Induction of Oxidative Stress by Myco- and Endotoxins

Induction of oxidative stress by myco- and endotoxins

By Josep Garcia-Sirera, technical accountmanager, and Kevin Vanneste, product manager Agrimprove

Mycotoxins and endotoxins are naturally occuring toxins that pose a serious threat to animal production due to their negative impact on animal performance and health. Many negative effects are linked to their capacity to induce oxidative stress.

Different types of toxins

Mycotoxins are toxic compounds naturally produced by certain types of molds (fungi). Molds that can produce mycotoxins grow on numerous foodstuffs such as cereals, dried fruits, nuts and spices. Mold growth can occur either before harvest or after harvest, during storage, on/in the food itself and often under warm and humid conditions. Most mycotoxins are chemically stable and survive food processing. Generally known symptoms are vomiting, decreased feed intake and growth, depressed immunity, fertility problems and increased mortality.

Endotoxins or lipopolysaccharides (LPS) are structural components of bacteria. They are part of the outer membrane of Gram-negative bacteria, which are released mainly when bacteria are lysed, due to use of antibiotics, or because of the body’s defense mechanism. LPS consists of three structural elements (Figure 1). One is a hydro- phobic component, called lipid A, which serves to anchor the molecule into the membrane. The second is a core oligosaccharide and the third component is a hydrophilic O-polysaccharide projecting into the extracellular space of intact bacterial cells. More than 150 different variants of the third component are known.

Figure 1. LPS Structure Mechanisms of action

After ingestion of feed contaminated with mycotoxins, the gastrointestinal tract is the first target organ of mycotoxins. Effects on epithelial cells include, among others, changed mucus production, altered cytokine production, decreased cell proliferation and compromised intestinal barrier function. After crossing the epithelial cells, mycotoxins are taken up in the blood and transported to the liver. In the liver, mycotoxins are metabolized into secondary metabolites. Aflatoxin B1 for example is converted into Aflatoxin M1, Zearalenone (ZEA) is mainly converted into α-ZEA and β-ZEA. Although the liver is known to detoxify toxic components, the liver does not always succeed in detoxifying mycotoxins. α-ZEA for example is 100 times more toxic than the initial form. After passing the liver, mycotoxins are systemically distributed throughout the body impacting the immune system and all organs.

Endotoxins are normally not present in feed, but they are produced in the gastrointestinal tract when there is an overgrowth of Gram-negative bacteria. The physiological activities of LPS are mediated mainly by the Lipid A component of LPS. Lipid A is a powerful biological response modifier that can stimulate the mammalian immune system. LPS stimulate localized or systemic inflammation via the activation of receptors. Additionally, LPS and associated inflammation can regulate intestinal epithelial function by altering epithelial barrier integrity as well as nutrient transport and utilization

Induction of oxidative stress

The detrimental action of both endotoxins and mycotoxins have some common elements. One is the induction of oxidative stress. Under normal conditions free radicals or ‘Reactive Oxygen Species’ (ROS) are produced as part of standard metabolic processes. These highly unstable and chemically reactive molecules are rapidly eliminated by the natural antioxidant system in order to avoid potential damage. When exposed to mycotoxins and/or endotoxins however, cellular ROS concentrations exceed the level of naturally occurring antioxidants resulting in oxidative stress. Excess ROS will induce a damaging chain reaction causing serious damage to nucleic acids, proteins and lipids. As these components are the basic molecules in all metabolic processes, ROS directly affects viability of cells and consequently animal health and performance. Oxidative stress in animals can be determined using different parameters such as the half haemolysis time of blood cells (HT50), glutathione (GSH) and glutathione peroxidase (GSH-Px), malondialdehyde (MDA) or superoxide dismutase activity (SOD).

Table 1 shows the impact of mycotoxins on oxidative stress levels in piglets. Piglets exposed to the maximum allowed EU level of feed contamination with DON (0.9 ppm) experienced more oxidative stress as indicated by different blood parameters. These higher oxidative stress levels resulted in lower feed intake and growth performance and had a negative effect on profitability.

