Toxicological and immunological effects of DON and LPS at the intestinal barrier

Toxicological and immunological effects of DON and LPS at the intestinal barrier

Thesis

Submitted in partial fulfilment of the requirements for the degree -Doctor of Veterinary Medicine-

Doctor medicinae veterinariae ( Dr. med. vet. )

by

Amal Abd El-Moniem Ali Halawa

El-Mahalla El-Kubra/Egypt Hannover, Germanny

2012

University of Veterinary Medicine Hannover

1. Referee: Prof. Dr. Gerhard Breves Institute of Physiology

University of Veterinary Medicine Hannover

2. Referee: Prof. Dr. Pablo Steinberg

Institute of Food Toxicology and Analytical Chemistry University of Veterinary Medicine Hannover

Day of the oral examination: 24.05.2012

TABLE OF CONTENTS List of abbreviations

1 Introduction ...11

2 Review of literature ...13

2.1 Trichothecenes ...13

2.1.1 Fungal growth and production of trichothecenes ...13

2.1.2 Types and characters of trichothecenes...14

2.1.3 Action of trichothecenes...16

2.1.4 Toxicokinetics of trichothecenes...17

2.2 Deoxynivalenol ...18

2.2.1 Occurrence and production ...18

2.2.2 Physical and chemical characters ...19

2.2.3 Biochemical mode of action...20

2.2.4 Toxicokinetics and animal sensitivity ...21

2.2.5 Acute toxicity ...23

2.2.6 Chronic and subchronic toxicity ...23

2.2.7 Neural effects of DON ...24

2.2.8 Cytotoxicity of DON...24

2.2.9 Effects of DON on nutrient uptake ...25

2.2.10 Effect of DON on body weight ...27

2.2.11 Immunological effects of DON...28

2.3 Lipopolysaccharide ...30

2.3.1 Occurrence of LPS ...30

2.3.2 Characters and functions of LPS ...31

2.3.3 Chemical structure of LPS ...31

2.3.3.1 Lipid A part ...32

2.3.3.2 The oligosaccharide core...33

2.3.3.3 O-antigen ...33

2.3.4 Mode of action of LPS ...34

2.3.4.1 LPS pathway in mCD14-bearing cells ...34

2.3.4.2 LPS pathway through sCD14 ...36

2.3.4.3 LPS pathway in intestinal epithelial cells ...37

2.3.4.4 Neutralization of LPS...38

2.3.5 LPS and the intestinal barrier ...39

2.3.6 LPS and cytokines ...41

2.4 The interaction between DON and LPS ...41

3 Material and methods ...43

3.1 Animals ...43

3.2 Experimental design ...43

3.3 Electrophysiological measurements ...45

3.3.1 Chemicals and solutions for the electrophysiological experiments ...45

3.3.2 Tissue sampling and preparation...46

3.3.3 Ussing chamber technique ...47

3.3.4 Calculations and Statistical analysis ...49

3.4 DON transport study ...49

3.4.1 Sampling...49

3.4.2 Determination of DON in the buffer samples of Ussing-chambers ...49

3.4.3 Analysis of buffer samples using LC/MSMS technique...50

3.4.4 Determination of plasma DON concentration...51

3.4.5 Determination of plasma TNF-α concentration ...51

3.4.5 Calculations and Statistical analysis ...52

4 Chapter 1: Effects of deoxynivalenol and lipopolysaccharide on electrophysiological parameters in growing pigs ... 53

4.1 Abstract ...55

4.2 Introduction ...55

4.3 Material and Methods...57

4.3.1 Experimental design ...57

4.3.2 Animals ...57

4.3.3 Chemicals ...57

4.3.4 Intestinal tissue sampling and preparation ...58

4.3.5 Ussing technique ...58

4.3.6 Calculations and Statistics...59

4.4 Results ...59

4.4.1 Basic electrophysiological parameters along the small intestines ...59

4.4.2 Acute in vitro effects of DON on nutrient stimulated electrophysiological parameters60 4.4.3 The main effect of DON in feed...60

4.4.4 The main effect of intraperitoneal LPS ...60

4.4.5 Interactions between DON in feed, intraperitoneal LPS challenge and DON in vitro61 4.5 Discussion ...61

4.6 Tables ...66

5 Chapter 2: Intestinal transport of deoxynivalenol across porcine small intestines ..69

5.1 Abstract ...71

5.2 Introduction ...71

5.3 Material and Methods...73

5.3.1 Experimental design ...73

5.3.2 Animals experiment and procedures ...73

5.3.3 Analyses ...74

5.3.3.1 Chemicals ...74

5.3.3.2 Buffer samples...74

5.3.4 Blood samples ...75

5.3.4.1 DON ...75

5.3.4.2 Tumor necrosis factor-alpha (TNF-α) ...75

5.3.5 Calculations and Statistics...76

5.4 Results ...76

5.4.1 Effect of feeding DON on live weight gain ...76

5.4.2 Plasma DON and TNF-α concentrations...76

5.4.3 DON transport study ...76

5.4.3.1 The main effect of in vitro DON concentration ...76

5.4.3.2 The main effect of DON in feed...76

5.4.3.3 The main effect of ip LPS ...78

5.4.3.4 Interaction effects ...78

5.5 Discussion ...78

5.5.1 Effect of oral DON on live weight gain ...78

5.5.2 Plasma DON and TNF-α...78

5.5.3 DON transport study ...79

5.6 Figures and Tables ...83

6 General discussion...84

7 Conclusion...89

8 Summary ...90

9 Zusammenfassung...92

10 References ...94

11 List of figures ...115

12 List of tables...116

List of abbreviations

aw water activity

BPI bactericidal-permeability increasing protein

BW body weight

ºC degree Celsius

D dalton

DAS diacetoxyscirpenol

DNA deoxy-ribonuclic acid

DOM-1 de-epoxy deoxynivalenol

DON deoxynivalenol

elF2α eukaryotic initiation factor 2 α-subunit GPI

glycosyl-phosphatidylinositol

HDL high-density lipoprotein

HPLC high performance liquid chromatography

HPLC-DAD HPLC with diode-array detection

HT-2 HT-2 toxin

IAC immunoaffinity columns

IC₅₀ inhibiting concentration of 50%

IgA immunoglobulin A

IGF1 insulin-like growth factor 1

IGFALS insulin-like growth factor acid-labile subunit

IgG immunoglobulin G

IgM immunoglobulin M

Ikk IκB kinase complex

IL-1 interleukin 1

IL-6 interleukin 6

Ip intraperitoneal

IRAK IL-1 receptor accessory protein kinase

Isc short circuit current

Iv intravenous

IκBα & β inhibitor kappa B proteins- alpha and beta

JNK c-Jun N-terminal kinase

Kd kilodalton

KDO 2-keto-3-deoxyoctonic acid

Kg kilogram

LBP LPS-binding protein

LC-ESI-MS/MS liquid chromatography-electrospray ionization

tandom mass spectrometry

LD50 lethal dose of 50%

LPS lipopolysaccharide

MAPK mitogen activated protein kinase

Mbr millibar

mCD14 cluster of differentiation No.14

(membrane form)

min minutes

ml milliliter

MyD88 the myeloid differentiation factor 88 protein

N number

NAG phosphorylated N-acetylglucosamine

NF-кB nuclear factor kappa B

Ng nanogram

NHLF normal human lung fibroblasts

NIV nivalenol

PAMPs pathogen-associated molecular patterns

PKR double stranded RNA-activated protein kinase

PRRs pattern-recognition receptors

rpm rounds per minute

RPTEC renal proximal tubule epithelial cells

rRNA ribosomal Ribonuclic Acid

SAP serum amyloid P component

sCD14 cluster of differentiation No.14 (soluble form)

SD standard deviation

SGLT1 sodium-glucose/galactose transporter 1

T-2 T-2 toxin

TEER trans-epithelial electrical resistance

TLRs Toll-like receptors

TNF-α tumor necrosis factor alpha

TRADD TNFR1-associated death domain protein

TRAF6 TNF-receptor-associated factor 6

µg microgram

1. Introduction

Trichothecenes are a major group of mycotoxins produced as secondary metabolites mainly by Fusarium species considered as the most economically relevant source of trichothecenes besides other field fungi (ERIKSEN and PETTERSSON 2004). They are commonly found in temperate regions throughout the world and mainly in contaminated cereals such as maize, wheat and corn (ERIKSEN and PETTERSSON 2004; ROTTER et al. 1996) and can induce toxic effects in both, humans and animals (BIAGI 2009). Trichothecenes are potent inhibitors of protein synthesis (ROCHA et al. 2005; FEINBERG and MCLAUGHLIN 1989) and mainly affect the active tissues and cells such as intestinal mucosa, skin and bone marrow (STEYN 1995;

ERIKSEN 2003). The main clinical signs observed from the toxic effect of trichothecenes are feed refusal, weight loss, emesis, skin lesions and immunomodulation.

