 (±2.74)

18.63 (±4.20) p=0.35

Ileum 40.17 (±22.39)

19.85 (±5.09)

26.71 (±3.54)

24.97 (±5.03)

22.32 (±10.69)

39.99 (±10.43)

84.54 (±21.80)*

39.01 (±4.55)* p=0.02


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(C) Treatment X Sex (p=0.91) (E) Treatment X Genotype (♂&♀) (p=0.70)

Figure 5.5: MPO activity in heart tissues of NEIL1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.), sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. MPO activity was significantly higher in ♀ animals (vs. ♂; Panel A; p<0.01). No other significant effects observed.


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Figure 5.6: MPO activity levels in lung tissues of NEIL1 transgenic mice

exposed to LPS Mice were treated with LPS (20 mg/kg i.p.), sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. MPO activity were significantly higher in vehicle treated animals (vs. treatment; Panel A; p<0.01) no other significant effects were observed.


123

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Figure 5.7: MPO activity in liver tissues of NEIL1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.), sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. An treatment X sex interaction was observed (Panel C; p=0.05). MPO activity was significantly higher in ♀ animals (vs. ♂; Panel A; p<0.01). No other significant effects were observed.


124

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Figure 5.8: MPO activity in kidney tissues of NEIL1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.), sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. No significant effects were observed.


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Figure 5.9: MPO activity in ileum tissues of NEIL1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.), sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. A treatment X sex interaction was observed (Panel C); a significantly lower increase in MPO activity in LPS treated KO ♀’s (vs. treated WT ♀’s; p=0.04). MPO activity was significantly higher in vehicle treated animals (vs. treatment; p=0.03). No other significant differences were observed.


126

there was no significant difference in MPO activity due to genotype,

however LPS treated female NEIL1+/+ mice (84.5 ± 21.8 mU/mg) had

greater MPO activity than NEIL-/- mice (39.0 ± 4.5 mU/mg; Figure 5.9E;

p=0.02).

5.2.3. Malondialdehyde content in LPS challenged NEIL1-/- mice

The MDA levels in various tissues of male and female WT and NEIL1 KO

mice following treatment with a vehicle or LPS are shown in Table 5.3.

Figures 5.10 - 5.14 present MDA levels in the heart, lung, liver, kidney and

ileum respectively. Panel A of these figures shows the main effects of

treatment (vehicle vs. LPS), genotype (WT vs. NEIL1 KO) and sex (male

vs. female). Panels B - D show the main effect interactions of (B)

treatment and genotype, (C) treatment and sex and (D) genotype and

sex. Panel E shows the three way interaction between treatment,

genotype and sex.

There were no significant three- or two-way interactions in heart tissues.

There was a sex difference in that female mouse heart tissues had more

MDA (6.71 ±0.98 nmol/mg protein) than male mice (2.99 ±0.99 nmol/mg

protein; Figure 5.10A; p=0.01). There were no significant differences

found in lung tissue (Figure 5.11).

In the liver there was no significant three-way interaction. However there

was a treatment x genotype interaction observed. Both genotypes had

increases in MDA levels when treated with LPS, the MDA content of

NEIL1+/+ (1.1 fold) increased less than NEIL1-/- (1.6 fold; Figure 5.12B;

p<0.01). There was also a sex difference observed between MDA levels in

which male liver MDA levels (0.78 ±0.15 nmol/mg protein) were lower

than those in the liver of female mice (1.21 ±0.15 nmol/mg protein;

Figure 5.12A; p=0.02).


127

Table 5.2: MDA levels in tissues of NEIL1 transgenic mice exposed to LPS

Mice were treated with LPS (20 mg/kg i.p.) or a vehicle (PBS), sacrificed after 12 hours and tissues removed for analysis. Results are shown as tissue MDA levels (mU/mg protein; mean ± SEM; n=6).

Vehicle Treated Treatment x Tissue Male Female Male Female Genotype x

WT KO WT KO WT KO WT KO Sex

Heart 3.10 (±0.54)

2.64 (±0.61)

4.45 (±0.71)

7.35 (±2.53)

2.81 (±0.46)

3.41 (±0.90)

6.79 (±1.52)

8.25 (±3.03) p=0.55

Lung 4.78 (±0.47)

4.25 (±0.63)

5.37 (±0.53)

4.96 (±0.64)

5.75 (±0.42)

4.99 (±0.22)

4.74 (±1.11)

4.45 (±0.74) p=0.80

Liver 0.51 (±0.11)

0.27 (±0.03)

1.14 (±0.35)

0.94 (±0.30)

0.80 (±0.18)

1.56 (±0.45)

0.99 (±0.37)

1.78 (±0.17) p=0.98

Kidney 2.34 (±0.55)

2.42 (±0.28)

1.79 (±0.45)

2.33 (±0.47)

3.50 (±0.74)

3.35 (±0.59)

1.80 (±0.16)

3.83 (±1.19) p=0.30

Ileum 7.02 (±1.65)

7.28 (±0.85)

6.75 (±1.02)

11.69 (±2.51)

10.51 (±2.23)

10.05 (±1.78)

6.84 (±1.70)

9.86 (±1.97) p=0.80


128

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Figure 5.10: MDA levels in heart tissues of NEIL1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.), sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. MDA levels were significantly higher in ♀ (vs. ♂; Panel A p<0.01). No other significant effects were observed.


129

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Figure 5.11: MDA levels in lung tissues of NEIL1 mice exposed to LPS Mice were treated with LPS (20 mg/kg i.p.), sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. No significant effects were observed.


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Figure 5.12: MDA levels in liver tissues of NEIL1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.), sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. A treatment x genotype interaction was observed (Panel B; p<0.01). MDA levels were significantly higher in treated animals (vs. vehicle treated; Panel A p<0.01), and ♀ (Panal A vs. ♂). No other significant effects were observed.


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Figure 5.13: MDA levels in kidney tissues of NEIL1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.), sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. MDA levels were significantly higher in treated animals (vs. vehicle treated; Panel A; p<0.01). No other significant effect was observed.


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(p=0.34) (p=0.10) (p=0.95)



Figure 5.14: MDA levels in ileum tissues of NEIL1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.), sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. No significant effect was observed.


133

In the kidney and liver tissues there were no significant three- or two-way

interactions. In the kidney there was a significant difference observed

between vehicle treated and LPS treated mouse tissues as vehicle treated

animals (2.22 ±0.02 nmol/mg protein) have lower MDA levels than LPS

treated animals (3.12 ±0.37 nmol/mg protein; Figure 5.13A; p=0.03).

There were no significant interactions or differences observed in MDA

levels in ileum tissues (Figure 5.14).

5.2.4. Glutathione levels in LPS challenged NEIL1-/- mice

The GSH levels in various tissues of male and female WT and OGG1 KO


Figures 5.15 – 5.19 present GSH levels in the heart, lung, liver, kidney and

ileum respectively. Panel A of these figures shows the main effects

comparing treatment (vehicle vs. LPS), genotype (WT vs. NEIL1 KO) and

sex (male vs. female). Panels B - D show the interactions of (B) treatment

and genotype, (C) treatment and sex and (D) genotype and sex. Panel F

shows the three-way interaction between treatment, genotype and sex.

There were no three-way interactions observed in the results for any

tissue. There was a genotype x sex interaction in the heart. Whilst GSH

levels were lower in NEIL-/- animals than NEIL1+/+ animals there was a

greater reduction in the female animals (0.6 fold) than the males (0.8

fold; Figure 5.15D; p<0.01). There was also a decrease in GSH levels

observed due to treatment, with vehicle treated mice (0.34 ±0.03

nmol/mg protein) having greater GSH levels than LPS treated mice (0.27

±0.04 nmol/mg protein; Figure 5.15A; p=0.01).


134

Table 5.3: GSH levels in tissues of NEIL1 transgenic mice exposed to LPS

Mice were treated with LPS (20 mg/kg i.p.) or a vehicle (PBS), sacrificed after 12 hours and tissues removed for analysis. Results are shown as mean tissue GSH levels (mU/mg protein; ± SEM; n=6).

Vehicle Treated Genotype x Tissue Male Female Male Female Treatment x


Heart 0.22 (±0.01)

0.23 (±0.02)

0.56 (±0.04)

0.35 (±0.03)

0.18 (±0.03)

0.08 (±0.03)

0.53 (±0.03)

0.30 (±0.06) p=0.35

Lung 4.13 (±0.65)

3.53 (±0.45)

5.12 (±0.38)

4.27 (±0.40)

4.52 (±1.51)

2.01 (±0.84)

2.57 (±0.22)

2.69 (±0.39) p=0.13

Liver 3.18 (±1.09)

1.25 (±0.32)

4.60 (±1.04)

4.10 (±0.92)

0.15 (±0.00)

0.13 (±0.01)

2.15 (±0.33)

1.06 (±0.13) p=0.14

Kidney 0.24 (±0.06)

0.43 (±0.06)

0.23 (±0.14)

0.09 (±0.03)

0.45 (±0.06)

0.66 (±0.09)

0.08 (±0.02)

0.23 (±0.13) p=0.22

Ileum 0.97 (±0.35)

1.84 (±0.80)

0.68 (±0.10)

0.83 (±0.18)

1.40 (±0.41)

2.36 (±0.74)

0.28 (±0.04)

0.59 (±0.05) p=0.94


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Figure 5.15: GSH levels in heart tissues of NEIL1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.), sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. (Panel A) An genotype X sex interaction was observed (Panel D; p<0.01). GSH levels were significantly lower (p<0.01) in vehicle KO ♂’s (vs. vehicle wt ♂’s) and in treated KO ♂’s and ♀’s (vs. treated WT ♂’s and ♀’s). Panel A: GSH levels were significantly higher (p<0.01) in vehicle treated animals (vs. treatment), WT (vs. KO) and ♀ (vs. ♂).


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Figure 5.16: GSH levels in lung tissues of NEIL1 transgenic mice exposed to LPS

Mice were treated with LPS (20 mg/kg i.p.), sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. GSH levels were significantly higher (Panel A; p<0.05) in vehicle treated animals (vs. treatment), and ♂ (vs. ♀). No other significant effect was observed.


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(A) Main Effects

(p=0.01) (p=0.05) (p=0.01)



Figure 5.17: GSH levels in liver tissues of NEIL1 transgenic mice exposed to LPS

Mice were treated with LPS (20 mg/kg i.p.), sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. GSH levels were significantly higher (Panel A; p<0.05) in vehicle treated animals (vs. treatment), WT (vs. KO) and ♀ (vs. ♂). No other significant effect was observed.


138

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(A) Main Effects

(p=0.06) (p=0.07) (p=0.01)



Figure 5.18: GSH levels in kidney tissues of NEIL1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.), sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. A treatment X sex interaction was observed (Panel C; p=0.05). GSH levels were significantly lower in ♀ (vs. ♂; Panel A; p<0.01). No other significant effect was observed.


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(p=0.78) (p=0.05) (p=0.01)



Figure 5.19: GSH levels in ileum tissues of NEIL1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.), sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. GSH levels were significantly lower (Panel A; p<0.05) in WT animals (vs. KO) and ♀ (vs. ♂). No other significant effect was observed.


140

In lung tissue there were no two-way interactions but there was a

difference observed upon treatment and with genotype. LPS treated mice

(2.95 ±0.44 nmol/mg protein) had lower GSH levels than vehicle treated

mice (3.12 ±0.30 nmol/mg protein; Figure 5.16A; p=0.01) and NEIL1-/-

mice (3.12 ±0.37 nmol/mg protein) had lower GSH levels than NEIL1+/+

mice (4.08 ±0.42 nmol/mg protein; Figure 5.16A; p=0.05).

In liver tissue there were again no significant two-way interactions.

However, there were significant differences with treatment, genotype and

sex (Figure 5.17A). There was a significantly lower level of GSH in LPS

treated mouse liver (3.28 ±0.48 nmol/mg protein) when compared to the

liver of vehicle treated mice (0.87 ±0.19 nmol/mg protein; p=0.01). There

was significantly less GSH in the liver of NEIL1-/- (1.63 ±0.38 nmol/mg

protein) mice when compared to NEIL1+/+ mice (2.52 ±0.48 nmol/mg

protein; p=0.04), and male mouse livers (1.18 ±0.36 nmol/mg protein)

had less GSH than female livers (2.97 ±0.44 nmol/mg protein; p=0.01).

The treatment x sex interaction was significant in kidney. Whilst there was

an increase in GSH levels in male mice (1.6 fold) due to LPS treatment,

there was no real change in female mice (0.93 fold; Figure 5.18C;

p=0.05).

In the ileum there were no significant two-way interactions, but there

were significant differences with genotype and sex. There was significantly

greater GSH in the ileum of NEIL1-/- (1.41 ±0.29 nmol/mg protein) mice

when compared to NEIL1+/+ mice (0.83 ±0.15 nmol/mg protein; p=0.05),

and male mouse ileal tissues (1.64 ±0.29 nmol/mg protein) had greater

GSH levels than female tissues (0.59 ±0.06 nmol/mg protein; p=0.01).


141

5.2.5. Age related cytokine output NEIL1-/- mice

Figures 5.20 – 5.23 detail IL concentrations in mouse blood of animals

sacrificed at 6-8 weeks and 12 months. Panel A shows the age x genotype x

sex interaction, panels B – D detail age x genotype, age x sex and genotype x

sex interactions respectively and panel E shows the main effects.

There were no significant differences observed in IL-4 concentrations although

there was evidence of a significant effect in the sex x age interaction as female

mouse IL-4 levels increased with age (2.20 fold), whilst male IL-4 levels

decreased slightly (0.78 fold; p=0.07).

There was no evidence of an age x genotype x sex interaction in Il-6 levels.

The genotype x age interaction was significant in that whilst the blood IL-6

concentration was similar at 12 months, it was significantly higher at 6-8

weeks in WT mice (28.14 ± 7.54 pg/ml) than NEIL1 KO mice (15.88 ± 9.78

pg/ml), meaning that the reduction due to age was considerably greater in WT

mice (p>0.01). There is a significant genotype x sex interaction observed in

IL-10 levels as whilst IL-10 levels reduced due to disruption of the NEIL1 gene

in female mice (0.54 fold) levels it actually increased in male mice (1.73 fold;

p=0.02). There were no significant differences in IL-12 concentration.

5.3. Discussion

Previous work has identified several BER related genes that are associated

with modulating the inflammatory response (Ando et al., 2008; Mabley et al.,

2005a; Touati et al., 2006; Virag and Szabo 2002). Here we show that the

disruption of the NEIL1 gene reduces initial IL-6 production, but levels of pro-

and anti-inflammatory cytokines indicate an overall increase


142

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IL-4

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(A) Genotype x Age (♀&♂) (p=0.63) (C) Sex x Age (p=0.07)

(B) Genotype x Age (p=0.45) (D) Genotype x Sex (p=0.14)

(E) Main Effects (p=0.32) (p=0.70) (p=0.75)

Figure 5.20: IL-4 concentrations in blood serum taken from NEIL1 transgenic mice Mice were sacrificed at the appropriate time and blood removed by cardiac puncture for analysis by ELISA (mean ± SEM; n=6). Panel A shows 3-way interactions between age, genotype and sex, panels B–D show interactions between age x genotype, age x sex and genotype x sex respectively and panels E shows main effects of age, genotype and sex. No significant interactions were observed in the IL-4 results although there was evidence of a sex x age interaction (Panel C; p=0.07)


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GenotypeIL

-6 (

pg

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Male

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(A) Genotype x Age (♂&♀) (p=0.30) (C) Sex x Age (p=0.87)

(B) Genotype x Age (p<0.01)

(D) Genotype x Sex (p=0.55)

(E) Main Effects (p<0.01) (p=0.03) (p=0.56)

Figure 5.21: IL-6 concentrations in blood serum taken from NEIL1 transgenic mice Mice were sacrificed at the appropriate time and blood removed by cardiac puncture for analysis by ELISA (mean ± SEM; n=6). Panel A shows 3-way interactions between age, genotype and sex, panels B–D show interactions between age x genotype, age x sex and genotype x sex respectively and panels E shows main effects of age, genotype and sex. There was a significant age x genotype interaction (p<0.01), there were also significant differences in the main effects of age (p<0.01) and genotype (p=0.03).


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Female

(A) Genotype x Age

(♂&♀) (p=0.80)

(C) Sex x Age (p=0.21)

(B) Genotype x Age (p=0.97)



Figure 5.22: IL-10 concentrations in blood serum taken from NEIL1 transgenic

mice Mice were sacrificed at the appropriate time and blood removed by cardiac puncture for analysis by ELISA (mean ± SEM; n=6). Panel A shows 3-way interactions between age, genotype and sex, panels B–D show interactions between age x genotype, age x sex and genotype x sex respectively and panels E shows main effects of age, genotype and sex. There was a significant genotype and sex interaction (p=0.02).


145

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Male WT Male KO Female WT Female KO

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(A) Genotype x Age (♂&♀) (p=0.94) (C) Sex x Age (p=0.61)

(B) Genotype x Age (p=0.36)



Figure 5.23: IL-12 concentrations in blood serum taken from NEIL1 transgenic mice

Mice were sacrificed at the appropriate time and blood removed by cardiac puncture for analysis by ELISA (mean ± SEM; n=6). Panel A shows 3-way interactions between age, genotype and sex, panels B–D show interactions between age x genotype, age x sex and genotype x sex respectively and panels E shows main effects of age, genotype and sex. No significant differences were observed.


146

in inflammation after 6 h. There are tissue specific differences in tissue

GSH content: in the heart, lung and liver of NEIL1-/- mice there is less GSH

implying more ROS are produced, but in the ileum tissues GSH levels rise

(Table 5.5).

Table 5.4 Summary of NEIL1 transgenic mouse blood serum cytokine results

** = p≤0.01, * = 0.05≥p>0.01, - = p≥0.10, ↓/↑ = comparison to WT. The detection of LPS by monocytes initiates an immune signalling

cascade; this is made up of many cytokines, and other signalling proteins

(Moller and Villiger 2006). The upregulation of IL-6 is one of the primary

elements in this cascade which goes on to stimulate increased serum

levels of other cytokines including the Th1 cytokine, IL-12, and the Th2 cell

stimulating anti inflammatory cytokines, IL-4 and 10 (Moller and Villiger

2006; Reed and Milton 2001; Wan et al., 2000). Here we have found that

the basal levels of IL-6 are significantly lower in male NEIL1-/- mice, and

that in response to the LPS challenge the level of IL-6 at 1 h in the serum

was lower in NEIL1-/- female mice, indicating a slower initial output of IL-

6. 6 h after treatment the levels of IL-12 (pro-inflammatory) in male

animals had increased but levels of the anti-inflammatory cytokines (IL-4

and 10) in female mice had decreased. These results may indicate

differing immune responses to compensate for the initially slowed IL-6

signalling. These differences in Th1/Th2 responses between male and

female mice can be explained by oestrogens ability to modulate the

immune response reducing Th1 activity and increasing that of Th2 (Salem,

2004). Alternatively, the disruption of the NEIL1 protein could be

modulating the

Time (h)

0 1 6 24

Cytokine Male Female Male Female Male Female Male Female

IL-4 - - - - - **↓ - -

IL-6 **↑ - *↓ *↑ - - **↓ **↓

IL-10 - - - - - **↓ - *↓

IL-12 - - - - **↑ - - -


147

Table 5.5: Summary of NEIL1 organ damage results

** = p≤0.01, * = 0.05≥p>0.01, ~ = 0.10>p>0.05, - = p≥0.10, ↓/↑ = comparison.

Primary interactions Secondary Interactions Tertiary

Interaction

Treatment (LPS Treated vs. Vehicle)

Genotype (KO vs. WT)

Sex (♀ vs. ♂)

Treatment x

Genotype (Increase in KO

vs. WT)

Treatment x Sex

(Increase in ♀ vs. ♂)

Genotype x Sex


Treatment x Genotype x

Sex

MPO

Heart - - **↓ - - - -

Lung *↑ - - - - - -

Liver - - *↓ ~↓ *↑ - -

Kidney - - - - - - -

Ileum *↑ - - - *↓ - *

MDA

Heart - - **↑ - - - -

Lung - - - - - - -

Liver **↑ - *↑ **↑ - - -

Kidney *↑ - - - - - -

Ileum - ~↑ - - ~↓ ~↓ -

GSH

Heart **↓ **↓ **↑ - - **↓ -

Lung **↓ *↓ - - - - -

Liver **↓ *↓ **↑ - - - -

Kidney ~↑ ~↑ **↓ - *↓ ~↓ -

Ileum - *↑ **↓ - - - -


148

activation of transcription factors such as NF-κβ and AP-1,

maybe via. PARP1 or APE1. In order to discern if this is the case

electrophoretic mobility-shift assays could be performed using cell extracts

from cultured cells (Abdel-Latif et al., 2004).

