Influence of Food-Derived Maillard Reaction Products on the Cellular

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Inﬂuence of Food-Derived Maillard Reaction Products on the Cellular Response of Macrophages

Den Naturwissenschaftlichen Fakult¨aten

der Friedrich-Alexander Universit¨atErlangen-N¨urnberg

zur

Erlangung des Doktorgrades

vorgelegt von Sonja Muscat

aus

N¨urnberg

M¨arz2007 Als Dissertation genehmigt von den Naturwissenschaftlichen Fakult¨atender

Friedrich-Alexander Universit¨atErlangen-N¨urnberg

Tag der m¨undlichen Pr¨ufung: 25. April 2007

Vorsitzender der Pr¨ufungskomission: Prof Dr. E. B¨ansch

Erstberichterstatter: Prof Dr. M. Pischetsrieder

Zweitberichterstatter: PD Dr. G. M¨unch F¨urmeine Eltern und Alex Parts of this work have been previously published:

Muscat, S., Pelka, J., Hegele, J., Weigle, B., M¨unch, G. and Pischetsrieder, M. (200x). Cof-

fee and Maillard Products activate NF-κB in macrophages via H2O2 production. Mol

Nutr Food Chem, In Press

Muscat, S., M¨unch, G., Weigle, B., and Pischetsrieder, M. (2005). Inﬂuence of Maillard re-

action products on the inﬂammatory cellular response of macrophages. DFG-Symposium,

Senate Commission on Food Safety (SKLM), Kaiserslautern

Muscat, S., M¨unch, G., and Pischetsrieder, M. (2006). Immunmodulierende Wirkung von

Kaﬀee und Melanoidinen. 35. Deutscher Lebensmittelchemikertag , Dresden Danksagung i

Danksagung

Mein großer und ganz herzlicher Dank gilt meiner Doktormutter Frau Prof. Dr. M. Pischets- rieder fur¨ die Aufnahme in Ihre Arbeitsgruppe, die engagierte Betreuung der Arbeit und die wissenschaftliche und pers¨onliche Unterstutzung¨ in den Jahren der Promotion sowie des

Studiums. Besonders möchte ich mich fur¨ die Unterstutzung¨ bei den Stipendienanträgen und die Ermöglichung eines Forschungsaufenthaltes in Australien bedanken.

Des weiteren m¨ochte ich mich bei Herrn PD Dr. G. Munch¨ fur¨ die Ubernahme¨ des Ko- referats und die Betreuung w¨ahrend meines Aufenthaltes an der James Cook University in Australien bedanken. Mein Dank gilt ferner Herrn Prof. Dr. med. M. Raithel fur¨ die

Ubernahme¨ der Nebenfachprufung¨ und Herrn Prof. Dr. G. Lee fur¨ die Ubernahme¨ des

Prufungsvorsitzes.¨

Fur¨ das Korrekturlesen der Arbeit und die Vermittlung australischer Lebensfreude m¨ochte ich mich ganz herzlich bei Ursula Tems bedanken. An dieser Stelle sei auch Alex fur¨ seine

Hilfe bei der Arbeit gedankt.

Mein herzlicher Dank geht an Dr. Jens Landwehr, Andreas Paul, Peter Lassner und Dr.

Carlo C. Peich fur¨ die Freundschaft, Unterstutzung¨ und sch¨one gemeinsame Zeit in der

Schuhstr. 19 w¨ahrend Studium und Promotion.

Weiterhin m¨ochte ich mich bei meinen lieben Kolleginnen und Kollegen, u.a. Andrea

Wuhr,¨ Bianca Knobloch, Jasmin Meltretter, Christine Willemann, Eva Karg, Clemens Bid- mon, Dr. Matthias Frischmann, Dr. J¨org Hegele, Taleb Khider, Rainer B¨auerlein, Martin

Tutsch und Grant Stuchbury bedanken. Zudem m¨ochte ich mich bei Joanna Pelka und Me- ii Danksagung lanie Deckert fur¨ die Mithilfe im Labor bedanken. Auch m¨ochte ich mich bei Frau Christine

Meißner fur¨ die Hilfe bei allen Verwaltungsvorg¨angen, Herrn A. Zillich-Balthasar und Herrn

C. Fischer aus der Werkstatt und der Materialausgabe bedanken.

Im Besonderen möchte ich mich bei meiner Freundin Jele fur¨ die große Unterstutzung¨ während der schönen gemeinsamen Erlanger Zeit bedanken. Auch gilt mein Dank Anne

Wittenberg fur¨ die lebenslange Freundschaft.

Der gr¨oßte Dank geht an meinen Herzensbub Alex, meine lieben Eltern und an meinen

Bruder mit Familie sowie Oma Anni! Table of Contents I

Table of Contents

1 Introduction 1

1.1 Formation of Maillard Reaction Products (MRPs) ...... 1

1.2 Formation of Advanced Glycation End Products (AGEs) ...... 2

1.3 Pathophysiological Signiﬁcance of AGEs ...... 4

1.3.1 AGE-Receptors ...... 4

1.3.2 AGE-RAGE-Induced Signalling Pathways ...... 5

1.4 Reactive Oxygen Species as Second Messengers in Signal Transduction . . . 7

1.5 Physiological Properties of Food-Derived MRPs ...... 8

1.6 Aims of the Work ...... 11

2 Results and Discussion 12

2.1 Food-Derived MRPs (Melanoidins) Induce NF-κB Activation in Macrophages

by a RAGE-Independent Mechanism ...... 12

2.1.1 Introduction ...... 12

2.1.1.1 Coﬀee ...... 13

2.1.1.2 Macrophages ...... 13

2.1.1.3 Transcription Factor NF-κB...... 14

2.1.2 Results ...... 17

2.1.2.1 Coﬀee Induces the Activation of NF-κB in Macrophages . . 17

2.1.2.2 MRPs Induce the Activation of NF-κB in Macrophages . . 17 II Table of Contents

2.1.2.3 High Molecular Weight Melanoidins are the Active Fraction

of the Maillard Reaction Mixture ...... 20

2.1.2.4 MRP-Induced NF-κB Activation is RAGE-Independent . . 23

2.1.2.5 MRP-Induced NF-κB Activation can be Inhibited by Catalase 24

2.1.2.6 Coﬀee-Induced NF-kappaB Activation can be Inhibited by

Catalase ...... 29

2.1.2.7 Hydrogen Peroxide Induces NF-κB Activation ...... 30

2.1.3 Discussion ...... 33

2.2 Food-Derived MRPs Induce NF-κB Activation through Cell-Independent

Generation of Hydrogen Peroxide ...... 35

2.2.1 Introduction ...... 35

2.2.1.1 Determination of Hydrogen Peroxide Concentration . . . . 36

2.2.2 Results ...... 37

2.2.2.1 Coﬀee Generates Hydrogen Peroxide in a Cell Free Matrix 37

2.2.2.2 MRPs Generate Hydrogen Peroxide in a Cell Free Matrix . 38

2.2.2.3 Generation of Hydrogen Peroxide by MRPs does not Require

Free Metal Ions ...... 39

2.2.2.4 MRP-Induced NF-κB Activation is not Due to Components

of the Cell Culture Medium ...... 42

2.2.3 Discussion ...... 44

2.3 Identiﬁcation of C4-Aminoreductone as a Signal Active MRP-Structure . . 48

2.3.1 Introduction ...... 48

2.3.2 Results ...... 48

2.3.2.1 C4-Aminoreductone Induces NF-κB Activation through Hy-

drogen Peroxide Generation ...... 48

2.3.2.2 AGEs do not Induce NF-κB Activation ...... 51

2.3.2.3 AGEs do not Augment LPS-Induced NF-κB Activation . . 54 Table of Contents III

2.3.2.4 AGEs Induce NO Generation through a RAGE-Dependent

Mechanism ...... 55

2.3.3 Discussion ...... 58

2.4 Cellular Reaction on MRP-Induced NF-κB Activation ...... 63

2.4.1 Introduction ...... 63

2.4.1.1 Cytokines ...... 63

2.4.1.2 NO ...... 64

2.4.2 Results ...... 64

2.4.2.1 MRP-Induced NF-κB Activation does not Promote the Pro-

duction of Inﬂammatory Cytokines ...... 64

2.4.2.2 MRP-Induced NF-κB Activation does not Cause the Gen-

eration of NO ...... 67

2.4.2.3 MRP-Induced Hydrogen Peroxide Generation Causes Cell

Death ...... 68

2.4.2.4 MRPs do not Induce Apoptosis ...... 69

2.4.3 Discussion ...... 71

2.4.4 Summary ...... 74

2.5 Screening of the Inﬂuence of Amino Acid Precursor on the Cytotoxicity of

Melanoidins by a Peptide Spot Library ...... 75

2.5.1 Introduction ...... 75

2.5.2 Results ...... 75

2.5.2.1 Comparison of Amino Acid Reactivity Toward Melanoidin

Formation Using a Dipeptide Spot Library ...... 75

2.5.2.2 Inﬂuence of Membrane-Bound Melanoidins on Cell Survival 79

2.5.3 Discussion ...... 85

3 Summary 89 IV Table of Contents

4 Deutsche Zusammenfassung 94

5 Materials and Methods 99

5.1 Materials and Equipment ...... 99

5.2 Buﬀers and Solutions ...... 105

5.3 Methods ...... 109

5.3.1 Preparation of Maillard Reaction Mixtures, AGEs, Coﬀee and MRPs 109

5.3.2 Fractionation of Maillard Reaction Mixture via SEC ...... 111

5.3.2.1 Calibration of SEC ...... 111

5.3.2.2 Fractionation of Maillard Reaction Mixture ...... 112

5.3.3 Cell Culture ...... 113

5.3.4 SDS Polyacrylamide Gel Electrophoresis and Western Blot . . . . . 115

5.3.5 Determination of NF-κB Translocation ...... 116

5.3.6 Detection of RAGE Expression ...... 119

5.3.7 Determination of Caspase-3 Activation ...... 120

5.3.8 Cell Viability Assays ...... 121

5.3.8.1 MTT Assay ...... 121

5.3.8.2 Alamar Blue Assay ...... 122

5.3.9 Determination of NO Production ...... 122

5.3.10 Cytokine Analysis ...... 123

5.3.10.1 Bio-Plex Cytokine Assay ...... 123

5.3.10.2 IL-6 Elisa ...... 123

5.3.11 Determination of Hydrogen Peroxide Concentration (FOX Assay) . . 125

5.3.12 Dipeptide Spot Library and Dipeptide/Amino Acid Spot Membranes 125

5.3.12.1 Preparation of MRP-Modiﬁed Dipeptide Spot Library and

Dipeptide/Amino Acid Spot Membranes ...... 127

5.3.12.2 Determination of Cell Proliferation on MRP-Modiﬁed Dipep-

tide/Amino Acid Spot Membranes ...... 127 Table of Contents V

5.4 Statistical Analysis ...... 128

Bibliography 129

List of Tables 145

List of Figures 146 VI Nomenclature

Nomenclature

•OH hydroxyl radical, page 7

AGEs advanced glycation end products, page 2

AR C4-aminoreductone, page 49

Bcl-2 B-cell leukemia/lymphoma-2, page 73

BSA bovine serum albumin, page 52

CML N-carboxymethyllysine, page 3

CML-BSA BSA-bound CML, page 53

DMEM Dulbecco’s Modiﬁed Eagle Medium, page 114

ECL enhanced chemiluminescence, page 115

ELISA enzyme-linked immunosorbent assay, page 66

FCS fetal calf serum, page 42

FOX assay ferrous oxidation xylenol orange assay, page 36

H2O2 hydrogen peroxide, page 7

HEK cells human embryonic kidney cells, page 23

HEK RA cells stably RAGE transfected HEK cells , page 23 Nomenclature VII

HEK ut cells untransfected HEK cells, page 23

HMW high molecular weight, page 9

HRP horseradish peroxidase, page 115

HSC T6 hepatic stellate cells, page 53

IκB inhibitory subunit family IkappaB, page 15

IKK IκB kinase, page 15

IL-1 interleukin-1, page 6

IL-10 interleukin-10, page 63

IL-12 interleukin-12, page 63

IL-6 interleukin-6, page 6

IL-8 interleukin-8, page 63

INF-γ interferon-gamma, page 13 iNOS inducible nitric oxide synthase, page 6

LDL low density lipoproteins, page 4

LMW low molecular weight, page 9

LPS lipopolisaccharide, page 13

MAPK mitogen-activated protein kinase, page 5

MEM Minimum Essential Medium, page 114

MRPs Maillard reaction products, page 1

MSR class A macrophage scavenger receptor, page 5 VIII Nomenclature

NADPH nicotinamide adenine dinucleotide phosphate, page 7

NF-κB nuclear factor-kappaB, page 5

NO nitric oxide, page 6

− NO2 nitrite, page 55

NOS nitric oxide synthase, page 64

•− O2 superoxide, page 7

PBS phosphate buﬀered saline, page 17

RAGE receptor for advanced glycation end products, page 4

ROS reactive oxygen species, page 7

SDS-PAGE sodium-dodeclysulfate polyacrylamide gel electrophoresis, page 115

SEC size exclusion chromatography, page 21 sRAGE soluble RAGE, page 5

TNF-α tumor necrosis factor-alpha, page 6

VCAM-1 vascular cell adhesion molecule-1, page 6 CHAPTER 1. INTRODUCTION 1

1 Introduction

1.1 Formation of Maillard Reaction Products (MRPs)

Maillard reaction products (MRPs) are formed by a non-enzymatic reaction between reducing sugars and proteins or amino acids. The Maillard reaction can occur in heat-treated foods such as bakery products, roasted meat, fried potatoes, beer and coffee. The reaction rate accelerates as the temperature increases. A wide range of MRPs are responsible for the formation of aroma and flavour in cooked foods. The colour of many kinds of food, for example coffee, has its origin in the Maillard reaction. Furthermore, heat-treated food shows increased shelf-life among other things due to antioxidative compounds (reductones), which are formed in the course of the Maillard reaction [Belitz et al., 2001].

The Maillard reaction was ﬁrst described by the chemist L.C. Maillard, who reported the formation of brown products upon heating a solution of amino acid and sugar [Maillard,

1912]. The initial step of the non-enzymatic browning is the reaction between carbonyl groups of reducing sugars and free amino groups of amino acids or proteins such as the - amino group of lysine. The amino group reacts in a nucleophilic addition with the carbonyl group to form an unstable Schiﬀ-base, which leads to the Amadori product after Amadori- rearrangement (Fig. 1.1). The Amadori product is an early MRP that undergoes further reactions like dehydration, oxidation or cross-linking to form a variety of stable MRPs.

In the final stage of the Maillard reaction, brown coloured polymers (melanoidins) with molecular weights of up to 100 kDa are formed. The molecular structures of the melanoidins are complex and remain largely unknown besides the identification of phenolic groups in coffee melanoidins [Bekedam et al., 2006; Hofmann, 1998]. 2 CHAPTER 1. INTRODUCTION

Figure 1.1: The early events of the Maillard reaction. The aldehyde group of the reducing sugar d-glucose reacts with the free amino group of the protein-bound lysine side chain to form an unstable Schiﬀ-base. This leads to the Amadori product after Amadori-rearrangement. The Amadori product undergoes further reactions (dehydration, oxidation or crosslinking) to form a variety of stable Maillard reaction products.

1.2 Formation of Advanced Glycation End Products (AGEs)

It is well known that MRPs are also formed in the human body by the Maillard reaction between intra- or extracellular proteins and sugar components. The resulting endogenously formed glycated proteins are termed advanced glycation end products (AGEs). High AGE- levels can be detected in the serum and tissues of patients with diabetes mellitus (increased glucose levels) [Brownlee et al., 1985; Ono et al., 1998] or renal failure (diminished excretion)

[Henle and Miyata, 2003; Schwenger et al., 2001], and in the tissues of elderly people (long incubation time) [Verzijl et al., 2000]. AGEs are believed to play a prominent role in the pathology of diabetic complications such as atherosclerosis, retinopathy, impaired wound healing and nephropathy [Ahmed, 2005; Vlassara and Palace, 2002]. AGEs are a very heterogenic group of reaction products and thus far only several speciﬁc AGEs, mostly CHAPTER 1. INTRODUCTION 3 protein-bound lysine- and/or arginine side chain derivates have been identiﬁed. These

Maillard-modiﬁed proteins have been isolated from model mixtures, tissues or serum and structurally characterized using chromatography or immunochemical methods. The AGEs exemplarily listed below were not only detected in vivo, but also in food.

One of the dominant AGEs, N-carboxymethyllysine (CML) [Reddy et al., 1995], can be formed, for example through metal-catalyzed oxidative degradation of the Amadori product

-fructoselysine (Fig 1.2) [Ahmed et al., 1986]. CML and a pentose-derived lysine-arginine cross-linking AGE, pentosidine [Sell and Monnier, 1989], were recognized to accumulate in tissue-proteins with age, especially in patients with diabetes mellitus [Dyer et al., 1993]. In the serum proteins of patients with diabetes mellitus, increased levels of a lysine derivate, formed from the reaction with 3-deoxyglucosone, pyrraline, was detected [Hayase et al.,

1989].

Figure 1.2: Chemical structures of AGEs. Lysine-derived AGEs: N-carboxymethyllysine (CML) and pyrraline.

Pentose-derived crosslink between lysine and arginine: pentosidine. 4 CHAPTER 1. INTRODUCTION

Through analogous reaction steps these AGEs can be formed in food. CML is present in processed malt products, condensed milk or heated milk products and can be used as a marker to determine the progress of the Maillard reaction [Hartkopf et al., 1994; Tauer et al., 1999]. Additionally, the amount of pyrraline was shown to increase with time and temperature in dry food products [Chiang, 1988]. Low amounts of pentosidine were detected in roasted coﬀee suggesting a minor role for pentosidine in protein crosslinking in heated food [Henle et al., 1997].

1.3 Pathophysiological Signiﬁcance of AGEs

Protein glycation in vivo modiﬁes the structural and physiological properties of proteins and is suggested to lead to vascular dysfunction and inﬂammation. The precise role of AGEs in the pathophysiology of diseases such as diabetes mellitus is not yet fully understood, however besides the ability of AGEs to change protein structures, receptor-dependent mechanisms are of special interest.

Pathophysiological eﬀects of AGEs, which are receptor-independent, can be observed in the progress of atherosclerosis. AGE-modiﬁcations of arterial collagen have been shown to trap low density lipoproteins (LDL) or other serum proteins and may accelerate atheroma formation [Brownlee et al., 1985]. However, a pivotal role of AGEs in atherogenesis stems from the interaction with the receptor for advanced glycation end products (RAGE), a cell surface AGE-binding protein (AGE-receptor) [Basta et al., 2004; Park et al., 1998].

1.3.1 AGE-Receptors

Macrophages were ﬁrst recognized to uptake and degrade AGEs via speciﬁc AGE-receptors

[Vlassara et al., 1985, 1986]. AGE-binding molecules have been identiﬁed on monocytes, macrophages, endothelial cells and cells of the central nervous system (astroglia and microglia) [Thornalley, 1998]. These molecules include oligosaccharyl transferase complex

(OST-48, p60, AGE-R1), 80K-H phosphoprotein (p90, AGE-R2) [Li et al., 1996], galectin-3 CHAPTER 1. INTRODUCTION 5

(AGE-R3) [Vlassara et al., 1995], RAGE [Schmidt et al., 1992; Neeper et al., 1992] and the class A macrophage scavenger receptor (MSR) [Araki et al., 1995].

The AGE-binding proteins OST-48, 80K-H phosphoprotein and galectin-3 are suggested to be associated in an AGE-receptor complex, with galectin-3 showing signiﬁcant AGE- binding capacity [Vlassara et al., 1995]. However, the function of this AGE-receptor complex is largely unknown. AGE-modiﬁed proteins, S100/calgaranulin [Hofmann et al., 1999] and

β-sheet ﬁbrils like amyloid-beta peptide [Yan et al., 1996] have been identiﬁed as ligands of

RAGE. However, CML is the only speciﬁc AGE shown to bind to RAGE [Kislinger et al.,

1999]. Whereas MSR is important in AGE-endocytosis [Araki et al., 1995] the interaction of RAGE with its ligands rather leads to cellular activation [Mackic et al., 1998].

1.3.2 AGE-RAGE-Induced Signalling Pathways

The best-characterized AGE-receptor is RAGE. This receptor was ﬁrst described as a 35 kDa

AGE-binding protein belonging to the immunoglobulin superfamily [Schmidt et al., 1992].

The cytosolic domain, which mediates cellular signal transduction [Kislinger et al., 1999], is associated with the extracellular ligand-binding domain (sRAGE), through the hydrophobic transmembrane domain (Fig 1.3) [Neeper et al., 1992]. RAGE is expressed on the surface of endothelial cells, smooth muscle cells, monocytes, macrophages, podocytes, astroglia and migroglia [Thornalley, 1998].

Binding of in vitro or in vivo-derived AGEs to RAGE activates cellular signalling pathways including the p21ras and mitogen-activated protein kinase (MAPK) pathways [Lander et al., 1997; Yeh et al., 2001; Chang et al., 2004]. Further down in the signal transduction cascade transcription factors like nuclear factor-kappaB (NF-κB) are activated (Fig 1.3)

[Yan et al., 1994; Lander et al., 1997; Yeh et al., 2001]. The cascade of signal transduction depends on the binding of AGEs to RAGE, as blocking RAGE with either an excess of sRAGE or anti-RAGE antibody prevented cellular activation. 6 CHAPTER 1. INTRODUCTION

Figure 1.3: AGE-RAGE induced signal transduction leads to intracellular oxidative stress, which triggers key signalling pathways resulting in the activation of the transcription factor NF-κB. The transcription of NF-κB regulated genes redirects the release of inﬂammatory cytokines from macrophages or the expression of adhesion molecules on endothelial cells.

AGE-induced activation of the transcription factor NF-κB redirects the transcription of inflammatory and prothrombotic genes that contribute to accelerated vascular and inflammatory complications. These are characteristic for the pathology of, e.g. atherosclerosis or impaired wound healing. The stimulation of cells in vitro with AGEs results in the release of the proinflammatory cytokines interleukin-1 (IL-1) , interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in macrophages [Yeh et al., 2001; Iida et al., 1994; Neumann et al.,

1999]. Also, the production of the inﬂammatory mediator nitric oxide (NO) occurs upon expression of the inducible nitric oxide synthase (iNOS) [Dukic-Stefanovic et al., 2003].

Furthermore, the expression of adhesive molecules like vascular cell adhesion molecule-1

(VCAM-1) on endothelial cells results from AGE stimulation [Schmidt et al., 1995]. Con- sequently, at sites of AGE-RAGE interaction in vivo, circulating monocytes migrate into tissues where they diﬀerentiate into macrophages and become activated. Macrophages are important mediators of the immune response and their activation leads to an inﬂammatory reaction. The relevance of AGE-RAGE-induced signalling pathways in vivo has been shown in the prevention of diabetic complications by administration of sRAGE. Intraperitoneal CHAPTER 1. INTRODUCTION 7 administration of sRAGE in diabetic mice completely suppressed diabetic atherosclerosis

[Park et al., 1998] or diabetes associated disordered wound healing [Goova et al., 2001] in a dose-dependent manner.

1.4 Reactive Oxygen Species as Second Messengers in Signal

Transduction

•− Reactive oxygen species (ROS) include oxygen radicals such as superoxide (O2 ) and hy-

• droxyl radical ( OH) and non-radical oxygen derivates such as hydrogen peroxide (H2O2).

In living organisms ROS play an important role in intra- and extracellular cell signalling.

However, during mitochondrial respiration ROS are also accidentally generated. An excessive overspill of oxidants can lead to the modiﬁcation of lipids, proteins and nucleic acids resulting in cell damage or cell death. Thus, the formation of ROS is controlled by the presence of antioxidants (e.g. glutathione or ascorbic acid) and enzymes (e.g. superoxide dismutase, catalase). The situation of an imbalance between oxidants and antioxidant defense mechanisms with the former prevailing is called ”oxidative stress” [Sies, 1997].

It is known that receptor-dependent signalling pathways use superoxide or hydrogen peroxide as second messengers [Suzuki et al., 1997; Schreck and Baeuerle, 1991]. Thereby a plasma membrane associated enzyme, nicotinamide adenine dinucleotide phosphate-oxidase

(NADPH oxidase), is suggested to play a central role in ROS-generation. The activated enzyme complex generates superoxide, which can then form hydrogen peroxide by the enzyme superoxide dismutase or non-enzymatical by disproportionation. Exemplarily, immunomodulatory molecules such as TNF-α and IL-1 induce the synthesis of superoxide and hydrogen peroxide leading to the activation of the transcription factor NF-κB [Schreck et al., 1991].

Also, the AGE-induced cellular signal transduction is triggered by a RAGE-mediated induction of oxidative stress (Fig 1.3). Increased amounts of thiobarbituric acid-reactive substances (TBRAS) were shown to form upon AGE-RAGE interaction as a result of enhanced oxidative stress [Yan et al., 1994]. Additionally, signalling pathways could be blocked by the 8 CHAPTER 1. INTRODUCTION administration of antioxidants (NF-κB, [Yan et al., 1994; Yeh et al., 2001]), or enhanced by the depletion of intracellular antioxidant (p21ras and MAPK, [Lander et al., 1997]). AGE- induced generation of ROS was suppressed by administration of NADPH oxidase inhibitors

[Wautier et al., 2001].

Besides their role in the intracellular signal transduction, ROS are also known to be extracellular signal molecules. As part of an inﬂammatory response macrophages release hydrogen peroxide in the extracellular space as host defense against invading bacteria (oxidative burst). Moreover, extracellular hydrogen peroxide, which can passively diﬀuse through cell membranes, can increase intracellular ROS in the surrounding cells, redirecting a cellular response, e.g. up-regulating the expression of adhesion molecules in endothelial cells

[Halliwell et al., 2000; Halliwell and Gutteridge, 1999].

1.5 Physiological Properties of Food-Derived MRPs

It is known that the Maillard reaction has an inﬂuence on the nutritional quality of the diet. Maillard-modiﬁcation of proteins has been shown to reduce their nutritional value

[O’Brien and Morrissey, 1989] and food toxicants can be formed in the course of the Maillard reaction [Jagerstad and Skog, 2005]. For example, the genotoxic compound acrylamide has been recently detected in high levels in heat-treated food such as French fries. However, the risk to human health by food-derived acrylamide is not yet clariﬁed because of lacking information about the bioavailability of dietary acrylamide [Tuohy et al., 2006].

Besides these undesirable eﬀects, MRPs are suggested to be health promoting. In vitro studies showed that food-derived MRPs, particularly the high molecular weight melanoidins, possess antioxidative activity [Lindenmeier et al., 2002; Borrelli et al., 2002]. Exemplarily, a

Maillard mixture was shown to inhibit metal-ion-induced oxidation of isolated human LDL, which could in vivo reduce atherosclerotic plaque formation and therefore the risk of vascular diseases [Dittrich et al., 2003]. The antioxidative activity of MRPs, demonstrated in vitro, is assumed to be health beneﬁt in vivo, however, little is known about the metabolization CHAPTER 1. INTRODUCTION 9 and absorption of the Maillard reaction-derived antioxidants in the human body [Somoza,

2005]. MRPs are also proposed to directly influence the gut health. The high molecular weight MRPs, namely melanoidins, are suggested to be prebiotics. In in vitro studies using experimental conditions to mimic the gut environment, melanoidins, isolated from coffee silverskin and bread crust, have been shown to increase the growth of potentially beneficial bacteria such as bifidobacteria [Borrelli et al., 2004, 2005].