Deoxynivalenol is considered as one of the most important trichothecenes due to its wide distribution and common occurrence in feedstuffs especially in wheat and barley (SCHOTHORST and EGMOND 2004). It is commonly found accompanied with its derivatives 3- and 15-acetyl DON (ERIKSEN and PETTERSSON 2004). Pigs are the most susceptible animal to DON toxicity (ROTTER et al. 1996) with feed refusal, vomiting and weight losses as the main clinical signs due to their exposure to DON-contaminated feed (PRELUSKY 1997).

Lipopolysaccharide is a component of the outer bacterial membrane of most of Gram- negative bacteria and is responsible for most of pathophysiological disorders accompanied with their infections (MAYEUX 1997). Its releasing in bloodstream can result in immunomodulation, inflammatory reactions and septic shock in severe cases (CHABY 1999).

LPS stimulates monocytes, macrophages and endothelial cells to release the specific mediators cytokines such as TNF-α, IL-1 and IL-6. These mediators play an important role in inflammatory response (CHABY 1999).

The effects of DON and LPS on nutrients absorption were studied in many species like broilers, swine, rabbits, mice and rats (ROTTER et al. 1996; TOMLINSON and BLAKSLAGER

2004; AWAD et al. 2004; ABAD et al. 2001).These studies revealed the ability of each to inhibit intestinal transport of some sugars and amino acids. Other researches examined the synergistic effect between DON and LPS to induce apoptosis or cytokine secretion (PESTKA

and ZHOU 2006; ZHOU et al. 2000).

The present study aimed at:

1- Examination of the effects of different concentrations of DON on electrogenic ion transport of alanine and glucose across different intestinal segments of pigs in vitro.

2- Evaluation of oral as well as in vitro DON and intraperitoneal LPS on the electrogenic ion transport of glucose and alanine across jejunal epithelia of growing pigs.

3- Estimation of DON transport across porcine jejunal tissues in vitro at two different DON concentrations.

2. Review of literature 2.1 Trichothecenes

2.1.1 Fungal growth and production of trichothecenes

More than 150 trichothecenes and their derivatives were identified (GUTLEB et al. 2002). They are produced by a number of fungal genera especially Fusarium species such as F.

sporotrichioides, F. graminearum, F. poae and other genera such as Myrothecium, Stachybotrys, Cephalosporium, Trichoderma and Trichothecium (BIAGI 2009; VISCONTI and BOTTALICO 1983; BENNETT 1987; BENNETT AND KLICH 2003; BRÄSE et al. 2009; SOBROVA et al. 2010), which evoke a toxic response when introduced in low concentrations to higher vertebrates and other animals by a natural route. Fusarium species considered one of the most significant fungal species, because they produce a large number of toxic secondary metabolites called mycotoxins (Table 1), which may affect animal health and production due to its incorporation into animal feed (COULOMBE 1993; MORGAVI and RILEY 2007).

Infection of the plant with fungi mostly occurs in the field and is then carried to the storage units depending on the pre- and post-harvest strategies (CHRISTENSEN andKAUFMANN 1965).

Fungal growth as well as production of trichothecenes is affected by many factors such as the water activity (aw) in the infected plant, the environmental temperatures and atmospheric humidity. Competition with other fungi, insect infestation, oxygen content and rainfall conditions are other factors influencing fungal growth and mycotoxins production (D’MELLO

and MACDONALD 1997; DOOHAN et al. 2003; PATERSON and LIMA 2010). F. graminearum and F. culmorum are important plant pathogens which grow on the crop in the field such as wheat and appear to be related to climatic conditions. F. graminearum grows in warm conditions (EFSA, 2004); it needs about 0.99 aw at 25°C and 0.98 aw at 15°C for its growth, while the optimum growth of F. culmorum is at 0.98 aw and 15°C and at 0.90 aw and 25°C (HOPE et al.

2005).

Trichothecenes distribute worldwide in grains and are detected in corn, wheat, barley, maize and other crops (BINDER et al. 2007; SCHWARZER 2009). Many surveys were conducted for detection of the natural occurrence of trichothecenes in feed and crops (BROGGI et al. 2007;

SCHOTHORST and EGMOND 2004).

Table 1: Some Fusarium species and their related mycotoxins:

Fusarium species Mycotoxins

F. graminearum (Gibberella zeae) DON, ZEN, NIV, FUS, AcDON; DAcDON

F. culmorum DON, ZEN, NIV, FUS, ZOH, AcDON

F. sporotrichioides T2, HT2, NEO, MAS, DAS

F. verticillioides FB1, FB2, FB3

F. poae DAS, NIV, FUS, MAS, T2, HT2, NEO

F. equiseti ZEN, ZOH, MAS, DAS, NIV, DAcNIV,

FUS

F. cerealis NIV, FUS, ZEN, ZOH

F. proliferatum FB1, BEA, MON, FUP, FB2

Modified from LOGRIECO et al. (2002)

Abbreviations: AcDON – Mono-acetyldeoxynivalenols (3-AcDON, 15-AcDON); AcNIV – Mono-acetylnivalenol (15-AcNIV); BEA – Beauvericin; DAcDON – Di- acetyldeoxynivalenol (3,15-AcDON); DAcNIV – Diacetylnivalenol (4,15-AcNIV); DAS – Diacetoxyscirpenol; DON – Deoxynivalenol; FB1 – Fumonisin B1; FB2 – Fumonisin B2;

FB3 – Fumonisin B3; FUS – Fusarenone-X (4-Acetyl-NIV; HT2 – HT-2 toxin; MAS – Monoactoxyscirpenol; MON – Moniliformin; NEO – Neosolaniol; NIV – Nivalenol T2 – T-2 toxin; ZEN – Zearalenone; ZOH – zearalenols (α and β isomers).

2.1.2 Types and characters of trichothecenes

The trichothecene mycotoxins are low molecular weight compounds (about 200-500 D) (PESTKA 2010) containing sesquiterpene rings characterized by a 12, 13-epoxy-trichothec-9- ene nucleus (HUSSEIN and BRASEL 2001) and most of them have a double bond at C9-C10 position (EHRLICH and DAIGLE 1987; GUTLEB et al. 2002) with different numbers of acetyl and hydroxyl groups (ERIKSEN et al. 2002) (Figure 1). They are heat-stable products, not degraded under processing and are not hydrolysed in the stomach after ingestion (ERIKSEN

2003; ZHOU et al. 2008).

