Can Mycotoxins Effect Gastro-Intestinal Tract Function?

Abstract

We often think of mycotoxin’s effect on the animal at dosages that would affect performance and cause visual lesions (e.g. oral and dermal lesions from T-2 toxin or fatty liver from aflatoxin). However, recent literature and work from our lab has implicated physiological and immunological effects at lower and more common levels of contamination that ultimately affects gastro-intestinal tract (GIT) functionality. These effects range from aflatoxin (AFLA) impacting endogenous nutrient loss from the intestinal tract, deoxynivalenol (DON) triggering tight junction protein degradation, and fumonisin (FUM) increasing coccidial lesion severity (in broiler chicks) and prolonged recovery due to alterations to regulation of inflammatory processes. Thus, this talk will address our historical to present knowledge of these three mycotoxins effects on the GIT.

Introduction

A healthy gastrointestinal tract (GIT) plays a vital role in ensuring the health and welfare of an animal. Being responsible for multiple crucial functions including gut barrier, nutrient digestion and absorption, gut immunity, and microbial activity, the GIT can consume 20% of all incoming energy of an animal (Cant et al., 1996). A great portion of the consumed energy can be attribute to the rapid protein turnover rate of intestinal cells, which can reach as high as 50 to 77% per day in chickens. The physiological and metabolic activity of the GIT determines the nutrient supply to all other tissues in the body, and also ensures both passive barriers and active immunological processes for pathogen clearance (Murugesan et al., 2015). As livestock production relies heavily on feed efficiency, the health of the GIT must be guaranteed in order to optimize the utilization of dietary nutrients and thus maximize the performance and welfare of the animal (Chen, 2016).

Although often neglected, the GIT is the first organ coming into contact with mycotoxins of dietary origin, and can be affected by mycotoxins with greater potency as compared to any other organs. Most of the mycotoxin absorption occurs at the upper part of the GIT, but passage of mycotoxins across the intestinal barrier varies considerably. In poultry, deoxynivalenol (DON) and fumonisins (FUM) are poorly absorbed at a rate of approximately 10 and 1%, respectively (as reviewed by Grenier and Applegate, 2013). Consequently, the entire gut is exposed to the remaining mycotoxins at high concentrations. Although aflatoxin B1 (AFB1) is highly absorbed in poultry (80%) and that the bioactivation of AFB1 to its toxic form aflatoxin B1-8,9-epoxide (AFBO) occurs primarily in the liver, a recent study revealed that this activation also takes place in the intestinal tract (Sergent et al., 2008). Meanwhile, enterohepatic cycling of mycotoxins can occur, allowing reentrance of absorbed toxins into the lower gut. Additionally, many of the mycotoxins are known as potent inhibitors of protein synthesis and activity through their interaction with DNA, RNA, and proteins, and thus the rapidly dividing intestinal enterocytes with high protein turnover can become a major target. Therefore, the GIT is likely at high risk of mycotoxin toxicity, yet our understanding on the gut health aspect of mycotoxicoses is not thorough. Based on a comprehensive review by Grenier and Applegate (2013), only 83 studies in total were published (as of 2013) on the topic of mycotoxins impact on intestinal processes; this included all mycotoxins on all animal species as well as in vitro studies. Nevertheless, recent literature and results from our work began to implicate that even at lower or more common levels of contamination, major mycotoxins, primarily DON, FUM, and AFB1, can lead to negative physiological and immunological changes on the GIT, including that on the intestinal barrier function, nutrient digestion and absorption, and gut immunity.

Mycotoxin on gut barrier function

As the first barrier against ingested contaminants, the intestinal barrier, formed primarily by tight junction complex, protects the luminal end of the intercellular space and regulates ions, water, and molecular transport through this paracellular route (Anderson and Van Itallie, 1995). The tight junctions (TJ) act as a fence that blocks the free diffusion of protein and lipids between the apical and basolateral membranes. Three integral proteins are components of TJ: claudin, occludin, and the junctional adhesion molecule (JAM). Among them, multiple isoforms of the claudin family are the key components of TJ and major determinants of paracellular characteristics (Gonzalez-Mariscal et al., 2003). Alteration in claudin expression and activity can lead to impairment of the tight junction network, and consequently increase permeability and allow higher translocation of luminal antigens (Chen, 2016).