Food-derived MRPs are formed through reaction steps that are analogous to AGE formation. The structural similarities of endogenous AGEs and exogenous MRPs suggested that food-derived MRPs may exert systemic eﬀects after intestinal absorption, which would contribute to AGE-induced pathophysiological events. Intestinal absorption of food-derived

MRPs does occur as demonstrated in diﬀerent animal studies. The absorbed MRPs are urinary excreted or incorporated into tissue, whereas the non-digestible MRPs are excreted in the faeces or metabolised by bacteria in the gut. Exemplarily, 60 - 80 % of ingested

Amadori product was shown to be urinary excreted, whereas 1 - 3 % was recovered in the faeces [Faist and Erberdobler, 2001]. In another study the metabolic transit of MRPs from a heated casein-14C-glucose mixture, which was fractionated into low molecular weight

(LMW) and high molecular weight (HMW) MRPs was elucidated. 61 % of the LMW products were excreted in the faeces and 27 % in the urine. In contrast, 87 % of the HMW melanoidins were excreted in the faeces and only 4.3 % in the urine. The MRPs were not retained or utilised in the body since little radioactivity [1.1 % (HMW) - 2.6 % (LMW)] could be detected in the carcass. Thus, the results indicated that the absorption rate of

LMW MRPs is much higher compared to HMW MRPs [Faist and Erberdobler, 2001; So- moza, 2005]. Also in humans, a significant increase in plasma AGE levels was found after oral administration of dietary AGEs, which were not further characterized [Koschinsky et al., 1997]. Animal studies have shown a significant role for dietary AGEs in promoting atherosclerosis in genetically hypercholesterolemic diabetic mice [Lin et al., 2003], as well as contributing to progressive diabetic nephrophathy and higher mortality in mice mod- 10 CHAPTER 1. INTRODUCTION els of type 1 and type 2 diabetes [Zheng et al., 2002]. The animals were fed a high-AGE diet with an average five-fold higher AGE content than the low-AGE diet. The observed accelerated diabetic complications in animals on the high-AGE diet were associated with increased AGE-levels in the serum. In human diabetic patients an increase in AGE serum levels and inflammatory markers (TNF-α, VCAM-1) corresponded with a high-AGE diet compared to the control group. In accordance with this result, a decrease in patients on a low-AGE diet was observed [Vlassara et al., 2002]. These results suggest a strong influence of food-derived MRPs on vascular dysfunctions and inflammation, especially in disorders such as diabetes mellitus, by increasing the AGE pool of the body [Uribarri et al., 2005].

However, the chemical structures of the physiologically active food-derived MRPs are unknown. As a speciﬁc AGE, only CML was identiﬁed to induce cellular perturbation upon binding to RAGE.

Besides systemic effects it is also likely that MRPs have a local effect on the intestine through similar mechanism as AGEs. There is evidence that dietary MRPs induce a cellular response through the induction of oxidative stress. Food-derived AGEs extracted by affinity chromatography could activate cells in culture by increasing glutathione peroxidase activity, depleting cellular glutathione in endothelial cells and inducing the release of TNF-α in macrophages [Cai et al., 2002].

The innermost membrane of the intestine, the intestinal mucosa, which comes into direct contact with the ingested food consists of an epithelial layer (lamina epithelialis), connective tissue (lamina propria) and smooth muscle ﬁbers (muscularis mucosae). Immune cells such as macrophages, located in the lamina propria, are responsible for the intestinal immune defense, rendering the intestine the largest immunobiological organ [Smith et al., 2005].

Macrophages mediate the inflammatory reaction in response to bacteria, protecting the mucosa. However, in inflammatory bowel diseases like Crohn´s disease and ulcerative colitis, a large number of activated macrophages can be detected in the intestinal mucosa triggering an inflammatory response, which mediates chronic mucosal inflammation [Mahida, 2000]. CHAPTER 1. INTRODUCTION 11

In studying the influence of food-derived MRPs on the cellular response of macrophages in vitro, the immunomodulating influence of MRPs in the intestine can be estimated. A MRP- rich diet could induce a cellular response in macrophages, thus modulating the intestinal immune response, which could, particularly in inflammatory bowel diseases, lead to an enhanced inflammatory reaction.

1.6 Aims of the Work

The aims of the present work were

• to investigate whether food-derived MRPs induce a cellular reaction in macrophages,

particularly the activation of the transcription factor NF-κB.

• to study the signal transduction of the MRP-induced NF-κB activation.

• to identify signal active structures in MRPs, which are responsible for the MRP-

induced activation of macrophages.

• to elucidate the cellular reaction on MRP-induced NF-κB activation. 12 CHAPTER 2. RESULTS AND DISCUSSION

2 Results and Discussion

2.1 Food-Derived MRPs (Melanoidins) Induce NF-κB Activation

in Macrophages by a RAGE-Independent Mechanism

2.1.1 Introduction

Interactions of AGEs with RAGE have been shown to induce cellular signalling pathways, leading to an inflammatory response. Food-derived MRPs are formed through reaction steps that are analogous to AGE formation and specific AGEs, e.g. CML have been identified in food.

The ﬁrst aim of the project was to investigate whether food-derived MRPs induce an intracellular signalling pathway, which is involved in the inﬂammatory cellular response.

Macrophages are key players in the immune response mediating inﬂammatory reactions.

Thereby, NF-κB has a central role in the signal transduction. The activation of NF-κB upon stimulation of macrophages with coffee or MRPs was immunochemically detected and used to indicate the induction of a signalling pathway, which is linked to the inflammatory cellular response. An extract of coffee was used as a model for food-derived MRPs, as this contains high levels of MRPs.

The second aim was to determine the cell signalling pathway by which coﬀee and MRPs induce NF-κB activation. CHAPTER 2. RESULTS AND DISCUSSION 13

2.1.1.1 Coﬀee

Coffee is a popular and worldwide consumed beverage, possessing various physiological effects. The biologically active compounds of coffee include, e.g. caffeine, which stimulates the central nervous system. Caffeine acts as an antagonist to the adenosine-receptor, reducing the calming effect of adenosine-receptor interaction [Forth et al., 1996]. Also, caffeine inhibits the enzyme phosphodiesterase leading to increased cyclic adenosine monophos- phate (cAMP) levels, which stimulates adrenaline-like effects. Additionally, the diterpenes cafestol and kahweol, present in the coffee lipid fraction, have been shown to increase the serum cholesterol level [Ranheim and Halvorsen, 2005]. Moreover, coffee contains phenolic compounds such as chlorogenic acid [Yen et al., 2005] and MRPs, particularly melanoidins

[Borrelli et al., 2002], which may exert beneﬁcial antioxidative eﬀects in vivo.

MRPs are formed during the coﬀee roasting process, which requires temperatures up to

230 °C leading to the typical formation of coﬀee aroma, ﬂavour and colour [Belitz et al.,

2001]. During the roasting process carbohydrates, proteins and chlorogenic acid are de- graded and the Maillard reaction takes place. Melanoidins account for up to 25 % of dry matter of coﬀee beverage.

2.1.1.2 Macrophages

Macrophages are cells of the innate immune system (non-specific defence) and have their origin in the bone marrow. The macrophage progenitor cells of the bone marrow exit into the peripheral blood and turn into monocytes. After a bacterial infection or a physical/chemical damage of tissue, monocytes leave the blood stream, migrate to the inflammation sites and mature into macrophages [Alberts et al., 2002]. Signals leading to the activation of macrophages include lipopolisaccharide (LPS) or T-cell derived cytokines like interferon- gamma (INF-γ). The major task of macrophages in the inflamed tissue is to phagocytose and digest the invading microorganisms, foreign bodies or damaged cells. Besides their role as phagocytes, they mediate the inflammatory response to immune cells as well as 14 CHAPTER 2. RESULTS AND DISCUSSION to non-immune cells, e.g. endothelial cells, by releasing chemotactic and inflammatory cytokines. Consequently, immune cells are recruited to the infection sites and become activated. Another feature of macrophages is the ability to present the processed antigen to cells of the adaptive immune system (specific defence). The involvement of cells of the adaptive immune system such as T- and B-lymphocytes provides a more specific immune response with a higher efficiency in the clearance of the pathogen.

2.1.1.3 Transcription Factor NF-κB

NF-κB is an important transcription factor, which is ubiquitously located in diﬀerent cell types and rapidly activated by a wide range of stimuli. Exogenous agents like bacterial and viral components, or endogenous stimuli like cytokines and growth factors activate

NF-κB, and therefore it plays a crucial role in immune and inﬂammatory responses. NF-

κB activation promotes the transcription of immune- and inﬂammatory-related proteins like immunoreceptors, cytokines, adhesion molecules and growth factors [Lee and Burckart,

1998]. Additionally, NF-κB provides a rapid stress response to environmental challenges including oxidative (e.g. hydrogen peroxide), physical (e.g. ionizing radiation) and chemical

(e.g. heavy metal) stress by activating acute phase and stress response genes [Pahl, 1999].

In the context of cellular stress the activation of NF-κB has been shown to exert both pro- and anti-apoptotic eﬀects, depending on the NF-κB-inducer [Kaltschmidt et al., 2000]. In a stress-associated environment NF-κB can be involved in the strongest stress-defending process by the induction of programmed cell death (apoptosis) or the promotion of cell survival by inducing the expression of anti-apoptotic proteins. The molecular mechanism switching NF-κB from a pro- to an anti-apoptotic mediator or vice versa is unknown.

NF-κB is composed of homo or heterodimeric combinations of NF-κB/Rel proteins. The

NF-kappaB proteins include p50 (NF-κB1) and p52 (NF-κB2) and the Rel proteins p65

(RelA), RelB and c-Rel. The most common combination is a heterodimeric form, consisting of p50 and p65 (Rel A) and this is generally referred to as NF-κB. The inactive form CHAPTER 2. RESULTS AND DISCUSSION 15 of NF-κB is associated with a member of the inhibitory subunit family IkappaB (IκB) and is located as a molecular complex in the cytoplasm. NF-κB-inducers activate NF-κB by initiating the dissociation of IκB from the complex (Fig. 2.1). IκB kinase (IKK) phosphory- lates IκB, which leads to its ubiquitination, resulting in degradation in the 26S proteasome.

Thereby, the nuclear translocation signal of NF-κB becomes exposed, which enables the transcription factor to translocate into the nucleus. Binding of NF-κB to regulatory ele- ments of its target genes leads to their transcription. The selectivity in the transcription of more than 150 NF-κB controlled genes is achieved through the diﬀerent NF-κB dimers as well as the presence of co-activators or further transcription factors, which are activated by other signalling pathways [Perkins, 2000].

In this work the method to determine activation of NF-κB is based on the immunochemical detection of the subunit p65 (65 kDa) in isolated nuclei by means of a Western Blot.

Nuclear NF-κB concentration was normalized to β-actin, which was detected in the same extract, migrating as a band at 41 kDa. The NF-kappaB translocation is expressed as a fold-increase of p65 to β-actin in comparison to the untreated control. 16 CHAPTER 2. RESULTS AND DISCUSSION

Figure 2.1: Cellular NF-κB activation. The NF-κB-inducer initiates the dissociation of the inhibitory subunit IκB from NF-κB. The nuclear translocation signal of NF-κB becomes exposed. NF-κB translocates into the nucleus and binds to the promoter regions of NF-κB target genes. The transcription occurs in combination with co-activators or further transcription factors, which are activated by other signalling pathways. The transcription and translation of

NF-κB regulated genes leads to their expression. CHAPTER 2. RESULTS AND DISCUSSION 17

2.1.2 Results

2.1.2.1 Coﬀee Induces the Activation of NF-κB in Macrophages

The influence of coffee on NF-κB activation in macrophages was investigated. The coffee roasting process requires temperatures up to 230 °C and leads to the formation of MRPs in the coffee beans. This is the key step in the typical formation of coffee aroma and

flavour. For the experiments, coffee was freshly brewed under normal conditions, using a coffee plunger with 13.5 g ground coffee per cup (180 mL). Boiling water was added to the ground coffee and allowed to steep for 10 min. Instead of plunging the coffee grounds to the bottom, the suspension was filtered through a fluted filter. The coffee was cooled to room temperature, adjusted to pH 7.4 and diluted 1:10 in physiological phosphate buffered saline (PBS, pH 7.4) to a final extract concentration of 2 mg/mL. Raw coffee, which does not contain MRPs, was extracted under the same conditions from ground raw coffee beans and diluted to the same extract concentration (1:7). The cells were incubated with the coffee extracts for 2 h and NF-κB levels were determined by Western Blot analysis of the isolated nuclei. While raw coffee extract lacking MRPs did not change nuclear NF-κB levels in the nuclei, freshly brewed coffee led to a significant 13-fold increase compared to the cells incubated only with PBS (Fig. 2.2). The results suggest that coffee-derived MRPs are responsible for the observed activation of macrophages.

2.1.2.2 MRPs Induce the Activation of NF-κB in Macrophages

It was hypothesized that MRPs formed during the roasting procedure were responsible for the coﬀee-induced activation of macrophages. To test the hypothesis, a model Maillard reaction mixture was prepared by heating d-ribose and l-lysine for 30 min at 120 ◦C. The preparation conditions for the model MRPs were similar to a general preparation protocol for model melanoidins using high temperatures (>100 °C) and amino acid/sugar mixtures

[Argirova, 2005], but the reaction mixture, which was used in the present work, was heated in aqueous solution and not dry heated (roasted). The Maillard reaction mixture was 18 CHAPTER 2. RESULTS AND DISCUSSION

Figure 2.2: (A) Coﬀee-induced NF-κB activation in NR8383 macrophages compared to the PBS treated control

(mean and standard deviation of four independent experiments). Cells were stimulated in PBS for 2 h with 2 mg/mL of raw coﬀee extract and 2 mg/mL of coﬀee extract. A nuclear extract was prepared and the NF-κB subunit p65 was immunochemically detected by Western Blot. Bands were obtained by enhanced chemiluminescence and densitometrically evaluated. The intensity of p65 signal was related to the β-actin content; *** p<0.001. (B)

Representative Western Blot of p65 and β-actin. CHAPTER 2. RESULTS AND DISCUSSION 19

Figure 2.3: MRP-induced NF-κB activation in NR8383 macrophages compared to the control, which was maintained in medium alone (mean and standard deviation of at least two independent experiments). Cells were stimulated in medium for 2 h with 250 µL PBS (equivalent to the largest added volume of the Maillard reaction mixture), 25 mM of heated ribose (ribose 30 min), 25 mM of heated lysine (lysine 30 min), 25 mM of unheated ribose-lysine mixture

(rib-lys 0 h) and 25 mM of Maillard reaction mixture consisting of ribose and lysine heated for 30 min at 120 °C (rib-lys 30 min); *** p<0.001. (B) Representative Western Blot of p65 and β-actin.

added in a ﬁnal concentration of 25 mM (d-ribose and l-lysine concentrations prior to heating) in medium to the cells and incubated for 2 h. After preparation of nuclear protein extract, the NF-κB concentration was determined by Western Blot analysis. A signiﬁcant 6- fold increased NF-κB translocation was detected compared to the medium treated control

(Fig. 2.3). The 6-fold increase in NF-κB translocation was due to MRP-dependent cell activation. Stimulation of the macrophages with the same concentration of the unheated reaction mixture (rib-lys 0 h), ribose and lysine, which were heated alone (ribose 30 min, lysine 30 min) or 250 µL PBS, equivalent to the largest added volume of the Maillard reaction mixture, did not lead to NF-κB translocation.

NF-κB activation was also tested with Maillard reaction mixtures that had been heated for longer time periods (1 h, 3 h, 5 h or 24 h, respectively). Already with the mixture heated for 30 min, a more than 6-fold increase in nuclear translocation was observed (Fig. 2.4). 20 CHAPTER 2. RESULTS AND DISCUSSION

Figure 2.4: Heating time-dependent MRP-induced NF-κB activation in NR8383 macrophages compared to the untreated control (mean and standard deviation of at least two independent experiments; mean without indicated standard deviation of one experiment measured in duplicate). Cells were stimulated in medium for 2 h with 25 mM of Maillard reaction mixture consisting of ribose and lysine (rib-lys) heated for 0.5 h, 1 h, 3 h, 5 h and 24 h at 120 °C; *** p<0.001, ** p<0.01.

After a heating time of 1 h an 8-fold increase was detected, which was not further augmented by prolonged heating up to 24 h.

The activation of NF-κB was shown to be dependent on the MRP concentration. While a 15 mM Maillard reaction mixture consisting of ribose and lysine heated for 1 h increased the nuclear translocation of NF-κB 6-fold, a 25 mM Maillard reaction mixture increased

NF-κB translocation 15-fold, whereas 5 mM did not induce NF-κB activation (Fig. 2.5).

The results using Maillard reaction mixtures conﬁrm the hypothesis that MRPs in coﬀee are responsible for the NF-κB activation in macrophages.

2.1.2.3 High Molecular Weight Melanoidins are the Active Fraction of the Maillard

Reaction Mixture

In addition, the signal active structures of the MRPs, leading to the translocation of NF-κB, should be identiﬁed. Coﬀee consists of a complex matrix containing several physiologically active compounds. Thus, the Maillard reaction mixture was used for the experiment. Dur- ing the Maillard reaction, a very heterogeneous mixture of products is formed. In order to identify the physiologically active MRPs, the Maillard reaction mixture of ribose and lysine CHAPTER 2. RESULTS AND DISCUSSION 21

Figure 2.5: Concentration-dependent MRP-induced NF-κB activation in NR8383 macrophages compared to the untreated control (mean and standard deviation of one experiment measured in duplicate). Cells were stimulated in medium for 2 h with 5 mM, 15 mM and 25 mM of Maillard reaction mixture consisting of ribose and lysine (rib-lys) heated for 1 h at 120 °C.

that had been heated for 30 min at 120 °C was fractionated by size exclusion chromatography (SEC). In chapter 5.3.2.2 (Fig. 5.2) the fractionation of the Maillard reaction mixture is illustrated. The SEC was calibrated using phenylalanine (0.165 kDa) and insulin (5.777 kDa) (Chapter 5.3.2.1, Fig. 5.1). The fractions of the Maillard reaction mixture were then tested for their ability to induce NF-κB activation.

A gel with a cut-oﬀ of 1.8 kDa yielded a low molecular fraction (LMW, <1.8 kDa) and a high molecular fraction (HMW, >1.8 kDa), the latter containing mostly complex melanoidins. When reconstituted to the original volume, the 25 mM HMW fraction contained 3.5 mg/mL dry matter and the 25 mM LMW fraction 3.3 mg/mL, indicating that the Maillard reaction mixture contains equal amounts of LMW products and high molecular weight melanoidins. Interestingly, NF-κB activation activity, similar to the Maillard reaction mixture, was only observed in the HMW fraction, but not in the LMW fraction

(Fig. 2.6). The activation of NF-κB by the HMW fraction is similar to that observed with the crude Maillard reaction mixture. There was no synergistic increase in cell activation when the fractions were recombined. Thus, it can be concluded that only high molecular 22 CHAPTER 2. RESULTS AND DISCUSSION

Figure 2.6: Melanoidin-induced NF-κB activation in NR8383 macrophages compared to the control, which was maintained in PBS (mean and standard deviation of two independent experiments). Cells were stimulated in PBS for

2 h with the HMW fraction (25 mM HMW = 3.5 mg/mL substance), LMW fraction (25 mM LMW = 3.3 mg/mL substance), combined HMW and LMW fraction (25 mM HMW + 25 mM LMW) of 25 mM Maillard reaction mixture consisting of ribose and lysine heated for 30 min at 120 °C and 25 mM Maillard reaction mixture (25 mM rib-lys 30 min = 7.2 mg/mL substance); *** p<0.001, ** p<0.01. (B) Representative Western Blot of p65 and β-actin. CHAPTER 2. RESULTS AND DISCUSSION 23 weight melanoidins are the active components leading to NF-κB activation in macrophages.

Due to the heterogeneous composition of melanoidins, which does not allow isolation of deﬁned products, further fractionation of the HMW MRPs was not performed.

2.1.2.4 MRP-Induced NF-κB Activation is RAGE-Independent

Afterwards, the signal transduction of the MRPs should be clariﬁed. It is well established that AGEs, which are Maillard products formed from sugars and proteins in vivo, can cause NF-κB activation in several cell types by interaction with RAGE. Therefore, it was investigated whether the Maillard reaction products used in this study also cause NF-κB translocation via RAGE. Human embryonic kidney (HEK) cells were stably transfected with RAGE (HEK RA) [Bartling et al., 2005] resulting in high levels of RAGE expression, whereas the expression in the untransfected cells (HEK ut) was below detection limit as determined by Western Blot analysis with a monoclonal anti-RAGE antibody (Fig. 2.7).

The HEK RA and HEK ut cells were obtained from Dr. Weigle (TU Dresden).

Figure 2.7: RAGE-Western Blot. Protein lysates of RAGE-transfected HEK cells (HEK RA) or untransfected HEK cells (HEK ut) were prepared and RAGE was immunochemically detected by a Western Blot using a monoclonal anti-RAGE antibody. As a positive control for RAGE-detection sRAGE was used.

Human RAGE has a molecular mass of 42 kDa, but migrates at 60 kDa in the West- ern Blot. This could be due to post-translational modification, e.g. glycosylation of the extracellular domain [Neeper et al., 1992; Thornalley, 1998]. The band corresponding to 24 CHAPTER 2. RESULTS AND DISCUSSION sRAGE, which was used as a positive control migrates to 60 kDa, but has a molecular mass of 35 kDa. This effect is due to the sRAGE-histidine-tag, used for sRAGE-purification, which affects the migration of sRAGE in the gel.

A Maillard reaction mixture consisting of d-ribose and l-lysine, which had been heated for 24 h at 120 ◦C, induced a 2-fold increase in NF-κB activation in the HEK RA cells

(Fig. 2.8 A, B). However, stimulation of the HEK ut cells by the MRPs also led to a 2-fold increase in NF-κB activation (Fig. 2.8 C, D). Furthermore, a Maillard reaction mixture consisting of d-ribose and l-arginine, which was prepared under the same conditions as the ribose-lysine MRPs, showed the same eﬀect.

Thus, it can be concluded that NF-κB activation by the MRPs takes place in human embryonic kidney cells via a similar mechanism as macrophages, but is not RAGE mediated.

This suggests that a second mechanism of NF-κB activation by MRPs must exist, which does not involve RAGE. In comparison to the MRP-induced NF-κB activation in macrophages

(6-fold) the cellular activation in the HEK cells by MRPs was less intense (2-fold). The

Western Blot showed that the basal level of translocated NF-κB in the HEK cells is much higher than in the macrophages, but the diﬀerence between the two cell lines is not yet described in the literature or cell line database.

2.1.2.5 MRP-Induced NF-κB Activation can be Inhibited by Catalase

AGEs have been shown to induce oxidative stress leading to the activation of the transcription factor NF-κB [Yan et al., 1994; Yeh et al., 2001]. Food-derived MRPs may interact with AGE-binding proteins, which are diﬀerent from RAGE, leading to the generation of

ROS. To determine the involvement of ROS in MRP-induced NF-κB activation, the stimulation of macrophages with the Maillard reaction mixture was repeated in the presence of catalase, which converts hydrogen peroxide into water and oxygen. Catalase, which was simultaneously added with the MRPs to the cell culture, fully abolished MRP-induced

NF-κB activation, whereas heat inactivated catalase did not show an eﬀect (Fig. 2.9 A, B). CHAPTER 2. RESULTS AND DISCUSSION 25

Figure 2.8: MRP-induced NF-κB activation in RAGE-transfected HEK (HEK RA) and untransfected HEK (HEK ut) cells . (A) MRP-induced NF-κB activation in HEK RA cells compared to cells maintained in medium alone (mean and standard deviation of four independent experiments; mean without indicated standard deviation of one experiment with repeat determination). Cells were starved for 24 h and stimulated in medium for 2 h with 25 mM of unheated control reaction mixtures (rib-lys 0 h, rib-arg 0 h), 25 mM of heated ribose (ribose 24 h), 25 mM of heated lysine

(lysine 24 h), 25 mM of heated arginine (arginine 24 h), 25 mM of Maillard reaction mixtures consisting of ribose and lysine, which was heated at 120 °C (rib-lys 24 h) and consisting of ribose and arginine, which was heated at 120 °C (rib-arg 24 h); *** p<0.001, (B) Representative Western Blot of p65 and β-actin. (C) MRP-induced NF-κB activation in HEK ut cells compared to the untreated control (mean and standard deviation of four independent experiments; mean without indicated standard deviation of one experiment with repeat determination). Cells were treated and stimulated according to the HEK RA experiment; *** p<0.001, ** p< 0.01. (D) Representative Western Blot of p65 and β-actin. 26 CHAPTER 2. RESULTS AND DISCUSSION

Figure 2.9: Catalase inhibits the MRP-induced NF-κB activation in macrophages. (A) MRP-induced NF-κB activation in NR8383 macrophages compared to control, which was incubated with medium alone (mean and standard deviation of three independent experiments). Cells were stimulated in medium for 2 h with 25 mM of Maillard reaction mixture consisting of ribose and lysine, which was heated at 120 °C (rib-lys 30 min). Catalase (150 U/mL) or heat inactivated catalase (5 min at 95 °C; 150 U/mL) was added simultaneously with the Maillard reaction mixture to the cells; *** p<0.001, ** p<0.01. (B) Representative Western Blot of p65 and β-actin. (C) NF-κB activation in

NR8383 macrophages compared to control, which was incubated with medium alone (mean and standard deviation of two independent experiments). Cells were stimulated in medium for 21 h with 10 ng/mL LPS. Catalase (150 U/mL) was added simultaneously with LPS to the cells. (D) Representative Western Blot of p65 and β-actin; ** p<0.01. CHAPTER 2. RESULTS AND DISCUSSION 27

When catalase is added to the cell culture medium, it remains in the extracellular space and can abolish extracellular hydrogen peroxide or hydrogen peroxide, which is drained out from the intracellular space by passive diﬀusion through the cell membrane [Halliwell, 2003].

To exclude that the amount of catalase used in the experiment suppressed NF-κB activation in general by blocking hydrogen peroxide-dependent signalling pathways, macrophages were stimulated with the NF-κB-inducer LPS in the presence of catalase. Catalase did not abolish the signiﬁcant LPS-induced NF-κB activation in the macrophages (Fig. 2.9 C, D).

This suggests that hydrogen peroxide is involved in MRP-induced NF-κB activation but is not required in LPS-triggered signal transduction leading to NF-κB activation.

Similarly to macrophages, catalase was able to inhibit MRP-induced cell activation in

HEK RA as well as HEK ut cells (Fig. 2.10). The inhibition of the cellular response upon stimulation with MRPs by catalase indicates that MRP-induced NF-κB activation is mediated by hydrogen peroxide in macrophages and in the HEK cells. 28 CHAPTER 2. RESULTS AND DISCUSSION

Figure 2.10: Catalase inhibits the MRP-induced NF-κB activation in HEK RA and HEK ut cells. MRP-induced

NF-κB activation in HEK RA (A) and HEK ut (B) cells compared to the cells maintained in medium alone (mean and standard deviation of at least two independent experiments). Cells were starved for 24 h and stimulated in medium for 2 h with 25 mM of a Maillard reaction mixture consisting of ribose and lysine or ribose and arginine, which was heated for 24 h (rib-lys 24 h, rib-arg 24 h) in the absence and presence of catalase (150 U/mL) or heat inactivated catalase (5 min at 95 °C; 150 U/mL); *** p<0.001, ** p<0.01. CHAPTER 2. RESULTS AND DISCUSSION 29

2.1.2.6 Coﬀee-Induced NF-kappaB Activation can be Inhibited by Catalase

The previous results suggest that MRPs in coﬀee are responsible for the coﬀee-induced

NF-κB activation. Thus, it was investigated whether hydrogen peroxide plays also a role in the coffee-induced signal transduction. Catalase, which was simultaneously added with the coffee to the cell culture, but not heat-inactivated catalase, efficiently reversed coffee-induced

NF-κB activation in macrophages (Fig. 2.11). Similar to MRP-induced NF-κB activation the coﬀee-induced NF-κB activation was blocked by catalase. The results indicate that the cellular activation of macrophages, induced by coﬀee and Maillard reaction mixture, is mediated by hydrogen peroxide.

Figure 2.11: Catalase inhibits the coffee-induced NF-κB activation in macrophages. (A) Coffee-induced NF-κB activation in NR8383 macrophages compared to the PBS treated control (mean and standard deviation of two independent experiments). Cells were stimulated in PBS for 2 h with 2 mg/mL of coffee extract. Catalase (150 U/mL) or heat inactivated catalase (5 min at 95 °C; 150 U/mL) was added simultaneously with the coffee extract to the cell culture; *** p<0.001. (B) Representative Western Blot of p65 and β-actin. 30 CHAPTER 2. RESULTS AND DISCUSSION

2.1.2.7 Hydrogen Peroxide Induces NF-κB Activation

It should be further conﬁrmed that hydrogen peroxide can induce NF-κB activation. Extra- cellular hydrogen peroxide, which can passively diﬀuse through the cell membrane into the cytoplasm [Halliwell and Gutteridge, 1999], or intracellular generated hydrogen peroxide, are suggested to increase the amount of ROS, resulting in the activation of NF-κB [Schreck et al., 1991]. To verify if hydrogen peroxide could induced NF-κB activation in the cell lines used, the translocation of the transcription factor upon stimulation with hydrogen peroxide was analysed.