The chemoattractants such as MIP-1α released as part of this signaling

cascade induce leukocytes, in particular neutrophils, to the area of

invasion. These cells produce an array of bactericidal compounds,

including ROS and RNS. These oxidizing compounds are produced via. a

network of enzymes including superoxide dismutase (SOD) and

myeloperoxidase (MPO), the activity of which can be measured as an

indication of neutrophil infiltration. In mice treated with LPS (80 mg/kg

i.p.) a significant increase in MPO activity can be measured after 4 h and

this activity continues to increase up to 24 h (Kabir et al., 2002; Yamashita

et al., 2000). In the case of these NEIL1-/- mice there was no significant

difference in MPO levels compared to NEIL1+/+ mice, this indicated a

reduced chemoattractant activity as suggested in the results from the

NEIL1-/- cells (section 4.2).

ROS produced by neutrophils, of course, are not specifically bactericidal

and a build up of these compounds can cause cellular damage. One

measure of this damage is MDA levels. Previous studies have shown that

when mice are treated with LPS the MDA concentrations in the liver,

kidney, lung, gut and heart tissues are raised significantly, although not

significantly so in brain tissue (Mabley et al., 2005a; Sebai et al.,

2010)accumulation of ROS within the system, which are then neutralised

via the GSH pathway. Interestingly there have been cases observed when

the immune response has shown markers of oxidative damage to increase

without an increase in neutrophil aggregation, in these cases it is thought

to be due to an increase in NO secondary messengers released as part of

inflammation (Pheng et al., 1995).


149

Raised levels of NO have been shown to increase the creation of Tg in

replicated DNA significantly. TG can block the progress of repair or

replicative DNA polymerases along the DNA strand (Aller et al., 2007).

NEIL1 has been implicated in the S-phase and repair within the replication

fork. Thus the reduced release of Tg from the ssDNA, due to the knockout

of NEIL1, could potentially be extending the S-phase and disrupting

production of both cytokines and neutrophils (Orr et al., 2007; Orren et

al., 1997).

NEIL1 has also been shown to interact with several proteins that have no

effect on the activity of NEIL1 including DNA ligase IIIα, polβ and OGG1

(Mokkapati et al., 2004a). DNA ligase and polβ both interact with XRCC1,

and whilst XRCC1 has no catalytic properties of its own it does interact

with both PARP-1 and APE1 (Mutamba et al., 2011). Mutations in XRCC1

have been shown to be associated with various inflammation-associated

malignancies including an increase the prevalence of breast cancer (Duell

et al., 2001) and reduced susceptibility to oesophageal (Lee et al., 2001),

lung (Ratnasinghe et al., 2001) and bladder cancers (Stern et al., 2001).

Additionally these proteins also interact with PARP-1 (Mutamba et al.,

2011), the knockout of which has been shown to reduce the immune

response to endotoxin (Virag and Szabo 2002). However, this inhibition is

thought to be due to a blanket reduction in all cytokine activity due to the

inhibition of NF-κβ and AP-1 with which it has been shown to be a co-

factor with (Kiefmann et al., 2004). This was not observed in the

developed NEIL1-/- mice which exhibited both increases and decreases in

cytokine production.

A number of proteins have been shown to interact with NEIL1 increasing

its activity including FEN-1, PCNA, CSB and WRN (Das et al., 2007a; Dou

et al., 2008; Hegde et al., 2008; Muftuoglu et al., 2009). WRN has also


150

been shown to be linked to inflammation; siRNA inhibition of this protein

has been shown to increase both IL-6 production and the activation of NF-

κβ (Turaga et al., 2009). As NEIL1 is activated by WRN and there was no

overall increase in inflammatory damage it is probable that this is not the

pathway by which inflammation is modulated.

FEN-1 as with many of these proteins does also interact with PARP-1

(Bouchard et al., 2003) and has been implicated in autoimmunity

disorders, chronic inflammation and cancer formation (Kucherlapati et al.,

2002; Lam et al., 2006; Zheng et al., 2007). More recently it was

suggested that the upregulation of this protein was responsible for the

increased inflammatory mediated cancer risk. The mechanism implicated

in this rise in inflammatory signals was the hypomethylation of key

promoter signals, leading to the hyperexpression of genes (Singh et al.,

2008). The lack of NEIL1 protein to bind with may therefore increase free

FEN-1 proteins within the cell reducing promoter methylation, and thus

increasing these inflammatory signals.

PCNA interacts with NEIL1 during the S-phase as it plays a role in DNA

replication at this point (Kisielewska et al., 2005). Additionally, due to the

positive correlation between PCNA concentration and severity of

inflammation, PCNA has been used as a marker for inflammation in

inflammatory bowel disease and several types of cancer including liver

(Ding et al., 2005; Harrison et al., 1993).

Little is known how the CSB protein may influence inflammatory response

but in an experiment in which CSB-/- mice were exposed to ozone, the

basal IL-6 levels in these mice were higher and the IL-6 response of the

mice in question was accelerated whilst the accumulation of oxidative

damage was decreased (Kooter et al., 2008). Whilst there was an

increased basal IL-6 level in the male NEIL1-/- mice, the IL-6 production

was not accelerated as it was in the CSB-/- mice. Although no mechanism


151

for CSB affecting the immune response has been forthcoming, the

removal of the NEIL1 protein may result in excess “free” CSB within the

cell.

Each of these proteins, including NEIL1 itself, can be linked to

inflammation via APE1. This enzymes expression is activated by the

presence of ROS such as those released by the innate immune response

(Yang et al., 2007). APE1 activates transcription factors NF-κΒ and AP-1

by the reduction of active cystine residues (Ando et al., 2008). The down

regulation of the disruption of APE1 activity has been shown to decrease

the activation of transcription factors NF-κΒ and AP-1 (Daily et al., 2001;

Fung and Demple 2005).

As the mice got older, basal levels of the cytokine IL-6 decreased,

however aging is more often cited as causing increased IL-6 production

(Daynes et al., 1993; Ershler et al., 1993), but there is controversy on this

point as cases exist where no change (Beharka et al., 2001) or a decrease

is observed (Boehmer et al., 2004; Goodman and Stein 1994; Sharman et

al., 2001). These NEIL1-/- mice had initially reduced basal levels of IL-6

which reduced to similar levels as WT mice at 12 months. A decrease in

basal IL-6 is thought to be due to reduced efficiency in the transcription

pathways, particularly the JNK and p38 pathways (Boehmer et al., 2004;

Goodman and Stein 1994) or due to the deregulation of IL-6 with age,

due to inhibition of NF-κβ by an increase in Th2 activity (Ye and Johnson

2001). Another hypothesis is that in younger mice, IL-6 plays a role in

developmental processes such as osteoclastogenesis, vascular

development and the development of the brain and that the basal level of

IL-6 reduces when it is no longer needed (Sharman et al., 2001).

The large numbers of proteins that may link through NEIL1 to

inflammation indicate that the BER repair system and inflammation may

be linked at several points, however there are many points where the BER


152

system “bottlenecks” i.e. after APE1 has cleaved the 3' PUA leaving a gap

that can be repaired by Pol β and DNA ligase III/XRCC1.

In conclusion, the knockout of the NEIL1 gene seems to slow the initiation

of the immune response, although this is compensated for by an increase

in the proinflammatory cytokine, IL-12, in males and a decrease in the

anti-inflammatory ILs 10 and 4 in the blood serum. Additionally there was

no difference in levels of MPO or MDA between NEIL1-/- and NEIL1+/+

mice, although there was differences observed between overall GSH,

indicating a mild ROS increase. The results shed more light on how

previously identified BER proteins may have modulated inflammation but

more work need to be done to identify the specific pathway.


153

6. Effects of OGG1 Gene Knockout on Endotoxin Induced Inflammation

6.1. Introduction

As outlined previously base excision repair proteins such as PARP-1, APE-

1 and MUTYH have been implicated in the regulation of the immune

system in response to endotoxin (Sections 1.3.1. & 1.3.2.) (Casorelli et al.,

2010; Virag and Szabo 2002). There is also evidence that another base

excision repair protein, OGG1, may have potentially protective effects

against endotoxin or helicobacter pylori induced inflammation (Mabley et

al., 2005a; Touati et al., 2006). This was first reported in 2005 when it

was shown that after LPS treatment (55 mg/kg i.p.) the mortality in OGG1-

/- mice was lower than that in wildtype animals. The levels of the IL-12

and TNF-α in blood plasma were reduced in OGG1-/- mice when compared

with OGG1+/+ mice as were levels of MIP-1α. Additionally, levels of anti-

inflammatory cytokines IL-4 and IL-10 were increased in the plasma of the

OGG1-/- mice when compared to OGG1+/+ mice, indicating an overall

suppression of the immune system. MPO activity in heart and lung tissues

was significantly reduced in OGG1-/- mice, compared to OGG1+/+ mice

after the mice were challenged with 80 mg/kg LPS (i.p.). Additionally MDA

levels were also reduced in the lung, heart and liver of OGG1-/- mice when

compared to their OGG1+/+ counterparts. These results suggest that the

reduction in levels of pro-inflammatory cytokines and chemokines, and the

increase in anti-inflammatory cytokines reduced neutrophil recruitment

and activation, which in turn attenuated the production of MDA (Mabley et

al., 2005a).

The same strain of OGG1-/- mice, this time bred onto a Big blue mouse

background useful for the observation of mutations (Hill et al., 1999),

infected with the gram-negative bacteria helicobacter pylori (H. pylori)

exhibited less severe histological lesions of the gastric mucosa than those

of OGG1+/+ mice. Additionally, infiltration of polymorphonuclear cells


154

(neutrophils, basophils and eosinophils) was observed in the gut tissue of

33% of OGG1-/- mice treated with H. Pylori , compared to 100% of the

OGG1+/+ mice, indicating that the recruitment of these cells was also

attenuated (Touati et al., 2006).

These studies support the hypothesis that there is a link between OGG1

and the inflammatory response, but this evidence has not been confirmed

by other sources.

6.1.1. Aims To identify the extent to which OGG1 determines the immune response to

LPS induced inflammation by:

1. Establishing whether levels of IL-6, IL-12, IL-10 and IL-4 differ

between OGG1-/- and OGG1+/+ mouse serum, at baseline and

after LPS treatment.

2. Identify whether changes in MPO activity, GSH levels and MDA

levels induced by LPS differ between OGG1-/- and OGG1+/+ mice.

6.2. Results

6.2.1. Cytokine Output in LPS Challenged OGG1-/- Mice

Figures 6.1 – 6.4 present interleukin concentrations measured in blood

serum of mice after 0, 1, 6 and 24 h after injection with LPS (2 mg/kg i.p.;

mean ± SEM; n=6). There was a significant difference in IL-4 levels

between WT and KO male mice at 6 h, here the OGG1+/+ (18.1 ± 0.9

pg/ml) mice having more IL-4 in their serum than OGG1-/- mice (12.9 ±

2.4 pg/ml; Figure 6.1A; p=0.05). IL-4 concentrations were not

significantly different at any time point when comparing female OGG1+/+

and OGG1-/- mice (p>0.55).

Serum IL-6 concentrations were significantly different between male WT

and KO mice at baseline (WT 5.2 ± 1.4 pg/ml vs. KO 1.8 ± 0.6 pg/ml;


155

p<0.01), at which point the KO concentration of IL-6 was higher than WT

levels (Figure 6.2A).

(A) Male IL-4 levels

0

50

100

150

200

250

300

350

0 1 6 24

Time (h)

IL-4

(pg

/ml)

WT

KO


0

50

100

150

200

250

300

350

0 1 6 24

Time (h)

IL-4

(pg

/ml)

WT

KO

Figure 6.1: IL-4 concentrations in blood serum taken from OGG1 transgenic

mice Mice were treated with LPS (2 mg/kg i.p.), sacrificed at the appropriate time and blood removed by cardiac puncture for analysis by ELISA (mean ± SEM; n=6). Panels A and B show serum IL-4 levels at 0, 1, 6 and 24 h in male and female respectively. (* - p≤0.05; ** - p≤0.01). A significant difference was observed between WT and KO ♂ mice at 1 (p=0.04) and 6 h (p=0.05). There was no significant difference in IL-4 levels between ♀ WT and KO mice (p>0.55).

There were significant differences between concentrations of IL-6 in the

serum of female WT and KO mice at 1 h (WT 414.3 ± 46.5 pg/ml vs. KO

311.2 ± 23.1 pg/ml; p<0.01) at which point the IL-6 levels in WT serum

was higher than that of the KO mice. After 24 h (WT 2.6 ± 2.5 pg/ml vs.

KO 23.8 ± 1.2 pg/ml; p<0.01; Figure 6.2B).

*

*


156


0

1500

3000

4500

6000

7500

9000

0 1 6 24

Time (h)

IL-6

(pg

/ml)

WT

KO


0

1500

3000

4500

6000

7500

9000

0 1 6 24Time (h)

IL-6

(pg

/ml)

WT

KO


mice Mice were treated with LPS (2 mg/kg i.p.), sacrificed at the appropriate time and blood removed by cardiac puncture for analysis by ELISA (mean ± SEM; n=6). Panels A and B show serum IL-6 levels at 0, 1, 6 and 24 h im male and female mice respectivly. (* - p≤0.05; ** - p≤0.01). a significant difference was observed between WT and KO ♂ mice at 0 h (p<0.01). There was aaso significant difference in IL-6 levels between ♀ WT and KO mice at 1 (p<0.01) and 24 h (p<0.01).

There were significant differences between concentrations of IL-10 in the

serum of WT and KO male mice at 6 h (WT 2356.6 ± 414.7 pg/ml vs. KO

3730.1 ± 381.9 pg/ml; p=0.02) and 24 h (WT – 60.2 ± 52.7 pg/ml vs. KO

1498.2 ± 228.9 pg/ml; p<0.01). In both of these cases IL-10 levels were

greater in KO serum than WT serum (Figure 6.3A). Similarly there were

significant differences between concentrations of IL-10 in the serum of

female WT and KO mice at 6 h (WT 5447.7 ± 103.8 pg/ml vs. KO 4323.5

**

**

**


157

± 321.1 pg/ml; p<0.01) and 24 h (WT 63.1 ± 1.8 pg/ml vs. KO 45.7 ±

1.9 pg/ml; p<0.01). However, in these cases the levels of IL-10 in KO

mice were lower than those of the WT mice (Figure 6.3B).


02000400060008000

100001200014000160001800020000

0 1 6 24

Time (h)

IL-1

0 (p

g/m

l)

WT

KO


02000400060008000

100001200014000160001800020000

0 1 6 24

Time (h)

IL-1

0 (p

g/m

l)

WT

KO


mice Mice were treated with LPS (2 mg/kg i.p.), sacrificed at the appropriate time and blood removed by cardiac puncture for analysis by ELISA (mean ± SEM; n=6). Panels A and B show serum IL-10 levels at 0, 1, 6 and 24 h in male and female mice respectvely. (* - p≤0.05; ** - p≤0.01). A significant difference was observed between WT and KO ♂ mice at 6 (p=0.02) and 24 h (p<0.01). There was a significant difference in IL-10 levels between ♀ WT and KO mice at 6 and 24 h (p<0.01).

Whilst there was no significant differences in serum IL-12 concentrations

between OGG1+/+ and OGG1-/- mice in either male or female mice, there

was evidence of a significant difference between OGG1+/+ and OGG1-/- in

* **

**

**


158

IL-12 levels at 1 h in female mice (WT 77.4 ± 68.2 pg/ml vs. KO 50.7 ±

24.9 pg/ml; Figure 6.4B; p=0.06).


050

100150200250300350400450500

0 1 6 24

Time (h)

IL-1

2 (p

g/m

l)

WT

KO

(B) Female IL-12 levels

050

100150200250300350400450500

0 1 6 24

Time (h)

IL-1

2 (p

g/m

l)

WT

KO


mice Mice were treated with LPS (2 mg/kg i.p.), sacrificed at the appropriate time and blood removed by cardiac puncture for analysis by ELISA (mean ± SEM; n=6). Panels A and B show serum IL-12 levels at 0, 1, 6 and 24 h in male and female mice respectively. (* - p≤0.05; ** - p≤0.01). No significant difference was observed between WT and KO ♂ mice at any time point (p>0.42). There was evidence of a significant difference in IL-12 levels between ♀ WT and KO mice at 1 h (p=0.06).


159

6.2.2. Myeloperoxidase Activity in LPS Challenged OGG1-/- Mice

MPO activity in various tissues of male and female OGG1+/+ and OGG1-/-


Figures 6.5 – 6.9 present MPO activity levels in the heart, lung, liver,

kidney and ileum respectively. Panel A of these figures shows the main

effects of treatment (vehicle vs. LPS), genotype (WT vs. OGG1 KO) and


and genotype, (C) treatment and sex and (D) genotype and sex. Panel E

shows the three way interaction between treatment, genotype and sex.

There was no significant treatment x genotype x sex, genotype x sex or

treatment x genotype interaction in determining heart MPO activity (Figure

6.5). There was evidence of a treatment x sex interaction (Figure 6.5C;

p=0.08) in that treatment decreased MPO activity in female mice from

28.1 (± 5.3) mU/mg protein to 15.8 (±2.2) mU/mg protein whereas there

was no effect of treatment on MPO activity in male mice (i.e. both WT and

Ogg1 KO mice combined). MPO activity in the heart did not vary with

genotype.


160

Table 6.1: MPO activity in tissues of OGG1 transgenic mice exposed to LPS

Mice were treated with LPS (20 mg/kg i.p.) or vehicle (PBS), sacrificed after 12 hours and tissues removed for analysis. Tissue MPO activity (mU/mg protein) results are shown as mean ± SEM (n=6).

Vehicle Treated Treatment X Tissue Male Female Male Female Genotype X


Heart 18.71 (±1.06)

16.54 (±2.32)

25.48 (±6.78)

30.65 (±9.22)

18.02 (±.39)

15.34 (±2.19)

17.67 (±4.48)

14.57 (±2.02) p=0.53

Lung 29.75 (±2.96)

45.01 (±4.49)

33.10 (±7.04)

27.50 (±4.59)

122.96 (±13.61)*

195.86 (±29.61)*

119.52 (±11.54)

120.04 (±11.47) p=0.16

Liver 10.75 (±1.93)

13.13 (±1.76)

9.66 (±2.06)

9.37 (±1.62)

46.32 (±5.73)

64.41 (±10.18)

45.26 (±4.01)

35.07 (±2.48) p=0.05

Kidney 6.69 (±1.91)

5.29 (±1.33)

9.20 (±1.95)

8.64 (±0.99)

13.26 (±2.04)

20.50 (±5.63)

13.44 (±1.48)

13.35 (±1.58) p=0.24

Ileum 11.05 (±1.04)

12.94 (±2.77)

28.33 (±8.82)

25.62 (±5.88)

19.38 (±4.95)

16.73 (±5.67)

9.07 (±3.66)

27.84 (±5.05) p=0.07


161

0

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(A) Main Effects

(p=0.40) (p=0.82) (p=0.11)



Figure 6.5: MPO activity in heart tissues of OGG1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.) or vehicle only, sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. No significant effect were observed.


162

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(A) Main Effects



Figure 6.6: MPO activity in lung tissues of OGG1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.) or vehicle only, sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. A significant genotype X sex interaction was observed. MPO activity was significantly higher in treated WT ♂ (vs. vehicle treated WT ♂; p<0.02). MPO activity was significantly lower (p<0.03) in vehicle treated animals (vs. treatment), WT (vs. KO) and ♀ (vs. ♂).