There are four types of trichothecenes according to their structures. Type A trichothecenes which lacks a carbonyl group at C8 position (ZHOU et al. 2008) such as T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS), 15-monoacetoxyscirpenol (MAS), neosolaniol (NEO) and scirpentriol (SCP) and have a hydrogen or ester type side chain in stead (BENNETT and KLICH

2003; ZHOU et al. 2008). Type B trichothecenes which have a carbonyl group at C8 position such as deoxynivalenol (DON) including its acetylated derivatives, nivalenol (NIV) and fusarenone-X (4-acetylnivalenol, FUS) (ROTTER et al. 1996; PESTKA 2007; ROCHA et al. 2005;

BOTTALICO 1998). Type C trichothecenes are characterised by a second epoxy group at C-7, 8 or C-9, 10 positions such as crotocin and are produced by Trichotheceum roseum and Myrothecium verrucaria (ROCHA et al. 2005). Type D trichothecenes or macrocyclic trichothecenes have a macrocyclic ring between C4 and C15 position such as verrucorins and satratoxins and are produced mainly by Myrothecium, Stachybotrys and Trichothecium species (GUTLEB et al. 2002; PESTKA 2007; ZHOU et al. 2008; JARVIS and MAZZOLA 1982;

BENNETT and KLICH 2003; BRÄSE et al. 2009).

Figure 1: General chemical structure of trichothecenes (SCHOTHORST and JEKEL 2001).

Substituents from R1 to R5 are given in table 2.

Table 2: Chemical structures of substituents from R1 to R5 of type A and B trichothecenes

Type Trichothecene R1 R2 R3 R4 R5 Chemical

formula

A Diacetoxyscirpenol OH OCOCH3 OCOCH3 H H C19H26O7

A Neosolaniol OH OCOCH₃ OCOCH₃ H OH C₁₉H₂₆O₈

A T-2 Toxin OH OCOCH3 OCOCH3 H OCOCH2CH(CH3)2 C24H34O9

A HT-2 Toxin OH OH OCOCH₃ H OCOCH₂CH(CH₃)₂ C₂₂H₃₂O₈

B Deoxynivalenol OH H OH OH =O C15H20O6

B 3-Acetyldeoxynivalenol OCOCH3 H OH OH =O C17H22O7

B Fusarenon X OH OCOCH₃ OH OH =O C₁₇H₂₂O₈

B Nivalenol OH OH OH OH =O C₁₅H₂₀O₇

2.1.3 Action of trichothecenes

Trichothecenes are potent inhibitors of eukaryotic protein synthesis (BENNETT and KLICH

2003; SCHWARZER 2009; EFSA 2004; ROCHA et al. 2005) affecting mainly on rapidly dividing cells like gastrointestinal and immune cells (ERIKSEN 2003). Depending on the substituents, trichothecenes have different effects on protein synthesis in whole cells. One group inhibits the initial step of protein synthesis (I-type) and the other group inhibits the elongation and termination steps of protein synthesis (E-type) (EHRLICH and DAIGLE 1987).

Trichothecenes with substituents at both C-3 and C-4 predominantly inhibit polypeptide chain initiation; whereas substitution at either C3 or C4 position enhances inhibition of peptidyl transferase or called inhibitors of peptide chain elongation (FEINBERG and MCLAUGHLIN

1989; ROTTER et al. 1996; EHRLICH and DAIGLE 1987). Other effects of trichothecenes were reported such as their ability to activate mitogen activated protein kinase (MAPK), induction of apoptosis (SHIFRIN and ANDERSON 1999), production of cytokines (DONG et al. 1994;

MEKY et al. 2001), suppression of immunity (JOHANNISSON et al. 1999) and inhibition of both DNA and RNA synthesis (ROCHA et al. 2005).

2.1.4 Toxicokinetics of trichothecenes

Trichothecenes, in general, are rapidly and efficiently absorbed from the gastrointestinal tract (CAVRET and LECOEUR 2006; HEIDTMANN-BEMVENUT et al. 2011) and may rapidly appear in plasma indicating the stomach and small intestines as the major abosorption sites (DÄNICKE et al 2004a). Their metabolism occurs in liver and digestive tract (CAVRET and LECOEUR 2006).

De-oxygenation or de-epoxydation is a significant method by which trichothecenes lose its toxicity (ERIKSEN et al. 2004; ZHOU et al. 2008) (Figure 2). DAS was completely biotransformed by the intestinal microorganisms from rats, cattle and swine to de-acetylated de-epoxydated products (SWANSON et al. 1988). Intestinal microflora transforms DON to de- epoxy-DON and DAS to de-epoxy-MAS and de-epoxy-scirpentriol and transforms T-2 toxin to de-epoxy-HT-2 (SWANSON et al. 1988). In farm pigs, the faecal incubate were able to convert DON and NIV to de-epoxy-products and de-acetylate 3-acetylDON (ERIKSEN et al.

2002). De-epoxydation is an effective reaction through which the toxicity of the trichothecene is reduced or lost. De-epoxy T-2 toxin was found to be 400 times less toxic than T-2 toxin in the rat skin irritation assay (SWANSON et al. 1988). The IC₅₀ of de-epoxy DON was 55 times higher than that of DON and the IC₅₀ of de-epoxy NIV was 54 times higher than that of NIV (ERIKSEN et al. 2004).

In ruminants, detoxification reactions occur in rumen mediated by ruminal microorganisms that make ruminants tolerate the toxic effect of trichothecenes (YIANNIKOURIS and JOUANY

2002). In chickens, the intestinal microorganisms transformed DAS into MAS and SCP via deacetylation reactions (SWANSON et al. 1988) and that of the large intestines transformed DON to DOM-1 (HE et al. 1992) via de-epoxydation reactions. Elimination of trichothecenes occurs mainly in urine and faeces without accumulation in tissues (ERIKSEN and PETTERSSON

2004).

Figure 2: General chemical structure of De-epoxy trichothecene (ERIKSEN et al. 2004)

2.2 Deoxynivalenol

2.2.1 Occurrence and production

Deoxynivalenol is a secondary metabolite produced mainly by Fusarium fungi especially Fusarium graminearum and Fusarium culmorum (VISCONTI and BOTTALICO 1983; BAKAN et al. 2002; BOTTALICO 1998; COULOMBE 1993). Those fungi grow on cereals such as corn, wheat, barley, maize and rice and frequently contaminate the grains either in the field or in the storage unit (TANAKA et al. 1990; ABBAS et al. 1985; MEGALLA et al. 1986; EFSA, 2004;

RICHARD 2007; FDA 2010; ERIKSEN and PETTERSSON 2004; PESTKA 2010). DON was the predominant type of Fusarium toxins that exist in cereals and cereal-products (REINHOLD and REINHARDT 2011). HOPE et al. (2005) estimated the ecological requirements for production of DON in fungal cultures and found that DON was produced at 0.99 aw and 15°C and at 0.98 aw

and 25°C by F. graminearum and at 0.97 aw and 15°C and at 0.99 aw and 25°C by F.

culmorum.

DON has two acetylated derivatives; 3- and 15-mono-acetyl DON (EFSA 2004) which are found in lower concentrations in accompany with DON and are mostly produced from Fusarium strains that produced DON. In vitro studies showed that the acetylated forms of DON were less toxic than DON (ERIKSEN 2003).

2.2.2 Physical and Chemical characteristics

DON is a polar organic compound. Its chemical name is 12, 13-epoxy-3α,7α,15- trihydroxytrichothec-9-en-8-one with 3 free hydroxyl groups in its molecule (Figure 3). DON is stable at high temperatures ranging from 120 to 210°C and can resist processing and boiling (SOBROVA et al. 2010; LARSEN et al. 2004). The amount of DON reduction to less toxic products during thermal food processing is highly variable and depends on the respective processing conditions (BRETZ et al. 2006; FDA 2010). The Food and Drug Administration (FDA) established the advisory levels of DON in food and feed as shown in table 4.