HEK RA and HEK ut cells were stimulated with diﬀerent concentrations of hydrogen peroxide for 2 h and the translocation of NF-κB into the nucleus was observed. The administration of 250 µMH2O2 into the cell culture medium leads to a clear translocation of

NF-κB in HEK RA and HEK ut cells (Fig 2.12). Higher concentrations of hydrogen peroxide did not amplify the eﬀect in HEK ut cells and only slightly increased the translocation of NF-κB in the HEK RA cells. The results indicate that concentrations of 250 µMH2O2 or more leads to an approximately 2-fold increase in NF-κB activation in the HEK cells, which is similar to the results observed by stimulating the cells with 25 mM of the Maillard reaction mixture. Hydrogen peroxide could induce NF-κB activation similar to the well known NF-κB inducer TNF-α.

The hydrogen peroxide-induced NF-κB activation in the HEK ut cells was also observed to be dependent on the stimulation time (Fig 2.13). The translocation of NF-κB reached its maximum after 120 min and decreased afterwards.

In a previous study, human myeloid KBM-5 cells were stimulated with diﬀerent concentrations of hydrogen peroxide for 2 h, and NF-κB activation could be observed upon stimulation of the cells with 250 µMH2O2 or more. The hydrogen peroxide-induced NF- kappaB activation reached a maximum at 120 min and decreased at 240 min [Takada et al.,

2003]. The results obtained here are in agreement with these ﬁndings.

Hydrogen peroxide could also induce the translocation of NF-κB in the macrophages. CHAPTER 2. RESULTS AND DISCUSSION 31

Figure 2.12: H2O2-induced NF-κB activation in HEK RA (A) and HEK ut (B) cells compared to the cells maintained in medium alone (mean and standard deviation of two independent experiments; mean without standard deviation of one experiment with repeat determination). Cells were starved for 24 h and stimulated in medium for 2 h with diﬀerent concentrations of hydrogen peroxide, ranging from 20 – 750 µMH2O2 or 10 ng/mL human TNF-α. 32 CHAPTER 2. RESULTS AND DISCUSSION

Figure 2.13: H2O2-induced NF-κB activation in HEK ut cells compared to the cells maintained in medium alone is dependent on the stimulation time (mean and standard deviation of one experiment with repeat determination).

Cells were starved for 24 h and stimulated with 250 µMH2O2 in medium for between 30 min and 240 min.

Figure 2.14: (A) H2O2-induced NF-κB activation in NR8383 macrophages compared to the cells maintained in PBS alone (mean and standard deviation of two independent experiments). Cells were stimulated in PBS for 2 h with

500 µMH2O2; * p<0.05. (B) Representative Western Blot of p65 and β-actin. CHAPTER 2. RESULTS AND DISCUSSION 33

The cells were maintained in PBS and stimulated with 500 µMH2O2 for 2 h. A 13-fold increase in NF-κB translocation could be observed compared to the cells maintained in PBS alone (Fig 2.14).

It could be shown that hydrogen peroxide can induce NF-κB activation in the cell lines that were used to study the physiological activity of food-derived MRPs. The results suggest a possible role for hydrogen peroxide in food-derived MRP-induced NF-κB activation.

2.1.3 Discussion

Freshly brewed coffee was shown to activate nuclear translocation of NF-κB 13-fold in macrophages compared to untreated cells maintained in PBS. The extract of raw coffee beans, which does not contain MRPs, did not induce NF-κB activation, which led to the hypothesis that MRPs formed during the coffee roasting process are responsible for the activation of macrophages. A Maillard reaction mixture consisting of ribose and lysine heated for 30 min at 120 °C induced a 6-fold translocation of NF-κB compared to untreated cells maintained in medium. The NF-κB activation was shown to be due to MRPs because the unheated mixture or ribose or lysine heated alone did not lead to NF-κB activation.

The Maillard reaction mixture was assumed to contain melanoidins as well as low molecular weight MRPs. To identify the signal active fraction of MRPs, the Maillard reaction mixture was fractionated, using size exclusion chromatography, into low molecular weight

(<1.8 kDa) and high molecular weight (>1.8 kDa) MRPs. The high molecular weight fraction of the Maillard reaction mixture - namely melanoidins - was shown to be the active fraction, inducing NF-κB activation. The result is in accordance with the observed coffee-induced NF-κB translocation, as melanoidins are one of the major components in roasted coffee, accounting up to 25 % to the dry matter of coffee beverage [Belitz et al.,

2001]. Melanoidins derived from coﬀee, bread crust or a roasted sugar-amino acid reaction mixture, have been shown to exert antioxidative eﬀects in vitro [Borrelli et al., 2002;

Lindenmeier et al., 2002; Wagner et al., 2002]. The results of the present work further 34 CHAPTER 2. RESULTS AND DISCUSSION suggest that food-derived MRPs - namely melanoidins - can, similar to AGEs, induce a cell signalling pathway, which is linked to an inﬂammatory response.

It has been previously shown that AGEs, which are also found in food, e.g. N-carboxymethyllysine, can induce cell signalling pathways and cause the activation of NF-κB through binding to RAGE [Kislinger et al., 1999]. RAGE-mediated generation of oxidative stress has been discussed as a possible mechanism for signal transduction [Yan et al., 1994]. To investigate if the food-derived MRP-induced NF-κB activation is also RAGE-dependent, stimulation was performed with HEK cells that had been stably transfected with RAGE.

RAGE expression in the untransfected cells was below detection limit as determined by a Western Blot. However, NF-κB activation was similar in transfected and untransfected cells indicating that MRPs exert their activity independently of RAGE. On the other hand,

MRPs induced oxidative stress, which was shown to contribute to NF-κB activation, as the effect of MRPs was blocked by adding catalase to the cell culture medium. Hydrogen peroxide was also shown to play a pivotal role in coffee-induced NF-κB activation. Furthermore, it was confirmed that catalase had no influence on the ability of cells to activate NF-κB.

The activation of NF-κB by LPS, a well known NF-κB inducer, was not blocked by adding catalase to the cell culture medium. The results indicate that hydrogen peroxide is involved in MRP-induced cell signalling in macrophages.

Also, the involvement of hydrogen peroxide in NF-κB activation was veriﬁed. Stimulation of the used cell lines with hydrogen peroxide led to a concentration- and time-dependent activation of NF-κB. The result conﬁrms a possible role of hydrogen peroxide in the MRP- induced NF-κB activation in macrophages.

It can be concluded that MRP-induced NF-κB activation is mediated by hydrogen peroxide in a RAGE-independent manner. It is not clear whether MRPs induce NF-κB activation through interactions with AGE-binding proteins, which are diﬀerent from RAGE [Thornal- ley, 1998], or if a receptor-independent mechanism of cell activation exists. CHAPTER 2. RESULTS AND DISCUSSION 35

2.2 Food-Derived MRPs Induce NF-κB Activation through

Cell-Independent Generation of Hydrogen Peroxide

2.2.1 Introduction

It is known that receptor-dependent signalling pathways use ROS as cellular signalling molecules [Suzuki et al., 1997].

Another source of ROS, independent of the presence of cells, is the Maillard reaction

[Rahbar and Figarola, 2003]. Oxidation of Maillard reaction products such as the Amadori product have been shown to generate superoxide and hydrogen peroxide [Mossine et al.,

1999; Ortwerth et al., 1998]. The mechanism of ROS-generation is suggested to proceed through enolization following oxidation (Fig 2.15). Molecular oxygen exists as a diradical with two unpaired electrons (triplet oxygen), preferably undergoing reactions with radicals. Thus, transition metal ions are assumed to catalyse the one-electron oxidation of, e.g. Amadori product, which leads to the formation of superoxide. Further reduction of superoxide presents hydrogen peroxide and hydroxyl radical (Fig. 2.16).

Figure 2.15: Mechanism of superoxide radical formation by Amadori product [Mossine et al., 1999]. Sugar (Sug).

So far the results of the present work indicate that the cellular activity of food-derived

MRPs is mediated by hydrogen peroxide. However, it is not known whether MRPs lead 36 CHAPTER 2. RESULTS AND DISCUSSION

•− Figure 2.16: Reduction of molecular oxygen (O2) leads to the formation of superoxide (O2 ), hydrogen peroxide

• (H2O2) and hydroxyl radical ( OH). to the generation of hydrogen peroxide extracellular or in the cytoplasm by a receptor- dependent induction of oxidative stress. Thus, hydrogen peroxide concentration were measured in coﬀee and Maillard reaction mixture in the absence of cells. Hydrogen peroxide was measured directly in the solutions, which were applied to the cell culture [Pelka, 2005].

The second aim of this part of the work was to investigate the role of metal ions in the

MRP-induced generation of hydrogen peroxide in the cell free matrix as well as in the cell culture experiments. Metal-catalysed oxidation of MRPs has been shown to enhance ROS generation. Exemplarily, in the presence of Cu2+ the generation of superoxide by Amadori product increased 100-fold [Mossine et al., 1999]. However, in the strict absence of metal ions the generation of superoxide by MRPs could be observed as well [Ortwerth et al., 1998;

Mossine et al., 1999].

2.2.1.1 Determination of Hydrogen Peroxide Concentration

The perchloric acid (PCA)-FOX (ferrous oxidation xylenol orange) assay [Gay and Gebicki,

2002] was used to determine hydrogen peroxide concentrations. The principle of the assay is based on the hydrogen peroxide-induced oxidation of ferrous (Fe2+) to ferric (Fe3+), which forms a coloured complex with xylenol orange. A blank was subtracted from each sample, which was treated in the same way except for the addition of catalase (150 U/mL). CHAPTER 2. RESULTS AND DISCUSSION 37

2.2.2 Results

2.2.2.1 Coﬀee Generates Hydrogen Peroxide in a Cell Free Matrix

The concentration of hydrogen peroxide was determined in coffee, which was diluted 1:10 in PBS (pH 7.4), to a final extract concentration of 2 mg/mL. Raw coffee was diluted to the same extract concentration (1:7). Coffee and raw coffee extract were prepared in the same way as described before in the cell culture experiments (Chapter 2.1.2.1). Briefly, the solutions were cooled to room temperature, adjusted to pH 7.4 and the coffee or raw coffee extract was diluted to the final extract concentration. Afterwards, hydrogen peroxide concentration was immediately determined (time = 0 h) in the absence of cells (Fig. 2.17).

Figure 2.17: H2O2 (µM) concentration in coffee extract and in raw coffee extract (mean of three independent experiments with indicated standard deviation). H2O2 was measured in coffee and raw coffee extract, which were diluted in

PBS to the final extract concentration of 2 mg/mL. The H2O2 concentration was measured immediately after coffee or raw coffee preparation (time = 0 h) and after 1, 2, 3, 4 and 5 h incubation at 37 °C.

A concentration of 81.1 µMH2O2 was measured in freshly brewed coﬀee (time = 0 h) whereas in raw coﬀee (time = 0 h) hydrogen peroxide was not detected. Additionally, hydrogen peroxide concentration was determined after incubation of the test solutions at

37 °C under the same conditions as applied in the cell culture experiments, but in the absence of cells. The concentration of hydrogen peroxide increased when coﬀee was incubated at

37 °C reaching its maximum after 2 h. Incubation of raw coﬀee extract at 37 °C for between 38 CHAPTER 2. RESULTS AND DISCUSSION

1 and 5 h did not lead to hydrogen peroxide generation. After 2 h, a 2-fold increase in hydrogen peroxide concentration was observed in coffee (156.8 µMH2O2) whereas raw coffee extract did not lead to hydrogen peroxide generation. It can be hypothesized that hydrogen peroxide generation in coffee is due to MRPs, since raw coffee extract, lacking

MRPs did not produce hydrogen peroxide.

2.2.2.2 MRPs Generate Hydrogen Peroxide in a Cell Free Matrix

The result implies that MRPs, which are formed during the coﬀee roasting process, are responsible for the generation of hydrogen peroxide. Thus, the production of hydrogen peroxide in the Maillard reaction mixture prepared by heating ribose and lysine for 30 min at 120 °C was determined (rib-lys 30 min). The Maillard reaction mixture was diluted in

PBS (pH 7.4) to the ﬁnal concentrations of 5 - 40 mM (d-ribose and l-lysine concentration prior to heating) and the hydrogen peroxide concentration was immediately measured

(time = 0 h). The amount of hydrogen peroxide in the Maillard reaction mixture was shown to be concentration-dependent (Fig. 2.18). Additionally, the concentration of hydrogen peroxide was determined in the Maillard reaction mixture incubated at 37 °C for 2, 4, and 6 h under the same conditions as applied in the cell culture experiments, but in the absence of cells. Hydrogen peroxide generation increased in the Maillard reaction mixture incubated at 37 °C reaching a maximum after 4 h (Fig. 2.18). The hydrogen peroxide concentration in a 25 mM Maillard reaction mixture increased from 287 µMH2O2 to 366 µMH2O2 after two hours incubation at 37 °C, further rising after two more hours to 423 µMH2O2 (Ta- ble 2.1). An incubation of 6 h did not lead to a further increase. The hydrogen peroxide generation is dependent on the presence of MRPs. Hydrogen peroxide was not detected in the unheated reaction mixture (rib-lys 0 h) or ribose and lysine, which were heated alone

(ribose 30 min, lysine 30 min), immediately after dilution in PBS to the ﬁnal concentration of 25 mM (d-ribose and/or l-lysine) (Table 2.1). Incubation of 25 mM ribose and 25 mM lysine, both heated alone, at 37 °C did not lead to hydrogen peroxide generation either. CHAPTER 2. RESULTS AND DISCUSSION 39

Figure 2.18: H2O2 (µM) concentration in a Maillard reaction mixture consisting of ribose and lysine heated at 120 °C (rib-lys 30 min) (mean of three independent experiments with indicated standard deviation). The Maillard reaction mixture was diluted in PBS to concentrations ranging from 5 – 40 mM. H2O2 was measured immediately after diluting the Maillard reaction mixture (time = 0 h) and after 2, 4 and 6 h incubation at 37 °C.

However, the unheated reaction mixture was shown to produce hydrogen peroxide during the incubation at 37 °C for between 2 h and 6 h. It is possible that this eﬀect is due to the formation of MRPs at 37 °C. The ﬁndings show that the Maillard reaction mixture produces hydrogen peroxide independent of the presence of cells. Incubation at 37 °C led to the production of hydrogen peroxide in the unheated reaction mixture or increased the hydrogen peroxide concentration in the Maillard reaction mixture.

2.2.2.3 Generation of Hydrogen Peroxide by MRPs does not Require Free Metal Ions

Furthermore, the mechanism of ROS generation by MRPs should be elucidated. It is known that metal ions catalyze the oxidation of MRPs leading to ROS generation [Mossine et al., 1999]. Metal ions could be present in PBS [Buettner and Jurkiewicz, 1996], which was used to prepare and dilute the Maillard reaction mixture in the experiments. To investigate whether the production of hydrogen peroxide by MRPs is dependent on the presence of free metal ions the Maillard reaction mixture and PBS were treated with Chelex. 40 CHAPTER 2. RESULTS AND DISCUSSION

Table 2.1: H2O2 (µM) concentration in 25 mM Maillard reaction mixture consisting of ribose and lysine heated at 120 °C (rib-lys 30 min), 25 mM unheated reaction mixture (rib-lys 0 h), 25 mM heated ribose (ribose 30 min) and 25 mM heated lysine (lysine 30 min) (mean of three independent experiments with indicated standard deviation). H2O2 was measured in the solutions immediately after diluting with PBS to the ﬁnal concentration (25 mM).

Furthermore, the test solutions were incubated for 2, 4 or 6 more hours at 37 °C.

H2O2 conc. H2O2 conc. H2O2 conc. H2O2conc. after 0 h after 2 h after 4 h after 6 h

µM, mean ± S.D. µM, mean ± S.D. µM, mean ± S.D. µM, mean ± S.D.

rib-lys (25 mM, 30 min) 286.9 ± 10.9 366.4 ± 8.2 423.0 ± 25.6 429.0 ± 27.6

rib-lys (25 mM, unheated) 13.2 ± 8.8 35.0 ± 9.5 45.6 ± 9.3 28.2 ± 5.2

ribose (25 mM, 30 min) 4.2 ± 5.1 2.5 ± 2.8 5.5 ± 4.4 6.1 ± 8.4

lysine (25mM, 30 min) 2.5 ± 3.4 0.6 ± 1.0 0.0 ± 0.0 0.7 ± 1.2

Chelex is a chelator, which eﬃciently removes free metal ions. A Chelex-treated Maillard reaction mixture was diluted to the ﬁnal concentrations of 5 - 40 mM (d-ribose and l- lysine concentration prior to heating) in Chelex-treated PBS and the test solutions were incubated for 2 h at 37 °C. A similar increase in hydrogen peroxide concentration during the incubation at 37 °C for 2 h was found in the Chelex-treated Maillard reaction mixture, diluted in Chelex-treated PBS compared to the untreated Maillard reaction mixture, which was dissolved in PBS (Fig. 2.19). These results indicate that hydrogen peroxide can be generated by MRPs in the absence of free metal ions.

Additionally, it was conﬁrmed that the generation of hydrogen peroxide by MRPs is not dependent on PBS. Coﬀee and Maillard reaction mixture were diluted with water, instead of

PBS, to their ﬁnal concentrations and the generation of hydrogen peroxide was determined.

Ground coffee was extracted as described before (Chapter 2.1.2.1) and diluted 1:10 in water to the final extract concentration of 2 mg/mL. The concentration of hydrogen peroxide was immediately measured (time = 0 h). Similar to the result observed when coffee was diluted with PBS, freshly brewed coffee contains 88.9 µMH2O2 (Table 2.2). Furthermore, the

Maillard reaction mixture was diluted in water and incubated for 2 h at 37 °C. A slightly higher concentration of hydrogen peroxide could be detected when the Maillard reaction mixture was diluted in water compared to the Maillard reaction mixture diluted in PBS. CHAPTER 2. RESULTS AND DISCUSSION 41

Figure 2.19: Free metal ion-independent H2O2 (µM) generation in Maillard reaction mixture consisting of ribose and lysine heated at 120 °C (rib-lys 30 min) (mean of at least two independent experiments with indicated standard deviation). The Maillard reaction mixture was diluted in PBS to concentrations ranging from 5 – 40 mM and H2O2 was measured immediately (time 0 h) or after incubation at 37 °C for 2 h (time 2 h). (•) Chelex-treated Maillard reaction mixture, diluted in Chelex-treated PBS after incubation at 37 °C for 2 h (Chelex-treated; time 2 h).

Table 2.2: H2O2 (µM) concentration in coﬀee and Maillard reaction mixture consisting of ribose and lysine heated at

120 °C (rib-lys 30min) dependent on the presence of PBS (mean of at least two independent experiments with indicated standard deviation). H2O2 was determined in coffee diluted 1:10 with water or PBS to the final concentration of 2 mg/mL and in the Maillard reaction mixture, diluted 1:20 in water or PBS to the final concentration of 25 mM, following incubation at 37 °C for 2 h.

H2O2 conc. after 0 h H2O2 conc. after 2 h µM, mean ± S.D. µM, mean ± S.D.

coﬀee (2 mg/mL) rib-lys (25 mM, 30 min)

diluted in water 88.9 ± 2.5 407.5 ± 1.5

diluted in PBS 81.1 ± 9.4 366.4 ± 8.2 42 CHAPTER 2. RESULTS AND DISCUSSION

2.2.2.4 MRP-Induced NF-κB Activation is not Due to Components of the Cell

Culture Medium

Cell culture medium, containing metal ions that are required for cell growth, is suggested to exert pro-oxidative eﬀects [Halliwell, 2003]. For example, antioxidants added to cell culture medium have been shown to undergo metal-catalyzed oxidation leading to the formation of

ROS [Long et al., 2000]. This could induce the activation of cells, which is however, limited to cell culture conditions. Therefore, the inﬂuence of the solvent in MRP-induced NF-κB activation in macrophages was investigated. The Maillard reaction mixture was added to the cells, which were maintained in medium, physiological PBS or Chelex-treated PBS. For some experiments, the Maillard reaction mixture was also treated with Chelex. While the

MRPs in medium produced a 6-fold elevated NF-κB activation in macrophages, the MRPs in PBS increased nuclear NF-κB concentration 18-fold (Fig. 2.20).

The boosted eﬀect of MRP-induced NF-κB activation in cells maintained in PBS compared to those maintained in medium could be due to the presence of fetal calf serum (FCS) in medium. FCS contains low levels of catalase and therefore weakens MRP-induced NF-κB activation, which is mediated by hydrogen peroxide. Similar to the experiments in medium, catalase inhibited MRP-induced NF-κB activation in PBS. Pre-treatment of PBS and/or

MRPs with Chelex did not attenuate NF-κB activation.

The results indicate that MRP-induced NF-κB activation is not an artifact caused by the components of the cell culture medium, since cellular activation by MRPs is even more enhanced in PBS. Additionally, the removal of metal ions by pre-treating PBS and Maillard reaction mixture with a chelator did not inﬂuence NF-κB activation. The ﬁndings imply that MRPs generate hydrogen peroxide under quasi-physiological conditions leading to NF-

κB activation in macrophages. CHAPTER 2. RESULTS AND DISCUSSION 43

Figure 2.20: MRP-induced NF-κB activation in NR8383 macrophages compared to the control, which was maintained in medium for experiments performed in medium or PBS for experiments performed in PBS or Chelex-treated PBS

(Chelex-PBS) (mean and standard deviation of three independent experiments). The Maillard reaction mixture was prepared by heating ribose and lysine at 120 °C (rib-lys 30 min). Cells were stimulated in medium, PBS or Chelex-treated PBS for 2 h with 10 mM, 25 mM of Maillard reaction mixture or 25 mM Maillard reaction mixture, which had been pre-treated with Chelex (Chelex-rib-lys 30 min). Where indicated, catalase (150 U/mL) was added simultaneously with the Maillard reaction mixture to the cell culture; *** p<0.001. (B) Representative Western Blot of p65 and β-actin. 44 CHAPTER 2. RESULTS AND DISCUSSION

2.2.3 Discussion

Food-derived MRPs induce NF-κB activation in macrophages, which could be blocked by adding catalase. The present investigations revealed that coﬀee and the Maillard reaction mixture, used to stimulate the macrophages for 2 h, produced hydrogen peroxide in the absence of cells. Freshly brewed coﬀee contained 89 µMH2O2. After incubation at 37 °C for 2 h, the hydrogen peroxide concentration increased up to 157 µM. 25 mM of the Mail- lard reaction mixture contained 287 µMH2O2, which increased up to 366 µMH2O2 after incubation at 37 °C for 2 h. The initial concentration of hydrogen peroxide was shown to be dependent on the concentration of the Maillard reaction mixture, however in a non-linear way. Hydrogen peroxide concentration in the Maillard reaction mixtures ≥ 30 mM, reached a maximum of ≈ 280 µM. Thus, the hydrogen peroxide generation by MRPs seems to be limited by other factors than MRPs, e.g. concentration of dissolved oxygen.

It can be hypothesized that MRPs are responsible for hydrogen peroxide generation as raw coﬀee extract and the unheated reaction mixture, both lacking MRPs, did not produce hydrogen peroxide. The results indicate that the cellular activity of food-derived MRPs is mediated through a receptor-independent generation of hydrogen peroxide in the extracellular space. The concentration of hydrogen peroxide, measured after 2 h incubation of coﬀee or Maillard reaction mixture under conditions as applied in the cell culture experiments, is likely to induce oxidative stress in macrophages, culminating in NF-κB activation. As reported in chapter 2.1.2.7, macrophages were shown to activate NF-κB upon stimulation with H2O2 for 2 h.

The formation of hydrogen peroxide in coffee has been described previously. Freshly brewed coffee was shown to contain 150 µMH2O2 [Long et al., 1999], while in another study 80 µMH2O2 was reported [Akagawa et al., 2003]. The studies used different conditions for the coffee preparation, e.g. coffee-extraction time, which does not allow a direct comparison of the hydrogen peroxide concentration in coffee, but shows consistent production of hydrogen peroxide during coffee brewing. After incubation at room temperature or CHAPTER 2. RESULTS AND DISCUSSION 45 at 37 °C the hydrogen peroxide concentration slightly increased in the beverages. It was suggested that the generation of hydrogen peroxide was caused by the polyphenol fraction

[Akagawa et al., 2003]. However, in the present work raw coﬀee extract, which is rich in phenols did not generate any hydrogen peroxide, indicating that roasting products, mainly

MRPs and melanoidins, are responsible for the observed effect. This result is in accordance with a previous report of the generation of hydrogen peroxide in roasted coffee but not in raw coffee [Fujita et al., 1985].

It is known that ROS are generated in the course of the Maillard reaction. Direct formation of superoxide anions and hydrogen peroxide during the Maillard reaction has been demonstrated [Ortwerth et al., 1998]. Other studies showed that the Amadori product can generate superoxide and hydrogen peroxide [Mossine et al., 1999]. The mechanism of hydrogen peroxide-generation by MRPs has not yet been elucidated. Metal ions are suggested to catalyze the oxidation of MRPs, leading to the reduction of oxygen to reactive oxygen species. However, the generation of ROS by MRPs was also observed in the absence of metal ions [Ortwerth et al., 1998; Mossine et al., 1999]. To investigate the involvement of metal ions in the observed hydrogen peroxide generation by MRPs, traces of metal ions in PBS and the Maillard reaction mixture were removed by pre-treatment of the solutions with Chelex. Removal of free metal ions, however, did not inﬂuence the generation of hydrogen peroxide by MRPs during the incubation at 37 °C for 2 h in the cell free matrix.

This indicates that hydrogen peroxide can be generated in the absence of free metal ions.

Furthermore coﬀee and Maillard reaction mixture were diluted with water instead of PBS and approximately the same amount of hydrogen peroxide was detected.

Cell culture media is known to contain metal ions and therefore exhibits a pro-oxidative eﬀect [Halliwell, 2003]. To further investigate whether NF-κB activation in macrophages, due to the presence of a Maillard reaction mixture, is an artifact derived from the cell culture media, the stimulation experiments were performed in physiological PBS. A threefold higher activation in PBS compared to medium was observed, which is probably due to the presence 46 CHAPTER 2. RESULTS AND DISCUSSION of FCS in the medium. FCS contains low levels of catalase and therefore weakens the

MRP-induced NF-κB activation caused by hydrogen peroxide. Furthermore, treatment of

PBS and Maillard reaction mixture with Chelex had no inﬂuence on MRP-induced NF-κB activation in the macrophages. The results indicate that the observed eﬀect of the MRPs is not an artifact of the cell culture media, depending on metal ions. Thus, it can be expected that MRPs can induce NF-κB activation in macrophages under physiological conditions.

The results confirm that hydrogen peroxide is present in coffee, most likely due to the presence of MRPs. There is strong evidence that hydrogen peroxide, which is formed in coffee is of relevance in vivo, because increased urinary hydrogen peroxide levels were measured after the consumption of coffee [Hiramoto et al., 2002; Long and Halliwell, 2000].

This means that either hydrogen peroxide is directly consumed with the coffee beverage and not fully detoxified, or that active components of the coffee continue hydrogen peroxide generation in vivo.

These ﬁndings show that MRPs in model mixtures as well as in food generate hydrogen peroxide, leading to NF-κB activation in macrophages. In chapter 2.1.2.3 melanoidins have been identiﬁed to be the signal active fractions of the Maillard reaction mixture.

Therefore, the Maillard reaction mixture was fractionated into low and high molecular weight compounds. The cells were stimulated with the fractions, which were reconstituted to the original volume after freeze-drying. Hydrogen peroxide, which is formed during the reaction between lysine and ribose at 120 °C is present in the Maillard reaction mixture

(Chapter 2.2.2.1) and will elute with the low molecular weight fraction. However, only the

HMW fraction was shown to induce NF-κB activation in the macrophages. No eﬀect was observed after stimulating the cells with the LMW fraction. It is likely that the hydrogen peroxide, present in the low molecular weight fraction, partly evaporates during the freeze-

1 1 drying step. This suggestion was conﬁrmed in an experiment showing that only /3 to /4 of hydrogen peroxide could be recovered after freeze-drying (data not shown).