163

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(A) Main Effects

(p=0.01) (p=0.43) (p=0.01)



Figure 6.7: MPO activity levels in liver tissues of OGG1 transgenic mice

exposed to LPS Mice were treated with LPS (20 mg/kg i.p.) or vehicle only, sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. Significant group X sex and genotype X sex interactions (p<0.05) were observed. MPO activity was significantly lower (p=0.01) in vehicle treated mice (vs. treated) and ♀ (vs. ♂). No other significant interactions were observed.


164

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Figure 6.8: MPO activity in kidney tissues of OGG1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.) or vehicle only, sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. MPO activity was significantly lower in vehicle treated animals (vs. treatment; p<0.01). No other significant effect was observed.


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Figure 6.9: MPO activity in ileum tissues of OGG1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.) or vehicle only, sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. A significant treatment X sex interaction was observed in (p=0.04). MPO activity was significantly higher in ♀ (vs. ♂; p=0.03). No other significant effect was observed.


166

In the lung there was no significant three way interaction observed in

MPO activity, although, there was a significant interaction between

genotype and sex (Figure 6.6D; p=0.01). Whilst there was an increase in

MPO activity in male animals due to the knockout of the Ogg1 gene (1.6

fold) there was no significant difference in MPO activity in female mouse

lung tissue (1.1 fold). The treatment x genotype (Figure 6.6B; p=0.08)

and treatment x sex (Figure 6.6D; p=0.08) interactions both approached

significance in that whilst in each case MPO activity increased due to

treatment that increase was greater in Ogg1-/- (4.7 fold vs. 3.9 fold)

animals and male mice (4.3 fold vs. 4.0 fold).

In the liver there was a significant three way interaction between

treatment, genotype and sex in determining MPO activity (Figure 6.7E;

p=0.05). In male and female mice, treatment resulted in a significant

increase in MPO activity in both Ogg1+/+ and Ogg1-/- mice. Whereas in

both male and female WT mice this increase was similar (4.3 fold in male

and 4.7 in female), in male Ogg1-/- mice this increase was 6.7 fold and in

female mice only 3.7 fold.

In the kidney there was no significant three way interaction between

treatment, genotype and sex (Figure 6.8E). There was an interaction

approaching significance between treatment and sex (Figure 6.8C;

p=0.07) in that whilst treated animals had greater MPO activity, these

levels rose more in male mice (2.8 fold) when compared to female mice

(1.5 fold). Interestingly whilst the female mice initially had lower levels of

MPO activity, after treatment MPO activity levels were higher than those of

the males (Figure 6.8C).

In the ileum there was no significant three way interaction, although it did

approach significance (Figure 6.9E; p=0.07). The treatment x sex

interaction was significant (Figure 6.9C; p=0.04), indicating that whilst

MPO activity of LPS treated male (18.1 ±3.5 mU/mg protein) and female


167

(20.3 ±4.6 mU/mg protein) mice were not significantly different, vehicle

treated female mice had significantly higher MPO activity levels (27.0 ±4.8

mU/mg protein) when compared to that of the males (12.0 ±1.4 mU/mg

protein) leading to a drop in MPO activity levels when treated with LPS.

Mean MPO activity was significantly higher in female mice (22.0 ±3.3

mU/mg protein) than males (15.0 ±1.9 mU/mg/protein; Figure 6.9A;

p=0.03).

6.2.3. Malondialdehyde Content in LPS Challenged OGG1-/- Mice

The MDA levels in various tissues of male and female OGG1+/+ and OGG1-

/- following treatment with a vehicle or LPS is shown in Table 6.3. Figures

6.10 – 6.14 present MDA levels in the heart, lung, liver, kidney and ileum

respectively. Panel A of these figures shows the main effects of treatment

(vehicle vs. LPS), genotype (WT vs. OGG1 KO) and sex (male vs. female).

Panels B - D show the interactions of (B) treatment and genotype, (C)

treatment and sex and (D) genotype and sex. Panel E shows the three

way interaction between treatment group, genotype and sex.

There were no three way interactions observed in the results of the MDA

assays, neither were there many two way interactions. Within the heart

there were no significant differences at all (Figure 6.10). In the lung there

were significant differences in MDA levels observed between vehicle

treated animals (5.5 ±0.6 nmol/mg protein) and those treated with LPS

(7.4 ±0.8 nmol/mg protein; Figure 6.11A; p=0.05). There was also a

significant difference observed between MDA levels in male (5.0 ±0.4

nmol/mg protein) and female mice (7.7 ±0.8 nmol/mg protein; Figure

6.11A; p=0.01).


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Table 6.2: MDA levels in tissues of OGG1 transgenic mice exposed to LPS

Mice were treated with LPS (20 mg/kg i.p.) or a vehicle (PBS), sacrificed after 12 hours and tissues removed for analysis. Tissue MPO activity (mU/mg protein) results are shown as mean ± SEM (n=6).



Heart 0.40 (±0.11)

0.94 (±0.37)

1.08 (±0.21)

0.64 (±0.10)

1.07 (±0.19)

1.00 (±0.30)

0.70 (±0.10)

0.79 (±0.10) p=0.06

Lung 6.48 (±1.71)

6.02 (±1.68)

5.16 (±0.95)

4.27 (±0.51)

6.93 (±1.00)

11.39 (±1.97)

5.58 (±0.76)

5.04 (±0.78) p=0.19

Liver 2.11 (±0.16)

2.87 (±0.60)

1.93 (±0.32)*

0.76 (±0.20)*

3.33 (±0.41)

4.69 (±0.56)

1.08 (±0.13)

0.37 (±0.07) p=0.90

Kidney 1.20 (±0.14)

1.11 (±0.18)

1.10 (±0.14)

1.68 (±0.19)

1.57 (±0.19)

2.12 (±0.24)

2.12 (±0.40)

2.91 (±0.97) p=0.69

Ileum 3.21 (±0.75)

6.25 (±2.12)

5.54 (±0.48)

4.77 (±0.45)

6.29 (±0.97)

6.36 (±1.68)

7.37 (±0.82)

8.85 (±1.22) p=0.11


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Figure 6.10: MDA levels in heart tissues of OGG1 transgenic mice exposed to LPS

Mice were treated with LPS (20 mg/kg i.p.) or vehicle only, sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. No significant effects were observed.


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Figure 6.11: MDA levels in lung tissues of OGG1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.) or vehicle only, sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. MDA levels were significantly higher (p<0.01) in treated animals (vs. vehicle treated), and ♀ (vs. ♂). No other significant effects were observed.


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Figure 6.12: MDA levels in liver tissues of OGG1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.) or vehicle only, sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. A significant treatment x sex and genotype X sex interactions were observed (p<0.01). Significantly lower levels of MDA were detected in vehicle treated WT ♀’s (vs. treated WT ♀’s; p<0.01). MDA levels were significantly lower in ♀ (vs. ♂; p<0.01).


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Figure 6.13: MDA levels in kidney tissues of OGG1 mice exposed to LPS Mice were treated with LPS (20 mg/kg i.p.) or vehicle only, sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. MDA levels were significantly higher in vehicle treated animals (vs. treatment; p<0.01). No other significant effects were observed.


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Figure 6.14: MDA levels in ileum tissues of OGG1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.) or vehicle only, sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. MDA levels were significantly higher in treated animals (vs. vehicle treated; p<0.01). No other significant effects were observed.


174

In liver tissues significant treatment x sex (Figure 6.12C; p=0.01) and

genotype x sex (Figure 6.12D; p=0.01) interactions were observed. The

treatment x sex interaction reflects that whilst the male animals

responded to the LPS treatment with an increase in MDA (1.6 fold), there

was a decrease observed in the female animals (0.5 fold). Similarly whilst

there was an increase in MDA levels (1.4 fold) in liver tissues from male

mice due to the knockout of the OGG1 gene, the female animals seemed

to have a decrease in MDA levels (0.4 fold) in the liver.

In the kidney, there was only one significant difference and that was

between the two treatment groups (Figure 6.13A), the vehicle treated

animals had significantly lower levels of MDA (1.3 ±0.1 nmol/mg protein)

than those of the animals treated with LPS (2.2 ±0.3 nmol/mg protein;

p=0.01). Similarly this was the only difference in the ileum tissues

showing again that vehicle treated animals had lower levels of MDA (4.9

±1.0 nmol/mg protein) than those treated with LPS (7.2 ±0.6 nmol/mg

protein; Figure 6.14A; p=0.01). There were no other significant

differences at any level in both organs.

6.2.1. Glutathione Activity in LPS Challenged OGG1-/- Mice

The GSH levels in various tissues of male and female OGG1+/+ and OGG1-

/- mice following treatment with a vehicle or LPS is shown in Table 6.4.

Figures 6.15 – 6.19 present GSH activity levels in the heart, lung, liver,

kidney and ileum respectively. Panel A of these figures shows the main

effects of treatment (vehicle vs. LPS), genotype (WT vs. OGG1 KO) and


and genotype, (C) treatment and sex and (D) genotype and sex. Panel E

shows the three way interaction between treatment, genotype and sex.


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Table 6.3: GSH levels in tissues of OGG1 transgenic mice exposed to LPS

Mice were treated with LPS (20 mg/kg i.p.) or vehicle (PBS), sacrificed after 12 hours and tissues removed for analysis. Tissue MPO activity (mU/mg protein) results are shown as mean ± SEM (n=6).



Heart 0.49 (±0.04)

0.29 (±0.04)

0.44 (±0.05)

0.48 (±0.01)

1.10 (±0.77)

0.50 (±0.36)

0.45 (±0.01)*

0.14 (±0.01)* p=0.96

Lung 4.90 (±0.98)

6.74 (±1.13)

5.11 (±0.38)

4.27 (±0.40)

2.44 (±1.18)

4.00 (±0.48)

2.55 (±0.29)

2.69 (±0.39) p=0.53

Liver 4.01 (±1.56)

4.72 (±0.57)

4.55 (±0.32)

3.32 (±0.44)

2.44 (±0.62)

2.55 (±0.53)

4.18 (±0.40)

1.97 (±0.81) p=0.85

Kidney 0.47 (±0.10)

0.59 (±0.11)

1.14 (±0.06)

1.11 (±0.05)

0.47 (±0.05)

0.86 (±0.17)

1.00 (±0.07)

0.98 (±0.20) p=0.40

Ileum 1.35 (±0.07)

2.21 (±0.32)

1.19 (±0.37)

1.70 (±0.65)

1.29 (±0.08)

0.97 (±0.09)

0.86 (±0.49)

0.85 (±0.17) p=0.44


176

There were no three way interactions detected when comparing GSH

levels in the mice. Indeed, in the heart there were no significant

differences identified at any level (Figure 6.15). In the lung treatment x

genotype and treatment x sex were not significant (Figures 6.16B & C).

There was, however, a significant genotype x sex interaction, as whilst

GSH levels in female mice remained similar in WT and Ogg-/- lung tissues

(0.9 fold), it increased significantly in male tissues (1.5 fold; Figure

6.16D). There was also a difference observed between the treatment

groups as vehicle treated mice (5.3 ±0.4 nmol/mg protein) had higher

GSH levels than those of LPS treated mice (3.0 ±0.4 nmol/mg protein;

Figure 6.16A; p=0.01).

There was an interaction between genotype x sex, as whilst GSH levels in

female mice were reduced when comparing WT and Ogg-/- lung tissues

(0.6 fold) it remained similar when comparing male tissues (1.1 fold;

Figure 6.17D). Within the liver treatment x genotype and treatment x sex

interactions were not significant. There was a difference between the

treatment groups as vehicle treated mice (4.2 ±0.4 nmol/mg protein) had

higher GSH levels in their liver tissues than those of LPS treated mice (2.7

±0.3 nmol/mg protein; Figure 6.17A; p=0.01).

In the kidney, there was only one significant difference found, male mice

had lower GSH levels within their kidney (0.6 ±0.1 nmol/mg protein) than

the female mice (1.1 ±0.1 nmol/mg protein; Figure 6.18A; p=0.01). In

the ileum tissues assayed there was a treatment x genotype interaction

which approached significance (Figure 6.15B; p=0.06). The reduction in

GSH levels due to treatment was greater in Ogg-/- animals (0.7 fold) than

those of their WT counterparts (0.9 fold). There were no other significant

differences observed.


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Figure 6.15: GSH levels in heart tissues of OGG1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.), sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. GSH levels were significantly higher in vehicle treated WT ♂ (vs. treated KO ♀; p<0.01). No other significant effects were observed.


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Figure 6.16: GSH levels in lung tissues of OGG1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.), sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. A genotype X sex interaction was observed in (p=0.05). GSH levels were significantly higher in vehicle treated animals (vs. treatment; p<0.01). No other significant effects were observed.


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Figure 6.17: GSH levels in liver tissues of OGG1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.), sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. A genotype X sex interaction was observed (p<0.01). GSH levels were significantly higher in vehicle treated animals (vs. treatment; p<0.01). No other significant effects were observed.


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(A) Main Effects

(p=0.95) (p=0.15) (p=0.01)



Figure 6.18: GSH levels in kidney tissues of OGG1 transgenic mice exposed to

LPS Mice were treated with LPS (20 mg/kg i.p.), sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. A genotype X sex interaction was observed (p<0.01). GSH levels were significantly higher in ♀ animals (vs. ♂; p<0.01). No other significant effects were observed.


181

0

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(p=0.01) (p=0.25) (p=0.17)



Figure 6.19: GSH levels in ileum tissues of OGG1 mice exposed to LPS Mice were treated with LPS (20 mg/kg i.p.), sacrificed after 12 hours and tissues removed for analysis (mean ± SEM; n=6). Panel A Main effects of treatment, genotype and sex, panels B – D Interactions between treatment and genotype, treatment and sex and genotype and sex respectively. Panel E shows 3-way interactions between treatment, genotype and sex. GSH levels were significantly higher in vehicle treated animals (vs. treated; p<0.01).


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6.3. Discussion Previous work has indicated that the disruption of the OGG1 gene in mice

had a protective effect against high dose LPS induced inflammation

(Mabley et al., 2005a). Here we show that at lower levels of LPS toxicity,

OGG1 disruption can vary levels of cytokine production and other markers

of endotoxin induced inflammation in lung and liver tissues, but whether it

is a protection from the damaging effects of inflammation varies according

to the endpoint measured. Furthermore, there is a strong genotype x sex

interaction indicating that whilst many endpoints in male mice indicate

increased oxidative damage due to inflammation, female animals showed

little or no change (Figure 6.20). As the OGG1-/- mice that were previously

reported to have been protected from inflammation have been male this

contradicts the previously published data (Mabley et al., 2005a; Touati et

al., 2006). These results suggest that OGG1 may play a role in cytokine

transcription or production, but that this effect may be tempered by a sex

linked effect.

Table 6.4 Summary of OGG1 transgenic mouse blood serum cytokine results

** = p≤0.01, * = 0.05≥p>0.01, - = p≥0.10, ↓/↑ = comparison to WT.

Whilst these results are being compared to previous mouse studies using

LPS to induce inflammation it is important to note the dose of LPS given to

the mice in previous studies. Previous work on OGG1-/- animals report

administering LPS at 80 mg/kg and incubating for 12 h as a model of

septic shock (Mabley et al., 2005a). When trying to duplicate these

conditions a large proportion of the mice dosed with these levels of LPS

Time (h)

0 1 6 24

Cytokine Male Female Male Female Male Female Male Female

IL-4 - - *↓ - *↓ - - -

IL-6 **↓ - *↓ - - - - **↓

IL-10 - - - - *↑ **↓ **↑ **↓

IL-12 - - - - - - - -


183

Table 6.5: Summary of OGG1 organ damage results

** = p≤0.01, * = 0.05≥p>0.01, ~ = 0.10>p>0.05, - = p≥0.10, ↓/↑ = comparison.

Primary interactions Secondary Interactions Tertiary

Interaction

Treatment (LPS Treated vs. Vehicle)

Genotype (KO vs. WT)

Sex (♀ vs. ♂)

Treatment x

Genotype (Increase in KO

vs. WT)

Treatment x Sex


Genotype x Sex


Treatment x Genotype x

Sex

MPO

Heart - - - - ~↓ - -

Lung **↑ *↑ **↓ ~↑ ~↓ **↓ -

Liver **↑ - **↓ - *↓ *↓ *

Kidney **↑ - - - ~↓ - -

Ileum - - *↑ - *↓ - ~

MDA

Heart - - - - - - ~

Lung *↑ - **↓ - - - -

Liver ~↑ - **↓ - **↓ **↓ -

Kidney **↑ - - - - - -

Ileum **↑ - - - - - -

GSH

Heart - - - - - - -

Lung **↓ - ~↓ - - *↓ -

Liver **↓ - - - - *↓ -

Kidney - - **↑ - - - -

Ileum **↓ - - ~↓ - - -


184

died before the treatment period was complete, and the remaining

animals exhibited signs of severe hypothermia. Furthermore, it has been

reported elsewhere that this dose of LPS was fatal in 90% of C57BLK/6

mice, although in this study no mice died before 12 h (Yamashita et al.,

2000). To ensure that mortality did not confound the results of the

experiment a lower dose of LPS was used. In order to identify levels of

LPS toxicity that altered levels of cytokine production, it was found that

much lower (~1 mg/kg) levels elicited a response in cytokine production

(Hasko et al., 1996; Izeboud et al., 1999).

Figure 6.20 Pathways involved in the generation and degradation of oxidants

and the effects of OGG1 knockout at key endpoints In normal mice a rise in myeloperoxidase indicates increased phagocyte activation which release ROS into the affected area. This rise in ROS leads to a drop in GSH as the cell attempts to lessen the oxidative load. Increased ROS leads to the formation of more oxidised lipids in the cell membrane. Key results from lung and liver tissues taken from new OGG1-/- mice coloured blue. Here this model is not followed by either the male or female mice. Phagocyte enzymes coloured red. GSH, Glutathione; GSSG, Glutathione Disulphide; GPX, Glutathione peroxidase. Adapted from (Jaber et al., 2005)

In order to identify changes in MPO, MDA and GSH dose of 20 mg/kg LPS

was considered an appropriate amount as it had been used previously to

obtain a significant difference in MPO and MDA levels, between treated

and untreated animals (Koizumi et al., 2003; Li et al., 2005). This use of a


185

lower dose of LPS may have contributed to the lack of a treatment effect

in the heart , kidney and ileum that had been noted in a previous study of

OGG1-/- mice (Mabley et al., 2005a), and may explain why less significant

differences were observed overall.

In OGG1-/- male mice the observed significant decrease in IL-6 levels at 1

and 6 h indicate an increase Th1 response, whilst increased levels of the

anti-inflammatory cytokine between 6 and 24 h would suggest a reduced

inflammatory response, the reduction in IL-4 would suggest a lessened

anti-inflammatory response. The increase in MPO activity and MDA also

suggests that a larger number of neutrophils were attracted to the lung

and liver. These endpoints together indicate that whilst there was an

overall reduction in measured pro-inflammatory signals, larger amounts of

neutrophils were being attracted to the area. Furthermore, greater

amounts of GSH were present where suggesting that either (i) less ROS

were being neutralised by (GPX) or SOD (ii) more GSH was being

produced within the tissues. As in all tissues except the heart, basal levels

of GSH were higher in OGG1-/- male mice than OGG1+/+ male mice

(Figures 6.16E – 6.19E; non-significant) hypothesis (ii) seems plausible.

Alternatively in female mouse serum samples, the low basal levels of IL-6

and a slower initial build up of this cytokine leads to reduced levels of both

IL-4 and 10, signifying that the initiation of the inflammatory response

would be depressed but the decrease in the regulatory anti-inflammatory

cytokines IL-4 and IL-10 would suggest that overall innate inflammatory

response may be unchanged. The lack of significant change in MPO

activity does indeed indicate this (Figure 6.6D & 6.7D). A decrease in GSH

however does suggest that either (i) more ROS are being neutralised by

glutathione peroxidise (GPX) or that (ii) less glutathione is being

produced within the tissues. Basal levels of GSH are lower in all tissues

from female mice except the heart (Figures 6.16E – 6.19E; non-

significant), leading to a hypothesis that the differing male and female


186

GSH levels are an adaptation to the scale of the immune response within

the animals. Indeed oxidative stress has been shown to up regulate GPX

mRNA transcription and activity (Esposito et al., 1999). The lowered MDA

levels in the lung and liver tissues from female mice would then be

another indication of an increase in ROS neutralisation through the

GSH/GSSG pathway.