Table 3: Physico-chemical characters of Deoxynivalenol (SOBROVA et al. 2010):

Property Information

Name Deoxynivalenol (DON), Vomitoxin

IUPAC name 12,13-epoxy-3α,7α,15-trihydroxytrichothec-

9-en-8-one

Molecular formula C15 H20 O6

Molar mass 296.32 g/mol

Physical state Colourless fine needles

Boiling point 543.9±50.0°C

Melting point 151-153°C

Flash point 206.9±2.5°C

Vapour pressure 4.26x10^-14 @ 25°C

Soluble in: Polar organic solvents (e.g., aqueous

methanol, ethanol, chloroform, acetonitrile, and ethyl acetate) and water

Figure 3: Chemical Structure of Deoxynivalenol (ROTTER et al. 1996)

Table 4: The advisory levels of DON for grains and grain by-products consumed by animals according to FDA, 2010 are:

Animal species Advisory guidelines of DON

- Ruminating beef and feedlot cattle older than 4 months

- Not exceed 10 ppm in total ration

- Ruminating dairy cattle older than 4 months - Not exceed 5 ppm

- Chickens - 10 ppm, with recommendation that such ingredient not exceed 50% of chickens’ diet

- Swine - 5 ppm, with recommendation that such ingredient not exceed 20% of their diet

- Other animals - 5 ppm, with recommendation that such ingredient not exceed 40% of the diet

2.2.3 Biochemical mode of action

DON is known to affect the gastrointestinal tract and the immune system (ROTTER et al.

1996). The inhibitory activity of DON requires both the presence of an unsaturated bond at C9-C10 position and the integrity of the 12, 13-epoxy ring (ROTTER et al 1996).

Deoxynivalenol binds to 60S subunit of eukaryotic ribosome (FEINBERG and MCLAUGHLIN

1989; KOUADIO et al. 2005) resulting in interfering with the activity of peptidyl transferase and inhibition of protein synthesis (ROTTER et al.1996; AWAD et al. 2008b; EFSA 2004; DÖLL

et al. 2009b; VAN DE WALLE et al. 2010a). It inhibits peptide-chain elongation due to lack of

substituents at C-4 position (EHRLICH and DAIGLE 1987). There are other mechanisms which were suggested to explain the inhibitory effect of DON on protein synthesis, such as induction of damage in 28s rRNA (LI and PESTKA 2008) or induction of double stranded RNA-activated protein kinase (PKR) and eukaryotic initiation factor 2 α-subunit (eIF2α) phosphorylation resulting in degradation of the latter protein in RAW 264.7 murine macrophage cells (ZHOU et al. 2003b). The IC50 of DON in protein synthesis and DNA synthesis were 5 and 1.7 µM, respectively in Caco-2 cells (KOUADIO et al. 2005). DON increases the activities of NF-кB resulting in releasing of the pro-inflammatory cytokines such as TNF-α and IL-6 (EFSA 2004) and inhibited cell proliferation (SERGENT et al. 2006).

2.2.4 Toxicokinetics and Animal sensitivity

The metabolism, absorption, distribution, and elimination of DON differ among different species (PESTKA 2007; KÖNIGS et al. 2007). DON loses its 12, 13-epoxide ring and is transformed by rumen microorganisms to a non toxic metabolite (DOM-1) which was detected in the urine and faeces of animals (Figure 4) (KING et al. 1984). De-epoxy-DON is produced via the activity of intestinal or ruminal microorganisms (YOSHIZAWA et al. 1983;

PESTKA 2007; HE et el. 1992; CộTÉ et al. 1986). In pigs, the absorption of DON was very rapid after intragastric dosing of ¹⁴C labelled-DON; it could be detected in plasma within 15- 30 min (PRELUSKY et al. 1988). Other studies agreed with this result and detected DON in plasma after 20 min of feeding pigs (ERIKSEN 2003) and the peak plasma concentration reached 3h after feeding. The majority of ingested DON in pigs was absorbed rapidly from the proximal parts of the small intestine (DÄNICKE et al. 2004a); the maximum serum concentration of DON reached 4.1h after oral exposure of DON naturally-contaminated wheat (4.2 mg DON/kg feed). In mice, the plasma concentration of DON peaked within the first hour (AMUZIE et al. 2009) then declined rapidly. DON reached peak plasma concentrations within 15-30 min after oral administration in mice (AMUZIE et al. 2008; PRELUSKY et al.

1988). The amount of ¹⁴C DON recovered in plasma of rats was highest at 8h after a single oral dose (MEKY et al. 2003). Regarding to elimination of DON, most of the orally administered DON in pigs is eliminated in urine as DON metabolites and much smaller amounts of de-epoxy DON metabolites (DÄNICKE et al. 2004b). When DON was applied

either orally or intravenously to pigs, residues do not appear to accumulate in tissues to any appreciable extent (PRELUSKY and TRENHOLM 1991; 1992). The microflora of the lower gut of the pigs (ceacum, colon and rectum) are able to transform DON to a less toxic metabolite (KOLLARCZIK et al. 1994), while duodenum and jejunum showed no transforming activity.

Another form of DON detoxification is through glucuronide conjugation in liver (ERIKSEN

2003). The glucuronide conjugate was found in plasma and urine of pigs fed on 3-acetyl DON, and the de-epoxydated form was detected in their faeces (ERIKSEN 2003). The glucuronide conjugated form of DON was detected also in human urine samples as well as in rat urine (MEKY et al. 2003). At the cellular level, the conjugated form was found in porcine hepatocytes (DÖLL et al. 2009b). After intragastric and intravenous administration of ¹⁴C- labelled DON in swine, a small amount of DON-glucuronide conjugate (< 5%) was detected in urine and bile (PRELUSKY et al. 1988).

The susceptibility of animals to DON vary according to many factors, such as dose, duration of exposure, the method of administration and the species involved (ROTTER et al.1996;

KÖNIGS et al. 2007). Swine are the most sensitive species to DON and poultry are more tolerant (ROTTER et al. 1996; KOLF-CLAUW et al. 2009; ERIKSEN 2003). This tolerance could be attributed to the lower degree of absorption into plasma and tissues as well as the rapid clearance without accumulation in tissues and eggs (ROTTER et al. 1996; PRELUSKY et al.

1986). Ruminants are relatively insensitive to DON, probably due to the ability of ruminal micro-organisms to detoxify DON under anaerobic conditions (ROTTER et al. 1996; CộTÉ et al. 1986; SWANSON et al. 1987). The order of decreasing sensitivity generally is pigs > mice >

rats > poultry ~ ruminants (PESTKA 2007).

Figure 4: Chemical Structure of DON-metabolite (DOM-1) (YOSHIZAWA et al. 1983)

2.2.5 Acute and subacute toxicity

Extremely high doses of DON can cause shock-like death (PESTKA 2007; SOBROVA et al.

2010). The toxicity of DON is characterized by vomiting (FORSYTH et al. 1977; WILLIAMS et al. 1988; PESTKA et al. 1987), diarrhea and feed refusal (EFSA 2004). DON is one of the more potent trichothecenes that has emetic activity (COPPOCK and JACOBSEN 2009). The minimum emetic oral dose in swine is about 50-200 µg/kg BW (LARSEN et al. 2004). After ip injection the minimum emetic dose was 50 µg/kg BW and 100 to 200 µg/kg BW after oral administration (FORSYTH et al., 1977). Necrosis was observed in gastrointestinal tract, bone marrow and lymphoid organs after acute intoxication (FORSELL et al. 1987). Pestka et al.

(1987) found that the minimum dose that induced emesis was 100.µg/kg BW following oral and ip administration for both DON and 15-acetyl DON. LD₅₀ for oral DON in mice was 78 mg/kg and that after ip administration was 49 mg/kg and for 15-acetylDON was 34 and 113 mg/kg, respectively (FORSELL et al. 1987). While THOMPSON and WANNEMACHER (1986) estimated LD₅₀ for DON after ip injection at 43 mg/kg and that after s/c injection was 45 mg/kg. The respective values for 3-acetyl DON were 54 and 59 mg/kg, respectively.