The majority of previous research on this topic focused on the effects of DON, a commonly encountered mycotoxin produced by Fusarium.graminearum and Fusarium. Culmorum. According to a recent 3-year survey on worldwide occurrence of mycotoxins, DON presented in 59% of animal feed samples tested, with an average contamination concentration of 1,104 mg/kg (Rodrigues and Naehrer, 2012). Using intestinal segments from broiler chicks and laying hens in Ussing chambers, Awad and co-workers have consistently observed a reduced trans-epithelial electrical resistance (TEER), an indication of altered paracellular permeability, when exposed to DON ex vivo; similar effects were also noted in intestinal tissues from birds fed DON (Awad et al., 2004, 2005, 2007, 2008, 2009). Consistently, in Caco-2 cells, DON decreased claudin expression and impaired intestinal barrier (McLaughlin et al., 2004).

As pigs are known to be rather susceptible to DON, a recent study from our lab explored the effects of DON in porcine IPEC-J2 cells. When the cells were treated with graded concentrations of purified DON, 4000 ng/ml (which is equivalent to 4 mg/kg) DON significantly reduced TEER at 24 h; at 48 h, 2000 ng/ml DON also exerted significant effect. Correspondingly, the FITC-dextran flux, representing the paracellular tracer flux, was significantly increased by 4000 ng/ml DON, indicating impaired gut barrier function. In the same study, the mRNA expression of claudin1, 3, and 4 were increased by 1000 to 4000 ng/ml starting at 24 h, while its protein expression decreased with increasing DON. Results of the time-response curve showed that reduction of protein expression stated to decrease at 2 h, while their respective gene expression started to increase from 4 to 6 h after treatment, suggesting that the increased claudin mRNA expression is a compensation effect to the disrupted protein synthesis and/or activity. A subsequent experiment using ubiquitin inhibitor revealed that DON inhibited the claudin protein expression by increasing the ubiquitin-facilitated protein degradation. The question remains as to if protein synthesis pathways are also affected, how DON affects barrier integrity, and what signaling pathways are involved. Interestingly, when DOM-1, an inactivated metabolite of DON, was used as the treatment, gene expression and protein expression of tight junctions were not affected, suggesting that degrading DON to DOM1 in animal feed may be an effective approach to minimize the adverse effects of DON on intestinal barrier (Zhao et al., unpublished data).

Although AFB1 is highly prevalent and is considered the most toxic mycotoxin worldwide to livestock animals, studies exploring AFB1’s effects on gut barrier has been lacking. The only available literature was an in vitro study, where the researchers found that AFB1 impaired the integrity of Caco-2 cells by measuring the trans-epithelial resistance (Gratz et al., 2007). To fill in the gaps, a recent study from our lab assessed the effects of cultured AFB1 on gut permeability in 20 d broilers using the dual sugar gut permeability test. Briefly, birds were given a non-metabolizable sugar solution of one mL (0.25 g/mL lactulose and 0.05g/mL L-rhamnose dissolved in dd H2O) by oral gavage, and blood was collected after 1 h of gavage. The serum concentration of lactulose and L-rhamnose were then determined. Because lactulose is absorbed via the paracellular pathway when tight junctions are impaired while rhamnose is absorbed transcellularly in the small intestine, an increase in blood lactulose:rhamnose (L:R) ratio is an indication of increased gut permeability. In this study, 1.5 mg/kg AFB1 showed a significant main effect of increasing the L:R ratio (P = 0.04), which is a direct indication that feeding AFB1-contaminated diet can lead to impaired intestinal barrier in vivo. Therefore, increased entrance of luminal antigens, including unabsorbed mycotoxins, is allowed; consequently, higher susceptibility to various infections would not be a surprise. Additionally, AFB1 also affected jejunal claudin 1 and 2 mRNA expression in vivo. These results suggest a high possibility that either the non-absorbed AF or the AF metabolites that are secreted back into the gut through the entero-hepatic circulation can have a direct impact on the gut epithelium, and thus are partly responsible for the physiological and metabolic disorders during aflatoxicosis. In addition, an increased gut permeability may also facilitate the absorption of any presented mycotoxins through the paracellular route that are normally absorbed at a less efficient rate, leading to synergistic toxicity effects when the feed is contaminated with multiple mycotoxins (Chen et al., 2016).