Incubation at 37 °C led to the production of hydrogen peroxide in the unheated reaction CHAPTER 2. RESULTS AND DISCUSSION 47 mixture or increased the hydrogen peroxide concentration in the Maillard reaction mixture as well as in coﬀee. These results indicate that MRPs, particularly melanoidins, contain structures that continually generate hydrogen peroxide during the stimulation of cells at

37 °C, leading to NF-κB activation. 48 CHAPTER 2. RESULTS AND DISCUSSION

2.3 Identiﬁcation of C4-Aminoreductone as a Signal Active

MRP-Structure

2.3.1 Introduction

In the present work, MRPs derived from coffee or a heated sugar/amino acid mixtures have been shown to induce NF-κB activation through hydrogen peroxide generation. Melanoidins have been identified as the signal active fraction of the Maillard reaction mixture, but the heterogeneous composition of melanoidins does not allow the isolation of defined products.

Therefore, signal active structures in MRPs, leading to activation of NF-κB through hydrogen peroxide generation should be identiﬁed.

AGEs are glycated proteins, formed through reaction steps analogous to MRP formation.

Signal active chemical constituents, present in melanoidins, may also be found in AGEs. It is well known that AGEs induce cell signalling pathways through interaction with RAGE

[Lander et al., 1997; Yan et al., 1994]. However, it is likely that exogenous ROS generation by AGEs also contributes, at least in part, to the AGE-induced activation of cells. The second aim of this part of the work was to investigate the AGE-induced NF-κB activation or expression of NF-κB regulated genes in order to determine whether the AGE-induced cell activation could also be mediated by the extracellular production of ROS.

2.3.2 Results

2.3.2.1 C4-Aminoreductone Induces NF-κB Activation through Hydrogen Peroxide

Generation

Early products of the Maillard reaction such as the Amadori product [Mossine et al., 1999] or

3-deoxyosones [Ortwerth et al., 1998] have been shown to generate superoxide. Superoxide- dismutation could then lead to the formation of hydrogen peroxide, causing NF-κB translocation in macrophages. Aminoreductones or reductones are compounds with antioxidative properties generated in the course of the Maillard reaction [Pischetsrieder et al., 1998; Lin- CHAPTER 2. RESULTS AND DISCUSSION 49

Figure 2.21: Structures of MRPs. (1) Amadori product, (2) 3-deoxy-glucosone, (3) C4-aminoreductone and (4) reductone pyranone. The aminoreductone and reductone structures are printed in bold type. denmeier et al., 2002]. However, in the presence of metal ions, aminoreductones as well as reductones have been shown to exert pro-oxidative eﬀects [Pischetsrieder et al., 1998].

The following MRPs, Amadori product (N-(1-deoxy-d-fructose-1-yl)l-lysine) and 3-deoxy- glucosone (3-deoxy-d-erythro-hexose-2-ulose), and compounds containing an aminoreductone structure, C4-aminoreductone (3-hydroxy-4-(morpholino)-3-buten-2-on) or a reductone ether, reductone pyranone (2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-on) were tested for their ability to induce NF-κB activation in the bioassay (Fig. 2.21) [Degel, 2005]. The compounds were added at a ﬁnal concentration of 10 mM in PBS to the macrophages and incubated for 2 h. The C4-aminoreductone induced a signiﬁcant 7-fold increased NF-κB translocation compared to the PBS treated control (Fig. 2.22). The reductone pyranone and the early MRPs, Amadori product and 3-deoxy-glucosone did not induce NF-κB activation.

The results suggest that C4-aminoreductone (AR) induces NF-κB activation. It was hypothesized that MRPs, which have been shown to induce NF-κB activation through hydrogen peroxide generation in the present work, contain aminoreductone structures, formed in the course of the Maillard reaction. To investigate whether hydrogen peroxide also plays a role in the C4-aminoreductone-induced NF-κB activation the experiment was repeated in 50 CHAPTER 2. RESULTS AND DISCUSSION

Figure 2.22: Aminoreductone-induced NF-κB activation in NR8383 macrophages compared to the control, which was maintained in PBS alone (mean and standard deviation of repeat determination). Cells were stimulated in PBS for

2 h with 10 mM (1) Amadori product, (2) 3-deoxy-glucosone, (3) C4-aminoreductone or (4) reductone pyranone; * p<0.05. (B) Representative Western Blot of p65 and β-actin. CHAPTER 2. RESULTS AND DISCUSSION 51

Figure 2.23: (A) Catalase inhibits C4-aminoreductone-induced NF-κB activation in NR8383 macrophages compared to control, which was incubated with PBS alone (mean and standard deviation of three independent experiments).

Cells were stimulated in PBS for 2 h with 5 mM or 10 mM C4-aminoreductone (AR). Catalase (150 U/mL) was added simultaneously with the C4-aminoreductone to the cells; *** p<0.001, * p<0.05. (B) Representative Western Blot of p65 and β-actin.

the presence of catalase. Catalase fully abolished the C4-aminoreductone-induced NF-κB activation (Fig. 2.23). A signiﬁcantly increased translocation of NF-κB was observed using

5 mM or 10 mM C4-aminoreductone. The ﬁndings show that similar to the results obtained with MRPs or coﬀee, the C4-aminoreductone-induced NF-κB activation is mediated by hydrogen peroxide.

2.3.2.2 AGEs do not Induce NF-κB Activation

Maillard-modiﬁed proteins (AGEs) are formed through reaction steps analogous to MRP- formation. Thus, AGEs could contain aminoreductone structures, that cause NF-κB activation through hydrogen peroxide generation. It is well known that AGEs induce NF-κB activation through binding to RAGE, which is expressed on the surface of macrophages

[Thornalley, 1998]. It should be investigated whether the AGE-induced cell activation could also be mediated by the extracellular production of ROS. By blocking RAGE with 52 CHAPTER 2. RESULTS AND DISCUSSION

Figure 2.24: AGEs do not induce NF-κB activation in macrophages. NF-κB activation in NR8383 macrophages compared to the control, which was maintained in medium alone (mean and standard deviation of repeat determination).

Cells were stimulated in medium for 2 h with 100 µM AGE-BSA(rib), 21 h with 70 µM AGE-BSA(rib) and 24 h with

20 µM AGE-BSA(rib) or AGE-BSA(glu). As positive control cells were stimulated with 10 or 100 ng/mL LPS for

21 h. an anti-RAGE antibody the AGE-induced NF-κB activation can be clearly assigned to an involvement of RAGE. Macrophages were stimulated with in vitro prepared AGEs. These

Maillard-modiﬁed proteins were prepared by heating bovine serum albumin (BSA) with ribose for 3 weeks at 37 °C under sterile conditions [AGE-BSA(rib)]. AGE-BSA(rib) was added at a ﬁnal concentration of 100 µM in medium to the macrophages and incubated for 2 h. Unexpectedly, AGEs did not induce NF-κB activation in the cells (Fig. 2.24).

The incubation time was extended to 21 h or 24 h and also lower concentrations of the

AGEs (70 µM and 20 µM) were used. The macrophages were also stimulated with 20 µM of an AGE reaction mixture, which was prepared by heating BSA with glucose for 8 weeks at 37 °C [AGE-BSA(glu)]. However, none of the AGE reaction mixtures induced NF-κB activation in the macrophages known to express RAGE (Fig. 2.24). The positive control stimuli LPS induced NF-κB activation in a concentration-dependent manner.

It was considered whether the absence of the biological activity of AGEs was speciﬁc for macrophages. Therefore, RAGE-transfected HEK cells (HEK RA) and hepatic stellate cells CHAPTER 2. RESULTS AND DISCUSSION 53

Figure 2.25: AGEs do not induce NF-κB activation in HEK RA and HSC T6 cells (A) NF-κB activation in HEK

RA cells compared to the control, which was maintained in medium alone (mean and standard deviation of two independent experiments). Cells were starved for 24 h and stimulated in medium for 4.5 h with 1.5 µM AGE-

BSA(glu) or 10 ng/mL human TNF-α; *p<0.05. (B) NF-κB activation in HSC T6 cells compared to the untreated control (mean and standard deviation of two independent experiments). Cells were treated and stimulated according to the HEK RA experiment.

(HSC T6) were incubated with 1.5 µM AGE-BSA(glu) for 4.5 h and the translocation of

NF-κB was determined. AGEs have been shown to induce NF-κB activation under similar experimental conditions, in particular AGE concentration and incubation time [Lander et al., 1997; Yan et al., 1994]. However, AGEs failed to induce NF-κB activation in HEK

RA (Fig. 2.25 A) and HSC T6 (Fig. 2.25 B) cells. The positive control stimuli TNF-α induced NF-κB in the cell lines used. Hepatic stellate cells were shown to express RAGE

[Fehrenbach et al., 2001] and RAGE expression in the HEK RA cells is demonstrated in chapter 2.1.2.4 (Fig. 2.7). The results show that the AGEs, which were prepared in the present study, do not induce NF-κB activation neither through interacting with RAGE nor through exogenous hydrogen peroxide generation.

CML is the only speciﬁc AGE, which was identiﬁed to induce cellular activation upon binding to RAGE [Kislinger et al., 1999]. Thus, the activation of NF-κB by protein-bound

CML (CML-BSA) [Hasenkopf, 2002] or CML [Degel, 2005] was investigated. CML-BSA was added at a ﬁnal concentration of 20 µM in medium to the macrophages and incubated for

24 h. Free CML was added at a ﬁnal concentration of 9 mM in PBS to the cells and incubated 54 CHAPTER 2. RESULTS AND DISCUSSION for 2 h. Neither CML-BSA nor CML induced NF-κB activation in the macrophages (data not shown). The unexpected results indicate that AGEs despite containing CML structure, do not induce NF-κB activation in contrast to other reports [Kislinger et al., 1999].

2.3.2.3 AGEs do not Augment LPS-Induced NF-κB Activation

Two previous studies by Valencia et al. and Reznikov et al. have reported that AGEs are not suﬃcient to induce inﬂammatory cellular signalling pathways alone [Valencia et al., 2004a;

Reznikov et al., 2004]. However, AGEs were shown to augment LPS-induced expression of cytokines suggesting that AGEs rather contribute to an inﬂammatory response than inducing signalling pathways on their own [Reznikov et al., 2004]. Thus, the stimulation of the macrophages with 70 µM AGEs was repeated in the presence of 10 ng/mL LPS, using a similar experimental design in terms of AGE concentration and incubation time as described in the literature [Reznikov et al., 2004]. However, in the present study AGEs did not augment LPS-induced NF-κB activation (Fig. 2.26). Moreover, BSA and AGE-

BSA(rib) appeared to suppress the LPS-induced NF-κB activation in macrophages.

Figure 2.26: AGEs do not augment LPS-induced NF-κB activation in NR8383 macrophages compared to the control, which was maintained in medium alone (mean and standard deviation of two independent experiments). Cells were incubated with 10 ng/mL LPS in the presence or absence of 70 µM BSA or AGE-BSA(rib) for 24 h. LPS was added

30 min after applying BSA or AGE-BSA(rib) to the cell culture; ** p<0.01. CHAPTER 2. RESULTS AND DISCUSSION 55

2.3.2.4 AGEs Induce NO Generation through a RAGE-Dependent Mechanism

Two diﬀerent AGE reaction mixtures prepared at 37 °C did not to induce NF-κB activation in several RAGE-expressing cells. Therefore, it was further investigated whether the conditions under which the AGE reaction mixtures were prepared, e.g. the temperature, have an inﬂuence on the biological activity of AGEs. In contrast to the previous prepa- rations of AGEs at 37 °C, BSA was incubated with glucose for 5 days (AGE-BSA #1) or 3 weeks (AGE-BSA #2) or 6 weeks (AGE-BSA #3) at 60 °C under sterile conditions.

AGE-BSA #3 was prepared under the same conditions as AGE-BSA #0, which was kindly donated from the workgroup Münch (Comparative Genomics Centre, James Cook Univer- sity, Australia) and has been shown before to exert inflammatory effects. It is known that

AGEs induce the expression of NF-κB regulated genes such as iNOS leading to the release of high amounts of NO as an inﬂammatory cellular response [Dukic-Stefanovic et al., 2003].

Thus, the release of NO in response to AGEs was measured instead of determining AGE-

− induced translocation of NF-κB. The Griess reaction was used to measure nitrite (NO2 ), which is spontaneously formed from NO [Dusse et al., 2005]. Macrophages derived from rat (NR8383) or mouse (RAW 264.7) were stimulated with AGE-BSA #0 for 24 h and

48 h and NO production was measured in the cell culture supernatant. AGE-BSA #0 induced a concentration-dependent generation of NO in the rat (Fig. 2.27 A) and mouse

(Fig. 2.27 B) macrophages. The NO-generation was AGE-dependent since BSA, incubated in the absence of sugar under the same conditions as AGE-BSA #0, did not induce a cellular response. The AGEs #1 - #3 were diluted in medium to their final concentrations and the mouse macrophages were incubated with the AGEs for 48 h. The cells only showed a significant release of NO in response to AGE-BSA #2. Lower modified AGE-BSA #1 and, unexpectedly, AGE-BSA #3, prepared under similar conditions as AGE-BSA #0, did not lead to an inflammatory response. The result indicates that AGEs can induce the release of NO. However, the biological activity of AGEs depends on unknown parameters during the preparation of AGEs. 56 CHAPTER 2. RESULTS AND DISCUSSION

Figure 2.27: AGE-induced NO expression. (A) AGE-induced NO expression in NR8383 macrophages (mean and standard deviation of at least two independent experiments). Cells were stimulated for 24 h or 48 h in medium with

750 µg/mL BSA #0 or 50, 350 and 750 µg/mL AGE-BSA #0. (B) AGE-induced NO expression in RAW 264.7 macrophages (mean and standard deviation of at least two independent experiments). Cells were stimulated for 48 h in medium with 750 µg/mL BSA #0 or 50, 350 and 750 µg/mL AGE-BSA #0, #1, #2 or #3; *** p<0.001, * p<0.05. CHAPTER 2. RESULTS AND DISCUSSION 57

The biological active AGE-BSA reaction mixture #2 was used to investigate whether the observed NO expression depends on AGE-RAGE interactions or at least in part on exogenously generated hydrogen peroxide, which could stem from Maillard-derived aminoreductone structures. Mouse macrophages were stimulated with 750 µg/mL AGE-BSA #2 for

15 h in the presence or absence of anti-RAGE antibody. Where indicated, the anti-RAGE antibody was added 3 h prior AGE-stimulation to the cells. Blocking RAGE with anti-

RAGE antibody caused an 84 % inhibition of AGE-induced NO production (Fig. 2.28). In each experiment, the cell viability was measured after stimulating the cells to conﬁrm that the anti-RAGE antibody did not inhibit AGE-induced NO-generation by causing cell death

(data not shown). The outcome of this experiment indicates that the biologically active

AGE reaction mixture completely exert its eﬀect through a RAGE-dependent mechanism and not through exogenously generated hydrogen peroxide.

Figure 2.28: Anti-RAGE antibody inhibits AGE-induced NO production (mean and standard deviation of two independent experiments). RAW 264.7 macrophages were stimulated with 750 µg/mL AGE-BSA #2 for 15 h in the presence or absence of anti-RAGE antibody (1 µg/mL) , which was added 3 h prior AGE-stimulation to the cells;

*** p<0.001. 58 CHAPTER 2. RESULTS AND DISCUSSION

2.3.3 Discussion

C4-Aminoreductone is a Signal Active MRP-Structure Leading to Activation of NF-κB through the Generation of Hydrogen Peroxide

C4-aminoreductone, which can be formed in the course of the Maillard reaction was iden- tiﬁed as a signal active MRP-structure (Chapter 2.3.2.1). The C4-aminoreductone could induce the activation and nuclear translocation of NF-κB 10-fold in macrophages compared to untreated cells maintained in PBS. In accordance with the cellular reaction caused by food-derived MRPs (Chapter 2.1), the eﬀect could be blocked by adding catalase to the cells, indicating hydrogen peroxide as signal mediator. It can be concluded that MRPs containing aminoreductone structures induce NF-κB activation through hydrogen peroxide generation in macrophages.

The induction of oxidative stress could be due to metal-catalyzed oxidation of aminoreductones. Maillard-derived aminoreductones are known to exert anti-oxidative activities

[Pischetsrieder et al., 1998; Lindenmeier et al., 2002]. However, in the presence of metal ions a pro-oxidative eﬀect was observed [Pischetsrieder et al., 1998]. The pro-oxidative activity of aminoreductones is suggested to be due to the formation of a complex with metal ions and oxygen, which causes reduction of oxygen to reactive oxygen species [Pischetsrieder et al., 1998]. A postulated mechanism based on Pischetsrieder et al. of ROS-generation by aminoreductone autoxidation is illustrated in ﬁgure 2.29.

Figure 2.29: Postulated mechanism based on Pischetsrieder et al. of aminoreductone autoxidation in the presence of metal ions (here ferric) [Pischetsrieder et al., 1998].

Additionally, it is known that reductones such as ascorbic acid, which have a similar structure and reducing properties as aminoreductones, can become pro-oxidative in the CHAPTER 2. RESULTS AND DISCUSSION 59 presence of metal ions and oxygen, generating hydrogen peroxide [Buettner and Jurkiewicz,

1996; Chen et al., 2005]. On the other hand polyphenols, which are antioxidants containing reductone structures, were shown to generate hydrogen peroxide also in the absence of free metal ions. This ﬁnding was explained by the direct reaction of oxygen with the polyphenols

[Akagawa et al., 2003]. However, it is questionable whether this reaction mechanism takes place taking into account the spin restriction between biomolecules - e.g. polyphenols - and oxygen, which reacts as a diradical [Buettner and Jurkiewicz, 1996].

The present result suggests that MRPs with aminoreductone structures become pro- oxidative producing hydrogen peroxide, which then induces cell signalling pathways. The autoxidation of MRPs may be metal ion-catalyzed in contrast to the present ﬁnding that

MRPs exert their biological activity also in the absence of free metal ions (Chapter 2.2.2.4).

MRPs are known to be chelators [Wijewickreme et al., 1997] and Chelex, which was used to remove free metal ions in the experiments, may have less chelating ability than MRPs.

Thus, metal ions strongly bound to MRPs could catalyze the oxidation of speciﬁc sites in MRPs leading to a hydrogen peroxide generation. As an important antioxidant defence mechanism, free metal ions are restricted in vivo [Halliwell and Gutteridge, 1999]. However, since free metal ions are not involved in the mechanism of hydrogen peroxide generation by MRPs it can be concluded that MRPs with aminoreductone structure can exert their biological eﬀect under physiological conditions.

The identiﬁcation of aminoreductones as signal active structures in MRPs is also in agreement with the previous results of the present work. Melanoidins have been shown to induce

NF-κB translocation in macrophages (Chapter 2.1.2.3) and similar to aminoreductones, melanoidins have been shown to possess reducing properties [Borrelli et al., 2002; Linden- meier et al., 2002; Wagner et al., 2002]. Moreover an aminoreductone could be identiﬁed in melanoidins, isolated from bread crust [Lindenmeier et al., 2002]. Thus, melanoidins containing aminoreductone structures could become pro-oxidative by generating hydrogen peroxide leading to the induction of NF-κB activation in macrophages. 60 CHAPTER 2. RESULTS AND DISCUSSION

A MRP with reductone structure was tested in the bioassay under the same experimental setup as applied for the aminoreductone, but the reductone did not induce NF-κB activation. The exchange of nitrogen by a oxygen in the reductone reduces the electron density and thus the reducing potential of the reductone structure. This may explain why the reductone did not activate NF-κB by the generation of ROS. It was further investigated if the early MRPs, Amadori product and 3-deoxy-glucosone induce NF-κB activation. Both structures did not induce NF-κB activation in the macrophages. The result is in accordance with the observed NF-κB activation by melanoidins (Chapter 2.1.2.3) since the MRPs, Amadori product and 3-deoxy-glucosone, account for the low molecular weight fraction of the Mail- lard reaction mixture.

AGEs do not Induce NF-κB Activation but RAGE-Dependent Generation of NO

AGEs are formed through reaction steps analogous to to MRP formation. Thus, Maillard- derived aminoreductones could be present in AGEs. It is known that AGEs induce NF-κB activation through interaction with RAGE [Lander et al., 1997; Yan et al., 1994; Yeh et al.,

2001]. The AGE-induced NF-κB activation was investigated to elucidate a possible role of exogenous hydrogen peroxide generation by AGEs, contributing to their biological activity.

Surprisingly, AGEs, which have been prepared by heating BSA with glucose or ribose at

37 °C did not induce NF-κB activation in RAGE-expressing cells. In contrast, the positive control stimulants LPS and TNF-α induced NF-κB activation. The biological activity of

AGEs has been described using AGEs derived from the incubation of BSA with glucose

[Lander et al., 1997; Yan et al., 1994; Mamputu and Renier, 2004; Dukic-Stefanovic et al.,

2003; Neumann et al., 1999]. High-aﬃnity RAGE binding AGEs have been described using ribose for protein glycation [Valencia et al., 2004b]. However, both AGE reaction mixtures failed to induce NF-κB activation in the present work. In the experiments, several AGE concentrations and incubation times for cell stimulation were used. CHAPTER 2. RESULTS AND DISCUSSION 61

CML is the only deﬁned AGE, known to induce NF-κB activation [Kislinger et al., 1999].

Thus, it was further investigated whether CML could lead to NF-κB activation. Surpris- ingly, neither protein-bound CML nor CML led to NF-κB translocation in the macrophages.

Furthermore, it was considered that the failure of AGEs to induce NF-κB activation via RAGE could be cell line-dependent. Similar to the experiments using macrophages,

AGEs did not show biological activity in hepatic stellate cells or RAGE-transfected HEK cells. Both cell lines have been shown to express RAGE (Chapter 2.1.2.4) [Fehrenbach et al., 2001]. The result indicates that the AGEs prepared at 37 °C neither induce NF-κB activation through interaction with RAGE nor through the generation of hydrogen peroxide.

It has been described before that AGEs do not induce cell signalling pathways [Valencia et al., 2004a], but increase endotoxin-mediated cellular reaction [Reznikov et al., 2004].

Reznikov et al. showed for example that AGEs augment an inflammatory cellular reaction in the presence of LPS [Reznikov et al., 2004]. Thus, it was investigated whether the failure of AGEs to induce NF-κB activation is due to the strict absence of LPS in the AGE reaction mixtures. However, incubation of the macrophages with AGEs in the presence of LPS did not lead to an augment of LPS-induced NF-κB activation. Moreover, LPS was less effective causing NF-κB translocation in the presence of AGEs. Similar to Valencia et al. the results of the present work show that AGEs do not lead to an inflammatory cellular reaction, e.g. the activation of NF-κB in macrophages [Valencia et al., 2004a].

The literature describes diﬀerent basic conditions for the preparation of AGEs, which vary, e.g. in the heating temperature and duration of heating [Lander et al., 1997; Yan et al., 1994;

Yeh et al., 2001; Dukic-Stefanovic et al., 2003]. This implies that the generation of signal active AGEs may vary as well. Thus, the biological activity of AGEs prepared by heating

BSA with glucose at 60 °C for between 1 and 6 weeks was investigated. The expression of

NF-κB regulated iNOS in response to AGEs was measured in form of released NO from macrophages. Among the AGEs prepared at 60 °C, the reaction mixture heated for 3 weeks caused a signiﬁcant release of NO. However, the generation of signal active AGEs by heating 62 CHAPTER 2. RESULTS AND DISCUSSION

BSA with glucose at 60 °C for 6 weeks was not reproducible, when a second batch of AGE was prepared under the same conditions. Thus, the biological activity of the AGEs did not correspond to specific conditions used for the AGE-preparation. The result indicates that the formation of biologically active AGEs depends on unknown parameters. Also, the finding underlines the lack of defined AGE structures, known to induce cell signalling pathways via RAGE.

The second aim of this part of the work was to investigate whether hydrogen peroxide generation by AGEs could play a role in AGE-induced cell signalling. Blocking RAGE with an anti-RAGE-antibody revealed that the observed AGE-induced NO production was almost fully depending on RAGE. Thus, the exogenous generation of hydrogen peroxide by

AGEs, if at all, plays only a minor part in AGE-induced cell activation. CHAPTER 2. RESULTS AND DISCUSSION 63

2.4 Cellular Reaction on MRP-Induced NF-κB Activation

2.4.1 Introduction

Food-derived MRPs have been shown to induce NF-κB activation in macrophages, which are key players in the immune response (Chapter 2.1). Besides their role as phagocytes, activated macrophages have immunomodulatory functions [Fujiwara and Kobayashi, 2005].

Thereby, NF-κB activation promotes the transcription of immune- and inﬂammatory-related proteins such as cytokines or enzymes like iNOS [Lee and Burckart, 1998]. In addition to immune related reactions, NF-κB activation can as well be involved in apoptotic or anti-apoptotic processes depending on the NF-κB-inducer as a cellular stress response

[Kaltschmidt et al., 2000; Pahl, 1999].

The aim of this part of the work was to investigate the cellular reaction on MRP-induced

NF-κB activation in macrophages. The release of inﬂammatory mediators especially cytokines and iNOS-derived NO was measured upon stimulation with MRPs. Also the inﬂu- ence of MRPs on cell survival was investigated and the involvement of apoptotic processes in MRP-induced cytotoxicity was elucidated.

2.4.1.1 Cytokines

Cytokines are able to promote cell growth, differentiation and functional activation of different cell types. Activated macrophages produce a range of pro-inflammatory cytokines including IL-1, IL-6, interleukin-8 (IL-8) , interleukin-12 (IL-12) and TNF-α. In a later event of the immune response, anti-inflammatory interleukin-10 (IL-10) is produced, deac- tivating the inflammatory process [de Waal Malefyt et al., 1991]. Exemplarily, IL-1 and

IL-6 increase the adaptive immune response. IL-1 activates T- and B-lymphocytes and

IL-6 promotes the proliferation and differentiation of B-lymphocytes. TNF-α induces the expression of adhesion molecules on the surface of endothelial cells resulting in an enhanced binding of immune cells to the endothelium. Consequently, a large number of immune cells migrate at infection sites into the tissue and become activated. Excessive inflammatory 64 CHAPTER 2. RESULTS AND DISCUSSION reactions are limited by the production of IL-10, which inhibits inflammatory mediators such as cytokines, chemokines and NO, avoiding damage to the host defence itself [Grutz,

2005].

2.4.1.2 NO

Diﬀerent cell types express nitric oxide synthase (NOS) leading to the production of NO, which acts as a neurotransmitter or vascular relaxing agent. Besides these physiological roles, high amounts of NO are generated as an inﬂammatory response through the expression of iNOS, e.g. in activated macrophages. In this context, NO is involved in the innate immune response, acting as a toxic agent towards invading organisms [Coleman, 2001].

Excessive NO production can also lead to host tissue damage [Janeway and Travers, 1994;

Coleman, 2001]. Additionally, NO is suggested to be an immune regulatory mediator, e.g. by inhibiting T-cell proliferation [Guzik et al., 2003]. However, the exact role of NO in the regulation of immune cell activity is not yet known.

2.4.2 Results

2.4.2.1 MRP-Induced NF-κB Activation does not Promote the Production of

Inﬂammatory Cytokines

In the present work, MRPs have been shown to induce NF-κB activation. The release of inflammatory cytokines from macrophages in response to MRP-induced NF-κB activation was screened using the Bio-Plex Cytokine assay (Bio-Rad). This system enables the simultaneous analysis of different cytokines such as IL-1α, IL-1β, IL-6, IL-10 and TNF-α in a single sample. The Maillard reaction mixture was added in a final concentration of 25 mM

(d-ribose and l-lysine concentration prior to heating) in medium to the cells and incubated for 4 h. Compared to cells maintained in medium alone, the Maillard reaction mixture induced a slight production of IL-1α, IL-1β and IL-6 (Fig. 2.30), but the eﬀect was only partly due to MRPs. Stimulation of the macrophages with the same concentration of the CHAPTER 2. RESULTS AND DISCUSSION 65

Figure 2.30: MRP-induced production of IL-1α, IL-1β, IL-6, IL-10 and TNF-α in NR8383 macrophages (single experiment). Cells were maintained in medium alone or stimulated with 25 mM of the unheated reaction mixture

(rib-lys 0 h), 25 mM of the Maillard reaction mixture consisting of ribose and lysine heated at 120 °C (rib-lys 30 min) or with 100 ng/mL LPS for 4 h. Supernatants were analyzed for cytokines using Bio-Plex Cytokine assay.

unheated reaction mixture also caused the expression of IL-1α, IL-1β and IL-6. Compared to the macrophages, treated with the unheated reaction mixture, a slightly higher amount of these inflammatory cytokines were released in response to MRPs (1.5-fold for IL-1α, 10- fold for IL-1β and 1.7-fold for IL-6). The production of pro-inflammatory TNF-α was not induced. In contrast to LPS, the inflammatory effect of MRPs is weak. Stimulation of the macrophages with 100 ng/mL LPS for 4 h caused high expression of the pro-inflammatory cytokines IL-1α, IL-1β, IL-6 and TNF-α and the presence of anti-inflammatory IL-10 indicates that the macrophages already started to suppress the inflammatory response. Un- treated cells or cells stimulated with the Maillard reaction mixture and especially with the unheated reaction mixture also led to a slight production of IL-10. The screening indicated that MRPs induce a slight release of the inflammatory cytokines IL-1α, IL-1β and particularly IL-6.