Table 1: Impact of mycotoxins on oxidative stress levels in piglets (Research center Agrifirm, The Netherlands)

Negative control 0.9 ppm DON

HT50 (min)* 90.92 87.51 (-3.8%) GSH-Px, U/mg protein** 6.26 6.62 (+5.8%) MDA, nmol/g protein*** 41.75 44.86 (+7.4%)

* HT50 is the time required to destroy or rupture 50% of blood cells exposed to a free radical attack as measured by the KRL-test. A lower HT50 indicates that blood cells have been exposed to higher levels of oxidative stress. ** MDA in blood is an end product of lipid peroxidation. Higher MDA blood values indicate more oxidative stress *** In its reduced form glutathione can neutralise free radicals converting glutathione to its oxidized form. The higher the ratio ‘oxidized over total glutathione’, the more oxidative stress the animal was exposed to. A similar effect on oxidative stress is shown in table 2. LPS injection resulted in reduced activities of SOD and GSH- Px in the jejunum of pigs. A greater (P < 0.05) content of MDA in the jejunal mucosa was observed in LPS group compared with control group.

Table 2. Effects of LPS injection on antioxidant enzyme activities and MDA content in jejunal mucosa of piglets (Chin et al., 2006)

Control LPS

SOD, U/mg protein* 95.85 + 3.13a 63.12 + 2.97b GSH-Px, U/mg protein** 72.11 + 3.51a 53.99 + 3.26b MDA, nmol/g protein*** 0.95 + 0.16a 1.51 + 0.25b a,b Different letters in one row indicate statistical significant differences (P<0.001) * SOD (superoxide dismutase) is an antioxidant enzyme responsible for the dismutation of superoxide radicals into oxygen and hydrogen peroxide. Lower SOD values indicate lower antioxidant capacity. ** GSH-Px (glutathione peroxidase) is responsible for the reduction of hydrogen peroxide to water and oxygen. Lower values of this enzyme equals a lower antioxidant activity *** MDA in jejunal mucosa is an end product of lipid peroxidation. Higher MDA values indicate more oxidative stress

Intestinal barrier function

After absorption or contact with epithelial cells, the gastrointestinal tract is highly impacted by the induction of oxidative stress by mycotoxins and endotoxins. Besides the negative effects on all cellular processes, oxidative stress has an enormous impact on intestinal barrier function (Figure 2). The intestinal barrier is mainly formed by a layer of epithelial cells covered with mucus. Tight junction proteins form a physical barrier between two adjacent epithelial cells preventing paracellular absorption of undesired substances such as toxins or pathogens. Oxidative stress has a negative effect on this intestinal barrier function due to the modification of certain cellular proteins. In vitro and ex vivo studies measuring trans-epithelial electrical resistance (TEER) have shown that DON and fumonisin B1 for example are able to increase the permeability of the intestinal epithelial layer of pigs and poultry. As a result, the compromised intestinal barrier function results in increased permeability for toxins, pathogens and feed-associated antigens. As per endotoxins, Aschenbach et al. (2003), used intestinal epithelial cell lines and concluded that an abnormal increase in luminal endotoxin induces cell apoptosis, which subsequently disrupts tight junction protein zonula occludens-1 as well as increases the production of nitric oxide, leading to increased mucosal permeability.

Figure 2. Oxidative stress results in im- paired intestinal barrier function

Countering toxins

Control of mycotoxins starts already in the field and with good quality control of raw materials. It seems however inevitable that mycotoxins contaminate feeds as shown by many research studies. Besides this, a good quality management system does not avoid the risk of endotoxins, which are naturally produced within the animals gas- tro-intestinal tract. A strategy of binding toxins, protecting the intestinal barrier function and supporting the animal to cope with toxins enables feed producers and farmers to avoid excessive oxidative stress caused by myco- and endotoxins resulting in optimal performance and peace of mind.

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