Oestrogen can act as an antioxidant and this has been shown ex vivo and

in vivo (Leal et al., 1998; Prokai et al., 2003; Sugioka et al., 1987; Zhang

et al., 2007). During pregnancy, where oestrogen production is increased

IL-10 and IL-4 (Th2) activity is observed (Ito et al., 2001). After

intratracheal LPS administration hypothermia and airway hyper

responsiveness are greater in male mice than female mice (Card et al.,

2006). The response markers to LPS induced inflammation (TNFα and

MIP1α) in female mice are reduced when compared to those in male mice

(Mabley et al., 2005b). Additionally it has been shown that the removal of

the ovaries in WT mice nullifies this protection with cytokine activity

becoming comparable to that of their male counterparts (Speyer et al.,

2005). In our study we see that the knockout of the OGG1 gene seems to

be causing increased oxidative damage in males, but this is reduced in

females due to the protective effects detailed.

In order to identify mechanisms that may be causing this differing effect

of the OGG1 knockout it is important to note that during a state of

inflammation the activity of OGG1 is subject to NO mediated inhibition,

whilst there is a simultaneous increase in cellular 8-OxoG (Jaiswal et al.,

2001). This is due to the redox mediated down regulation of OGG1, and it

may be that under the duress of inflammation the cell prioritises the

accumulation of 8-OxoG to OGG1 activity (Jaiswal et al., 2000; Jaiswal et

al., 2001). One reason proposed for this by Radek and Bodogh (2010) is

that in the process of DNA repair OGG1 removes a base causing the

creation of single stranded gaps and nicks in the DNA backbone. These


187

gaps and nicks may then be recognised by DNA-damage-dependent

kinases, which can trigger an inflammatory response. Thus in OGG1-/-

mice there are fewer gaps created and inflammation may be curtailed.

Indeed in the pancreatic islet cells of patients with the inflammatory

disease, type II diabetes, there is reduced OGG1 activity in the nucleus

whilst mitochondrial DNA repair continues as normal (Radak and Boldogh

2010; Tyrberg et al., 2002).

It has been argued that the increase of 8-oxodG within the cell can reduce

inflammation by inhibiting Rac1/2, subsequently regulating the redox-

sensitive NF-κβ pathway by the activation of the NADPH oxidase complex

(Ock et al., 2011b). Additionally it was shown that the inhibition of Rac1

can affect the STAT3 signalling pathway which is associated with chronic

inflammatory diseases (Kim et al., 2006; Ock et al., 2011a).

It has already been shown that female mice are more resistant to

oxidative stress than those of males (Du et al., 2004). Here we report that

female OGG1-/- mice are more resistant to LPS induced inflammation than

male OGG1-/- mice. Could the reduced inflammatory response that is

reported in other female mice be reducing the oxidative damage to the

cell further so that there are fewer strand breaks, reducing the feed back

from gap dependant kinases and thus reducing inflammation more? In

order to identify if inflammation increased in the presence of increased

single strand breaks, SSB’s could be induced in vivo utilising increased

concentrations of LPS or with cells in vitro using H2O2 or UV radiation to

induce the breaks (Frankenberg-Schwager et al., 2008).

Additionally this observed effect could be the result of OGG1 interacting

with other proteins as gender differences have also been observed in the

protective effect of the PARP-1 inhibite mice. Whilst PARP-1 inhibited male

mice showed a marked decrease in TNFα output (~50%) after LPS

challenge (1 mg/kg i.p.) compared to non-inhibited animals (~1025 vs.


188

~450 pg/ml). The TNFα output of female mice that were not PARP-1

inhibited began at a lower level and the inhibition of PARP-1 did not

change this level (both ~4000 pg/ml). In order to identify if this was a due

to the low uninhibited amount of TNFα a further set of female mice were

treated with a more robust amount of LPS (30 mg/kg i.p.). Again no

significant difference was observed between normal and PARP-1 inhibited

serum TNFα concentrations (Mabley et al., 2005b).

In gel shift assays it was further shown that PARP-1 cooperatively interacts

with oestrogen receptor-α (ERα), this cooperation is further enhanced in

the presence of oestrogen. A model was proposed in which the PARP-1 –

Erα – oestrogen complex inhibits the activity of PARP-1 (Figure 6.21;

(Mabley et al., 2005b).

Figure 6.21 Proposed model of PARP-1 inhibition by oestrogen Whilst the PARP-1 and ERα proteins interact stably on single stranded DNA breaks (top), the addition of oestrogen causes a conformational change in ERα which binds PARP-1 more tightly causing the zinc-finger locating portion of the protein to become less able to locate on the DNA strand reducing the efficiency of DNA repair. Adapted from (Mabley et al., 2005b).

To date no physical interaction between OGG1 and PARP-1 has been

identified, but OGG1 acts in the base excision repair pathway ahead of


189

PARP-1. If the inflammatory effect of PARP-1 is triggered by its detection

of DNA breaks then the removal of the OGG1 protein and the subsequent

reduction in these breaks would reduce its inflammatory effect. In order to

identify if this were the case PARP-1 activity could be assessed by the

quantity of poly (ADP-ribose) (PAR) polymers produced in OGG1-/- extracts

or in peripheral blood mononuclear cells from OGG1-/- mice (Tentori et al.,

2003). If no link were found between OGG1 and PARP-1 it may be

interesting to determine if transcription factor activation, such as NF-κβ,

was altered in OGG1-/- mice, and proceed to identify the process by which

inflammation is mediated by OGG1.

If proved to be a modifying factor in inflammation the OGG1 protein could

conceivably be targeted as a therapy for combating inflammation based

disease. It has the advantage that (in mice) OGG1s disruption has few

short term side effects (Klungland et al., 1999). Care would have to be

taken however in long term use as older mice experienced higher rates of

cancer (Sakumi et al., 2003) and polymorphisms of the gene in human

populations have shown variable links with various cancers including lung

(Kohno et al., 2006), gastric (Farinati et al., 2008) and prostate cancers

(Chen et al., 2003).


190

7. Overall Discussion

Previously documented literature has demonstrated that the disruption of

certain genes connected with the base excision repair pathway reduce the

inflammatory response to endotoxin induced inflammation (Mabley et al.,

2005a; Virag and Szabo 2002). Here we have investigated the hypothesis

that if DNA glycosylases, specifically NEIL1, NEIL2, OGG1 and NTH1, are

involved in the regulation of endotoxin induced inflammation, then the

knockout of these genes, in murine models, will result in a reduction in the

inflammatory response to endotoxin measured by cytokine response and

certain oxidative stress markers. In order to investigate this hypothesis a

new strain of NEIL1-/- mouse was created. Using MEFs derived from these

NEIL1-/- mice, and other DNA glycosylase deficient mice, a reduction in

MIP-1α production was observed in both untreated and LPS treated

NEIL1-/-, OGG1-/-, NTH1-/- and [OGG1/NTH1]-/- cells. Additionally, OGG1-/-

cells had higher basal IL-10 levels, and NEIL1-/- cells had lower basal IL-6

output.

In vivo at lower levels of LPS toxicity (20 mg/kg), OGG1 disruption varied

cytokine production (as measured in blood serum), but this difference

differed according to sex: basal IL-6 levels and LPS-induced IL-10 levels

were higher but LPS-induced IL-4 levels lower in male OGG1-/- mice. In

female OGG1-/- mice there was a decrease in LPS treated levels of IL-10,

and in the rate of IL-6 release. There was no significant genotype x

treatment interaction in MPO, MDA and GSH results for OGG1-/- mice

(Table 7.2). However there was a strong genotype x sex interaction in

lung and liver indicating that whilst many endpoints on male OGG1-/- mice

indicate increased oxidative damage due to inflammation, female OGG1-/-

animals showed little or no change. Male NEIL1-/- mice had higher basal

levels of IL-6, and although IL-6 production was slowed, its maximum

level was similar to that of WT animals, additionally, after LPS treatment

serum IL-12 levels were increased. There were no significant differences


191

Table 7.1: Summary and comparison of NEIL1 and OGG1 cytokine output results when compared with those of WT animals

** = p≤0.01, * = 0.05≥p>0.01, ~ = 0.10>p>0.05, - = p≥0.10, ↓/↑ = comparison.

Time (h)

0 1 6 24

Male Female Male Female Male Female Male Female

Cyto-kine

NEIL1-/- OGG1-/- NEIL1-/- OGG1-/- NEIL1-/- OGG1-/- NEIL1-/- OGG1-/- NEIL1-/- OGG1-/- NEIL1-/- OGG1-/- NEIL1-/- OGG1-/- NEIL1-/- OGG1-/-

IL-4 - - - - - *↓ - - - *↓ **↓ - - - - -

IL-6 **↑ **↓ - - *↓ *↓ *↑ - - - - - **↓ - **↓ **↓

IL-10 - - - - - - - - - *↑ **↓ **↓ - **↑ *↓ **↓

IL-12 - - - - - - - - **↑ - - - - - - -


192

Table 7.2: Summary and comparison of NEIL1 and OGG1 organ damage results

** = p≤0.01, * = 0.05≥p>0.01, ~ = 0.10>p>0.05, - = p≥0.10, ↓/↑ = comparison.

Primary interactions Secondary Interactions

Tertiary Interaction

Treatment Genotype Sex Treatment x Genotype

Treatment x Sex

Genotype x Sex

Treatment x

Genotype x

Sex

(LPS Treated vs. Vehicle)

(KO vs. WT) (♀ vs. ♂) (Increase in KO vs.

WT) (Increase in ♀ vs.

♂) (Increase in ♀ vs.

♂)

NEIL1-/- OGG1-/- NEIL1-/- OGG1-/- NEIL1-/- OGG1-/- NEIL1-/- OGG1-/- NEIL1-/- OGG1-/- NEIL1-/- OGG1-/- NEIL1-/- OGG1-/-

MPO

Heart - - - - **↓ - - - - ~↓ - - - -

Lung *↑ **↑ - *↑ - **↓ - ~↑ - ~↓ - **↓ - -

Liver - **↑ - - *↓ **↓ ~↓ - *↑ *↓ - *↓ - *

Kidney - **↑ - - - - - - - ~↓ - - - -

Ileum *↑ - - - - *↑ - - *↓ *↓ - - * ~

MDA

Heart - - - - **↑ - - - - - - - - ~

Lung - *↑ - - - **↓ - - - - - - - -

Liver **↑ ~↑ - - *↑ **↓ **↑ - - **↓ - **↓ - -

Kidney *↑ **↑ - - - - - - - - - - - -

Ileum - **↑ ~↑ - - - - - ~↓ - ~↓ - - -

GSH

Heart **↓ - **↓ - **↑ - - - - - **↓ - - -

Lung **↓ **↓ *↓ - - ~↓ - - - - - *↓ - -

Liver **↓ **↓ *↓ - **↑ - - - - - - *↓ - -

Kidney ~↑ - ~↑ - **↓ **↑ - - *↓ - ~↓ - - -

Ileum - **↓ *↑ - **↓ - - ~↓ - - - - - -


193

with genotype in the MPO and MDA levels of NEIL1-/- mice although there

was an indication that GSH levels were raised in the heart, lung and liver

tissues and decreased in the ileum. These in vivo results indicate that at

this level of LPS there was a variable change in cytokine production, but

that this change is not great enough to change neutrophil recruitment

enough to reduce oxidative damage markers.

When cytokine release between OGG1-/- and NEIL1-/- mice was compared

(Table 7.1) it can be seen that the deletion of DNA glycosylases NEIL1 and

OGG1 has similar effects reducing the concentration of the cytokines in IL-

6 (male 1 h and female 24 h) and IL-10 (female 6 and 24 h) in serum

samples. This indicates that both Th1 and Th2 responses were affected by

the knockout of these DNA glycosylases. The differences in male and

female responses shown are probably due to the differences in oestrogen

levels, as higher physiological levels of oestrogen modulate the immune

from a Th1 mediated response to that of Th2 mediated response (Gilmore et

al., 1997; Salem, 2004; Whitacre, 2001).

Additionally the interaction between genotype x sex was only observed

strongly in the OGG1-/- mice. There was however, no genotype x

treatment interaction observed in the case of either NEIL1-/- or OGG1-/-

mice, indicating that whilst there was a general change in cytokine

activity, neutrophil aggregation and ROS production were not affected

significantly. Perhaps this can be explained somewhat by the reduction in

MIP-1α production noted in all knockout MEF cell types, as a reduction in

the production of this chemokine would reduce the accumulation of

leukocytes.

The pathway by which the NEIL1 and OGG1 proteins modulate the

immune system can be surmised to be a different from that of PARP1 as

while PARP1 knockout resulted in a reduced output of all measured

cytokines, this was not apparent in OGG1-/- and NEIL1-/- mice as cytokine


194

output also increased. The effect of PARP1 may be due to it’s interaction

with NF-κβ (Virag and Szabo 2002). Similarly APE1 can also be discounted

as it activates NF-κβ via redox reactions meaning a lack of this enzyme

would result in a complete reduction in all cytokines (Ando et al., 2008).

It would be interesting to study if the NEIL1 protein is inhibited during

inflammatory processes as is OGG1 by NO (Jaiswal et al., 2000; Jaiswal et

al., 2001), but currently there is no literature on the subject. If it were

found to be so it would add weight to the theory that damage dependant

kinases are responsible for the increase in inflammation (Hofseth et al.,

2003). The disruption of the OGG1 gene has previously been reported to

reduce the inflammatory response (Mabley et al., 2005a; Touati et al.,

2006). OGG1 is inhibited in the inflammatory reaction by NO, it is thought

that this inhibition is preferable to the creation of gaps in the DNA strand

due to the removal of oxidised bases (Jaiswal et al., 2001). These gaps

have been implicated in the inflammatory response as their presence

actives DNA damage dependent kinases which can trigger inflammation

through the activation of p53 (Gudkov et al., 2011). As NEIL1 has been

shown to interact with OGG1, indeed it is suggested that OGG1 activity is

stimulated in the presence of NEIL1 (Mokkapati et al., 2004a), then the

disruption of the NEIL1 gene may reduce inflammatory activity in a similar

manner to NO, but on a permanent basis. This may come someway to

explaining the change in IL-6 basal and 1 h levels due to reduced initial

immune signalling. It may also explain the reduction in MPO activity as

there would be less MIP-1α produced during the immune response.

Disruption of bifunctional DNA glycosylases was also found to reduce the

basal and LPS induced output of MIP-1α from all BER KO MEFs when

compared to WT cells. As MIP-1α is a potent chemokine that attracts

neutrophils to the area of infection, this would suggest a decrease in

neutrophil activity in vivo. It is interesting to note that basal and LPS

stimulated MPO activity was not affected by genotype in either the


195

NEIL1-/- or OGG1-/- mouse models. It could be the case that in the in vivo

model there are other regulatory systems available to overcome the

effects a potential loss in MIP-1α output. Additionally, OGG1-/- cells had

higher basal IL-10 levels, whilst in the whole animal model this was also

not observed. Indeed NEIL1-/- cells had lower basal IL-6 output, whilst the

male animals showed a marked increase in basal rates of IL-6. In line with

previously published material detailing the use of MEF’s, the cells were not

used after passage 10 so as to reduce the risk of possible mycoplasma

infections and mutations being the cause of any reported effects (Khobta

et al., 2009).

During the course of this study two new strains of mice were created,

namely a NEIL1-/- mouse and a putative NEIL2-/- mouse. Assays examining

the NEIL1-/- genomic DNA (PCR), mRNA (RT-PCR) and protein (western

blot) have confirmed the success of this model. As a previous colony of

NEIL1-/- mice had displayed a sporadic phenotype that shared similar

symptoms with the human metabolic syndrome, a colony of these animals

was kept for an extended period of time in order to discern if a similar

phenotype would be observed (Vartanian et al., 2006). Whilst there was

no significant difference in overall weight between the NEIL1+/+ and

NEIL1-/- mice, a significant difference was observed in the weight of

ommentum fat surrounding the intestinal tract of the NEIL1-/- male mice.

This indicates less overall fat in these animals, showing that the obese

phenotype did not appear in these NEIL1-/- mice. There is however data

indicating that the size of the adrenal glands was increased in NEIL1-/-

mice and that the reproductive organs of the NEIL1-/- female mice was

increased whilst the testes size of male NEIL1-/- mice was decreased. This

suggests that there may be hormonal differences in the mice including

raised oestrogen levels in the NEIL1-/- female knockout animals and

lowered testosterone levels in the NEIL1-/- male mice as changes in the

production of the appropriate hormone effects the size of these organs

(Kenagy and Trombulak 1986; Wood, 2000). Interestingly it has been


196

observed that in cases of obesity in humans the concentration of blood

born testosterone decreases and oestrogen increases (Kley et al., 1979).

It was not discerned in this study whether the increase in adrenal gland

size raised adrenaline output, as some diseases such as

Phenochromocytoma do increase adrenal size and output (Strong et al.,

2008), whereas Addison’s disease does not but it increases the cortex

layer around the gland reducing the size of the adrenaline producing

medulla (Zelissen et al., 1995).

There were overall differences in the tissue GSH content in that levels

were lower in the heart, lung and liver of NEIL1-/- mice, but elevated in

the ileum. In order to investigate why there could be changes in the GSH

levels but not in MPO or MDA levels, other known differences in the

NEIL1-/- mice were examined. As both male and female animals produce

both testosterone and oestrogen, the reduction in male testes weight and

increase in female sex organ weight suggest a shift in the ratio of

testosterone:oestrogen toward a larger proportion of oestrogen in both

male and female NEIL1 mice. However as oestrogen has been shown to

be an antioxidant this would not account for lowered levels of GSH (Leal

et al., 1998; Prokai et al., 2003; Sugioka et al., 1987; Zhang et al., 2007).

It is also possible that a change in the levels of serum adrenaline,

suggested by the increased adrenal gland size, could alter the levels of

ROS and therefore GSH within the tissues mentioned. As adrenaline

affects the fight or flight response an increase in its output affects

different organs and systems in different ways. Heart rate and lung

respiration increase, causing a raised generation of ROS in those organs

(Sarker et al., 2009). In the liver an increase in adrenaline stimulates

glycogenolysis, and increases mitochondrial ROS creation via. NADPH

oxidase (Diaz-Cruz et al., 2007) The presence of adrenaline additionally

reduces the metabolic activity in ileum tissues by restricting blood flow to

the area potentially reducing the creation of ROS (Konstantinidis et al.,


197

2011). This suggests GSH levels may be altered by changes in metabolism

due to an increased production of adrenaline (Figure 7.1). If an

overproduction of adrenaline is the reason for the changes in GSH levels,

it does not explain a lack in an increase in MDA. Perhaps the increase of

adrenaline is great enough to elicit a change in GSH levels, but that this

level of oxidation is neutralised by cellular antioxidants before significant

lipid oxidation can occur.

Figure 7.1: Alterations in organ activity due to adrenaline and observed levels

of GSH in NEIL1-/- mice An increase in adrenalin activates the fight or flight responses in mammalian systems, triggering increased activity in organs such as the heart, lung and liver and reducing activity in areas that are not required for immediate gain such as the gut. In organs where activity is increased one would expect an increase in ROS production as a by product of metabolism, resulting in a reduction in cellular GSH. Conversely in the gut where activity is decreased one may expect oxidative stress to be reduced due to a reduction in metabolism.

Classically, raised adrenalin levels lead to hypertension and eventually

kidney failure (Franco-Morselli et al., 1977), Phenochromocytoma, a rare

cancer of the adrenal gland, which also causes raised adrenaline levels

results in an increased heart rate, increased blood glucose and weight loss

(Strong et al., 2008). In order to identify if there are raised adrenalin


198

levels in the NEIL1-/- mice serum levels of adrenaline should be measured.