2.2.6 Chronic and subchronic toxicity

The most common effects of prolonged feeding of DON to experimental animals are anorexia, reduced weight gain and dysregulation of the growth hormone (PESTKA 2010).

DON at 1-2 ppm resulted in partial feed refusal in pigs ingesting naturally-contaminated feedstuffs, whereas 12 ppm caused complete feed refusal (ROTTER et al 1994; YOUNG et al.

1983). No lesions were observed in the gut of pigs fed up to 43 ppm dietary DON for 21 day (YOUNG et al. 1983). Subchronic exposure to 10 ppm DON for 6 weeks resulted in reduced body weights in mice (HUNDER et al. 1991). AMUZIE and PESTKA (2010) suggested that the retardation of growth that accompanied with subchronic exposure of mice to DON (20 ppm) in feed was due to downregulation of hepatic insulin-like growth factor acid-labile subunit (IGFALS) mRNA expression and suppression the levels of circulating insulin-like growth factor 1 (IGF1) and IGFALS with increased levels of DON in plasma.

2.2.7 Neural effects of DON

DON is transported to the brain and stimulates dopaminergic receptors inducing vomiting, hence the name vomitoxin (SOBROVA et al. 2010). Emesis and anorexia are mediated by the serotonergic system in the central nervous system (CNS) or via the peripheral action on serotonin receptors (LARSEN et al. 2004).

2.2.8 Cytotoxicity of DON

At the cellular level both, lymphocytes and fibroblasts were found to be the most DON- sensitive cell types (ROCHA et al. 2005) as well as epithelial cells have also been considered to be highly susceptible to trichothecenes (ERIKSEN 2003). DON was found to reduce the viability of human intestinal Caco-2 cells at concentration of 1 µM and significantly at 10 µM in a dose-dependent manner (KOUADIO et al. 2005). DON induced cell death to the epithelial cell line HEp-2 (HeLa-derived larynx epithelium) over 80% after 2 days at a concentration of 100 ng/ml (CALVERT et al. 2005), however, the inhibitory effect of DON reached more than 80% at 1000 ng/ml for 2 days and at 300 ng/ml for 4 days in HeLa cell line (cervical epithelium), reflecting higher sensitivity of HEp-2 cells to DON than HeLa cell line. A reduction in cell counts was observed at in vitro DON concentration of 2000 and 4000 ng/ml in IPEC-1 and IPEC-2 cell lines (non-transformed intestinal porcine epithelial cell lines) (DIESING et al. 2011a). In IPEC-J2 cell line the basolateral application of DON at the same concentrations significantly reduced the cell counts after 72h incubation period with significant reduction in TEER after 24h incubation time (DIESING et al. 2011b). Reduction in the living cells was observed in renal proximal tubule epithelial cells (RPTEC) and in normal human lung fibroblasts (NHLF) after exposure to different concentrations of DON (KÖNIGS et al. 2007), with reduction approximately 50% in cell viability after 48h in both RPTEC and NHLF at DON concentration of 100 µM. In the same study, DON induced apoptosis in NHLF at concentrations from 1 to 25 µM DON. On the contrary, the renal proximal tubule epithelial cells (RPTEC) showed necrotic signs with increasing DON concentrations and time of incubation but the normal lung fibroblasts did not. The viability of unstimulated porcine

PBMC cells was reduced at DON concentration of 1.4 and 2.7 µM with maximum drop down to 19% at the latter concentration (DÄNICKE et al. 2010)

DON inhibited the cellular activity of the porcine alveolar macrophages in a dose-dependent manner (DÖLL et al. 2009a) and induced apoptosis in murine lymphatic cells through induction of TNF-α (UZARSKI et al. 2003) and in RAW 264.7 murine macrophages cells (ZHOU et al. 2005b) via intrinsic mitochondrial pathway. Oral (5.7 mg/kg diet) and intravenous (53 µg/kg BW) administration of DON did not affect lymphocyte proliferation (IgA⁺, CD3⁺, CD4⁺ and CD8⁺ cells) in pigs (DÖLL et al. 2006). DON inhibited T- and B- lymphocyte proliferation in a dose-dependent manner in human peripheral blood monocytes at concentrations of 100, 1000 and 5000 ng/ml for 4h (BEREK et al. 2001). No changes were observed in lymphocyte subsets (CD3, CD4, CD8, CD79 alpha and IgA) in piglets treated with oral DON at 0.5 ppm/pig for a week and 1 ppm/pig for another 5 weeks (FERRARI et al.

2009). It was observed that DON concentrations at 50-500 ng/ml at day 5 and at 100-500 ng/ml at day 7 strongly inhibited murine CD4⁺ cell proliferation and showed super induction of interleukins (AZCONA-OLIVERA et al. 1995a).

2.2.9 Effects of DON on nutrient uptake

Deoxynivalenol could cross the intestinal barrier by the transcellular pathway (VIDEMANN et al. 2007) or through the paracellular route probably by passive diffusion (AWAD et al. 2007a;

SERGENT et al. 2006). It modified the paracellular pathway through its effect on the intestinal barrier function as well as a reduction in the expression of the tight junction proteins, claudins, such as claudin-4 (PINTON et al. 2009; 2010) that are involved in the maintenance of the intestinal epithelial barrier via activation of mitogen activated protein kinase (MAPK) dependent pathway. DON altered the gut function of chickens and decreased the absorption of both glucose and amino acids (AWAD et al. 2008c). It inhibited the uptake of glucose across the jejunal epithelium of laying hens (AWAD et al. 2007a) in a mechanism similar to that of phlorizin (a specific SGLT1-inhibitor). DON at 10 mg/kg feed impaired intestinal glucose transport in broilers (AWAD et al. 2004). Whereas addition of microbial feed additives to DON-contaminated feed resulted in increasing the short circuit currents (Isc) after addition of D-glucose to the luminal side of the jejunal mucosa of young chickens, while the Isc in the

DON treated group decreased (AWAD et al. 2009). DON had a local irritant effect on the gastrointestinal tract in rats and mice (ARNOLD et al. 1986). It had a negative effect on the active transport of glucose in the small intestine of broilers (AWAD et al. 2008c). The pre- incubation of the isolated mucosal tissues from jejunum and colon with 10 µg DON /ml omitted the stimulated effect of glucose on Isc (AWAD et al. 2008b). After addition of DON to the mucosal side of intestinal tissues in Ussing chambers, DON reduced the maximal Isc response to glucose across jejunum of broiler chickens and laying hens (AWAD et al. 2008a;

AWAD et al. 2005a). The short circuit currents were not affected by the addition of D-glucose after pre-incubation of tissues with B-trichothecenes (AWAD et al. 2008a). The rapid increase in Isc after addition of glucose to the mucosal side reflected that the active transport of glucose stimulated the active transport of Na⁺ in the isolated rabbit ileum (SCHULTZ and ZALUSKY 1963). But this increase in Isc after addition of glucose was diminished with time (SCHULTZ and ZALUSKY 1964a) due to a gradual decrease in the active sugar and Na⁺ transport interaction. SCHULTZ and ZALUSKY (1964b) cited that the active transport of sugar across the small intestine was a saturated function of the mucosal sugar concentration. The stimulated response of glucose across intestinal mucosa of laying hens was observed in all tissue segments with greater increase in the small intestine especially in jejunum than in the large intestine compared with basal values, suggesting that jejunum was most segment responsible for Na⁺ /D-glucose co-transport (AWAD et al. 2007b). In the same study, a reduction in Isc was observed after addition of 10 µg DON/ml in all segments but especially in duodenum and mid jejunum. The inhibiting effect of DON was greater in duodenum and jejunum than in other intestinal segments. DON inhibited intestinal cell proliferation (HT-29- D4 cells) (MARESCA et al. 2002) in a dose-dependent manner and was absorbed through the intestinal epithelium via simple diffusion without difference in the transport rate of DON from the apical to basolateral direction and vice versa (SERGENT et al. 2006). DON at concentrations of 1, 5 and 10 µg/ml decreased the Isc values across the jejunal mucosa of laying hens in a dose-dependent manner (AWAD et al. 2005a). DON decreased the transepithelial electrical resistance (TEER) after 24h incubation of Caco-2 monolayer at different in vitro DON concentrations with a significant reduction in TEER at 0.5 µg DON /ml and a maximal effect at 4 µg DON /ml (SERGENT et al. 2006). The TEER was decreased by 57% in treated HT-29-D4 cells at 10 µmol DON /L and was abolished at 100 µmol DON /

L (MARESCA et al. 2002). TEER was inhibited in IPEC-1 DON-treated cells (porcine cell lines) by 61% after 48h incubation period (PINTON et al. 2010).

