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 ¹⁴ C-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 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 1β, 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).