Alternatively studies could be performed around the previously mentioned

symptoms, namely the measurement of the mouse’s blood pressure, blood

glucose and serum creatine (to measure kidney function) which would all

be good measures of systemic damage due to over production of

adrenaline (Dunn et al., 2004). Our NEIL1-/- mice have shown no overt

change in weight, though previously described NEIL1-/- mice have shown

increases in weight and additionally the previous mice were noted to be

less active, spending fewer hours each day in voluntary exercise (Sampath

et al., 2011; Vartanian et al., 2006).

If the absence of the NEIL1 protein does indeed cause an increase in

adrenal output it could suggest links with the activation of

adrenocorticotropic hormone (ACTH), implicating a connection with the

central nervous system (Wilson III and McNeill 2007). NEIL1 mRNA is

transcribed in brain tissue in a moderate amount when compared to other

tissues (Hazra et al., 2002a). It has been reported that whilst the

expression of other BER proteins, OGG1 and APE-1, decline by 40 - 50%

in rat brain from the embryonic (1 day prior to birth) to the young adult

stage (3 months), in the same period NEIL1 and NEIL2 expression

increased (1.5 and 2.5 fold respectively (Englander and Ma 2006).

Additionally in mice, mitochondrial NEIL1 expression in the cortex

increased 1.7 fold between adulthood (5 months) and middle age (10

months), whilst there was no increase in its expression in the

hippocampus over the same time period, indicating that if there were

some link between NEIL1 and brain activity it would most likely be

connected to the cortex region. This was not specific to NEIL1 and in the

same mice similar increases were detected in NEIL2, NTH1, OGG1 and

APE-1 expression. These changes in expression have been suggested as

being a response to a build up in oxidative damage as the brain ages, and

could indicating why mRNA instability and neurodegenerative diseases are

more common in the hippocampus (Gredilla et al., 2010). Whilst the first


199

of the two studies implicates that NEIL1 and NEIL2 are important in the

mature brain, the second study indicates that the specific difference is not

in the mitochondria of either the cortex or hippocampus, suggesting that

the ability to repair within the bubble structure genomic DNA is important

to the mature brain. It would be interesting to identify which areas of the

brain, rather than just general brain tissue, show marked difference in

BER protein expression, and how the disruption of DNA glycosylases

affects their function.

When attempting to confirm the success of the NEIL2 knockout only the

PCR of genomic DNA suggested that NEIL2-/- mice had been generated

and that the Tk/Neo cassette was present in the genomic DNA, and in the

correct position. However, RT-PCR indicated that undisrupted mRNA was

being produced and western blots indicated that a protein of the

appropriate size was present that bound to anti-NEIL2 IgG. This is a

discrepancy that at the moment we are unable to explain with any

certainty. Further investigation into the positioning of the Tk/Neo cassette

could be performed using techniques such as fluorescence in situ

hybridization, this may not result in this NEIL2 model being validated as

the western blot and RT-PCR confirm the presence of NEIL2 protein, but it

would confirm the chromosomal positioning of the insert perhaps leading

to greater understanding of the reason that the knockout was

unsuccessful.

Further understanding of the role of DNA glycosylases in the inflammatory

response to endotoxin could be elucidated by the performing of further

experiments upon the organs that had already been analysed for MPO,

MDA and GSH. Portions of these organs have been fixed in formalin

solution which would make it possible for histological analysis of the

samples to be performed. This would be useful in identifying and

phenotyping the infiltrating cells to the areas of inflammation.


200

In order to further examine if a DNA glycosylase affects the inflammation

response to endotoxin it may be useful to perform some additional work

on double DNA glycosylase knockout mice. This is due to the redundancy

built into the base excision repair system. Interesting pairings to look at

may would be those with similar substrates such as [OGG1/NEIL1]-/- and

[NEIL1/NTH1]-/-. A [NEIL1/NTH1]-/- mouse has already been produced and

has shown particular susceptibility to pulmonary and hepatic tumours

(Chan et al., 2009).

As a new NEIL1-/- model has been described that does not seem to exhibit

the same phenotype as previously presented models it may be interesting

to identify if the DNA damage response is effected in a similar fashion. In

MEF cells treated with DNA damaging agents such as H2O2 this could be

examined using comet assays. To compare the accumulation of oxidised

bases, such as FapyA, FapyG and 8-oxo-G, over the course of a lifetime

gas chromatography/mass spectrometry could be used.

In conclusion we have found that the deletion of DNA glycosylases has led

to reduction in cytokine outputs (IL-6 and IL-10) when challenged with

LPS but also had very little effect on the physiological markers MPO, MDA

and GSH. This indicates that whilst the disruption of these genes may

reduce inflammatory signalling this reduction is not great enough to lessen

the oxidative stress caused during the inflammatory response. This could

be due to the levels of LPS used to elicit the response in our experiments.

Indeed the sex of the subjects seemed to have a more protective effect

on inflammatory response. These results point to DNA glycosylases being

a poor target for anti-inflammatory therapies in all but the severest of

cases.

Furthermore the previously reported obesity phenotype in the NEIL1-/-

mice was not noted in this strain of mice, although there is evidence of

possible hormonal changes, specifically increased oestrogen and


201

adrenaline, in the animals. The results for tissue GSH in NEIL1-/- mice we

observed a pattern similar to that of raised adrenal output.


202

8.0 References

Abdel-Latif MM, O'Riordan J, Windle HJ, Carton E, Ravi N, Kelleher D and Reynolds JV (2004) NF-kappaB activation in esophageal adenocarcinoma: relationship to Barrett's metaplasia, survival, and response to neoadjuvant chemoradiotherapy. Ann. Surg. 239 (4) 491 - 500

Abdel-Rahman WM, Ollikainen M, Kariola R, Jarvinen HJ, Mecklin JP, Nystrom-Lahti M, Knuutila S and Peltomaki P (2005) Comprehensive characterization of HNPCC-related colorectal cancers reveals striking molecular features in families with no germline mismatch repair gene mutations. Oncogene. 24 (9) 1542 - 1551

Aller P, Rould MA, Hogg M, Wallace SS and Doublie S (2007) A structural rationale for stalling of a replicative DNA polymerase at the most common oxidative thymine lesion, thymine glycol. Proc. Natl. Acad. Sci. U. S. A. 104 (3) 814 - 818

Ando K, Hirao S, Kabe Y, Ogura Y, Sato I, Yamaguchi Y, Wada T and Handa H (2008) A new APE1/Ref-1-dependent pathway leading to reduction of NF-kappaB and AP-1, and activation of their DNA-binding activity. Nucl. Acids. Res. 36 (13) 4327 - 4336

Arai K, Morishita K, Shinmura K, Kohno T, Kim SR, Nohmi T, Taniwaki M, Ohwada S and Yokota J (1997) Cloning of a human homolog of the yeast OGG1 gene that is involved in the repair of oxidative DNA damage. Oncogene. 14 (23) 2857 - 2861

Arai T, Kelly VP, Minowa O, Noda T and Nishimura S (2006) The study using wild-type and Ogg1 knockout mice exposed to potassium bromate shows no tumor induction despite an extensive accumulation of 8-hydroxyguanine in kidney DNA. Toxicology. 221 (2-3) 179 - 186

Arbour NC, Lorenz E, Schutte BC, Zabner J, Kline JN, Jones M, Frees K, Watt JL and Schwartz DA (2000) TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nat. Genet. 25 (2) 187 - 191

Aspinall GO, Monteiro MA, Pang H, Walsh EJ and Moran AP (1996) Lipopolysaccharide of the Helicobacter pylori type strain NCTC 11637 (ATCC 43504): structure of the O antigen chain and core oligosaccharide regions. Biochemistry. 35 (7) 2489 - 2497

Audebert M, Radicella JP and Dizdaroglu M (2000) Effect of single mutations in the OGG1 gene found in human tumors on the substrate specificity of the Ogg1 protein. Nucleic Acids Research. 28 (14) 2672 - 2678


203

Banerjee A and Verdine GL (2006) A nucleobase lesion remodels the interaction of its normal neighbor in a DNA glycosylase complex. Proc. Natl. Acad. Sci. U. S. A. 103 (41) 15020 - 15025

Beharka AA, Meydani M, Wu D, Leka LS, Meydani A and Meydani SN (2001) Interleukin-6 production does not increase with age. J. Gerontol. A Biol. Sci. Med. Sci. 56 (2) B81 - B88

Berczi I, Bertok L and Bereznai T (1966) Comparative studies on the toxicity of Escherichia coli lipopolysaccharide endotoxin in various animal species. Can. J. Microbiol. 12 (5) 1070 - 1071

Berliner JA and Watson AD (2005) A role for oxidized phospholipids in atherosclerosis. N. Engl. J. Med. 353 (1) 9 - 11

Beutler B and Rietschel ET (2003) Innate immune sensing and its roots: the story of endotoxin. Nat. Rev. Immunol. 3 169 - 176

Beutler B (2004) Innate immunity: an overview. Molecular Immunology. 40 (12) 845 - 859

Bhakat KK, Hazra TK and Mitra S (2004) Acetylation of the human DNA glycosylase NEIL2 and inhibition of its activity. Nucleic Acids Research. 32 (10) 3033 - 3039

Boehmer ED, Goral J, Faunce DE and Kovacs EJ (2004) Age-dependent decrease in Toll-like receptor 4-mediated proinflammatory cytokine production and mitogen-activated protein kinase expression. J. Leukoc. Biol. 75 (2) 342 - 349

Bohr VA (2002) Repair of oxidative DNA damage in nuclear and mitochondrial DNA, and some changes with aging in mammalian cells. Free Radic. Biol. Med. 32 (9) 804 - 812

Bolger MS, Ross DS, Jiang H, Frank MM, Ghio AJ, Schwartz DA and Wright JR (2007) Complement levels and activity in the normal and LPS-injured lung. Am. J. Physiol Lung Cell Mol. Physiol. 292 (3) L748 - L759

Bouchard VJ, Rouleau M and Poirier GG (2003) PARP-1, a determinant of cell survival in response to DNA damage. Exp. Hemat. 31 (6) 446 - 454

Braun-Fahrlander C, Riedler J, Herz U, Eder W, Waser M, Grize L, Maisch S, Carr D, Gerlach F, Bufe A, Lauener RP, Schierl R, Renz H, Nowak D and von ME (2002) Environmental exposure to endotoxin and its relation to asthma in school-age children. N. Engl. J Med. 347 (12) 869 - 877

Bravard A, Vacher M, Moritz E, Vaslin L, Hall J, Epe B and Radicella JP (2009) Oxidation status of human OGG1-S326C polymorphic variant determines cellular DNA repair capacity. Cancer Res. 69 (8) 3642 - 3649


204

Brown KL, Adams T, Jasti VP, Basu AK and Stone MP (2008) Interconversion of the cis-5R,6S- and trans-5R,6R-thymine glycol lesions in duplex DNA. J. Am. Chem. Soc. 130 (35) 11701 - 11710

Bruskov VI, Malakhova LV, Masalimov ZK and Chernikov AV (2002) Heat-induced formation of reactive oxygen species and 8-oxoguanine, a biomarker of damage to DNA. Nucleic Acids Research. 30 (6) 1354 - 1363

Buer J and Balling R (2003) Mice, microbes and models of infection. Nat. Rev. Genet. 4 (3) 195 - 205

Butterfield TA, Best TM and Merrick MA (2006) The dual roles of neutrophils and macrophages in inflammation: a critical balance between tissue damage and repair. J. Athl. Train. 41 (4) 457 - 465

Caldecott KW (2003) DNA Single-Strand Break Repair and Spinocerebellar Ataxia. Cell. 112 (1) 7 - 10

Campalans A, Amouroux R, Bravard A, Epe B and Radicella JP (2007) UVA irradiation induces relocalisation of the DNA repair protein hOGG1 to nuclear speckles. J. Cell Sci. 120 (Pt 1) 23 - 32

Card JW, Carey MA, Bradbury JA, DeGraff LM, Morgan DL, Moorman MP, Flake GP and Zeldin DC (2006) Gender differences in murine airway responsiveness and lipopolysaccharide-induced inflammation. J. Immunol. 177 (1) 621 - 630

Casorelli I, Pannellini T, de Luca G, Degan P, Chiera F, Iavarone I, Giuliani A, Butera A, Boirivant M, Musiani P and Bignami M (2010) The Mutyh Base Excision Repair Gene Influences the Inflammatory Response in a Mouse Model of Ulcerative Colitis. PLoS. One. 5 (8)

Cavagna G, Foa V and Vigliani EC (1969) Effects in man and rabbits of inhalation of cotton dust or extracts and purified endotoxins. Br. J. Ind. Med. 26 (4) 314 - 321

Chaby R (1999) Strategies for the control of LPS-mediated pathophysiological disorders. Drug Discov. Today. 4 (5) 209 - 221

Chan MK, Ocampo-Hafalla MT, Vartanian V, Jaruga P, Kirkali G, Koenig KL, Brown S, Lloyd RS, Dizdaroglu M and Teebor GW (2009) Targeted deletion of the genes encoding NTH1 and NEIL1 DNA N-glycosylases reveals the existence of novel carcinogenic oxidative damage to DNA. DNA Repair. 8 (7) 786 - 794

Cheadle JP and Sampson JR (2003) Exposing the MYtH about base excision repair and human inherited disease. Hum. Mol. Genet. 12 R159 - R165


205

Chen L, Elahi A, Pow-Sang J, Lazarus P and Park J (2003) Association between polymorphism of human oxoguanine glycosylase 1 and risk of prostate cancer. J. Urol. 170 (6 Pt 1) 2471 - 2474

Christmann M, Tomicic MT, Roos WP and Kaina B (2003) Mechanisms of human DNA repair: an update. Toxicology. 193 (1-2) 3 - 34

Church DM, Goodstadt L, Hillier LW, Zody MC, Goldstein S, She X, Bult CJ, Agarwala R, Cherry JL, DiCuccio M, Hlavina W, Kapustin Y, Meric P, Maglott D, Birtle Z, Marques AC, Graves T, Zhou S, Teague B, Potamousis K, Churas C, Place M, Herschleb J, Runnheim R, Forrest D, mos-Landgraf J, Schwartz DC, Cheng Z, Lindblad-Toh K, Eichler EE and Ponting CP (2009) Lineage-specific biology revealed by a finished genome assembly of the mouse. PLoS. Biol. 7 (5) e1000112 -

Cirl C, Wieser A, Yadav M, Duerr S, Schubert S, Fischer H, Stappert D, Wantia N, Rodriguez N, Wagner H, Svanborg C and Miethke T (2008) Subversion of Toll-like receptor signaling by a unique family of bacterial Toll/interleukin-1 receptor domain-containing proteins. Nat. Med. 14 (4) 399 - 406

Cleaver JE (1968) Defective repair replication of DNA in xeroderma pigmentosum. Nature. 218 (5142) 652 - 656

Conlon KA, Zharkov DO and Berrios M (2004) Cell cycle regulation of the murine 8-oxoguanine DNA glycosylase (mOGG1): mOGG1 associates with microtubules during interphase and mitosis. DNA Repair (Amst). 3 (12) 1601 - 1615

Cortazar D, Kunz C, Selfridge J, Lettieri T, Saito Y, MacDougall E, Wirz A, Schuermann D, Jacobs AL, Siegrist F, Steinacher R, Jiricny J, Bird A and Schar P (2011) Embryonic lethal phenotype reveals a function of TDG in maintaining epigenetic stability. Nature. 470 (7334) 419 - 423

Daily D, Vlamis-Gardikas A, Offen D, Mittelman L, Melamed E, Holmgren A and Barzilai A (2001) Glutaredoxin protects cerebellar granule neurons from dopamine-induced apoptosis by activating NF-kappa B via Ref-1. J. Biol. Chem. 276 (2) 1335 - 1344

Dalton TP, Dieter MZ, Yang Y, Shertzer HG and Nebert DW (2000) Knockout of the mouse glutamate cysteine ligase catalytic subunit (Gclc) gene: embryonic lethal when homozygous, and proposed model for moderate glutathione deficiency when heterozygous. Biochem. Biophys. Res. Commun. 279 (2) 324 - 329

Das A, Boldogh I, Lee JW, Harrigan JA, Hegde ML, Piotrowski J, de Souza PN, Ramos W, Greenberg MM, Hazra TK, Mitra S and Bohr VA (2007a) The human Werner syndrome protein stimulates repair of oxidative DNA base


206

damage by the DNA glycosylase NEIL1. J. Biol. Chem. 282 (36) 26591 - 26602

Das S, Chattopadhyay R, Bhakat KK, Boldogh I, Kohno K, Prasad R, Wilson SH and Hazra TK (2007b) Stimulation of NEIL2-mediated oxidized base excision repair via YB-1 interaction during oxidative stress. J. Biol. Chem. 282 (39) 28474 - 28484

Daynes RA, Araneo BA, Ershler WB, Maloney C, Li GZ and Ryu SY (1993) Altered regulation of IL-6 production with normal aging. Possible linkage to the age-associated decline in dehydroepiandrosterone and its sulfated derivative. J. Immunol. 150 (12) 5219 - 5230

De Bont R and van Larebeke N (2004) Endogenous DNA damage in humans: a review of quantitative data. Mutagenesis. 19 (3) 169 - 185

Dherin C, Radicella JP, Dizdaroglu M and Boiteux S (1999) Excision of oxidatively damaged DNA bases by the human alpha-hOgg1 protein and the polymorphic alpha-hOgg1(Ser326Cys) protein which is frequently found in human populations. Nucleic Acids Research. 27 (20) 4001 - 4007

Dianov GL, Sleeth KM, Dianova II and Allinson SL (2003) Repair of abasic sites in DNA. Mutat. Res. 531 (1-2) 157 - 163

Diaz-Cruz A, Guinzberg R, Guerra R, Vilchis M, Carrasco D, Garcia-Vazquez FJ and Pina E (2007) Adrenaline stimulates H2O2 generation in liver via NADPH oxidase. Free Radic. Res. 41 (6) 663 - 672

Ding X, Hiraku Y, Ma N, Kato T, Saito K, Nagahama M, Semba R, Kuribayashi K and Kawanishi S (2005) Inducible nitric oxide synthase-dependent DNA damage in mouse model of inflammatory bowel disease. Cancer Sci. 96 (3) 157 - 163

DiStasi MR and Ley K (2009) Opening the flood-gates: how neutrophil-endothelial interactions regulate permeability. Trends Immunol. 30 (11) 547 - 556

Dizdaroglu M, Karahalil B, Senturker S, Buckley TJ and Roldan-Arjona T (1999) Excision of products of oxidative DNA base damage by human NTH1 protein. Biochemistry. 38 (1) 243 - 246

Dobson AW, Grishko V, LeDoux SP, Kelley MR, Wilson GL and Gillespie MN (2002) Enhanced mtDNA repair capacity protects pulmonary artery endothelial cells from oxidant-mediated death. Am. J. Physiol Lung Cell Mol. Physiol. 283 (1) L205 - L210

Donadelli R, Abbate M, Zanchi C, Corna D, Tomasoni S, Benigni A, Remuzzi G and Zoja C (2000) Protein traffic activates NF-kB gene signaling and promotes MCP-1-dependent interstitial inflammation. Am. J. Kidney Dis. 36 (6) 1226 - 1241


207

Dou H, Mitra S and Hazra TK (2003) Repair of oxidized bases in DNA bubble structures by human DNA glycosylases NEIL1 and NEIL2. J. Biol. Chem. 278 (50) 49679 - 49684

Dou H, Theriot CA, Das A, Hegde ML, Matsumoto Y, Boldogh I, Hazra TK, Bhakat KK and Mitra S (2008) Interaction of the human DNA glycosylase NEIL1 with proliferating cell nuclear antigen. The potential for replication-associated repair of oxidized bases in mammalian genomes. J. Biol. Chem. 283 (6) 3130 - 3140

Doublie S, Bandaru V, Bond JP and Wallace SS (2004) The crystal structure of human endonuclease VIII-like 1 (NEIL1) reveals a zincless finger motif required for glycosylase activity. Proc. Natl. Acad. Sci. U. S. A. 101 (28) 10284 - 10289

Douwes J, Zuidhof A, Doekes G, van der Zee SC, Wouters I, Boezen MH and Brunekreef B (2000) (1-->3)-beta-D-glucan and endotoxin in house dust and peak flow variability in children. Am. J. Respir. Crit. Care Med. 162 (4 Pt 1) 1348 - 1354