IL-6 production in response to MRPs was further tested using enzyme-linked immunosor- 66 CHAPTER 2. RESULTS AND DISCUSSION

Figure 2.31: IL-6 expression in NR8383 macrophages determined with ELISA (mean and standard deviation of at least four independent experiments). Cells were stimulated in medium for 17 h with 25 mM or 50 mM of the unheated reaction mixture (rib-lys 0 h), 10 mM, 25 mM or 50 mM of the Maillard reaction mixture consisting of ribose and lysine heated at 120 °C (rib-lys 30 min), 500 µM or 750 µMH2O2, 10 mM C4-aminoreductone (AR), 2 mg/mL of coffee extract or raw coffee extract and with LPS as positive control (n = 2); ** p<0.01, * p<0.05. bent assay (ELISA) technique. Macrophages were incubated in the presence of different concentrations of the Maillard reaction mixture in medium for 17 h and IL-6 was measured in the cell culture supernatant. Compared to cells maintained in medium alone or treated with the same concentrations of the unheated reaction mixture, MRPs did not increase IL-6 expression (Fig. 2.31).

Likewise, stimulation of the macrophages with hydrogen peroxide, which has been identi-

ﬁed as signal mediator for MRP-induced NF-κB activation (Chapter 2.1.2.5) did not lead to

IL-6 production. Furthermore, macrophages were stimulated with the C4-aminoreductone, which has been identiﬁed as a signal active MRP-structure, leading to the activation of NF-

κB (Chapter 2.3.2.1). No increase in IL-6 production was measured in response to 10 mM

C4-aminoreductone. Coﬀee has been shown to activate NF-κB translocation in contrast to raw coﬀee extract, lacking MRPs (Chapter 2.1.2.1). Incubation of the macrophages with

2 mg/mL coffee extract for 17 h caused a moderate significant 1.4-fold increase in IL-6 production (109 pg/mL) compared to cells maintained in medium alone. The effect can CHAPTER 2. RESULTS AND DISCUSSION 67 not be related to coffee-derived MRPs. Compared to cells treated with coffee extract, raw coffee extract induced a 30-fold increased expression of IL-6 (3325 pg/mL). In summary, the coffee- and MRP-induced activation of NF-κB by a RAGE-independent and hydrogen peroxide-dependent mechanism does not redirect the expression of inflammatory cytokines.

2.4.2.2 MRP-Induced NF-κB Activation does not Cause the Generation of NO

It was further investigated whether MRPs lead to an enhanced inﬂammatory response, which is mediated besides others (e.g. cytokines) by the release of reactive nitrogen species, like NO [Coleman, 2001]. The macrophages were treated with the Maillard reaction mixture for 3 h and further incubated for 48 h before NO concentrations were measured in the cell culture supernatants. However, stimulation of the cells with 5 mM, 10 mM or 25 mM of the Maillard reaction mixture did not induce a signiﬁcant generation of NO in contrast to

INF-γ, which was used as a positive control stimulant (Fig. 2.32).

Figure 2.32: NO expression in MRP- and INF-γ-treated NR8383 macrophages (mean and standard deviation of three independent experiments). Cells were stimulated for 3 h in medium with 5 mM, 10 mM or 25 mM of the Maillard reaction mixture consisting of ribose and lysine heated at 120 °C (rib-lys 30 min) or 10, 50, 100, 250 or 500 U/mL INF-γ. Cells were washed twice and further incubated in media for 48 h; ** p<0.01, * p<0.05. 68 CHAPTER 2. RESULTS AND DISCUSSION

Figure 2.33: MRPs and INF-γ-induced NO (µM) release in NR8383 macrophages (mean and standard deviation of

ﬁve independent experiments). The cells were stimulated for 3 h in medium with 10 mM or 25 mM of the Maillard reaction mixture consisting of ribose and lysine heated at 120 °C (rib-lys 30 min). Afterwards the cells were washed and stimulated with indicated concentrations of INF-γ for 48 h in medium.

It has been shown before that AGEs did not activate cytokine-expression in mononuclear cells by itself, but augmented LPS-induced inﬂammatory response [Reznikov et al., 2004].

Synergistic enhancement of iNOS transcription has also been described for INF-γ and NF-

κB activating stimuli like LPS [Saura et al., 1999]. Therefore, it was also tested if MRPs show a similar enhancing eﬀect to INF-γ-induced NO expression. However, neither 10 mM nor 25 mM Maillard mixture added to 10 - 500 U/mL INF-γ caused a signiﬁcant increase in NO release (Fig. 2.33).

The results show that the MRP-induced NF-κB activation is not suﬃcient to promote the expression of the NF-κB regulated genes, iNOS and IL-6 on its own.

2.4.2.3 MRP-Induced Hydrogen Peroxide Generation Causes Cell Death

The transcription factor NF-κB is a central switch in redox-sensitive signal transduction pathways of cells and is involved in multiple cellular reactions. In addition to immune related reactions, NF-κB can also be involved in apoptotic or anti-apoptotic processes [Kaltschmidt CHAPTER 2. RESULTS AND DISCUSSION 69

Figure 2.34: MRP-induced cytotoxicity in NR8383 macrophages determined with the MTT assay (mean and standard deviation of at least two independent experiments). The cells were stimulated in PBS for 2.5 h with the Maillard reaction mixture consisting of ribose and lysine heated at 120 °C (rib-lys 30 min) in the presence or absence of catalase (150 U/mL) and with the unheated reaction mixture (rib-lys 0 h). et al., 2000; Pahl, 1999]. Thus, the inﬂuence of the MRPs on cell survival was investigated.

Stimulation of macrophages in PBS with MRPs for 2.5 h led to a concentration-dependent cell death as determined with the MTT assay (Fig. 2.34). The MRP-induced cytotoxicity was fully blocked by the addition of catalase indicating hydrogen peroxide as the toxic agent. Furthermore, the results show that the addition of catalase to the cells, stimulated with MRPs, even led to a slightly increased cell proliferation compared to cells maintained in PBS alone. Stimulation of the cells with 17.5 mM of the unheated reaction mixture or more also caused cell death. This eﬀect can be explained by the formation of hydrogen peroxide during the incubation of ribose and lysine at 37 °C for 2.5 h (Chapter 2.2.2.2), which then leads to cell demise. The results indicate that MRPs have a cytotoxic eﬀect on macrophages, which is mediated by hydrogen peroxide.

2.4.2.4 MRPs do not Induce Apoptosis

Hydrogen peroxide has diﬀerent physiological roles. It acts, e.g. as a second messenger in cell signalling and macrophages can release hydrogen peroxide as a cytotoxic agent in 70 CHAPTER 2. RESULTS AND DISCUSSION the host defence [Halliwell et al., 2000]. The present work revealed that the MRP-induced generation of hydrogen peroxide leads to the translocation of NF-κB into the nucleus and causes cell death. The latter can occur through the induction of signal cascades leading to programmed cell death (apoptosis) or less orderly through external factors causing accidental cell death (necrosis). Thereby, NF-κB could be involved in the cellular stress response promoting pro- or anti-apoptotic processes rather than inducing the expression of immune- and inﬂammatory-related genes. Thus, it was investigated whether MRP-induced cell demise involves apoptosis, which would then lead to the conclusion that the activation of NF-κB promotes pro-apoptotic signals. The activated fragment of the protease caspase-3

- cleaved caspase-3 (17 - 19 kDa) - resulting from cleavage of caspase-3 (35 kDa) plays a central role in the processing of apoptosis and can be detected in protein lysates of cells undergoing apoptosis by means of a Western Blot [Yin and Dong, 2003]. The macrophages were maintained in PBS and stimulated with 5 mM Maillard reaction mixture for 4 h or 6 h and the presence of cleaved caspase-3 was determined in the protein lysates. Stimulation with 5 mM Maillard reaction mixture has been shown before to cause 66 % cell death, compared to cells maintained in PBS alone (Chapter 2.4.2.3). In contrast to the positive control stimulant staurosporine (Fig. 2.35), activation of caspase-3 in response to 5 mM

Maillard reaction mixture was not detected (Western Blot not shown).

Likewise, stimulation of the cells with 1 mM Maillard reaction mixture did not lead to the cleavage of caspase-3 (Fig. 2.35). Furthermore, cells treated with hydrogen peroxide concentrations ranging from 25 µMH2O2 (Fig. 2.35) to 100 µMH2O2 (Western Blots not shown) did not undergo apoptosis.

The ﬁndings indicate that the generation of hydrogen peroxide by MRPs does not induce apoptosis, but necrotic cell death. Thus, it can be speculated that NF-κB activation promotes the expression of anti-apoptotic proteins and not pro-apoptotic signals. CHAPTER 2. RESULTS AND DISCUSSION 71

Figure 2.35: Cleaved caspase-3 Western Blot. NR8383 macrophages were maintained in (1) PBS and stimulated with

(2) 1 µM staurosporine, (3) 25 µMH2O2 and (4) 1 mM Maillard reaction mixture consisting of ribose and lysine heated for 30 min at 120 °C, for between 4 h and 6 h. Caspase-3 and cleaved caspase-3 were immunochemically detected in the protein lysates using monoclonal anti-caspase-3 antibody.

2.4.3 Discussion

The activation of NF-κB by food-derived MRPs does not cause an inﬂammatory cellular reaction. The expression pattern of cytokines involved in the immune reaction of macrophages was investigated in response to MRPs. A screening assay revealed that, compared to the

LPS-induced release of cytokines, the Maillard reaction mixture led only to a slight production of IL-1α, IL-1β and IL-6. A similar eﬀect was observed when the cells were treated with the unheated reaction mixture. This could stem from the Maillard reaction-derived production of hydrogen peroxide in the unheated reaction mixture during the incubation at 37 °C as demonstrated in chapter 2.2.2.2. However, further investigations using an IL-6

ELISA revealed that neither hydrogen peroxide nor MRPs or the unheated reaction mixture induced a significant release of the inflammatory cytokine. Likewise, stimulation of the cells with the aminoreductone, which has been identified as a signal active MRP-structure

(Chapter 2.3.2.1) did not lead to an inflammatory cellular reaction. Among the tested substances only coffee induced a moderate significant 1.4-fold increase in IL-6 expression compared to the medium treated control. A positive relation between coffee and inflamma- 72 CHAPTER 2. RESULTS AND DISCUSSION tory reactions has been described before. Compared to coffee non-drinkers an increase in inflammatory markers such as IL-6 and TNF-α has been detected in serum of healthy volunteers after coffee consumption [Zampelas et al., 2004]. However, the coffee-induced IL-6 expression (109 pg/mL) in the present work is not due to MRPs since raw coffee extract, lacking MRPs, induced the release of high amounts of IL-6 (3325 pg/mL). The effect of raw coffee extract modulating immune reactions has not yet been described in the literature.

In summary, coffee can be suggested to induce inflammatory reactions through ingredients different from food-derived MRPs.

Hydrogen peroxide has been identified as a signal mediator in the MRP-induced activation of cells. However, the hydrogen peroxide-induced NF-κB activation does not culminate in an inflammatory reaction in the macrophages. A ROS-stimulated release of inflammatory cytokines has been shown in mast cells and differentiated muscle cells [Frossi et al., 2003;

Kosmidou et al., 2002], but not in endothelial cells or ﬁbroblasts [Kosmidou et al., 2002;

Yoshida et al., 1999] and therefore seems to be cell-speciﬁc. Thus, macrophages itself may not release cytokines in response to MRPs, but other cell types could react with an inﬂammatory response to the induction of oxidative stress by food-derived MRPs.

Similar to IL-6, NO production was not signiﬁcantly increased after activation of NF-κB by MRPs alone or applied as co-stimulants in the presence of INF-γ. This means that

MRPs do not increase inﬂammatory reactions in macrophages.

On the other hand, MRPs showed a concentration-dependent cytotoxicity. 68 % of the macrophages maintained in PBS died in the presence of 25 mM Maillard reaction mixture after 2.5 h. Similar to MRP-induced NF-κB activation, cytotoxicity was mediated by hydrogen peroxide, because catalase completely inhibited cell demise. The addition of catalase to cells stimulated with the Maillard products even led to a slightly increased cell proliferation compared to cells maintained in PBS alone. This eﬀect could originate from unreacted sugar, which is still present in the Maillard model mixture. It is well known that hydrogen peroxide leads to cell death in diﬀerent cell types involving both, apoptosis and CHAPTER 2. RESULTS AND DISCUSSION 73 necrosis [Hampton and Orrenius, 1997; Palomba et al., 1999]. It can be concluded that

MRPs induce oxidative stress in macrophages through the generation of hydrogen peroxide culminating in cell death. Thereby, the translocation of NF-κB could be involved in the immediate stress response rather than promoting immune and inﬂammatory processes.

In the presence of oxidative stress, NF-κB could promote pro- or anti-apoptotic signals.

NF-κB has been shown to play a pro-apoptotic role in hydrogen peroxide-induced apoptosis [Kaltschmidt et al., 2000; Jones et al., 2000]. On the other hand, it is known that

NF-κB promotes also anti-apoptotic signals, e.g. in TNF-α-induced cell death as a cy- toprotective response [Natoli et al., 1998]. Apoptosis is triggered by the activation of a proteolytic enzyme family termed caspases [Yin and Dong, 2003]. The MRP-induced generation of hydrogen peroxide and hydrogen peroxide alone did not induce apoptosis in the macrophages as determined by the absence of cleaved active caspase-3 in the cell lysates.

The result suggests that the macrophages undergo necrosis in response to MRP-induced oxidative stress. Thereby, NF-κB activation may be involved in anti-apoptotic processes, e.g. inducing the expression of B-cell leukemia/lymphoma-2 (Bcl-2) [Tamatani et al., 1999].

HeLa-cells have been shown to undergo apoptosis in response to hydrogen peroxide, but in

Bcl-2-transfected cells continuously expressing the anti-apoptotic protein the type of cell demise switched from apoptosis to necrosis [Du et al., 2006]. The activation of NF-κB by

MRPs may be involved in the regulation of MRP-induced cell death in macrophages promoting the expression of anti-apoptotic proteins, which would suppress apoptosis. This effect increase cell survival in a stress associated environment. However, as the oxidative stress increases, the excess of ROS leads to necrotic cell death rather than apoptosis. On a long term, food rich in melanoidins may contribute to the intestinal immune response through the induction of necrotic cell death. Apoptosis leads to the safe disposal of the cytosol and cellular fragments by phagocytes. In contrast, necrotic cell death causes loss of membrane integrity redirecting the spill of cytosolic constituents into the extracellular space. This leads to inflammatory reactions in the surrounding tissue by efflux of intracellular contents with immunomodulatory functions [Proskuryakov et al., 2003]. 74 CHAPTER 2. RESULTS AND DISCUSSION

2.4.4 Summary

The aim of the present work was to study the inﬂuence of food-derived MRPs on the cellular response of macrophages.

Coﬀee and Maillard reaction mixture have been shown to activate NF-κB. The activation of NF-κB by food-derived MRPs does not depend on RAGE. Instead, MRPs have been shown to generate extracellular hydrogen peroxide under physiological conditions, which acts as second messenger in MRP-induced NF-κB activation. As signal active MRP- structure melanoidins, particularly containing aminoreductone structures, could be identi-

ﬁed. As cellular reaction to the MRP-induced hydrogen peroxide generation, macrophages were shown to undergo necrotic cell death.

In summary, food-derived MRPs have been shown to induce a cellular reaction in macrophages in vitro. The activation of NF-κB by food-derived MRPs in vivo may modulate the intestinal immune response. NF-κB plays a crucial role in the transcription of more than 150 immune- and inflammation-related genes. The effect of food-derived MRPs may be particularly enhanced in patients with inflammatory bowel disease, where NF-κB activation in macrophages has an important role in pathogenesis. Also, the induction of necrotic cell death in macrophages by MRP-derived hydrogen peroxide, could lead or enhance an inflammatory response in the surrounded tissue. In contrast to apoptosis, necrotic cell death causes loss of membrane integrity redirecting the spill of cytosolic constituents with immunomodulatory functions into the extracellular space. The in vitro results of the present work indicate that MRPs may induce a cellular reaction in vivo, modulating the intestinal immune response. CHAPTER 2. RESULTS AND DISCUSSION 75

2.5 Screening of the Inﬂuence of Amino Acid Precursor on the

Cytotoxicity of Melanoidins by a Peptide Spot Library

2.5.1 Introduction

In the present work, melanoidins have been shown to activate NF-κB as an intermediate stress response and cause cytotoxic eﬀects. As model for melanoidins, a Maillard reaction mixture consisting of lysine-derived MRPs was used. Food-derived physiological active

MRPs/melanoidins could stem from the reaction between sugar and reactive side chains or α-amino groups of the 20 proteinogenic amino acids. The aim of this part of the work was to investigate, which of the proteinogenic amino acids forms cytotoxic melanoidins.

The reactivity of the 20 proteinogenic amino acid side chains in the Maillard reaction was simultaneously investigated using a membrane-bound dipeptide library (dipeptide spot library). This method enables the screening of amino acids reactivities toward melanoidin formation under the same experimental setup in a time-saving manner. The browning of the

Maillard-modified dipeptides was determined by densitometric analysis and used as a marker for melanoidin formation. The influence of the membrane-bound melanoidins derived from the different dipeptides and amino acids on cell survival was further investigated.

2.5.2 Results

2.5.2.1 Comparison of Amino Acid Reactivity Toward Melanoidin Formation Using a

Dipeptide Spot Library

Peptides can be synthesized punctual on a planar cellulose membrane with the SPOT- method. This type of solid phase peptide synthesis allows the parallel synthesis of peptides on defined membrane areas (spots). A dipeptide library containing all 400 combinations of the 20 proteinogenic amino acids was synthesized on a cellulose membrane (dipeptide spot library) (InterdisziplinäresZentrum fürklinische Forschung, UniversitätLeipzig). The configuration of the dipeptides is illustrated in detail in figure 2.36. 76 CHAPTER 2. RESULTS AND DISCUSSION

Figure 2.36: (A) Conﬁguration of dipeptide spot library. The single-letter amino acid code is used with alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine

(H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V). (B) Section of dipeptides in row y9 and column x21, x22 and x23. The left letters indicates the C-terminal amino acid. The right letter the N-terminal amino acid. (C) Membrane-bound dipeptide with acetylated N-terminal amino group and side chains exposed to glycation. CHAPTER 2. RESULTS AND DISCUSSION 77

Figure 2.37: Maillard-modiﬁed dipeptide spot library. Membrane-bound dipeptides and methylglyoxal were heated for two weeks at 60 °C. The colour intensity of the spots, quantiﬁed using Image MasterTM 2D Platinum software are indicated on the spots. Colour intensity values range from 7 (weakest intensity) to 30 (strongest intensity).

To compare the reactivity of the amino acid side chains toward melanoidin formation, a dipeptide spot library with acetylated N-terminal amino groups and exposed side chains was used (Fig. 2.36 C). The reactive amino acid side chains were Maillard-modiﬁed using methylglyoxal as carbonyl compound. The dipeptide spot library was heated with methylglyoxal for two weeks at 60 °C and the intensity of brown coloured melanoidin-formation was determined by densitometric analysis (Fig. 2.37).

The colour intensity pattern of Maillard-modiﬁed dipeptides consisting of the same amino acid, e.g. alanine-alanine (AA) revealed that the nucleophilic groups of the amino acid side 78 CHAPTER 2. RESULTS AND DISCUSSION

Table 2.3: Colour intensity of Maillard-modiﬁed dipeptide spots consisting of arginine (R), aspartic acid (D), cysteine

(C), lysine (K) and tryptophan (W). The colour intensities of the spots were determined by densitometric analysis.

Values range from 7 (weakest intensity) to 30 (strongest intensity).

N-terminal amino acid

R D C K W

R 19 20 13 22 19

D 12 10 10 22 15

C 11 12 7 22 15

K 26 22 22 25 30

C-terminal amino acid W 21 17 20 29 26 chains of lysine (K), tryptophan (W) and arginine (R) led to melanoidin formation with the ranking of reactivity lysine ≈ tryptophan > arginine (Fig. 2.37, Table 2.3).

In comparison to these side chains, the thiol group of cysteine (C) was less reactive.

Interestingly, aspartic acid (D) also formed brown-coloured products under the conditions applied. The side chains of the other proteinogenic amino acids did not generate melanoidins by heating with methylglyoxal (Fig. 2.37). In combination with the Maillard-reactive amino acids lysine, tryptophan, arginine, and aspartic acid, all other proteinogenic amino acids led to the formation of melanoidins, except arginine in combination with methionine or serine, and aspartic acid in combination with glutamic acid. Cysteine caused weak MRP-formation only in combination with histidine, isoleucine and tyrosine and, as mentioned above, with the reactive amino acids. Comparison of the colour intensities of the 400 dipeptides revealed that the dipeptides containing lysine were most reactive toward melanoidin formation. The reactivity ranking order was lysine > tryptophan (except in combination with valine tryptophan was more reactive than lysine) >> arginine ≈ aspartic acid. The highest reactivity toward melanoidin formation was observed with the two dipeptides consisting of lysine and tryptophan (Fig. 2.37, Table 2.3).

In summary, the comparison of amino acid side chain reactivity toward melanoidin formation using a dipeptide spot library with blocked N-terminal amino groups revealed that among the 20 proteinogenic amino acids lysine, tryptophan and, to a lesser degree, arginine and aspartic acid are most reactive. CHAPTER 2. RESULTS AND DISCUSSION 79

2.5.2.2 Inﬂuence of Membrane-Bound Melanoidins on Cell Survival

The second goal was to investigate the inﬂuence of the membrane-bound melanoidins on cell survival. Common cell assays for studying biological activities of substances use soluble products to stimulate cells, which are cultured on plastic material. Instead of using soluble dipeptides for the generation of model melanoidins, a membrane-based cell viability assay was established to investigate the inﬂuence of membrane-bound melanoidins on cell survival.

The method enables the screening of the inﬂuence of the amino acid pecrursor on the cytotoxicity of melanoidins, using the dipeptide spot library.

The principle of the assay is illustrated in figure 2.38. The Maillard-modified dipeptide spots were cut out from the membrane as peptide discs and embedded in individual wells of a 96-well plate. The cells were directly cultured on the peptide discs and cell survival could be monitored online by a fluorescence-based cell viability assay.

Figure 2.38: Schematic illustration of the membrane-based cell viability assay, which was used to investigate the inﬂuence of membrane-bound melanoidins on cell survival.

First, the applicability of the cellulose membrane for culturing adherent macrophages

(RAW 264.7) was tested. The macrophages were seeded on a membrane disc (not containing peptides) in an individual well of a 96-well plate and cell growth was monitored with the

Alamar blue cell viability assay over a 12 day period. Alamar blue is a non-toxic dye and the cells can be further grown after determining cell viability by reexchanging Alamar blue 80 CHAPTER 2. RESULTS AND DISCUSSION solution with cell culture medium. The bioassay is based on the ability of viable cells to reduce non-fluorescent resazurin into a fluorescent product (resorufin) resulting in a linear relationship between fluorescence and cell number.

The ﬂuorescent signal of the macrophages cultured on the cellulose membrane disc increased dependent on the culture time indicating proliferation (Fig. 2.39 A).

Furthermore, the cells were stained on the membrane discs with MTT. Viable cells reduce the yellow tetrazolium salt MTT to purple formazan crystals, which leads to blue stained cells on yellow-coloured membrane discs. During the culture period of 12 days an increase of the cell density on the membrane discs was detected, which further demonstrates macrophage proliferation (Fig. 2.39 B, C).

Lysine and arginine have been determined with the dipeptide spot library as reactive amino acids toward melanoidin formation. To establish the membrane-based cell viability assay, the inﬂuence of melanoidins on cell survival was investigated using membrane-bound

MRP-modiﬁed dipeptides, exemplarily consisting of lysine and/or arginine. The dipeptide spots lysine-arginine (KR), arginine-lysine (RK), lysine-lysine (KK) and arginine-arginine

(RR) or spots containing the single amino acids lysine (K) or arginine (R) were heated with methylglyoxal, glyceraldehyde or ribose at 60 °C for two weeks. Both the N-terminal amino groups and functional side chains of the amino acids or dipeptides were subjected to glycation. The amino acids and dipeptides were also incubated with PBS under the same conditions as a control. The dipeptide spot consisting of lysine-lysine was highly reactive toward melanoidin formation with all three carbonyl compounds used (Fig. 2.40). Among them, methylglyoxal caused the strongest melanoidin formation. The spots containing single amino acids did not generate coloured MRPs. Membranes without dipeptides or amino acids

(blank) were also incubated with methylglyoxal, glyceraldehyde, ribose or PBS respectively as controls.

The dipeptide spots, amino acid spots and cellulose membranes without spots (blank), which have been heated with methylglyoxal, glyceraldehyde, ribose or PBS respectively, CHAPTER 2. RESULTS AND DISCUSSION 81

Figure 2.39: (A) Cell growth on cellulose membrane disc (not containing peptides) of two independent experiments with indicated standard deviation. The RAW 264.7 macrophages were seeded in a density of 1x104 cells/membrane disc and cultured for 12 days. Cell growth is expressed as amount of fluorescent product resorufin generated by incubation of the cells with 100 µL non-fluorescent resazurin for 1.5 h. (B) MTT-stained cells on the cellulose membrane discs. (C) Microscope image of MTT-stained cells cultured on a membrane disc. 82 CHAPTER 2. RESULTS AND DISCUSSION

Figure 2.40: Membrane-bound dipeptides consisting of lysine (K) and/or arginine (R) and the respective membrane- bound single amino acids were heated with 0.5 M methylglyoxal, 0.5 M glyceraldehyde, 0.5 M ribose or PBS at 60 °C for two weeks. Membranes without peptides (blank) were incubated under the same conditions.

were cut out from the membranes as peptide or membrane discs. The macrophages were seeded on the dipeptide-, amino acid-, and membrane discs and allowed to attach for 2 h.

Afterwards the discs were transferred into new wells and the number of attached cells was determined with the Alamar blue assay. After a 24 h and 48 h culture period, the number of viable cells on the discs was again determined by ﬂuorescence intensity measurement.

An increase of the fluorescent signal after 24 h and 48 h compared to the signal after 2 h was recorded on all discs indicating cell growth (data not shown). However, compared to the PBS-treated control discs, a significantly lower cell proliferation on methylglyoxal- incubated dipeptide and amino acid discs was detected after 24 h (Fig. 2.41 A). The effect was enhanced after a 48 h culture period. Lysine-lysine-derived melanoidins reduced 64 % of cell number on the membrane discs compared to the PBS-treated lysine-lysine dipeptide after 48 h. Compared to the other dipeptides and amino acids, the lysine-lysine-derived

MRPs had the strongest inﬂuence on cell growth. CHAPTER 2. RESULTS AND DISCUSSION 83

As shown in figure 2.41 B, the glyceraldehyde-modified dipeptides, but not amino acids inhibited cell proliferation. In correspondence to the results of the methylglyoxal-modified dipeptides, the lysine-lysine-derived melanoidins had the strongest effect on cell proliferation among the glyceraldehyde-incubated dipeptides. After a 48 h culture period, 51 % less cells grew on the lysine-lysine-derived melanoidins compared to the PBS-treated dipeptide.

Among the MRPs formed during the incubation of dipeptides or amino acids with ribose, only the lysine-lysine-derived melanoidins signiﬁcantly inhibited cell growth (Fig. 2.41 C).

Compared to cells grown on the PBS-incubated lysine-lysine dipeptide spot for 48 h, only

71 % were detected on the ribose-incubated dipeptide spot.