In a study applied on different intestinal segments of White Leghorn chickens, an increase in Isc was observed after addition of 5 mmol/L of D-glucose in all segments with similar behaviour of jejunum, ileum and proximal cecum (20% relative to basal values). Rectum was significantly higher than the rest of the intestine (AMAT et al. 1999).Like sugars, addition of 1 mmol /L of proline on the luminal side resulted in increase in the Isc values across the jejunal mucosa of laying hens (AWAD et al. 2005b). While after addition of 10 µ g DON /ml and after addition of L-proline, the Isc was decreased and returned to the basal values. In mice, glucose transport was reduced significantly at 10 ppm DON without effect on L-leucine uptake (HUNDER et al. 1991).

2.2.10 Effect of DON on body weight

Dietary exposure to 3 ppm DON-naturally contaminated diet reduced feed consumption as well as weight gain in castrated male pigs (PRELUSKY et al. 1994). While in pigs fed on diet with 3 ppm purified DON, the aforementioned signs observed only at the first 2 days and the animals were able to compensate sufficiently during subsequent days. In weanling piglets fed on a naturally DON-contaminated diet containing 2.8 mg DON/kg feed for 4 weeks, a reduction in weight gain was observed only in the first week of the experiment when compared to control animals (WACHÉ et al. 2009). This variance was due to the presence of other compounds in the naturally contaminated diet that act synergistically with DON to induce such effect (PESTKA 2007). Pigs fed on a diet containing 4 mg DON/kg showed 20%

lower feed intake and 13% lower weight gain compared to control ad libitum-fed pigs (ROTTER et al. 1995). Reduction in weight gains throughout the experimental period and reduced feed utilization efficiency were observed in pigs fed on 3.5 mg/kg DON-naturally contaminated oat (BERGSJØ et al. 1993). A significant reduction in body weight gain was observed at the 16^th day and final day in pigs fed on a mixture of 1 ppm DON and 250 ppm ZON for 6-weeks experimental period with a reduction in feed intake (CHENG et al. 2006). In a feeding trial on pigs kept on DON-spiked diet at 4 and 9 ppm for 7 days, feed consumption was reduced by 14% and 46% and weight gain was reduced by 8% and 65%, respectively

especially in the first 3 days (PRELUSKY 1997). On the other hand, no differences were observed in weight gain of pigs fed on 5.7 mg/kg DON-contaminated diet for 5 weeks (ZERULL et al. 2005) and no variation in weight gain in the pigs treated orally with pure 0.5 ppm DON/pig for one week and 1 ppm/pig for another 5 weeks (FERRARI et al. 2009). There was also no great variation in weight gain of piglets on a diet containing up to 1.2 mg DON/kg feed in a feeding trial for 56 day (DROCHNER et al. 2004).

2.2.11 Immunological effects of DON

Trichothecenes are either immunostimulants or immunosuppressors depending on the dose and exposure frequencies (PESTKA et al. 2004; ROTTER et al. 1996). Leukocytes are the target cells for trichothecenes (ZHOU et al. 2003a). Low doses of trichothecenes induce immunostimulatory effects represented by transient upregulation of proinflammatory cytokines and chemokines and elevated serum IgA levels (LARSEN et al. 2004). Exposure of animals to acute high doses of trichothecenes induced severe injuries to certain tissues such as bone marrow, lymph nodes, spleen and intestinal mucosa, resulted in immunosuppression which was evidenced by decreased leukocyte count, reduced serum IgG and IgM levels, inhibition of antibody response and decreased the resistance to pathogens (LARSEN et al.

2004; PESTKA et al. 2004). In pigs treated with both DON and zearalenone for 6 weeks, a decrease in the expression of IFN-γ, TNF-α, IL-1β, IL-2 and IL-6 was observed (CHENG et al.

2006). However, the levels of the proinflammatory cytokines IL-1β, IL-6, IFN-γ, TNF-α, IL- 2, IL-4 and IL-10 mRNA expressions were increased after a single oral exposure to DON at 5 and 25 mg/kg BW in mice (ZHOU et al. 1997). Subchronic levels of DON at 0, 10 and 25 ppm in mice induced the expression of IL-2, IFN-γ, TNF-α and IL-10 (ZHOU et al. 1998). DON induced gene expression for a number of interleukins such as IL-4, IL-6, IL-2 and IL-5 mRNA (AZCONA-OLIVERA et al. 1995a; 1995b; DONG et al. 1994). It was found that DON at concentrations from 2 to 25 mg/kg BW induced plasma IL-1β in mice, and doses at 12.5 and 25 mg/kg BW induced plasma TNF-α level (ISLAM and PESTKA 2006). DON induced mRNA expression of TNF-α and IL-6 upregulation within 1h and peaked within 2h in murine liver (AMUZIE et al. 2009). In murine macrophage cell line, oral DON at 0.1-12.5 mg/kg BW induced TNF-α, IL-6, IFN-γ and IFN-β mRNA at 2 h and fewer amounts at 7h (CHUNG et al.

2003). The expression of IL-8 in transfected-U937 human monocytes was upregulated when the cells were incubated with 1 µg/ml DON for 11h (GRAY and PESTKA 2007). DON was found to upregulate both, IL-2 and IL-8 in Jurkat T-cell line (PESTKA et al. 2005) and to induce phosphorylation of MAPK (CHUNG et al. 2003) in a dose-dependent manner as a mechanism to induce pro-inflammatory cytokine-expression after ribosomal binding in murine spleen, monocytes and macrophages (ZHOU et al. 2003a; BAE and PESTKA 2008). It was proved that DON relied in its mechanism to induce inflammatory mediators on the phosphorylation of both MAPKs (SERGENT et al. 2006; VAN DE WALLE et al. 2010b) and NF- кB (VAN DE WALLE et al. 2010b) evidenced by increase in IL-8 secretion and PGE-2 synthesis capacity of Caco-2 intestinal cells.

The mechanism by which DON can induce proinflammatory cytokine secretion involves inhibition of protein synthesis and results in alteration in MAPK pathway in a process known as a ribotoxic stress response (LASKIN et al. 2002; ZHOU et al. 2005a) that involves disruption or cleavage mainly at the 3’-end of the large 28S ribosomal RNA (rRNA) that functions in peptidyl transferase activity and ribosomal translocation (LI and PESTKA 2008; BEA and PESTKA 2008) resulting in expression of some important genes that integrated in cell proliferation, differentiation and survival. After its binding to the ribosome, DON activates p54 and p46 c-Jun N-terminal kinase (JNK), p38 and extracellular signal-regulated protein kinase 1/2 (ERK1/2) (IORDANOV et al. 1997; MOON and PESTKA 2002; PESTKA et al. 2005;

ZHOU et al. 2005a; 2005b; ISLAM and PESTKA 2006). DON is able to phosphorylate IκB in human Caco-2 cells (VAN DE WALLE et al., 2008); resulting in release of the transcription factor NF-кB from its inhibitor IкB, then NF-кB translocates into nucleus and binds to its binding sites and activates the transcription of specific genes (FINCO and BALDWIN 1995) (A schematic diagram for the action of DON is shown in figure 5). Deoxynivalenol was found to increase the binding activity of NF-кB in vitro (OUYANG et al. 1996) through inhibition of resynthesis of IкBα, a member of IкB family. When the concentrations of DON increased it would convey the cells to apoptosis rather than cytokine upregulation (PESTKA et al. 2005).