Droge W (2002) Free radicals in the physiological control of cell function. Physiol Rev. 82 (1) 47 - 95

Du L, Bayir H, Lai Y, Zhang X, Kochanek PM, Watkins SC, Graham SH and Clark RS (2004) Innate gender-based proclivity in response to cytotoxicity and programmed cell death pathway. J. Biol. Chem. 279 (37) 38563 - 38570

Duell EJ, Millikan RC, Pittman GS, Winkel S, Lunn RM, Tse CK, Eaton A, Mohrenweiser HW, Newman B and Bell DA (2001) Polymorphisms in the DNA repair gene XRCC1 and breast cancer. Cancer Epidemiol. Biomarkers Prev. 10 (3) 217 - 222

Dunn SR, Qi Z, Bottinger EP, Breyer MD and Sharma K (2004) Utility of endogenous creatinine clearance as a measure of renal function in mice. Kidney Int. 65 (5) 1959 - 1967

Eder W, Klimecki W, Yu L, von Mutius E, Riedler J, Braun-Fahrlander C, Nowak D and Martinez FD (2004) Toll-like receptor 2 as a major gene for asthma in children of European farmers. J. Allergy Clin. Immunol. 113 (3) 482 - 488

Elahi A, Zheng Z, Park J, Eyring K, McCaffrey T and Lazarus P (2002) The human OGG1 DNA repair enzyme and its association with orolaryngeal cancer risk. Carcinogenesis. 23 (7) 1229 - 1234

Elder RH and Dianov GL (2002) Repair of dihydrouracil supported by base excision repair in mNTH1 knock-out cell extracts. J. Biol. Chem. 277 (52) 50487 - 50490


208

Elder RH, Jansen JG, Weeks RJ, Willington MA, Deans B, Watson AJ, Mynett KJ, Bailey JA, Cooper DP, Rafferty JA, Heeran MC, Wijnhoven SW, van Zeeland AA and Margison GP (1998) Alkylpurine-DNA-N-glycosylase knockout mice show increased susceptibility to induction of mutations by methyl methanesulfonate. Mol. Cell Biol. 18 (10) 5828 - 5837

Elder RH, Rosenquist TA and Parsons JL (2004) Characterisation of [NTH1,OGG1]-/- mice. Proc Amer Assoc Cancer Res. 45

Engelward BP, Weeda G, Wyatt MD, Broekhof JL, de WJ, Donker I, Allan JM, Gold B, Hoeijmakers JH and Samson LD (1997) Base excision repair deficient mice lacking the Aag alkyladenine DNA glycosylase. Proc Natl. Acad. Sci. U. S. A. 94 (24) 13087 - 13092

Englander EW and Ma H (2006) Differential modulation of base excision repair activities during brain ontogeny: Implications for repair of transcribed DNA. Mech. Age. Dev. 127 (1) 64 - 69

Enterline PE, Sykora JL, Keleti G and Lange JH (1985) Endotoxins, cotton dust, and cancer. Lancet. 2 (8461) 934 - 935

Ershler WB, Sun WH, Binkley N, Gravenstein S, Volk MJ, Kamoske G, Klopp RG, Roecker EB, Daynes RA and Weindruch R (1993) Interleukin-6 and aging: blood levels and mononuclear cell production increase with advancing age and in vitro production is modifiable by dietary restriction. Lymphokine Cytokine Res. 12 (4) 225 - 230

Esposito LA, Melov S, Panov A, Cottrell BA and Wallace DC (1999) Mitochondrial disease in mouse results in increased oxidative stress. Proc. Natl. Acad. Sci. U. S. A. 96 (9) 4820 - 4825

Fan H and Cook JA (2004) Molecular mechanisms of endotoxin tolerance. J. Endotoxin Res. 10 (2) 71 - 84

Farinati F, Cardin R, Bortolami M, Nitti D, Basso D, de BM, Cassaro M, Sergio A and Rugge M (2008) Oxidative DNA damage in gastric cancer: CagA status and OGG1 gene polymorphism. Int. J. Cancer. 123 (1) 51 - 55

Faurschou M and Borregaard N (2003) Neutrophil granules and secretory vesicles in inflammation. Microbes Infect. 5 (14) 1317 - 1327

Franco-Morselli R, Elghozi JL, Joly E, Di GS and Meyer P (1977) Increased plasma adrenaline concentrations in benign essential hypertension. Br. Med. J. 2 (6097) 1251 - 1254

Frankenberg-Schwager M, Becker M, Garg I, Pralle E, Wolf H and Frankenberg D (2008) The role of nonhomologous DNA end joining, conservative homologous recombination, and single-strand annealing in


209

the cell cycle-dependent repair of DNA double-strand breaks induced by H2O2 in mammalian cells. Radiat. Res. 170 (6) 784 - 793

Fromme JC, Banerjee A, Huang SJ and Verdine GL (2004) Structural basis for removal of adenine mispaired with 8-oxoguanine by MutY adenine DNA glycosylase. Nature. 427 (6975) 652 - 656

Fromme JC and Verdine GL (2004) Base excision repair. Adv. Protein Chem. 69 1 - 41

Fung H and Demple B (2005) A vital role for Ape1/Ref1 protein in repairing spontaneous DNA damage in human cells. Mol. Cell. 17 (3) 463 - 470

Gilmore W, Weiner LP and Correale J (1997) Effect of estradiol on cytokine secretion by proteolipid protein-specific T cell clones isolated from multiple sclerosis patients and normal control subjects. J. Immunol. 158 (1) 446 - 451

Girard PM, D'Ham C, Cadet J and Boiteux S (1998) Opposite base-dependent excision of 7,8-dihydro-8-oxoadenine by the Ogg1 protein of Saccharomyces cerevisiae. Carcinogenesis. 19 (7) 1299 - 1305

Gloire G, Legrand-Poels S and Piette J (2006) NF-[kappa]B activation by reactive oxygen species: Fifteen years later. Biochem. Pharmacol. 72 (11) 1493 - 1505

Golyasnaya NV and Tsvetkova NA (2006) Mismatch repair. Mol. Biol. 40 (2) 183 - 193

Goodman L and Stein GH (1994) Basal and induced amounts of interleukin-6 mRNA decline progressively with age in human fibroblasts. J. Biol. Chem. 269 (30) 19250 - 19255

Goodrick CL, Ingram DK, Reynolds MA, Freeman JR and Cider N (1990) Effects of intermittent feeding upon body weight and lifespan in inbred mice: interaction of genotype and age. Mech. Age. Dev. 55 (1) 69 - 87

Graff G, Gamache DA, Brady MT, Spellman JM and Yanni JM (1998) Improved myeloperoxidase assay for quantitation of neutrophil influx in a rat model of endotoxin-induced uveitis. J. Pharacol. Toxicol. 39 (3) 169 - 178

Gredilla R, Garm C, Holm R, Bohr VA and Stevnsner T (2010) Differential age-related changes in mitochondrial DNA repair activities in mouse brain regions. Neurobiol. Aging. 31 (6) 993 - 1002

Grin IR, Rieger RA and Zharkov DO (2010) Inactivation of NEIL2 DNA glycosylase by pyridoxal phosphate reveals a loop important for substrate binding. Biochem. Biophys. Res. Commun. 394 (1) 100 - 105


210

Gros L, Saparbaev MK and Laval J (2002) Enzymology of the repair of free radicals-induced DNA damage. Oncogene. 21 (58) 8905 - 8925

Gudkov AV, Gurova KV and Komarova EA (2011) Inflammation and p53: A Tale of Two Stresses. Gene. Canc. 2 (4) 503 - 516

Gupta SC, Kim JH, Kannappan R, Reuter S, Dougherty PM and Aggarwal BB (2011) Role of nuclear factor kappaB-mediated inflammatory pathways in cancer-related symptoms and their regulation by nutritional agents. Exp. Biol. Med. 236 (6) 658 - 671

Hanaoka T, Sugimura H, Nagura K, Ihara M, Li XJ, Hamada GS, Nishimoto I, Kowalski LP, Yokota J and Tsugane S (2001) hOGG1 exon7 polymorphism and gastric cancer in case-control studies of Japanese Brazilians and non-Japanese Brazilians. Cancer Lett. 170 (1) 53 - 61

Hansen R, Saebo M, Skjelbred CF, Nexo BA, Hagen PC, Bock G, Bowitz L, I, Johnson E, Aase S, Hansteen IL, Vogel U and Kure EH (2005) GPX Pro198Leu and OGG1 Ser326Cys polymorphisms and risk of development of colorectal adenomas and colorectal cancer. Cancer Lett. 229 (1) 85 - 91

Harrison RF, Reynolds GM and Rowlands DC (1993) Immunohistochemical evidence for the expression of proliferating cell nuclear antigen (PCNA) by non-proliferating hepatocytes adjacent to metastatic tumours and in inflammatory conditions. J. Pathol. 171 (2) 115 - 122

Hasday JD, Bascom R, Costa JJ, Fitzgerald T and Dubin W (1999) Bacterial endotoxin is an active component of cigarette smoke. Chest. 115 (3) 829 - 835

Hashiguchi K, Stuart JA, de Souza-Pinto NC and Bohr VA (2004) The C-terminal alphaO helix of human Ogg1 is essential for 8-oxoguanine DNA glycosylase activity: the mitochondrial beta-Ogg1 lacks this domain and does not have glycosylase activity. Nucleic Acids Res. 32 (18) 5596 - 5608

Hasko G, Szabo C, Nemeth ZH, Kvetan V, Pastores SM and Vizi ES (1996) Adenosine receptor agonists differentially regulate IL-10, TNF-alpha, and nitric oxide production in RAW 264.7 macrophages and in endotoxemic mice. J. Immunol. 157 (10) 4634 - 4640

Hayes RC and LeClerc JE (1986) Sequence dependence for bypass of thymine glycols in DNA by DNA polymerase I. Nucleic Acids Res. 14 (2) 1045 - 1061

Hazra TK, Izumi T, Boldogh I, Imhoff B, Kow YW, Jaruga P, Dizdaroglu M and Mitra S (2002a) Identification and characterization of a human DNA glycosylase for repair of modified bases in oxidatively damaged DNA. Proc. Natl. Acad. Sci. U. S. A. 99 (6) 3523 - 3528


211

Hazra TK, Kow YW, Hatahet Z, Imhoff B, Boldogh I, Mokkapati SK, Mitra S and Izumi T (2002b) Identification and characterization of a novel human DNA glycosylase for repair of cytosine-derived lesions. J. Biol. Chem. 277 (34) 30417 - 30420

Hazra TK, Das A, Das S, Choudhury S, Kow YW and Roy R (2007) Oxidative DNA damage repair in mammalian cells: A new perspective. DNA Repair. 6 (4) 470 - 480

Hegde ML, Theriot CA, Das A, Hegde PM, Guo Z, Gary RK, Hazra TK, Shen B and Mitra S (2008) Physical and functional interaction between human oxidized base-specific DNA glycosylase NEIL1 and flap endonuclease 1. J. Biol. Chem. 283 (40) 27028 - 27037

Hewitt SC and Korach KS (2003) Oestrogen receptor knockout mice: roles for oestrogen receptors alpha and beta in reproductive tissues. Reproduction. 125 (2) 143 - 149

Hill JW, Hazra TK, Izumi T and Mitra S (2001) Stimulation of human 8-oxoguanine-DNA glycosylase by AP-endonuclease: potential coordination of the initial steps in base excision repair. Nucleic Acids Research. 29 (2) 430 - 438

Hill KA, Buettner VL, Glickman BW and Sommer SS (1999) Spontaneous mutations in the Big Blue-« transgenic system are primarily mouse derived. Mutat. Res. -Rev. Mut. R. 436 (1) 11 - 19

Hirano S, Tominaga Y, Ichinoe A, Ushijima Y, Tsuchimoto D, Honda-Ohnishi Y, Ohtsubo T, Sakumi K and Nakabeppu Y (2003) Mutator phenotype of MUTYH-null mouse embryonic stem cells. J. Biol. Chem. 278 (40) 38121 - 38124

Hoffler U, Hobbie K, Wilson R, Bai R, Rahman A, Malarkey D, Travlos G and Ghanayem BI (2009) Diet-induced obesity is associated with hyperleptinemia, hyperinsulinemia, hepatic steatosis, and glomerulopathy in C57Bl/6J mice. Endocrine. 36 (2) 311 - 325

Hofseth LJ, Saito S, Hussain SP, Espey MG, Miranda KM, Araki Y, Jhappan C, Higashimoto Y, He P, Linke SP, Quezado MM, Zurer I, Rotter V, Wink DA, Appella E and Harris CC (2003) Nitric oxide-induced cellular stress and p53 activation in chronic inflammation. Proc. Natl. Acad. Sci. U. S. A. 100 (1) 143 - 148

Hung RJ, Hall J, Brennan P and Boffetta P (2005) Genetic polymorphisms in the base excision repair pathway and cancer risk: a HuGE review. Am. J. Epidemiol. 162 (10) 925 - 942


212

Ikeda S, Kohmoto T, Tabata R and Seki Y (2002) Differential intracellular localization of the human and mouse endonuclease III homologs and analysis of the sorting signals. DNA Repair (Amst). 1 (10) 847 - 854

Imlay JA (2003) Pathways of oxidative damage. Annu. Rev. Microbiol. 57 395 - 418

Imlay JA, Chin SM and Linn S (1988) Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro. Science. 240 (4852) 640 - 642

Ito A, Bebo BF, Jr., Matejuk A, Zamora A, Silverman M, Fyfe-Johnson A and Offner H (2001) Estrogen treatment down-regulates TNF-alpha production and reduces the severity of experimental autoimmune encephalomyelitis in cytokine knockout mice. J. Immunol. 167 (1) 542 - 552

Ivor J (1994) The ins and outs of fibroblast growth factors. Cell. 78 (4) 547 - 552

Izeboud CA, Monshouwer M, van Miert AS and Witkamp RF (1999) The beta-adrenoceptor agonist clenbuterol is a potent inhibitor of the LPS-induced production of TNF-alpha and IL-6 in vitro and in vivo. Inflamm. Res. 48 (9) 497 - 502

Izumi T, Wiederhold LR, Roy G, Roy R, Jaiswal A, Bhakat KK, Mitra S and Hazra TK (2003) Mammalian DNA base excision repair proteins: their interactions and role in repair of oxidative DNA damage. Toxicology. 193 (1-2) 43 - 65

Jaber BL, Pereira BJ, Bonventre JV and Balakrishnan VS (2005) Polymorphism of host response genes: implications in the pathogenesis and treatment of acute renal failure. Kidney Int. 67 (1) 14 - 33

Jaiswal M, LaRusso NF, Burgart LJ and Gores GJ (2000) Inflammatory cytokines induce DNA damage and inhibit DNA repair in cholangiocarcinoma cells by a nitric oxide-dependent mechanism. Cancer Res. 60 (1) 184 - 190

Jaiswal M, LaRusso NF, Nishioka N, Nakabeppu Y and Gores GJ (2001) Human Ogg1, a protein involved in the repair of 8-oxoguanine, is inhibited by nitric oxide. Cancer Res. 61 (17) 6388 - 6393

Jin H, Jiang B, Tang J, Lu W, Wang W, Zhou L, Shang W, Li F, Ma Q, Yang Y and Chen M (2008) Serum visfatin concentrations in obese adolescents and its correlation with age and high-density lipoprotein cholesterol. Diabetes Res. Clin. Prac. 79 (3) 412 - 418


213

Kabir K, Gelinas JP, Chen M, Chen D, Zhang D, Luo X, Yang JH, Carter D and Rabinovici R (2002) Characterization of a murine model of endotoxin-induced acute lung injury. Shock. 17 (4) 300 - 303

Kalam MA, Haraguchi K, Chandani S, Loechler EL, Moriya M, Greenberg MM and Basu AK (2006) Genetic effects of oxidative DNA damages: comparative mutagenesis of the imidazole ring-opened formamidopyrimidines (Fapy lesions) and 8-oxo-purines in simian kidney cells. Nucleic Acids Res. 34 (8) 2305 - 2315

Kano A, Wolfgang MJ, Gao Q, Jacoby J, Chai GX, Hansen W, Iwamoto Y, Pober JS, Flavell RA and Fu XY (2003) Endothelial cells require STAT3 for protection against endotoxin-induced inflammation. J. Exp. Med. 198 (10) 1517 - 1525

Karahalil B, de Souza-Pinto NC, Parsons JL, Elder RH and Bohr VA (2003) Compromised incision of oxidized pyrimidines in liver mitochondria of mice deficient in NTH1 and OGG1 glycosylases. J. Biol. Chem. 278 (36) 33701 - 33707

Kenagy GJ and Trombulak SC (1986) Size and function of mammalian testes in relation to body size. J. Mam. 67 (1) 1 - 22

Khobta A, Kitsera N, Speckmann B and Epe B (2009) 8-Oxoguanine DNA glycosylase (Ogg1) causes a transcriptional inactivation of damaged DNA in the absence of functional Cockayne syndrome B (Csb) protein. DNA Repair. 8 (3) 309 - 317

Kiefmann R, Heckel K, Doerger M, Schenkat S, Kupatt C, Stoeckelhuber M, Wesierska-Gadek J and Goetz AE (2004) Role of PARP on iNOS pathway during endotoxin-induced acute lung injury. Intensive Care Med. 30 (7) 1421 - 1431

Kim HS, Ye SK, Cho IH, Jung JE, Kim DH, Choi S, Kim YS, Park CG, Kim TY, Lee JW and Chung MH (2006) 8-hydroxydeoxyguanosine suppresses NO production and COX-2 activity via Rac1/STATs signaling in LPS-induced brain microglia. Free Radic. Biol. Med. 41 (9) 1392 - 1403

Kisielewska J, Lu P and Whitaker M (2005) GFP-PCNA as an S-phase marker in embryos during the first and subsequent cell cycles. Biol. Cell. 97 (3) 221 - 229

Kley HK, Solbach HG, McKinnan JC and Kruskemper HL (1979) Testosterone decrease and oestrogen increase in male patients with obesity. Acta Endocrinol. (Copenh). 91 (3) 553 - 563

Klungland A, Rosewell I, Hollenbach S, Larsen E, Daly G, Epe B, Seeberg E, Lindahl T and Barnes DE (1999) Accumulation of premutagenic DNA


214

lesions in mice defective in removal of oxidative base damage. Proc. Natl. Acad. Sci. U. S. A. 96 (23) 13300 - 13305

Kohno T, Kunitoh H, Toyama K, Yamamoto S, Kuchiba A, Saito D, Yanagitani N, Ishihara S, Saito R and Yokota J (2006) Association of the OGG1-Ser326Cys polymorphism with lung adenocarcinoma risk. Cancer Sci. 97 (8) 724 - 728

Koizumi K, Poulaki V, Doehmen S, Welsandt G, Radetzky S, Lappas A, Kociok N, Kirchhof B and Joussen AM (2003) Contribution of TNF-alpha to leukocyte adhesion, vascular leakage, and apoptotic cell death in endotoxin-induced uveitis in vivo. Invest Ophthalmol. Vis. Sci. 44 (5) 2184 - 2191

Konstantinidis A, Valatas V, Ntelis V, Balatsos V, Karoumpalis I, Hatzinikoloaou A, Manolakopoulos S, Vafiadis I, Archimandritis A and Skandalis N (2011) Endoscopic treatment for high-risk bleeding peptic ulcers: a comparison of epinephrine alone with epinephrine plus ethanolamine. Annals of Gastro. 24 (2) 101 - 107

Kooter IM, Frederix K, Spronk HM, Boere AJ, Leseman DL, van SH, ten CH and Cassee FR (2008) Lung inflammation and thrombogenic responses in a time course study of Csb mice exposed to ozone. J. Appl. Toxicol. 28 (6) 779 - 787

Krishnamurthy N, Haraguchi K, Greenberg MM and David SS (2008) Efficient removal of formamidopyrimidines by 8-oxoguanine glycosylases. Biochemistry. 47 (3) 1043 - 1050