Furthermore, the cells were grown on membrane discs not containing dipeptides or amino acids (blank), but incubated with all three carbonyl compounds, respectively. Proliferation of cells on the membrane discs incubated with methylglyoxal, glyceraldehyde or ribose was similar to that on PBS-incubated membrane discs (Fig. 2.41 A, B, C). The results conﬁrm that the observed inhibition of cell proliferation by sugar carbonyl-incubated dipeptides and amino acids was due to the formation of MRPs.

Finally, the cells, which have been grown on the membrane or peptide discs for 48 h were stained with MTT. Exemplarily, figure 2.42 shows MTT-stained cells on the peptide spots consisting of lysine-lysine incubated with PBS or methylglyoxal or glyceraldehyde or ribose at 60 °C for two weeks. The membrane-bound lysine-lysine-derived melanoidins have been shown before to inhibit cell proliferation, which was determined with the Alamar blue assay. However, only on the methylglyoxal-derived melanoidin spot less cells can be visually detected compared to the PBS-incubated spot. The difference in the number of cells on the glyceraldehyde- or ribose-derived melanoidin spot, compared to the PBS-incubated spot, could not be observed visually. It was also investigated whether the influence of melanoidins

- bound to the dipeptide spot library (Fig. 2.37) - on cell proliferation could be visually detected by staining viable cells, which were grown for 48 h on the Maillard-modiﬁed dipeptide spot library. However, visual irregularities in the cell layer on the Maillard-modiﬁed 84 CHAPTER 2. RESULTS AND DISCUSSION

Figure 2.41: Melanoidin-induced inhibition of cell proliferation (mean and standard deviation of three independent experiments). The RAW 264.7 macrophages (5.5x104 cells/well) were seeded on methylglyoxal, glyceraldehyde, ribose or PBS-heated dipeptide- (KR, RK, KK, RR), amino acid- (K, R) and membrane (blank) -discs. The number of cells was monitored with the Alamar blue assay after 2 h, 24 h and 48 h. The intensity of the ﬂuorescent signal after 24 h or 48 h was related to the one after 2 h. Cell growth on the methylglyoxal (A), glyceraldehyde (B) and ribose (C)

-incubated discs is compared to the PBS-incubated controls, respectively; *** p<0.001, ** p<0.01,* p<0.05. CHAPTER 2. RESULTS AND DISCUSSION 85

Figure 2.42: Cell growth on lysine-lysine-derived melanoidins. The cells were stained with MTT on the lysine-lysine spots, incubated with PBS or methylglyoxal or glyceraldehyde or ribose at 60 °C for two weeks, after a culture period of 48 h. dipeptide spot library could not be detected (data not shown). The result indicates that the inﬂuence of membrane-bound melanoidins on cell proliferation can not be determined by staining viable cells on the spots with MTT, which could be due to the lack of sensitivity in the visual detection.

2.5.3 Discussion

The reactivity of the proteinogenic amino acids toward melanoidin formation was determined with a membrane-bound combinatorial dipeptide library (dipeptide spot library).

This method enables the screening of amino acids reactivities toward melanoidin formation under the same experimental setup in a time-saving manner. The dipeptides with free functional side chains and N-terminal blocked amino groups were Maillard-modiﬁed by heating with methylglyoxal at 60 °C for two weeks. This sugar degradation product can be found in food, e.g. coﬀee [Nagao et al., 1986] and is a reactive carbonyl compound taking part in the

Maillard reaction [Thornalley, 2005]. The formation of the brown-coloured melanoidins was quantiﬁed by densitometric analysis. Among the proteinogenic amino acids, the side chains of lysine, tryptophan, arginine and cysteine are reactive toward melanoidin formation. Sur- prisingly, aspartic acid also led to the formation of brown-coloured products by heating with methylglyoxal. Dipeptides without functional side chains, which can be regarded as 86 CHAPTER 2. RESULTS AND DISCUSSION negative controls, did not cause melanoidin formation. The ranking order of reactivity was lysine ≈ tryptophan > arginine ≈ aspartic acid > cysteine. The results indicate that the amino group of lysine, the indole amine of tryptophan and the guanidine group of arginine led to melanoidin formation. In comparison to that the thiol group of cysteine was less reactive.

It is well known that methylglyoxal reacts with the functional side chain of lysine forming, e.g. the lysine-derived MRPs N-carboxyethyllysine (CEL) [Ahmed et al., 1997]. Also, the guanidine group of arginine is known to participate in the Maillard reaction. For example, in the reaction of arginine residues with methylglyoxal imidazolone is formed [Henle et al.,

1994]. Furthermore, the thiol-group of cysteine has been shown to react with methylglyoxal.

In the Maillard reaction of Nα-acetylcysteine and methylglyoxal the hemithioacetal adduct could be detected [Lo et al., 1994]. The results of the present work further revealed that the functional side chain of tryptophan leads to the formation of melanoidins. However, the reactivity of the indole-NH-group of Nα-protected tryptophan in the Maillard reaction has not yet been described.

In another study, a similar dipeptide spot library was used to characterize anti-AGE antibody affinities [Dukic-Stefanovic et al., 2002]. For that purpose, the dipeptide spot library was incubated with glucose at 50 °C for two weeks and dipeptide-derived AGE-epitopes were detected with different anti-AGE antibodies. Similar to the results of the present, work the functional side chains of lysine, tryptophan and arginine, but not cysteine, were shown to participate in the Maillard reaction. Interestingly, the functional side chain of asparagine was identified to generate AGE-epitopes. In the present work, surprisingly, aspartic acid has been shown to form brown-coloured products. The reactivity of the proteinogenic amino acids with sugar was also investigated using a dipeptide spot library, which was glycated for four days with radiolabeled sugar [Munch et al., 1999]. The autoradiography of the dipeptide spot library showed that the functional side chains of lysine, tryptophan, arginine, cysteine and the imidazolyl group of histidine reacted with the sugar. CHAPTER 2. RESULTS AND DISCUSSION 87

In summary, the dipeptide spot library is an excellent tool to investigate the reactivity of the proteinogenic amino acids toward Maillard-modiﬁcation. The method enables the simultaneous detection of melanoidin-formation, under the same experimental setup, in a time-saving manner. The functional side chains of lysine, tryptophan, and arginine are reactive toward glycation processes leading to the generation of melanoidins. The reactivity of cysteine toward glycation, as reported by Munch et al., did not result in a noteworthy melanoidin formation in the present work [Munch et al., 1999]. Additionally, previously glycation of histidine does not lead to the generation of melanoidins in this study.

To investigate the influence of the membrane-bound Maillard-modified peptides on cell survival, a membrane-based cell viability assay was established. The method allows to record cell proliferation on defined membrane areas using a fluorescence-based cell viability assay. The bioassay was established with a combinatorial dipeptide library, consisting of lysine-arginine, arginine-lysine, lysine-lysine and arginine-arginine dipeptides, which was

Maillard-modiﬁed using methylglyoxal. Furthermore, glyceraldehyde and ribose were used as carbonyl compounds.

The results showed that cell proliferation on Maillard-modified dipeptides, which were heated with methylglyoxal, was reduced compared to the non-modified dipeptides, respectively. Melanoidins derived from lysine-lysine dipeptides incubated with methylglyoxal had the strongest effect reducing 66 % of cell growth. Similar results were obtained with the glyceraldehyde-modified dipeptides. However, the melanoidin-induced inhibition of cell growth was less intense. Among the glyceraldehyde-modified dipeptides the melanoidins derived from lysine-lysine spots showed the strongest reduction of cell growth (51 %). Ex- cept of melanoidins originating from lysine-lysine dipeptides, the ribose-derived MRPs had no influence on cell proliferation. The ribose-glycated lysine-lysine dipeptide spot inhibited

29 % of cell growth compared to the PBS-treated control.

In addition to the dipeptides also the amino acids lysine and arginine were Maillard- modified using the different carbonyl compounds. Only the methylglyoxal-incubated amino 88 CHAPTER 2. RESULTS AND DISCUSSION acid spots showed an inhibitory effect on cell proliferation. However, the effect can not be attributed to melanoidins since no colour formation was detected on the amino acid spots. It is possible that besides melanoidins, early MRPs have also an influence on cell proliferation. The difference in melanoidin-formation by amino acids and dipeptides may be due to the different concentration of educts - amino acid : dipeptide (1:2) - taking part in the Maillard reaction.

The results show that independently of the carbonyl compound used for glycation, melanoidins derived from lysine-lysine dipeptides exert an inhibitory eﬀect on cell proliferation.

In the present work the Maillard reaction mixture containing soluble MRPs, which was prepared by heating lysine with ribose, was shown to cause cell death (Chapter 2.4.2.3).

However, the membrane-bound lysine-lysine-derived melanoidins did not induce cytotoxicity, but inhibited cell proliferation. This eﬀect can be explained by the relatively low concentration of membrane-bound Maillard-modiﬁed dipeptides per spot (17 nmol) compared to the concentration of Maillard reaction mixture (5 mM - 25 mM) used to determine

MRP-induced cytotoxicity.

The present work also shows that the biological eﬀect of the MRPs is dependent on the carbonyl compound used for the glycation. Methylglyoxal-derived MRPs showed the strongest eﬀect on cell proliferation. To a lesser degree glyceraldehyde-derived MRPs also inhibited cell proliferation whereas only ribose-derived lysine-lysine MRPs reduced cell growth.

The results of this part of the work indicate that the biological activity of melanoidins depends on the amino acid precursor and the carbonyl compound used for glycation. CHAPTER 3. SUMMARY 89

3 Summary

Maillard reaction products (MRPs) are formed through a non-enzymatical reaction between reducing sugars and proteins or amino acids. This so called Maillard reaction takes place in heated foods such as bakery products and roasted meat. A wide range of MRPs are responsible for aroma- and flavour-formation in cooked food. The colour of many kinds of food, e.g. coffee has its origin in the Maillard reaction. Furthermore, so called ”advanced glycation end products” (AGEs) are formed through analogous reaction steps in the human body. Highly increased AGE-levels can be detected in patients with diabetes mellitus (increased glucose level) or renal failure (diminished excretion). It is well known that AGEs can induce an inflammatory cellular response, most likely, by a receptor-dependent induction of oxidative stress, e.g. via RAGE (receptor for advanced glycation end products). So far, however little is known about the question if food-derived MRPs can directly trigger inflammatory processes in the intestine, or if they have inflammatory activity in the human body after resorption. In this regard, macrophages which are important mediators of the immune response are of particular interest. MRPs could induce a cellular reaction in macrophages, which are widely spread in the intestine, modulating the intestinal immune response. In the pathogenesis of inflammatory bowel disease, a large number of activated macrophages can be detected in the intestinal mucosa, triggering an inflammatory response.

Thus, particularly in inﬂammatory bowel diseases, the activation of macrophages by MRPs could lead to an enhanced inﬂammatory reaction.

The transcription factor NF-κB plays a central role in cellular signal transduction promoting the expression of more than 150 immune- and inflammation-related genes. Macrophages 90 CHAPTER 3. SUMMARY were stimulated with MRPs and the translocation of NF-κB into the nucleus was immunochemically detected. NF-κB translocation was used to indicate the activation of a signalling pathway, which is linked to the cellular immune response. Coffee, which contains high levels of MRPs, was used for the stimulation of macrophages. Whereas an extract of raw coffee beans did not lead to NF-κB activation, freshly brewed coffee increased the translocation of NF-κB 13-fold in macrophages. To confirm the hypothesis that MRPs formed during the coffee roasting process are responsible for the activation of macrophages, a Maillard reaction mixture consisting of lysine and ribose heated for 30 min at 120 °C was prepared.

It could be shown that stimulation of macrophages with the Maillard reaction mixture led to a signiﬁcant 18-fold increased NF-κB activation compared to the control maintained in

PBS. The observed eﬀect was due to MRPs because stimulation with the unheated reaction mixture or ribose and lysine heated alone did not cause NF-κB activation. The Maillard reaction mixture was fractionated by size exclusion chromatography. Afterwards, the low molecular weight (LMW, <1.8 kDa) and high molecular weight fraction (HMW, >1.8 kDa), the latter containing mostly complex melanoidins, were tested for their biological activity.

The melanoidins, but not the LMW-compounds were shown to induce NF-κB activation, which is in good accordance with the observed NF-κB activation by coﬀee extract since melanoidins account for up to 25 % of dry matter of coﬀee bevarage.

Using RAGE-transfected and untransfected human embryonic kidney (HEK) cells for the stimulation experiments, the activation of NF-κB by food-derived MRPs was shown to be

RAGE-independent. The stimulation of the HEK cells with MRPs caused a 2-fold increased

NF-κB translocation in both cell lines. On the other hand, similar to the AGE-RAGE signal transduction, the food-derived MRPs induced oxidative stress, leading to the activation of the signalling pathway. NF-κB activation by MRPs, either derived from coﬀee or Mail- lard reaction mixture, was completely inhibited by co-incubation with catalase indicating hydrogen peroxide as signal mediator. Further investigations revealed that MRPs induce

NF-κB activation through the induction of oxidative stress by an extracellular generation CHAPTER 3. SUMMARY 91

of hydrogen peroxide. Freshly brewed coﬀee was shown to contain 89 µMH2O2 and 25 mM

Maillard reaction mixture 287 µMH2O2. Hydrogen peroxide was not generated in solutions lacking MRPs including raw coffee extract, the unheated reaction mixture or ribose and lysine, which have been heated alone. The incubation of coffee or Maillard reaction mixture under the same conditions as applied in the cell culture experiments led to an increase in hydrogen peroxide generation up to 157 µM in coffee and 366 µM in 25 mM Maillard reaction mixture. It is suggested, that free metal ions catalyse the MRP-induced reactive oxygen species-generation. However, as an important antioxidative defence mechanism, free metal ions are restricted in vivo. Using chelator-treated solutions, the MRP-induced hydrogen peroxide generation and NF-κB activation was shown to be independent on the presence of free metal ions. Thus, the hydrogen peroxide generation by food-derived MRPs may not only take place in food, but also in vivo after ingestion, leading to the activation of signalling pathways in immune cells. The present work revealed that besides the known receptor-mediated reaction, a second mechanism of cell activation by MRPs exists, through the generation of hydrogen peroxide.

Investigations regarding the reaction of macrophages on MRP-induced NF-κB activation revealed that the expression of inflammatory mediators, cytokines and NO, was not pro- moted. However, the induction of oxidative stress by MRPs caused cell death, which could be totally blocked by catalase. The MRPs did not induce programmed cell death (apoptosis) as determined by the absence of cleaved caspase-3 in the lysates of MRP-treated cells. The active form of caspase-3 (cleaved caspase-3) plays a central role in the processing of apoptosis. Thus, it can be concluded that MRP-induced cytotoxicity leads to necrotic cell death. Thereby, MRP-induced NF-κB activation may contribute to necrotic cell death by promoting anti-apoptotic signals. Necrotic cell death caused by food-derived MRPs in vitro, may redirect an inflammatory response in vivo. Whereas apoptotic processes lead to the safe disposal of cells, necrotic cells spill cytosolic constituents with immunomodulatory activity into the extracellular space causing inflammatory reactions in the surrounded tissue. 92 CHAPTER 3. SUMMARY

The heterogeneous composition of melanoidins did not allow the isolation of deﬁned signal active products. Among several synthesized MRPs, which were tested for their ability to induce NF-κB activation in the bioassay, a MRP with aminoreductone structure, was shown to be physiological active. Similar to the experiments with coﬀee or Maillard reaction mixture, the C4-aminoreductone-induced NF-κB activation was blocked by catalase.

Food-derived MRPs are formed through reaction steps analogous to AGE-formation, which leads to the possibility that AGEs also contain signal active aminoreductone structures.

Thus, a possible role of hydrogen peroxide in AGE-induced cell activation was investigated.

Surprisingly, in vitro prepared AGEs did not induce NF-κB activation neither through interacting with RAGE nor through hydrogen peroxide generation. Finally, the expression of the NF-κB-regulated enzyme, inducible nitric oxide synthase (iNOS), was shown to be dependent on the AGE preparation. Thereby, however, an influence of specific external factors could not be identified. The signal transduction of the physiological active AGE-batch was fully dependent on RAGE as shown by blocking RAGE with anti-RAGE antibody.

In the last part of this work, the influence of amino acid precursor on the cytotoxicity of melanoidins was investigated using a dipeptide spot library. The membrane-bound dipeptides of the spot library were Maillard-modified and the formation of brown coloured melanoidins was determined by densitometric analysis. Among the amino acids forming melanoidins the order of reactivity was lysine ≈ tryptophan > arginine ≈ aspartic acid > cysteine. The influence of the membrane-bound melanoidins on the survival of cells, grown on the membrane, was investigated with a membrane-based cell viability assay. The assay combines the conditions of the membrane-bound dipeptide spots with a cell viability assay. The method was successfully established with a Maillard-modified dipeptide spot library, exemplarily consisting of lysine and/or arginine. The membrane-bound melanoidins, especially those derived from lysine were identified to inhibit cell proliferation.

In the present work, coffee and MRPs were shown to produce hydrogen peroxide and lead to the activation of NF-κB as an immediate stress response. Activation of NF-κB, CHAPTER 3. SUMMARY 93 however, did not result in an inflammatory reaction. On the other hand, MRP-induced generation of hydrogen peroxide caused necrotic cell death. On a long term, foods rich in melanoidins, such as coffee, may influence the function of immune cells in the intestine.

Activation of NF-κB plays a crucial role in the transcription of immune-related genes. Also, the induction of necrotic cell death in macrophages by MRPs, could lead to an inﬂamma- tory response in the surrounded tissue in the intestine. In contrast to apoptosis, necrotic cell death causes loss of membrane integrity redirecting the spill of cytosolic constituents with immunomodulatory functions into the extracellular space. The eﬀect of food-derived

MRPs may be particularly enhanced in patients with inﬂammatory bowel disease, where

NF-κB activation in macrophages has an important role in pathogenesis, mediating chronic mucosal inﬂammation. 94 CHAPTER 4. DEUTSCHE ZUSAMMENFASSUNG

4 Deutsche Zusammenfassung

Maillardprodukte (Maillard reaction products, MRPs) werden durch eine nicht enzymati- sche Reaktion zwischen reduzierenden Zuckern und Proteinen oder Aminos¨auren gebildet.

Diese so genannte Maillard Reaktion läuft in stark erhitzten Lebensmitteln wie z.B. Back- waren und gebratenen Fleisch ab. Eine Vielzahl der Maillard Produkte ist an der Aroma- und Geschmacksbildung beteiligt. Weiterhin hat die Farbe vieler Lebensmittel z.B. von Kaf- fee, ihren Ursprung in der Maillard-Reaktion. Auch im menschlichen Körper werden durch analoge Reaktionsschritte sogenannte ”Advanced glycation end products” (AGEs) gebildet. Stark erhöhte AGE-Spiegel lassen sich vor allem in Patienten mit Diabetes mellitus

(erh¨ohter Glucosespiegel) oder Niereninsuﬃzienz (verminderte Ausscheidung) feststellen.

Es ist bekannt, dass AGEs eine inﬂammatorische Zellantwort hervorrufen k¨onnen, wahr- scheinlich uber¨ eine Rezeptor-vermittelte Induktion von oxidativem Stress z.B. uber¨ RAGE

(Rezeptor fur¨ AGEs). Bisher ist jedoch nur wenig daruber¨ bekannt, ob MRPs die aus der

Nahrung aufgenommen werden uber¨ ¨ahnliche Mechanismen direkt im Darm entzundliche¨

Prozesse auslösen oder nach Resorption im gesamten Körper zu einer entzundlichen¨ Zel- lantwort fuhren¨ können. Im Besonderen interessieren dabei Makrophagen, die wichtige Me- diatoren von Immunreaktionen sind. MRPs könnten zelluläre Reaktionen in Makrophagen, die im Darm weit verbreitet sind, auslösen und somit die intestinale Immunantwort beeinflussen. In der Pathogenese von entzundlichen¨ Darmerkrankungen kann eine Vielzahl an aktivierten pro-inflammatorischen Makrophagen in der Darmmukosa detektiert werden.

Hierbei ist es denkbar, dass die zus¨atzliche Aktivierung von Makrophagen durch MRPs die

Entzundungsreaktion¨ verst¨arkt. CHAPTER 4. DEUTSCHE ZUSAMMENFASSUNG 95

Der Transkriptionsfaktor Nuclear Factor-κB (NF-κB) stellt ein fruhes¨ und zentrales

Glied in der Signaltransduktion von Entzundungs-¨ und Immunreaktionen dar und induziert die Expression von mehr als 150 Genen. Als Parameter einer Zellaktivierung in MRP- stimulierten Makrophagen, die im Zusammenhang mit einer Immunreaktion steht, wurde deshalb die Translokation von NF-κB in den Zellkern immunochemisch nachgewiesen. Kaf- fee wurde fur¨ die Stimulation der Makrophagen verwendet, da dieser einen hohen Anteil an MRPs enthält. Im Gegensatz zu Rohkaffeeextrakt induzierte frisch gebruhter¨ Kaffee eine 13-fach erhöhte Translokation von NF-κB in den Makrophagen. Um die Hypothese zu bestätigen, dass MRPs die erst bei der Röstung von Rohkaffeebohnen enstehen fur¨ die Ak- tivierung der Makrophagen verantwortlich sind, wurde eine Maillard-Modellmischung aus

Lysin und Ribose hergestellt die bei 120 °C fur¨ 30 min erhitzt wurde. Es konnte gezeigt werden, dass die Stimulation mit der Maillard-Modellmischung zu einer signiﬁkanten, 18-fachen

Aktivierung von NF-κB gegenuber¨ der nicht-stimulierten Kontrolle in Makrophagen fuhrte.¨

Der beobachtete Eﬀekt wurde durch Produkte der Maillard-Reaktion hervorgerufen, da

Versuche mit einer nicht erhitzten Ribose-Lysin Mischung gleicher Konzentration oder den erhitzten Einzelkomponenten, Ribose und Lysin zu keiner Aktivierung von NF-κB fuhrten.¨

Die Maillard-Modellmischung wurde durch eine Gr¨oßenausschlusschromatographie, unter

Verwendung einer Porengr¨osse fur¨ Molekulmassen¨ bis zu 1,8 kDa, fraktioniert. Es zeigte sich, dass die hochmolekulare (HMW) Fraktion (>1,8 kDa) der Maillard-Modellmischung, deren Produkte als Melanoidine bezeichnet werden, im Gegensatz zur niedermolekularen

(LMW) Fraktion (<1,8 kDa) fur¨ die NF-κB Aktivierung verantwortlich ist. Das Ergebnis ist in guter Ubereinstimmung¨ mit der beobachteten Kaﬀee-induzierten Zellaktivierung, da die Trockenmasse von Kaﬀee bis zu 25 % aus Melanoidinen besteht.

Die Stimulationsversuche wurden weiterhin sowohl in stabil RAGE-transformierten als auch in untransformierten humanen embryonalen Nierenzellen (HEK-Zellen) durchgefuhrt.¨

Dabei zeigte sich, dass die MRP-induzierte Aktivierung von NF-κB unabh¨angig von der

Expression von RAGE ist, da die Stimulation mit der Maillard-Modellmischung zu einer 96 CHAPTER 4. DEUTSCHE ZUSAMMENFASSUNG

2-fach erh¨ohten Translokation von NF-κB in beiden Zelllinien fuhrte.¨ Auf der anderen Seite wurde durch die Maillard-Modellmischung, ¨ahnlich wie bei der AGE-RAGE Signaltrans- duktion, oxidativer Stress induziert, der zur Aktivierung des Signalweges fuhrte.¨ Die durch

Kaffee oder Maillard-Modellmischung induzierte NF-κB Aktivierung konnte durch Katalase vollständig inhibiert werden, was Wasserstoffperoxid als Signalmolekul¨ identifiziert. Weitere

Versuche zeigten, dass im Lebensmittel gebildete MRPs durch die extrazellul¨are Bildung von Wasserstoﬀperoxid oxidativen Stress induzieren, der zur Aktivierung von NF-κB fuhrt.¨

In Kaffee konnte 89 µMH2O2 und in 25 mM Maillard-Modellmischung 287 µMH2O2, un- abhängig von der Anwesenheit von Zellen, nachgewiesen werden. In Lösungen, die keine

MRPs enthielten, nämlich Rohkaffeeextrakt, nicht erhitzte Ribose-Lysin Mischung und erhitzte Einzelkomponenten, Ribose und Lysin, wurde kein Wasserstoffperoxid gebildet. Die

Inkubation der MRPs unter den Bedingungen der Zellkulturexperimente fuhrte¨ zu einer

Erhöhung des Wasserstoffperoxid Gehaltes von bis zu 157 µM in Kaffee und 366 µM in

25 mM Maillard-Modellmischung. Es wird vermutet, dass Metallionen die MRP-induzierte

Bildung von reaktiven Sauerstoﬀspezien katalysieren. In vivo sind Metallionen jedoch nur in gebundener Form, als wichtiger Abwehrmechanismus gegenuber¨ Oxidationen, zu ﬁnden. Die

Verwendung von Lösungen die zuvor mit einem Chelator behandelt wurden zeigten, dass die MRP-induzierte Wasserstoffperoxid-Bildung und Aktivierung von NF-κB unabhängig von der Anwesenheit freier Metallionen ist. Somit ist es denkbar, dass die Bildung von

Wasserstoﬀperoxid durch MRPs nicht nur in Lebensmitteln stattﬁndet sondern auch nach

Aufnahme der MRPs uber¨ die Nahrung in vivo und zu einer Aktivierung von Signalwegen in Immunzellen fuhrt.¨ Die vorliegenden Ergebnisse zeigen, dass MRPs, neben der bekann- ten Rezeptor-vermittelten Reaktion, auch uber¨ einen zweiten Mechanismus, n¨amlich der

Bildung von Wasserstoﬀperoxid, Zellen aktivieren k¨onnen.

Weitere Experimente zeigten, dass die Expression von pro-inflammatorischen Signalen, wie Cytokinen oder NO, durch die MRP-induzierte NF-κB Aktivierung in Makrophagen nicht nachgewiesen werden konnte. Auf der anderen Seite fuhrte¨ der MRP-induzierte oxi- CHAPTER 4. DEUTSCHE ZUSAMMENFASSUNG 97 dative Stress zu einem Zellsterben, dass durch die Zugabe an Katalase vollständig blockiert werden konnte. Die Maillardprodukte lösten jedoch keinen programmierten Zelltod (Apop- tose) aus, was durch die Abwesenheit von proteolytisch aktivierter Caspase-3 in Zelllysaten von MRP-stimulierten Zellen bestimmt wurde. Die aktivierte Form von Caspase-3 (proteolytisch aktivierte Caspase-3) spielt eine zentrale Rolle in der apoptotischen Signalkaskade.

Dehalb kann man annehmen, dass die MRP-induzierte Zytotoxizit¨at zu einem nekrotischen

Zelltod fuhrt.¨ Dabei ist es denkbar, dass die Aktivierung von NF-κB anti-apoptotische Si- gnale unterstutzt,¨ die einen nekrotischen Zelltod f¨ordern. Die in vitro durch Lebensmittel-

MRPs induzierte Nekrose könnte in vivo eine Entzundungsreaktion¨ auslösen. Im Gegensatz zu apoptotischen Prozessen, die zu einer sicheren Beseitigung von Zellen fuhrt,¨ kommt es in nekrotischen Zellen zum Ausfluss von cytosolischen Bestandteilen, mit u.a. immunmodulie- render Aktivität, in den extrazellulären Raum, was zu einer Entzundung¨ im umliegenden

Gewebe fuhren¨ kann.

Auf Grund der Heterogenität der Melanoidine war es nicht möglich signalaktive Einzel- strukturen zu isolieren. Unter einigen synthetisierten Maillardprodukten, die im Bioassay auf ihre Eigenschaft NF-κB zu aktivieren getestet wurden, erwies sich ein Maillardprodukt mit Aminoreduktonstruktur als signalaktiv. Ahnlich¨ wie bei den Experimenten mit Kaffee und Maillard-Modellmischung, konnte die C4-Aminoredukton-induzierte NF-κB Aktivie- rung vollständig durch Katalase inhibiert werden. Die im Lebensmittel gebildeten Maillard- produkte enstehen durch analoge Reaktionsschritte wie AGEs. Es ist denkbar, dass AGEs signalaktive Aminoreduktonstrukturen enthalten. Deshalb wurde untersucht ob Wasserstoff- peroxid bei der AGE-induzierten Zellaktivierung beteiligt sein könnte. Uberraschenderweise¨ induzierten in vitro hergestellte AGEs weder uber¨ die Interaktion mit RAGE noch uber¨ die Bildung von Wasserstoffperoxid NF-κB. Letztendlich konnte gezeigt werden, dass die

Expression des NF-κB regulierten Enzyms, induzierbare NO-Synthase (iNOS), bei sonst gleichen Reaktionsbedingungen, von der AGE-Charge abh¨angt. Dabei konnte jedoch kein

Einfluss von bestimmten äusseren Faktoren identifiziert werden. Durch Blockierung von 98 CHAPTER 4. DEUTSCHE ZUSAMMENFASSUNG

RAGE mit einem anti-RAGE Antikörper konnte nachgewiesen werden, dass die Signal- transduktion der signalaktiven AGE-Charge vollständig von RAGE abhängig ist.