Figure 5: A schematic diagram of the mechanisms by which DON can exert its effects

2.3 Lipopolysaccharide (LPS or Bacterial endotoxin)

2.3.1 Occurrence of LPS

LPS is a component of the outer membrane of most Gram-negative bacteria such as Escherichia coli, Salmonella enterica and Haemophilus influenzae (HODGSON 2006;

TOMLINSON and BLIKSLAGER 2004; ALEXANDER and RIETSCHEL 2001; MAYEUX 1997;

ULEVITCH and TOBIAS 1995; WYCKOFF et al. 1998) and is released during bacterial growth (RIETSCHEL et al. 1994; ROSENFELD et al. 2006) or when the bacteria undergo autolysis or lyses. Both humans and animals are susceptible to LPS (ROTH et al. 1997) at different degrees, depending on the disease state, age, presence of a xenobiotic agent and other factors (GANEY and ROTH 2001). The name endotoxin is due to the biological activities that induced by LPS after entering the host (KABANOV and PROKHORENKO 2010). The quantity of LPS

molecules present in each bacterium approximately 2x10⁶ molecules of LPS / Bacterium (RAETZ 1986; RICHARD 1999; MAYEUX 1997).

2.3.2 Characters and Functions of LPS

LPS, as a component of the outer bacterial membrane, acts as a stimulator of the innate immunity of the host but in high doses it can induce septic shock (ALEXANDER and RIETSCHEL 2001). LPS stimulates the immune cells to produce specific mediators in response to its systemic effect like reduced oxygen species, bioactive lipids and proteins such as interleukins (SCHLETTER et al. 1995). The bacterial membrane plays an important role in nutrient transport and mediates the interaction between the bacteria and the host organism (RIETSCHEL et al. 1994). Also LPS acts as a permeability barrier against different external factors like antibiotics (ALEXANDER and RIETSCHEL 2001). LPS is a pyrogenic molecule which may cause increase in the host temperature approximately from 1 to 1.5 °C (TAYLOR et al. 1991; NORIMATSU et al. 1995; WRIGHT et al. 2000; GARCIA-HERRERA et al. 2004; ABAD et al. 2002; JOHNSON and BORELL 1994). LPS is able to form extremely stable aggregates in aqueous environment (GUTSMANN et al. 2007).

2.3.3 The chemical structure of LPS

The lipopolysaccharide consists of a lipophilic hydrophobic region called lipid A part through which LPS is connected to the bacterial cell membrane (KIRSCHNING and BAUER 2001;

ULEVITCH and TOBIAS 1995) and a hydrophilic region called polysaccharide side chain (HITCHCOCK et al. 1986; TOMLINSON and BLIKSLAGER 2004; HODGSON 2006; SCHLETTER et al. 1995; RIETSCHEL et al. 1994). The polysaccharide side chain consists of a core oligosaccharide and the terminal O-specific chain antigen (Figure 6).

Figure 6: The general chemical structure of LPS of Enterobacteria (ALEXANDER and

RIETSCHEL 2001). Abbreviations: GlcN, glucosamine; Kdo, 2-keto-3-deoxyoctonic acid; Hep, D-glycero-D-manno-heptose.

2.3.3.1 Lipid A part

Lipid A region is the lipid component of LPS molecule that is responsible for the biological activity of LPS (RAETZ et al. 2006). It consists of a hydrophilic carbohydrate region and a hydrophobic fatty acid region (KABANOV and PROKHORENKO 2010). The hydrophobic membrane-anchoring region makes LPS forms aggregates in aqueous environment without biological activity (HODGSON 2006). The most active form of lipid A consists of two phosphorylated N-acetylglucosamine (NAG) molecules at positions 1´ and 4´ (Figure 7) (HODGSON 2006; RICHARD 1999); the molecules are connected via a β 1-6 linkage. Lipid A molecule carries about 6 β-hydroxy fatty acids (FAs) as that found in the pathogenic bacteria like Escherichia coli and Salmonella species and attached on carbon 2 via amide linkage and on carbon 3 via ester linkage (RIETSCHEL et al. 1994; HODGSON 2006). All the fatty acids in Lipid A are saturated. Some FAs are attached directly to the NAG dimer and others are esterified to the 3-hydroxy fatty acids that are characteristically present. The structure of Lipid A is highly conserved among Gram-negative bacteria, and is responsible for peptide mediator induction (RIETSCHEL et al. 1994) or the endotoxic activity of Gram-negative bacteria (GALANOS et al. 1985) either the synthetic or the natural lipid A portion.

Figure 7: General chemical structure of lipid A. R1 to R4 represent β-OH fatty acids (HODGSON 2006)

2.3.3.2 The oligosaccharide core

It is a hetero-oligosaccharide portion and in turn subdivided into an outer core and an inner core (RIETSCHEL et al. 1994; MAYEUX 1997). The outer core contains hexoses or neutral sugars (D-glucose, D-galactose and N-acetyl-D-glucosamine). It is the attachment site for O- antigen (RAETZ and WHITFIELD 2002). While the inner core consists of heptoses (Hep) and 2- keto-3-deoxyoctonic acid (Kdo) that considered as a characteristic marker of the LPS molecule and is connected directly with the lipid A part (MAYEUX 1997; ALEXANDER and RIETSCHEL 2001; WYCKOFF et al. 1998).

2.3.3.3 O-antigen

It is a long polysaccharide chain consisting of repeating units of hexoses (up to 50 units) bound together by α1-4 or β1-6 linkages forming either homopolymers or heteropolymers (i.e.

from one to eight monosaccharides) (HODGSON 2006; HITCHCOCK et al. 1986; ALEXANDER

and RIETSCHEL 2001; RAETZ and WHITFIELD 2002). The structure of these repeating units varies among bacterial strains and thus allows structural variability (RIETSCHEL et al. 1994).

So that it acts as a surface antigen that protects the bacteria from phagocytosis and serum

complement, and determines the serological specificity or the bacterial serotype (SCHLETTER

et al. 1995; ALEXANDER and RIETSCHEL 2001). O-antigen chain also differ in length upon which LPS was divided into three types. Smooth LPS in which the O-antigen specific chain shows a full length, semi-rough LPS with restricted O-antigen and rough LPS without O- antigen such as in H. influenzae and is called lipooligosaccharide or LOS (HODGSON 2006;

ALEXANDER and RIETSCHEL 2001). LOS contains inner core from which one or more mono- or oligosaccharide branches extended (RAETZ and WHITFIELD 2002).

2.3.4 Mode of action of LPS

LPS is considered as a molecular pattern related to the pathogen-associated molecular patterns (PAMPs) of the bacterial cell wall structure through which the bacteria can be recognized via specific host receptors called pattern-recognition receptors (PRRs) that detect the pathogenic bacterial structure and specifically bind to the PAMPs (ECKMANN 2004;

COLLIER-HYAMS and NEISH 2005). One group of the PRRs are the toll-like receptors (TLRs) which activate cellular signal transduction pathways after binding with LPS and trigger innate defence mechanisms.