Krokan HE, Nilsen H, Skorpen F, Otterlei M and Slupphaug G (2000) Base excision repair of DNA in mammalian cells. FEBS Letters. 476 (1-2) 73 - 77

Kucherlapati M, Yang K, Kuraguchi M, Zhao J, Lia M, Heyer J, Kane MF, Fan K, Russell R, Brown AM, Kneitz B, Edelmann W, Kolodner RD, Lipkin M and Kucherlapati R (2002) Haploinsufficiency of Flap endonuclease (Fen1) leads to rapid tumor progression. Proc. Natl. Acad. Sci. U. S. A. 99 (15) 9924 - 9929

Kurt-Jones EA, Sandor F, Ortiz Y, Bowen GN, Counter SL, Wang TC and Finberg RW (2004) Use of murine embryonic fibroblasts to define Toll-like receptor activation and specificity. J. Endotoxin. Res. 10 (6) 419 - 424

Lam JS, Seligson DB, Yu H, Li A, Eeva M, Pantuck AJ, Zeng G, Horvath S and Belldegrun AS (2006) Flap endonuclease 1 is overexpressed in prostate cancer and is associated with a high Gleason score. BJU. Int. 98 (2) 445 - 451


215

Lange JH, Mastrangelo G, Fadda E, Priolo G, Montemurro D, Buja A and Grange JM (2005) Elevated lung cancer risk shortly after smoking cessation: Is it due to a reduction of endotoxin exposure? Med. Hypotheses. 65 (3) 534 - 541

Lange JH, Mastrangelo G, Fedeli U, Fadda E, Rylander R and Lee E (2003) Endotoxin exposure and lung cancer mortality by type of farming: is there a hidden dose-response relationship? Ann. Agric. Environ. Med. 10 (2) 229 - 232

Larsen E, Meza TJ, Kleppa L and Klungland A (2007) Organ and cell specificity of base excision repair mutants in mice. Mutat. Res. -Fund. Mol. M. 614 (1-2) 56 - 68

Le Page F., Klungland A, Barnes DE, Sarasin A and Boiteux S (2000) Transcription coupled repair of 8-oxoguanine in murine cells: the ogg1 protein is required for repair in nontranscribed sequences but not in transcribed sequences. Proc. Natl. Acad. Sci. U. S. A. 97 (15) 8397 - 8402

Leal AM, Ruiz-Larrea MBa, Mart-¦¦ünez R and Lacort M (1998) Cytoprotective actions of estrogens against tert-butyl hydroperoxide-induced toxicity in hepatocytes. Biochem. Pharmacol. 56 (11) 1463 - 1469

Lee FD (1992) The role of interleukin-6 in development. Dev. Biol. 151 (2) 331 - 338

Lee JM, Lee YC, Yang SY, Yang PW, Luh SP, Lee CJ, Chen CJ and Wu MT (2001) Genetic polymorphisms of XRCC1 and risk of the esophageal cancer. Int. J. Cancer. 95 (4) 240 - 246

Lefévre N, Corazza F, Duchateau J, Desir J and Casimir G (2012) Sex Differences in Inflammatory Cytokines and CD99 Expression Following In Vitro LPS Stimulation. Shock.

Lehmann AR (2003) DNA repair-deficient diseases, xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Biochimie. 85 (11) 1101 - 1111

Li L, Bhatia M, Zhu YZ, Zhu YC, Ramnath RD, Wang ZJ, Anuar FB, Whiteman M, Salto-Tellez M and Moore PK (2005) Hydrogen sulfide is a novel mediator of lipopolysaccharide-induced inflammation in the mouse. FASEB J. 19 (9) 1196 - 1198

Liu AH (2007) Hygiene theory and allergy and asthma prevention. Paediatr. Perinat. Epidemiol. 21 Suppl 3 2 - 7

Lloyd DR and Phillips DH (1999) Oxidative DNA damage mediated by copper(II), iron(II) and nickel(II) fenton reactions: evidence for site-specific mechanisms in the formation of double-strand breaks, 8-


216

hydroxydeoxyguanosine and putative intrastrand cross-links. Mutat. Res. 424 (1-2) 23 - 36

Luna L, Bjoras M, Hoff E, Rognes T and Seeberg E (2000) Cell-cycle regulation, intracellular sorting and induced overexpression of the human NTH1 DNA glycosylase involved in removal of formamidopyrimidine residues from DNA. Mutat. Res. 460 (2) 95 - 104

Mabley JG, Pacher P, Deb A, Wallace R, Elder RH and Szabo C (2005a) Potential role for 8-oxoguanine DNA glycosylase in regulating inflammation. FASEB J. 19 (2) 290 - 292

Mabley JG, Horvath EM, Murthy KGK, Zsengeller Z, Vaslin A, Benko R, Kollai M and Szabo C (2005b) Gender Differences in the Endotoxin-Induced Inflammatory and Vascular Responses: Potential Role of Poly(ADP-ribose) Polymerase Activation. Journal of Pharmacology And Experimental Therapeutics. 315 (2) 812 - 820

Maloney G, Schroder M and Bowie AG (2005) Vaccinia virus protein A52R activates p38 mitogen-activated protein kinase and potentiates lipopolysaccharide-induced interleukin-10. J. Biol. Chem. 280 (35) 30838 - 30844

Marenstein DR, Chan MK, Altamirano A, Basu AK, Boorstein RJ, Cunningham RP and Teebor GW (2003) Substrate specificity of human endonuclease III (hNTH1). Effect of human APE1 on hNTH1 activity. J. Biol. Chem. 278 (11) 9005 - 9012

Marnett LJ (1999) Lipid peroxidation-DNA damage by malondialdehyde. Mutat. Res. 424 (1-2) 83 - 95

Martin J, Han C, Gordon LA, Terry A, Prabhakar S, She X, Xie G, Hellsten U, Chan YM, Altherr M, Couronne O, Aerts A, Bajorek E, Black S, Blumer H, Branscomb E, Brown NC, Bruno WJ, Buckingham JM, Callen DF, Campbell CS, Campbell ML, Campbell EW, Caoile C, Challacombe JF, Chasteen LA, Chertkov O, Chi HC, Christensen M, Clark LM, Cohn JD, Denys M, Detter JC, Dickson M, mitrijevic-Bussod M, Escobar J, Fawcett JJ, Flowers D, Fotopulos D, Glavina T, Gomez M, Gonzales E, Goodstein D, Goodwin LA, Grady DL, Grigoriev I, Groza M, Hammon N, Hawkins T, Haydu L, Hildebrand CE, Huang W, Israni S, Jett J, Jewett PB, Kadner K, Kimball H, Kobayashi A, Krawczyk MC, Leyba T, Longmire JL, Lopez F, Lou Y, Lowry S, Ludeman T, Manohar CF, Mark GA, McMurray KL, Meincke LJ, Morgan J, Moyzis RK, Mundt MO, Munk AC, Nandkeshwar RD, Pitluck S, Pollard M, Predki P, Parson-Quintana B, Ramirez L, Rash S, Retterer J, Ricke DO, Robinson DL, Rodriguez A, Salamov A, Saunders EH, Scott D, Shough T, Stallings RL, Stalvey M, Sutherland RD, Tapia R, Tesmer JG, Thayer N, Thompson LS, Tice H, Torney DC, Tran-Gyamfi M, Tsai M, Ulanovsky LE, Ustaszewska A, Vo N, White PS, Williams AL, Wills PL, Wu JR, Wu K, Yang J, Dejong P, Bruce D, Doggett NA, Deaven L, Schmutz J,


217

Grimwood J, Richardson P, Rokhsar DS, Eichler EE, Gilna P, Lucas SM, Myers RM, Rubin EM and Pennacchio LA (2004) The sequence and analysis of duplication-rich human chromosome 16. Nature. 432 (7020) 988 - 994

Martin SP, Hortelano S, Bosca L and Casado M (2006) Cyclooxygenase 2: understanding the pathophysiological role through genetically altered mouse models. Front Biosci. 11 2876 - 2888

Martin TR (2000) Recognition of Bacterial Endotoxin in the Lungs. Am. J. Respir. Cell Mol. Biol. 23 128 - 132

Martinez FO, Sironi M, Vecchi A, Colotta F, Mantovani A and Locati M (2004) IL-8 induces a specific transcriptional profile in human neutrophils: synergism with LPS for IL-1 production. Eur. J. Immunol. 34 (8) 2286 - 2292

Mathiak G, Kabir K, Grass G, Keller H, Steinringer E, Minor T, Rangger C and Neville LF (2003) Lipopolysaccharides from different bacterial sources elicit disparate cytokine responses in whole blood assays. Int. J. Mol. Med. 11 (1) 41 - 44

Matsukawa A, Yoshimura T, Miyamoto K, Ohkawara S and Yoshinaga M (1997) Analysis of the inflammatory cytokine network among TNF alpha, IL-1 beta, IL-1 receptor antagonist, and IL-8 in LPS-induced rabbit arthritis. Lab Invest. 76 (5) 629 - 638

Maynard S, Schurman SH, Harboe C, de Souza-Pinto NC and Bohr VA (2009) Base excision repair of oxidative DNA damage and association with cancer and aging. Carcinogenesis. 30 (1) 2 - 10

Mberikunashe J, Banda S, Chadambuka A, Gombe NT, Shambira G, Tshimanga M and Matchaba-Hove R (2010) Prevalence and risk factors for obstructive respiratory conditions among textile industry workers in Zimbabwe, 2006. Pan Afr. Med. J. 6 (1)

McElvenny DM, Hurley MA, Lenters V, Heederik D, Wilkinson S and Coggon D (2011) Lung cancer mortality in a cohort of UK cotton workers: an extended follow-up. Br J Cancer. 105 (7) 1054 - 1060

McKERROW CB, McDERMOTT M, GILSON JC and SCHILLING RS (1958) Respiratory function during the day in cotton workers: a study in byssinosis. Br. J. Ind. Med. 16 75 - 83

Millar CB, Guy J, Sansom OJ, Selfridge J, MacDougall E, Hendrich B, Keightley PD, Bishop SM, Clarke AR and Bird A (2002) Enhanced CpG mutability and tumorigenesis in MBD4-deficient mice. Science. 297 (5580) 403 - 405


218

Miller H, Fernandes AS, Zaika E, McTigue MM, Torres MC, Wente M, Iden CR and Grollman AP (2004) Stereoselective excision of thymine glycol from oxidatively damaged DNA. Nucleic Acids Res. 32 (1) 338 - 345

Mitchell JR, Hoeijmakers JH and Niedernhofer LJ (2003) Divide and conquer: nucleotide excision repair battles cancer and ageing. Curr. Opin. Cell Biol. 15 (2) 232 - 240

Mokkapati SK, Wiederhold L, Hazra TK and Mitra S (2004a) Stimulation of DNA glycosylase activity of OGG1 by NEIL1: functional collaboration between two human DNA glycosylases. Biochemistry. 43 (36) 11596 - 11604

Mokkapati SK, Wiederhold L, Hazra TK and Mitra S (2004b) Stimulation of DNA glycosylase activity of OGG1 by NEIL1: functional collaboration between two human DNA glycosylases. Biochemistry. 43 (36) 11596 - 11604

Moller B and Villiger PM (2006) Inhibition of IL-1, IL-6, and TNF-alpha in immune-mediated inflammatory diseases. Springer Semin. Immunopathol. 27 (4) 391 - 408

Moore RN, Goodrum KJ and Berry LJ (1976) Mediation of an endotoxic effect by macrophages. J Reticuloendothel. Soc. 19 (3) 187 - 197

Morland I, Rolseth V, Luna L, Rognes T, Bjoras M and Seeberg E (2002) Human DNA glycosylases of the bacterial Fpg/MutM superfamily: an alternative pathway for the repair of 8-oxoguanine and other oxidation products in DNA. Nucleic Acids Res. 30 (22) 4926 - 4936

Muftuoglu M, de Souza-Pinto NC, Dogan A, Aamann M, Stevnsner T, Rybanska I, Kirkali G, Dizdaroglu M and Bohr VA (2009) Cockayne syndrome group B protein stimulates repair of formamidopyrimidines by NEIL1 DNA glycosylase. J. Biol. Chem. 284 (14) 9270 - 9279

Mutamba JT, Svilar D, Prasongtanakij S, Wang XH, Lin YC, Dedon PC, Sobol RW and Engelward BP (2011) XRCC1 and base excision repair balance in response to nitric oxide. DNA Repair. 10 (12) 1282 - 1293

Naik E and Dixit VM (2011) Mitochondrial reactive oxygen species drive proinflammatory cytokine production. J. Exp. Med. 208 (3) 417 - 420

Netea MG, van Deuren M, Kullberg BJ, Cavaillon JM and Van der Meer JWM (2002) Does the shape of lipid A determine the interaction of LPS with Toll-like receptors? Trends Immunol. 23 (3) 135 - 139

Nilsen H, Stamp G, Andersen S, Hrivnak G, Krokan HE, Lindahl T and Barnes DE (2003) Gene-targeted mice lacking the Ung uracil-DNA glycosylase develop B-cell lymphomas. Oncogene. 22 (35) 5381 - 5386


219

Nilsen H, Rosewell I, Robins P, Skjelbred CF, Andersen S, Slupphaug G, Daly G, Krokan HE, Lindahl T and Barnes DE (2000) Uracil-DNA Glycosylase (UNG)-Deficient Mice Reveal a Primary Role of the Enzyme during DNA Replication. Mol. Cell. 5 (6) 1059 - 1065

Nilsson BO (2007) Modulation of the inflammatory response by estrogens with focus on the endothelium and its interactions with leukocytes. Inflamm. Res. 56 (7) 269 - 273

Nusbaum C, Mikkelsen TS, Zody MC, Asakawa S, Taudien S, Garber M, Kodira CD, Schueler MG, Shimizu A, Whittaker CA, Chang JL, Cuomo CA, Dewar K, FitzGerald MG, Yang X, Allen NR, Anderson S, Asakawa T, Blechschmidt K, Bloom T, Borowsky ML, Butler J, Cook A, Corum B, DeArellano K, DeCaprio D, Dooley KT, Dorris L, III, Engels R, Glockner G, Hafez N, Hagopian DS, Hall JL, Ishikawa SK, Jaffe DB, Kamat A, Kudoh J, Lehmann R, Lokitsang T, Macdonald P, Major JE, Matthews CD, Mauceli E, Menzel U, Mihalev AH, Minoshima S, Murayama Y, Naylor JW, Nicol R, Nguyen C, O'Leary SB, O'Neill K, Parker SC, Polley A, Raymond CK, Reichwald K, Rodriguez J, Sasaki T, Schilhabel M, Siddiqui R, Smith CL, Sneddon TP, Talamas JA, Tenzin P, Topham K, Venkataraman V, Wen G, Yamazaki S, Young SK, Zeng Q, Zimmer AR, Rosenthal A, Birren BW, Platzer M, Shimizu N and Lander ES (2006) DNA sequence and analysis of human chromosome 8. Nature. 439 (7074) 331 - 335

Oakley AJ (2005) Glutathione transferases: new functions. Curr. Opin. Struc. Biol. 15 (6) 716 - 723

Ocampo MT, Chaung W, Marenstein DR, Chan MK, Altamirano A, Basu AK, Boorstein RJ, Cunningham RP and Teebor GW (2002) Targeted deletion of mNth1 reveals a novel DNA repair enzyme activity. Mol. Cell Biol. 22 (17) 6111 - 6121

Ock CY, Kim EH, Hong H, Hong KS, Han YM, Choi KS, Hahm KB and Chung MH (2011a) Prevention of colitis-associated colorectal cancer with 8-hydroxydeoxyguanosine. Cancer Prev. Res. (Phila). 4 (9) 1507 - 1521

Ock CY, Hong KS, Choi KS, Chung MH, Kim Ys, Kim JH and Hahm KB (2011b) A novel approach for stress-induced gastritis based on paradoxical anti-oxidative and anti-inflammatory action of exogenous 8-hydroxydeoxyguanosine. Biochem. Pharmacol. 81 (1) 111 - 122

Orr Y, Wilson DP, Taylor JM, Bannon PG, Geczy C, Davenport MP and Kritharides L (2007) A kinetic model of bone marrow neutrophil production that characterizes late phenotypic maturation. Am. J. Physiol Regul. Integr. Comp Physiol. 292 (4) R1707 - R1716

Orren DK, Petersen LN and Bohr VA (1997) Persistent DNA damage inhibits S-phase and G2 progression, and results in apoptosis. Mol. Biol. Cell. 8 (6) 1129 - 1142


220

Osterod M, Hollenbach S, Hengstler JG, Barnes DE, Lindahl T and Epe B (2001) Age-related and tissue-specific accumulation of oxidative DNA base damage in 7,8-dihydro-8-oxoguanine-DNA glycosylase (Ogg1) deficient mice. Carcinogenesis. 22 (9) 1459 - 1463

Oyama M, Wakasugi M, Hama T, Hashidume H, Iwakami Y, Imai R, Hoshino S, Morioka H, Ishigaki Y, Nikaido O and Matsunaga T (2004) Human NTH1 physically interacts with p53 and proliferating cell nuclear antigen. Biochem. Biophys. Res. Commun. 321 (1) 183 - 191

Panduri V, Liu G, Surapureddi S, Kondapalli J, Soberanes S, de Souza-Pinto NC, Bohr VA, Budinger GRS, Schumacker PT, Weitzman SA and Kamp DW (2009) Role of mitochondrial hOGG1 and aconitase in oxidant-induced lung epithelial cell apoptosis. Free Radic. Biol. Med. 47 (6) 750 - 759

Parsons JL and Elder RH (2003) DNA N-glycosylase deficient mice: a tale of redundancy. Mutat. Res. 531 (1-2) 165 - 175

Paulos CM, Kaiser A, Wrzesinski C, Hinrichs CS, Cassard L, Boni A, Muranski P, Sanchez-Perez L, Palmer DC, Yu Z, Antony PA, Gattinoni L, Rosenberg SA and Restifo NP (2007) Toll-like receptors in tumor immunotherapy. Clin. Cancer Res. 13 (18 Pt 1) 5280 - 5289

Perez-Campo FM, Spencer HL, Elder RH, Stern PL and Ward CM (2007) Novel vectors for homologous recombination strategies in mouse embryonic stem cells: An ES cell line expressing EGFP under control of the 5T4 promoter. Exp. Cell. Res. 313 (16) 3604 - 3615

Pettus BJ, Chalfant CE and Hannun YA (2002) Ceramide in apoptosis: an overview and current perspectives. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 1585 (2-3) 114 - 125

Pheng LH, Francoeur C and Denis M (1995) The involvement of nitric oxide in a mouse model of adult respiratory distress syndrome. Inflammation. 19 (5) 599 - 610

Prins JM (1996) Antibiotic induced release of endotoxin - clinical data and human studies. J. Endotoxin Res. 3 (3) 269 - 273

Prokai L, Prokai-Tatrai K, Perjesi P, Zharikova AD, Perez EJ, Liu R and Simpkins JW (2003) Quinol-based cyclic antioxidant mechanism in estrogen neuroprotection. Proc. Natl. Acad. Sci. U. S. A. 100 (20) 11741 - 11746

Purmal AA, Kow YW and Wallace SS (1994) Major oxidative products of cytosine, 5-hydroxycytosine and 5-hydroxyuracil, exhibit sequence context-dependent mispairing in vitro. Nucleic Acids Res. 22 (1) 72 - 78


221

Qureshi ST, Lariviere L, Leveque G, Clermont S, Moore KJ, Gros P and Malo D (1999) Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4). J. Exp. Med. 189 (4) 615 - 625

Radak Z and Boldogh I (2010) 8-Oxo-7,8-dihydroguanine: Links to gene expression, aging, and defense against oxidative stress. Free Radic. Biol. Med. 49 (4) 587 - 596

Radicella JP, Dherin C, Desmaze C, Fox MS and Boiteux S (1997) Cloning and characterization of hOGG1, a human homolog of the OGG1 gene of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U. S. A. 94 (15) 8010 - 8015

Radon K (2006) The two sides of the "endotoxin coin". Occup. Environ. Med. 63 (1) 73 - 8, 10