Im letzten Teil der Arbeit wurden der Einﬂuss der 20 proteinogenen Aminos¨auren hinsichtlich der Bildung von zytotoxischen Melanoidinen mit Hilfe einer Dipeptid-Spot-

Bibliothek untersucht. Die Membran-gebundenen Dipeptide der Spot Bibliothek wurden

Maillard-modifiziert und die Bildung an braun-gefärbten Melanoidinen densitometrisch aus- gewertet. Die Reaktivität der Aminosäuren hinsichtlich der Bildung von Melanoidinen nahm in der Reihenfolge Lysin ≈ Tryptophan > Arginin ≈ Asparaginsäure > Cystein ab. Der

Einﬂuss der Membran-gebundenen Melanoidine auf das Wachstum von Zellen, kultiviert auf der Membran, wurde mit Hilfe eines Membran-gebundenen Zell-Vitalit¨atstest bestimmt. Der

Bioassay kombiniert die Bedingungen der Membran-gebundenen Dipeptide mit einem Zell-

Vitalit¨atstest. Die Methode konnte erfolgreich mit einer Maillard-modiﬁzierten Dipeptid-

Spot-Bibliothek etabliert werden, die exemplarisch aus Lysin und/oder Arginin bestand. Es konnte gezeigt werden, dass die Melanoidine besonders solche die aus Lysin gebildet werden zur St¨orung des Zellwachstums fuhren.¨

In der vorliegenden Arbeit konnte gezeigt werden, dass Kaﬀee und Maillardprodukte

Wasserstoffperoxid produzieren und als akute Stressantwort die Aktivierung von NF-κB auslösen. Die Aktiverung von NF-κB fuhrte¨ nicht zu einer entzundlichen¨ Zellantwort. Je- doch löste die MRP-induzierte Bildung von Wasserstoffperoxid Nekrose aus. Auf lange Sicht könnten stark melanoidinhaltige Lebensmittel, wie z.B. Kaffee, die Funktion von Immun- zellen im Darm beeinflussen. Die Aktivierung von NF-κB spielt eine entscheidende Rolle in der Transkription von Genen, die an der Immunantwort beteiligt sind. Auch könnte die

MRP-induzierte Nekrose von Makrophagen Entzundungen¨ im umliegenden Darmgewebe auslösen. Im Gegensatz zur Apoptose fuhrt¨ der nekrotische Zelltod zum Ausfluss von cytosolischen Bestandteilen mit immunmodulierenden Funktionen in den extrazellulären Raum.

Der Eﬀekt von MRPs aus Lebensmitteln k¨onnte vor allem in Patienten mit entzundlichen¨

Darmerkrankungen von Bedeutung sein, da in der Pathogenese die Aktivierung von NF-κB in Makrophagen eine wichtige Rolle spielt. CHAPTER 5. MATERIALS AND METHODS 99

5 Materials and Methods

5.1 Materials and Equipment

General Materials

Tubes 15 mL and 50 mL (Greiner)

Microcentrifuge tubes (Eppendorf)

96-well plate (Nunc-immuno plate Maxisorp 96 well, Nunc)

Preparation of Maillard Reaction Mixtures, AGEs and Coﬀee

Sterile ﬁlter 0.22 µm, PVDF (Roth) l-lysine, purum, crystallized, >98 % (Roth) l(+)-arginine, 98+ % (Acros) d(-)-ribose, >99 % (HPLC, sum of enantiomers) (Fluka)

PBS Dulbecco´s (Biochrom)

Drying oven (Ehret)

654 pH meter (Metrohm)

BSA (Sigma-Aldrich or Boehringer Mannheim ) d-glucose (Sigma-Aldrich or Merck)

Visking dialysis semi-permeable membrane, molecular weight cut-oﬀ 14 kDa (Roth)

Dialysis tubing cellulose, molecular weight cut-oﬀ 10 kDa (Sigma-Aldrich)

Bio-Rad protein assay (Bio-Rad)

Sodium chloride (Acros) 100 CHAPTER 5. MATERIALS AND METHODS

Di-Sodium hydrogen phosphate dihydrate (Fluka)

Sodium dihydrogen phosphate dihydrate (Fluka)

Sodium dihydrogen phosphate monohydrate (Merck)

Potassium chloride (Merck)

Microplate reader model 550 (Bio-Rad)

Ground coﬀee ”Erlanger Meistermischung”, 100 % arabica beans, (Koenigmanns Kaf- feeroesterei, Erlangen)

Raw coﬀee beans, 100 % arabica beans, (Koenigmanns Kaﬀeeroesterei, Erlangen)

Ultraturax homogenizer (IKA Works)

Fluted ﬁlter (Macherey-Nagel)

Fractionation of Maillard Reaction Mixture via SEC

D-SaltTM polyacrylamide desalting column (107 mm x 12 mm), cut-oﬀ 1.8 kDa (Perbio,

Pierce®

UV/Vis spectrophotometer Lambda2 (Perkin-Elmer)

Freeze dryer Novalyphe-NL 150 (Savant)

Phenylalanine (Sigma-Aldrich)

Insulin (Sigma-Aldrich)

Sodium azide (Sigma-Aldrich)

Cell Culture

Cell incubator (Binder)

25 cm2 or 75 cm2 cell culture ﬂasks (Biochrom)

12-, 24- or 96- well plates (Biochrom)

Cell scraper (Sarstedt)

Autoclave (Systec)

Penicillin and Streptomycin [10.000 IU/10.000 µg/mL] (Biochrom)

HAM´s F12 [1.176 g/L NaHCO3, stable glutamine] (Biochrom) CHAPTER 5. MATERIALS AND METHODS 101

DMEM [4.5 g/L glucose, GlutaMAX I , sodiumpyruvate] (Invitrogen, GIBCO®)

Geneticin (Invitrogen, GIBCO®)

MEM with Earl´s salts [2.2 g/L NaHCO3, without l-glutamine] (Biochrom)

DMEM [0.168 g/L l-arginine] (Tropbio Pty. Ltd., JCU Townsville, Australia)

FCS (Biochrom)

EDTA in PBS (Biochrom)

Trypsin (0.25 %)/EDTA (0.02 %) in PBS (Biochrom)

SDS Polyacrylamide Gel Electrophoresis and Western Blot

Acrylamide/Bis solution (Bio-Rad)

Tris base (Fluka)

SDS (Fluka)

TEMED (Bio-Rad)

Ammoniumpersulfate (Bio-Rad)

Glycine (Sigma-Aldrich)

Tween 20 (Sigma-Aldrich)

Ponceau red S (Sigma-Aldrich)

Trichloroacetic acid (Acros)

Skim milk powder (Fluka)

Molecular weight marker for proteins (Sigma-Aldrich)

ChemiBlotTM molecular weight marker (Chemicon)

Blot apparatus: standard power pack p25 (Biometra)

Nitrocellulose transfer membrane, pore size 0.45 µm (Schleicher & Schuell)

Gel-blotting paper, GB002 (Schleicher & Schuell)

ECL Western Blotting detection reagents (Amersham biosciences)

HyperﬁlmTM ECL (Amersham biosciences)

Densitometer, BioDoc Analyzer (Bio-Rad) 102 CHAPTER 5. MATERIALS AND METHODS

Roller mixer SRT1 (Stuart Scientiﬁc)

Determination of NF-κB Translocation

LPS from Escherichia coli 055:B5 (Sigma-Aldrich)

TNF-α (Biochrom)

Catalase from bovine liver (Sigma-Aldrich)

Chelex 100 resin, 100-200 mesh (Bio-Rad)

HEPES (Merck)

Potassium chloride (Merck)

Magnesium chloride hexahydrate (Merck)

Glycerol anhydrous (Fluka)

EDTA (Roth)

EGTA (Sigma-Aldrich)

Protease Inhibitor Tablet Complete (Roche)

PMSF (Fluka)

DTT (Fluka)

Sodium chloride (Acros)

NP-40 (Fluka)

Vortexer (Heidolph)

Microcentrifuge (Neolab)

Centrifuge Universal 16R (Hettich Zentrifugen)

Dc-protein assay (Bio-Rad)

Polyclonal rabbit anti-p65 antibody (Santa Cruz, sc-109)

Monoclonal mouse anti-p65 antibody (Santa Cruz, sc-8008)

Monoclonal mouse anti-β-actin antibody (Sigma-Aldrich, A5541)

HRP-conjugated anti-rabbit antibody (Sigma, A6154) CHAPTER 5. MATERIALS AND METHODS 103

HRP-conjugated anti-mouse antibody (Sigma, A6782)

Tris-HCl (Fluka)

Urea (Riedel-de-Ha¨en)

Bromophenolblue (Merck)

Detection of RAGE Expression

Monoclonal mouse anti-RAGE antibody (A11) (Dr. Weigle, TU Dresden)

HRP-conjugated anti-mouse antibody (Sigma-Aldrich, A6782) sRAGE (Dr. Weigle, TU Dresden)

Determination of Caspase-3 Activation

Staurosporine (Sigma-Aldrich)

Monoclonal rabbit anti-caspase-3 antibody (Cell Signaling, 8G10)

HRP-conjugated anti-rabbit antibody (Sigma-Aldrich, A6154)

Cell Viability Assays

MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] (Merck)

Isopropanol (BASF)

Resazurin sodium salt (Sigma-Aldrich)

Fluorescence reader, Wallac 1420 VICTOR3TM V (Perkin Elmer)

Determination of NO Production

INF-γ (CytoLab Ltd.)

LPS from Escherichia coli 055:B5 (Sigma-Aldrich) 104 CHAPTER 5. MATERIALS AND METHODS

N-(1-naphthyl)ethylenediaminedihydrochloride (Sigma-Aldrich)

Sulfanilamide (Sigma-Aldrich)

Sodium nitrite (Ajax Chemicals)

Microplate reader Multiskan Ascent (Labsystem)

Polyclonal goat anti-RAGE antibody (Santa Cruz, sc-8230)

Cytokine Analysis

Bio-Plex System and Cytokine reagent kit (Bio-Rad)

Bio-Plex rat Cytokine 9-Plex A panel (Bio-Rad)

Rat IL-6 DuoSet ELISA Development kit (R & D Systems)

Freeze dryer Novalyphe-NL 150 (Savant)

LPS from Escherichia coli 055:B5 (Sigma-Aldrich)

Determination of Hydrogen Peroxide Concentration (FOX Assay)

Xylenol orange (Fluka)

Ammonium iron (II) sulfate hexahydrate (Fluka)

Perchloric acid (Gr¨ussing)

Catalase from bovine liver (Sigma-Aldrich)

30 % hydrogen peroxide (Degussa AG)

Peptide Spot Library

Peptide spot library and dipeptide/amino acid spot membranes (Dr. Rothemund, IZKF

Leipzig)

Petri dishes (Sarstedt)

Tubes 10 mL (Sarstedt) CHAPTER 5. MATERIALS AND METHODS 105 d(-)-ribose (Sigma-Aldrich)

Methlyglyoxal solution, 40 % in water (Sigma-Aldrich) dl-glyceraldehyde (Sigma-Aldrich)

Image MasterTM 2D Platinum (Amersham Biosciences)

5.2 Buﬀers and Solutions

Phosphate Buﬀered Saline (PBS)

137 mM NaCl

8.1 mM Na2HPO4 x 2H2O

2.7 mM KCl

1.6 mM NaH2PO4 x 2H2O

100 mM Phosphate Buﬀer

22.6 mM NaH2PO4 x H2O

77.4 mM Na2HPO4 x 2H2O

200 mM Phosphate Buﬀer

100 mM NaH2PO4 x H2O

100 mM Na2HPO4 x 2H2O

10x Electrophoresis Buﬀer

0.25 M Tris base

1.89 M glycine

0.035 M SDS 106 CHAPTER 5. MATERIALS AND METHODS

Transfer Buﬀer

25 mM Tris base

200 mM glycine

20 % methanol

Ponceau S Solution

2.6 mM ponceau S

180 mM trichloroacetic acid

PBS/Tween

0.1 % (v/v) Tween 20 in PBS

Chelex-PBS and Chelex-Maillard Reaction Mixture

To remove metal ions, PBS and the Maillard reaction mixture were incubated with

50 mg/mL Chelex 100 resin for 1 h. Chelex 100 resin was removed by centrifugation and the solutions were ﬁlter sterilized.

Blocking Buﬀer

5 % (w/v) skim milk powder in PBS/Tween

Molecular Weight Marker for Proteins

1:4 dilution of stock solution in lysis buﬀer heat for 5 min at 95 °C, store in aliquots at -20 °C

ChemiBlotTM Molecular Weight Marker

1:150 dilution of stock solution in lysis buﬀer heat for 5 min at 95 °C, store in aliquots at -20 °C CHAPTER 5. MATERIALS AND METHODS 107

100x Protease Inhibitor Solution

1 Protease Inhibitor Tablet Complete made up to a ﬁnal volume of 0.5 mL PBS store in aliquots at -20 °C

Hypotonic Lysis Buﬀer A

10 mM HEPES-KOH pH 7.4

10 mM KCl

1 mM MgCl2 x 6H2O

5 % glycerol

0.5 mM EDTA

0.1 mM EGTA

To be added before use (per mL)

1 % protease inhibitor solution

2 mM PMSF

0.5 mM DTT

Nuclear Extraction Buﬀer B

20 mM HEPES-KOH pH 7.4

1 % NP-40

400 mM NaCl

10 mM KCl

1 mM MgCl2 x 6H2O

20 % glycerol

0.5 mM EDTA

0.1 mM EGTA

To be added before use (per mL)

1 % protease inhibitor solution 108 CHAPTER 5. MATERIALS AND METHODS

2 mM PMSF

0.5 mM DTT

5x Lysis Buﬀer

0.3 M Tris-HCl

0.35 M SDS

5.2 M urea

0.32 M DTT

35 % glycerol

Bromophenolblue adjust to pH 7.4 with 2 N NaOH

MTT Solution

1 mg/mL MTT in PBS

Alamar Blue Solution

2.5 mg/L resazurin sodium salt in cell culture medium

Griess Reagent

To be mixed before use

Equal volumes of 0.1 % N-(1-naphthyl)ethylenediaminedihydrochloride in water and 1 % sulfanilamide in 5 % HCL

FOX Reagent

0.45 mM xylenol orange

0.45 mM (NH4)2Fe(SO4)2 x 6H2O in 0.11 M HClO4 CHAPTER 5. MATERIALS AND METHODS 109

5.3 Methods

5.3.1 Preparation of Maillard Reaction Mixtures, AGEs, Coﬀee and MRPs

Synthesis of Maillard Reaction Mixtures and Control Reaction Mixtures

Maillard reaction mixtures consisting of either Maillard-modiﬁed lysine or Maillard-modiﬁed arginine (rib-lys or rib-arg) were produced by incubation of 0.5 M lysine or 0.5 M arginine in equimolar ratio with ribose in PBS (pH 7.4, Biochrom) at 120 °C for between 30 min and

24 h under sterile conditions in an airtight glass container. The unheated solutions (pH 9.6) were used as control reaction mixtures (rib-lys 0 h, rib-arg 0 h). As further controls, lysine, arginine, and ribose were heated alone under the same conditions. Before further use in cell culture experiments, mixtures were adjusted to pH 8.0 with 2 N HCl or 2 N NaOH. All solutions were ﬁlter sterilized and stored at -20 °C.

Synthesis of AGE-Modiﬁed BSA for Determination of NF-κB Activation

AGE-modiﬁed BSA was prepared by heating 0.6 mM BSA (Sigma-Aldrich) with 0.4 M glucose (Sigma-Aldrich) in PBS at 37 °C under sterile conditions in an airtight tube for

8 weeks while shaking [AGE-BSA(glu)]. Furthermore, BSA (Sigma-Aldrich) was glycated with ribose under the same conditions for 3 weeks [AGE-BSA(rib)]. Unbound sugar was removed via dialysis three times against PBS for 24 h using tubing with a molecular weight cut-oﬀ of 14 kDa (Roth). BSA was also incubated in the absence of sugar under the same conditions to serve as a control. Protein concentration was determined with the Bio-Rad protein assay. AGE-BSA and BSA were ﬁlter sterilized and stored at -20 °C.

Synthesis of AGE-Modiﬁed BSA for Determination of NO Production

AGE-modiﬁed BSA was prepared by heating 0.1 mM BSA (Boehringer Mannheim) with

0.1 M glucose (Merck) in 100 mM phosphate buﬀer (pH 7.4) or 200 mM phosphate buﬀer

(pH 6.8) at 60 °C (Table 5.1). Unbound sugar was removed via dialysis three times against 110 CHAPTER 5. MATERIALS AND METHODS

Table 5.1: List of conditions used for AGE-modiﬁed BSA preparation

AGE-BSA Incubation time pH adjusted to Phosphate Dialysis

at 60 °C buﬀer after

#1 5 days pH 7.4 every day 100 mM 5 days

#2 6 weeks pH 7.4 every week 100 mM 3 weeks

#3 6 weeks pH 6.8 every week 200 mM 6 weeks either 100 mM or 200 mM phosphate buffer for 24 h using tubing with a molecular weight cut-off of 10 kDa (Sigma-Aldrich). Protein concentration was determined with the Bio-Rad protein assay. AGE-BSA were filter sterilized and stored at -20 °C.

Bio-Rad Protein Assay

The Bio-Rad protein assay is based on the Bradford dye-binding procedure. 15 µL of standard, sample or blank was mixed with 750 µL dye reagent (1:5 diluted with H2O).

Within 1 h the absorbance was read in a 96-well plate in triplicate with a microplate reader at 595 nm (200 µL reaction mixture/well). The standard curve was prepared in a range between 0.2 and 1.4 mg/mL BSA in PBS.

Coﬀee and Raw Coﬀee Preparation

For the coﬀee preparation, 50 mL hot water (100 °C) was added to 3.75 g ground coﬀee.

The suspension was allowed to stand for 10 min and then filtered through a fluted filter.

The filtrate was cooled to room temperature, adjusted to pH 7.4 with KOH, and for use in cell culture experiments filter sterilized. The coffee was used for further experiments exactly one hour after the preparation was started. Raw coffee extract was obtained in the same way from raw coffee beans, which were ground before use with an Ultraturax.

The described procedure yielded 20 mg/mL dry matter from roasted coffee and 14 mg/mL from raw coffee as determined by weighing after freeze drying. The extract concentration in coffee was calculated for further experiments from these extraction rates. CHAPTER 5. MATERIALS AND METHODS 111

Maillard Reaction Products

The MRPs Amadori product (N-(1-deoxy-d-fructose-1-yl)l-lysine), 3-deoxy-glucosone (3- deoxy-d-erythro-hexose-2-ulose), N-carboxymethyllysine (CML), and compounds containing aminoreductone structure, C4-aminoreductone (3-hydroxy-4-(morpholino)-3-buten-2- on) or a reductone ether, reductone pyranone (2,3-dihydro-3,5-dihydroxy-6-methyl-4H- pyran-4-on) were synthesized and kindly donated from Andrea Degel, workgroup Pischet- srieder [Degel, 2005]. Protein-bound CML (CML-BSA) was synthesized and kindly donated from Dr. Katrin Hasenkopf [Hasenkopf, 2002].

5.3.2 Fractionation of Maillard Reaction Mixture via SEC

SEC uses porous particles to separate molecules of diﬀerent sizes. Molecules that are smaller than the pore size can enter the particles and therefore have a longer path and longer transit time than larger molecules that cannot enter the particles.

5.3.2.1 Calibration of SEC

The D-SaltTM polyacrylamide desalting column (cut-oﬀ 1.8 kDa) was used to fractionate the Maillard reaction mixture into low molecular fraction (LMW, <1.8 kDa) and a high molecular fraction (HMW, >1.8 kDa). After the column was equilibrated with 5 x 5 mL

PBS, the column was calibrated using phenylalanine (0.17 kDa) and insulin (5.8 kDa) as reference substances. 100 mM phenylalanine dissolved in distilled water was used as the low molecular weight marker and 0.625 mM insulin dissolved in 2 N HCl was used as the high molecular weight marker. After loading 0.5 mL sample, the column was washed with either distilled water or 2 N HCl and 0.5 mL fractions were collected. Before applying insulin the column was equilibrated with 2 x 5 mL 2 N HCl. The concentration of phenylalanine in each fraction was determined by measuring absorbance at 257 nm, while insulin was detected at

276 nm. The column was regenerated with 10 x 5 mL PBS and stored in 0.02 % sodium azide. Insulin was almost totally emerged after applying 3.0 mL eluant while phenylalanine 112 CHAPTER 5. MATERIALS AND METHODS

Figure 5.1: Calibration of SEC. Emerge of phenylalanine and insulin from D-SaltTM polyacrylamide column in 0.5 mL fractions started to emerge after additional 0.5 mL eluant (Fig. 5.1).

5.3.2.2 Fractionation of Maillard Reaction Mixture

After equilibration the D-SaltTM polyacrylamide desalting column with 5 x 5 mL PBS,

275 µL of a 500 mM Maillard reaction mixture was applied and eluted stepwise with distilled water. Fractions above and below 1.8 kDa were combined to produce the HMW and

LMW fraction of the Maillard reaction mixture. According to the calibration (Fig. 5.1) the HMW fraction is almost totally emerged from the column after applying 3.25 mL water. The LMW fraction is eluted after applying further 5 mL water. The HMW fraction was lyophilized and constituted to the original volume then separated a second time using the same procedure (Fig. 5.2). The LMW from this step was combined with the LMW of the ﬁrst fractionation, lyophilized, reconstituted to the original volume and separated again using the same procedure. The resulting HMW from this step was combined with the HMW of the second fractionation. Finally, the combined HMW and LMW fractions were lyophilized and reconstituted to the original volume yielding solutions of 69.6 mg/mL

(HMW fraction) or 66.5 mg/mL (LMW fraction). For cell culture experiments the solutions were ﬁlter sterilized. Since the exact molecular weight of the compounds is not known, it CHAPTER 5. MATERIALS AND METHODS 113

Figure 5.2: Schematic illustration of Maillard reaction mixture (rib-lys)-fractionation into low molecular fraction

(LMW, <1.8 kDa) and high molecular fraction (HMW, >1.8 kDa) using SEC, cut-oﬀ 1.8 kDa. The HMW is emerged from the column after applying 3.25 mL water. The LMW is eluted with further 5 mL water. was assumed that the concentration was still 500 mM and this value was used for all further calculations.

5.3.3 Cell Culture

The cell lines listed in table 5.2 were cultivated for the present studies.

NR8383 macrophages and RAW 264.7 macrophages were obtained from American Type

Culture Collection (ATCC) and HSC T6 cells were obtained from the faculty of medicine

FAU Erlangen-N¨urnberg. HEK293 RA and HEK293 ut cells were provided from Dr. Weigle,

Technische Universit¨atDresden. HEK293 cells stably expressing RAGE were prepared as described before [Bartling et al., 2005]. 114 CHAPTER 5. MATERIALS AND METHODS

Table 5.2: List of cultivated cell lines

Designation Characteristic Organ/Tissue Species

NR8383 macrophages lung rat

HEK293 RA epithelial cells transfected with full length RAGE kidney human

HEK293 ut epithelial cells untransfected kidney human

HSC T6 stellate cells liver rat

RAW 264.7 macrophages ascites mouse

Cultivation of Cell Lines

All cell lines were cultivated in a cell incubator at 37 °C in a humidiﬁed atmosphere con-

2 2 taining 5 % CO2 in 5 mL (25 cm ﬂask) or 10 mL medium (75 cm ﬂask). The media were supplemented with penicillin (100 IU/mL) and streptomycin (100 µg/mL) to avoid bacteria contamination.

The NR8383 suspension cell line consisting of adherent and suspension cells was maintained in HAM’s F12 medium supplemented with 15 % FCS. Conﬂuent NR8383 cells

(5x105 cells/mL) were passaged by diluting the suspension cells 1:5 in medium in a new

ﬂask.

HEK293 RA and HEK293 ut cells were maintained in Dulbecco’s Modiﬁed Eagle Medium

[4.5 g/L glucose, GlutaMAX I, sodiumpyruvate] (DMEM) supplemented with 10 % FCS.

For selectively culturing stable RAGE-transfected cells, 0.5 mg/mL Geneticin was added.

Confluent HEK cells were passaged by diluting 1:5. To detach the cells from the flask they were washed with PBS and 2 mL (25 cm2 flask) or 4 mL (75 cm2 flask) of 1 mM EDTA in

PBS was added. After 3 min the solution was removed and cells were incubated in the cell incubator for 5 min before resuspending them in medium.

HSC T6 cells were maintained in Minimum Essential Medium (MEM) with Earl´s salts containing 20 % FCS. The cells were passaged (1:10) and detached with 1 mL of a Trypsin

(0.25 %)/EDTA (0.02 %) solution for 1 min.

RAW 264.7 cells were cultivated in DMEM [0.168 g/L l-arginine] containing 3 % FCS. CHAPTER 5. MATERIALS AND METHODS 115

Conﬂuent cells were mechanically detached from the ﬂask by scraping, resuspended in medium and split 1:10.

For use in experiments, cells were suspended as described above, counted with a Neubauer counting chamber and the indicated amounts of cells described in the experiments were seeded in 12-, 24- or 96- well plates, or 25 cm2 or 75 cm2 ﬂasks. The total number of

NR8383 macrophages was calculated by duplicating the number of cells in suspension.

5.3.4 SDS Polyacrylamide Gel Electrophoresis and Western Blot

Proteins were separated according to their molecular mass with sodium-dodeclysulfate polyacrylamide gel electrophoresis (SDS-PAGE). The principle of the method is based on the molecular mass-dependent migration of denaturated and negatively charged proteins through a polyacrylamide gel in an electric ﬁeld. Afterwards, the separated proteins can be transferred onto a membrane (Western Blot) and immunochemically detected.

12 % SDS-polyacrylamide gels were used and the proteins were separated for 20 min at

80 V (stacking gel) followed by 75 min at 120 V (separation gel). The transfer of proteins onto a nitrocellulose membrane was performed in transfer buﬀer at 150 mA for 1 h. To control protein transfer the membrane-bound proteins were stained with ponceau S solution for 1 min, which could be destained with PBS. The molecular weights of separated protein bands were determined with molecular weight markers (Sigma-Aldrich, molecular weights:

66, 45, 36, 29, 24, 20 and 14 kDa), which could be stained with ponceau S. Non-speciﬁc binding sites were blocked with 15 mL blocking buﬀer for 1 h. The membrane was washed with

10 mL PBS/Tween for 5 min followed by incubation with the primary antibody overnight at 4 °C. Afterwards, the membrane was washed three times with 10 mL PBS/Tween for

10 min followed by incubation with the secondary antibody at room temperature for 1 h.

Using horseradish peroxidase (HRP)-conjugated secondary antibodies, the protein bands can be visualized with an enhanced chemiluminescence (ECL) reaction. HRP catalyzes a chemiluminescence reaction, which is enhanced by performing the reaction in the presence 116 CHAPTER 5. MATERIALS AND METHODS of phenol. Before incubating the membrane with ECL Western Blotting detection reagents according to the instructions of the manufacturer (Amersham biosciences) for 1 min, the membrane was washed four times with 10 mL PBS/Tween for 10 min. The light emission was detected on an autoradiographic ﬁlm, which was exposed for between 10 sec and 5 min.

The ﬁlm was developed and the relative intensity of the bands could be determined by densitometric analysis. Using ChemiBlotTM molecular weight markers (Chemicon, molecular weights: 14.8, 28.2, 41.6, 55.0, 68.4, and 81.8 kDa), which have IgG-binding capacities of various species the molecular weights of protein bands visualized on the ﬁlm could be determined.