The lyses of Gram-negative bacteria cause them to release LPS from the outer membrane of their cell wall. The monomer form of LPS binds to LPS-binding protein (LBP) forming LPS- LBP complex. LBP is a 60-Kd glycoprotein, synthesized in hepatocytes and released into the blood circulation (TOMLINSON and BLIKSLAGER 2004). LBP presents in normal human serum at a concentration of 5-10 µg/ml; this level increases to approximately 200 µg/ml in case of acute phase response (GUTSMANN et al. 2001). The low concentrations of LBP enhance LPS- induced activation of mononuclear cells while acute concentrations inhibit such effect (GUTSMANN et al. 2007). It facilitates the diffusion of LPS and catalyzes its binding to the receptor antigen CD14, either the membrane-bound form (mCD14) or the soluble form (sCD14) (YU et al. 1997; ULEVITCH andTOBIAS 1994; SZALMÁS 2000).

2.3.4.1 LPS pathway through mCD14-bearing cells

LPS-LBP complex is recognized by a receptor membrane-bound molecule, mCD14 or cluster differentiation antigen 14 and is bound to it through the lipid A portion. mCD14 is a 55-KD

glycoprotein (MAYEUX 1997; TOMLINSON and BLIKSLAGER 2004; GUTSMANN et al. 2007;

SZALMÁS 2000) linked to the membrane of myeloid cells (tissue macrophages and peripheral monocytes) by a glycosyl-phosphatidyl-inositol (GPI) anchor (YU et al. 1997). In case of mCD14-expressing cells, the GPI tail lacks a transmembrane region and can not transmit a signal into the cell (RICHARD 1999), so that mCD14 must conjugate to another cell membrane component in order to form a functional LPS receptor able to transmit signals (TOMLINSON

and BLIKSLAGER 2004; GUTSMANN et al. 2007). The toll like receptor (TLR) is a transmembrane structure (TOMLINSON and BLIKSLAGER 2004), expressed on the surface of the intestinal epithelial cells (CARIO et al., 2002) and approximately 9 to 10 different TLRs were identified in mammals (BRIGHTBILL and MODLIN 2000; HERSHBERG 2002), one of them is TLR4 which is the major receptor mediating the LPS signaling pathway (TOMLINSON and BLIKSLAGER 2004) in association with myeloid differentiation protein 2 (MD2) (BRIGHTBILL

and MODLIN 2000; DZIARSKI et al. 2001; WERLING and JUNGI 2003; SHIMAZU et al. 1999;

ABREU et al. 2001; 2003; ECKMANN 2004) that enabled TLR4 to respond to LPS molecule.

Activation of this complex results in triggering of a signal transduction through a serial of events including myeloid differentiation factor 88 (MyD 88), interleukin-1 (IL-1) receptor- associated kinase (IRAK) and TNF-α receptor-associated factor 6 (TRAF6).This is followed by activation of mitogen activated protein kinase (MAPK) pathway (WERLING and JUNGI

2003; ALEXANDER and RIETSCHEL 2001; CARIO et al. 2000) in which NF-кB-inducing kinase (NIK) is activated. The activated NIK activates IκB-α and β kinases resulting in proteolytic degradation of IκB and activation of the nuclear factor kappa B (NF-кB). NF-кB normally and in a steady state located in the cytoplasm in association with the inhibitory IκB protein in an inactive form (SCHEIDEREIT 1998). The activated transcription factor (NF-кB) then translocates to the nucleus of the cell (BRIGHTHILL and MODLIN 2000; CHANG and KARIN

2001; DAVIS 2000; ABREU et al. 2001; CARIO et al. 2000; ELEWAUT et al. 1999) and switches on gene expression for cytokines (i.e. activates the transcription of pro-inflammatory genes) and consequent release of proinflammatory mediators (HODGSON 2006) such as tumor necrosis factor-α (TNF-α) and interleukins (GUTSMANN et al. 2007) (Figure 8).

2.3.4.2 LPS pathway through sCD14

Another form in which GPI anchor is absent is sCD14. sCD14 is released in serum by mature phagocytes either by secretion or by proteolytic cleavage (ALEXANDER and RIETSCHEL 2001;

MAYEUX 1997) at concentrations of 3-4 µg/ml in normal human serum (BAŽIL et al. 1986;

SCHÜTT 1999). sCD14 affords recognition of LPS by non-CD14-bearing cells such as endothelial cells, dendritic cells, epithelial cells and smooth muscle cells ((SCHLETTER et al.

1995; ALEXANDER and RIETSCHEL 2001; MAYEUX 1997; FREY et al. 1992), but at the same time expressing toll-like receptor 4 (TLR4) and facilitate the systemic action of low serum LPS concentrations. While at higher concentrations, LPS follows the CD14-independent pathway (GUTSMANN et al. 2007).

TNF-α and IL-1β that are released in response to LPS cellular activation from CD14-bearing cells can in turn activate NF-κB even in CD14-non-bearing cells via cytokine receptors on the target cells such as IL-1R1 and TNFR1 or endothelial cytokine receptors and initiate inflammation and activate both the complement pathways and the coagulation pathway and this is considered as an indirect way for cellular response to LPS (VAN DE WALLE et al. 2008;

TOBIAS et al. 1997; STANCOVSKI and BALTIMORE 1997) (Figure 9).

Figure 8: A schematic diagram for the mechanism of action of LPS 2.3.4.3 LPS pathway and intestinal epithelial cells

Intestinal epithelial cells are the first line of defence against microbial invasion (TOMLINSON

and BLIKSLAGER 2004); at the same time the intestinal lumen contains many bacteria that the normal intestinal epithelial cells do not respond to. This could be explained by the intestinal epithelial cells express low levels of TLR4 and MD2 (ABREU et al. 2001; 2003) make the intestinal cells unresponsive to LPS. This can explain why LPS from commensally bacteria do not cause a frank inflammation to the exposed mucosa (ECKMANN 2004). So that increasing the expression of both TLR4 and MD2 is needed to make the intestinal epithelia responsive to LPS stimulation. It was found that interferon gamma (IFN-γ) increased the expression of MD2 and TLR4 in intestinal cell lines, also tumor necrosis factor alpha (TNF-α) increased the expression of TLR4 (ABREU et al. 2002; 2003).

The epithelial cells are devoid of membrane-bound form of CD14 (mCD14) antigen receptors (BÄCKHED et al. 2002) which is important for binding to LPS molecule and conveying it to TLR4-MD2 complex. So that there are two mechanisms suggested explaining the responsiveness of the intestinal cells to the LPS molecules. Firstly, it could be bound to LPS- LBP complex through the soluble form of CD14 (sCD14) forming sCD14-LPS-LBP complex then binding to the surface receptor resulting in cellular activation (ULEVITCH and TOBIAS

1995). Secondly, the LPS molecule could interact directly with TLR4 which are found on the apical surface of the intestinal epithelial cells (IMAEDA et al. 2002).

2.3.4.4 Neutralization of LPS

Neutralization of LPS results in inhibition in mononuclear cell-activation and thus reduces cytokine-release (GUTSMANN et al. 2001; NETEA et al. 1998). It is carried out via different pathways, including binding with LBP or sCD14 or by binding to the endogenous lipoproteins. LBP catalyzes the transfer of LPS directly to high-density lipoprotein (HDL) which is known to bind and inactivate LPS (WURFEL et al. 1994; FLEGEL et al. 1993).

Indirectly, LBP convey LPS to sCD14 which in turn transfers LPS to HDL (SCHÜTT 1999; DE

HAAS et al. 2000). While at low concentrations of LBP, it was suggested that serum amyloid P component (SAP) bound to HDL preventing its binding to LPS and played a role in balancing the amount of LPS (DE HAAS et al. 2000). Another protein, Bactericidal-permeability increasing protein (BPI) is found in neutrophil granules (ULEVITCH and TOBIAS 1995) and can bind to the cell wall of Gram-negative bacteria via the N-terminal region causing membrane disruption and subsequent death with releasing of its LPS. The released LPS then is bounded by the BPI protein and be neutralized (CANNY et al. 2002).