Raetz CR, Guan Z, Ingram BO, Six DA, Song F, Wang X and Zhao J (2009) Discovery of new biosynthetic pathways: the lipid A story. J. Lipid Res. 50 Suppl S103 - S108

Rahman I, Kode A and Biswas SK (2006) Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat. Protoc. 1 (6) 3159 - 3165

Ratnasinghe D, Yao SX, Tangrea JA, Qiao YL, Andersen MR, Barrett MJ, Giffen CA, Erozan Y, Tockman MS and Taylor PR (2001) Polymorphisms of the DNA repair gene XRCC1 and lung cancer risk. Cancer Epidemiol. Biomarkers Prev. 10 (2) 119 - 123

Reardon JT, Bessho T, Kung HC, Bolton PH and Sancar A (1997) In vitro repair of oxidative DNA damage by human nucleotide excision repair system: possible explanation for neurodegeneration in xeroderma pigmentosum patients. Proc. Natl. Acad. Sci. U. S. A. 94 (17) 9463 - 9468

Reed CE and Milton DK (2001) Endotoxin-stimulated innate immunity: A contributing factor for asthma. J. Allergy Clin. Immunol. 108 (2) 157 - 166

Rietschel ET, Kirikae T, Schade FU, Mamat U, Schmidt G, Loppnow H, Ulmer AJ, Zahringer U, Seydel U, Di PF and . (1994) Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J. 8 (2) 217 - 225

Risom L, Dybdahl M, Moller P, Wallin H, Haug T, Vogel U, Klungland A and Loft S (2007) Repeated inhalations of diesel exhaust particles and oxidatively damaged DNA in young oxoguanine DNA glycosylase (OGG1) deficient mice. Free Radic. Res. 41 (2) 172 - 181

ROACH SA and SCHILLING RS (1960) A clinical and environmental study of byssinosis in the Lancashire cotton industry. Br. J. Ind. Med. 17 1 - 9


222

Robertson AB, Klungland A, Rognes T and Leiros I (2009) DNA repair in mammalian cells: Base excision repair: the long and short of it. Cell Mol. Life Sci. 66 (6) 981 - 993

Rosenquist TA, Zaika E, Fernandes AS, Zharkov DO, Miller H and Grollman AP (2003) The novel DNA glycosylase, NEIL1, protects mammalian cells from radiation-mediated cell death. DNA Repair (Amst). 2 (5) 581 - 591

Rosenquist TA, Zharkov DO and Grollman AP (1997) Cloning and characterization of a mammalian 8-oxoguanine DNA glycosylase. Proc. Natl. Acad. Sci. U. S. A. 94 (14) 7429 - 7434

Ruchko M, Gorodnya O, LeDoux SP, Alexeyev MF, Al-Mehdi AB and Gillespie MN (2005) Mitochondrial DNA damage triggers mitochondrial dysfunction and apoptosis in oxidant-challenged lung endothelial cells. Am. J. Physiol Lung Cell Mol. Physiol. 288 (3) L530 - L535

Rylander R (2002) Endotoxin in the environment--exposure and effects. J. Endotoxin Res. 8 (4) 241 - 252

Sakumi K, Tominaga Y, Furuichi M, Xu P, Tsuzuki T, Sekiguchi M and Nakabeppu Y (2003) Ogg1 knockout-associated lung tumorigenesis and its suppression by Mth1 gene disruption. Cancer Res. 63 (5) 902 - 905

Salem ML (2004) Estrogen, a double-edged sword: modulation of TH1- and TH2-mediated inflammations by differential regulation of TH1/TH2 cytokine production. Curr. Drug Targets. Inflamm. Allergy. 3 (1) 97 - 104

Sampath H, Batra AK, Vartanian V, Carmical JR, Prusak D, King IB, Lowell B, Earley LF, Wood TG, Marks DL, McCullough AK and Stephen R (2011) Variable penetrance of metabolic phenotypes and development of high-fat diet-induced adiposity in NEIL1-deficient mice. Am. J. Physiol Endocrinol. Metab. 300 (4) E724 - E734

Sarker AH, Ikeda S, Nakano H, Terato H, Ide H, Imai K, Akiyama K, Tsutsui K, Bo Z, Kubo K, Yamamoto K, Yasui A, Yoshida MC and Seki S (1998) Cloning and characterization of a mouse homologue (mNthl1) of Escherichia coli endonuclease III. J. Mol. Biol. 282 (4) 761 - 774

Sarker K, Hashim MA, Hossain MA and Lucky NS (2009) Caudal epidural analgesia in calves using different local analgesics. J. Bangladesh Agri. Uni. 7 (1) 53 - 56

Schromm AB, Brandenburg K, Loppnow H, Zahringer U, Rietschel ET, Carroll SF, Koch MH, Kusumoto S and Seydel U (1998) The charge of endotoxin molecules influences their conformation and IL-6-inducing capacity. J. Immunol. 161 (10) 5464 - 5471


223

Sebai H, Sani M, Ghanem-Boughanmi N+ and Aouani E (2010) Prevention of lipopolysaccharide-induced mouse lethality by resveratrol. Food Chem. Toxicol. 48 (6) 1543 - 1549

Sharman K, Sharman E, Campbell A and Bondy SC (2001) Reduced basal levels and enhanced LPS response of IL-6 mRNA in aged mice. J. Gerontol. A Biol. Sci. Med. Sci. 56 (12) B520 - B523

Sheppard TL, Ordoukhanian P and Joyce GF (2000) A DNA enzyme with N-glycosylase activity. Proc. Natl. Acad. Sci. U. S. A. 97 (14) 7802 - 7807

Shinmura K, Tao H, Goto M, Igarashi H, Taniguchi T, Maekawa M, Takezaki T and Sugimura H (2004) Inactivating mutations of the human base excision repair gene NEIL1 in gastric cancer. Carcinogenesis. 25 (12) 2311 - 2317

Shirota H, Ishii KJ, Takakuwa H and Klinman DM (2006) Contribution of interferon-beta to the immune activation induced by double-stranded DNA. Immunology. 118 (3) 302 - 310

Shrivastav M, De Haro LP and Nickoloff JA (2008) Regulation of DNA double-strand break repair pathway choice. Cell Res. 18 (1) 134 - 147

Sidorenko VS, Nevinsky GA and Zharkov DO (2008) Specificity of stimulation of human 8-oxoguanineΓÇôDNA glycosylase by AP endonuclease. Biochem. Bioph. Res. Co. 368 (1) 175 - 179

Singh P, Yang M, Dai H, Yu D, Huang Q, Tan W, Kernstine KH, Lin D and Shen B (2008) Overexpression and hypomethylation of flap endonuclease 1 gene in breast and other cancers. Mol. Cancer Res. 6 (11) 1710 - 1717

Slupphaug G, Kavli B and Krokan HE (2003) The interacting pathways for prevention and repair of oxidative DNA damage. Mutat. Res. 531 (1-2) 231 - 251

Smart DJ, Chipman JK and Hodges NJ (2006) Activity of OGG1 variants in the repair of pro-oxidant-induced 8-oxo-2'-deoxyguanosine. DNA Repair. 5 (11) 1337 - 1345

Smith JA (1994) Neutrophils, host defense, and inflammation: a double-edged sword. J. Leukoc. Biol. 56 (6) 672 - 686

Smith RS, Smith TJ, Blieden TM and Phipps RP (1997) Fibroblasts as sentinel cells. Synthesis of chemokines and regulation of inflammation. Am. J. Pathol. 151 (2) 317 - 322

Soriano FG, Liaudet L, Szabo E, Virag L, Mabley JG, Pacher P and Szabo C (2002) Resistance to acute septic peritonitis in poly(ADP-ribose) polymerase-1-deficient mice. Shock. 17 (4) 286 - 292


224

Speyer CL, Rancilio NJ, McClintock SD, Crawford JD, Gao H, Sarma JV and Ward PA (2005) Regulatory effects of estrogen on acute lung inflammation in mice. Am. J. Physiol Cell Physiol. 288 (4) C881 - C890

Starcevic D, Dalal S and Sweasy JB (2004) Is there a link between DNA polymerase beta and cancer? Cell Cycle. 3 (8) 998 - 1001

Stern MC, Umbach DM, van Gils CH, Lunn RM and Taylor JA (2001) DNA repair gene XRCC1 polymorphisms, smoking, and bladder cancer risk. Cancer Epidemiol. Biomarkers Prev. 10 (2) 125 - 131

Stivers JT and Jiang YL (2003) A mechanistic perspective on the chemistry of DNA repair glycosylases. Chem. Rev. 103 (7) 2729 - 2759

Strong VE, Kennedy T, Al-Ahmadie H, Tang L, Coleman J, Fong Y, Brennan M and Ghossein RA (2008) Prognostic indicators of malignancy in adrenal pheochromocytomas: clinical, histopathologic, and cell cycle/apoptosis gene expression analysis. Surgery. 143 (6) 759 - 768

Sugioka K, Shimosegawa Y and Nakano M (1987) Estrogens as natural antioxidants of membrane phospholipid peroxidation. FEBS Letters. 210 (1) 37 - 39

Svilar D, Goellner EM, Almeida KH and Sobol RW (2011) Base excision repair and lesion-dependent subpathways for repair of oxidative DNA damage. Antioxid. Redox. Signal. 14 (12) 2491 - 2507

Takao M, Kanno S, Kobayashi K, Zhang QM, Yonei S, van der Horst GT and Yasui A (2002a) A back-up glycosylase in Nth1 knock-out mice is a functional Nei (endonuclease VIII) homologue. J. Biol. Chem. 277 (44) 42205 - 42213

Takao M, Kanno S, Shiromoto T, Hasegawa R, Ide H, Ikeda S, Sarker AH, Seki S, Xing JZ, Le XC, Weinfeld M, Kobayashi K, Miyazaki J, Muijtjens M, Hoeijmakers JH, van der HG and Yasui A (2002b) Novel nuclear and mitochondrial glycosylases revealed by disruption of the mouse Nth1 gene encoding an endonuclease III homolog for repair of thymine glycols. EMBO J. 21 (13) 3486 - 3493

Takeda K, Tanaka T, Shi W, Matsumoto M, Minami M, Kashiwamura S, Nakanishi K, Yoshida N, Kishimoto T and Akira S (1996) Essential role of Stat6 in IL-4 signalling. Nature. 380 (6575) 627 - 630

Tenopoulou M, Doulias PT, Barbouti A, Brunk U and Galaris D (2005) Role of compartmentalized redox-active iron in hydrogen peroxide-induced DNA damage and apoptosis. Biochem. J. 387 (Pt 3) 703 - 710

Tentori L, Leonetti C, Scarsella M, D'Amati G, Vergati M, Portarena I, Xu W, Kalish V, Zupi G, Zhang J and Graziani G (2003) Systemic administration of GPI 15427, a novel poly(ADP-ribose) polymerase-1


225

inhibitor, increases the antitumor activity of temozolomide against intracranial melanoma, glioma, lymphoma. Clin. Cancer Res. 9 (14) 5370 - 5379

Tominaga K, Kirikae T and Nakano M (1997) Lipopolysaccharide (LPS)-induced IL-6 production by embryonic fibroblasts isolated and cloned from LPS-responsive and LPS-hyporesponsive mice. Mol. Immunol. 34 (16-17) 1147 - 1156

Touati E, Michel V, Thiberge JM, Ave P, Huerre M, Bourgade F, Klungland A and Labigne A (2006) Deficiency in OGG1 protects against inflammation and mutagenic effects associated with H. pylori infection in mouse. Helicobacter. 11 (5) 494 - 505

Tremblay S, Gantchev T, Tremblay L, Lavigne P, Cadet J and Wagner JR (2007) Oxidation of 2'-deoxycytidine to four interconverting diastereomers of N1-carbamoyl-4,5-dihydroxy-2-oxoimidazolidine nucleosides. J. Org. Chem. 72 (10) 3672 - 3678

Tsuzuki T, Egashira A, Igarashi H, Iwakuma T, Nakatsuru Y, Tominaga Y, Kawate H, Nakao K, Nakamura K, Ide F, Kura S, Nakabeppu Y, Katsuki M, Ishikawa T and Sekiguchi M (2001) Spontaneous tumorigenesis in mice defective in the MTH1 gene encoding 8-oxo-dGTPase. Proc Natl. Acad. Sci. U. S. A. 98 (20) 11456 - 11461

Turaga RV, Paquet ER, Sild M, Vignard J, Garand C, Johnson FB, Masson JY and Lebel M (2009) The Werner syndrome protein affects the expression of genes involved in adipogenesis and inflammation in addition to cell cycle and DNA damage responses. Cell Cycle. 8 (13) 2080 - 2092

Turrens JF (2003) Mitochondrial formation of reactive oxygen species. J. Physiol. 552 (Pt 2) 335 - 344

Tyrberg B, Anachkov KA, Dib SA, Wang-Rodriguez J, Yoon KH and Levine F (2002) Islet Expression of the DNA Repair Enzyme 8-oxoguanosine DNA Glycosylase (OGG1) in Human Type 2 Diabetes. BMC Endocr. Disord. 2 2 - 2

Vaara M (1999) Lipopolyscharride and the Permeability of the Bacterial Outer Membrane. 10 (2) 31 - 38

Van SJ (1990) Interleukin-6: an overview. Annu. Rev. Immunol. 8 253 - 278

Vartanian V, Lowell B, Minko IG, Wood TG, Ceci JD, George S, Ballinger SW, Corless CL, McCullough AK and Lloyd RS (2006) The metabolic syndrome resulting from a knockout of the NEIL1 DNA glycosylase. Proc. Natl. Acad. Sci. U. S. A. 103 (6) 1864 - 1869


226

Vernooy JHJ, Dentener MA, van Suylen RJ, Buuman WA and Wouters EFM (2001) Intratracheal instillation of lipopolysaccharide in mice induces apoptosis in bronchial epithelial cells: No role for tumor necrosis factor-a and infiltrating neutrophils. Am. J. Respir. Cell Mol. Biol. 24 569 - 576

Vilenchik MM and Knudson AG, Jr. (2000) Inverse radiation dose-rate effects on somatic and germ-line mutations and DNA damage rates. Proc. Natl. Acad. Sci. U. S. A. 97 (10) 5381 - 5386

Virag L and Szabo C (2002) The therapeutic potential of poly(ADP-ribose) polymerase inhibitors. Pharmacol. Rev. 54 (3) 375 - 429

Vogel U, Nexo BA, Olsen A, Thomsen B, Jacobsen NR, Wallin H, Overvad K and Tjonneland A (2003) No association between OGG1 Ser326Cys polymorphism and breast cancer risk. Cancer Epidemiol. Biomarkers Prev. 12 (2) 170 - 171

Wallace SS (2002) Biological consequences of free radical-damaged DNA bases. Free Radic. Biol. Med. 33 (1) 1 - 14

Wan GH, Li CS and Lin RH (2000) Airborne endotoxin exposure and the development of airway antigen-specific allergic responses. Clin. Exp. Allergy. 30 (3) 426 - 432

Wang JP, Kurt-Jones EA, Shin OS, Manchak MD, Levin MJ and Finberg RW (2005) Varicella-zoster virus activates inflammatory cytokines in human monocytes and macrophages via Toll-like receptor 2. J. Virol. 79 (20) 12658 - 12666

Wang X and Quinn PJ (2010) Lipopolysaccharide: Biosynthetic pathway and structure modification. Prog. Lipid. Res. 49 (2) 97 - 107

Watson J and Riblet R (1974) Genetic control of responses to bacterial lipopolysaccharides in mice. I. Evidence for a single gene that influences mitogenic and immunogenic respones to lipopolysaccharides. J. Exp. Med. 140 (5) 1147 - 1161

Watson J and Riblet R (1975) Genetic control of responses to bacterial lipopolysaccharides in mice. II. A gene that influences a membrane component involved in the activation of bone marrow-derived lymphocytes by lipipolysaccharides. J. Immunol. 114 (5) 1462 - 1468

Whitacre CC (2001) Sex differences in autoimmune disease. Nat. Immunol. 2 (9) 777 - 780

Williams TJ (1979) Prostaglandin E2, prostaglandin I2 and the vascular changes of inflammation. Br. J. Pharmacol. 65 (3) 517 - 524

Wilson III DM and McNeill DR (2007) Base excision repair and the central nervous system. Neuroscience. 145 (4) 1187 - 1200


227

Wolfer DP, Crusio WE and Lipp HP (2002) Knockout mice: simple solutions to the problems of genetic background and flanking genes. Trends Neurosci. 25 (7) 336 - 340

Wood C (2000) Future trends in human reproduction. Aust. N. Z. J. Obstet. Gynaecol. 40 (2) 127 - 132

Wood RD, Mitchell M, Sgouros J and Lindahl T (2001) Human DNA repair genes. Science. 291 (5507) 1284 - 1289

Xanthoudakis S, Smeyne RJ, Wallace JD and Curran T (1996) The redox/DNA repair protein, Ref-1, is essential for early embryonic development in mice. Proc. Natl. Acad. Sci. U. S. A. 93 (17) 8919 - 8923

Xie Y, Yang H, Cunanan C, Okamoto K, Shibata D, Pan J, Barnes DE, Lindahl T, McIlhatton M, Fishel R and Miller JH (2004) Deficiencies in mouse Myh and Ogg1 result in tumor predisposition and G to T mutations in codon 12 of the K-ras oncogene in lung tumors. Cancer Res. 64 (9) 3096 - 3102

Yamashita T, Kawashima S, Ohashi Y, Ozaki M, Ueyama T, Ishida T, Inoue N, Hirata K, Akita H and Yokoyama M (2000) Resistance to endotoxin shock in transgenic mice overexpressing endothelial nitric oxide synthase. Circulation. 101 (8) 931 - 937

Yang D, Elner SG, Bian ZM, Till GO, Petty HR and Elner VM (2007) Pro-inflammatory cytokines increase reactive oxygen species through mitochondria and NADPH oxidase in cultured RPE cells. Exp. Eye Res. 85 (4) 462 - 472

Ye SM and Johnson RW (2001) An age-related decline in interleukin-10 may contribute to the increased expression of interleukin-6 in brain of aged mice. Neuroimmunomodulation. 9 (4) 183 - 192

Zelissen PMJ, Bast EJEG and Croughs RJM (1995) Associated Autoimmunity in Addison's Disease. J. Autoimmun. 8 (1) 121 - 130

Zhang L, Fujii S and Kosaka H (2007) Effect of oestrogen on reactive oxygen species production in the aortas of ovariectomized Dahl salt-sensitive rats. J. Hypertens. 25 (2) 407 - 414

Zheng L, Dai H, Zhou M, Li M, Singh P, Qiu J, Tsark W, Huang Q, Kernstine K, Zhang X, Lin D and Shen B (2007) Fen1 mutations result in autoimmunity, chronic inflammation and cancers. Nat. Med. 13 (7) 812 - 819

Zody MC, Garber M, Adams DJ, Sharpe T, Harrow J, Lupski JR, Nicholson C, Searle SM, Wilming L, Young SK, Abouelleil A, Allen NR, Bi W, Bloom T, Borowsky ML, Bugalter BE, Butler J, Chang JL, Chen CK, Cook A, Corum B, Cuomo CA, de Jong PJ, DeCaprio D, Dewar K, FitzGerald M, Gilbert J,


228

Gibson R, Gnerre S, Goldstein S, Grafham DV, Grocock R, Hafez N, Hagopian DS, Hart E, Norman CH, Humphray S, Jaffe DB, Jones M, Kamal M, Khodiyar VK, LaButti K, Laird G, Lehoczky J, Liu X, Lokyitsang T, Loveland J, Lui A, Macdonald P, Major JE, Matthews L, Mauceli E, McCarroll SA, Mihalev AH, Mudge J, Nguyen C, Nicol R, O'Leary SB, Osoegawa K, Schwartz DC, Shaw-Smith C, Stankiewicz P, Steward C, Swarbreck D, Venkataraman V, Whittaker CA, Yang X, Zimmer AR, Bradley A, Hubbard T, Birren BW, Rogers J, Lander ES and Nusbaum C (2006) DNA sequence of human chromosome 17 and analysis of rearrangement in the human lineage. Nature. 440 (7087) 1045 - 1049

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The Role of Base Excision Repair in Regulating Endotoxin