5.3.5 Determination of NF-κB Translocation

Cell Stimulation

NR8383 macrophages (2.0x106) were stimulated in medium or PBS in 25 cm2 flasks for between 2 h and 24 h. The test substances were added to the medium or PBS to a total volume of 5 mL in the cell-containing flasks. When experiments were performed in PBS, the medium was removed from the adherent cells in the flask and floating cells were collected by centrifugation (1500 rpm, 2 min). Floating cells and remaining adherent cells in the

flask were washed with 5 mL PBS. The floating cells were re-suspended in 4 mL PBS and transferred back into the flask with the adherent cells. For experiments where the influence of metals was excluded Chelex-treated PBS (Chelex-PBS) was used instead of PBS. As a positive control for NF-κB translocation the macrophages were stimulated with 10 or

100 ng/mL LPS in medium for 21 h. HEK or HSC T6 cells (6.0x106) were starved for 24 h with medium containing 0.2 % FCS (HEK cells) or medium without FCS (HSC T6 cells) in 75 cm2 ﬂasks. The cells were then stimulated for between 30 min and 4.5 h in starving medium yielding a total volume of 10 mL. As a positive control for NF-κB translocation

HEK or HSC T6 cells were stimulated with 10 ng/mL human TNF-α in medium for between

2 h and 4.5 h. To inhibit hydrogen-peroxide induced NF-κB activation, 150 U/mL catalase CHAPTER 5. MATERIALS AND METHODS 117 dissolved in PBS, or heat inactivated catalase was added simultaneously with the test substances to the cell culture. For heat inactivation sterile catalase solution was heated at 95 °C for 5 min.

Nuclear Extraction

Adherent cells were washed with 5 mL ice cold PBS, mechanically detached by scraping , suspended in 5 mL ice cold PBS and collected by centrifugation (4 °C, 1500 rpm, 4 min).

The cells were washed with 5 mL ice cold PBS and collected in a 15 mL tube by centrifugation. Floating cells of the macrophage suspension cell line (NR8383) were collected by centrifugation in a 15 mL tube, washed with 5 mL ice cold PBS and resuspended in

1 mL ice cold PBS after centrifugation. The resuspended cells were combined with the corresponding adherent cell pellet and collected by centrifugation. Further centrifugations of less than 30 sec were carried out in a microcentrifuge. Between the steps samples were kept on ice. Cells were resuspended with 1 mL ice cold PBS, transferred into a 1.5 mL microcentrifuge tube and collected by centrifugation. For nuclear extraction, a previously described method was used with some modiﬁcations [Andrews and Faller, 1991]. Brieﬂy,

NR8383 macrophages were resuspended in 0.5 mL hypotonic lysis buffer A, whereas HSC T6 cells were resuspended in 1 mL. After 15 min incubation, 0.65 % of NP-40 was added and cells were vortexed for 10 sec. The supernatant containing the cytoplasmatic proteins was removed after centrifugation and the pellet was washed with 0.5 mL hypotonic lysis buffer A to ensure a complete separation of cytoplasmatic proteins from cell nuclei. Nuclear proteins were extracted for 1 h with 30 µL (NR8383) or 100 µL (HEK or HSC T6 cells) nuclear extraction buffer B. The pellet was resuspended in nuclear extraction buffer and vortexed during the extraction every 20 min. Cellular debris was removed by centrifugation for

2 min and the supernatant, containing DNA binding proteins, was stored at -80 °C. Pro- tein concentration was determined with the Dc-Protein assay (Bio-Rad) using BSA as a standard. 118 CHAPTER 5. MATERIALS AND METHODS

Dc-Protein Assay

The Dc-Protein assay is suitable to determine protein concentrations following detergent solubilization and is similar to the Lowry procedure. Brieﬂy, 6 µL nuclear protein extract was diluted 1:1 with nuclear extraction buﬀer B. To 5 µL standard/blank or sample, 20 µL reagent A (alkaline copper tartrate solution, 1 mL was supplemented with 20 µL reagent

S before use) and 200 µL reagent B (Folin reagent) was added in a 96-well plate. Each measurement was performed in duplicate. After 15 min the absorbance was read at 630 nm with a microplate reader. The standard curve was prepared in a range between 0.2 and

1.4 mg/mL BSA in nuclear extraction buﬀer B.

Immunochemical Detection of p65

10 µg nuclear proteins and 2 µL of 5-fold concentrated lysis buﬀer were adjusted to a total volume of 10 µL with water. 10 µL of lysate was then denaturated by heating at 95 °C for

5 min before proteins were separated by SDS-PAGE and transferred to membrane (Chap- ter 5.3.4). The membrane was stained with ponceau S to visualize protein bands. β-actin

(41 kDa) was used as an internal standard to control the amount of proteins subjected to the electrophoresis and so the membrane was cut between 41 kDa and 65 kDa with a scalpel before incubating with blocking buﬀer (Fig. 5.3). The NF-κB subunit p65 was detected on membrane (>65 kDa) with anti-p65 antibody diluted in 2.5 mL blocking buﬀer. Pri- mary antibodies used to detect p65 included: polyclonal rabbit anti-p65 antibody (sc-109,

Lot C1804) 1:200 in blocking buﬀer, polyclonal rabbit anti-p65 antibody (sc-109, Lot J1804)

1:500 in blocking buffer, monoclonal mouse anti-p65 antibody (sc-8008, Lot J1404) 1:100 in blocking buffer. On the other membrane (<41 kDa) β-actin was detected with a monoclonal mouse anti-β-actin antibody, which was diluted 1:20000 in 4 mL blocking buffer.

As secondary antibodies HRP-conjugated anti-rabbit antibody, diluted 1:1500 in 2.5 mL blocking buffer or HRP-conjugated anti-mouse antibody, diluted 1:2000 in 2.5 mL blocking buffer were used. For p65 detection the film was exposed for approximately 1.5 min and for CHAPTER 5. MATERIALS AND METHODS 119

Figure 5.3: Schematic illustration of ponceau S stained Western Blot with molecular weight markers and nuclear protein lysate (lane 1). The membrane was cut between 66 kDa and 45 kDa. P65 was detected on membrane

>65 kDa and β-actin on membrane <41 kDa.

β-actin detection approximately 20 sec. The relative intensity of the bands was determined by densitometric analysis. The intensity of the p65 signal was related to the β-actin band.

The NF-κB translocation was expressed as fold increase of p65 to β-actin in comparison to the control, which was maintained in the solvent (medium or PBS) alone. The activation of NF-κB in each extract was measured in duplicate.

5.3.6 Detection of RAGE Expression

Cell Stimulation

Cell lysates of HEK RA and HEK ut cells were prepared by lysing 1.6x105 cells in a 24-well plate. The cells were washed with PBS, blotted dry and 80 µL protease inhibitor solution was added followed by the addition of 20 µL 5-fold concentrated lysis buﬀer. For complete protein denaturation the samples were heated for 5 min at 95 °C. Lysates were stored at

-20 °C.

Immunochemical Detection of RAGE

RAGE was detected in a Western Blot using a mouse monoclonal anti-RAGE antibody

(A11) which was obtained from Dr. Weigle, Technische Universit¨atDresden. The antibody 120 CHAPTER 5. MATERIALS AND METHODS was raised against the two constant-like domains (c1 and c2) of human RAGE [Srikrishna et al., 2002]. 5 µL cell lysate was subjected to gel electrophoresis and Western Blot as described above (Chapter 5.3.4). The membrane was incubated with the primary antibody,

1:100 diluted in 5 mL blocking buﬀer. As secondary antibody HRP-conjugated anti-mouse antibody, diluted 1:2000 in 2.5 mL blocking buﬀer was used. Human RAGE (42 kDa) migrating as a band at 60 kDa was visualized with ECL. sRAGE (35 kDa) was obtained from the same source like RAGE and was used as a positive control in the RAGE Western

Blot migrating as a band at 60 kDa.

5.3.7 Determination of Caspase-3 Activation

Cell Stimulation

NR8383 macrophages (2.0x106) were stimulated in PBS in a total volume of 1 mL in a

24-well for between 4 h and 6 h. As a positive control for cleaved caspase-3, the cells were stimulated with 1 µM staurosporine, which was dissolved in dimethylsulfoxide (DMSO).

Protein Extraction from Cell Culture

Floating cells of the NR8383 macrophages suspension cell line were collected by centrifugation and washed with 0.5 mL PBS. Cell lysates were prepared by resuspending the cells in

40 µL protease inhibitor solution followed by the addition of 10 µL 5-fold concentrated lysis buﬀer. The corresponding adherent cells were lysed by transferring the prepared ﬂoating cell lysate into the well. For complete protein denaturation the samples were heated for

5 min at 95 °C. Lysates were stored at -20 °C.

Immunochemical Detection of Cleaved Caspase-3

10 µL cell lysate was subjected to gel electrophoresis and Western Blot as described above

(Chapter 5.3.4). Caspase-3 (35 kDa) and active fragments of caspase-3 resulting from cleavage at aspartic acid 175 (cleaved Caspase-3, 17/19 kDa) were detected in a Western CHAPTER 5. MATERIALS AND METHODS 121 blot using a rabbit monoclonal anti-Caspase-3 antibody diluted 1:1000 in 5 mL blocking buﬀer. HRP-conjugated anti-rabbit antibody, diluted 1:1500 in 5 mL blocking buﬀer was used as secondary antibody. Cleaved caspase-3 and caspase-3 were visualized with ECL.

5.3.8 Cell Viability Assays

MTT- and Alamar blue assay were used for measuring cell viability. The principle of the MTT assay is based on the reduction of MTT by mitochondrial enzymes of viable cells, which cleave the yellow tetrazolium salt to purple formazan crystals [Mosmann, 1983]. After dissolving the crystals the absorbance can be measured. In contrast to MTT, Alamar blue is a non-toxic dye and the assay is based on the ability of viable cells to reduce non-fluorescent resazurin into a fluorescent product (resorufin).

5.3.8.1 MTT Assay

For determination of MRP-induced cytotoxicity in NR8383 macrophages, 1.5x106 cells were stimulated in 1 mL PBS in a 24-well plate for 2.5 h. In some samples, the cells were pre- incubated with 150 U/mL catalase for 10 min before adding the MRPs. Floating cells were collected by centrifugation in a 15 mL tube (1500 rpm, 2 min), washed with 1 mL

PBS and resuspended in 0.5 mL MTT solution. The suspension was combined with the remaining adherent cells and incubated for 3 h at 37 °C. Afterwards, the MTT solution was removed after centrifugation (1500 rpm, 2 min) and the remaining formazan crystals were resuspended in 0.5 mL isopropanol/1N HCl (25:1), transferred back to the corresponding well with the formazan crystals from the adherent cells and dissolved while gently shaking for 10 min. The absorbance was determined at 595 nm in a 96-well plate using a microplate reader. Isopropanol/1N HCl was used as blank and control cells maintained in PBS were considered as 100 % cell viability. 122 CHAPTER 5. MATERIALS AND METHODS

5.3.8.2 Alamar Blue Assay

Cells were incubated with 0.1 mL Alamar blue solution for 1.5 h in a 96-well plate. The

ﬂuorescence was read at wavelengths of 545 nm for excitation and 595 nm for emission.

Where required, cells could be further grown by re-exchanging the Alamar blue solution with 0.2 mL medium. For that purpose the assay was performed under sterile conditions.

5.3.9 Determination of NO Production

− The Griess reaction was used to measure nitrite (NO2 ), which is spontaneously formed from NO [Dusse et al., 2005].

Cell Stimulation

NR8383 macrophages (3x104 cells/0.2 mL medium) or RAW 264.7 macrophages (5x104 cells/-

0.2 mL medium) were cultivated in a 96-well plate for 4 h. The cells were stimulated with

Maillard reaction mixture in medium for 3 h. As a positive control, INF-γ was added to the cells in medium (50 - 500 U/mL). Cells were washed twice with PBS and cultivated for additional 48 h in medium. In some experiments, the medium was removed after MRP- stimulation and INF-γ was added in the indicated concentrations for 48 h to the cells. To inhibit AGE-induced NO expression the cells were incubated prior to AGE-stimulation with

1 µg/mL polyclonal goat anti-RAGE antibody (Santa Cruz) for 3 h.

Quantiﬁcation of Nitrite

− NO2 was determined by mixing an equal amount of cell culture supernatant (50 µL) with

Griess reagent in a 96-well plate. After 10 min the absorbance was read at 540 nm using

− a microplate reader. NO concentrations were determined as NO2 , using NaNO2 dissolved in medium as a standard at concentrations ranging from 20 - 100 µM. CHAPTER 5. MATERIALS AND METHODS 123

5.3.10 Cytokine Analysis

5.3.10.1 Bio-Plex Cytokine Assay

NR8383 macrophages were stimulated according to the conditions used for determining

NF-κB translocation as described before (Chapter 5.3.5) for 4 h. As a positive control for an inflammatory response the cells were stimulated with 100 ng/mL LPS. Analysis of cytokines was performed using the Bio-Plex Cytokine assay (Bio-Rad). The Bio-Plex system enables the simultaneous analysis of different biomolecules, such as cytokines, in a single microplate well (http://www.bio-rad.com/bioplexsystem/). The assays contain dyed beads conjugated with monoclonal antibodies specific for a target antigen, which is allowed to react with a sample plus a secondary, or detection, antibody in a microplate well (Fig. 5.4).

Unbound detection antibodies are removed through the ﬁlter bottom of the microplate well.

Multiplex assays can be created by mixing bead sets with diﬀerent conjugated antibodies to simultaneously test for many analytes in one sample. The assay solution is drawn into the

Bio-Plex array reader, which illuminates and reads the sample (Fig. 5.5). When a red diode

”classification” laser (635 nm) in the Bio-Plex array reader illuminates a dyed bead, the bead´s fluorescent signature identifies it as a member of the 100 possible sets (for example, an IL-2 capture antibody coupled to bead # 36). A green ”reporter” laser (523 nm) in the array reader simultaneously excites a fluorescent reporter tag bound to the detection antibody in the assay. The amount of green fluorescence is proportional to the amount of analyte captured in the immunoassay. In the present work the cell culture supernatants were simultaneously analyzed for IL-1α, IL-1β, IL-6, IL-10 and TNF-α with the Bio-Plex

Cytokine assay. The standard curves were prepared in medium with concentrations ranging from 0.49 and 32000 pg/mL cytokine.

5.3.10.2 IL-6 Elisa

For IL-6 determination 1x106 cells were cultivated for 4 h and stimulated in 2 mL medium in a 12-well plate for 17 h. As a positive control for IL-6 expression the cells were stimulated 124 CHAPTER 5. MATERIALS AND METHODS

Figure 5.4: Illustration of the Bio-Plex sandwich immunoassay in a filter bottom well. The dyed beads are conjugated with antibodies, which bind the antigens. Detection antibodies with fluorescent reporter tags are bound to the antigen whereas unbound detection antibodies are removed through the filter bottom.

Figure 5.5: Bio-Plex array reader with two lasers: A red ”classiﬁcation” laser for bead identiﬁcation and a green

”reporter” laser. CHAPTER 5. MATERIALS AND METHODS 125 with 10 or 100 ng/mL LPS. The cell culture supernatant (2 mL) was lyophilized, stored at

-80 °C and resuspended in 200 µL distilled water. Concentration of IL-6 in the reconstituted solution was determined in duplicate with a rat IL-6 ELISA according to the instruction manual (R & D Systems). The standard curve was prepared in a concentration range of

50 - 8000 pg/mL IL-6.

5.3.11 Determination of Hydrogen Peroxide Concentration (FOX Assay)

The perchloric acid (PCA)-FOX (ferrous oxidation xylenol orange) assay [Gay and Gebicki,

2002] was used to determine H2O2 concentrations in coﬀee or raw coﬀee extract, Maillard reaction mixtures and control reaction mixtures. The assay is based on the principle of hydrogen peroxide-induced oxidation of ferrous (Fe2+) to ferric (Fe3+) which forms a coloured complex with xylenol orange. A blank was subtracted from each sample, which was treated in the same way except for the addition of catalase. The samples were diluted in PBS to a total volume of 0.5 mL in a 24-well plate and where required incubated at 37 °C, 5 %

CO2 for the indicated times. H2O2 concentrations were measured in duplicate. 60 µL of diluted sample was placed in 0.5 mL microcentrifuge tube and 20 µL PBS or for preparing the blank 10 µL PBS and 10 µL catalase (1000 U/mL) were added and mixed. The blank was incubated for 15 min, allowing the catalase to convert H2O2 into water and oxygen.

20 µL of sample or blank were added in a 96-well plate in triplicate and diluted 1:10 with

FOX reagent. After 30 min incubation with shaking, the absorbance was read at 550 nm with a microplate reader. H2O2 content was calculated with an external calibration graph.

The standard curve was prepared in a range between 22 and 453 µMH2O2 in PBS.

5.3.12 Dipeptide Spot Library and Dipeptide/Amino Acid Spot Membranes

Peptides can be synthesized punctual on a planar cellulose membrane with the SPOT- method. This type of solid phase peptide synthesis allows the parallel synthesis of membrane-bound peptides on deﬁned spots. A dipeptide spot library containing all 400 combinations of the 20 natural l-amino acids was provided from Dr. Rothemund, Inter- 126 CHAPTER 5. MATERIALS AND METHODS

Figure 5.6: Selectively deprotection of the membrane-bound dipeptide side chains and the N-terminus [Munch et al.,

1999] disziplinäres Zentrum fürklinische Forschung, UniversitätLeipzig. The dipeptides with the amino acid sequences listed in chapter 2.5.2.1 (Fig. 2.36) were arranged in an array of 25 x 16 spots in a 9 x 7.5 cm format. The dipeptides were synthesized on membrane-coupled

β-alanin-β-alanin anchors using a semi-automatic pipetting workstation (Auto-Spot Robot

ASP 222, ABIMED). β-alanin was coupled spotwise on β-alanin derivatized Whatman 540

ﬁlter paper sheets providing linkers and a grind of spots. The spot-synthesis was conducted with side chain protected Fmoc-amino acids. The selectively deprotection and modiﬁca- tion of the functional groups yield to dipeptides with free amino groups at the N-terminus and blocked side chains (Fig. 5.6 II) or vice versa (Fig. 5.6 III) or free functional groups of the side chains and N-terminus (Fig. 5.6 IV). For dipeptides with blocked N-terminus and free side chains (Fig. 5.6 III) the Fmoc-protecting group of the N-terminus was selectively deprotected and blocked in a subsequent step by acetylation, allowing a selectively deprotection of the side chains.

The dipeptide/amino acid spot membranes containing 8 x 12 spots of membrane-bound arginine-arginine, arginine-lysine, lysine-arginine, lysine-lysine, or membrane-bound arginine, or lysine with deprotected functional groups were also provided from Dr. Rothemund,

IZKF, Universit¨atLeipzig. CHAPTER 5. MATERIALS AND METHODS 127

5.3.12.1 Preparation of MRP-Modiﬁed Dipeptide Spot Library and Dipeptide/Amino

Acid Spot Membranes

The cellulose membranes were washed three times with water, sterilised in 70 % ethanol for 5 min, and dried under laminar air flow. For the preparation of either MRP-modified side chains (dipeptide spot library) or MRP-modified side chains and N-terminus (dipeptide/amino acid spot membranes) the dipeptide spot library was incubated with 0.5 M ribose and the dipeptide/amino acid spot membranes with 0.5 M ribose, methylglyoxal, or glyceraldehyde in PBS for 2 weeks at 60 °C under sterile conditions. The dipeptide/amino acid spot membranes were also incubated with PBS under the same conditions to serve as a control. Membrane discs (side length 0.4 cm), not containing peptides, were incubated with ribose, methylglyoxal, glyceraldehyde, or PBS under the same conditions as the dipeptide/amino acid spot membranes to serve as a further control. The dipeptide spot library was incubated in a total volume of 10 mL in a petri dish ( 15 cm) and the dipeptides or amino acids from the spot membranes in a ratio of 6 - 8 spots/2 mL solution in an airtight falcon. Finally, the cellulose membranes were washed three times with water and sterilised as described above. The colour intensities of the brown melanoidin-modified spots were quantified by densitometric analysis using the Image MasterTM 2D Platinum software.

5.3.12.2 Determination of Cell Proliferation on MRP-Modiﬁed Dipeptide/Amino Acid

Spot Membranes

The brown coloured melanoidin-modiﬁed or UV-light-visual spots of the dipeptide/amino acid spot membranes were cut out from the membrane as peptide discs (side length 0.4 cm) as illustrated in chapter 2.5.2.2 (Fig. 2.38). The peptide discs with single spots of MRP- modiﬁed or PBS-incubated dipeptides or amino acids and control membrane discs (side length 0.4 cm), not containing peptides, were placed in a 96-well plate (one disc/well) under sterile conditions and 0.1 mL cell culture medium was added. The 96-well plate was incubated in the cell incubator for 10 min to soak the discs with medium prior seeding 128 CHAPTER 5. MATERIALS AND METHODS

5.5x104 cells per well. The cells were allowed to attach to the discs for 2 h before transferring each disc into a new well. Afterwards, cell viability was measured with the Alamar Blue assay as described before (Chapter 5.3.8.2). The number of viable cells on each disc can be expressed as ﬂuorescent signal (amount of resoruﬁn generated after 1.5 h). The Alamar blue solution was removed from the wells and the cells were further grown in 0.2 mL medium.

After a 24 h and 48 h culture period the number of viable cells on the discs was again determined by ﬂuorescence intensity measurement. The ﬂuorescent signal was related to the one after 2 h and cell proliferation on the PBS-incubated control discs was considered as 100 %.

Viable cells on the discs could be visualized with MTT solution resulting in blue stained cells on yellow-coloured membranes. Brieﬂy, the medium was removed from the cells and

0.1 mL MTT solution was added for 1 h.

5.4 Statistical Analysis

Each experiment was performed in duplicate. The data are reported as mean ± standard deviation of independent experiments or of one experiment (repeat determination). Statisti- cal significance of the data was calculated using unpaired, two-tailed t-test with significance levels, * p<0.05 moderate significant, ** p<0.01 significant, *** p<0.001 highly significant.

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List of Tables

2.1 H2O2 concentration in the unheated reaction mixture and ribose or lysine

heated alone ...... 40

2.2 H2O2 concentration in coﬀee and Maillard reaction mixture dependent on

the presence of PBS ...... 41

2.3 Colour intensity of Maillard-modiﬁed dipeptide spots ...... 78

5.1 AGE-modiﬁed BSA preparation ...... 110

5.2 List of cultivated cell lines ...... 114 List of Figures 147

List of Figures

1.1 The early events of the Maillard reaction ...... 2

1.2 Chemical structures of AGEs ...... 3

1.3 AGE-RAGE induced signal transduction ...... 6

2.1 Cellular NF-κB activation ...... 16

2.2 Coﬀee-induced NF-κB activation in macrophages ...... 18

2.3 MRP-induced NF-κB activation in macrophages ...... 19

2.4 Heating time-dependent MRP-induced NF-κB activation in macrophages . 20

2.5 Concentration-dependent MRP-induced NF-κB activation in macrophages . 21

2.6 Melanoidin-induced NF-κB activation in macrophages ...... 22

2.7 RAGE-Western Blot ...... 23

2.8 MRP-induced NF-κB activation in HEK RA and HEK ut cells ...... 25

2.9 Catalase inhibits the MRP-induced NF-κB activation in macrophages . . . 26

2.10 Catalase inhibits the MRP-induced NF-κB activation in HEK RA and HEK

utcells...... 28

2.11 Catalase inhibits the coﬀee-induced NF-κB activation in macrophages . . . 29

2.12 H2O2-induced NF-κB activation in HEK RA and HEK ut cells ...... 31

2.13 H2O2-induced NF-κB activation in HEK ut cells is dependent on the stimu-

lation time ...... 32

2.14 H2O2-induced NF-κB activation in macrophages ...... 32

2.15 Mechanism of superoxide radical formation by Amadori product ...... 35 148 List of Figures

2.16 Reduction of oxygen to ROS ...... 36

2.17 H2O2 concentration in coﬀee and in raw coﬀee extract ...... 37

2.18 H2O2 concentration in Maillard reaction mixture ...... 39

2.19 Free metal ion-independent H2O2 generation in Maillard reaction mixture . 41

2.20 Free metal-ion independent MRP-induced NF-κB activation in macrophages 43

2.21 Structures of MRPs ...... 49

2.22 C4-aminoreductone-induced NF-κB activation in macrophages ...... 50

2.23 Catalase inhibits C4-aminoreductone-induced NF-κB activation in macrophages 51

2.24 AGEs do not induce NF-κB activation in macrophages ...... 52

2.25 AGEs do not induce NF-κB activation in HEK RA and HSC T6 cells . . . 53

2.26 AGEs do not augment LPS-induced NF-κB activation in macrophages . . . 54

2.27 AGE-induced NO expression ...... 56

2.28 Anti-RAGE antibody inhibits AGE-induced NO production ...... 57

2.29 Postulated mechanism of aminoreductone autoxidation ...... 58

2.30 MRP-induced production of cytokines in macrophages determined with Bio-

Plex Cytokine assay ...... 65

2.31 IL-6 expression in macrophages determined with ELISA ...... 66

2.32 NO expression in MRP- and INF-γ treated macrophages ...... 67

2.33 MRPs do not show synergistic eﬀects on INF-γ-induced NO release . . . . . 68

2.34 MRP-induced cytotoxicity in macrophages ...... 69

2.35 Cleaved caspase-3 Western Blot ...... 71

2.36 Dipeptide spot library ...... 76

2.37 Maillard-modiﬁed dipeptide spot library ...... 77

2.38 Schematic illustration of the membrane-based cell viability assay ...... 79

2.39 Cell growth on cellulose membrane ...... 81

2.40 Maillard-modiﬁed dipeptide membranes ...... 82 List of Figures 149

2.41 Melanoidin-induced inhibition of cell proliferation determined with dipeptide

membranes ...... 84

2.42 Cell growth on lysine-lysine-derived melanoidins ...... 85

5.1 Calibration of SEC ...... 112

5.2 Schematic illustration of Maillard reaction mixture-fractionation via SEC . 113

5.3 Schematic illustration of ponceau S stained Western Blot ...... 119

5.4 Illustration of the Bio-Plex sandwich immunoassay ...... 124

5.5 Bio-Plex array reader ...... 124

5.6 Selectively deprotection of the membrane-bound dipeptide side chains and

the N-terminus ...... 126 150 Lebenslauf

Lebenslauf

Pers¨onliche Daten

Sonja Muscat

Wilhelmstr. 15

91054 Erlangen

Geb. am 11. 10. 1978 in Nurnberg¨

Schulbildung

1985–1989 Grundschule Lauf

1989–1994 Gymnasium Lauf

1994–1998 Labenwolf Gymnasium Nurnberg¨

(Leistungskurse Deutsch und Wirtschaft-Recht)

Praktikum

1998–1999 Sosioscan Recherche Marketing,

24/25 rue Yves Toudic, 75010 Paris, Frankreich

Hochschulbildung

1999–2003 Studium der Lebensmittelchemie and der Friedrich-Alexander-Universit¨at

Erlangen

2001 Lebensmittelchemische Vorprufung¨

2003 Erste Staatsprufung¨ fur¨ Lebensmittelchemiker Lebenslauf 151

Berufserfahrung

2003–2007 Promotion am Lehrstuhl fur¨ Lebensmittelchemie, Friedrich-Alexander-

Universit¨at Erlangen unter Prof. Dr. M. Pischetsrieder

2003–2005 Stipendium fur¨ Doktoranden der Erika-Giehrl Stiftung

2005–2006 Stipendium fur¨ Doktorandinnen aus dem Hochschul- und Wissenschafts-

programm (HWP)

2005–2006 Forschungsaufenthalt am Comparative Genomics Centre der James

Cook University, Townsville, Australien

2006–2007 Wissenschaftliche Mitarbeiterin am Lehrstuhl fur¨ Lebensmittelchemie,

Friedrich-Alexander-Universit¨at Erlangen

Fremdsprachen (nach Common European Framework of Reference for Languages, CEF)

Englisch - C2 (nahezu muttersprachliche Sprachbeherrschung)

Franz¨osisch - B1 (selbst¨andige Sprachverwendung)

EDV

Betriebssysteme Windows

Anwendungen Excel, Word, PowerPoint, MDL ISIS-Draw, Corel-DRAW,

Sprachen LATEX

Erlangen, im M¨arz 2007