Fumonisins (FUM), derived from Fusarium spp, are another major group of mycotoxins which have strong structural similarity to sphinganine, the backbone precursor of sphingolipids, and thus are known to lead to disruption of sphingolipid metabolism, resulting in impaired membrane formation. Fumonisin B1 (FB), the most toxic form of FUM, has been reported to lead to reduced TEER (Bouhet et al., 2004), in addition to reduced tight junction protein expression in pig ileum (Bracarense et al., 2012). Although the body of literature is still relatively small, especially for FUM and AFB1, current evidences suggest that gut barrier integrity can indeed be disrupted by major mycotoxins through interfering with protein synthesis, protein degradation, or membrane formation. Clearly, more in vivo studies are required in different animal species to better understand the effect of each mycotoxin as cell culture models may not perfectly mimic the complex in vivo conditions (e.g. enterohepatic recycling). Also, conclusions from mRNA expression data should be drawn carefully, as our recent study showed that increased tight junction mRNA expression upon DON exposure did not represent increased protein expression, but rather, a compensation effect to the disrupted protein activity.

Mycotoxins on nutrient digestion and absorption

Mycotoxin-induced disturbance of digestive enzyme and nutrient transporters may lead to intestinal disorders, resulting in alteration of nutrient digestibility and growth of animals. Previous researchers have noted changes in pancreatic digestive enzyme activities and nutrient digestibility upon AF exposure, yet it is difficult to reach a consensus based on the available data. Han et al. (2008) observed increased digestive enzyme activities (protease, amylase, chymotrypsin, and trypsin) yet decreased apparent digestibility of crude protein in 42 d ducks fed 0.02 and 0.04 mg/kg AFB1. Conversely, laying hens fed up to 2.5 mg/kg AFB1 for 14 d did not show altered apparent digestibility of dry matter and N, but apparent digestible energy was significantly reduced in those hens compared to control (Applegate et al., 2009). The discrepancies present in the literature can be due to the differences in experimental animals (species, genetic lines, and age), source and concentration of AF, exposure time, nutritional composition of the diets, sampling site, etc.

On the other hand, accurate estimation of protein and amino acid digestibility requires the correction for endogenous losses; the latter include gastric, pancreatic, and biliary secretions, sloughed intestinal cells, and mucosal cells. This endogenous loss might be altered with changes in health conditions induced by AFB1, hence, evaluation of the influence of AFB1 on endogenous amino acid loss is required in order to provide a more accurate estimation of nutrient digestibility during aflatoxicosis (Chen, 2016). In our recent study of Chen et al. (2016), the endogenous N and amino acid loss during aflatoxicosis was first determined; subsequently, standardized N and amino acid digestibility was calculated, which is a more accurate estimation of birds’ digestion capacity compared to apparent values. Results indicated that diet AFB1 contamination at 1.5 mg/kg has the potential of increasing endogenous N loss in broilers by 22%; similarly, amino acid losses from the AFB1-treated birds were all numerically higher than those of control birds by approximately 20 to 30%. Exposure to AFB1 can also significantly reduce standardized ileal N, amino acid, and energy digestibility. During aflatoxicosis, the increased endogenous N flow may come largely from sloughed mucus layer. As the major constituent of the mucus layer, mucin secretion is shown to be increased by anti-nutritional factors through abrasive interactions with the mucus layer (Montagne et al., 2000). Additionally, increased secretions from the pancreas and small intestine are likely to contribute to the N flow during AF exposure, as previous researchers suggested that pancreatic damage during aflatoxicosis may cause an increased release of proenzymes from pancreatic cells to the intestinal tract (Han et al., 2008; Matur et al., 2010). The intestinal epithelium cells have a very high protein turnover rate, thus it is essential that adequate substances are provided for the protein synthesis. However, a reduced N and amino acid digestibility and reduced ileal digestible energy may lead to an insufficient nutrient and energy supply to the intestinal cells, thus affecting normal activities and functions of these cells. Pastorelli et al. (2012) suggested an increased maintenance requirement of the gut upon challenges, which include metabolic costs for repairing damaged tissues and stimulation of the immune functions. Therefore, the increased maintenance cost along with the decreased ability of animals to utilize dietary nutrients are indeed factors that lead to the array of metabolic disturbances during aflatoxicosis (Chen et al., 2016).

Information regarding the effects of DON and FUM on nutrient digestion is relatively limited; nevertheless, several previous studies have looked at how they modulate nutrient absorptive processes. Reduced small intestinal villi height is mostly found after Fusarium toxins (FB and DON) challenge (Awad et al., 2006, 2011; Yunus et al., 2012; Girgis et al., 2010), which indicated intestinal epithelial damage, and may lead to decreased absorption of dietary nutrients. Consistently, utilizing the Ussing Chamber method in cell culture models, multiple studies have revealed that FB and DON could interfere with glucose absorption (Maresca et al., 2001, 2002; Lessard et al., 2009). Maresca et al. (2002) demonstrated that the inhibition of nutrient uptake by DON is through a specific modulation on intestinal transporters rather than nonspecific cell damage. Compared to DON and FUM, studies on AF effect on nutrient absorption is still scarce and not yet conclusive. Applegate et al. (2009) reported no changes in jejunum villus height in laying hens upon AFB1 challenge, while Wan et al. (2013) noticed decreased villus height and villus/crypt ratio in ducklings. In broiler chicks, AFB1 had no effect on jejunal villi histology (Chen et al., 2016). However, a very consistent trend of increased mRNA expression of jejunal peptide and amino acid transporters were observed in broiler chicks upon AFB1 challenge, both on the brush boarder side (b0,+AT, EAAT3, PepT1, rBAT) and the basolateral side (yLAT1 and 2). This might be a compensatory effect of transporter gene expression on two levels. First, a higher mRNA production is needed to increase translation process in order to restore the possible impaired protein activities by AFB1 or AFBO. Second, a higher absorption rate is necessary to compensate the decreased amino acid digestibility. In addition, this may also suggest an increased requirement for amino acids absorption and subsequent protein synthesis during aflatoxicosis (Chen et al., 2016).

In addition to individual mycotoxin, contamination of multiple mycotoxins is not uncommon and evaluation of multi-contamination effects in livestock animals is necessary. According to a worldwide mycotoxin survey from 2004-2013 (Streit et al., 2013), 72% feed and feed ingredient samples contained at least one mycotoxin and 38% contained multiple mycotoxins out of the 17,316 samples tested. In laying hens, feeding a diet contaminated with both AF and ochratoxin A resulted in a more pronounced effect on reducing dietary metabolizable energy than when either toxin was fed alone, which occurred through a significant increase in the maintenance energy requirement (Verma et al., 2007). Feeding hens a diet naturally contaminated with multiple Fusarium mycotoxins also led to further decreased nutrient digestibility and metabolizable energy (Danicke et al., 2002). In broiler chicks that were challenged with both DON and FB, a synergistic effects of DON and FB were observed on reducing N digestibility (Grenier et al., unpublished). Evidently, multi-contamination poses a bigger challenge for animals because simultaneous exposure may potentially lead to synergistic interactions. Thus there is a need to further assess their interactions in animals to better understand whether a particular combination may lead to antagonistic, additive, or synergistic effects. However, caution must be taken when conducting such studies, especially when evaluating nutrient digestion and absorption, that the results must not be confounded by the effects of mold(s) on the nutrient and energy content of the feed ingredient (Murugesan et al., 2015).

Mycotoxin on gut immunity

Besides nutrient digestion and absorption, the GIT also plays essential roles in immune responses as up to 70% of the immune defenses are located in the GIT (Grenier and Applegate, 2013). The major players in the intestinal immune responses include the GALT (gut-associated lymphoid tissue), Peyer’s patches (aggregated lymphoid nodule located in ileum), mesenteric lymph nodes, and cecal tonsils, which are responsible for producing immunocompetent cells upon infection. Additionally, localized responses along the GIT are facilitated by the mucus, intraepithelial immune cells, and epithelial cells (Grenier and Applegate, 2013). Although the immunosuppressive property of AF is well accepted, the modulation of GIT immune system has been less studied. The study by Watzl et al. (1999) in which no significant effects on the GALT and no damage on the intestinal epithelium were found in rats fed 1 mg/kg AFB1 represents the only published result regarding the direct effect of AF on gut immunity, while such effects have not been explored in poultry species. Considering the potent toxicity of AFB1 and its inhibitory effect on protein synthesis and activity, it is expected that feeding AF-contaminated feeds can interfere with gut immune responses similar with or more intense than those seen with other mycotoxins, and thus awaits further research. Indeed, a very recent study by Jiang et al. (2015) first revealed that AF exposure (0.6 mg/kg) can decrease the intestinal IgA cell numbers and negatively affect the mRNA expression of IgA, pIgR, IgM, and IgG in 21 day-old broilers.

In the meantime, studies using other mycotoxins have revealed modulation on the immune balance and intestinal immune responses during parasitic, bacterial, and viral infections. Altered cytokine expression within the intestine upon exposure to DON in vivo, ex vivo, or in vitro has been documented, where a consistent trend of upregulated pro-inflammatory cytokines (IL--1β, Il-6, IL-8, and TNF-α) as well as Th1, Th2, and T-regulatory cytokines can be observed (Grenier and Applegate, 2013). Similar responses have also been noted in pigs exposed to FUM (Bracarense et al., 2012). Thus the question remains as to whether mycotoxicoses may also influence the animal’s ability to mount an effective and timely immunological response to infections. Antonissen et al. (2014) examined the effect of DON on the development of necrotic enteritis (NE), a disease perpetuated from initial intestinal damage and secondary exposure to the gram positive bacterium Clostridium perfringens, in broiler chicks, and found that a low level of DON (3 to 4 mg/kg) increased the percentage of birds with NE from 20 to 47%. The authors concluded that DON predisposed the broilers to NE by reducing the intestinal epithelial tight junction integrity, thus increasing plasma protein/amino acid leakage into the intestinal lumen which provides the necessary nutrients for Clostridium perfringens proliferation. Similarly, broiler breeder pullets fed diets containing Fusarium mycotoxins (at a concentration that was lower than that could negatively affect performance) showed impaired intestinal recovery from enteric coccidial lesions (Girgis et al., 2010), whereas broilers exposed to Ochratoxin A had higher lesion and oocyst in the intestine upon coccidial challenge by Eimeria acervulina (Koynarski et al., 2007 a, b) and higher number of Salmonella typhimurium in duodenum and cecum, with the presence of acute enteritis (Fukata et al., 1996) compared to control birds. In a recent study by Grenier et al.

(unpublished), when broilers were fed a co-contaminated diet with DON and FB at subclinical concentrations (1.5 mg/kg DON and 20 mg/kg FB) while challenged with Eimeria spp, intestinal lesions and oocysts number in jejunum of challenged birds were more severe in birds fed mycotoxins than birds fed a control diet. Interestingly, the type of interaction of DON and FB was highly dependent on the endpoint evaluated, where synergistic effects were seen on nutrient digestibility, while additive effects were found on multiple pathogenicity markers (intestinal lesion and oocyst number) as well as markers for gut inflammation (IL-8, IL-10, and SOCS1).

Antagonistic effects were also observed on 3 endpoints assessed. The authors suggested while endpoints were crucial when investigating the interactions of mycotoxins, other factors may also influence the outcome, which may include animal species, exposure age, and concentrations used. Based on their results, even mycotoxins from the same fungi origin and similar mode of action may result in antagonistic effects; further experiments using different mixture of mycotoxins are therefore needed to better understand the effects of mycotoxin interaction.

Summary

Although the body of literature on mycotoxins’ effects on the GIT functionality is still relatively small, current evidence clearly suggest a direct and/or indirect impact of several major mycotoxins on gut barrier integrity, nutrient digestion and absorption, and gut immunity. The main mechanisms by which DON, AF, and FUM affect growth through the gut may include 1) interfering with protein synthesis and degradation, leading to impaired tight junction protein complex, and therefore impaired gut barrier; 2) increasing endogenous nutrient loss and interfering with digestive enzyme synthesis and/or activities, leading to reduced nutrient digestibility; 3) damaging intestinal villi and disrupting the normal activity of nutrient transporters, resulting in reduced nutrient absorption; 4) altering cytokine expression and providing nutrients for luminal pathogen proliferation (through impaired gut barrier which increases plasma nutrient leakage), resulting in an amplification of the severity of infection; 5) increasing the maintenance requirement of the gut while sparing nutrients for immune function needs, and thus decreasing nutrient availability.

Notably, the concentrations used in many of these aforementioned studies are considered low or sub-clinical. For instance, in several studies, DON at concentrations ≤ 4 mg/kg showed a significant negative effect on barrier integrity and nutrient digestibility, and increased the birds’ susceptibility to multiple infections in poultry species. However, poultry were traditionally considered quite tolerant to DON; the EU maximum guidance level of DON for poultry feed is 5 mg/kg. Therefore, these mycotoxins at even lower concentrations that may not lead to significant growth impairment may still pose a risk to GIT functions and may predispose the animals to other infections, leading to greater production loss. Clearly, this gut health aspect of mycotoxicoses is not yet fully understood and deserves further research. In particular, more in vivo studies are required to elucidate how mycotoxins modulate barrier functions and nutrient digestive and absorptive processes. Also, as it is a common practice to include multiple feedstuffs in practical diets, the risk of simultaneous exposure to multiple mycotoxins increases in the field compared to research settings. Future research should elucidate feeds that are co-contaminated to account for mycotoxin interactions.

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